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Basal forebrain cholinergic neurons: regulation by dopamine and responses to arousing stimuli Day, Jamie Catherine 1994

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BASAL FOREBRAIN CHOLINERGIC NEURONS: REGULATION BY DOPAMINEAND RESPONSES TO AROUSING STIMULIByJAMIE CATHERINE DAYB.Sc., University of Victoria, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESProgram in NeuroscienceWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAAugust 1994©Jamie Catherine DayIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)__________________________________Department of .6l44ufl tt n AJ..Q1MSC €A’(CQThe University of British ColumbiaVancouver, CanadaDate /kLkL2JAJi 1 (qc1LtDE-6 (2)88)11ABSTRACTThe regulation of forebrain cholinergic systems, specifically those neurons in thecholinergic basal nuclear complex (CBC) which project to the hippocampus and cortexare of great interest, given the involvement of acetyicholine (ACh) in cognitive function.To assess the activity of CBC neurons, in vivo microdialysis has been used in the presentexperiments to measure ACh release in the hippocampus and cortex of freely movingrats.Dialysate concentrations of ACh in the hippocampus and cortex (and striatum) offreely moving rats were found to correlate positively and significantly with locomotoractivity, a behavioural measure of arousal. Two arousing stimuli, injection of vehicleand onset of the rats’ dark phase, increased locomotor activity and ACh release in allthree brain regions, as did injection of the muscarinic antagonist scopolamine. Thesedata suggest that forebrain cholinergic neurons are responsive to arousing stimuli andthat ACh release in the crtex, hippocampus and striatum generally correlates witharousal.The dopaminergic regulation of CBC neurons was examined by determining theextent to which dopamine (DA) receptor agonists and antagonists affect cortical andhippocampal ACh release. The indirect DA agonist d-amphetamine (AMPH) and theDA receptor agonist apomorphine increased ACh release in both the cortex andhippocampus as did the selective D1 receptor agonist CY 208-243. D2 receptor agonists(quinpirole and/or PHNO) had no effect on ACh release in the cortex and producedslight decreases in the hippocampus. In addition, the AMPH-induced increases in AChrelease in both regions were attenuated more by the D1 receptor antagonist SCH 23390than by the D2 antagonists haloperidol and/or raclopride, as was the apomorphineinduced release of ACh in the cortex. That DA mediates AMPH-induced increases incortical ACh release was supported by the finding that prior selective lesions ofUiascending dopaminergic but not noradrenergic systems attenuated this effect of AMPH.These results suggest that CBC neurons are regulated in an excitatory manner by DAacting primarily at D1 receptors.The extent to which ACh release in the cortex and hippocampus is related to theperformance of a learned behavioural task was assessed in rats trained to anticipate andconsume a palatable liquid diet. Hippocampal ACh release increased during theanticipatory and consummatory periods of the task, but the increase observed in ratstrained with the liquid diet was not higher than the increases seen in rats trained withwater or in naive rats. In contrast, cortical ACh release increased to a greater extent inrewarded rats than it did in the two control groups. This suggests that cholinergicactivity in both the cortex and hippocampus is increased by a reward-independent aspectof the task, such as arousal or attention, while an additional reward-dependentcomponent is seen with respect to cortical ACh release.ivTable of ContentsPageAbstract iiTable of Contents ivList of Tables viList of Figures viiAcknowledgments ixI. Introduction 1(A) The Role of Acetylcholine in the CentralNervous System 1(B) In Vivo Microdialysis and Assay of ACh 7II. Acetyicholine Release in the Rat Hippocampus, Cortex andStriatum Correlates with Behavioural Arousal 13(A) Introduction 13(B) Materials and Methods 14(C) Results 18(D) Discussion 26III. Dopaminergic Regulation of Cortical Acetyicholine Release 35(A) Introduction 35(B) Materials and Methods 38(C) Results 39(D) Discussion 48IV. Attenuation of Amphetamine-Induced Increases of CorticalAcetyicholine Release by Forebrain Depletions of Dopamine,but not Noradrenaline 57(A) Introduction 57(B) Materials and Methods 58(C) Results 63(D) Discussion 70V. Dopaminergic Regulation of Hippocampal Acetyicholine Release 75(A) Introduction 75(B) Materials and Methods 77(C) Results 78(D) Discussion 84VII. Selective Enhancement of Cortical, but not Hippocampal,Acetyicholine Release during the Anticipation and Consumptionof a Palatable Meal 89(A) Introduction 89(B) Materials and Methods 90(C) Results 93(D) Discussion 101VPageVII. General Discussion 107(A) Technical Considerations of CholinergicResearch 107(B) Regulation of Basal ForebrainCholinergic Neurons 110VIII. References 114viList of TablesPageTable I Probe characteristics and baseline output of ACh and Chin the striatum, hippocampus and frontal cortex of rats 19Table 2 Correlation of regional ACh outputs and activity countsduring experimental treatments 25Table 3 Effects of 6-OHDA lesions of the mesotelencephalicdopamine system on regional tissue concentrations ofdopamine, noradrenaline and serotonin 64Table 4 Effects of 6-OHDA lesions of the dorsal noradrenergicbundle on regional tissue concentrations of dopamine,noradrenaline and serotonin 65Table 5 Consumption and latency to drinking during dialysis 96Table 6 Average acetylcholine release during anticipatory andconsummatory periods 98vi.’List of FiguresPageFigure 1 Schematic of in vivo microdialysis with on-line assay for ACh,and steps in the enzymatic conversion and electrochemicaldetection of ACh 12Figure 2 Dialysis probe placements in the frontal cortex, dorsalstriatum and dorsal hippocampus 16Figure 3 Striatal dialysate values of ACh and Ch after injections ofvehicle and scopolamine and exposure to dark 20Figure 4 Hippocampal dialysate values of ACh and Ch after injections ofvehicle and scopolamine and exposure to dark 21Figure 5 Frontal dialysate values of ACh and Ch after injections ofvehicle and scopolamine and exposure to dark 22Figure 6 Dialysate ACh concentrations and activity counts from threeindividual rats after injections of vehicle and scopolamineand exposure to dark 24Figure 7 Frontal cortex dialysate values of ACh after injections ofd-amphetamine, apomorphine or vehicle, and after localadministration of d-amphetamine through the dialysis probe 40Figure 8 Frontal cortex dialysate values of ACh after injections ofquinpirole and PHNO 41Figure 9 Frontal cortex dialysate values of ACh after injections ofCY 208-243 or a combination of quinpirole and CY 208-243 42Figure 10 Frontal cortex dialysate values of ACh after injections ofSCH 23390, d-amphetamine and vehicle, alone or in combination 44Figure 11 Frontal cortex dialysate values of ACh after injections ofhaloperidol or raclopride, d-amphetamine and vehicle, aloneor in combination 45Figure 12 Frontal cortex dialysate values of ACh after injections ofSCH 23390 or raclopride and apomorphine 46Figure 13 Tetrodotoxin-sensitivity of cortical ACh recovered by atransverse dialysis probe made of acrylonitrile fibre 67Figure 14 Dialysate values of ACh in the frontal cortex of rats havingunilateral 6-OHDA lesions of the mesotelencephalic dopaminesystem or of control rats after injection of d-amphetamine 68Figure 15 Dialysate values of ACh in the frontal cortex of rats havingbilateral 6-OHDA lesions of the dorsal noradrenergic bundleor of control rats after injection of d-amphetamine and vehicle 69vi”PageFigure 16 Hippocampal dialysate values of ACh after injection of apomorphine 79Figure 17 Hippocampal dialysate values of ACh after injections ofCY 208-243, quinpirole or vehicle 80Figure 18 Hippocampal dialysate values of ACh after injections ofd-amphetamine, SCH 23390, raclopride or vehicle, alone orin combination 82Figure 19 Effect of d-amphetamine applied through the dialysis membraneon hippocampal ACh output 83Figure 20 Volume of Sustacal consumed and latency to drink by trainedrewarded rats 95Figure 21 Dialysate concentrations of ACh in the hippocampus of trainedrewarded rats, trained non-rewarded rats and naive rats duringthe anticipatory and consummatory periods of the task 99Figure 22 Dialysate concentrations of ACh in the cortex of trainedrewarded rats, trained non-rewarded rats and naive rats duringthe anticipatory and consummatory periods of the task 100ixAcknowledgmentsIn addition to being a daunting role-model, my supervisor Dr. H.C. Fibiger hasprovided an atmosphere of scientific excellence in the Department of Psychiatry,Division of Neurological Sciences at U.B.C. My experiences in this department haveincluded: training by Dr. Geert Damsma, one of the world’s experts in AChmicrodialysis; superb technical (and moral) support from Chui-Se Tham and CatrionaWilson; statistical assistance from Dr. Campbell Clark; and valuable discussions andcollaborations with fellow students and postdoctoral fellows including Drs. Elio Acquas,Erin Brown, Lilli Collu, Fiona Inglis, Mark Klitenick, George Nomikos, GeorgeRobertson, and Julie Williams. I sincerely thank each of these people.My research has been supported by a Medical Research Council of CanadaStudentship. Some of the drugs for the experiments included in this thesis were kindlydonated by Astra, McNeil, Merck Sharp and Dohme, Sandoz, and Schering Plough.For my mother, Barbara Day, for always expecting my bestFor my husband, Alan Matheson, for accepting me at my worstFor Geert, an inspiring scientist and friend who is and will be greatly missed1I. INTRODUCTION(A) THE ROLE OF ACETYLCHOLINE IN THE CENTRAL NERVOUS SYSTEMAs a result of Loewi’s classic experiments in the early 1920’s, acetylcholine (ACh)was the first chemical demonstrated to act as a neurotransmitter. These studies involvedstimulating the vagus nerve of a frog, causing a decrease in heart rate, while perfusingthe heart with a physiological solution. The solution recovered, when perfused througha denervated frog heart also decreased stimulation-induced heart rate, suggesting that ahumoral factor was responsible for the effect of the nerve stimulation. This factor,termed “vagusstoff”, was later identified as ACh. Eccles subsequently demonstrated inthe 1950’s that ACh could also act as a transmitter in the mammalian central nervoussystem (CNS). Dale had previously identified cholinergic transmission at theneuromuscular junction; based on this information and the famous Dale’s principle,Eccles reasoned that ACh must also be the. neurotransmitter responsible for thecommunication between the spinal motorneuron and the electrophysiologically identifiedRenshaw interneuron. These landmark studies were among the first of a great body ofresearch on cholinergic systems which has helped to establish many of the basicprinciples of synaptic function and neurotransmission (see Karczmar, 1993).Since these early studies, extensive research into the anatomy, chemistry,pharmacology and physiology of central cholinergic systems has yielded several wellaccepted principles. For example, cholinergic neurons are now known to innervate muchof the mammalian brain: nuclei in the brainstem send projections mainly to thethalamus and other diencephalic structures, neurons in the forebrain innervate the entirecortical mantle including the hippocampus and amygdala, and intrinsic cholinergicinterneurons are found within the basal ganglia (Semba and Fibiger, 1989). Cholinergicneurons can be defined by the presence of the enzyme choline acetyltransferase (ChAT),2which catalyzes the synthesis of ACh from choline and acetylcoenzyme A (Tuek, 1984).Early investigations of cholinergic actions led to the discovery of two classes of receptorsfor ACh, nicotinic and muscarinic, which have more recently been subdivided basedboth on pharmacology and molecular biology (Ladinsky, 1993). The neurophysiologicaleffects of ACh, mediated by nicotinic cation channel/receptor macromolecules and/or byG—protein coupled muscarinic receptors, are determined by the cellular location of thereceptors and the specific second messenger systems and effectors to which they arecoupled (Krnjeviã, 1993).Given the complex anatomy of central cholinergic systems, the diversity ofreceptors which bind ACh, and the variety of ACh’s electrophysiological effects, it isnot suprising that ACh has complex and diverse roles in behaviour. Indeed, AChneurotransmission has been reported to participate in a variety of behavioural functionsincluding circadian rhythms (Rusak and Bina, 1990), antinociceptiOn (Iwamoto, 1989;Klamt and Prado, 1991), locomotion (Brudzynski et at., 1991; Flicker and Geyer, 1982),regulation of the sleep/wake cyOle (Baghdoyan et al., 1993; Steriade et a!., 1991), stress(Gilad, 1987; Imperato et a!., 1991) and electrocortical arousal (Semba, 1991). Stimulatedby the discovery that a consistent correlate of Alzheimer’s dementia is a loss ofcholinergic neurons in the basal forebrain (Coyle ci al., 1983), an intensively studiedfunction of ACh has been its proposed role in learning and memory. Along with thiscorrelational evidence of cholinergic loss in Alzheimer’s dementia, studies have shownthat administration of anticholinergic drugs to animals or humans or lesions of forebrainregions containing cholinergic cell bodies in animals cause cognitive impairments, whichmay be ameliorated by cholinergic agonists (Collerton, 1986; Hagan and Morris, 1988).These findings have supported a cholinergic hypothesis of cognition’, and haveencouraged much research into possible cholinergic therapies for the treatment ofcognitive deficits.3The excitement generated by this hypothesis has been tempered by the overallineffectiveness of cholinergic therapies in treating dementia (Holttum and Gershon,1992) and by criticisms of the above-mentioned supporting experimental evidence. Forexample, one major confound of the axon-sparing neurotoxic lesions used in animals tomodel Alzheimer’s dementia is that non-cholinergic neurons in the vicinity of the targetcholinergic neurons are lesioned as well (Arbogast and Kozlowski, 1988; Dunnett et al.,1987; Everitt et a!., 1987). In fact, the extent to which excitotoxic lesions of the basalforebrain decrease cortical ChAT activity does not correlate with the severity of theresulting behavioural deficits in several learning and memory tasks (Dunnett et al.,1987). Specifically, quisqualic acid lesions of the basal forebrain produce largerdecreases in cortical ChAT, but less severe deficits in the acquisition and performance ofseveral behavioural tasks, than do ibotenic acid lesions (Markowska et al., 1990; Robbinset a!., l989b). This suggests that the observed lesion-induced effects may be largelynon-cholinergic in origin. Along with the lack of a selective cholinergic neurotoxin,several other shortcomings of the data supporting the cholinergic hypothesis of dementiahave been outlined (Fibiger, 1991), including the idea that regionally specific effects ofperipherally administered cholinergic drugs have not been considered.Defining a cholinergic role in cognition is also complicated by the inherentdifficulties in designing and interpreting the results of behavioural tasks. It is possible,for example, that the influence of ACh on cognition may be more accurately defined bythe participation of forebrain cholinergic neurons in attentional processes rather than inlearning and memory per Se. Attention has various operational definitions but isgenerally assessed by a subject’s ability to use sensory information to follow some rule orcontingency; whereas a learning and memory task might assess the acquisition, retentionor recall of this rule, an attention task assesses the subject’s ability to follow the rule insituations where the initially salient sensory cue is unexpectedly switched to a new cue(attention reversal) or where the salient cue is presented among distractions (selective4attention). In support of a cholinergic involvement in attention, cholinergic agonistsimprove such measures of attention in Alzheimer’s patients (Sahakian et at., 1989; 1993)and the cholinergic receptor antagonist, scopolamine, disrupts attention in normal humansubjects (Dunne and Hartley, 1985; Wesnes and Warburton, 1983). Additionally,excitotoxic lesions or pharmacological inhibition of the basal forebrain in animals causesimpairments in attentional tasks (Olton et at., 1988; Pang et at., 1993; Robbins et at.,1989a; Voytko et al., 1994) which, in some cases, have been ameliorated by cholinergicagonists or tissue grafts (Muir et at., 1992a; 1992b; 1994). It is important to note,however, that these experiments suffer from the same limitations as those describedabove (for example, lack of a selective cholinergic neurotoxin), and that contradictorydata have also been reported (Roberts et at., 1992). It should also be noted that theseresults do not exclude a role for basal forebrain cholinergic neurons in learning andmemory. It has recently been suggested, for example, that excitotoxic lesions of basalforebrain regions more rostral than those which cause attention deficits may impairmemory (Muir et at., 1993).Learning, memory and attention are complex cognitive constructs, operationallydefined using behavioural measures. In contrast, electroencephalographic (EEG) arousalis an electrophysiological measure, defined by low voltage fast activity (LVFA), ordesynchrony, in the cortex and rhythmic slow activity (RSA), or theta rhythym, in thehippocampus. The importance of ACh in the generation of these averaged electricalpatterns is well supported and has been reviewed (Buzsáki and Gage, 1989; Riekkinen etat., l991a; Semba, 1991; Smythe et at., 1992; Vanderwolf, 1988). Briefly, the activity ofpresumed cholinergic neurons in the basal forebrain is increased during EEG arousal(Détári and Vanderwolf, 1987; Sweeney et a!., 1992); stimulation of cholinergic cell bodyregions induces LVFA (Metherate et a!., 1992); RSA can be induced or blocked byadministration of cholinergic agonists or antagonists, respectively, into the hippocampus(Golebiewski et at., 1992; Rowntree and Bland, 1986) or the basal forebrain (Monmaur5et a!., 1993); cortical ACh release is increased during LVFA (Casamenti et a!., 1986;Celesia and Jasper, 1966; Kanai and Szerb, 1965); and cholinergic antagonists or lesionsof cholinergic cell body regions disrupt EEG arousal (Buzsâki et a!., 1988; Ray andJackson, 1991; Riekkinen et a!., 1992) which in some cases can be ameliorated bycholinergic agonists (Riekkinen et a!., 1991a Vanderwolf et a!., 1993).Although the critical significance of ACh in EEG arousal is well established, themechanisms by which ACh and other neurotransmitters interact to create theelectrophysiological phenomena underlying LVFA and RSA are complex and still beingelucidated (Foote and Morrison, 1987; Smythe et a!., 1992, Steriade et a!., 1993;Vanderwoif, 1988). In addition, the behavioural significance of EEG arousal has notbeen definitively determined. It should be noted, for example, that LVFA and RSAoccur during rapid eye movement (REM) sleep. In the waking state, however, EEGarousal occurs during voluntary movement in laboratory animals (Vanderwolf, 1988) andduring periods of facilitated sensory processing (Livingstone and Hubel, 1981; Simon andEmmons, 1956). It is thus often speculated, but not universally accepted (Vanderwolf,1988), that EEG arousal may be a physiological representation of attentiveness, and maybe required for cognitive functioning. This theory is supported by correlationalevidence including reports that the degree of disruption of EEG patterns correlates withthe severity of cognitive deficits in Alzheimer patients (Penttilä et at., 1985) and thatcognition enhancers activate the EEG (Delacour et at., 1990a). The fact that LVFA andRSA also occur during REM sleep may not dispute the theory that aroused EEG patternsare required for cognitive functioning, given that REM sleep may be involved ininformation processing (Dujardin et a!., 1990).Consistent with the speculation that increased ACh release occuring during EEGarousal contributes to increased attentiveness, cholinergic mechanisms are involved infacilitating the activity of hippocampal (Markram and Segal, 1990) and cortical neuronsresponding to afferent neural transmission. Single or multiple unit responses of cortical6neurons to visual, auditory or somatic stimulation, or to stimulation of the auditorythalamus, are enhanced by iontophoretic application of ACh and/or by stimulation ofthe basal forebrain (Donoghue and Carroll, 1987; Hars et at., 1993; Metherate and Ashe,1993; Metherate et at., 1987; Sillito and Kemp, 1983; Tremblay et a!., 1990; Webster etat., 1991). In some cases, the facilitory effects of basal forebrain stimulation have beenblocked by intracortical iontophoretic or systemic administration of the muscarinicreceptor antagonist atropine (Hars et al., 1993; Metherate and Ashe, 1993; Tremblay eta!., 1990). Electrical stimulation of the basal forebrain may approximate an increase inthe firing of basal forebrain neurons, such as that which occurs in monkeys presentedwith stimuli that precede, or have been previously paired with reinforcement(Richardson and DeLong, 1990; Wilson and Rolls, 1990) or in rabbits presented with aconditioned stimulus which predicts a mildly aversive stimulus (Whalen et al., 1994).Accordingly, the increased cortical ACh release presumably accompanying this increasedfiring may facilitate cortical neuronal responses to other, co—incident afferent signals,thereby providing a mechanism by which the relevance of sensory stimuli could beneurally encoded. Cholinergic facilitation of neuronal responsiveness may alsoparticipate in sensory cortex plasticity (Delacour et at., 1990b), cellular associativeconditioning (Pirch et al., 1992; Rigdon and Pirch, 1986), long-term potentiation in boththe cortex and hippocampus (Blitzer et al., 1990; Hunter et at., 1994; Lin and Phillis,1991) and afferent stimulation-induced protein synthesis in the hippocampus (Feig andLipton, 1993), all of which have been proposed as cellular models or correlates oflearning and memory.Considered together, the research outlined above points to the importance offorebrain cholinergic systems in determining an animal’s perception of its environmentas to which aspects are deemed significant, attended to, and thus perhaps remembered.Although terms such as learning, memory, attention and arousal may be insufficientlyprecise to define the roles of ACh in the CNS, they do succeed in suggesting a role for7this neurotransmitter in cognitive function. To complement research aimed atdetermining the functional significance of forebrain cholinergic systems, basiccharacterizations of these neurons are required, including their response to environmentalstimuli and their regulation by endogenous neurotransmitters. Given the anatomy andproposed functions of cholinergic systems, such research should ideally include measuresof regionally specific cholinergic activity in awake, behaving animals.(B) IN VIVO MICRODIALYSIS AND ASSAY OF ACHIn vivo microdialysis allows the measurement of interstitial concentrations ofneurotransmitters in discrete brain regions of awake, behaving animals (Benveniste andHüttemeier, 1990; Ungerstedt, 1984; Westerink et at., 1987) and is presently the bestavailable technique for estimating ACh release. Microdialysis exploits the ability ofsmall molecules to diffuse down a concentration gradient from the brain into a solutionapproximating brain interstitial fluid perfused through a semi-permeable, tubular dialysismembrane. A neurochemical which is permeable to the dialysis membrane, and forwhich there is a sensitive assay, can then be measured in the collected dialysate.Similarly, using reverse dialysis, drugs included in the perfusion solution can be appliedby diffusion into the discrete sampling area of the dialysis probe, and this can beaccomplished concurrently with neurochemical measurement. An obvious advantage ofmicrodialysis over ex vivo techniques is the ability to monitor neuochemica1s frequently(several times an hour) and for many hours in each animal. Compared to earlier in vivosampling methods, such as push-pull perfusion (Philippu, 1984) and the cortical-cuptechnique (Moroni and Pepeu, 1984), microdialysis produces less tissue damage andperturbation, can be performed in most brain regions, and is amenable to use in awake,freely-moving animals. Microdialysis also has an advantage over another current in vivo8neurochemical sampling technique, in vivo electrochemistry (Kawagoe et at., 1993),which can only be used to detect electroactive neurochemicals.Despite its many advantages, in vivo microdialysis has several limitations whichmust be considered. First, the concentrations of neurotransmitters measured usingmicrodialysis do not represent absolute synaptic concentrations. Factoring into thecalculations the in vitro recovery of the dialysis probe succeeds in estimating theconcentration of molecules reaching the dialysis probe, but neglects the influence ofimportant in vivo factors (Amberg and Lindefors, 1989; Benveniste et at., 1989; Justice,1993) such as metabolism, active uptake and tortuosity, which is a descriptor of theimpedance of the interstitial pathway through which the transmitter diffuses between itsrelease site and the probe. Because the absolute synaptic concentration of transmittermolecules can not yet be accurately determined, most microdialysis data are interpretedwith regard to relative changes in interstitial concentrations of transmitter arising,presumably, from synaptic overflow.The possibile origin of the recovered transmitter from activity-independent releasefrom neurons or from non-neuronal sources is the second limitation of microdialysis thatmust be considered. To assess the degree to which diaysate concentrations of aneurochemical are dependent on activity-dependent release, the sodium channel blockertetrodoxin (TTX) can be added to the perfusion solution, effectively blocking theconduction of action potentials in the vicinity of the dialysis probe. In optimalmicrodialysis conditions, TTX treatment reduces the concentrations of severalneurotransmitters by at least 80%, and often to below detectable levels (Damsma et at.,1987b;1987c; Santiago and Westerink, 1990; van Veldhuizen et a!., 1990). Removal ofcalcium ions from the perfusion solution, or their replacement with competitive bivalentcations, can also be used to estimate the proportion of dialysate transmitters originatingfrom calcium-dependent vesicular release (Benveniste and HUttemeier, 1990), although9non-vesicular, calcium-independent release may still be neuronal in origin and activityrelated (Levi and Raiteri, 1993).It has been demonstrated that the tissue trauma resulting from probe implantationaffects interstitial concentrations of neurochemicals (de Boer et a!., 1992; Reiriz et a!.,1989); the amount of time after implantation required to optimize recovery ofneurochemicals related to activity-dependent release, usually one to two days, depends inpart upon the type of dialysis probe used (Santiago and Westerink, 1990). In the case ofneurochemicals such as glutamate and adenosine, which have constitutive roles in cellularfunctioning as well as proposed roles as neurotransmitters, those molecules originatingfrom neurotransmission-related release may only represent a fraction of the total amountrecovered. Thus, for example, basal microdialysate concentrations of glutamate areunaffected or only partially reduced by TTX infusion (Girault et a!., 1986; Moghaddam,1993; Westerink et a!., 1987).A third important consideration to be made concerning microdialysis is whetherbasal, unstimulated neurotransmitter release is being measured. Dialysate concentrationsof neurotransmitters have been shown to be dependent on the ionic composition of theperfusate (Moghaddam and Bunney, 1989; Osborne et aL, 1991). Perfusate solutionscontaining higher concentrations of calcium or potassium than those found in theinterstitial space surrounding the probe would be expected to artificially stimulatetransmitter release by affecting the respective ion gradients and currents across theneuronal membrane. Indeed, it has been shown that perfusion solutions differing incalcium concentration can yield qualitatively different results in ACh microdialysisexperiments (de Boer et a!., 1990a). To ensure that basal release is being assessed, theionic composition of the perfusate should closely approximate that in the interstitialspace (Hansen, 1985).A final characteristic of microdialysis that must be considered is the basicfunctional principal of dialysis itself: that any molecule or ion which is permeable to10the dialysis membrane and which is not in equal concentrations in the brain and in theperfusion solution will cross the membrane, flowing down its concentration gradient.Although this allows the recovery and analysis of neurochemicals, it also disturbs thelocal chemical microenvironment by removing not only the molecular species of interestbut also many others. The brain tissue surrounding the probe for up to 1mm is drainedby this neurochemical “sink” (Benveniste, 1989; Blaha, 1991) and may be influenced bysuch factors as rate of perfusion, perfusate composition, and probe geometry. It musttherefore be recognized that microdialysis does not assess neurotransmitter release inentirely normal physiological conditions, but rather within a zone of artificialequilibrium between the tissue and the dialysis probe, where many dynamic neuronalprocesses are presumably affected. The recently developed technique of quantitativedialysis (Justice, 1993) attempts to deal with this issue, at least with respect to thespecies of interest, by including this neurochemical in the perfusate at a concentrationwhere no net flow occurs across the membrane. While this modification is prohibitivelylabour-intensive for most dialysis applications, it is informative about the dialysistechnique itself.A special consideration must be made in the case of microdialysis for recovery ofACh. Because the degradation of released ACh by acetylcholinesterase (AChE) activityis extremely efficient, an AChE inhibitor is usually included in the perfusion solution.The reversible, non-lipophilic, quaternary AChE inhibitor neostigmine has been arguedto be best suited for this purpose (Damsma and Westerink, 1991). Depending on thesensitivity of the specific assay, this adaptation is necessary in most cases to allow therecovery of reliably detectable amounts of ACh. High-maintenance assay procedures orlong sampling intervals have been used to carry out dialysis experiments in the absenceof an AChE inhibitor (Damsma et at., l987c; de Boer et at., 1990b; Messamore et at.,1993), although it is usually reported that release is not detectable in all animals tested.To increase the number of successful experiments, inclusion of low concentrations of an11AChE inhibitor is generally considered an acceptable compromise of the dialysistechnique. Indeed, presently it is not known to what extent the presence of AChEinhibition might actually ameliorate the above—mentioned dialysis “sink” effect, at leastwith respect to the local depletion of ACh surrounding the membrane.Although the above-mentioned issues must be considered in assessing microdialysisas an in vivo neurochemical sampling technique, the question of quantitative, assays forACh need only address two points: sensitivity and specificity. Several assays for AChhave been coupled with in vivo monitoring techniques, including bioassays (Celesia andJasper, 1966; Kanai and Szerb, 1965), gas chromatography/mass spectrometry (Marienand Richard, 1990), a radioenzymatic assay (Consolo et a!., 1987) and aradioimmunoassay (Kawashima et at., 1991). The highest sensitivities routinely reported,however, have been from chromatographic methods coupled to electrochemical detection(ECD; Damsma et at., 1987a’). ECD of the electrically inactive ACh molecule isachieved by post-chromatographic enzymatic conversion of ACh, via anacetylcholinesterase, to choline and acetic acid, and the subsequent conversion ofcholine, via choline oxidase, to betaine and hydrogen peroxide (Fig. 1). The hydrogenperoxide, stoichiometrically produced from ACh, is detected by its oxidation at aplatinum electrode. The chromatographic separation and subsequent enzymaticprocessing of the ACh fulfills the requirement of specificity in the assay.Given the above-mentioned considerations, the experiments reported here use invivo microdialysis coupled to on-line assay of ACh by high performance liquidchromatography with ECD (HPLC-ECD; Damsma and Westerink, 1991) to assessneuronal overflow of ACh in forebrain regions of awake, freely moving rats (Fig. 1).Experiments were conducted on the second day after implantation of a transversemicrodialysis probe and 100 nM neostigmine was included in the perfusate.Microdialysate concentrations of ACh using this methodology have previously beendemonstrated to be calcium-dependent and TTX-sensitive (Damsma et al., 1987c; 1988).12CH3\+ /,2CHN_CHrcOH 2 12°2CH3Fig. 1 Upper panel: Schematic of in vivo microdialysis with on-line assay foracetyicholine (ACh). a) Mobile phase reservoir, b) HPLC pump, c) pulse dampener, d)guard column, e) standard ioop and valve, f) sample loop and valve, g) timer, h)analytical column, i) immobilized enzyme reactor, j) electrochemical detector includingworking and reference electrodes and potentiostat, k) chart recorder and 1) perfusatesyringe driver. An average time delay of 20 mm occurs between the neurochemicalevent in the rat’s brain and its representation on the chart recorder. See Chapter II,Materials and Methods section for details.Lower panel: Steps in the enzymatic conversion and electrochemical detection of ACh,occuring in the enzyme reactor and on the working electrode (i and j above).bC dgaCH3,CH3CHN-CCOCCH3Acetlycholine CH3Esterase + ,CH3+ H20 -CHN-CH2CHO +HO—CCH3CH3 Choline\ + OxidaseCHi—N—CIk-CHPH + H20 + 202CH3PlatinumElectrode14202+ 500 mV+ 2W + 2e13II. ACETYLCHOLINE RELEASE IN THE RAT HIPPOCAMPUS, CORTEX ANDSTRIATUM CORRELATES WITH BEHAVIOURAL AROUSAL(A) INTRODUCTIONACh has been implicated in a variety of behavioural functions including learningand memory (Collerton, 1986; Hagan and Morris, 1988). Although the strength of thedata supporting the cholinergic hypothesis of dementia has been questioned (Fibiger,1991), forebrain ACh may participate in cognitive functions other than learning andmemory per Se, such as attention (Muir et al., 1994; Sahakian et al., 1993; Voytko et al.,1994) or arousal (Semba, 1991). Arousal can be operationally defined in laboratoryanimals either electroencephalographically or behaviourally. Central cholinergicneurotransrnission has been associated with both EEG and behavioural measures ofarousal. Using cortical cup and push/pull perfusion techniques, increased concentrationsof ACh have been recovered during spontaneous or stimulated EEG desynchronization incats (Celesia and Jasper, 1966; Kanai and Szerb, 1965; Phillis and Chong, 1965; Szerb,1967) and rats (Casamenti et al., 1986), during increased behavioural activity in rabbits(Collier and Mitchell, 1967), and during sensory stimulation or treadmill-running in rats(Dudar et al., 1979). More recently, significant correlations have been reported betweencortical, hippocampal and striatal dialysate ACh concentrations (Or whole brain tissueconcentrations) and motor activity counts after anticholinergic treatment (Frances andJacob, 1971; Toide, 1989; Watanabe and Shimizu, 1989).Microdialysate concentrations of ACh from awake, freely-moving rats in drug-freeconditions have been demonstrated to vary considerably (Damsma et al., 1987b), andpreliminary studies in this laboratory suggested that these variations may be due tochanges in animals’ activity levels. The experiments reported here were undertaken tofurther examine the possible relationship between ACh release and arousal, as measured14by locomotor activity. Photocell beam interruptions and in vivo microdialysateconcentrations of ACh and choline (Ch) in the dorsal hippocampus, frontal cortex ordorsal striatum of rats were measured simultaneously under three conditions: 1) after aninjection of vehicle; 2) after administration of the muscarinic receptor antagonist,scopolamine; and 3) before and after the beginning of the rats’ night cycle. The twonon-pharmacological stimuli are associated with increased locomotion, while muscarinicreceptor antagonists also increase locomotion and are known to increase ACh release inthe cortex, hippocampus, and striatum via the blockade of an inhibitory feedbackmechanism (Aquilonius et al., 1972; Dolezal and Wecker, 1990; Lefresne et al., 1978;Nordstrom and Bartfai, 1980; Rospars et a!., 1977; Szerb et a!., 1977).(B) MATERIALS AND METHODSExperimental protocol and drugsExperiments were performed on male Wistar rats (250-330g) two days after theimplantation of a microdialysis probe. Following surgery, rats were housed individuallyin Plexiglas cages (35x35x25 cm) and maintained on a 12:12 h light:dark schedule withfood and water available ad libitum. During the light phase of the rats’ daily cycle,subjects were first injected with water (1 mi/kg, s.c.), and then 3 h later with themuscarinic antagonist, scopolamine hydrobromide (0.4 mg/kg, Sigma). Experimentscontinued into the rats’ usual dark phase (when the room lights were turned off)approximately 4 h later. During each of these conditions, locomotor activity andhippocampal, cortical or striatal interstitial concentrations of ACh and Ch were measuredsimultaneously.15Surgery and microdialysisTrans-cerebral microdialysis sampling of ACh was performed according to themethodology of Damsma et a!. (1987b) which has been reviewed (Damsma andWesterink, 1991). Rats were stereotaxically implanted with a transverse dialysis probe(Fig. 2; Damsma et a!., 1987b; Imperato and Di Chiara, 1984) under pentobarbitalanesthesia (50-60 mg/kg, i.p.). The probe was placed in one of three sites according tothe atlas of Paxinos and Watson (1986), measured from bregma in mm: striatum A:+l.7,V:-4.75; hippocampus A:-4.3, V:-3.3; frontal cortex A:+2.7, V:-2.5. The probes weremade of saponified cellulose ester dialysis fibre (ID = 0.22 mm, OD = 0.27 mm,molecular weight cut off > 10 000 Dalton; Cordis Dow Medical), having an activesurface length of 6.4 mm, 6.8 mm, or 10.9 mm for the striatum, hippocampus, or frontalcortex, respectively. On completion of each experiment, the probe location was verifiedusing standard histological procedures.During microdialysis experiments the dialysis fibre was perfused at 5 uI/mm,controlled by a syringe pump (Carnegie Medicin). The syringe was connected to theprobe inlet by polyethylene tubing (800x0.28 mm); the probe outlet was connected to thesample loop (100 UI) of the analytical system by fused silica tubing (800x0.1 mm). Thesample valve was controlled by an adjustable timer (Valco), and samples (50 ul) werecollected and injected at ten minute intervals.The composition of the perfusion solution was selected to approximate the ioniccomposition of the interstitial fluid in the brain (Hansen, 1985) and contained NaCI (125mM), KC1 (3 mM), CaCl2 (1.3 mM), MgCI2 (1.0 mM), NaHCO3 (23 mM) in aqueousphosphate buffer (pH 7.3). To recover detectable dialysate concentrations of ACh, areversible acetylcholinesterase (AChE) inhibitor (neostigmine bromide, 0.1 uM; Sigma)was included in the perfusion solution. Thirty minutes of perfusion preceded samplecollection to allow perfusate concentrations of ACh and Ch to equilibrate with those inthe brain.16-‘-2.7Figure 2. Dialysis probe placements in the frontal cortex, dorsal striatum and dorsalhippocampus. Measurements are from Bregma, in mm. Thinner areas of the proberepresent those areas of the fibre open to dialysis, whereas the thicker areas are blockedby glue.6 /\÷1.7-4.317Assay of AChACh was assayed by HPLC-ECD in conjunction with an enzyme reactor (Damsmaet a!., 1987a; Fig. 1). ACh and Ch were separated on a reverse phase column (75x2.1mm) pretreated with lauryl sulphate. The eluent from this analytical column then passedthrough an enzyme reactor (10x2.1 mm) containing acetylcholinesterase (EC;Sigma, type VI-S) and choline oxidase (; Sigma), covalently bound toglutaraldehyde-activated Lichrosorb NH2 (10 urn; Merck). The separated ACh and Chreacted to give a stoichiometric yield of hydrogen peroxide, which was electrochemicallydetected at a platinum electrode at a potential of +500 mV versus an Ag/AgC1 referenceelectrode (BAS-LC4B). The mobile phase, 0.2 M aqueous potassium phosphate bufferpH 8.0, was delivered by a pump (LKB-2150) at 0.4 ml/min. The detection limit of theassay was approximately 50 fmol/injection. The time required to complete achromatogram was 4-5 mm. Standards of ACh/Ch were injected hourly.Motor activityDuring the microdialysis experiments, a Digiscan Animal Activity Monitor (modelRXYZCM(l6); Omnitech Electronics, Inc.) was used to measure locomotor activity in 10mm blocks corresponding to the 10 mm dialysate samples. Successive interruptions ofthe same photocell beam were categorized by the monitor as “stereotypy”, and thesestereotypy counts were subtracted from the total horizontal counts to yield a measure ofambulation.Statistical analysesBiochemical data were calculated as a percent of baseline concentrations, 100%baseline being defined as the average of the last three pre-scopolamine values. Aunivariate analysis of variance (ANOVA) with repeated measures was used to comparethe effects of scopolamine and vehicle on ACh and Ch output. ANOVAs were also18conducted to evaluate the effects of the vehicle injection and exposure to dark on AChand Ch. These analyses included the three samples prior to and the three samples afterthe initiation of these two treatments. Comparisons of the biochemical responses to thethree experimental variables between brain regions were also made using ANOVAs.Greenhouse-Geisser adjustments of the degrees of freedom were made to account forthe use of time as a repeated measure. All reported values refer to the interaction effectof time with experimental treatment. Pearson’s correlation coefficients between AChconcentrations (in fmol/min) and motor counts were determined for the three treatmentconditions in each individual rat. Group correlations were carried out as well, in whichthe data of all animals having probes in the same brain region were combined foranalysis. In this case, the biochemical measures were expressed as percentage ofbaseline. Because motor counts were not normally distributed, for all the correlationanalyses these values were normalized using logarithmic transformation.(C) RESULTSThe average baseline outputs of ACh and Ch in the three brain regions are shownin Table I and have been corrected for the differences in probe surface length.The neurochemical and motor effects of vehicle and scopolamine injections, andexposure to dark, are shown in Figs. 3, 4 and 5 for the striatum, hippocampus andcortex, respectively. Vehicle injections transiently increased extracellular ACh by 54%in the hippocampus (pczO.05) and by 161% in the frontal cortex (n.s.), while no changewas evident in the striatum. No significant effect of this treatment on dialysateconcentrations of Ch was observed in any brain region. The responses of ACh and Chin the three brain regions to vehicle treatment were not significantly different. Injectionof vehicle caused short lasting increases in motor activity.19Table 1. Probe characteristics and baseline output of ACh and Ch in the striatum,hippocampus and frontal cortex of ratsBaseline Dialysate Output Probe In vitroBrain (fmol/min/mm) Surface ProbeRegion ACh Ch Lenath (mml Recovery (%‘lStriatum 9.04 ± 0.67 588 ± 130 6.4 21.4 ± 6.2Hippocampus 2.10 ± 0.48 368 ± 82 6.8 22.5 ± 5.4Frontal cortex 2.30 ± 0.56 294 ± 82 10.9 33.3 ± 7.6Dialysate outputs are expressed as the mean (± S.E.M.) of the last 3 pre-scopolamine valuesfrom 4-6 rats. Outputs are corrected for the active surface length of the probes, as shown.Neostigmine (100 nM) was included in the perfusion solution.20500 -a,U,150 (Uw -400-JW 100CI) ID0 300 50 HD00.. 200F— 00o 100-.3000 >I-0- 2000 >I—1000 00TIME (hr)Figure 3. Striatal dialysate values of ACh (closed circles) and Ch (open circles) afterinjections of vehicle and scopolamine and exposure to dark. Activity counts (open bars)were measured concurrently. Vehicle (1 mI/kg) and scopolamine (0.4 mg/kg) wereinjected subcutaneously, and “lights off” occured at the appropriate time in the rats’daily cycle. Data points represent group means (n=4-6) ± S.E.M.%ooo999STRIATUMScopolamine Lights Off0 1 2 3 4 5 6 7 8 9 10 11211600-jI 1400-z --JW 1200-C,)fXl 1000-F— 800-600-0400-• 2000-0-120(U• --100I--80 D0-I—000HIPPOCAMPUS0 1 2 3 4 6 7 8 9TIME (hr)3000 >I-2000 >F-1000 00Figure 4. Hippocampal dialysate values of ACh (closed circles) and Ch (open circles)after injections of vehicle and scopolamine and exposure to dark. Activity counts (openbars) were measured concurrently. Vehicle (1 ml/kg) and scopolamine (0.4 mg/kg) wereinjected subcutaneously, and “Lights off’ occured at the appropriate time in the rats’daily cycle. Data points represent group means (n=4) ± S.E.M.222000 -1750-W 180z =j1500w (U(0-140aiI-1000I— 100 ft.I—I— 750 Qo 60- 500 0o 0• 2503000 >0 !z2000 >I—1000 00TIME (hr)Figure 5. Frontal dialysate values of ACh (closed circles) and Ch (open circles) afterinjections of vehicle and scopolamine and exposure to dark. Activity counts (open bars)were measured concurrently. Vehicle (1 mi/kg) and scopolamine (0.4 mg/kg) wereinjected subcutaneously, and lights off” occured at the appropriate time in the rats’daily cycle. Data points represent group means (n=4-5) ± S.E.M.FRONTAL CORTEX0 1 2 3 4 5 6 7 8 9 1023Scopolamine (0.4 mg/kg, s.c.) caused large increases in interstitial ACh and inmotor activity. Peak ACh increases of 400, 1200 and 1400% in the striatum,hippocampus and cortex, respectively, occurred within 20 mm and persisted forapproximately 1 h. Compared to vehicle values, scopolamine had a significant effect onACh output (striatum p<0.0O1, hippocampus and cortex p<0.05). The drug significantlyaffected Ch only in the striatum (p.cO.0l) where there was an increase in the secondpost-injection sample (159% of baseline). The effect of scopolamine on ACh wassignificantly smaller in the striatum than in either the cortex or hippocampus (p<0.05).The drug responses in the hippocampus and cortex did not differ significantly from eachother. Motor activity was greatly increased by scopolamine with approximately the sametime course as was observed for the increase in ACh.At the onset of the dark phase of the rats’ day-night cycle, motor activityincreases coincided with ACh increases of 58% in the striatum, 169% in thehippocampus, and 77% in the cortex. In the striatum, ACh showed a significantillumination by time interaction effect (p<0.05); the increases in the other areas failed toreach significance. The three brain regions did not differ significantly with respect tothe ACh nor Ch response to dark.To illustrate the nature of the relationship between behavioural activity and AChmeasured in either the striatum, hippocampus or cortex, Fig. 6 shows the motor anddialysate profiles of three individual animals. Most of the animals tested exhibitedsignificant correlations between locomotor activity and ACh release under all threeexperimental conditions. After vehicle injection, significant correlations (p<0.05) werefound in 2 of 4 animals with striatal probes (r=0.572-0.643), 3 of 4 animals withhippocampal probes (r=0.6ll-0.808) and in 3 of 5 animals with frontal cortex probes(r=0.665-0.704). Scopolamine treatment yielded significant values in all 6 animals withstriatal probes (r=0.532-0.867), in all 4 with hippocampal probes (r=0.517-0.736) and in 4of 5 animals with frontal cortex probes (r=0.741-0.877). During lights-off, motor counts24E4oEE300E20.010..STRIATUM40302010- 7505000-50 -2500Scopr=O.330[IIPPOCAMPUSLightsr=O.603*I>-F-1000>I—05000ScopFRONTALLights CORTEXI r=O.6650-403020ID0Figure 6. Dialysate ACh concentrations (closed circles) and activity counts (open bars)from three individual rats after injections of vehicle (1 mI/kg) and scopolamine (0.4mg/kg), and exposure to dark. Insets show Pearson’s correlation coefficients of the twomeasures for the time points within each experimental treatment. * p<0.05.1500100050003 4 5 6TIME (hr)7 8 925Table 2. Correlation of regional ACh outputs and activity counts during experimentaltreatmentsBrain TreatmentRegion Vehicle Scopolamine Lights Off* *Striatum 0.256 0.599 0.300* * *Hippocampus 0.472 0.630 0.595* *Frontal cortex 0.151 0.468 0.515Pearson’s correlation coefficients of ACh outputs (as % baseline) in the striatum, hippocanipusor cortex (4-6 animals/group) and locomotor activity are shown. Analyses included the twomeasures for all time points (10-26) within each experimental treatment. * p<0.05.26correlated significantly with dialysate ACh concentrations in the striatum (4 of 5animals, r=O.677-O.844), in the hippocampus (all 4 animals, r=O.493-O.926) and in thecortex (3 of 4 animals, r=O.696-O.899).As evident in Table 2, when the data were grouped across animals, the correlationvalues became less robust. This grouping facilitates comparisons between the brainregions across the experimental conditions. The highest correlation between motoractivity and ACh output occurred in the hippocampus under the scopolamine condition.The highest value for the striatum was also after scopolamine, while in the cortex AChconcentrations correlated best with motor activity during the dark phase. The lowestcorrelations for each group were seen after vehicle treatment.(D) DISCUSSIONSource of microdialysate ACh and ChThe source of ACh recovered in the microdialysates from the three studied brainregions is anatomically distinct. The striatum contains intrinsic cholinergic interneurons(Phelps et al., 1985; Semba et a!., 1987) while the hippocampus and cortex receivecholinergic projections from the basal forebrain. Cholinergic perikarya in the basalforebrain are located among other non-cholinergic neurons in nuclei including themedial septal nucleus, the nucleus of the diagonal band of Broca, the magnocellularpreoptic nucleus, substantia innominata and the globus pallidus. An alternativesubdivision of these cholinergic neurons, independent of these nuclear groups and basedinstead on their projection patterns, has also been suggested (Mesulam et a!., 1983).Although theoretical subdivisions of basal forebrain cholinergic neurons are tenable, ithas also been demonstrated that these neurons form an anatomical continuum (Schwaber27et a!., 1987), with hippocampal and cortical cholinergic projections originating mainly inthe anterior and posterior regions, respectively, of this cholinergic basal nuclear complex.The possibility exists that extrinsic cholinergic projections from the basal forebrainare not the sole source of ACh in the rat cortex and hippocampus.Immunohistochemical studies have suggested the presence of intrinsic cholinergicneurons in these regions (see Semba and Fibiger, 1989), a finding unique to rodents.However, a sensitive in situ hybridization technique failed to detect mRNA for cholineacetyltransferase in the rat hippocampus or cortex (Butcher et at., 1992). The possibilitythat cholinergic interneurons are located in these regions thus remains controversial.With regard to this controversy, attempts have been made to estimate the percentage ofACh in cortical and hippocampal microdialysates derived from basal forebrain projectionneurons. Mechanical deafferentations or excitotoxic lesions of cholinergic projectionneurons have decreased ACh in hippocampal microdialysates by 90% (Leanza et at.,1993) and in cortical microdialysates by up to 60% (Ammassari-Teule et a!., 1993;Herrera-Marschitz et at., 1990; Rosenblad and Nilsson, 1993). Although thecompleteness of the lesions may be questioned, these results may also be interpreted tosupport the possibility that cholinergic interneurons exist in the cortex and hippocampusand contribute to microdialysate concentrations of ACh.The source and significance of microdialysate concentrations of Ch are not wellunderstood. Ch is both a precursor of ACh and a product of its breakdown. Furthercomplicating any interpretation of changes in extracellular Ch is the fact that at least80% of the Ch turnover in the brain is thought to be involved in phospholipidmetabolism (Choi et al., 1975). Additional investigations of the source of extracellularCh are required before the meaning of the experimentally manipulated concentrations ofdialysate Ch can be determined.28Basal concentrations of microdialysate ACh and ChPrevious microdialysis studies comparing baseline outputs of ACh in the frontalcortex, hippocampus and striatum have reported values that are an order of magnitudehigher than those reported here (Ajima and Kato, 1988; Toide and Arima, 1989; Wu eta!., 1988). These discrepancies are likely due to differences in probe type and brainplacement, post-operative recovery times, type and concentration of the cholinesteraseinhibitor, and composition and pH of the perfusate. The lower concentration ofcholinesterase inhibitor used in this study, and the more physiological pH and calciumconcentration of the perfusion solution are the most likely sources of theabovementioned differences in basal outputs. Indeed, the values reported here formicrodialysate ACh in the individual brain regions are comparable to those reported inexperiments using more similar methodologies (Damsma et al., 1987b; Durkinet at.,1992; Mark et a!., 1992).The rank order of baseline concentrations measured in the three brain regionsgenerally compares well with those found previously in dialysates and tissuehomogenates: for ACh, striatum >> frontal cortex hippocampus; for Ch, striatum >hippocampus > frontal cortex (Ajima and Kato, 1988; Jope, 1982; Toide and Arima,1989; Wecker and Dettbarn, 1979; Wu et at., 1988). Incontrast, a very different rankorder for basal ACh (hippocampus >> frontal cortex striatum) was found in anexperiment using a perfusate lacking an AChE inhibitor (Xu et al., 1991). However,given that this result does not agree with earlier reports from these researchers, againusing AChE inhibitor-free perfusion (Ajima and Kato, 1988),further experiments willbe necessary to determine the possibly differential effects of AChE inhibition on basaldialysate concentrations of ACh in these three brain regions.29Effects of vehicle injection on ACh releaseSubcutaneous injection of vehicle tended to increase locomotion and dialysate AChconcentrations in the hippocampus and frontal cortex, although the effect in the latterstructure failed to reach significance due to high inter-subject variability. This findingis reliably replicated, for both cortical and hippocampal ACh release, in later chapters(Chapters III, IV, V). It has also been demonstrated that ACh release is increased in thehippocampus of rats being handled (Nilsson et a!., 1990). Arousing stimuli such ashandling and injecting the animals may involve stress, which has also been shown toincrease microdialysate concentrations of ACh in the hippocampüs (Imperato et a!., 1992;Nilsson and Björklund, 1992). The finding that a control injection increases ACh releasein the hippocampus and cortex emphasizes the necessity of including control vehicleinjections when studying the effects of drugs on cholinergic activity in these regions;this finding also suggests that it may be possible to study cholinergic correlates ofbehaviour in one or both of these structures.Effects of the muscarinic receptor antagonist on ACh releaseIt has been previously demonstrated, using in vitro techniques, that muscarinicreceptor antagonists including scopolamine increase ACh release in the striatum (Dolezaland Wecker, 1990; Lefresne et al., 1978), hippocampus (Nordstrom and Bartfai, 1980;Szerb et al., 1977), and cortex (Aquilonius et al., 1972; Rospars et a!., 1977). Suchantagonists are thought to block an inhibitory feedback mechanism. This phenomenonhas also been confirmed using microdialysis (de Boer et a!., 1990c; Marien and Richard,1990; Toide, 1989; Toide and Arima, 1989; Watanabe and Shimizu, 1989). However, itmust be noted that these dialysis experiments and those reported here have been carriedout in the presence of AChE inhibition. Decreasing the degradation of ACh in thesynapse with an AChE inhibitor may be expected to increase cholinergic autoreceptoroccupancy and thereby alter the effects of autoreceptor ligands on ACh release. Indeed,30when an AChE inhibitor is not included in the perfusate, the muscarinic antagonistatropine does not increase microdialysate concentrations of ACh in the cortex or striatum(de Boer et at., 1990b; Messamore et at., 1993). In addition, the muscarinic receptoragonist oxotremorine decreases striatal ACh release in the absence, but not in thepresence, of cholinesterase inhibition (de Boer et at., 1990b) leading the authors of thisstudy to conclude that under physiological conditions, muscarinic autoreceptors are notfully occupied. However, given that microdialysis depletes neurochemicals such as AChfrom the sampled tissue (Benveniste, 1989), it is unclear whether perfusion without anAChE inhibitor more accurately reflects physiological conditions than does perfusionwith AChE inhibitors.The differences in magnitude of scopolamine’s effect on ACh release (cortexhippocampus >> striatum) may reflect regional differences in muscarinic autoreceptorlocation and/or function. In the cortex and hippocampus, the effect of the antagonist onACh release is thought to be mediated by muscarinic autoreceptors located oncholinergic terminals (Marchi et a!., 1983; Molenaar and Polak, 1980; Sethy and Hyslop,1990). In contrast, evoked ACh release in the striatum is apparently not controlled bymuscarinic terminal autoreceptors (James and Cubeddu, 1987; Marchi et at., 1983;Raiteri et at., 1984). The necessary components for the effects of muscarinic antagonistson striatal ACh are known to be intrinsic to this region because intrastriatal applicationof antimuscarinics through the dialysis probe increases dialysate ACh concentrations (deBoer et at., 1990c; Marien and Richard, 1990). The increased release of ACh measuredin the striatum after scopolamine administration (this study, Toide and Arima, 1989;Watanabe and Shimizu, 1989) may therefore be mediated by multi-neuron circuits withinthe striatum, or possibly by dendritic or somal receptors located on the cholinergicinterneurons.31Effects of the onset of dark phase on ACh releaseExposure to dark at the appropriate time in the rats’ day-night cycle tended totransiently increase both locomotion and microdialysate concentrations of ACh in allthree brain regions. Only the increase in the striatum was statistically significant due tothe high variability of the locomotor responses and the neurochemical responses in theother two brain areas. The effect in the cortex has recently been confirmed in studieswhich used the onset of darkness as a stimulus to reliably increase cortical ACh release(Moore et a!., 1992; 1993). It has also been reported that sensory stimuli other thandarkness, both tactile and non-tactile, increase hippocampal but not cortical ACh releaseas assessed using a superfusion technique (Dudar et a!., 1979). This is in contrast to thelability of cortical ACh release found here and may be due to methodologicaldifferences.In addition to a transient increase in ACh release at the onset of the dark phase,cholinergic activity is reportedly higher on average over the course of the dark phasewhen compared to the light phase, as discussed below. Wholebrain ACh concentrationsexhibit diurnal oscillations; the lowest concentrations of ACh in tissue extracts, thoughtto reflect high cholinergic activity, have been found six hours after the onset of the darkperiod (Hanin et a!., 1970). Microdialysate concentrations of ACh in the cortex(Jimenez-Capdeville and Dykes, 1993; Kametani and Kawamura, 1991) and hippocampus(Mizuno et at., 1991) have also been demonstrated to be higher during the dark phase ofthe day/night cycle. Together, these data and the results of the experiments reportedhere suggest that environmental stimuli can influence cholinergic neurotransmission inthe hippocampus and frontal cortex.32Correlation between ACh release and behavioural arousal after non-pharmacologicalstimuliThe present results indicate that changes in dialysate concentrations of ACh in thestriatum, hippocampus and frontal cortex induced by the non-pharmocological stimulicorrelate with locomotor activity, a measure of behavioural arousal. It should be notedthat such a correlation has not been reported previously for other neurotransmitters.These findings corroborate previous suggestions of a relationship between ACh releaseand arousal, defined both electroencephalographically (Casamenti et at., 1986; Celesiaand Jasper, 1966; Kanai and Szerb, 1965; Phillis and Chong, 1965; Szerb, 1967) andbehaviourally (Collier and Mitchell, 1967). ACh release-has also been reported toincrease in the hippocampus of swimming rats (Nilsson and Björklund, 1992) and in thehippocampus (Dudar et al., 1979) and cortex (Kurosawa et al., 1993) of rats walking orrunning on a treadmill. It should be noted, however, that it is uncertain to what degreespontaneous locomotion, which is thought to reflect an animal’s level of arousal, issimilar to forced locomotion, which may involve stress.Although the accumulated evidence suggests that a correlation exists betweenbehavioural arousal and cholinergic activity in the brain regions examined, the source ofthis correlation is unknown. The locomotor measure of arousal defined here can bedissociated pharmacologically from ACh release in the cortex and hippocampus: -increased ACh release can occur in the absence of increased locomotion and the reverseis also true (see Chapters III, IV, V). This suggests that neither measure is absolutelynecessary for the occurence of the other, although they do coincide in the non-drugsituations described here. Discovering whether a cause-effect relationship is responsiblefor the observed correlation between behavioural arousal and ACh release, or on whatcommon element both measures are dependent, is a challenge for future research. Toaccomplish this goal, more detailed behavioural descriptions, coupled with an in vivoACh measurement with improved temporal resolution will be required.33Correlation between AC/i release and behavioural arousal after scopolamine administrationThe positive correlations between locomotor activity counts and ACh release in thethree brain regions after scopolamine administration extend previous findings thatmuscarinic receptor antagonist-induced increases in motor activity are associated withincreases in frontal and hippocampal dialysate ACh (Toide, 1989), and with decreases inwholebrain tissue levels of ACh (Frances and Jacob, 1971).The correlation evident after application of scopolamine may be qualitativelydifferent than that discussed above, for the non-pharmacological stimuli. In drug-freeconditions, increased ACh release is associated with increased chblinergicneurotransmission; the correlation between ACh release and behavioural arousal wouldtherefore suggest a correlation between cholinergic transmission and behavioural arousal.After injection of scopolamine, however, the increased ACh release occurs as a result of,and during, muscarinic receptor blockade. The behavioural hyperactivity caused by thisanticholinergic agent thus occurs in the presence of decreased muscarinic cholinergictransmission. Therefore, while behavioural arousal correlates with cholinergictransmission after the non—pharmacological stimuli, it does not appear to be associatedwith muscarinic cholinergic transmission in scopolamine-treated rats. This apparentinconsistency may be accounted for in several ways: 1) Pre- and postsynapticmuscarinic receptors may differ such that the dose of scopolamine used here mightpreferentially bind the presynaptic autoreceptors, resulting in increased ACh release,while the postsynaptic receptors remain unblocked. This would serve to increasecholinergic transmission. This possibility can be rejected, however, as Szerb et al. (1977)have reported that scopolamine has a ten fold lower efficacy at presynaptic than atpostsynaptic muscarinic receptors. 2) While scopolamine blocks muscarinic transmission,it would at the same time increase nicotinic cholinergic transmission by increasing AChrelease. The results obtained here could be explained if the action of ACh on nicotinicreceptors was responsible for the correlation between ACh release and behavioural34arousal. 3) As discussed above, the source of the correlation after the non-pharmacological stimuli is not known, and it is possible to pharmacologically dissociatethis correlation. This additional example of a pharmacological dissociation suggests thatthe correlation does not involve a direct cause-effect relationship between ACh releaseand behavioural arousal. Thus, pharmacological agents such as scopolamine, whichincrease locomotion while decreasing muscarinic tone, may affect these two measuresindependently, perhaps by acting at different loci within the central and peripheralnervous systems.Significance of the correlation between behavioural arousal and ACh release in the cortex,hippocampus and striatumGiven the previously suggested involvement of basal forebrain cholinergic neuronsin arousal (see Semba, 1991), the responsiveness of ACh release in the frontal cortex andhippocampus to arousing stimuli and the correlation of This release with a behaviouralmeasure of arousal is not surprising. The correlation of ACh release in the s.triatum withbehavioural arousal is perhaps more unexpected. In addition to the anatomicaldifferences between cholinergic interneurons and basal forebrain cholinergic neurons, thefunctions usually ascribed to the striatum are quite different from those of thehippocampus and cortex. The striatum is part of the basal ganglia, a group of nucleitraditionally thought to be involved in initiating and modulating motor output.However, the striatum is considered the most cognitive of the basal ganglia nuclei, beingclosely connected with the cortex. Indeed, mnemonic functions of the striatum havebeen suggested (Divac, 1972; Mishkin and Petri, 1984; Packard and McGaugh, 1992).In summary, these experiments illustrate the feasibility of carrying out AChmicrodialysis experiments of long duration, coupled to behavioural monitoring, indifferent brain regions. The data reported here indicate that arousing stimuli canincrease cholinergic transmission in a regionally selective manner.35III. DOPAMINERGIC REGULATION OF CORTICAL ACETYLCHOLINE RELEASE(A) INTRODUCTIONThe extrinsic cholinergic innervation of the cortex and hippocampus emanatesfrom posterior and anterior portions, respectively, of the cholinergic basal nuclearcomplex (CBC). The CBC is an anatomical continuum of cholinergic neurons which areinterspersed among non-cholinergic neurons within several classically defined nucleiincluding the medial septal nucleus, the nucleus of the diagonal band of Broca, themagnocellular preoptic area, the substantia innominata and the globus pallidus (Schwaberet a!., 1987; Semba and Fibiger, 1989). Direct synaptic contacts made with these ChAT-containing neurons are relatively sparse and generally localized to distal dendrites(Ingham et a!., 1985). However, afferents to the CBC arise from many brain regions, asoutlined below, suggesting that hippocampally- and cortically-projecting cholinergicneurons may be weakly innervated by multiple sources.The CBC receives many inputs among which are reciprocal projections from thecortex and hippocampus, as well as ascending projections from the hypothalamus andbrainstem nuclei including the ventral tegmental area, substantia nigra, dorsal andmedian raphe, locus coeruleus, pedunculopontine and laterdorsal tegmental nuclei (Sembaand Fibiger, 1989; Woolf, 1991). The neurotransmitter contents of some of theseafferents have been identified immunohistochemically. Catecholaminergic neuronsdefined by the presence of tyrosine hydroxylase (TH) have been retrogradely labelled,from the CBC, in regions known to contain dopaminergic and noradrenergic perikaryaincluding the ventral tegmental area, substantia nigra and locus coeruleus (Jones andCuello, 1989; Semba et al., 1988). Serotonin-containing neurons localized in severalnuclei including the dorsal raphe, and ChAT-positive neurons in the mesopontinetegmentum were also retrogradely labelled in these experiments.36Ultrastructural characterizations using electron microscopy have provided evidenceof direct synaptic contacts with basal forebrain neurons. Terminals immunoreactive forTH and ChAT synapse on ChAT-labeled neurons in the CBC (Milner, 1991). Serotonincontaining terminals synapse on hippocampally-projecting basal forebrain neurons, someof which may be cholinergic (Milner and Veznedaroglu, 1993). Synaptic contacts havealso been demonstrated between GABAergic terminals and ChAT containing neurons(TOth et at., 1993; Zaborszky et al., 1986b), or presumably cholinergic cortically-projecting neurons (Ingham et a!., 1988) in the basal forebrain. GABAergic synapsesmay arise from projections of the nucleus accumbens (Walaas and Fonnum, 1979) orhippocampus (TOth et al., 1993) or from intrinsic GABAergic neurons (KOhler and -Chan-Palay, 1983; Zaborszky et al., l986a). Recent electrophysiological studies ofidentified cholinergic neurons in the basal forebrain of guinea pig brain slices alsoprovide evidence of GABAergic, noradrenergic, as well as histami-nergic effects on thefiring rates of CBC neurons (Fort et at., 1993; Khateb et a!., 1993; Pegna et a!., 1993).Presumptive glutamate/aspartate projections to the basal forebrain from manybrain regions including the amygdala, thalamus and cortex have also been described(Carnes et at., 1990), although neurons using excitatory amino acid transmitters aredifficult to characterize anatomically. Evidence supporting a glutamatergic innervationof the CBC includes the finding that 1-glutamate binding sites represent a large portionof the total binding sites autoradiographically identified in the basal forebrain of the rat(Zilles et at., 1991) and that the AMPA subtype of glutamate receptor has beenimmunocytochemically localized to cholinergic basal forebrain neurons in the monkey(Martin et al., 1993). In addition, Rasmusson et at. (1994) have recently reported thatincreases in cortical ACh release caused by brainstem stimulation in anaesthetized ratscan be blocked by application of a glutamate antagonist into the basal forebrain. Withregard to the abovementioned proposition that the presence of appropriate receptorsfurther supports the regulatory role of a transmitter within a certain brain region, it37should be noted that receptors for ACh, serotonin, GABA, dopamine (DA) andnoradrenaline have also been autoradiographically identified in the basal forebrain (Zilleset at., 1991). Evidence of whether some of these receptors may be situated oncholinergic neurons awaits further ultrastructural studies.In addition to the classical neurotransmitters, a variety of neuropeptide transmittersand growth factors may regulate CBC neurons. There is evidence for interactionsbetween basal forebrain neurons, in some cases identified as cholinergic, and terminalscontaining substance P. enkephalin, somatostatin, neuropepetide Y, neurotensin orvasopressin (reviewed by Záborszky et at., 1991). In addition, receptors forneurotrophins and estrogen have been located in CBC neurons (Toran-Allerand et at.,1992).Considered together, the above anatomical data suggest that the activity of CBCneurons may be regulated by a great variety of neurotransmitters. The regulation ofthese cholinergic neurons, specifically those projecting to the cortex, by theneurotransmitter DA has been investigated in the experiments discussed in this chapter.To this end, in viva dialysate concentrations of ACh from the frontal cortex ofconscious, freely moving rats were measured after systemic administration of thepsychomotor stimulant d-amphetamine (AMPH) or the DA receptor agonistapomorphine, and after local application of AMPH through the dialysis probe. Todetermine which DA receptor subtype(s) may mediate the dopaminergic regulation ofcortically-projecting cholinergic neurons, the effects of selective D1-like and D2-likereceptor agonists on cortical ACh release were examined. In addition, the extent towhich selective D1-like and D2-like receptor antagonists attenuate AMPH- andapomorphine-induced effects on cortical ACh release was determined. Furtherreferences to “D1” and “D2 should be interpreted as “D1—like” and “D2-like” respectively(see Discussion).38(B) MATERIALS AND METHODSExperimental protocol and drugsThe size and strain of subject rats were as in Chapter II. Dialysis experimentswere conducted as described in Chapter II and involved the subcutaneous (s.c.) injectionof the following drugs, alone or in combination: d-amphetamine sulfate (AMPH, 2.0mg/kg; BDH), apomorphine hydrochloride (1.0 mg/kg; Sigma), CY 208-243 (1.0 mg/kgcontaining 0.4% acetic acid; Sandoz), quinpirole hydrochloride (0.2 or 0.5 mg/kg; RB!),(+)-4-propyl-9-hydroxynaphthoxazine (PHNO, 0.05 mg/kg; Merök, Sharp and Dohme),haloperidol base (0.15 mg/kg; McNeil Pharmaceutical), raclopride (1.0 mg/kg; Astra),and SCH 23390 (0.3 mg/kg; Schering Plough). The drugs were dissolved in distilledwater, and injected in a volume of 1 mI/kg. AMPH (10 uM) was also administereddirectly through the dialysis probe by its addition to- the perfusion solution. The dosesused in this study have significant effects on behaviour (Christensen et a!., 1984;Markstein et a!., 1988; Martin et a!., 1984; Ogren et a!., 1986) and/or have reliableneurochemical effects on dopaminergic systems (Imperato and DiChiara, 1985; Kuczenskiand Segal, 1989; Sharp et at., 1987) or on striatal cholinergic neurons (Bertorelli andConsolo, 1990; Damsma et at., 1990; 1991; Robertson et al., 1993; Westerink et at., 1990).Surgery and microdialysisTransverse dialysis probes were stereotaxically implanted into the frontal cortex ofrats as described in Chapter II. Microdialysis was carried out as previously describedwith the following exception: the probe outlet was connected to the sample valve by alength of polyethylene tubing, rather than fused silica tubing, containing 50 ul.Assay of AChACh was assayed by HPLC-ECD as described in Chapter II.39Statistical analysesBiochemical data were calculated as a percent of baseline concentrations, 100%baseline being defined as the average of the last three pre-drug values. UnivariateANOVAs with repeated measures were used to compare the effects of the differentdrugs on dialysate ACh concentrations. Reported values refer to the main group effectof the experimental treatment, unless otherwise noted as referring to the interactioneffect of time with experimental treatment. In the latter case, Huynh-Feldt adjustmentsof degrees of freedom were made to account for the use of time as a repeated measure.For the local application of AMPH, the last baseline sample and the six samples duringAMPH perfusion were used to test for an effect of the drug over time.(C) RESULTSThe average baseline output of ACh ( S.E.M.) from the frontal cortex in all ofthe animals was 46.66 ± 6.69 fmol/min (n=88). Systemically administered AMPH (2.0mg/kg) increased ACh output to 280% of baseline values within I h and the increaselasted for approximately 3 h. This was statistically significant [F(l,lO)=46.98, p<O.OO1]compared to the transient increase after injection of vehicle (149%, Fig. 7A). The DAreceptor agonist apomorphine (1.0 mg/kg) also significantly increased ACh for 1 h to amaximum of 220% baseline [F(l,9)=5.73, p=0.04; Fig. 7A]. Both AMPH andapomorphine caused behavioural hyperactivity (data not shown). Local application ofAMPH (10 uM) through the dialysis probe did not influence behavioural activity and didnot significantly affect interstitial concentrations of ACh in the frontal cortex[F(3.59,1O.76)=0.54; Fig. 7BJ.The effects of selective DA receptor agonists on cortical dialysate output of AChare shown in Figs. 8 and 9. The effect of the specific D2 agonist quinpirole, at 0.2 orwz-JwCoIa-I:D0-C)—1 0 1TIME (hr)40300 -250 -200 -150 -100-50-0-I I I • I—1 0 1 2200- B3[lOuM AMPHI150-100-50-0-_________________________I I I___ •2 3Figure 7. Frontal cortex dialysate values of ACh after A: injections of d-amphetamine(AMPH; 2.0 mg/kg; open circles), apomorphine (APO; 1.0 mg/kg; open squares), orvehicle (1.0 mI/kg; closed circles), and B: local administration of AMPH (10 uM)through the dialysis probe. Data points represent group means (n=4-6) ± S.E.M.41B 0 PHNO (0.05 mg/kg)• VEHICLE1TIME (hr)A• QUIN (0.2 mg/kg)o QUIN (0.5 mg/kg)250 -200 -150-100-50-0-250 -200 -150-100-50-0-1 I • I0 1 2 3wz-Jw(I)I—D0F0C)Figure 8. Frontal cortex dialysate values of ACh after injections of A: quinpirole(QUIN) 0.2 mg/kg (closed circles) or 0.5 mg/kg (open circles), and B: PHNO (0.05mg/kg; open circles) or vehicle (1.0 mI/kg; closed circles). Data points represent groupmeans (n=5-7) ± S.E.M.0 2TIME (hr)• I2 3-I--- • I2 3Figure 9. Frontal cortex dialysate values of ACh after injections of CY 208-243 (CY;1.0 mg/kg; open circles in A and B) or a combination of quinpirole (QUIN; 0.2 mg/kg)and CY (1.0 mg/kg; closed circles in B). The effect of vehicle injection from Fig. 7A isincluded in A (closed circles) for comparison. Data points represent group means(n=5—6) ± S.E.M.A42o CY (1.0 mg/kg)• VEHICLEwz-JwC,)I0I—0()250-200 -150-100-50-0-200 -150-100-50-0-10Bo CY (1.0 mg/kg)• CY (1.0 mg/kg) + QUIN (0.2 mg/kg)0 1430.5 mg/kg, was not significantly different from that of vehicle injections [F( 1,1 0)=0.97,and F(1,9)=l.03, respectively; Fig. 8A1. Similarly, the D2 agonist PHNO (0.05 mg/kg)did not have a significant effect on ACh output compared to vehicle-treated animals[F(1,1 1)=2.13; Fig. 8B]. Although a tendency for increasing ACh values was evident 1hour after administration of PHNO, the effect failed to reach significance due to highvariability in these drug-treated animals. Although the D2 agonists did not affectcortical ACh release, these drugs produced behavioural stereotypies that lasted forapproximately 1.5 h.The D1 receptor agonist CY 208-243 (1.0 mg/kg) significantly increased AChconcentrations to 180% of baseline within 30 mm, and this lasted approximately 1 hcompared to the transient increase observed after vehicle injections [F(l,9)=8.39,p=0.018; Fig. 9A]. This drug treatment did not appreciably increase the rats’ locomotion.The D1 agonist-induced increase in cortical ACh release was not altered by simultaneousadministration of the D2 agonist quinpirole [0.2 mg/kg; F( 1 ,8)= 1.00; Fig. 9B]. Thebehavioural effects of the co-administered D1 and D2 receptor agonists were similar tothat of the D2 agonist alone.Figs. 10 and 11 show the effects of the DA receptor antagonists on ACh effluxand on the ACh response to the subsequently administered AMPH. (The AMPHresponse from Fig. 7A is superimposed for comparison). Compared to the effect of thevehicle injection which is depicted in Fig. 7A, the selective D1 antagonist SCH 23390(0.3 mg/kg) maximally decreased ACh to 30% of baseline values [F(l,9)=22.27, p=0.OOl;Fig. 10]. When animals received AMPH 30 mm after SCH 23390, the ACh response wassignificantly lower than that after AMPH alone [130% vs 280% maximal values;F=(1,9)=42.82, p<0.OOl], significantly higher than that after SCH 23390 alone[F(l,8)=16.63, p=0.OO4; Fig. 10], and not significantly different from that after thevehicle injection [F(1,9)=0.13].Figure 10. Frontal cortex dialysate values of ACh. SCH 23390 (0.3 mg/kg) was injected30 mm prior to peripheral administration of d-amphetamine (AMPH; 2.0 mg/kg; closedsquares) or vehicle (1.0 mI/kg; open squares). The effect of AMPH alone (open circles)from Fig. 7A is included for comparison. Data points represent group means (n=5-6) ±S.E.M.44wz-JwC,)I—0F0()300-250-200-‘so.100•500-0AMPH (2.0 mg/kg)23390 (0.3 mg/kg)!AMPH (2.0 mg/kg)23390 (0.3 mg/kg)!VEHICLE0I -, I I1 2 3 4TIME (hr)45300-250-200-150-— 100-Uiz-jUiU)0Ia-I—00TIME (hr)Figure 11. Frontal cortex dialysate values of ACh. A: Haloperidol (HAL; 0.15 mg/kg)and B: raclopride (RAC; 1.0 mg/kg) were injected 30 mm prior to peripheraladminstration of d—amphetamine (AMPH; 2.0 mg/kg; closed squares) or vehicle (1.0mi/kg; open squares). The effect of AMPH alone (open circles) from Fig. 7A isincluded for comparison. Data points represent group means (n=5-6) ± S.E.M.AOAMPH (2.0 mg/kg)•HAL (0.15 mg/kg)!AMPH (2.0 mg/kg)OHAL (0.15 mg/kg)/VEHICLE50-I • I I • I0 1 2 3 4B300-250 -200-150-10050-aAMPH (2.0 mg/kg)•RAC (1.0 mg/kg)!AMPH {2.O mg/kg)URAC (1.0 mg/kg)/VEHICLE0-0 1I • I I2 3 446wz-JUiCl)F0I0C-)TIME (hr)250-200-150-II • I • I0 1 2 3B 0 APO (1.0 mg/kg)• RAC (1.0 mg/kg) I APO (1.0 mg/kg)100-50-0-25020015010050I • I • I I0 1 2 3Figure 12. Frontal cortex dialysate values of ACh. A: SCH 23390 (SCH; 0.3 mg/kg)and B: raclopride (RAC; 1.0 mg/kg) were injected 30 mm prior to peripheraladministration of apomorphine (APO; 1.0 mg/kg; closed circles). The effect of APOalone (open circles) is included for comparison. Data points represent group means(n=5-6) ± S.E.M.I0-47The somewhat selective D2 receptor antagonist haloperidol (0.15 mg/kg) and theselective antagonist raclopride (1.0 mg/kg) did not significantly affect dialysateconcentrations of ACh [F( 1 ,9)=0.04, F( 1 ,9)=3.42, respectively; Fig. 11], although atendency for both to result in decreasing concentrations was apparent. When AMPH wasadministered 30 mm after the neuroleptics, the ACh response was less than that ofAMPH alone and greater than the neuroleptic alone. Haloperidol did not completelyblock the effect of AMPH, because AMPH significantly increased ACh output afterhaloperidol, compared to haloperidol alone [drug x time interaction: F(5.48,43.8 1 )=2.54,p=0.038; the overlapping curve shapes yielded a nonsignificant main effect: F(1,8)=0A2;Fig. 1 1A]. Raclopride also did not completely block the effect of AMPH, becauseAMPH administration after raclopride caused a significant increase over raclopride alone[F(1 ,8)=6.0l, p=0.O4; Fig. 1 lB]. However, both D2 antagonists did significantly attenuatethe ACh response to AMPH, compared to the response of AMPH alone [F(1,9)=16.53,p=O.003 for haloperidol and F(l,9)=8.64, p=0.017 for raclopride]. The maximum increaseafter AMPH (280%) was decreased to 160% by haloperidol and 170% by raclopride. Theattenuated responses of ACh to AMPH injections after the neuroleptics did not differsignificantly from the vehicle condition [F(l ,9)=2.l6 for haloperidol and F(1,9)=3.50 forraclopridel. All three receptor antagonists completely blocked the AMPH-inducedbehavioural hyperactivity (data not shown).Fig. 12 shows the effects of selective DA receptor antagonists on the ability ofapomorphine, the non-selective DA receptor agonist, to increase ACh release. (Theapomorphine response from Fig. 7A is superimposed for comparison). Theapomorphine-induced increase in ACh output was completely blocked by the D1receptor antagonist SCH 23390 [0.3 mg/kg; F(1,8)=l0.22, p=0.013; Fig. l2A]. Althoughthe D2 receptor antagonist raclopride (1.0 mg/kg) appeared to partially block theapomorphine-induced increase in cortical ACh release, this effect was not statistically48significant [F(l,9)=O.02; Fig. 12B]. Both the D1 and D2 receptor antagonists blockedapomorphine-induced behavioural activation.(D) DISCUSSIONThe results presented here, that an indirect DA agonist (AMPH) and a DAreceptor agonist (apomorphine) increase cortical ACh release, suggest that DA regulatesthe activity of cortically-projecting cholinergic neurons. This effect is probablymediated outside of the cortex given that cortically applied AMPH did not increase AChrelease. Because D1 selective agonists and antagonists more robustly affected basal anddrug-stimulated release of ACh than did D2 selective drugs, dopaminergic regulation ofcortical ACh release is apparently mediated primarily via D1 receptors, although acontribution of D2 receptors cannot be excluded.Regulation of cortically-projecting cholinergic neurons by DAThe results summarized above confirm previous evidence that apomorphine andAMPH increase cortical ACh release as assessed using the cortical cup technique (Beaniand Bianchi, 1973; Casamenti et al., 1986; 1987; Hemsworth and Neal, 1968; Mantovaniet at., 1977; Pepeu and Bartolini, 1968). A microdialysis study has also confirmed thiseffect of AMPH on cortical ACh release (Okada, 1991). In addition, AMPH has beenreported to increase the turnover rate of cortical ACh although apomorphine does not(Robinson et al., 1978) suggesting either that the latter measure is not as sensitive asmicrodialysis or that the turnover of ACh is not always coupled to ACh release.AMPH is a psychomotor stimulant which induces behavioural hyperactivity andEEG desynchronization (Fairchild et at., 1967). The behavioural effects of this drug areusually ascribed to its ability to elevate synaptic concentrations of DA, noradrenaline49(NA) and, to a lesser extent, serotonin (5-HT) (Azzaro and Rutledge, 1973; Kuczenski,1983). The data presented here demonstrate that the AMPH-induced increases inmonoaminergic transmission have robust effects on cortical ACh release. It isnoteworthy that AMPH’s ability to desynchronize cortical EEG activity is, at least inpart, cholinergically mediated (Dren and Domino, 1968; White and Daigneault, 1959).DA could be responsible for AMPH’s stimulation of cortical ACh release given that theDA receptor agonist apomorphine, which increases the firing rate of unidentifiedneurons in the basal forebrain (Napier et a!., 1991), increases cortical ACh release(present results; Mantovani et a!., 1977).The other monoamines that are affected by AMPH, NA and 5-HT, have not beenexamined in these experiments, but existing evidence suggests that theseneurotransmitters are less likely candidates for mediating the AMPH-induced increasesin cortical ACh release. For example, while AMPH increases synaptic concentrations ofNE, this transmitter appears to inhibit cortical ACh release. Application of NA orstimulation of noradrenergic cell bodies in the locus coeruleus decreases cortical releaseof ACh, apparently via actions on alpha receptors (Beani et a!., 1978; Bianchi et a!.,1979; Vizi, 1980). Conversely, while alpha antagonists have been reported to blockAMPH-induced increases in cortical ACh turnover (Robinson et a!., 1978), Bartolini andPepeu (1970) found that beta, but not alpha, blockers attenuated the effect of AMPH oncortical ACh release. An understanding of these apparently contradictory results awaitsfurther study using the more selective drugs and the more sensitive procedures that arenow available.AMPH also increases synaptic concentrations of 5-HT (Kuczenski and Segal, 1989)but like NE, 5-HT has previously been reported to have an inhibitory influence oncortical ACh release as determined in slice, synaptosome or cortical cup preparations(Barnes et at., 1989; Bianchi et a!., 1990; Muramatsu et a!., 1990; Siniscaichi el a!., 1990;1991). Recent microdialysis results have revealed, however, that increasing serotonergic50tone via administration of the 5-HT releasing agent fenfluramine increases cortical AChrelease (Hirano, Day and Fibiger, submitted). Regardless of whether 5-HT may alsoregulate CBC neurons in an excitatory manner, the near complete blockade of theAMPH-induced increases in cortical ACh release by the D1 antagonist suggests that 5-HT does not mediate AMPH’s effect on CBC neurons. In support of the conclusion thatDA is the primary transmitter through which AMPH increases cortical ACh release,Casamenti et at. (1987) have reported that AMPH-induced increases in cortical AChrelease are not reduced by the 5-HT synthesis inhibitor, para-chlorophenylalanine, orthe NA depletor, N-(2-chloroethyl)-N-ethyl-bromobenzylamine (DSP4).Neuroanatomy of dopaminergic regulation of cortically-projecting cholinergic neuronsThe site of interaction between dopaminergic drugs and cortically-projectingcholinergic neurons has not been determined by these experiments. As outlined in theIntroduction, direct synaptic contact between catecholaminergic terminals and cholinergicperikarya in the CBC has been reported (Mimer, 1991). Given that catecholaminergicinnervation of the basal forebrain arises in part from nuclei containing dopaminergicperikarya (Jones and Cuello, 1989; Semba et at., 1988), it is possible that at least aportion of this innervation is dopaminergic. Indirect support of a dopaminergicinteraction with cholinergic perikarya includes the findings that unidentified neurons inthe basal forebrain exhibit increased firing rates in response to microiontophoreticapplication of DA (Napier et al., 1991) and receptors for DA are present in the basalforebrain (Zilles et at., 1991). Although forebrain cholinergic neurons apparently do notexpress D2 receptor mRNA (Le Moine et al., 1990), parallel evidence for other DAreceptor subtypes is unavailable.The possibility also exists that dopaminergic regulation of cortical ACh release ismediated by presynaptic control of cholinergic terminals in the cortex. Neuroanatomicalstudies have demonstrated a dopaminergic innervation of, and DA receptors in, the51cortex (Lindvall et a!., 1974; Richfield et at., 1989). However, the data presented hereindicate that AMPH does not act locally in the frontal cortex to increase ACh releaseand this is supported by previous findings (Beani and Bianchi, 1973; Pepeu andBartolini, 1968). It is unlikely that the lack of effect of locally applied AMPH is due toinsufficient dosage: AMPH is known to be permeable through the dialysis membraneused here (Nomikos et at., 1990), and the concentration of AMPH chosen for thisexperiment has potent effects on dialysate concentrations of both DA and ACh in thestriatum, as shown previously using identical methodology (Westerink et at., 1990).Future experiments using more selective dopaminergic drugs may help determine ifcortical DA can presynaptically regulate cortical ACh release.It is also possible that multisynaptic mechanisms mediate DA’s effects oncortically-projecting cholinergic neurons. For example, DA in the nucleus accumbensmay serve to disinhibit CBC neurons by inhibiting the activity of a GABA-containingprojection from the nucleus accumbens to the ventral pallidum (Casamenti et al., 1986).Previous evidence supporting this possibility includes: 1) a projection from the nucleusaccumbens terminates on CBC neurons (Zaborszky and Cullinan, 1992); 2) this projectionmay use GABA as a transmitter (Walaas and Fonnum, 1979); 3) peripheral administrationof a dopaminergic agonist decreases extracellular concentrations of GABA in the basalforebrain (Bourdelais and Kalivas, 1992); 4) microinjections of DA into the nucleusaccumbens increases the firing rate of the same unidentified basal forebrain neurons thatare stimulated by a GABA antagonist (Yang and Mogenson, 1989); and 5) GABAregulates cortically-projecting cholinergic neurons (Sarter et al., 1990).Previously, studies of interactions between DA and ACh have focussed mainly onthe striatum (Lehmann and Langer, 1983; Stoof et at., 1992) largely due to the putativeclinical relevance of such interactions to Parkinson’s disease and neuroleptic-inducedextrapyramidal side effects (Barbeau, 1962; Borison and Diamond, 1987; McGeer et al.,1961). Abundant evidence from striatal slice and homogenate studies has shown that52local DA receptors of the D2 subtype have inhibitory effects on striatal ACh release(Scatton, 1982; Stoof and Kebabian, 1982). Thus, at a superficial level, an excitatorydopaminergic regulation of cortical ACh release might be unexpected based on thepreviously reported inhibitory regulation of striatal ACh by DA. However, whenexamined in intact preparations using in vivo microdialysis, the effect of DA on striatalACh release has recently been shown to be more complex than earlier reports hadsuggested: excitatory actions at DA receptors of the D1 subtype appear to override localD2 mediated inhibitory actions of DA. There remains a controversy as to whether theD1 receptors mediating this effect on striatal ACh release are located in the striatum(Consolo et al., 1992; Zocchi and Pert, 1993) or extrastriatally (Damsma et a!., 1991).The results presented here suggesting that extracortical dopamine receptors stimulatecortical ACh release are not dissimilar to the suggestion that extrastriatal D1 receptorsstimulate striatal ACh release.DA receptor subtype(s) mediating dopaminergic regulation of cortically-projectingcholinergic neuronsReceptors for DA were subdivided in the early 1980’s into two classes based ontheir pharmacological, biochemical and physiological properties (Kebabian and Caine,1979). More recently, gene cloning techniques have led to the subdivision of DAreceptors into five genetically-defined subtypes. The D5 receptor is similar to the D1receptor, while the D3 and D4 receptors are more similar to the previously defined D2receptor. For example, quinpirole was previously described as a D2 agonist, but hasbeen shown to have high affinity at the D3 and D4 sites (Sokoloff et at., 1990; Tang etat., 1994). PHNO has been reported to bind D2 rather than D3 or D4 receptors (Seemanet a!., 1993). Studies of the D3, D4 and D5 receptors are in the early stages, and theexperiments presented here (and in Chapter V) do not attempt to characterize thedopaminergic regulation of cortically-projecting cholinergic neurons according to this53new classification of DA receptors. Therefore, for the purposes of this thesis, “D1” and“D2” should be interpreted as D1 -like and D2-like, respectively.The results of the pharmacological experiments presented here, using selectiveagonists and antagonists for D1 and D2’ type dopamine receptors, suggest that DAregulates the activity of cortically-projecting cholinergic neurons primarily via actions atD1 receptors. Thus, the selective D1 agonist significantly increased cortical ACh releasewhile the D2 agonists did not. In addition, the increased cortical ACh release inducedby AMPH or apomorphine was blocked to a greater degree by the D1 antagonist SCH23390 than by the D2 antagonists, and only the D1 antagonist significantly decreasedbaseline concentrations of ACh.There is indirect evidence from other sources suggesting that D1 receptors are ofgreater importance than D2 receptors in regulating the activity of cortically-projectingcholinergic neurons. Measures which are at least in part cholinergically mediated, suchas behavioural hyperactivity, EEG desynchrony, and reversal of narcosis, are affectedmore by D1 receptor stimulation and blockade (Bagetta et at., 1987; Ongini et at., 1985)than by D2 manipulations (Horita and Carino, 1991; Horita et at., 1991; Ongini andLongo, 1989). In addition, with regard to the possible anatomical sites of dopaminergicregulation of cortical ACh release discussed above, it is noteworthy that D1 receptors aremore numerous than D2 receptors in the basal forebrain (Zilles et at., 1991), in mostregions and laminae of the cortex (Richfield et at., 1989) and in the nucleus accumbens(Richfield et at., 1987). It has also been demonstrated recently that the D1 agonist usedin the present experiments induces expression of Fos-like immunoreactivity, which isoften considered a marker of neuronal activity, in cortically-projecting cholinergicneurons in the basal forebrain (Robertson and Staines, 1994). Existingelectrophysiological evidence, that a D1 agonist increases activity while a D2 agonistdecreases activity in a majority of basal forebrain neurons (Maslowski and Napier,541991), neither supports nor contradicts the data presented here given that the neuronsunder investigation were not identified as cholinergic or cortically-projecting.In contrast to the proposition that D1 receptors are of primary importance fordopaminergic regulation of cortical ACh relese, Casamenti et a!. (1987) have reportedthat AMPH-induced increases in cortical ACh release, as assessed by a cortical cuptechnique in immobilized rats, are blocked by a high dose of haloperidol (1.0 mg/kg)and not by a low dose of SCH 23390 (0.003 mg/kg). These researchers also reportedthat high doses of ciuinpirole (1.0 and 2.5 mg/kg) significantly increased cortical AChrelease to a peak of 44 and 51%, respectively, over baseline, while the partial D1 agonistSKF 38393 (1.25 and 10 mg/kg) was without effect. Interestingly, in pilot experimentsin this laboratory, SKF 38393 at 20 mg/kg robustly increased cortical ACh to 350% ofbaseline. The difference between the present results and these earlier observations maybe due to the use of different doses, and/or to the use of freely moving animals andmore physiologically relevant sampling techniques in the present study.Although the evidence presented here indicates that D1 receptors predominate overD2 receptors in mediating the effects of DA on CBC neurons, several observationssuggest that D2 receptor stimulation may also participate. D2 antagonists partiallyblocked the effect of AMPH and apomorphine on cortical ACh release and slightly, butinsignificantly, decreased basal dialysate values of cortical ACh. Thus, in attempting todetermine the relative contribution of D2 receptors in regulating cortical ACh release, aninconsistency has become apparent: D2 receptor antagonists are somewhat effectivewhile D2 agonists are not. It remains possible that D2 agonists have minor excitatoryactions on the basal forebrain cholinergic system that were not revealed by the presentexperiments. For example, although the effects of the D2 agonists were not statisticallysignificant, extracellular ACh concentrations were somewhat increased in the secondhour after administration of the drugs. This is in contrast to quinpirole- and PHNOinduced decreases of ACh release in the striatum (Bertorelli and Consolo, 1990;55Robertson et a!., 1993; Timmerman and Westerink, 1991), which are not larger in thesecond hour after administration than in the first. It is unlikely that the lack ofsignificant effect was due to insufficient dosage, because the doses of quinpirole andPHNO used here have been shown by others to have behavioural and/or neurochemicaleffects (Bertorelli and Consolo, 1990; Martin et al., 1984; Robertson et at., 1993). Thepresent experiments also demonstrated that the expression of an otherwise silent D2effect on cortical ACh release is not enabled by D1 receptor stimulation. Interactionsbetween D1 and D2 receptors have been proposed to explain certain other D2 mediatedeffects (Clark and White, 1987; Waddington and O’Boyle, 1989). However, quinpirolehad no effect when paired with the D1 agonist in the present experiments, and thereforesuch interactions do not appear to be important in the regulation of the basal forebraincholinergic system.If D2 receptors are not involved in regulating cortical ACh release, as some ofthese experiments suggest, it remains to be explained why D2 antagonists significantlyattenuate AMPH-induced increases in cortical ACh release. It is possible that a nondopaminergic contribution to AMPH’s effect on cortical ACh release is blocked by theseD2 antagonists by virtue of their effects on other monoamine receptors (Christensen etat., 1984; Hall et at., 1986; Ogren et al., 1986). In addition, it was demonstrated inChapter II that there is a significant relationship between locomotor activity and corticalACh release across a number of conditions. Therefore, because raclopride andhaloperidol both reduce AMPH-induced increases in locomotor activity, it is possiblethat these D2 receptor antagonists block that portion of AMPH’s effects on cortical AChrelease that is associated with increases in locomotor activity. It is noteworthy in thisregard that the increasing trend of cortical ACh release in the second hour afteradministration of the D2 agonists corresponds to the time of increased locomotionreported after injection of 0.5 mg/kg quinpirole (Eilam and Szechtman, 1989).56Given the previously demonstrated correlation between cortical ACh release andbehavioural activity, it is interesting that the DA receptor antagonists blocked theAMPH-induced locomotor activity while failing to antagonize completely the AMPHinduced increase in ACh release and that the D2 agonists stimulated behaviour withoutincreasing cortical ACh release. In addition, unlike more potent Dj agonists (Acquas eta!., 1994) the D1 agonist used in the present studies did not increase locomotion,although it did increase cortical ACh release. Thus, pharmacological treatments candissociate the usually positive correlation between ACh release and locomotion. Moreprecise behavioural characterizations might help reveal the basis of the correlation andits dissociation by pharmacological treatments.The experiments reported here indicate that D1 receptors primarily mediate theexcitatory effects of dopaminergic drugs on cortically-projecting CBC neurons. A minorrole of D2 receptors cannot be excluded, however, and the basis of the discrepancybetween the lack of effect of D2 agonists and the partial effects of D2 antagonistsremains to be determined.57IV. ATTENUATION OF AMPHETAMINE-INDUCED INCREASES OF CORTICALACETYLCHOLINE RELEASE BY FOREBRAIN DEPLETIONS OF DOPAMINE, BUTNOT NORADRENALINE(A) INTRODUCTIONAs discussed previously, cholinergic projections to the neocortex which arise fromthe basal forebrain (Lehmann et at., 1980) are critically involved in cortical activation(Semba, 1991). The stimulant drug AMPH desynchronizes the cortical EEG, causesbehavioural hyperactivity (Fairchild et a!., 1967), and increases the activity of neurons inthe CBC as measured by increased cortical ACh turnover (Robinson et al., 1978) andrelease (Chapter III; Casamenti et at., 1986; 1987; Hemsworth and Neal, 1968; Okada,1991; Pepeu and Bartolini, 1968). These effects of AMPH are likely mediated indirectlyby DA and/or NA, the neuronal release of which are increased by this drug (Azzaro andRutledge, 1973; Kuczenski and Segal, 1989). In support of this hypothesis, dopaminergicand noradrenergic neurons innervate the cortex (Fuxe et at., 1968; Lindvall et a!., 1974)and the region in the basal forebrain containing cholinergic perikarya (Semba et at.,1988; Zaborszky et al., 1991). Receptors for DA and NA are also present in both thecortex (Palacios and Kuhar, 1980; Richfield et at., 1989; Sargent Jones et a!., 1985) andbasal forebrain (Zilles et at., 1991). Multi-synaptic pathways may also account for theeffect of DA and/or NA on cortical ACh release. Of these possible sites of interaction,the cortex has been ruled out given the lack of effect of cortically-applied AMPH oncortical ACh release (Chapter III; Beani and Bianchi, 1973; Pepeu and Bartolini, 1968).Pharmacological experiments have also suggested an interaction of both DA andNA with CBC neurons. Nonselective DA- and D1- receptor agonists increase corticalACh release and D1 receptor antagonists decrease basal release and block AMPHinduced release of ACh in the cortex (Chapter III; Mantovani et al., 1977). In contrast,58although effects of noradrenergic agents on cortical ACh release have been reported,these seem unable to account for AMPH—induced increases in cortical ACh release.Thus, application of NA or stimulation of noradrenergic cell bodies in the locuscoeruleus decreases cortical ACh release, apparently via actions on alpha-noradrenergicreceptors (Beani et a!., 1978; Bianchi et at., 1979; Vizi, 1980). Conversely, while alphareceptor antagonists have been reported to block AMPH-induced increases in corticalACh turnover (Robinson et a!., 1978), Bartolini and Pepeu (1970) found that beta, butnot alpha, blockers attenuated the effect of AMPH on cortical ACh release. Morerecently, it has been reported that NA depolarizes identified CBC neurons in guinea pigbrain slices (Fort et a!., 1993). An understanding of these apparently contradictoryresults awaits further study using the more selective drugs and physiological techniquesthat are now available.The present experiments further assessed the extent to which forebrain DA and/orNA mediate AMPH-induced stimulation of CBC neurons. To this end, AMPH-inducedchanges in cortical microdialysate concentrations of ACh were measured in animalsdepleted of forebrain DA or NA by neurotoxic lesions. In addition, the effect oncortical ACh release of an arousing non-pharmacological stimulus, injection of vehicle(Chapter II), was examined in rats depleted of NA to test for a possible interaction oflocus coeruleus NA neurons and CBC neurons in cortical arousal (Berridge and Foote,1991; Foote et a!., 1980; Semba, 1991).(B) MATERIALS AND METHODSExperimental protocol and drugsMale Wistar rats (270-3 10 g) received unilateral 6-hydroxydopamine (6-OHDA)lesions of the mesotelencephalic dopamine system (MDS), bilateral 6-OHDA lesions of59the dorsal noradrenergic bundle (DNB), or control surgeries and were group housed for 2weeks with food and water available ad libitum. Following surgical implantation of amicrodialysis probe into the frontal cortex at the end of this 2 week period, all rats werehoused individually in Plexiglas cages (35x35x25 cm) and maintained on a 12:12 hlight:dark schedule with food and water available ad libitum. Dialysis was initiated twodays after probe implantation, during the rats’ normal “lights on” phase, and experimentsinvolved the subcutaneous injection of vehicle or AMPH (2.0 mg/kg; BDH). This doseof AMPH has significant behavioural effects and reliably increases ACh release in thefrontal cortex (Chapter III). An unlesioned group of rats with bilateral frontal cortexprobes was also dialyzed with perfusates containing the sodium channel blocker TTX(luM) or lacking calcium ions.Unilateral o-OHDA lesions of the mesotelencephalic DA systemAlthough theoretically preferable, bilateral lesions of the MDS were precluded byensuing aphagia and adipsia in bilaterally lesioned animals (Fibiger et a!., 1973).Therefore, unilateral lesions were performed as follows: rats were pretreated withdesmethylimipramine (20 mg/kg intraperitoneal (i.p.)) 30 mm prior to surgery,anaesthetized with sodium pentobarbital (50-60 mg/kg i.p.), and placed in a stereotaxicframe with the incisor bar positioned 4.2 mm below interaural zero. A 30 gaugestainless steel cannula was lowered through a burr hole in the skull to a site in the lateralhypothalamus containing axons of the MDS (A:+5.9, L:+2.3, D:+2.2 measured frominteraural zero). The cannula was attached by polyethylene tubing to a 5 ul Hamiltonsyringe driven by a syringe pump (Harvard Apparatus), all shielded from light. 6-OHDA HBr (3.0 mg/mI, Sigma) was dissolved in a vehicle consisting of 0.5 mg/miascorbic acid in 0.9% saline and kept on ice in the dark before use. Following a twominute delay after the cannula was positioned in the target site, the 6-OHDA solutionwas infused at 0.2 uI/mm over 20 mm to yield a dose of 8 ug base/4 ui/site. After a60further 5 mm to allow for diffusion, the cannula was removed and the wound stitchedclosed. Control animals were pretreated identically to the lesioned animals; the cannulawas aimed at the same co-ordinates, but lowered only into the cortex (D+8.0 frominteraural zero) and left there for 5 mm.Bilateral 6-OHDA lesions of the dorsal noradrenergic bundleRats were anaesthetized with sodium pentobarbital (50-60 mg/kg i.p.) and placedin a stereotaxic frame with the incisor bar 3.3 mm below interaural zero. Simultaneousbilateral injections of 6-OHDA were carried out as described above with the followingdifferences: the cannulae were positioned in the DNB (A:+2.6, L:±l .0, D:+3.6 measuredfrom interaural zero), and 6-OHDA (2.0 mg/ml) was infused at 0.2 ui/mm over 10 mmto yield a dose of 2.7 ug base/2 ui/side. For the surgical control group, the cannulaewere aimed at the same anterior/lateral co-ordinates, but lowered only into the cortex(D:+8.6 from interaural zero) and left there for 5 mm.Probe implantation and microdialysisBrain microdialysis was performed as in Chapter III with the following exceptions.The probes were made of acrylonitrile-sodium methallyl sulfonate fiber (inner diameter= 220 urn, outer diameter = 310 urn, molecular weight cut off> 60 000 Dalton; FiltralAN69, Hospal) and had an active surface length of 10.9 mm in the DNB lesioned andcontrol groups or 5.45 mm in the MDS lesioned and control groups. In the latter groups,only the half of the probe situated in the cortical hemisphere ipsilateral to the lesion wasleft unblocked and thus active for dialysis.To prevent twisting of inlet and outlet tubing due to excessive turning in the MDSlesioned rats treated with AMPH, the tubings of these rats and their controls wereattached to a liquid swivel which was anchored to a “vest” worn by the rat.61In vitro recovery of probesUnused bilateral cortical microdialysis probes were perfused with the neostigminecontaining perfusion solution while immersed in a standard solution of ACh/Chmaintained at 37°C. The recovery of ACh across the membrane was calculated as apercentage of the standard solution.Assay of AChACh was assayed by HPLC-ECD as described in Chapter II, with the followingadditions to the technical equipment: an HPLC pump (Shimadzu) and a platinumelectrode/reference electrode assembly (Antec).Tissue level analysisTo determine the efficacy of the neurotoxic lesions, representative dopaminergicterminal regions of the MDS (striatum and nucleus accumbens (NAc)) and representativenoradrenergic terminal regions of the DNB (frontal cortex (FCTX), parietal cortex(PCTX) and hippocampus) were assayed for biogenic amine content. Rats were killedby cervical dislocation and the brains were rapidly removed. To assess DA depletion inthe MDS lesioned rats, 2 coronal sections of forebrain were cut with a freezingmicrotome, spanning the following anterior coordinates (in mm) measured from bregma(from the atlas of Paxinos and Watson, 1986): 1.2-2.2 for NAc and 0.2-1.2 for striatum.To assess NA depletion in the DNB lesioned rats, 2 coronal sections were cut using aMcllwain tissue chopper, spanning 1.2-5.2 for the FCTX and (-l.8)-(+0.2) for thePCTX. From the remaining caudal portion of brain, the hippocampi were handdissected. The brain sections were placed on ice-cold, dampened filter paper where DAand NA terminal regions were hand-dissected from both hemispheres with the aid ofbinocular magnifiers. This procedure yielded unilateral tissue samples having meanweights of 9.6 mg for NAc, 13.2 mg for striatum, 26.4 mg for FCTX, 18.6 mg for62PCTX and 17.0 mg for hippocampus. Tissue samples were sonicated in ice-cold 0.2 MHC1O4 containing 0.15% Na2SO3and 0.05% Na2EDTA (NAc, FCTX and PCTX in 300ul, striatum in 400 ul, hippocampus in 500 ul). After centrifugation, samples were storedat -80°C until the time of thçir assay. Thawed samples were injected from a 100 ulloop on a valve (Valco) onto a reverse phase analytical column (nucleosil 5 urn C18;Phenomenex). The mobile phase consisted of 0.1 M sodium acetate buffer, 0.01 mMNa2EDTA, 0.75-0.85 mM octane sulfonic acid, 10-12% methanol, pH 3.6 and wasdelivered at 0.35-0.8 mi/mm by a pump (PM-48, BAS or Shimadzu LC-600). Soluteswere quantified by electrochemical detection using an LC-4B amperometric detector(BAS) with a glassy carbon working electrode at +0.7 V vs. a Ag/AgCl referenceelectrode. The detection limit for all compounds under these conditions was between 10and 100 fmol/injection.Retrograde labellingUnilateral cortical probes, in MDS lesioned and control rats which could not beused for dialysis experiments due to technical problems (n=3), were filled with a 4%solution of Fluoro-Gold (Fluorochrome, Inc.) in 0.9% saline, and flushed with saline 12h later. One week later these animals were sacrificed and coronal forebrain slices werecollected, processed and examined under ultraviolet light as described by Robertson eta!. (1991).Statistical analysesMicrodialysate outputs in each animal were calculated as a percent of baseline,100% baseline being defined as the average of the last six pre-drug concentrations(fmol/min). As an alternate method of correcting for inter-animal variability in baselineoutput, data from individual animals were also transformed into “delta fmol/min” bysubtracting the average of the last six pre-drug concentrations (fmol/min) from the63absolute concentration (fmol/min) of each dialysate sample. Univariate ANOVAs wereused to compare basal microdialysate outputs (fmol/min) of cortical ACh in lesion vs.control groups for the two lesion types. ANOVAs with repeated measures were used tocompare treatment-induced changes in cortical microdialysate outputs of ACh (in both %baseline and delta fmol/min scores) in surgical control vs. 6-OHDA lesioned rats, andthe main treatment effect is reported. Treatment x time interaction effects are alsoreported which include Huynh-Feldt adjustments of the degrees of freedom to accountfor the use of time as a repeated measure. One-way analyses of variance with Tukey“highly significant differences” post-hoc comparisons were used to assess differences inlesion-induced tissue concentrations of monoamines.(C) RESULTSEfficacy of lesionsTable 3 shows the effects of 6-OHDA lesions of the MDS on tissue concentrationsof DA, NA and 5-HT. DA was significantly depleted in the striatum and NAcipsilateral to the lesioned MDS, and decreased to a lesser extent in the contralateral NAc.NA concentrations were also significantly reduced ipsilaterally by these lesions. Tissueconcentrations of 5-HT were not affected in any region.The results of 6-OHDA DNB lesions on regional tissue concentrations of DA, NAand 5-HT are summarized in Table 4. NA was significantly depleted in all 3 regions,leaving tissue concentrations of DA and 5-HT unaffected.Site of neurons projecting to unilateral cortical probeApplication of Fluoro-Gold through the unilateral cortical probe retrogradelylabeled cell bodies in, among other areas, the basal forebrain (data not shown). While64Table 3. Effects of 6-OHDA lesions of the mesotelencephalic dopamine system on regionaltissue concentrations of dopamine, noradrenaline, and serotoninTissue concentrationpmol/mg tissue ± S.E.M. (% surgical control)Region Surgical Control Lesion Controla LesionDOPAMINEN. accumbens 39.1±2.8 27.9±1.5 (71.4%)# 0.19±0.06 (0.49%)*Striatum 57.6±3.3 57.6±2.5 (100.0%) 0.06±0.02 (0.10%)*NORADRENALINEN. accumbens 0.96±0.09 0.71±0.08 (74.0%) 0.36±0.07(37•5%)**Striatum 0.48±0.05 0.62±0.07 (129.2%) 0.11±0.05 (22.9%)SEROTONINN. accumbens 2.68±0.26 2.24±0.15 (83.7%) 2.59±0.26 (96.8%)Striatum 1.23±0.08 1.08+0.06 (87.6%) 1.04+0.13 (84.3%)Tissue was assayed from 6 surgical control rats and 10 6—OHDA lesioned rats.Lesion control samples are from the hemisphere contralateral to the lesion.Values are significantly different from both the surgical control and the lesion control, p<O.05.#Value is significantly different from both the surgical control and the lesion, p<0.05.65Table 4. Effects of 6-OHDA lesions of the dorsal noradrenergic bundle on regionaltissue concentrations of dopamine, noradrenaline, and serotoninTissue concentrationpmol/mg tissue ± S.E.M. (% surgical control)Region Surgical Control LesionDOPAMINEFrontal Cortex 0.33±0.02 0.34±0.03 (102.6%)Pariëtal Cortex 0.11±0.04 0.076±0.0 13 (70.2%)Hippocampus 0.047±0.010 0.037±0.005 (78.5%)NORADRENALINE*Frontal Cortex 1.44±0.09 0.048±0.009 (3.3%)*Parietal Cortex 1.07±0.06 0.022±0.005 (2.1%)*Hippocampus 1.85±0.14 0.10±0.03 (5.6%)SEROTONINFrontal Cortex 2.85±0.17 3.05±0.24 (106.7%)Parietal Cortex 1.19±0.09 1.41+0.11(118.5%)Hippocampus 1.87±0.13 2.10±0.16 (111.9%)Tissue was assayed from 7 surgical control rats and 9 6-OHDA lesioned rats, with theexception of basal forebrain tissue which was dissected from 5 surgical control rats and 76—OHDA lesioned rats.*Values are significantly different from the surgical control, p<0.O5.66the vast majority of the neurons labeled in this region were in the hemisphere ipsilateralto the open side of the probe, a few labeled neurons could be observed in thecontralateral basal forebrain.Characterization of probes made with acrylonitrile fiberThe effect of locally-perfused TTX (1 uM) on cortical ACh output recovered bythe acrylonitrile membrane is shown in Fig. 13. Dialysate concentrations of AChdecreased significantly during the 60 mm of TTX perfusion [F(1.06,4.24)=l 1.99,p=O.O23]. Calàium-free perfusion also reduced ACh output by 75-80% in two rats (datanot shown). The unlesioned rats used for these experiments had an average basal output(± S.E.M.) of 68.34 ± 10.79 fmol/min (n=7). In vitro recovery of the bilateral cortexprobe, using Hospal membrane, was 51.7 ± 2.5% (n=3).Effects of AMPH on cortical ACh release in DA- or NA-depleted ratsThe average outputs of ACh from the unilateral cortical probes in the MDSlesioned and control groups (in fmol/min ± S.E.M.) were 44.1 ± 4.9 and 31.6 ± 7.6respectively. The bilateral cortical probes yielded average outputs of 67.8 ± 10.3 in DNBlesioned rats, and 85.9 ± 13.4 in their surgical controls. These values were notsignificantly different in the MDS groups [F(l,l2)=2.25, p=O.16] or the DNB groups[F(l,14)=1.19, p=O.29].The effects of AMPH on ACh release in the cortex of DA-depleted rats and theirsurgical controls are shown in Fig. 14. In surgical control animals, AMPH increasedACh release to a maximum of approximately 270% of baseline (Fig. 14A) or by 40fmol/min (Fig. l4B), for 3 h. In contrast, ACh release increased to 160% of baseline, orby 25 fmol/min in the 6-OHDA MDS-lesioned rats. The AMPH-induced increases inACh release in the cortex of these two groups of animals differed significantly, usingdata calculated as percent baseline [F(l,l2)=8.92, p=0.Oll]. The more conservative67wz-JwC,)I01-0C)TIME (hr)Figure 13. Tetrodotoxin (TTX)-sensitivity of cortical ACh recovered by a transversedialysis probe made of acrylonitrile fibre. TTX (1 uM) was included in the perfusatefor 1 h. Data points represent group means (n=5) -- S.E.M.150-100-500-—1I0684)Ca)U,CuI—00CE0ECua)F00TIME (hours)AAMPH, 2 mg/kgI I • I I • I—1 0 1 2 3B4300 -250-200-150-100-050-60-40-20-0-o —20-• .Figure 14. Dialysate values of ACh in the frontal cortex of rats having unilateral6-OHDA lesions of the mesotelencephalic dopamine system (MDS; closed circles, n=9)or of control rats (open circles, n5) after injection of d-amphetamine (AMPH; 2.0mg/kg). Data points represent group means ± S.E.M. and are presented as A:percentage of baseline, or B: delta fmol/min (see Experimental Procedures).o CONTROL• MDS LESIONAMPH, 2 mg/kg—1 1 2 3a)a)U)I—0I—00CE0E‘I4-,a,-oIa0C-)• • I i ‘1—1 0 1 2 3TIME (hours)AVEHICLEAMPH, 2 mg/kg500 -400-300-200-100-0-200-100-0-I— • I • I • I • I • I • I I—1 0 1 2 3 4 5 6B696Figure 15. Dialysate values of ACh in the frontal cortex of rats having bilateral 6-OHDA lesions of the dorsal noradrenergic bundle (DNB; closed circles) or of control rats(open circles) after injection of d-amphetamine (AMPH; 2.0 mg/kg; DNB lesion n=9,control n=7) and vehicle (1.0 mI/kg; DNB lesion n=4-7, control n=3-5). Data pointsrepresent group means ± S.E.M. and are presented as A: percentage of baseline, or B:delta fmol/min (see Experimental Procedures).VEHICLEAMPH, 2 mg/kg4 570treatment of data, using delta fmol/min, yielded a main effect approaching significanceat the 5% level [F(1,12)=4.21, p=0.063] and a significant treatment x time interaction[F(8.74,104.87)=2.54, p=O.0l2]. Following AMPH administration, both groups of ratswere behaviourally hyperactive and the 6-OHDA lesioned rats rotated ipsilaterally.Fig. 15 illustrates the effects of AMPH and vehicle administration in NA-depletedrats and their surgical controls. When calculated as percent baseline (Fig. 15A), AChrelease in the cortex was increased by AMPH to a maximum of 350% for 3 h in thecontrol group and to a greater extent (430% of baseline) in the 6-OHDA DNB-lesionedgroup. The effect of AMPH was not significantly different between these two groups[F(l,14)=l.89, p=0.l9l] This result is more evident when the data are represented asdelta fmol/min (Fig. 15B). AMPH increased ACh release by 200 fmol/min in bothlesioned and control animals, with no significant difference between these groups[F(l,14)=1.19, p=0.29’I]. The effect of vehicle injections on cortical ACh release, atransient increase, was not significantly different between the NA-depleted and controlanimals using either form of data representation [F(1,l0)=l.73, p=0.218 for % baselinedata and F(l,lO)=1.46, p=0.255 for delta fmol/min].(D) DISCUSSIONThe results reported here point to a more important role of forebrain DA than NAin regulating cortically-projecting cholinergic neurons, given that 6-OHDA lesions of theMDS attenuated AMPH-induced increases in cortical ACh release, whereas 6-OHDAlesions of the DNB did not. The increase in cortical ACh release caused by an arousingstimulus was also unaffected by the forebrain NA depletion produced by the DNBlesion. The finding that DA mechanisms contribute significantly to AMPH’s effects oncortical ACh release supports the pharmacological experiments described in Chapter III.71Technical considerationsDialysate concentrations of cortical ACh were found to be calcium-dependent andTTX-sensitive using probes made of acrylonitrile membrane. Although the in vitrorecovery of this membrane (52%) is superior to that of the cellulose ester fiber reportedin Chapter 11(33%), the average basal output found here using the acrylonitrile fiber inthe cortex of unlesioned rats (68 ± 11 fmol/min) is only somewhat higher than thatfound in Chapter 111 using cellulose ester fiber (47 ± 7 fmol/min). This finding is notunreasonable in view of the theory that in vivo factors, rather than in vitro recoveries,are the limitting factors determining the recoveries of dialysis probes (Benveniste et al.,1989). The present results demonstrate that the acrylonitrile membrane is a suitablealternative for the construction of transverse microdialysis probes for recovery of ACh.An unexpected complication in the present experiments was that the 6-OHDAlesion of the MDS with desmethylimipramine pretreatment did not selectively depleteipsilateral DA. The contralateral NAc showed a small but significant depletion of DA,and NA was significantly depleted in both the striatum and NAc. However, because themore substantial NA depletion after DNB lesions did not affect AMPH—inducedincreases in cortical ACh release, it can be concluded that the attenuation of AMPH’seffects by the MDS lesion was not noradrenergically mediated.Technical factors may also have contributed to an underestimation of the effect ofunilateral MDS lesions on AMPH-induced cortical ACh release. Because Fluoro-Goldadministered through the unilateral probe retrogradely labelled a small number ofneurons in the contralateral basal forebrain, it is possible that this molecule diffusedfrom the open side of the probe to the cortex contralateral to the lesion; similarly, asmall amount of ACh released from the contralateral cortex may have been recovered by-the unilateral probe. Alternatively, contralateral Fluoro-Gold staining in the basalforebrain could be caused by crossed cholinergic projections; however, this possibility72has received no previous neuroanatomical support. Either possibility would account fora small portion of ACh in the dialysate being unaffected by the unilateral MDS lesion.A final technical consideration requires discussion. Because the differences inbasal output between lesioned vs. control groups were large but not statisticallysignificant, an alternate method for presenting the dialysis data was developed. Unlikethe percent baseline calculations, the delta fmol/min measure removes the potentiallymisleading effects that differences in average basal output can have on percent scores.This is well illustrated in Fig. 15: when presented in the conventional percent baselineformat, the DNB lesions appear to have potentiated ACh release compared to the controlgroup. It is obvious, however, using the delta fmol/min measure (Fig. 15B), that therewas no difference between the groups in AMPH-stimulated ACh release, and theapparent difference seen in Fig. 15A was entirely due to the non—significant baselinedifferences between the two groups. In addition, the error terms are reduced using the•latter measure. This example demonstrates that where moderate differences in basaloutput between experimental groups exist, conventional percent baseline datapresentation can yield potentially misleading results. This problem can be overcomeusing the delta fmol/min calculation.Regulation of cortical ACh release by monoarninesThe data reported here confirm that DA regulates cortically-projecting cholinergicneurons. In support of this conclusion, nonselective DA- and selective D1- receptoragonists increase cortical ACh release (Chapter Ill; Casamenti et al., 1986; Mantovani etal., 1977). In addition, it has recently been demonstrated that a population of cortically-projecting CBC neurons are stimulated by a D1 agonist, assessed using the expression ofFos-like immunoreactivity as a marker of neuronal activity; this same population ofneurons exhibits markedly reduced ChAT immunoreactivity after 6-OHDA lesions of theMDS (Robertson and Staines, 1994). These authors thus speculate that destruction of DA73neurons may have deprived CBC neurons of an excitatory drive. As outlined in theIntroduction, previous evidence of noradrenergic regulation of CBC neurons isinconsistent, given that both excitatory (Bartolini and Pepeu, 1970; Fort et a!., 1993;Robinson et a!., 1978) and inhibitory (Beani et al., 1978; Bianchi et a!., 1979; Vizi, 1980)interactions have been reported.Previous experiments using rats depleted of monoamines further support theconclusion that DA, but not NA, mediates the effect of AMPH on basal forebraincholinergic neurons. Casamenti et a!. reported that presumed depletions of DA (1986)but not NA (1987) blocked AMPH.-induced increases of cortical ACh overflow measuredusing the cortical cup technique. In addition, Nilsson et a!. (1992) reported that DA, butnot NA, depletions attenuate AMPH-induced increases of hippocampal microdialysateconcentrations of ACh.It is perhaps surprising that neither of the two lesion conditions produced largereffects on AMPH-induced cortical ACh release. AMPH is not entirely selective for therelease of NA and DA, and also increases synaptic concentrations of serotonin (5-HT)(Azzaro and Rutledge, 1973; Kuczenski and Segal, 1989). Because 5-HT also appears tostimulate cortical ACh release (Hirano, Day, and Fibiger, submitted), a serotonergiccontribution to the effect of AMPH on cortical ACh release cannot be excluded. Thispossibility should be explored further. It should be noted, however, that it is notsupported by previous cortical cup data which showed no effect of the 5-HT synthesisinhibitor, para-chiorophenylalanine, on the AMPH-induced increase in ACh release(Casamenti et a!., 1987).One possible explanation for the failure of the extensive unilateral MDS lesions tomore effectively attenuate AMPH-induced cortical ACh release is that AMPH-inducedDA release in the intact hemisphere may have mediated the lesion resistant effect of thedrug on cortical ACh release. The precise neuroanatomical systems mediating this effectof AMPH have not been delineated and may include crossed projections. This74possibility is supported by the fact that systemic pharmacological blockade of AMPHinduced cortical ACh release by the D1 receptor antagonist SCH 23390 (Chapter III) isgreater than that produced by the unilateral DA lesions used here.Although the experiments presented here provide no evidence of noradrenergicregulation of cortically-projecting cholinergic neurons, it remains possible that NA doesaffect this system but that the DNB lesions left the relevant NA projectionintact.Unlike the MDS, which accounts for almost all DA in the forebrain, the noradrenergicinnervation consists of a dorsal tract (DNB) and a ventral tract (VNB) (Ungerstedt,1971). Thus, before excluding NA as a regulator of cortically-projecting cholinergicneurons, it will be necessary to determine if VNB lesions can attenuate AMPH-inducedcortical ACh release. In this regard, it is noteworthy that AMPH-induced increases inhippocampal ACh release is not affected by VNB lesions (Nilsson et at., 1992).75V. DOPAMINERGIC REGULATION OF HIPPOCAMPAL ACETYCHOLINERELEASE(A) INTRODUCTIONCholinergic projections to the hippocampus originate from perikarya in the medialseptum and vertical limb of the diagonal band of Broca (Fibiger, 1982). Rather thanbeing arranged as discrete nuclei these cholinergic neurons, along with cholinergicneurons projecting to the cerebral cortex, appear to form an anatomical continuum inthe basal forebrain (Schwaber et aL, 1987). Given this anatomical organization, the CBCmay to some extent operate as a single functional unit. According to this hypothesis,measures of cholinergic neuronal activity in the hippocampus and cortex should besimilar under most circumstances. This hypothesis can be evaluated by examining theresponses of these spatially dispersed neurons to pharmacological and physiologicalchallenges.The hypothesis that the CBC acts as a functional unit is supported by evidence ofsimilar behavioural and pharmacological regulation of cholinergic neurons that project tothe cortex and hippocampus. For example, cholinergic activity in both the cortex andhippocampus is increased during arousal, defined behaviourally orelectroencephalographically (Chapter II; Kanai and Szerb, 1965; Nilsson et at., 1990),with greater ACh release occurring during waking and paradoxical sleep than duringslow wave sleep (Jasper and Tessier, 1971; Kametani and Kawamura, 1990).Furthermore, thyrotropin releasing hormone increases ACh release to a similar extent inthe two regions (Giovannini et at., 1991). ACh release in the cortex and hippocampus isalso similarly affected by alterations in serotonergic tone (Bertorelli et a!., 1992),although serotonin’s effects may be mediated by different receptor subtypes in the tworegions (Barnes et al., 1989; Maura and Raiteri, 1986). Benzodiazepine agonists decrease76ACh release in the hippocampus (Imperato et a!., 1993a); similarly, benzodiazepineinverse agonists increase ACh release in the cortex (Moore et a!., 1992). However, it hasalso been reported that the effects of benzodiazepine inverse agonists on high-affinitycholine uptake are different for the hippocampus and cortex (Miller and Chmielewskj,1990). Additional pharmacological evidence suggests that the trans-synaptic regulationof hippocampal and cortical cholinergic activity, although often similar, can bedifferentiated: local opiate—induced inhibition of ACh release in these two regions ismediated by different receptor subtypes (Lapchak et a!., 1989), adenosine is lesseffective in decreasing ACh release from cortical than hippocampal synaptosomes(Pedata et a!., 1986), and not all nootropics appear to affect both cortical andhippocampal cholinergic measures (Pepeu and Spignoli, 1989). Thus, althoughcholinergic neurons projecting to the hippocampus and cortex respond similarly duringthe physiological situations examined to date, it remains unclear if these neurons act as afunctional continuum, with identical regulation.The regulation of cortically-projecting basal forebrain cholinergic neurons by DA,acting primarily at receptors of the D1 subtype, has been described previously (ChapterIII, IV). The hypothesis that the CBC acts as a functional unit predicts thathippocampally-projecting cholinergic neurons would be similarly regulated by DA. Totest this hypothesis, in vivo microdialysate concentrations of ACh in the hippocampus ofconscious, freely moving rats were measured after systemic administration of the nonselective DA receptor agonist apomorphine, the D1-type receptor agonist CY 208-243,and the D2-type receptor agonist quinpirole. The catecholamine-releasing agent AMPHwas administered both locally through the dialysis probe and systemically; the extent towhich the D1 and D2 receptor antagonists (SCH 23390 and raclopride, respectively)attenuate the AMPH-induced increase in hippocampal ACh release was also investigated.77(B) MATERIALS AND METHODSExperimental protocol and drugsDialysis experiments and drug treatments were as described in Chapter III,including the size and strain of subject rats. For some experiments (see below), ratsreceiving two vehicle injections separated by 30 mm were used as a control group topermit appropriate comparison with each drug treatment.Surgery and microdialysisBrain microdialysis was performed as previously described (Chapter III).Transverse dialysis probes were stereotaxically implanted into the hippocampi of rats(A:-4.3, V:-3.3 measured from bregma, according to the atlas of Paxinos and Watson,1986). The dialysis probes were made of acrylonitrile-sodium methallyl sulfonate fibre(see Chapter IV), with tKe following exceptions: for the apomorphine and local AMPHexperiments, rats were implanted with saponified cellulose ester dialysis membrane (seeChapter II). All probes had an active surface length of 6.8 mm.Assay of AChACh was assayed by HPLC-ECD as described in Chapter IV.Statistical analysesData were analyzed as described in Chapter III.78(C) RESULTSThe average baseline output of ACh (± S.E.M.) from the hippocampi in all of theanimals implanted with acrylonitrile dialysis fibres was 28.77 ± 1.69 fmol/min (n=43).In animals implanted with cellulose ester fibre, the average basal output was 28.60 ±2.95 fmol/min (n=1 1). There was no difference in the baseline output between theexperimental groups having acrylonitrile probes [F(7,35)=0.81], or between those havingcellulose ester probes [F( 1 ,9)= 1.461.The effects of the non-selective DA receptor agonist apomorphine on hippocampaldialysate output of ACh are shown in Fig. 16. Apomorphine (1.0 mg/kg) increased AChrelease, to a maximum of 200% of baseline values, for approximately 90 mm. Thiseffect was significant compared to the transient increase measured after vehicle injection[F(l,8)=7.00, p=0.O29]. Behavioural hyperactivity was evident for approximately 90 mmafter apomorphine injection (data not shown).The effects of specific D1 and D2 receptor agonists on microdialysateconcentrations of ACh in the hippocampus are shown in Fig. 17. The specific D1agonist CY 208-243 (1 mg/kg) increased hippocampal ACh release for about 2 h, to amaximum of 230%; this effect was significant compared to vehicle injections[F(1,8)=10.81, p=0.0l1]. In contrast, the specific D2 agonist quinpirole (0.5 mg/kg)produced a small but statistically significant decrease in hippocampal ACh release[F(l,lO)=10.51, p=O.0O9]; examination of Fig. 17 suggests, however, that this may be aspurious statistical finding. Although CY 208-243 increased interstitial concentrations ofACh in the hippocampus, it did not appreciably activate the rats’ locomotion; incontrast, the D2 agonist had no obvious effect on ACh release while it elicitedbehavioural stereotypies that lasted approximately 1.5 h (data not shown).The indirect DA agonist AMPH (2.0 mg/kg) also increased hippocampal AChrelease compared to vehicle injections [F(l,7)=42.44, p<O.001J, to a maximum of 250%U-Iz-JUIC/)I0I0-0TIME (hr)Figure 16. Hippocampal dialysate values of ACh after injection of apomorphine (APO;1.0 mg/kg; closed circles; n=6) or vehicle (open circles; n=4). Data points representgroup means ± S.E.M.79-‘3250-200-150-100-500--I I I—1 0 21wz-JwC,)F0I—0-c()TIME (hr)Figure 17. Hippocampal dialysate values of ACh after injections of CY 208-243 (CY;1.0 mg/kg; open squares; n=5), quinpirole (QUIN; 0.5 mg/kg; open circles; n=7) orvehicle (closed circles; n=5). All groups were injected with vehicle 30 mm prior to thesecond injection. Data points represent group means ± S.E.M.80o CY (1.0 mg/kg)o QUIN (0.5 mg/kg)•VEHI300250-200150100-500.VEHI • I • I • I • I I—1 0 1 2 3 481baseline for about 3 h (Fig. 18). The D1 receptor antagonist SCH 23390 (0.3 mg/kg),injected 30 mm prior to AMPH, significantly attenuated AMPH’s effect on hippocampalACh release (170% vs. 250% maximal values; [F(1,8)=22.06, p=O.002; Fig 18A]). TheAMPH-induced response following SCH 23390 pretreatment was not significantlydifferent from that after either the control vehicle treatment [F( 1 ,9)<0.00 1] or the SCH23390/vehicle treatment [F( I ,8)= 1.79]. Compared to the control vehicle treatment (seeFig. 17), SCH 23390 significantly decreased hippocampal ACh release [F(l,7)=8.29,p=O.O24]. The D1 antagonist also blocked AMPH-induced behavioural hyperactivity(data not shown).In contrast, the D2 antagonist raclopride did not significantly attenuate the effectof AMPH on hippocampal ACh release (Fig. 1 8B). AMPH significantly increased AChrelease after raclopride pretreatment, compared to both raclopride alone [F(l,10)=16.60,p=0.OO2] and to the vehicle control [F(l,lO)=6.2l, p=0.O32]. The AMPH-inducedincrease in ACh release was not significantly decreased by raclopride pretreatment[F( 1 ,9)=2.02]; it should be noted, however, that a significant treatment x time interactionwas found [F(9.89,88.99)=3.84, p<0.OOl], possibly due to the larger blockade in the firsthour than than that later in the time course (Fig. l8B). Raclopride also significantlydecreased basal release of ACh, compared to the control vehicle injections [F(l,8)=13.54,p=0.006]. The D2 antagonist completely blocked AMPH-induced behaviouralhyperactivity in four of the seven rats, while three rats showed slight stimulationbeginning in the second hour after AMPH injection (data not shown).As shown in Fig. 19, AMPH (10 uM) applied locally through the dialysismembrane for 1 h did not significantly affect ACh release in the hippocampus[F(2.89,8.68)=0.86].82Figure 18. Hippocampal dialysate values of ACh after injections of d-amphetamine(AMPH; 2.0 mg/kg), SCH 23390 (SCH; 0.3 mg/kg; Fig. 3A), raclopride (RAC; 1.0mg/kg; Fig. 3B) or vehicle in the following combinations: AMPH 30 mm after vehicleinjection (closed circles in A and B; n=4), AMPH 30 mm after SCH 23390 (open circlesin A; n=6), AMPH 30 mm after raclopride (open circles in B; n=7), vehicle 30 mm afterSCH 23390 (open squares in A; n=4), and vehicle 30 mm after raclopride (open squaresin B; n=5). Data points represent group means ± S.E.M.A • VEH/AMPH (2 mg/kg)o SCH (0.3 mg/kg)/AMPH (2 mg/kg)o SCH (0.3 mg/kg)/VEHwz-JLUC’)I00-C-)300-250-200-150-100-50-0-300-250-200-150-100-50-I I- • I • I—1 0 1 2• I3 40-I • I I I •—1 0 1 2 3 4TIME (hr)83wz-jwC,)IDa-I0C)lOuM AMPHJTIME (hr)300-250 -200-150-100 -I500___________I -I I • I—1 0 1 2Figure 19. Effect of 10 uM d-amphetamine (AMPH) applied through the dialysismembrane for I h on hippocampal ACh output (n=4). Data points represent groupmeans ± S.E.M.84(D) DISCUSSIONThe results presented here suggest that the basal forebrain cholinergic projection tothe hippocampus is regulated by DA acting primarily at Dj -type receptors. Thisconclusion is based on the findings that the indirect DA agonist AMPH, the DA receptoragonist apomorphine, and the D1 receptor agonist CY 208-243 increased ACh release inthe hippocampus, whereas the D2 agonist quinpirole had a minor inhibitory effect.Furthermore, the D1 antagonist SCH 23390 attenuated AMPH-induced increases of AChrelease more effectively than did the D2 antagonist raclopride. Nevertheless, a minorrole of D2 receptors in regulating hippocampal ACh release cannot be ruled out in viewof the following findings: the D2 antagonist reduced basal release of ACh (as did theDj antagonist), and it attenuated the effect of AMPH on hippocampal ACh releaseparticularly during the first hour.Regulation of hippocampally-projecting cholinergic neurons by DAThe conclusion that DA stimulates septohippocampal cholinergic neurons isconsistent with recent reports of AMPH- and apomorphine-induced increases ofhippocampal ACh release (Nilsson et at., 1992; Imperato et al., l993b; 1993c). However,in contrast to the effect of AMPH on hippocampal ACh release observed here, Imperatoet al. (1993c) described a very different time course of action of AMPH at the samedose, using experimental methods closely approximating those reported here. Theseauthors reported AMPH-induced increases of hippocampal ACh release which returnrapidly to baseline within 2 h. Although different routes of AMPH administration and adifferent strain of rats were used in the two studies (i.p. and Sprague Dawley,respectively, by Imperato et al. vs. s.c. and Wistar in the present experiments), this doesnot appear to explain the difference in time course, given that other authors using i.p.administration and Sprague Dawley rats (Nilsson et a!., 1992) report a time course85similar to that demonstrated here. The source of this difference in time course ofAMPH’s effects on hippocampal ACh release remains to be determined.Although the data presented here are consistent with an important role of DA inregulating hippocampal ACh release, a role of other monoamines cannot be ruled out.As indicated earlier, AMPH releases noradrenaline and serotonin as well as DA(Kuczenski and Segal, 1989) and these other transmitters may therefore contribute toAMPH’s effects on hippocampal ACh release, and account for the incomplete blockadeof this effect by DA receptor antagonists. In support of this, it has been suggested thatserotonin may increase release of ACh in the hippocampus (Bertorelli et at., 1992; Mauraand Raiteri, 1986; Ohue et a?., 1992); less information is available regardingnoradrenergic regulation of hippocampal ACh.In contrast to the data reported here, the results of earlier investigations led to theconclusion that DA was inhibitory on septohippocampal cholinergic neurons. It has beenreported, for example, that apomorphine and a DA analogue decrease ACh turnover inthe hippocampus, while intraseptal injections of haloperidol or 6-OHDA lesions increasethis measure (Costa et al., 1983; Robinson et at., 1978; 1979). It is becomingincreasingly clear, however, that drug-induced changes in regional ACh release asestimated by in vivo microdialysis have no predictable relationship to the effects of thesame compounds on ACh turnover in the same brain structures (Chapter III; Costa et at.,1983; Nilsson et al., 1992; Robinson el at., 1978; 1979). This suggests that turnover rateis not necessarily coupled to release.Anatomical considerationsAnatomical evidence provides support for dopaminergic interactions withhippocampally-projecting CBC neurons. The medial septal nucleus and the nucleus ofthe diagonal band of Broca contain DA receptors, with a higher density of the D1receptor subtype than the D2 subtype (Zilles et a?., 1991). Catecholaminergic terminals86synapse on identified cholinergic perikarya in the medial septum/diagonal band (Milner,1991), although the majority of these afferents appear to be noradrenergic rather thandopaminergic (Lindvall and Stenevi, 1978; Moore, 1978). In addition, dopaminergicafferents innervate the lateral septum (Lindvall and Stenevi, 1978; Moore, 1978) whichcommunicates directly with the medial septum (Swanson and Cowan, 1979); thus, amultisynaptic pathway within the basal forebrain may mediate the effects of DA onhippocampally-projecting cholinergic neurons.DA may also have a transmitter role within the hippocampus (Bischoff et al.,1979). In support of this hypothesis, dopaminergic innervation (Scatton et a!., 1980;Verney et a!., 1985) along with D1 and D2 receptors (Bouthenet et a!., 1987; Dewar andReader, 1989; Mansour et at., 1991) have been identified in the hippocampus. However,it is unlikely that the actions of DA on septohippocampal cholinergic neurons aremediated within the hippocampus, because of the demonstrated lack of effect ofhippocampally-applied AMPH on ACh release. It is unlikely that this negative findingwas due to insufficient dosage: AMPH is known to be permeable through the dialysismembrane used here (Nomikos et a!., 1990), and the concentration of AMPH chosen forthis experiment has potent effects on dialysate concentrations of both DA and ACh inthe striatum, as shown previously using identical methodology (Westerink et at., 1990).Although it appears from the present experiment that DA released by AMPH is notacting within the hippocampus to increase ACh release, it has been reported previouslythat millimolar concentrations of locally applied DA increased hippocampal ACh release(Ohue et a!., 1992). The results of the latter experiment are suspect, however, given thehigh concentrations of DA applied, and the inappropriate perfusate and dialysisconditions (de Boer et al., 1990a; Benveniste and HUttemeier, 1990).87DA receptor subtype(s) mediating the dopaminergic regulation of septo-hippocampalcholinergic neuronsIn general, the effects of specific D1 and D2 agonists and antagonists onhippocampal ACh release are very similar to those observed in the cortex (Chapter III).One minor difference between the microdialysis results obtained in the hippocampus andcortex is the modest inhibition of hippocampal ACh release observed here, compared tothe lack of effect of D2 receptor agonists on cortical ACh release. Experimentscomparing dose-response effects of D2 agonists on cortical and hippocampal ACh releaseare needed to determine if this discrepancy is simply due to differential sensitivity ofthese cholinergic projections to D2 agonists, or whether it represents a qualitativedifference in the dopaminergic regulation of these systems. Although it is possible thatsuch differences exist, D1 receptors are clearly more important than D2 receptors inregulating ACh release in these brain regions.The importance of the D1 receptor subtype in mediating the effect of DA onhippocampal ACh release is supported by several other recent reports (Imperato et al.,1993b; 1993c). However, these authors also reported a significant excitatory effect ofthe D2 agonist quinpirole (Imperato et al., 1993a). Quinpirole (0.5 mg/kg) was found toincrease ACh release in the hippocampus to approximately 180% of basal release,whereas in the experiments reported here this same dose was found relativelyineffective, causing a small decrease of hippocampal ACh release. Given that a majorstrength of in vivo microdialysis is the ability to monitor the time course of action ofpharmacological treatments, it is unfortunate that Imperato and colleagues (Imperato etal., 1993b; 1993c) presented data from only one time point, which they described as thepeak effect. Without a comparison of the full time course of action of the D2 agonist,the basis of the difference between these previous data and those reported here cannotbe determined. Given the variability of hippocampal ACh release over time (seeChapter Ii and the response of vehicle injected rats in Fig. 17), it is not only desirable,88but perhaps indeed necessary, to compare the effect of a drug over time with referenceto a vehicle injection.In conclusion, the data reported here suggest that hippocampally-projecting CBCneurons, similar to those that are cortically-projecting, are stimulated by DA actingprimarily at D1-type receptors that are located outside the hippocampus. This isconsistent with the hypothesis that the CBC, which innervates both the hippocampus andcortex, may act as a functional unit at least with respect to its regulation by DA.89VI. SELECTIVE ENHANCEMENT OF CORTICAL, BUT NOT HIPPOCAMPAL,ACETYLCHOLINE RELEASE DURING THE ANTICIPATION AND CONSUMPTIONOF A PALATABLE MEAL(A) INTRODUCTIONPrevious authors have proposed that CBC neurons may play important roles inlearning and memory (Collerton, 1986; Hagan and Morris, 1988), attention (Muir et a!.,1994; Pang et at., 1993; Voytko et at., 1994), or arousal as definedelectroencephalographically (Semba, 1991; Smythe et at., 1992) or behaviourally (ChapterII; Collier and Mitchell, 1967). ACh released from these projections may influencecognition through facilitation of neuronal responsiveness to afferent signals, includingsensory stimuli (Hars et at., 1993; Metherate and Ashe, 1993; Tremblay et a!., 1990).ACh has also been implicated in the regulation of conditioning-related neuronalresponses. For example, frontal cortex neurons in the rat exhibit a discriminativeresponse to conditioned stimuli; cortically-applied muscarininc receptor antagonists orlesions of CBC neurons attenuate these responses (Pirch et at., 1992; Rigdon and Pirch,1986). Discriminative responses have also been measured in unidentified neurons in thebasal forebrain (Pirch, 1993). The conditioned stimulus (CS+) was a tone followed byrewarding medial forebrain bundle stimulation in this paradigm, although food followingthe CS+ could also be used to condition the discrimination in the cortex (as noted inRigdon and Pirch, 1986).More complex behavioural studies in monkeys have also suggested that responses ofneurons in the basal forebrain are “context-dependent” (Richardson and DeLong, 1990)or “reinforcement-related” (Wilson and Rolls, 1990). For example, the increased firingof nucleus basalis neurons during the choice phase of a rewarded task was not found tobe specific for the motor or sensory aspects of the task (Richardson and DeLong, 1990).-90These researchers thus concluded that the responses of basal forebrain neurons mayreflect transient increases in arousal or decision-making processes. Wilson and Rolls(1990) used a different task and described basal forebrain neurons which responded tosensory stimuli that, through learning of different contingencies, had come to signal theavailability of reinforcement (access to fruit juice). Together, these data suggest thatneurons in the basal forebrain may encode the learned reinforcement value, orsignificance, of stimuli. Although this theory is consistent with the proposed roles offorebrain ACh, the experiments described above did not identify the basal forebrainneurons under examination as being cholinergic, or as to their projection sites.To assess the possibility that cortically- and/or hippocampally-projecting cholinergicneurons may be involved in signalling the learned reinforcement value of stimuli, in vivobrain microdialysis was used in the present experiments to measure ACh release in thefrontal cortex and hippocampus of unrestrained, non-food-deprived rats trained to drinka palatable liquid chocolate meal. If CBC neurons are involved in learning about theavailability of a reward, ACh release in the frontal cortex and/or hippocampus oftrained rats might be expected to be augmented during anticipation and consumption ofthe reward, compared to naive or non-rewarded animals.(B) MATERIALS AND METHODSExperimental subjectsExperiments were performed on 40 male Long-Evans rats (Charles Rivers, Quebec).Rats which were subjected to behavioural training (n=27) weighed 275 ± 33g at the startof training, and 336 ± 30g at the time of surgery. Those run as naive controls (n=13)weighed 315 ± 12g at the time of surgery. The rats were housed individually under a12:12 h light-dark cycle, and unless otherwise stated, had free access to food and water.91Behavioural trainingRats were trained in a Plexiglas chamber (40x30x40 cm) to drink a chocolate-flavoured liquid meal (Sustacal; Mead Johnson). The chamber was divided into twocompartments (24x30x40 cm and 16x30x40 cm) by a removable plastic mesh screen. Atone end of the chamber, in the smaller compartment, Sustacal was available through a 30ml drinking tube.On initial exposure to the chamber, with the screen absent, each rat was given 1 hto explore the chamber and to taste the chocolate solution. The rat was then returnedhome and water-deprived overnight. On all subsequent training sessions, the rat wasplaced in the chamber for 20 mm (the “anticipatory” period), separated from the Sustacalby the mesh screen. The screen was then removed, and the rat was allowed 20 mm inwhich to drink the Sustacal (consummatory period) and explore the entire chamber. Atthe end of the session, the rat was returned to its home cage. With the exception of theovernight water-deprivation following the first training session, rats had free access tofood and water in their home cages throughout the period of the experiment. Rats werethus trained over a period of 14 days. Training took place once a day, between 1 lOOhand l400h. In vivo microdialysis was performed on day 14 of training.Two groups of rats were run concurrently as controls. A group of “non-rewarded”rats was trained as described above, the exception being that throughout training theseanimals had access to water when the screen was removed. Another group (naive)consisted of animals that were first introduced into the experimental chamber, whereSustacal was available, on the day of dialysis.Surgery and microdialysisBrain microdialysis was performed essentially as described in Chapters III and V.On the afternoon of the 12th day of training, the rat was anaesthetized with Equithesin,and a transverse microdialysis probe was implanted stereotaxically into the frontal cortex92or dorsal hippocampus. Dialysis probes were made of acrylonitrile fibre (Chapters IVand V) and had active surface lengths of 6.8mm (hippocampus) and 10.9mm (cortex).Following surgery, rats were housed individually in Plexiglas cages with free access tofood and water; they were allowed to recover and trained as previously. Dialysissampling was initiated two days after surgery (on the 14th day of training) while theanimal was in its home cage, approximately 3 h prior to the test session. The probe wasperfused for 30 mm before collection of samples to allow perfusate concentrations ofACh and Ch to equilibrate with those in the brain. After six baseline samples werecollected in the homecage (baseline was defined behaviourally, with the animal resting,sleeping, or displaying low levels of quiet, non-exploratory behaviour such as groomingor chewing food), the rat was placed in the experimental chamber, and training carriedout as described above. Dialysate samples were collected throughout this period, and for150 mm after the rat was returned to its home cage. Rats in the naive group weretreated as described above on the day of the experiment, which was two days afterprobe implantation, and which represented their first exposure to the experimentalchamber.Assay of ACh -ACh was assayed by HPLC-ECD as described in Chapter IV.Behavioural measuresLatency to drink Sustacal and volume consumed were recorded throughout training,and on the day of dialysis.Statistical analysesVolumes of liquid consumed and latencies to consumption on the day of dialysiswere compared using univariate ANOVAs. Differences between trained rewarded rats93and the control groups were measured using Student’s t-test, with Bonferroni correctionfor multiple comparisons.Biochemical data were expressed as a percentage of baseline concentrations, 100%baseline being defined as an average of the final six values before rats were introducedinto the experimental chamber. ANOVAs were used to test for group differences, timeeffects, and group x time interactions. In the latter two cases, Huynh-Feldt correctionswere made to account for the use of time as a repeated measure. Differences in AChrelease were assessed at individual time-points using univariate ANOVA. At each timepoint, ACh concentrations in trained rats which received Sustacal were compared -withthose in both the trained non-rewarded rats and the naive rats.(C) RESULTSBehavioural responsesOther than ingestion of liquid, rewarded and non-rewarded rats displayedqualitatively similar behaviours. Most animals explored the chamber on the first days oftraining, but froze for several minutes when the screen was removed. On the day ofdialysis, prior to removal of the screen, the rats displayed active exploratory behavioursuch as running, burrowing in the chamber, sniffing, rearing, and nose-poking at thescreen. On removal of the screen, rats in the trained rewarded and trained nonrewarded groups explored the previously closed area of the chamber. During the finalminutes of this “consummatory period,” many of the rats engaged in bed-makingbehaviour and began to sleep. Neither the latency to drink nor the amount of Sustacalconsumed on the day of dialysis was different from the previous session, suggesting thatcerebral perfusion did not adversely affect behaviour. Rats in the naive group displayedbehaviours similar to rats in the other groups during the first days of training, and94typically froze when the screen was removed, and were slow to enter the previouslyrestricted area of the cage.Latency to drinking and volume consumedLatencies to drink and volumes consumed for trained rewarded rats from day threeto day 14 are presented in Fig. 20. Behavioural data from rats with cortical orhippocampal probes were not found to differ significantly (two-way ANOVA) andtherefore these data were pooled. During the first seven days of training, the volume ofSustacal consumed increased gradually, and thereafter remained at an average ofapproximately 6m1 throughout the training. The latency to drinking decreased steadilyduring training. The volume of Sustacal consumed was reduced slightly on days 13 and14, while the latency on day 13 increased slightly, possibly due to trauma associated withthe dialysis surgery. All trained rewarded rats drank Sustacal during the trainingsessions post-surgery.Average latencies to drink and volumes consumed on the test (dialysis) session arepresented in Table 5. Trained rats which received Sustacal drank a significantly greatervolume of liquid than either the non-rewarded or the naive controls. The latency todrink was significantly greater for the naive rats than for the trained, rewarded rats, butwas not significantly different between trained rewarded and non-rewarded groups.Rats in the trained non-rewarded group frequently tasted the water in the drinkingtube, but drank little. The average volume consumed on days three to 12 was less than0.5ml; however, the volume of water consumed increased to an average (of the ratswhich drank) of 2ml and l.Sml on days 13 and 14, respectively. Of the six rats whichdrank on the day of dialysis, the latency ranged from 4-97s.Seven out of 13 naive rats placed in the experimental chamber for the first timeduring the dialysis test session consumed some Sustacal (average volume, of the rats95-Da)E(I)0C)0E2OO—0)cDC)‘00B Consumptionci Latency ProbeImplantation 3001086420 0Day of TrainingFigure 20. Volume of Sustacal consumed and latency to drink by trained rewarded ratsduring training days three to 14. Data are presented as mean ± S.E.M. (nl3).3 4 5 6 7 8 9 10 11 12 13 1496Table 5. Consumption and latency to drinking during dialysisGroup Volume (ml) Latency (s)*# *Sustacal, trained 5.4+0.7 ‘ (n=13) 15+3 (n=13)Water, trained 0.8+0.3 (n=14) 38±14 (n=6)Sustacal, naive 1.2±0.4 (n=13) 395±77 (n=7)Data for volume consumed (average of all rats) and latency to drink (of rats which drank)on the day of dialysis is expressed as mean ± S.E.M. Univariate ANOVA revealedsignificant differences between group volumes [F(2,37)=25.1, p<0.O011 and latencies[F(1,23)=35.6, p<0.001j.*Values are significantly different from the non-rewarded (water, trained) group. p.<0.Ol.#Value is significantly different from the naive group. p.<0.01.97which drank, 2m1). Of these animals, two had hippocampal probes, and five hadcortical probes. The latencies varied from 170—660s.ACh releaseThe average basal output of ACh (mean ± S.E.M.) of all the animals used in theseexperiments was 61.65 ± 7.4 fmol/min in the frontal cortex (n=21) and 37.70 ± 3.9fmol/min in the hippocampus (n=19). There was no significant difference in basal AChoutput between groups with hippocampal probes [F(2,16)=0.92, p=0.42] or betweengroups with cortical probes [F(2,18)=0.58, p=O.57]. Because the basal outputs betweengroups were very similar, delta fmol/min calculations yielded very similar figures andidentical statistical results to the percentage baseline calculations (data not shown).Mean values of ACh release for each group of animals during the anticipatory andconsummatory periods are presented in Table 6. It is evident from these averages, aswell as from Figs. 21 and 22, that introduction of the rats to the experimental chamberdifferentially affected ACh release in the frontal cortex and hippocampus. HippocampalACh release (Fig. 21) increased to approximately 220% of baseline concentrations in allexperimental groups, upon initial exposure to the chamber. No further increase occurredduring the consummatory period in any group; ACh release decreased gradually overtime and returned to baseline approximately 100 mm after the rats were returned totheir home cages. This effect of time was highly significant [F(9.02,144.26)=24.l6,p<0.OOl]. However, ANOVA revealed no significant group difference [F(2,l6)0.55,p=0.587]or group x time interaction [F(18.03,l44.26)=1.ll, p=O.352] in the hippocampus.In contrast, ACh release in the frontal cortex (Fig. 22) varied between the threegroups of rats. On initial exposure to the experimental chamber, ACh release increasedto approximately 300% of baseline in rats trained with Sustacal. ACh release continuedto increase and was maximal during the first sample after removal of the screen, whenSustacal consumption was greatest. ACh release in both the trained non-rewarded group98Table 6. Average acetylcholine release during anticipatory and consummatory periodsAcetyicholine release(mean % baseline ± S.E.M.)Group Anticipatory Period Consummatory PeriodHIPPOCAMPUSSustacal, trained (n=6) 211±28 190+28Water, trained (n=7) 204±8 181±10Sustacal, naive (n=6) 231±10 201±19CORTEXSustacal, trained (n=7) 314±46 312±49Water, trained (n=7) 224±30 187±33Sustacal, naive (n7) 184±7 169±15ACh release was averaged over the two samples in each period.a,a)C’,40z04-,30C)HIPPOCAMPUSHome A—1 0 1 2 3TIME (hr)99• Sustacal, Trainedo Water1 Trained• Sustacal, Naive400-300-200-100 -0Figure 21. Diaiysate concentrations of ACh in the hippocampus of trained rewarded rats(Sustacal; closed squares; n=6), trained non-rewarded rats (water; open squares; n=;7) andnaive rats (closed circles; n=6) during the anticipatory (A) and consummatory (C) periodsof the task. Data points represent group means ± S.E.M.a)a)(04-,04-,z0C)Figure 22. Dialysate concentrations of ACh in the cortex of trained rewarded rats(Sustacal; closed squares; n=7), trained non-rewarded rats (water; open squares; n=7) andnaive rats (closed circles; n=7) during the anticipatory (A) and consummatory (C) periodsof the task. Data points represent group means ± S.E.M. * significantly different fromnaive rats; + significantly different from trained non-rewarded (water) rats; p<0.O5.100CORTEX****+*• Sustacal, Trainedo Water, Trained• Sustacal, Naive*400-300-200-100 -0-Home lAid Home—1 0 1 21 J -1Time (hr)101and the naive group also increased when the rats were in the experimental chamber, butthese increases were smaller than that seen in the trained rewarded group. Two-wayANOVA revealed a significant difference in cortical ACh release between groups[F(2,18)=4.27, p=O.03O] but no group x time interaction [F(24.17,2l7.56)=l.21, p=0.237].The effect of time was again highly significant [F(12.09,217.56)=14.35, p<O.O01].Univariate ANOVA demonstrated differences between groups at several time-points, asnoted in Fig. 22.(D) DISCUSSIONThe present results demonstrate that different patterns of ACh release occur in thefrontal cortex and hippocampus of rats during the anticipation and consumption of ahighly palatable meal. While ACh release in the hippocampus increased similarly in eachgroup, regardless of previous training or the presence of a reward, ACh release in thefrontal cortex occurred in a differential manner. That is, in the frontal cortex AChrelease was greater in those rats trained to anticipate and consume a palatable reward.The increased ACh release occuring during this task can be separated into twocomponents. Both the hippocampus and cortex exhibit the first component, which is atraining- and reward-independent increase. Only the cortex, however, exhibits thesecond component which is an increase over and above Component 1 and appears to bereinforcement-related. These results are notably similar to an earlier report concerningenhanced cholinergic transmission in the cortex of rabbits trained to press a lever for awater reward in response to auditory or visual cues, on different operant schedules ofreinforcement (Rasmusson and Szerb, 1975; 1976). Cortical ACh release was increasedin response to performing the behavioural task, in the five tested combinations of twocortical regions, two cues of differing sensory modality and two schedules of102reinforcement. However, a second, larger increase was also noted in the sensorimotorcortex of light-cued rabbits reinforced for low response rates. The two components ofincreased ACh release found in the present task are discussed below.Component 1: Training- and reinforcement-independent increases of ACh release incortex and hippocampusThe increased ACh release evident during this task in the hippocampus and in thecortex of naive and non-rewarded rats may be due to a number of variables. Forexample, forebrain cholinergic neurons are involved in arousal defined byelectrophysiological (Semba, 1991; Smythe et at., 1992) or behavioural (Chapter II;Collier and Mitchell, 1967) measures. Arousing stimuli, such as handling or injectingthe animal, have also been shown to increase hippocampal and/or cortical ACh release(Chapter II; Dudar et a!., 1979; Nilsson et a!., 1990). The animals’ behaviours in thepreseni task suggested that they were aroused by being put into an environmentdifferent than their home cages (the experimental chamber), both when the situation wasnovel (naive group) and after they had repeated access to it (trained non-rewardedgroup). Thus, increases in arousal may explain the first component of the increasedACh release in the cortex and hippocampus..Attention, defined operationally in behavioural studies, is also thought to involveforebrain cholinergic neurons. For example, excitotoxic lesions or pharmacologicalinhibition of the basal forebrain causes impairments in measures of attention (Olton etal., 1988; Pang et at., 1993; Robbins et a!., 1989a; Voytko et at., 1994) which in somecases have been ameliorated by cholinergic agonists or tissue grafts (Muir et at., 1992a;1992b; 1994). In addition, cholinergic agonists improve measures of attention inAlzheimer’s patients (Sahakian et at., 1989; 1993) and the cholinergic receptor antagonist,scopolamine, disrupts attention in normal human subjects (Dunne and Hartley, 1985;103Wesnes and Warburton, 1983). It is possible, therefore, that increased attentiveness ofthe rats during the task may account for increased ACh release.Given that factors such as arousal or attention may increase ACh release duringbehavioural tasks, independent of learning or memory per se (as in the naive groups), itis evident that attempts to study cholinergic correlates of learning and memory mustcarefully control for these factors. However, while arousal and/or attention may havecontributed to the increased ACh release, these mechanisms cannot explain thesignificantly greater ACh release in the frontal cortex of those rats which were trainedwith Sustacal. This assertion is similar to that made by Rasmusson and Szerb (1975;1976) in the interpretation of their results, which have been described above: althoughthe general increase of cortical ACh release seen in all groups might be explained bysome shared factor, such as the behavioural activity or “work expended” by the animalduring the task, this factor could not account for the larger increase observed in thegroup which exhibited lower response rates.Cornponent 2: Reinforcement-related increase of ACh release in the cortexThe finding of an increase in cortical ACh release in the trained rewarded group,over and above the arousal/attention related increase, is consistent with reports ofreinforcement-related neurons in regions of the basal forebrain which contain cortically-projecting cholinergic neurons (Richardson et al., 1991; Wilson and Rolls, 1990). Studieshave demonstrated neurons within the hippocampus and cortex which respondspecifically during the delay between stimulus and reward in tasks which requireremembering the position of a cue (Kojima and Goldman-Rakic, 1982; Watanabe andNiki, 1985); in contrast, few basal forebrain neurons are active during such delays(Richardson and DeLong, 1990). This suggests that while there is a role for thehippocampus and frontal cortex in remembering information during a delay, the corticalprojection from the basal forebrain is involved specifically in recognition of those cues104which signal the reward. Thus, the context-specific alterations in cortical ACh releaseobserved in the present experiment are consistent with a specific role of cortically-projecting CBC neurons in the recognition or anticipation of an impending food reward.In this regard, it is noteworthy that the group of rabbits exhibiting the largest increasein cortical ACh release in the experiments of Rasmusson and Szerb (1976) were those toreceive significantly more reinforcements than any other group.The present results demonstrate differences in task—induced ACh release in thecortex and hippocampus. It is possible that these differences could be explained onanatomical grounds. For example, combined anterograde tracing andimmunohistochemical techniques have demonstrated that cholinergic neurons in the CBCand mesopontine tegmentum innervate the frontal cortex (Satoh and Fibiger, 1986). It isconceivable, therefore, that the group-dependent patterns of ACh release which occurredin the frontal cortex, but not in the hippocampus, were due in part to enhanceddischarge of mesopontine neurons in response to the anticipation and presentation of thereward. However, the distribution of terminals in the frontal cortex arising from thelaterodorsal tegmental nucleus is confined to the medial frontal cortex, at a level moreventral and posterior to the area of cortex dialysed in the present study. Further, thereare no data to suggest that mesopontine cholinergic neurons are active specifically duringthe anticipation and presentation of a reward. It should also be noted, with regard tothe anatomical specificity of “Component 2 of the ACh release in this task, that theresults presented here for the frontal cortex are not necessarily representative of theentire neocortex. For example, Rasmusson and Szerb (1976) reported differences in theeffects of a behavioural task on ACh release in the sensorimotor and visual cortices ofrabbits.Another possible explanation for the differences in cortical and hippocampal AChrelease in this task is that the activities of cortically- and hippocampally-projectingcholinergic neurons can be differentially regulated. Previously, it has been shown that105pharmacological and physiological stimuli have nearly identical effects on ACh release inthese structures (Chapters II, III and V) lending support to the hypothesis that thecontinuum of neurons that forms the CBC (Schwaber et a!., 1987) acts as a singlefunctional unit. The present results show that this is not the case, and argue for asubdivision of this continuum on functional grounds.The question then arises as to which input(s) to the basal forebrain differentiallyregulate cortically- and hippocampally-projecting CBC neurons in the present task.There is extensive evidence that dopaminergic mechanisms are involved in reward(Fibiger and Phillips, 1986). Dopaminergic mechanisms are also involved in theregulation of ACh release in the hippocampus and frontal cortex (Chapters III, IV, V).However, it seems unlikely that regulation of CBC neurons by DA can account for thedifferential ACh release in the cortex and hippocampus in this study, for the followingreasons. In rats trained in the same paradigm as that used here, DA release increases inthe nucleus accumbens; however, these increases occur during the consumption ratherthan during the anticipation of the reward (Fibiger, 1993). Given this difference, theincreased ACh release observed in the present study is not likely the indirect result ofincreased DA release in the nucleus accumbens. In addition, it has been demonstratedthat dopaminergic regulation of cortical and hippocampal ACh release is very similar(Chapters III and V).Although the anatomical or transmitter-specific basis for the differential regulationof cortical and hippocampal ACh release in the present task is unknown, it is possiblethat the different patterns observed may have been due to the type of behavioural taskemployed. While there is evidence that both the hippocampus and frontal cortex areinvolved in learning (Hagan and Morris, 1988), the hippocampus has been implicatedspecifically in spatial memory (O’Keefe and Nadel, 1978). It is possible, therefore, thata task which depends specifically upon learning about the spatial orientation of a rewardmight increase ACh release in the hippocampus, compared to a non-rewarded or naivecontrol group.106107VII. GENERAL DISCUSSIONThe experiments presented in this thesis have demonstrated that ACh release in thefrontal cortex and hippocampus varies depending on the animal’s level of arousal.Cortically- and hippocampally-projecting neurons in the CBC are regulated in anexcitatory manner by DA acting primarily at receptors of the D1 subtype, probablylocated outside of the terminal regions of these neurons. Although cortical andhippocampal ACh release are affected very similarly by dopaminergic drugs, theanatomically continuous CBC can be functionally subdivided based on differential AChrelease in the cortex and hippocampus of rats trained to anticipate and consume apalatable meal. The relevance of these findings with respect to both the technicalaspects of cholinergic research, and to theories of forebrain cholinergic function, arediscussed below.(A) TECHNICAL CONSIDERATIONS OF CHOLINERGIC RESEARCHMicrodialysis represents an improvement over previously used in vivo samplingtechniques, such as cortical cup or push-pull perfusion, by virtue of its ease ofapplicability to freely moving animals and its less traumatic tissue interaction. Thesensitivity of the HPLC-ECD assay for ACh has also allowed the use of morephysiological perfusion solutions (lower concentrations of cholinesterase inhibitor andcalcium ions), while the on-line assembly makes data available with as little as a fifteenminute time delay. This latter advance is highly desirable for in vivo determination ofACh, given the labile nature of hippocampal and cortical ACh release and given that itis advantageous to know that a relatively stable baseline has been achieved before anexperimental manipulation is initiated. The improvements noted above may account for108some discrepancies between the present results andthose found previously using corticalcup or push-pull techniques. For example, Dudar et a!. (1979) found that hippocampalbut not cortical ACh release was responsive to sensory stimuli and Casamenti et a!.(1987) reported D2- but notD1-mediated regulation of cortical ACh release.In contrast, the factors accounting for discrpancies between the present resultsand those found previously using ACh turnover rate assays remain poorly understood.These methods are based on the proposition that steady-state kinetics can be applied toACh turnover; however, the assumptions made underlying this proposition may beunfounded (Cheney and Costa, 1977) and may therefore limit the suitability of the AChturnover measure as an indicator of cholinergic neuronal activity. The presentmicrodialysis data and those of others, concerning the effects of dopaminergic drugs onhippocampal and cortical ACh release (Nilsson et a!., 1992), have no predictablerelationship to the effects of the same compounds on ACh turnover in the same brainstructures (Costa et a!., 1983; Robinson et a!., 1978; 1979). It is thus becoming apparentthat turnover rate is not necessarily coupled to release and that the functionalsignificance of ACh turnover is unknown.To augment the substantial advantages over previous in vivo sampling techniquesoffered by dialysis, methodological refinements are continually being made to thistechnique. Statistical treatment and interpretation of the data is one area which requiresimprovement and standardization, especially in the case of forebrain ACh release whichis highly labile and subject to large basal variations. Attempts to control for theindividual variabilities in basal dialysate concentrations of a neurotransmitter haveusually involved expressing these concentrations as percentages of baseline. Thismanipulation is probably appropriate if groups with substantially different average basaloutputs are not compared. If such comparisons are to be made, it is more appropriate touse calculations such as the delta fmol/min (presented in Chapter IV) or absolute outputsusing a basal value as a covariate of statistical comparisons (Moore et al., 1993).109Although the alternative data description techniques noted above may allow moreappropriate analyses of ACh microdialysis results, it remains necessary in any of thesecases to define a “basal” concentration, or output, of neurotransmitter recovered from thebrain region of interest in each animal. This is not a straightforward task in the case ofACh microdialysis given the correlation of ACh release and behavioural activity(Chapter II) and given the between-animal variability in behavioural activity over time.While “baseline” is usually defined as the average of three to six pre-treatmentconcentrations, the behavioural activity of the animals during this period is often notreported. Comparisons between research groups using ACh microdialysis would be -facilitated if the definition of baseline were standardized, ideally including averagedvalues from an hour of sampling during sleeping and quiet waking behaviours, as inChapter VI.Future challenges to the improvement of the microdialysis technique formeasurement -of ACh are manifold. Most investigations into the principles ofmicrodialysis have been carried out using catecholamine assays. The significance of theperfusion-related neurochemical “sink” (Benveniste, 1989; Blaha, 1991) remains to becharacterized in the case of ACh; the possibility that a low concentration of thereversible cholinesterase inhibitor neostigmine in the perfusion solution might counteractthis sink also requires examination. Further characterization of the correlation betweenACh release and behavioural activity should also be a priority of basic ACh dialysisresearch, given that the ultimate research objectives to which ACh microdialysis can beapplied include behavioural studies. An improved understanding of how ACh release isrelated to behaviour, how this relationship can be dissociated pharmacologically, andhow it can be controlled for to allow unconfounded measurements of cognition-relatedcholinergic activity will probably depend on improved sensitivity and temporal resolutionof the ACh assay coupled with more detailed behavioural characterizations.110(B) REGULATION OF BASAL FOREBRAIN CHOLINERGIC NEURONSThe results of Chapters II and VI are in agreement with the hypothesis that CBCneurons are involved in cognitive functions including arousal and attention. Thesefunctions may subserve the commonly accepted role of ACh in learning and memory(Hagan and Morris, 1988) and the proposed association of decreased cholinergic capacitywith dementia (Collerton, 1986; Coyle et a!., 1983). The huge social and economicimpact of dementing diseases such as Alzheimer’s, in a society with a growingpercentage of its population found in the most-affected age group, provides the mandatefor investigating the regulation of CBC neurons.Although the shortcomings of the cholinergic hypothesis of dementia arerecognized (Fibiger, 1991), and despite the overall ineffectiveness of cholinergictherapies in treating dementia (Hoittum and Gershon, 1992), the search continues for apharmacological agent which stimulates forebrain cholinergic systems to amelioratecognitive deficits. For example, it has been hypothesized that attempts to treatdementias with cholinergic receptor agonists or cholinesterase inhibitors may beunsuccessful because the normal spatial and temporal patterning of cholinergictransmission is not maintained by such treatments (Drachman et al., 1982). In normalcircumstances, ACh facilitates the activity of cortical neurons responding to afferentneural transmission, and an approximate temporal coincidence of the cholinergicstimulation with the afferent stimulus appears to be necessary for this increase in the“signal” (Hars et a!., 1993; Metherate et al., 1987; Tremblay et at., 1990). Conversely,using cholinergic agonists to improve cognitive deficits may increase the “noise” in thesystem by increasing cholinergic tone in a temporally non-selective manner. A similarsituation may apply in the hippocampus, where ACh facilitates long-term potentiation(Blitzer et at., 1990; Hunter et at., 1994) and enhances excitatory postsynaptic potentialselicited by afferent stimulation (Markram and Segal, 1990).111Based on the above arguments, it has been suggested that a more promisingpharmacological approach to the treatment of dementia would be to amplify the activityof remaining CBC neurons without disrupting the normal patterning of cholinergictransmission (Sarter et a!., 1990). These authors review evidence suggesting thatpharmacologically modulating the inhibitory GABAergic regulation of CBC neuronsmight have such a therapeutic action. Could similar arguments also apply to thedopaminergic regulation of CBC neurons? Together with the cholinergic hypothesis ofdementia, the results of Chapters 111-V suggest that dopaminergic agonists, especiallythose acting at the D1 DA receptor subtype, may enhance cognition via stimulation offorebrain cholinergic function.The above hypothesis, that dopaminergic drugs may act as cognition enhancers, hasnot received unequivocal support from the results of previous research. AlthoughAMPH improves the performance of both animals and humans in a variety of learningand memory tasks (Doty and Doty, 1966; McGaugh, 1973; Packard and White, 1989;Quartermain et a!., 1988; Soetens et a!., 1993; Strupp et at., 1991; Weingartner et at.,1982), AMPH has also been reported to have no effect or deleterious effects on theperformance of other tasks (Beatty and Rush, 1983; Beatty et at., 1984; Ennaceur, 1994;Kesner et a!., 1981). These apparently contradictory results may be reconciled inseveralways. Kesner and colleagues have suggested that arousal, such as that induced byAMPH (Fairchild et al., 1967), is detrimental for the induction of short-term memorywhile it facilitates the consolidation required for long—term memory (Kesner, 1973;Kesner et al., 1981). It has also been proposed that AMPH-induced changes insensorimotor functions or attention may actually account for what had been reported asAMPH-induced memory effects (Ennaceur, 1994; Beatty and Rush, 1983; Bureovã andBure, 1982).The results of more specific dopaminergic drugs on cognitive tasks are no lessambiguous. Cognitive deficits after withdrawal of L-dopa in Parkinson’s patients have112been reported (Lange et at., 1992), as have retention-improving effects of apomorphinein rats (Grecksch and Matthies, 1981). Conversely, Bureová and Bure (1982) andChrobak and Napier (1992) found no effect of apomorphine on rats’ performance. Morerecently, researchers have begun to question which DA receptor subtype(s) may beinvolved in cognition; given the inconsistencies noted above, it is perhaps not surprisingthat conflicting evidence has been reported (Castellano et at., 1991; Chrobak and Napier,1992; Levin et at., 1990). For example, although a D2 but not a D1 agonist improvedthe performance of rats in a radial maze (Packard and White, 1989), cortical injectionsof a D1 but not a D2 antagonist have recently been reported to cause cognitiveimpairments in monkeys (Sawaguchi and Goidman-Rakic, 1994).The lack of concensus as to the role of DA in cognition, as noted above, may bedue to variations among studies including the different dopaminergic drugs, cognitivetasks, experimental measures and interpretation of results. The ambiguities may alsoreflect a complex, regionally-specific involvement of DA in cognitive functions that hasnot been adequately assessed by the majority of behavioural studies thus far conductedusing systemic administration of drugs.Irrespective of whether or not systemically applied dopaminergic drugs might haveclinical applications as cognition enhancers, the significance of the dopaminergicregulation of CBC neurons in the normally functioning nervous system is of greattheoretical interest. As discussed previously, ACh in the cortex and hippocampusappears to act by accentuating the activity of afferent transmission with which its releaseis paired. Stimulation of cortical and hippocampal ACh release by DA might thereby bea means by which dopaminergic transmission could signal the saliency of afferenttransmission to the hippocampus and cortex. Indeed, mesotelencephalic DA systems areknown to be activated by stressful as well as rewarding stimuli (Abercrombie et at.,1989; Bertolucci-D’Angio et al., 1990; Deutch and Roth, 1990; Di Chiara and Imperato,1988; Fibiger and Phillips, 1986). The increased DA release may be partially responsible113for the reported activation of basal forebrain neurons during the presentation of cuespreviously paired with aversive or rewarding stimuli (Richardson and DeLong, 1990;Whalen et a!., 1994; Wilson and Rolls, 1990). The extent to which the dopaminergicregulation of CBC neurons is involved with cholinergic facilitation of afferenttransmission in the cortex and hippocampus remains to be determined. 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