AN JJi VIVO ELECTROCHEMICAL ANALYSIS OF THE ROLE OF DOPAMINE IN FEEDING BEHAVIORS By LORINDA JEAN HOLMES B . S c , U n i v e r s i t y o f V i c t o r i a , 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES N e u r o s c i e n c e We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF August ®Lorinda Jean BRITISH 1990 Holmes, COLUMBIA 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of T \ o XK^O^cXfi^t p The University of British Columbia Vancouver, Canada Date QuKj^h , t ^ f j DE-6 (2/88) Abstract The involvement of dopamine in ant ic ipatory and consummatory aspects of feeding behaviors was invest igated in the present thes is . A l l measurements of dopaminergic a c t i v i t y were taken by in vivo electrochemical techniques. In Experiment 1, dopamine ef f lux in the nucleus accumbens and caudate of male rats was monitored during sessions in which a smal l , unsignal led l i q u i d meal was consumed. Increases in the electrochemical measure of dopamine a c t i v i t y , which were of s imi lar temporal pattern and magnitude, were observed in both the nucleus accumbens and str iatum following meal consumption. These data suggest a poss ib le post ingest ional ro le of dopamine in these two bra in s tructures . In Experiment 2, a conditioned feeding paradigm was u t i l i z e d to study the role of dopamine during a d i scre te ant i c ipa tory phase of feeding. Rats were conditioned to d iscr iminate between a pos i t ive condit ioned stimulus (CS+) p r e d i c t i v e of meal d e l i v e r y , and a negative conditioned stimulus (CS-) that was not associated with food. Increases in dopamine a c t i v i t y , as determined by changes in e lectrochemical oxidat ion currents , were found to be greater during the CS+ than during the CS- in both the nucleus accumbens and caudate. In add i t ion , the magnitude of increase was greater i n the nucleus accumbens than the caudate, suggesting that the accumbens may be p r e f e r e n t i a l l y i i i involved in the processing of external incentive s t i m u l i . The re s u l t s support a role for dopamine in both the nucleus accumbens and caudate during appetitive or anticipatory responding for food in the male rat. iv TABLE OF CONTENTS Abstract i i L i s t of Figures v i Acknowledgements v i i i INTRODUCTION Experimental Background 1 Theoretical Background 8 Rationale for the application of electrochemistry to the study of feeding in the rat 14 GENERAL METHODS Electrode preparation 17 Surgery 17 Recording procedure 18 Histology 19 EXPERIMENT I Effects of an unsignalled l i q u i d meal on electrochemical measures of DA a c t i v i t y 22 Methods 23 Results 25 Discussion 42 V EXPERIMENT I I Elec t r o c h e m i c a l measures of dopamine a c t i v i t y during a n t i c i p a t o r y and consummatory phases of feeding behaviors i n the r a t 44 Methods 45 Results 52 Disc u s s i o n 81 GENERAL DISCUSSION 85 REFERENCES 92 v i L i s t of Figures Figure 1 Example of a ty p i c a l voltammetric sweep, ramped at 10 mv/s 20 Figure 2 Chronoamperometric signal recorded from the caudate and nucleus accumbens of rat #1 before and after the delivery of a lOcc Sustacal meal 26 Figure 3 Chronoamperometric signal recorded from the caudate and nucleus accumbens of rat #2 before and after the delivery of a lOcc Sustacal meal 28 Figure 4 Chronoamperometric signal recorded from the caudate and nucleus accumbens of rat #3 before and after the delivery of a lOcc Sustacal meal 30 Figure 5 Chronoamperometric signal recorded from the caudate and nucleus accumbens of rat #4 before and after the delivery of a lOcc Sustacal meal 32 Figure 6 Representative chronoamperometric record from the caudate and nucleus accumbens of a single rat on the f i r s t day of testing 34 Figure 7 Average chronoamperometric signal recorded from the caudate and nucleus accumbens of rats 1, 2, 3, and 4 before and af t e r the delivery of a lOcc Sustacal meal . . 36 Figure 8 Electrode placements directed at the l e f t nucleus accumbens and right caudate i n Experiment 1 39 Figure 9 Mean amount of time spent in the feeding niche during the CS+ period prior to the onset of food delivery 50 Figure 10 Mean latency to enter niche following CS onset 55 Figure 11 Mean changes in DA oxidation current i n the nucleus accumbens and caudate during CS+, CS- and pre-CS baseline periods . . 57 v i i Figure 12 Representative chronoamperometric records obtained from four d i f f eren t subjects during consecutive CS+ and CS- t r i a l s . . 59 Figure 13 Behavioral and electrochemical r e s u l t s from e x t i n c t i o n subject #1 64 Figure 14 Behavioral and electrochemical r e s u l t s from e x t i n c t i o n subject #2 67 Figure 15 Behavioral and electrochemical r e su l t s from e x t i n c t i o n subject #3 70 Figure 16 Representative CS+ sweep t r i a l from one subject 73 Figure 17 Mean peak, height r a t i o s from 10 CS+ and 10 CS- voltammetric sweep t r i a l s recorded from the nucleus accumbens of a s ing le rat 75 Figure 18 Electrode placements d irected at the l e f t nucleus accumbens and r i g h t caudate in Experiment 2 77 Acknowledgements I would l i k e to thank Chuck Blaha, Mike Jung, Fred LePiane, Jim Pfaus, and Tony P h i l l i p s for the generous assistance they provided during the preparation of this thesis. 1 INTRODUCTION Numerous studies have implicated dopaminergic systems in feeding behaviour, but c u r r e n t l y , there i s no c lear consensus as to the exact nature of t h i s involvement. The f i r s t sect ion of th i s introduct ion reviews experimental data that have associated dopaminergic a c t i v i t y with feeding. Theoret ica l models that have been proposed to expla in the ro le of dopamine i n feeding behaviors are discussed next. The introduct ion concludes with a b r i e f descr ip t ion of, and rat iona le for the use of, in vivo e lectrochemical measurements of e x t r a c e l l u l a r dopamine l eve l s during d i f f erent phases of feeding in the r a t . Experimental Background Select ive des truct ion of n i g r o s t r i a t a l dopamine (DA) neurons has been shown to produce a syndrome of aphagia and adips ia (F ib iger , Z i s , & McGeer, 1973; Ungerstedt, 1971). S i m i l a r l y , pharmacological blockade of dopamine receptors has resul ted in a d i s r u p t i o n of feeding behaviours. Xenakis and Sc la fani (1981) found that the DA antagonist pimozide suppressed consumption of a palatable saccharin-glucose so lu t ion i n a dose-re lated manner after a 4-hr food deprivat ion per iod . The DA antagonist sp i roper ido l also decreased food intake i n rats r e s t r i c t e d to a 4-hr feeding 2 period (Heffner, Zigmond, and Strieker, 1977). Because reduced dopaminergic a c t i v i t y has been linked with the attenuation of feeding, increased DA a c t i v i t y might be expected to f a c i l i t a t e feeding. Evans and Eikelboom (1987) observed s i g n i f i c a n t increases i n food intake and meal duration when they administered the DA agonists bromocriptine and d-amphetamine intracerebroventricularly. They also noted that the activation was selective to food intake, as other behaviours (grooming, drinking, a c t i v i t y ) were not affected s i g n i f i c a n t l y by the drugs. However, Heffner et a l . (1977) have shown that the DA agonist amphetamine and apomorphine reduce food intake i n food-deprived rats. E l e c t r i c a l stimulation of the ventral tegmental area (VTA) and the substantia nigra (SN) also increases synaptic a c t i v i t y of DA neurons (Fibiger, LePiane, Jakubovic, & P h i l l i p s , 1987; P h i l l i p s & Fibiger, 1979). In addition, feeding has been e l i c i t e d by e l e c t r i c a l stimulation of the VTA and SN, both o r i g i n s of ascending dopaminergic pathways ( P h i l l i p s Se Fibiger, 1973). The evidence presented so far deals solely with the role of DA systems in feeding behaviours of the rat under unnatural conditions. That i s , a l l subjects were tested either i n a state of reduced or elevated DA a c t i v i t y produced by the experimenter. More b i o l o g i c a l l y relevant data have emerged from studies that have attempted to correlate dopaminergic a c t i v i t y and feeding under more natural circumstances, without producing pharmacological or 3 physical a l t e r a t i o n s of brain a c t i v i t y . One such study was conducted by Heffner, Hartman, and Seiden (1980). Rats maintained on a feeding schedule consisting of 20 hours of food deprivation followed by 4 hours of access to food p e l l e t s were decapitated either before food delivery or after one hour of access to food. Dihydroxyphenylacetic acid (DOPAO/DA r a t i o s were determined by radioenzymatic assay to provide indices of dopaminergic a c t i v i t y . These researchers found elevated ratios in the nucleus accumbens, hypothalamus, and amygdala of deprived rats given access to food compared to animals that were not fed or to non-deprived controls. Ratios remained unchanged i n the striatum, o l f a c t o r y tubercle, frontal cortex, and septum. Chance, Foley-Nelson, Nelson, and Fischer (1987) reported elevated lev e l s of the DA metabolites DOPAC and HVA in the nucleus accumbens and striatum of ch r o n i c a l l y deprived rats s a c r i f i c e d following one hour of access to food, as compared to values from control rats s a c r i f i c e d i n a deprived state. In this l a t e r study, neurotransmitter and metabolite measurements were conducted on regional brain homogenates using high performance l i q u i d chromatography with electrochemical detection (HPLC/ED). Simansky, Bourbonais, and Smith (1985) also employed HPLC/ED to determine DOPAC/DA rat i o s i n rats under various feeding conditions. They observed that DOPAC/DA ra t i o s were increased in the hypothalamus of rats maintained on a d a i l y 4-hr food-access schedule when they were measured after one hour of access to 4 food or simply to food-related stimuli alone, as compared to nondeprived controls or deprived rats not exposed to food or food-related s t i m u l i . They did not observe any similar r a t i o changes in the nucleus accumbens, striatum, olfactory tubercle, or amygdala. A s i g n i f i c a n t advance in the analysis of dopaminergic a c t i v i t y and feeding behaviours occurred with the application of chronic in vivo techniques to measure e x t r a c e l l u l a r levels of neurotransmitters i n freely moving animals. One of these techniques, microdialysis with HPLC/ED, was employed by Church, Justice, and N e i l l (1987). These researchers found that rats trained on a 1-min fixed interval feeding schedule showed increased e x t r a c e l l u l a r s t r i a t a l DA lev e l s during and extending 15 to 20 min after the 15-min feeding periods. Using the same technique, Hernandez and Hoebel (1988) observed increased e x t r a c e l l u l a r DA lev e l s in the nucleus accumbens of food-deprived rats during feeding sessions. E x t r a c e l l u l a r l e v e l s of DA in the nucleus accumbens also were elevated, but no changes were seen in the striatum of deprived rats bar-pressing for food. In another study employing microdialysis and HPLC/ED, Radhakishun, van Ree, and Westerink (1988) observed an increase i n DA e f f l u x i n the nucleus accumbens that was correlated with the onset of a scheduled feeding session. Levels of DA remained elevated during the entire food intake period. Another i n vivo technique, voltammetry, has been used 5 to study the role of DA i n feeding behaviours. K e l l e r , Strieker, and Zigmond (1983) found a variety of exteroreceptive stimuli including t a i l shock, ice-water bath, and food after a 24 hour deprivation period, to increase voltammetric signals i n the rat striatum. Although the electrodes used in this study were not selective for DA ( i . e . , DOPAC, AA, and DA may have a l l contributed to the signal obtained), the res u l t s of pharmacological manipulations suggested that the changes in the electrochemical signal were due mainly to increased DA eff l u x . In vivo voltammetric analyses of ex t r a c e l l u l a r levels of the DA metabolite HVA (used as an index of DA a c t i v i t y ) were observed recently i n the rat caudate nucleus during lever pressing for food reward (Joseph, Hodges, and Gray, 1989). D'Angio and Scatton (1989) found feeding or exposure to food odors alone, both resulted in increased e x t r a c e l l u l a r DOPAC level s i n the anteromedial prefrontal cortex as determined by in vivo voltammetric measurements. In vivo voltammetric data obtained by Louilot, Le Moal, and Simon (198S), argued against the hypothesis that the observed increases i n dopaminergic a c t i v i t y are due simply to the induction of motor a c t i v i t y . Exposure of a test subject to an aggressive male caused an increase i n the DA a c t i v i t y of the test subject, even though i t t y p i c a l l y assumed a frozen stance. As indicated by the evidence reviewed so far, i t appears that dopaminergic systems are involved i n feeding 6 behaviors. However, there i s s t i l l uncertainty as to which aspects of feeding are associated with dopaminergic a c t i v i t y and which brain structures are involved. Blackburn, P h i l l i p s , and Fibiger (1987) stressed the u t i l i t y of factoring motivated behaviors into t h e i r component parts and analyzing the role of DA within each component. These authors emphasize the major d i s t i n c t i o n between consummatory and preparatory behaviours. With respect to feeding, they define consummatory behaviours as "those that occur after the animal has made contact with food and that r e s u l t in i t s ingestion" and give "biting, chewing, and swallowing" as examples of this category. Preparatory behaviours are defined as "appetitive acts that t y p i c a l l y lead to, and make possible, consummatory behaviours". Studies by these and other researchers suggest that dopaminergic a c t i v i t y may play a greater role in preparatory than consummatory feeding behaviours. Unlike the results of e a r l i e r studies with DA antagonists, Blackburn et a l . (1987) found that pimozide did not s i g n i f i c a n t l y attenuate food intake, but did a f f e c t preparatory responses. The procedure followed in thi s study was based on a conditioned feeding design by Weingarten (1984). Using this experimental procedure, the researchers found that appetitive responses to a conditioned stimulus (CS+) s i g n a l l i n g the subsequent onset of food delivery were attenuated by the administration of pimozide, whereas consummatory behaviours appeared normal when the food was 7 delivered. In a related study, the DA antagonists metoclopramide and thioridazine had d i f f e r e n t i a l e f f e cts on preparatory behaviour (Blackburn, P h i l l i p s , & Fibiger, 1989a). Like pimozide, metoclopramide attenuated preparatory responses to the conditioned stimulus, whereas thioridazine did not. It was suggested that the effects of metoclopramide r e f l e c t a p r e f e r e n t i a l action of this drug in the caudate nucleus. In another study employing the same conditioned feeding paradigm, increased DOPAC/DA and HVA/DA ra t i o s were observed in the nucleus accumbens and striatum (though s t r i a t a l values did not reach s t a t i s t i c a l significance) after exposure to the CS+ alone (Blackburn, P h i l l i p s , Jakubovic, & Fibiger, 1989b). No increases i n either r a t i o were found i n rats allowed to ingest an unsignalled meal. A l l measurements were made on postmortem tissue by HPLC/EC analysis. Further support for the hypothetical role of DA i n preparatory behaviours comes from the attenuation of hoarding, a form of preparatory feeding behaviour, following the disruption of mesolimbic DA neurons by 6-OHDA lesions (Kelley & Stinus, 1985). Others have found that food-related stimuli (odors, sights and sounds associated with scheduled feeding) in the absence of access to food, can e l i c i t increased DA turnover (D'Angio & Scatton, 1989; Simansky et a l . , 1985). Schultz (1986) used electrophysiological techniques to monitor a c t i v i t y i n dopaminergic midbrain neurons of monkeys during an 8 appetitive task. The majority of these neurons increased their f i r i n g rate when the monkeys were presented with visual and auditory cues that signalled the a v a i l a b i l i t y of food and also when the monkeys reached out toward the food. However, the f i r i n g rates of very few neurons increased above baseline levels when food was actually placed into the mouth. Theoretical Background Several hypotheses have been proposed to account for the experimental data r e l a t i n g changes in dopaminergic a c t i v i t y to feeding behaviors. The "sensorimotor hypothesis", was developed in response to observations following dopamine depletion caused by 6-OHDA lesions of the n i g r o s t r i a t a l pathway. The hypothesis relates ascending DA system involvement in sensorimotor f a c i l i t a t i o n (Marshall, Richardson, & Teitelbaum, 1974; White, 1986). That i s , DA a c t i v i t y can modulate the a b i l i t y of sensory stimuli to i n i t i a t e motor responses. Numerous studies have shown that destruction of this pathway results i n locomotor and postural d e f i c i t s , an i n a b i l i t y to orient to external stimuli and a disruption of spontaneously i n i t i a t e d natural behaviors (c.f. Berridge, Venier, & Robinson, 1989). With respect to feeding behaviors, the sensorimotor hypothesis predicts that DA a c t i v i t y would be required for the 9 p r o d u c t i o n o f a p p e t i t i v e and consummatory responses to e x t e r n a l f o o d - r e l a t e d cues such as food odors. An a l t e r n a t i v e h y p o t h e s i s , the "response i n i t i a t i o n h y p o t h e s i s " , proposes t h a t DA i s e s s e n t i a l f o r response p r o d u c t i o n ( F i b i g e r , Zis, & P h i l l i p s , 1975; P o s l u n s , 1962). T h i s h y p o t h e s i s proposes t h a t an a n i m a l t r e a t e d w i t h a n e u r o l e p t i c i s c a p a b l e o f u n d e r s t a n d i n g the s i g n i f i c a n c e o f a b i o l o g i c a l l y r e l e v a n t e n v i r o n m e n t a l cue, but i s unable t o i n i t i a t e a motor response t o t h i s cue. T h i s h y p o t h e s i s was d e v e l o p e d to account f o r the o b s e r v a t i o n t h a t n e u r o l e p t i c -t r e a t e d a n i m a l s c o u l d not p e r f o r m a v o i d a n c e responses t o a s t i m u l u s t h a t p r e d i c t e d f o o t s h o c k , but c o u l d perform s h o r t l a t e n c y escape r e s p o n s e s once the shock began (Hunt, 1956). D u r i n g p r e s e n t a t i o n o f the p r e d i c t i v e s t i m u l u s , the a n i m a l s s q u e a l e d and d e f e c a t e d , i n d i c a t i n g t h a t they r e a l i z e d the r e l e v a n c e o f the cue, y e t were u n a b l e to perform a response to a v o i d the impending shock. The a b i l i t y to escape once the shock began demonstrates t h a t the animal had the p h y s i c a l c a p a c i t y to p e r f o r m the response. F i b i g e r e t a l . (1975) e x p l a i n t h i s p r e s e r v e d a b i l i t y by s u g g e s t i n g t h a t even though an animal i s unable t o p e r f o r m an avoidance r e s p o n s e , i n v o l u n t a r y motor a c t i v i t y caused by the shock i t s e l f may be s u f f i c i e n t t o i n i t i a t e an escape r e s p o n s e . B l a c k b u r n e t a l . (1987) s u g g e s t e d t h a t both a v o i d a n c e and a p p e t i t i v e b e h a v i o r s may f a l l under the g e n e r a l c a t e g o r y o f p r e p a r a t o r y r e s p o n s e s , whereas escape and i n g e s t i o n may be c l a s s i f i e d as consummatory r e s p o n s e s . Given t h i s 10 d i s t i n c t i o n , the response i n i t i a t i o n d e f i c i t hypothesis derived from shock experiments could be applied to feeding behaviors. DA a c t i v i t y may be required for the i n i t i a t i o n of any preparatory response, whether this response i s made to avoid shock or to approach food. In 1978, Wise, Spindler, DeWit, and Gerber proposed the "anhedonia hypothesis" to explain the action of neuroleptic drugs and possibly the role of ascending dopamine systems. This hypothesis suggested that dopaminergic neurons mediate the rewarding or "hedonic" impact of positive r e i n f o r c e r s , such as food (Wise, 1982, 1985; Wise et a l . , 1978a). Accordingly, disruption of ascending dopaminergic pathways attenuates the hedonic properties of various positive reinforcers. The anhedonia hypothesis i s consistent with the reduction in food-rewarded responses observed in animals treated with the DA antagonist pimozide (Geary & Smith, 1984; Wise, Spindler, & Legault, 1978b; Xenakis & Sclaf a n i , 1981). Therefore, i f the reinforcer loses i t s hedonic impact, the response associated with i t w i l l be extinguished. However, th i s apparent "extinction" e f f e c t r e s u l t i n g from DA receptor blockade i s not equivalent to the ty p i c a l extinction seen following a period of nonreinforcement. S p e c i f i c a l l y , more rapid ext i n c t i o n i s observed as the e f f e c t s of neuroleptics sum with those of extinction by nonreinforcement (Gray & Wise, 1980; P h i l l i p s & Fibiger, 1979; Tombaugh et a l . , 1980). Results obtained by Blackburn, P h i l l i p s , Jakubovic, and 11 Fibiger (1986) also do not support the anhedonia hypothesis. In this study, increased DA a c t i v i t y was observed following ingestion of a n u t r i t i v e l i q u i d diet or lab chow, but not after ingestion of a palatable saccharin solution. These re s u l t s suggest that since saccharin solution i s avidly consumed by rats and has been shown to maintain high rates of operant responding, the apparent "reward" associated with saccharin ingestion i s not s u f f i c i e n t to increase DA a c t i v i t y . As mentioned above, Blackburn et a l . (1987) noted that pimozide disrupted responses to a conditioned stimulus s i g n a l l i n g food, but not responses to the food i t s e l f . They argued that the anhedonia hypothesis does not adequately explain why the response to a secondary reinforcer (the conditioned stimulus) should be more susceptible to disruption by pimozide than a primary reinforcer (food). In addition, the hypothesis cannot account for the results obtained from a second experiment i n which pimozide-treated animals were given unlimited quantities of food in their home cages. Under th i s condition, the anhedonia hypothesis would predict that following pimozide treatment, the food would be less rewarding, r e s u l t i n g in decreased consumption. However, this prediction was not confirmed. In a recent study, Berridge et a l . (1989) provided evidence that argues against both the anhedonia and the sensorimotor hypotheses. These researchers employed a taste r e a c t i v i t y paradigm to examine the effects of mesostriatal 12 6-OHDA lesions. Rats emit positive and aversive responses naturally, depending on the p a l a t a b i l i t y of an ingested substance. By studying the reaction of both lesioned and control rats to solutions over a range of taste p a l a t a b i l i t y and intensity, the researchers sought to discriminate between the sensorimotor and anhedonia hypotheses. They proposed that a decrease i n sensorimotor arousal would lead to a general reduction i n taste r e a c t i v i t y whereas anhedonia would s e l e c t i v e l y reduce positive reactions. However, taste r e a c t i v i t y remained unchanged after the lesions, a r e s u l t that supported neither hypothesis. Berridge et a l . (1989) argued that their study "provides evidence that the capacity for hedonics can be neurologically dissociated from motivated appetitive behavior". They proposed an alternative theory based on incentive a t t r i b u t i o n . The concept of "incentive motivation" emphasizes the a b i l i t y of environmental stimuli associated with b i o l o g i c a l l y s i g n i f i c a n t events to e l i c i t behavioral responses (Bindra, 1978; Bolles, 1972; Toates, 1981). Preparatory behavior, including both appetitive and aversive responses, may be i n i t i a t e d by formerly neutral stimuli that have become incentives through experience. The strength of an incentive stimulus i s determined by the degree of association between this stimulus and a b i o l o g i c a l l y relevant stimulus (e.g., food, shock), and also by the salience of the b i o l o g i c a l stimulus. That i s , although an internal condition such as "hunger" cannot i n i t i a t e a c t i v i t y 13 i t s e l f , i t can potentiate the effectiveness of a given food-related environmental stimulus in i n i t i a t i n g future responses (Wise, 1987). Bindra (1978) argued that physiological conditions are important because they "serve as 'gates' or l i m i t s within which cert a i n p a r t i c u l a r incentive stimuli become e f f e c t i v e " , but without an incentive or a stimulus predictive of an incentive, there i s no motivation or response i n i t i a t i o n . It has been suggested that incentive stimuli not only predict hedonic events, but also acquire some of the hedonic properties of the b i o l o g i c a l l y s i g n i f i c a n t events they are associated with (Berridge & Schulkin, 1989; Berridge et a l . , 1989; Stewart, de Wit, & Eikelboom, 1984). In a study c i t e d by Stewart et a l . (1984), opiate-like effects were reported by former addicts s e l f - i n j e c t i n g saline under high-drug-expectancy conditions. Berridge and Schulkin (1989) found that positive palatability-dependent reactions to a taste previously paired with s a l t , were enhanced only when a rat was deprived of sodium. This enhancement of p a l a t a b i l i t y occurred even though the conditioned taste became associated with s a l t during "sodium-balanced" t r i a l s , a condition under which rats show aversive reactions to hypertonic s a l t . The s h i f t in p a l a t a b i l i t y of the conditioned taste^ resembled the s h i f t that would have occurred with s a l t i t s e l f during sodium depletion, indicating that the conditioned taste had acquired some of the hedonic q u a l i t i e s of the s a l t . It has been proposed that the neural substrate of 14 incentive motivation involves ascending DA systems (Blackburn et a l . , 1987; Fibiger & P h i l l i p s , 1986; Mogenson and P h i l l i p s , 1976). According to this hypothesis, dopaminergic a c t i v i t y i s required when motivational incentive significance i s assigned to neutral stimuli through association with b i o l o g i c a l l y relevant events. Disruption of the neural system involved in the a t t r i b u t i o n of motivational salience would disrupt motivated behavior even i f motor, sensory, and affective systems were intact. The incentive motivation model of DA function asserts that exposure to an incentive stimulus can lead to an increase in the release of DA in the forebrain, which i n turn results i n a preparatory response (Blackburn et a l . , 1987). This hypothesis i s supported by the observation of increased dopaminergic a c t i v i t y during appetitive behaviors (Schultz, 1986; Blackburn et a l . , 1989b). It i s also consistent with the selective disruption of preparatory, but not consummatory feeding behaviors observed following pimozide treatment (Blackburn et a l . , 1987). Rationale for the application of electrochemistry to the study of feeding i n the rat The purpose of the present thesis was to study the role of DA in feeding behaviors in vivo by employing the technique of electrochemistry with stearate-modified carbon 15 paste electrodes. This technique offers several advantages. Electrochemistry does not depend on postmortem tissue analysis, but can be performed on unanesthetized, f r e e l y -moving subjects. Electrochemical signals can be obtained from one animal over an extended period of time ( i . e . , during both baseline and experimental phases), allowing i t to serve as i t s own control. This also enables the researcher to observe the time course of an observed neurochemical response. In addition, the technique can be used to study neurochemical and behavioral correlations without the need for experimentally manipulated DA lev e l s obtained by e l e c t r i c a l stimulation, lesions, or pharmacology. Although a l l of the points mentioned so far could apply equally to both electrochemistry and microdialysis, each technique has i t s own unique advantages. The temporal resolution of chronoamperometry i s superior to microdialysis because samples can be taken every few seconds (usually 15 to 20 s) as opposed to the 5 to 20 min sampling time i n microdialysis. Also, chronically implanted electrochemical electrodes tend to remain viable for much longer periods than microdialysis probes (often, weeks as opposed to days), thus f a c i l i t a t i n g r e p l i c a t i o n . F i n a l l y , electrochemical electrodes are generally smaller than d i a l y s i s probes and as a r e s u l t , cause less damage during implantation. However, the exact nature of the substance contributing to signals obtained by electrochemical methods i s not firmly established, whereas the results obtained by microdialysis with HPLC/ED analysis are unequivocal. Stearate-modified carbon-paste electrodes have been developed for the selective monitoring of e x t r a c e l l u l a r DA levels in "DA-rich brain regions (Blaha & Lane, 1983). This gives them an advantage over other electrochemical electrodes that are either nonselective, or that monitor DA metabolites as indices of DA turnover. 17 GENERAL METHODS Electrode preparation A three-electrode electrochemical detection system was used i n a l l experiments. Recording electrodes were made from Teflon-coated s t a i n l e s s steel wire (0.008" bare, 0.011" coated). The sta i n l e s s steel was withdrawn approximately 0.5mm through the Teflon coating leaving a well at one end. This well was packed with a graphite paste mixture consisting of graphite powder, s t e a r i c acid, and l i q u i d p a r a f f i n (Nujol). Reference electrodes were made from Teflon-coated s i l v e r wires. These electrodes had the Teflon coating stripped for l-2mm at one end, and the exposed s i l v e r wire was s i l v e r - c h l o r i d e d . The t h i r d type of electrode used, was the au x i l i a r y electrode, consisting of a bare s t a i n l e s s steel wire anchored to a s k u l l screw. The free ends of each type of electrode were soldered to gold connector pins. Surgery A l l rats used in the following experiments had two cra n i a l electrodes implanted under sodium pentobarbital anesthesia (Somnitol, 60mg/kg i.p.) One recording electrode was implanted s t e r e o t a x i c a l l y in the nucleus accumbens, 18 1.2mm a n t e r i o r t o bregma, 1.2mm l a t e r a l ( l e f t h e m i s p h e r e ) , and 6.5mm v e n t r a l to the s u r f a c e o f the c o r t e x w i t h the s i n c i s o r bar s e t a t -3.3. The second r e c o r d i n g e l e c t r o d e was im p l a n t e d i n t o the s t r i a t u m , 1.0mm a n t e r i o r t o bregma, 1.7mm l a t e r a l ( r i g h t h e m i s p h e r e ) , and 4.0mm v e n t r a l to the c o r t i c a l s u r f a c e . For each r a t , a s i n g l e r e f e r e n c e e l e c t r o d e was i m p l a n t e d a p p r o x i m a t e l y 2-3mm p o s t e r i o r and l a t e r a l t o bregma i n t o the r i g h t hemisphere. The r e f e r e n c e e l e c t r o d e was lowered u n t i l the c h l o r i d e d t i p d i s a p p e a r e d i n t o the c o r t e x . A s e p a r a t e a u x i l i a r y e l e c t r o d e was wrapped s e v e r a l t imes around one s k u l l screw. Three t o fou r a d d i t i o n a l screws were anchored t o the s k u l l . The g o l d p i n from each e l e c t r o d e was snapped i n t o a p l a s t i c h o l d e r . T h i s whole assembly was cemented t o g e t h e r w i t h d e n t a l a c r y l i c . The upper edge o f the p l a s t i c h o l d e r c o n t a i n i n g the g o l d p i n s was t h r e a d e d so t h a t a l e a d used f o r e l e c t r o c h e m i c a l r e c o r d i n g c o u l d be screwed on. The r a t s were a l l o w e d t o r e c o v e r from s u r g e r y f o r a t l e a s t 2 days b e f o r e b e i n g used i n an experiment. R e c o r d i n g procedure Chronoamperometric r e c o r d i n g s were ta k e n by a p p l y i n g square-wave p o t e n t i a l p u l s e s t o each r e c o r d i n g e l e c t r o d e a t 20-s ( E x p t . 1) or 30-s (Expt. 2) i n t e r v a l s and measuring the 19 current a f t e r 1 s. The voltage parameters for the pulses were determined by f i r s t obtaining voltammetric sweep records. In a sweep, the applied voltage was ramped at +10mV/s, and the res u l t i n g current was recorded. When the • record was semidifferentiated, any current due to the oxidation of a given substance at the electrode t i p showed up as an exaggerated peak superimposed upon a background curve (Figure 1). The pulse parameters for chronoamperometry were set by determining the window within which the peak of interest lay (e.g., -lOOmV to +175mV for the proposed DA peak in Figure 1). When voltammetric sweep records themselves were used to record changes in DA levels during a test session (in Expt. 2 only), a sweep interval of 10 min was chosen to allow s u f f i c i e n t time for e q u i l i b r a t i o n of DA at the electrode t i p . Peak height was used as an ind i c a t i o n of ex t r a c e l l u l a r DA l e v e l s . Histology At the conclusion of an experiment, each rat was s a c r i f i c e d in a C02 chamber and i t s brain was removed and stored in a 10% formalin solution for several days. Later, each brain was frozen, sectioned, and mounted in order to allow h i s t o l o g i c a l confirmation of electrode placements in accordance with the Paxinos and Watson atlas (1982). 20 F i g u r e 1 Example o f a t y p i c a l v o l t a m m e t r i c sweep, ramped a t lOmV/s (-150 t o +450mV). The peak, o c c u r r i n g a t a p p r o x i m a t e l y lOOmV r e p r e s e n t s the p u t a t i v e DA peak. 21 (yu) ^uojjno uoftDpjxo EXPERIMENT 1 Effects of an unsiqnalled l i q u i d meal on electrochemical measures of DA a c t i v i t y Experiment 1 was designed to determine whether any changes in the dopaminergic electrochemical signal could be detected in the rat caudate and nucleus accumbens before, during, and after the delivery and consumption of a small unsignalled meal of a palatable l i q u i d diet. The simple procedure followed in this experiment allowed observation o the time course of any e f f e c t s of feeding on e x t r a c e l l u l a r DA l e v e l s . One advantage of t h i s experiment over past studies correlating feeding and DA release was that food deprivation was not a factor here. Each rat was allowed free access to food except during the actual test sessions, r e s u l t i n g in a more normal physiological, and possibly neurophysiological, state during testing. Based on the re s u l t s of other feeding studies mentioned e a r l i e r , increases in DA a c t i v i t y would be predicted to occur i n response to the meal presented i n the current study. Methods Subjects \ Four male hooded Long-Evans rats from Charles River Laboratories were used i n th i s study. At the beginning of the experiment, the rats weighed between 350g and 450g. Apparatus During the da i l y recording sessions, individual rats were placed inside a Plexiglas testing chamber located within a larger Faraday cage to reduce e l e c t r i c a l noise. The bottom of the chamber consisted of a wire gri d floor with a bed of San-i-cel beneath i t . At one side of the compartment, a hole was d r i l l e d and a brace attached to accommodate a removable Richter tube containing the l i q u i d d i e t , Sustacal (Mead Johnson), used in the study. A commutator for electrochemical recording extended down from the roof of the Faraday cage to just above the roof of the Plexiglas compartment. A recording lead was attached to this commutator at one end and to the rat's electrode assembly at the other. Outside of the Faraday cage, leads o r i g i n a t i n g from the commutator were connected to an electrochemical recording box. Recorded electrochemical 24 signals were translated by, and viewed on, a micro-computer. On each t r i a l , the r a t s ' behavior was observed via a video camera in front of the test chamber. The room was l i t by a bulb turned away from the rats and dimmed as much as possible while s t i l l allowing an adequate video recording of the r a t s ' behavior. Procedure In order to prevent neophobia during test sessions, a l l rats were preexposed i n their home cages to 10-20ml of the chocolate-flavored l i q u i d diet in addition to their usual diet of lab chow p e l l e t s and water. Rats that consumed the l i q u i d diet overnight or upon a second exposure, were used in the experiment. During a given experimental session, an individual rat was connected to a recording lead and placed inside the test chamber. At the beginning of a test session, the rat had no access to food nor water. Each day, voltammetric sweep records were f i r s t obtained and chronoamperometric parameters were then determined from these sweep data. After a stable baseline (less than 1 nA peak, to peak fluctuation) of at least 10 min was recorded at a 20 s pulse in t e r v a l rate, the test box was opened and a 10ml portion of the l i q u i d diet was placed inside. The electrochemical signal from the rat was recorded for another 18-30 min. During a l l phases of the experiment, the rat remained undisturbed in a closed room except during the brief food delivery period. Each session was videotaped and the tapes were used to analyze the time spent on feeding behavior. A the conclusion of a test session, the rat was returned to i ts home cage and given free access to lab chow and water unti l the following session. Each rat was tested in this manner, once per day, for seven consecutive days. Results Each of the 4 rats used in this experiment showed an increase in chronoamperometric oxidation current of approximately 0.5 to 2.0 nA over baseline levels from both the n. accumbens and caudate in the period following the introduction of Sustacal inside the test chamber (Figures 2 3, 4, and 5). Each figure represents data obtained from 1 of the 4 single rats averaged over test days 2 through 7. Data from the f i r s t test day for each rat were not included in this average because the feeding response was typical ly not immediate nor as vigorous on this day as on the following sessions, when the rat was more familiar with the test chamber and procedure. In fact, none of the four subjects showed any recorded increases in either the n. accumbens or caudate on the f i r s t day of testing (Figure 6) Aside from the differences observed between day 1 and days 26 Figure 2 Chronoamperometric signals recorded from the caudate (squares) and nucleus accumbens (crosses) before and after the delivery of a lOcc Sustacal meal. The figure represents averaged data obtained from rat #1 over days 2-7. The point at time zero was adjusted to OnA to f a c i l i t a t e comparison of oxidation current changes between the two recording channels. The average meal duration from days 2-7 is bounded by time zero (the point of food delivery) and the arrow indicating the end of the feeding bout. 27 (yu) }uexino uoftDpjxo 28 F i g u r e 3 C h r o n o a m p e r o m e t r i c s i g n a l r e c o r d e d f r o m t h e c a u d a t e ( s q u a r e s ) and n u c l e u s accumbens ( c r o s s e s ) b e f o r e and a f t e r t h e d e l i v e r y o f a l O c c S u s t a c a l m e a l . The f i g u r e r e p r e s e n t s a v e r a g e d d a t a o b t a i n e d f r o m r a t #2 o v e r d a y s 2-7. The p o i n t a t t i m e z e r o was a d j u s t e d t o OnA t o f a c i l i t a t e c o m p a r i s o n o f o x i d a t i o n c u r r e n t c h a n g e s b e t w e e n t h e two r e c o r d i n g c h a n n e l s . The a v e r a g e m eal ' d u r a t i o n f r o m d a y s 2-7 i s bounded by t i m e z e r o ( t h e p o i n t o f f o o d d e l i v e r y ) and t h e a r r o w i n d i c a t i n g t h e end o f t h e f e e d i n g b o u t . 29 (Vu) *u©xmo uoftDpixo 30 Figure 4 Chronoamperometric signal recorded from the caudate (squares) and nucleus accumbens (crosses) before and after the delivery of a lOcc Sustacal meal. The figure represents averaged data obtained from rat #3 over days 2-7. The point at time zero was adjusted to OnA to f a c i l i t a t e comparison of oxidation current changes between the two recording channels. The average meal ' duration from days 2-7 i s bounded by time zero (the point of food delivery) and the arrow indicating the end of the feeding bout. (yu) *u©xino uoftDpjxo 32 F i g u r e 5 C h r o n o a m p e r o m e t r i c s i g n a l r e c o r d e d f r o m t h e c a u d a t e ( s q u a r e s ) and n u c l e u s accumbens ( c r o s s e s ) b e f o r e and a f t e r t h e d e l i v e r y o f a l O c c S u s t a c a l m e a l . The f i g u r e r e p r e s e n t s a v e r a g e d d a t a o b t a i n e d f r o m r a t #4 o v e r d a y s 2-7. The p o i n t a t t i m e z e r o was a d j u s t e d t o OnA t o f a c i l i t a t e c o m p a r i s o n o f o x i d a t i o n c u r r e n t c h a n g e s b e t w e e n t h e two r e c o r d i n g c h a n n e l s . The a v e r a g e meal ' d u r a t i o n f r o m d a y s 2-7 i s bounded by t i m e z e r o ( t h e p o i n t o f f o o d d e l i v e r y ) and t h e a r r o w i n d i c a t i n g t h e end o f t h e f e e d i n g b o u t . 34 F i g u r e 6 Representative chronoamperometric r e c o r d from the caudate (squares) and nucleus accumbens (cr o s s e s ) of r a t #2 on the f i r s t day of t e s t i n g . Time zero i n d i c a t e s the p o i n t of d e l i v e r y of a lOcc S u s t a c a l meal and the arrow i n d i c a t e s the end of the f i r s t feeding bout. oxidation current (nA) 36 Figure 7 Chronoamperometric signal recorded before and after the delivery of a lOcc Sustacal meal. The figure represents, averaged data obtained from rats 1, 2, 3, and 4 over days 2-7. The point at time zero was adjusted to OnA to f a c i l i t a t e comparisons of oxidation current changes between the 2 recording channels. The average meal duration is bounded by time zero (the point of food delivery) and the arrow indicating the end of the feeding bout. The symbols (crosses = n. accumbens, squares = caudate) represent averaged data from the 4 r a t s , and the s o l i d l i n e s represent standard error of the mean. a) n. accumbens b) caudate TIME (min) 38 39 Figure 8 Electrode placements d i r e c t e d at the l e f t nucleus accumbens (n=4) and r i g h t caudate (n=4) i n Experiment 1. 40 +L2mm +0.7mm 41 2-7, no f u r t h e r developments across sessions were observed. For a given r a t , the time course and the magnitude of the recorded increases i n chronoamperometric current were very s i m i l a r i n both the n. accumbens and caudate. In a d d i t i o n , the p a t t e r n of change seen i n each of the four r a t s was comparable. The average change i n recorded o x i d a t i o n current across a l l 4 subjects i s presented i n Figure 7. The time for a r a t to complete i t s f i r s t intense feeding bout was defined as ending when the r a t had spent at l e a s t 30s away from the feeder ( g e n e r a l l y , the r a t had consumed most or a l l of the meal at t h i s p o i n t ) . In three of the four f i g u r e s (Figures 2-4), the recorded current appears to be r e t u r n i n g to b a s e l i n e l e v e l s approximately 25-30 minutes a f t e r food d e l i v e r y . Although Figure 5 does not i n d i c a t e a complete r e t u r n to b a s e l i n e , t h i s f i g u r e represents data c o l l e c t e d for l e s s than 20 minutes, which may not have been s u f f i c i e n t to observe a r e t u r n to baseline i n t h i s subject. A n a l y s i s of the h i s t o l o g i c a l data revealed that a l l of the e l e c t r o d e t i p s were lo c a t e d e i t h e r i n the r i g h t dorsomedial s t r i a t u m or the l e f t nucleus accumbens (Figure 8) . 42 Discuss ion The r e s u l t s of Experiment 1 i n d i c a t e that feeding has an e f f e c t on dopaminergic a c t i v i t y i n both the nucleus accumbens and s t r i a t u m of the r a t . In both cases, estimates of e x t r a c e l l u l a r DA l e v e l s derived from chronoamperometric records increased a f t e r consumption of a l i q u i d meal. Both the magnitude and time course of the recorded increases were s i m i l a r i n the n. accumbens and s t r i a t u m , suggesting that the two recording s i t e s may share a s i m i l a r r o l e i n feeding behaviors. Two f a c t o r s i n t h i s study i n d i c a t e that the DA response may be a s s o c i a t e d with a p o s t i n g e s t i v e stage of feeding. F i r s t , there appeared to be a l a t e n c y between the i n i t i a t i o n of food comsumption and the observation of a notable increase i n the chronoamperometric s i g n a l . Second, the DA l e v e l s remained elevated between 20 and 30 min a f t e r the meal had been completed. One p o t e n t i a l l y confounding f a c t o r i n the present study a r i s e s from the p o s s i b i l i t y that the " b a s e l i n e " values recorded at the beginning of each t e s t session represent an e x t r a c e l l u l a r DA concentration that has already been elevated simply by p l a c i n g the r a t i n t o the t e s t chamber. The combination of handling, exposure to a novel environment complete with odors from previous feeding experiments i n the same t e s t chamber, and p o s s i b l y , a n t i c i p a t i o n of a p a l a t a b l e meal, could have r e s u l t e d i n an increase i n DA a c t i v i t y . 43 This e l e v a t i o n could have inf l u e n c e d the observation of any f u r t h e r increase i n DA a c t i v i t y due to the p r e s e n t a t i o n and consumption of food. That i s , the changes from true " b a s e l i n e " l e v e l s may have been even l a r g e r than those that were a c t u a l l y observed. One aspect of feeding that t h i s study does not explore i s the r o l e of DA i n a n t i c i p a t o r y stages of feeding. In Experiment 1, the r a t s began to consume the u n s i g n a l l e d meal w i t h i n seconds of d e l i v e r y . As noted i n the I n t r o d u c t i o n , e a r l i e r s t u d i e s have i n d i c a t e d that DA systems may be s e l e c t i v e l y involved i n preparatory aspects of feeding behaviors (Blackburn et a l . , 1987, 1989a). This d i s t i n c t i o n between preparatory and consummatory feeding stages was i n v e s t i g a t e d i n Experiment 2. EXPERIMENT 2 Ele c t r o c h e m i c a l measures of dopamine a c t i v i t y during a n t i c i p a t o r y and consummatory phases of feeding behaviors i n the r a t This experiment was designed to study changes i n e l e c t r o c h e m i c a l measurements of e x t r a c e l l u l a r DA during d i f f e r e n t phases of a conditioned feeding paradigm developed by Weingarten (1983, 1984). As reviewed i n the I n t r o d u c t i o n , DA has been associated with a p p e t i t i v e responding for food (Blackburn et a l . , 1987, 1989b; Sc h u l t z , 1986). In order to study t h i s proposed c o r r e l a t i o n , i t was necessary to observe the a p p e t i t i v e phase independently of the consummatory response. Weingarten's paradigm accomplished t h i s separation by c o n d i t i o n i n g the subjects to an e x t e r n a l stimulus cue (CS+) that p r e d i c t e d the d e l i v e r y of a meal. This cue, consisted of a buzzer and a b r i g h t l i g h t , preceded, and then continued throughout the food d e l i v e r y period. In the present experiment, during the CS+ period preceding food d e l i v e r y , the r a t ' s behavior (nosepokes i n t o the feeding niche) was monitored as an i n d i c a t i o n of a p p e t i t i v e responding. Simultaneously, chronoamperometric records were obtained. As i n Experiment 1, the r a t ' s electrochemical s i g n a l from both the caudate and nucleus accumbens was recorded during a l l stages of the feeding bout, i n c l u d i n g a d i s t i n c t " a p p e t i t i v e " phase. One i n t e r p r e t a t i o n of the i n c e n t i v e m o t i v a t i o n hypotheses of DA f u n c t i o n p r e d i c t s that the CS+ alone should act as an i n c e n t i v e stimulus r e s u l t i n g i n increased DA release and subsequently, an approach to the feeding niche (Blackburn et a l . , 1989a). Methods Subj ects A l l 40 subjects used i n t h i s study were male hooded Long-Evans r a t s weighing 350 to 450g at the beginning of the exper iment. Apparatus Rats were housed i n d i v i d u a l l y w i t h i n four separate c o n d i t i o n i n g chambers. Each chamber c o n s i s t e d of a sound atte n u a t i n g modified Coleman cooler (Sno-Lite Low Boy model) co n t a i n i n g a smaller P l e x i g l a s compartment i n which the r a t was housed. The f l o o r of the inner compartment was made of a wire g r i d with a bed of absorbent S a n - i - c e l below i t . Each compartment had two holes and two R i c h t e r tube braces s i t u a t e d at one end w a l l . Water was f r e e l y a v a i l a b l e to the 46 rat at a l l times through one of these two holes. The opposite wall contained a recessed feeding niche with a 1 cm l i p enabling i t to hold the l i q u i d d i e t . A small hole was d r i l l e d at the back of the feeding niche to accommodate the end of a length of S i l a s t i c tubing. Sustacal was del ivered through th is tubing by a Cole-Parmer p e r i s t a l t i c pump. The sides of the feeding niche were f i t t e d with a photoce l l . Interrupt ion of the photocel l beam indicated a r a t ' s entry into the niche. Outside of the P lex ig las compartment, each cooler was f i t t e d with a dim housel ight (on at 11:00 am, o f f at 11:00 pm) and a fan (always on). For condi t ioning purposes, a buzzer, bright cue l i g h t and tone generator were also mounted near the c e i l i n g of the coo ler . The cues, pump, and photocel ls were c o n t r o l l e d i n i t i a l l y by a Nova and subsequently by an INI computer using Manx software. Two of the four chambers were set up for electrochemical recording . In these chambers, a Faraday cage was placed between the cooler and the smaller Plexig las compartment. Each cage was f i t t e d with a commutator extending down to the roof of the P lex ig las chamber. A recording lead was attached between th i s commutator and the r a t ' s head apparatus. Cables were extended from the opposite end of the commutator to an electrochemical recording box outside of the cooler . Recorded s ignals were translated by and viewed on an IBM compatible personal computer. 47 Procedure The procedure followed i n Experiment 2 was based on a c o n d i t i o n e d f e e d i n g paradigm developed by Weingarten (1983, 1984). The present procedure i n v o l v e d exposing i n d i v i d u a l l y housed r a t s to an average of e i g h t s i g n a l l e d 9ml l i q u i d meals per day, a volume s u f f i c i e n t to keep the animals between 90% and 100% f r e e - f e e d i n g body weight. P r e s e n t a t i o n of a meal c o i n c i d e d with the f i n a l minute of a 330 second b u z z e r - l i g h t c o n d i t i o n e d stimulus (CS+). A l l meals were d e l i v e r e d i n t o the feeding niche a t one end of the cage. When the r a t entered the niche, a p h o t o c e l l beam was i n t e r r u p t e d and t h i s event was recorded by computer. A second s t i m u l u s (CS-), presented as a pure tone, was not p a i r e d with food d e l i v e r y . The i n t e r t r i a l i n t e r v a l ranged from 43 to 105 minutes with a mean of 90 minutes. A f t e r s e v e r a l days of t r a i n i n g on an a l t e r n a t i n g CS+/CS- schedule, the r a t s r e l i a b l y d i s p l a y e d the a b i l i t y to d i s c r i m i n a t e between the two cues by spending much of the CS+ p e r i o d i n the n i c h e , and l i t t l e or no time i n the niche d u r i n g the CS-p e r i o d . The amount of time and number of "nosepokes" i n t o the niche d u r i n g p r e s e n t a t i o n of the CS+ stimulus were co n s i d e r e d to be i n d i c a t i o n s of p r e p a r a t o r y responding i n a n t i c i p a t i o n of a meal. Subjec t s were c o n d i t i o n e d to d i s c r i m i n a t e between the two s t i m u l u s cues e i t h e r before or a f t e r e l e c t r o d e 48 implantat ion. In both cases, condit ioned rats were monitored chronoamperometrically and behavioral ly (via nosepoke data) continuously except during a b r i e f d a i l y , and more extensive weekly, maintenance and data c o l l e c t i o n per iod . Once a week, the rats were removed temporarily from the condit ioned feeding boxes to be weighed and to have the ir cages cleaned. After i n i t i a l hook-up to the recording apparatus, a 2-day period was allowed for adaptation to the lead, the novel test box, and for those rats removed for implantation after t r a i n i n g , to become reacquainted with the condit ioned feeding schedule. I f the subject was returned to the colony for any reason, an add i t i ona l day of adaptation was allowed following reconnection. Event-marked chronomperometric records were scored b l i n d l y for any changes in s i g n a l , and l a t e r , they were matched with computer data ind ica t ing whether event markers represented CS+, C S - , houselights on (11:00 p .m. ) , or housel ights o f f (11:00 a.m.). For each t r i a l , a chronoamperometric current value ( in nanoamps) was obtained by determining the change in oxidat ion current r e l a t i v e to the basel ine l e v e l preceding the event. For one subject , which was showing good behavioral (nosepoke data) and chronoamperometric CS d i scr iminat ion , add i t i ona l voltammetric sweep records were obtained during a ser ies of condit ioned feeding t r i a l s . In the 10 CS+ and 10 CS- t r i a l s conducted, the sweep i n t e r v a l was set at 10 min with a voltage ramp of +10mV/s. Each t r i a l was timed such 49 that the end of one sweep occurred approximately 270 s after the onset of a CS. The actual sweeps lasted between 40 and 60 s depending on the selected voltage parameters. This timing was chosen so that one sweep could be obtained as la te as possible i n the "CS alone" period which ended with the onset of food d e l i v e r y , 270 s af ter the CS+ began. In addi t ion to the sweep recorded during the CS per iod , at least three sweeps were obtained both before and after this per iod. Three subjects from which several days of good behavioral and chronoamperometric data had been c o l l e c t e d , were placed on an e x t i n c t i o n schedule. During th i s cond i t ion , two changes were made. F i r s t , the food de l ivery tubing was disconnected from the back, of the feeding niche, and second, a Richter tube with a d a i l y supply of Sustacal was f i t t e d into the extra brace in the wall opposite the niche. Behavioral and electrochemical measurements were continued throughout the ext inct ion period. In one of the three subjects , these measurements were continued into an addi t iona l r e i n i t i a t i o n period in which the ad l i b food supply was removed and the food de l ivery tubing reconnected. Of the to ta l of 40 rats that began th i s experiment, the records from 10 rat s (3 with 2 good channels) were used in the s t a t i s t i c a l a n a l y s i s . Electrochemical recording channels were e l iminated from the analys is i f any one of the three following condit ions occurred before s u f f i c i e n t data were co l l ec t ed : noisy chronoamperometric records, small 50 (<l/2 nA) or absent voltammetric peaks, or a p u l l e d e l e c t r o d e cap. Of the r a t s that developed noisy s i g n a l s or p u l l e d caps a f t e r only a few recording sessions, several showed promising r e s u l t s up u n t i l the s i g n a l was l o s t . The three c r i t e r i a discussed above accounted f o r the records of a l l but 2 of the discarded s u b j e c t s . Although these 2 r a t s had reasonable voltammetric peak amplitudes and chronoamperometric noise l e v e l s , showed behavioral CS+/CS-d i s c r i m i n a t i o n , and h a d . h i s t o l o g i c a l l y confirmed electrode placements, for unknown reasons, they d i d not appear to e x h i b i t chronoamperometric responses to the CS s t i m u l i . Because no chronoamperometric responses were ever obtained from these 2 r a t s , they were not included i n the f o l l o w i n g s t a t i s t i c a l analyses. S t a t i s t i c a l Analyses For each v i a b l e electrochemical recording s i t e (accumbens, n=8; caudate, n=5), a s e r i e s of 20 consecutive " s c o r a b l e " CS+ and 20 CS- t r i a l s were analyzed. A t r i a l was not scored i f i n the 10 minutes preceding the event, there was a t r a n s i e n t increase i n the noise of the s i g n a l , a r i s i n g b a s e l i n e , or a spike i n the record. Excluding the adaptation days, the f i r s t 20 scorable t r i a l s were used i n the s t a t i s t i c a l a n a l y s i s . The mean change from bas e l i n e was c a l c u l a t e d for each set of 20 CS t r i a l s . 51 The chronoamperometric measures were analyzed using a two-way w i t h i n subjects ANOVA for repeated measures, with implant l o c a t i o n (accumbens vs. caudate) and event (CS+ vs. CS-) as the two f a c t o r s . In order to estimate baseline v a r i a b i l i t y , a b a s e l i n e d i f f e r e n c e score was c a l c u l a t e d for each recording s i t e . D i f ference scores were determined by using baseline data preceding the f i r s t CS+ and CS- t r i a l s i n a s e r i e s . The mean baseline value over f i v e points beginning 540 s (30 s i n t e r v a l s ) p r i o r to the CS onset was subtracted from the mean value beginning 270 s before the CS. These b a s e l i n e d i f f e r e n c e scores were then averaged for each b r a i n s i t e / e v e n t combination. Behavioral d i s c r i m i n a t i o n was analyzed with respect to two measures: t o t a l time spent i n niche during the f i r s t 270 s of a CS and l a t e n c y to enter niche f o l l o w i n g CS onset. Values for these measures were obtained from the same t r i a l s that were used i n the chronoamperometric a n a l y s i s . Average values w i t h i n each set of 20 t r i a l s were c a l c u l a t e d and then analyzed i n two separate two-way ANOVAs; one for the amount of time spent i n niche, and the other for entrance latency. For both measures, the two f a c t o r s analyzed were implant l o c a t i o n (accumbens vs. caudate) and event (CS+ vs. CS-). Voltammetric sweep records from a s i n g l e subject were analyzed with respect to peak, height r a t i o s . The height of a peak was determined by f i r s t drawing a l i n e between the two minimum values on e i t h e r side of the proposed DA peak. 52 The peak, height value was subsequently obtained by measuring the v e r t i c a l distance from the peak t i p to t h i s l i n e . R atios were determined between peak height measured during the CS pe r i o d and peak height i n the previous 10 min period. The peak height r a t i o s for CS+ and CS- (10 t r i a l s each) were analyzed using an independent v a r i a b l e s t - t e s t . Results Behavioral d i s c r i m i n a t i o n between the two CS c o n d i t i o n s i s i n d i c a t e d by the r e s u l t s from the two behavioral measures shown i n Figures 9 and 10. Whereas during the CS+, the average la t e n c y to enter the feeding niche was r e l a t i v e l y s h ort, with a s u b s t a n t i a l amount of time spent i n the niche, the reverse was true during the CS-. These r e s u l t s were supported s t a t i s t i c a l l y . The ANOVA for the amount of time spent i n the feeding niche showed no s i g n i f i c a n t e f f e c t of implant l o c a t i o n (accumbens vs. caudate) and no s i g n i f i c a n t l o c a t i o n by event i n t e r a c t i o n . However, there was a s i g n i f i c a n t w i t h i n subjects e f f e c t ( F ( l , l l ) = 56.55, p<.00001). The amount of time spent i n the niche was s i g n i f i c a n t l y greater during the CS+ than the CS- for both groups. The ANOVA for latency to enter the niche a l s o showed no s i g n i f i c a n t e f f e c t of implant l o c a t i o n or l o c a t i o n by event i n t e r a c t i o n . Again, there was a s i g n i f i c a n t w i t h i n subjects e f f e c t ( F ( l , l l ) = 152.19, p<.00001). Here, the 5 3 Figure 9 Mean amount of time spent in the feeding niche during the CS+ and CS- periods p r i o r to the onset of food d e l i v e r y . Data are the average of 20 consecutive t r i a l s on which a good chronoamperometric record was obtained. Error bars represent standard error of the mean. *p<.0001 60 50 H 40 H caudate n. accumbens 30 20 H 1(H C S -55 Figure 10 Mean latency to enter niche following CS+ or CS-onset on the same t r i a l s as shown in Figure 8. Error bars represent standard error of the mean. *p<.0001 57 Figure 11 Mean changes i n DA o x i d a t i o n current i n the nucleus accumbens (n=8) and caudate (n=5) during CS+, CS- and pre-CS b a s e l i n e periods (bCS+, bCS-). S i g n i f i c a n t d i f f e r e n c e s were found between values obtained from the nucleus accumbens and caudate (p<.03) and between the CS+ and CS- (p<.0004). The v e r t i c a l l i n e d i v i d i n g baseline and event r e s u l t s i n d i c a t e s that no s t a t i s t i c a l comparison was made betwen the two c o n d i t i o n s , due to large sample s i z e d i s c r e p a n c i e s . Error bars represent standard e r r o r of the mean. 59 Figure 12 Representative chronoamperometric records obtained from four d i f f e r e n t subjects during consecutive CS+ and CS- t r i a l s . Arrows indicate CS+, C S - , or meal onset. a) ,b) n. accumbens c) ,d) caudate TIME (min.) 61 (yu) }u©xino uop,opjxo (yu) }u©jjno uoftDpjxo 64 F i g u r e 13 B e h a v i o r a l and e l e c t r o c h e m i c a l r e s u l t s from e x t i n c t i o n s u b j e c t #1. E = f i r s t day o f e x t i n c t i o n a) mean amount o f time spent i n f e e d i n g n i c h e d u r i n g CS+ p e r i o d b) mean change i n o x i d a t i o n c u r r e n t r e c o r d e d from the n u c l e u s accumbens d u r i n g CS+ 65 (s) oijom UJ euni i 1 1 1 r LO O LO O LO O CVJ C\J t— o o (VU) juej jno uoijepjxo in e6ueqo 67 Figure 14 Behavioral and electrochemical results from extinction subject #2. E = f i r s t day of extinctio n a) mean amount of time spent in feeding niche during CS+ period b) mean change in oxidation current recorded from the nucleus accumbens and caudate during CS+. time in niche (s) 89 69 ( VU ) juej jno uojiepjxo UJ e6uei|o 70 Figure 15 Behavioral and electrochemical resul t s from ex t inc t ion subject #3. E = f i r s t day of e x t i n c t i o n , R = f i r s t day of r e i n i t i a t i o n to conditioned feeding a) mean amount of time spent in feeding niche during CS+ period b) mean change in oxidat ion current recorded from the nucleus accumbens and caudate during CS+. Electrochemical s ignal from the caudate was los t on day 8. (S) 9 i p j U Uj 9UJI1 (VU) juej jno uoji&pixo m e6ueqo 73 Figure 16 Representative CS+ sweep t r i a l from one subject . The f igure presents nine consecutive sweeps ramped at lOmV/s with a 10-min inter-sweep i n t e r v a l . The arrow indicates the DA peak that was obtained during the CS+ period p r i o r to food d e l i v e r y . oxidation current (nA) 75 Figure 17 Mean peak height r a t i o s from 10 CS+ and 10 CS-voltammetric sweep t r i a l s recorded from the nucleus accumbens of a single rat. Error bars represent standard error of the mean. *p=.001 76 o s - c s * 77 Figure 18 Electrode placements directed at the l e f t nucleus accumbens (n=8) and r i g h t caudate (n=5) in Experiment 2. 78 + 1.2mm +0.7mm latency to enter the niche f o l l o w i n g CS onset was s i g n i f i c a n t l y greater during the CS- than the CS+ for both groups. Figure 11 shows the r e s u l t s from the corresponding e l e c t r o c h e m i c a l measurements. I t i s evident from the f i g u r e that the CS+ has a greater e f f e c t on DA o x i d a t i o n currents than the CS-. In a d d i t i o n , the chronoamperometric response i n the nucleus accumbens appears to be greater than i n the caudate. Consistent with t h i s , the ANOVA detected a s i g n i f i c a n t e f f e c t of implant l o c a t i o n ( F ( l , l l ) = 6.82, p<.03; accumbens > caudate), and of event ( F ( l , l l ) = 30.78, p<.0004; CS+ > CS-), but no s i g n i f i c a n t l o c a t i o n by event i n t e r a c t i o n . Figure 11 a l s o presents average pre-CS base l i n e d i f f e r e n c e scores. Although these baseline values were not compared s t a t i s t i c a l l y to event scores because of the discrepancy i n sample s i z e , they are included to give an i n d i c a t i o n of b a s e l i n e f l u c t u a t i o n . The average magnitude of change i n o x i d a t i o n current observed for these baseline d i f f e r e n c e scores was s i m i l a r to the average scores i n the CS- c o n d i t i o n , both of which were s u b s t a n t i a l l y smaller than the values obtained for the CS+. Figure 12 d e p i c t s r e p r e s e n t a t i v e chronoamperometric records during CS+ and CS- t r i a l s from four d i f f e r e n t subjects (2 n. accumbens, 2 caudate). These records i n d i c a t e an immediate increase i n DA o x i d a t i o n current at CS+ onset, with no apparent change i n response to the CS-. I n d i v i d u a l data from successive d a i l y t e s t s are 80 presented f o r each of the three " e x t i n c t i o n " s u b j e c t s i n Fi g u r e s 13, 14, and 15. In Figures 13a, 14a, and 15a, dramatic decreases i n the average amount of time spent i n the niche d u r i n g the CS+ alone p e r i o d , can be seen on the f i r s t day of, and c o n t i n u i n g throughout, the e x t i n c t i o n p e r i o d . Although l e s s pronounced, corresponding decreases are found i n the average o x i d a t i o n c u r r e n t measures (Fi g u r e s 13b, 14b, and 15b). F i g u r e 15 i l l u s t r a t e s that when the CS+/food c o n d i t i o n was r e - e s t a b l i s h e d , the amount of time spent i n the niche immediately i n c r e a s e d to l e v e l s t h a t were even hig h e r than those observed dur i n g the 2 days p r i o r to e x t i n c t i o n . However, i n the one remaining v i a b l e channel, there i s an apparent 2-day l a g before any notable increase i s d e t e c t e d f o r the average change i n o x i d a t i o n c u r r e n t . A r e p r e s e n t a t i v e t r i a l i n which l i n e a r sweep voltammetric records were obtained, i s presented i n Figure 16. An i n c r e a s e i n peak, height can be seen f o l l o w i n g CS + onset and c o n t i n u i n g f o r two sweeps. Average peak height r a t i o s were c a l c u l a t e d from a s e r i e s o f 10 CS+ and 10 CS-rec o r d s o f t h i s type (CS+ r a t i o = 1.09, CS- r a t i o =0.95) and are presented i n F i g u r e 17. The r e s u l t s are presented i n F i g u r e 17. The t - t e s t performed on t h i s data detected a s i g n i f i c a n t d i f f e r e n c e between the CS+ and CS- c o n d i t i o n s , with the CS+ peak he i g h t r a t i o s being s i g n i f i c a n t l y greater than the CS- r a t i o s (t(18)=3.95, p=.001). H i s t o l o g i c a l c o n f i r m a t i o n of the caudate and nucleus accumbens e l e c t r o d e placements i s presented i n Figure 18. 81 Discussion The behavioral results from this experiment provide a clear demonstration of the subjects' a b i l i t y to discriminate between CS+ and CS- conditions. The average amount of time spent in the niche during the CS alone period was substantially greater during the CS+ t r i a l s and latency to enter the niche upon CS onset was much greater during the CS- t r i a l s . In fact, most rats rarely entered the niche at a l l during the CS- period. The results of both measures suggest that the rats were responding in anticipation of a meal predicted by the CS+, but not the CS-. The corresponding electrochemical re s u l t s indicated that changes in the neurochemical signal also reflected the discrimination between CS+ and CS-. Increased DA release following CS+ onset was indicated by changes in the chronoamperometric records from both the nucleus accumbens and caudate. Generally, the increased oxidation currents in the caudate p a r a l l e l l e d those in the n. accumbens, although the magnitude of these increases were lower in the caudate. The re s u l t s provide evidence that e x t r a c e l l u l a r levels of DA may be affected s e l e c t i v e l y by sa l i e n t external environmental cues. Although the magnitude of change for a given t r i a l varied both within and between subjects, a c h a r a c t e r i s t i c pattern could be c l e a r l y seen in the chronoamperometric 82 record. In t r i a l s during which an increase could be observed unambiguously, the record t y p i c a l l y showed an immediate and r a p i d ascent to peak l e v e l s f o l l o w i n g CS+ onset. The latency from CS+ onset to peak current approximated 5 min. A more gradual decrease to baseline was observed, u s u a l l y w i t h i n 20 to 30 min a f t e r stimulus onset. The e x t i n c t i o n experiment c o n s i s t e d of p r o v i d i n g ad 1ibiturn access to the l i q u i d d i e t i n a Richter tube, c o i n c i d e n t with stopping the d e l i v e r y of food on a l l CS+ t r i a l s . Three r a t s were tested i n e x t i n c t i o n and on each e x t i n c t i o n day, both the time spent i n the niche and the average change i n the o x i d a t i o n current decreased to l e v e l s below those obtained for the 2 preceding baseline days. As expected, on the f i r s t day of e x t i n c t i o n , the amount of time spent i n the niche during the CS+ decreased over successive t r i a l s . However, even during the f i r s t few t r i a l s on t h i s day, behavioral responses were s u b s t a n t i a l l y reduced r e l a t i v e to the p r e - e x t i n c t i o n period. One f a c t o r that could account for t h i s observation i s s a t i e t y . Since the r a t s were given free access to the l i q u i d d i e t , they may have fed to s a t i a t i o n and as a r e s u l t , may have been less responsive to e x t e r n a l cues s i g n a l l i n g food. Although changes were observed i n both the behavioral and e l e c t r o c h e m i c a l measures, the behavioral changes were much more pronounced. I f DA plays a r o l e i n the processing of s a l i e n t e x t e r n a l cues and the i n i t i a t i o n of g o a l - d i r e c t e d behaviors, i t i s p o s s i b l e that some degree of processing was 83 s t i l l o c c u r r i n g i n response to the CS+ during the e x t i n c t i o n phase. The r a t s that were placed under e x t i n c t i o n had been exposed to the conditioned feeding program for several weeks, a l l o w i n g for the development of a strong a s s o c i a t i o n between the CS+ and food. During e x t i n c t i o n , the neural response to the CS+ may have p e r s i s t e d to some extent, although i t may not have been s u f f i c i e n t to generate a behavioral approach to the niche. The i n d i v i d u a l sweep t r i a l s showed r e l a t i v e l y small changes i n peak height f o l l o w i n g CS+ onset. However, these changes were c o n s i s t e n t with the chronoamperometric records i n that the trends were i n the same d i r e c t i o n . That i s , increases were observed during CS+, but not CS- t r i a l s . One advantage of a sweep t r i a l i s that i t allows the observation of p o t e n t i a l changes i n the s i z e , shape, and p o s i t i o n , of the proposed DA peak. However, there are al s o d e f i n i t e disadvantages to the use of t h i s technique. F i r s t , the temporal r e s o l u t i o n i s l i m i t e d . I t i s p o s s i b l e that any change i n DA rel e a s e c o r r e l a t e d with the CS+ may be masked by subsequent re-uptake and degradation occuring i n the 10 min i n t e r v a l between sweeps. F o r t u n a t e l y , the neural changes examined i n t h i s experiment tended to l a s t between 20 and 30 min, as mentioned above, a l l o w i n g the c o l l e c t i o n of 2 or 3 sweeps during t h i s period. In the sample sweep t r i a l shown i n Figure 15, the two consecutive sweeps f o l l o w i n g CS+ onset showed increased peak heights, whereas, the t h i r d peak was considerably diminished. This temporal 84 p a t t e r n of change i n peak current i s c o n s i s t e n t with the changes observed i n the chronoamperometric records. A second disadvantage of l i n e a r sweep voltammetry at the sweep rate of 10 mV/s employed here, involves the i n f l u e n c e of a s c o r b i c a c i d (AA) on DA o x i d a t i o n . Although the o x i d a t i o n of AA i t s e l f does not c o n t r i b u t e to the DA peak, AA a m p l i f i e s the DA peak by c a t a l y z i n g a r e a c t i o n that allows repeated regeneration of DA from the orthoquinone, and subsequent r e o x i d a t i o n at the electrode surface (Stamford, 1986). This e f f e c t i s pronounced at the slow sweep r a t e , leading to an a m p l i f i c a t i o n of the peak. Therefore, the c o n t r i b u t i o n of AA during slow voltammetric sweeps may mask d e t e c t i o n of changes i n e x t r a c e l l u l a r DA l e v e l s . Echizen and Freed (1986) provide evidence that the r e l a t i v e c oncentration of catecholamine ( i n t h i s case, DA) to AA determines the amount of a m p l i f i c a t i o n taking place. They found that the a m p l i f i c a t i o n f a c t o r decreased as the catechol concentration increased r e l a t i v e to the AA c o n c e n t r a t i o n . As a r e s u l t , they proposed that recorded changes i n catecholamine concentrations were a c t u a l l y much lower than the r e a l changes that were occu r r i n g . During square-wave pulse chronoamperometry, c a t a l y t i c a m p l i f i c a t i o n i s minimized because of the short d u r a t i o n of e l e c t r o c h e m i c a l measurement (Blaha & Jung, i n preparation; Stamford, 1986). 85 GENERAL DISCUSSION The experiments presented i n t h i s t h e s i s provide f u r t h e r evidence for the involvement of dopamine i n feeding. The combined behavioral and e l e c t r o c h e m i c a l analyses revealed d i f f e r e n t i a l changes i n DA a c t i v i t y i n the s t r i a t u m and nucleus accumbens during a p p e t i t i v e and consummatory phases of feeding. This work represents an extension of the conditioned feeding s t u d i e s conducted p r e v i o u s l y by Blackburn et a l . (1987, 1989a, 1989b). While the behavioral paradigm followed i n both t h i s t h e s i s and the Blackburn studies i s s i m i l a r , the approach used to analyze dopaminergic a c t i v i t y i s very d i f f e r e n t . Blackburn's experiments involved pharmacological manipulations and ex. vivo t i s s u e analyses, whereas the present i n v e s t i g a t i o n employed the technique of i n vivo e l e c t r o c h e m i s t r y with c h r o n i c a l l y implanted s t e a r a t e - m o d i f i e d carbon paste e l e c t r o d e s . This technique permitted the recording of ongoing changes i n e x t r a c e l l u l a r DA l e v e l s of f r e e l y moving subjects. To begin t h i s d i s c u s s i o n , a summary of the major r e s u l t s obtained i n the current study i s presented. In Experiment I , increases i n DA a c t i v i t y were observed i n both the nucleus accumbens and the caudate f o l l o w i n g the p r e s e n t a t i o n of a l i q u i d meal. Changes observed for each of the four subjects were very s i m i l a r i n magnitude and temporal p a t t e r n for both b r a i n s t r u c t u r e s . The s i m i l a r i t y 86 of responses at both recording s i t e s might i n d i c a t e a common r o l e for the caudate and accumbens i n feeding. The f a c t that the increases i n the chronoamperometric record lagged behind the i n i t i a t i o n of meal consumption suggests that the observed e f f e c t s may be due to p o s t i n g e s t i v e f a c t o r s . Experiment I I allowed a more d e t a i l e d a n a l y s i s of dopaminergic a c t i v i t y during feeding by f a c t o r i n g the behaviors i n t o separate a n t i c i p a t o r y and consummatory phases. With t h i s approach, i t was discovered that changes i n e l e c t r o c h e m i c a l estimates of e x t r a c e l l u l a r DA l e v e l s corresponded to the pr e s e n t a t i o n of an exte r n a l cue (CS+) which, through previous experience, had become a r e l i a b l e p r e d i c t o r of meal d e l i v e r y . S i g n i f i c a n t increases i n DA o x i d a t i o n currents were observed i n both the nucleus accumbens and the caudate, but the magnitude of change observed i n the nucleus accumbens during s t i m u l i p resentations was s i g n i f i c a n t l y greater than i n the caudate. When the behavioral paradigm was a l t e r e d such that the CS+ was no longer p r e d i c t i v e of a meal (the e x t i n c t i o n phase), the DA response to the e x t e r n a l stimulus diminished a c c o r d i n g l y . The r e s u l t s from Experiment I I were c o n s i s t e n t with hypotheses a s s o c i a t i n g DA a c t i v i t y and "i n c e n t i v e m o t i v a t i o n " as discussed i n the In t r o d u c t i o n . As pr e d i c t e d by i n c e n t i v e m o t i v a t i o n a l t h e o r i e s of DA f u n c t i o n , increases i n DA a c t i v i t y were observed f o l l o w i n g onset of an e x t e r n a l stimulus p r e d i c t i v e of a meal. In a d d i t i o n , increased 87 e n t r i e s i n t o the f e e d i n g n i c h e , assumed to be a measure o f a p p e t i t i v e r e s p o n d i n g , were observed d u r i n g t h i s " i n c e n t i v e s t i m u l u s " (CS+) p e r i o d . A l s o c o n s i s t e n t wi th i n c e n t i v e t h e o r i e s was the f i n d i n g tha t there were s i g n i f i c a n t d i f f e r e n c e s between the DA responses observed f o l l o w i n g CS+ and C S - onse t . A l though both the CS+ and the C S - presumably began as n e u t r a l s t i m u l i , a f t e r repeated p r e s e n t a t i o n o f the CS+ wi th food , the CS+ a lone became a s s o c i a t e d wi th i n c r e a s e s i n o x i d a t i o n c u r r e n t s from the accumbens and caudate tha t were s i g n i f i c a n t l y g r e a t e r than those f o l l o w i n g C S - o n s e t . When the r e s u l t s from Experiments I and II are compared, i t can been seen t h a t the average magnitude o f change i n DA l e v e l s , as e s t i m a t e d from abso lu te changes i n the chronoamperometr ic s i g n a l , was g r e a t e r with the c o n d i t i o n e d f eed ing p r o c e d u r e . There are a v a r i e t y o f f a c t o r s which c o u l d have c o n t r i b u t e d to t h i s d i f f e r e n c e . F i r s t , s u b j e c t s i n Exper iment I may have had e l e v a t e d e x t r a c e l l u l a r l e v e l s o f DA before meal d e l i v e r y due to d a i l y h a n d l i n g , placement i n t o a t e s t box that had p r e v i o u s l y been used for f eed ing e x p e r i m e n t s , or to the a n t i c i p a t i o n o f a h i g h l y p a l a t a b l e meal . T h e r e f o r e , any observed i n c r e a s e f o l l o w i n g meal d e l i v e r y may r e p r e s e n t a b l u n t e d response because o f e l e v a t e d b a s e l i n e l e v e l s . T h i s was not a f a c t o r i n Exper iment II as the s u b j e c t s l i v e d i n the r e c o r d i n g chambers. A second e x p l a n a t i o n for the d i f f e r e n c e i n magnitude may be t h a t the two experiments were i n v e s t i g a t i n g the r o l e of DA i n r e l a t i o n to two d i f f e r e n t aspects of feeding. In Experiment I, subjects were provided with a small l i q u i d meal that was s u f f i c i e n t l y p a l a table to i n i t i a t e immediate i n g e s t i o n even i n non-deprived animals. Small increases i n DA a c t i v i t y , which tended to occur f o l l o w i n g meal consumption, may have been due to post-i n g e s t i o n a l f a c t o r s . In Experiment I I , a d i s t i n c t a n t i c i p a t o r y phase was added to the feeding sessions. The r e l a t i v e l y large increases observed during t h i s phase suggest that DA may play a d i f f e r e n t , and p o s s i b l y more important r o l e during a p p e t i t i v e responding than during subsequent consummatory and p o s t - i n g e s t i v e stages of feeding. The preceding d i s c u s s i o n of magnitudes of change i n DA o x i d a t i o n currents must be approached with caution. What e x a c t l y does the magnitude represent? Although i t may r e f l e c t a c t u a l q u a n t i t a t i v e d i f f e r e n c e s i n e x t r a c e l l u l a r DA l e v e l s , i t may also be a f f e c t e d by f a c t o r s such as the q u a l i t y of the r e c o r d i n g electrode surface, and the amount of scar t i s s u e surrounding an implant. Also, the placement of the electrode would have an i n f l u e n c e . I t i s w e l l e s t a b l i s h e d that the s t r i a t u m , for example, i s not a homogeneous system operating i n synchrony. The p r o x i m i t y of an e l e c t r o d e to an a c t i v e s i t e of t r a n s m i t t e r release would be expected to a f f e c t the magnitude of any recorded changes. In a d d i t i o n , a small change i n one s t r u c t u r e may have greater f u n c t i o n a l s i g n i f i c a n c e than a large change at a 89 d i f f e r e n t n e u r a l s i t e . These f a c t o r s s h o u l d be kept i n mind when g e n e r a l i z a t i o n s a re made c o n c e r n i n g r e l a t i v e magnitudes of change, e s p e c i a l l y when they are based on s m a l l sample s i z e s . Comparison o f the caudate and n u c l e u s accumbens r e c o r d s a c r o s s both e x p e r i m e n t s r e v e a l s f u r t h e r i n t e r e s t i n g r e s u l t s . Whereas the p a t t e r n and magnitude o f i n c r e a s e i n DA r e l e a s e f o l l o w i n g meal d e l i v e r y a re s i m i l a r f o r both s t r u c t u r e s i n Experiment I , t h e r e i s a s i g n i f i c a n t d i f f e r e n c e between the two b r a i n s i t e s i n Experiment I I . T h i s r e s u l t a g a i n s u g g ests t h a t we may be i n v e s t i g a t i n g two d i f f e r e n t a s p e c t s o f f e e d i n g b e h a v i o r s . D e s p i t e the afor e m e n t i o n e d problems w i t h comparisons o f magnitude, i t i s tempting to p r e s e n t a few s p e c u l a t i o n s . Suppose we assume t h a t the i n c r e a s e s o b t a i n e d i n Experiment I are p o s t - i n g e s t i o n a l and Experiment I I a r e , a t l e a s t i n i t i a l l y , a p p e t i t i v e . I t may be t h a t the caudate and n. accumbens share a s i m i l a r r o l e d u r i n g p o s t -i n g e s t i v e s t a g e s o f f e e d i n g , but t h a t the n u c l e u s accumbens i s p r e f e r e n t i a l l y i n v o l v e d i n a p p e t i t i v e r e s p o n d i n g . DA t e r m i n a l s i n the n. accumbens may be i n v o l v e d i n the p r o c e s s i n g o f , and the i n i t i a t i o n o f motor responses t o , e x t e r n a l cues. C o u l d the magnitude o f i n c r e a s e i n DA l e v e l s r e f l e c t the s a l i e n c e o f the cue? T h i s might e x p l a i n why s m a l l i n c r e a s e s ( l . l n A ) were observed i n the n u c l e u s accumbens i n response to the CS-. Whereas some degree o f a n t i c i p a t o r y a r o u s a l i n v o l v i n g DA c o u l d have o c c u r r e d i n response to t h i s cue, i t may not have been s u f f i c i e n t t o 90 m e r i t an approach toward the n i c h e . A s i m i l a r e x p l a n a t i o n would account f o r the p e r s i s t e n t , though d i m i n i s h e d , d o p a m i n e r g i c r e s p o n s e s to the CS+ under e x t i n c t i o n c o n d i t i o n s . The p a t t e r n o f change i n e x t r a c e l l u l a r l e v e l s o f DA o b t a i n e d i n these e x p e r i m e n t s deserves f u r t h e r mention. Whereas Experiment I I s u g g e s t s t h a t DA t e r m i n a l s may be a c t i v e d u r i n g a p p e t i t i v e r e s p o n d i n g , the r e s u l t s a l s o i n d i c a t e t h a t DA l e v e l s remain e l e v a t e d t hroughout and even p a s t the c o m p l e t i o n o f a f e e d i n g s e s s i o n . Another i n t e r p r e t a t i o n o f the p r e s e n t r e s u l t s i s t h a t i n c r e a s e d DA a c t i v i t y d u r i n g a n t i c i p a t o r y s t a g e s i s a s s o c i a t e d w i t h an "aroused" m o t i v a t i o n a l s t a t e induced by i n c e n t i v e s t i m u l i . T h i s s t a t e o f a r o u s a l c o u l d r e s u l t i n an enhanced s e n s i t i v i t y , and i n c r e a s e d l i k e l i h o o d of r e s p o n s e , t o b i o l o g i c a l l y r e l e v a n t cues. Would the enhanced m o t i v a t i o n a l s t a t e make an a n i m a l more r e s p o n s i v e to any b i o l o g i c a l cues, or would i t be s p e c i f i c to the e v e n t a s s o c i a t e d w i t h the i n c e n t i v e s t i m u l i ? R e s u l t s from o t h e r s t u d i e s have demonstrated the i n v o l v e m e n t o f DA i n d i f f e r e n t t y p e s o f a n t i c i p a t o r y r e s p o n d i n g . I n p a r t i c u l a r , Pfaus (1990) has shown t h a t DA a c t i v i t y i s i n c r e a s e d d u r i n g a n t i c i p a t o r y s t a g e s o f s e x u a l b e h a v i o r i n male r a t s . In a d d i t i o n , an i n v e s t i g a t i o n by B l a c k b u r n (1989) showed DA a c t i v i t y t o be a s s o c i a t e d w i t h p r e p a r a t o r y r e s p o n s e s to a cue s i g n a l l i n g impending shock. P o s s i b l y , i n c r e a s e d DA a c t i v i t y r e s u l t s i n a g e n e r a l s t a t e o f a r o u s a l 91 that i s common to s u b j e c t s exposed to i n c e n t i v e cues p r e d i c t i n g d i f f e r e n t consummatory goals. Summary This t h e s i s examined the r o l e of DA i n feeding behaviors i n the male r a t . The approach used has allowed the i n v e s t i g a t i o n of DA involvement i n d i f f e r e n t aspects of feeding. This approach combined the d i v i s i o n of f e e d i n g behaviors i n t o a p p e t i t i v e , consummatory, and p o s t -i n g e s t i o n a l phases, with an e l e c t r o c h e m i c a l r e c o r d i n g technique. The temporal r e s o l u t i o n of the e l e c t r o c h e m i c a l r e c o r d s allowed c o r r e l a t i o n of ongoing b e h a v i o r a l and neural responses. The r e s u l t s of t h i s t h e s i s provide support f o r t h e o r i e s based on i n c e n t i v e m o t i v a t i o n . 92 R e f e r e n c e s B e r r i d g e , K.C., and S c h u l k i n , J . (1989). P a l a t a b i l i t y s h i f t o f a s a l t - a s s o c i a t e d i n c e n t i v e d u r i n g sodium d e p l e t i o n . The Q u a r t e r l y J o u r n a l o f E x p e r i m e n t a l P s y c h o l o g y , 41B(2), 121-138. B e r r i d g e , K.C., V e n i e r , I . L . , and Robinson, T.E. (1989). Taste r e a c t i v i t y a n a l y s i s o f 6-hydroxydopamine-induced a p h a g i a : i m p l i c a t i o n s f o r a r o u s a l and anhedonia hypotheses o f dopamine f u n c t i o n . B e h a v i o r a l n e u r o s c i e n c e , 1 0 3 ( 1 ) , 36-45. B i n d r a , D. (1978). 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An in vivo electrochemical analysis of the role of dopamine in feeding behaviors Holmes, Lorinda Jean 1990
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