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Effects of in-vivo stimulation of the rat ventral subiculum on the expression of presynaptic proteins… Lam, Clayton 2003

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E F F E C T S OF IN-VIVO S T I M U L A T I O N OF T H E R A T V E N T R A L S U B I C U L U M O N T H E E X P R E S S I O N OF P R E S Y N A P T I C P R O T E I N S I N T H E N U C L E U S A C C U M B E N S , T H E P R E F R O N T A L C O R T E X , A N D T H E D O R S A L S T R I A T U M by C L A Y T O N L A M B . S c , The University of British Columbia, 1998 B M L S c . , The University of British Columbia, 2000 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R S OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Graduate Program in Neuroscience) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A June 2003 © Clayton Lam, 2003 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 The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT The present project focuses on changes on the expression of presynaptic proteins at synaptic junctions in the core of the nucleus accumbens ( N A c ) , the shell of the N A c , the prefrontal cortex (PFC), and the dorsal striatum (STR) after repeated electrical stimulation of the subicular projection from the ventral hippocampus of rats. Related research in Dr. Phillips' laboratory has shown that comparable stimulation caused an increased release of dopamine in the N A c and hyperlocomotion. The purpose of this thesis is to investigate possible changes in presynaptic protein concentration in response to repeated stimulation of the ventral subiculum (vSub) at parameters shown previously to potentiate locomotor activity and D A efflux in the N A c . The first series of experiments confirmed that repeated vSub stimulation caused an increase and potentiation of locomotor activity. The second series of experiments investigated the effect of repeated vSub stimulation on the expression of presynaptic proteins in the four brain regions. Five presynaptic proteins were quantified by enzyme-linked immunoabsorbent assay ( E L I S A ) technique and they were synaptophysin, syntaxin, S N A P - 2 5 , complexin I and complexin II. The analysis of variance ( A N O V A ) was used to analyze group and regional differences in the expression of each presynaptic protein. A l l presynaptic proteins showed a significant difference between the stimulation group and the control group. Regional differences in the expression o f most presynaptic proteins including synaptophysin, complexin I, complexin II and complexin II/I ratio were highly significant except for syntaxin. The group x region interaction of complexin II/I ratios was significant. This interaction indicated that regional differences in the relative expression of the two complexin proteins may be caused by vSub stimulation. The last analysis examined the correlation between the expression of presynaptic proteins and locomotor activity. The immunoreactivity of syntaxin in the core of the N A c showed a significantly high correlation with locomotor activity. This finding suggests that presynaptic proteins may be part of the subcellular mechanism in regulating vSub-induced locomotor activity. i i TABLE OF CONTENTS Abstract i i Table of Contents i i i List of Tables v List of Figures v i Acknowledgements v i i C H A P T E R I I N T R O D U C T I O N 1 1.1 The Origin of Adaptive Voluntary Movements 1 1.1.1 The Anatomy of the Thalamocortical Pathway 4 1.1.2 The Anatomy of the Motor Circuit in the Basal Ganglia 7 1.2 The Role of Mesolimbic D A System in Locomotion 8 1.2.1 The Role of Glutamatergic Efferents from the Ventral Hippocampus in Locomotion 11 1.2.2 The Role of D A in Modulating the Electrophysiological State of Medium-sized Spiny Neurons 12 1.2.3 Functional Interaction between Glutamatergic Efferents from the Ventral Hippocampus and the Mesolimbic D A System 13 1.2.4 The Role of Dopamine in Long-term Potentiation 14 1.3 The Role of Dopamine and Presynaptic Proteins in Learning and Memory 15 1.4 The Role of Presynaptic Proteins in Neurotransmission 16 1.4.1 The Role of Presynaptic Proteins in Docking and Priming of Synaptic Vesicles 16 1.4.2 The Role of Presynaptic Proteins in Synaptic Vesicle Fusion and Exocytosis 20 1.5 The Role of Presynaptic Proteins in Plasticity of the Brain 22 1.6 Hypotheses and Objectives of This Study 24 C H A P T E R II M A T E R I A L S A N D M E T H O D S 26 2.1 Animals 26 2.2 Surgical Procedures 26 i i i 2.3 Measurement of Locomotor Activity 26 2.4 Application of Electrical Stimulation 27 2.5 Verification of Electrode Placements by Histology 27 2.6 Homogenization and Detergent Compatible (DC) Assay Technique 28 2.7 Enzyme-Linked Immunoabsorbent Assay (ELISA) Technique 29 2.8 Statistical Methods ...31 C H A P T E R III E F F E C T S OF E L E C T R I C A L S T I M U L A T I O N O F T H E V S U B 33 3.1 Effects of Repeated vSub Stimulation on Locomotor Activi ty 33 3.2 Effects of Repeated vSub Stimulation on Presynaptic Proteins 33 3.3 The Relationship between the Expression of Syntaxin and Synaptophysin 39 3.4 The Relationship between the Expression of Complexin I and Complexin II.. .50 3.5 Correlation between Locomotor Activity and the Expression of Presynaptic Proteins Induced by Electrical Stimulation of the vSub 50 C H A P T E R IV D I S C U S S I O N A N D C O N C L U S I O N 59 4.1 Effects of vSub Stimulation on Locomotor Activity 59 4.2 Effects of vSub Stimulation on the Expression of Presynaptic Proteins 60 4.3 Limitations 66 4.4 Future Directions 68 4.5 Conclusions 70 Bibliography 71 LIST OF T A B L E S Table 1. Summary of statistical analyses with A N O V A comparisons of the amount of presynaptic proteins among different brain regions (NAc core, N A c shell, P F C , and STR) of the stimulation group and the control group 38 Table 2. Quantification and percentage differences of the presynaptic proteins concentration at a fixed half-maximal optical density in different brain regions between the stimulation and the control groups 40 Table 3. Quantification and percentage differences between the average optical density of complexin presynaptic proteins in different regions of the brain of the stimulation and the control groups 41 Table 4. Spearman rank correlation of presynaptic protein concentration in different brain regions vs. locomotor activity 58 v L I S T OF F I G U R E S Fig. 1. The neurocircuitry of the meso-cortico-limbic system 5 Fig. 2. The circuitry of the basal ganglia 9 Fig. 3. The structure of S N A R E core complex 18 Fig. 4. Indirect log-log analysis 30 Fig. 5. Change in locomotor activity following single trains of electrical stimulation of vSub given daily for four days 34 Fig. 6. Histology of stimulating electrode placements 36 Fig. 7. Immunoreactivity (IR) of presynaptic proteins in the core of the nucleus accumbens in the stimulation group relative to control values (100%; no difference) 42 Fig. 8. Immunoreactivity (IR) of presynaptic proteins in the shell of the nucleus accumbens in the stimulation group relative to control values (100%; no difference) 44 F ig . 9. Immunoreactivity (IR) of presynaptic proteins in the prefrontal cortex in the stimulation group relative to control values (100%; no difference) 46 F ig . 10. Immunoreactivity (IR) o f presynaptic proteins in the dorsal striatum in the stimulation group relative to control values (100%; no difference) 48 Fig . 11. Comparison among syntaxin to synaptophysin ratios in different brain regions in the ipsilateral hemisphere of the stimulation and the control groups 51 Fig . 12. Comparison among syntaxin to synaptophysin ratios in different brain regions in the contralateral hemisphere of the stimulation and the control groups 53 Fig . 13. Comparison among complexin II/I (CxII/I) ratios in different brain regions in the contralateral hemisphere of the stimulation and the control groups 55 vi A C K N O W L E D G E M E N T S The author would like to acknowledge his awesome supervisor, Dr. Anthony G . Phillips, whose vision and enthusiasm have inspired the author. The author is grateful to Dr. Wil l iam G. Honer whose expertise and insight are important to this thesis project. The author would like to thank Dr. John R. O ' K u s k y and Dr. Joanne Weinberg for providing valuable constructive suggestions to this project. Furthermore, the author wants to express his deep appreciation to Dr . Pornnarin Taepavarapruk, M r . Fredrick LePiane and Miss . Christina Cheng for their technical assistance in animal cares. Dr. L i l y H u is gratefully acknowledged for teaching the author the E L I S A technique, which is crucial to the present project. Last but not least, the author would like to thank M s . Isabella Ghement and M r . Weiliang Qiu in providing their expert opinions on statistical analyses of this project. v i i C H A P T E R I I N T R O D U C T I O N 1.1 The Origin of Adaptive Voluntary Movements A n i m a l species evolve by adapting to changes according to the law of natural selection. In 1858, Darwin (1890) suggested 'that the mental qualities of animals of the same kind, born in a state of nature, vary much, could be shown by many facts occasional and strange habits in wi ld animals, which, i f advantageous to the species, might have given rise, through natural selection, to new instincts.' A s animal species evolved, the number of initiator of movements increased in addition to the complexity of motor skills for adaptation and survival (Mogenson et al., 1980). Beside the internal drive for homeostasis, motivation, a state reflecting incentive stimuli from the external environment, plays a particularly important role in triggering movements. This has led neuroscientists to search for a mechanism for producing adaptive voluntary movements governed by "motives". In 1890, Wi l l i am James postulated that a motive or a w i l l was 'a supply o f ideas of the various movements that are possible, left in the memory by experiences of their involuntary performance, was thus the first prerequisite of the voluntary l ife ' (Mi l le r , 1983). This postulation may be reinterpreted in light of recent neuroanatomical and cognitive findings and may reflect physical and functional connections between the hippocampus, an important memory center, and the extrapyramidal motor system (Groenewegen et a l , 1996; Sesack and Pickel , 1990; Taepavarapruk et al., 2000). These specific data w i l l be discussed in later sections. The mesolimbic dopamine (DA) system is known to play an active role in modulating locomotor activity and this may be related to the involvement of this system in motivation 1 and reward (Mogenson et al., 1980). Reward is mediated by the five senses, namely sight, sound, taste, smell, and touch, and also by direct electrical or chemical stimulation of the brain (Wise, 2002). The rate responding for intracranial self-stimulation (ICS) from electrodes in the ventral tegmental area ( V T A ) was decreased by lesions of the mesotelencephalic D A projections with 6-hydroxydopamine ( 6 - O H D A ; Phillips and Fibiger, 1978). Furthermore, the mesolimbic D A system has been implicated in psychiatric disorders l ike schizophrenia (Car l sson , 2002). A d m i n i s t r a t i o n o f radiolabeled 3,4-hydroxyphenylalanine ( D O P A ) or f luoro-DOPA, a precursor of dopamine revealed that the labeled dopamine or fluorodopamine was increased in drug-naive patients with schizophrenia measured by positron emission tomography (PET; Hietala et al., 1994). Tonic D A release is a continuous release of D A to establish a steady-state baseline level and has been proposed to be involved in the etiology of schizophrenia (Grace, 1991). Tonic D A release in the nucleus accumbens (NAc) is probably decreased by a decrement in prefrontal cortical activity leading to an increase in sensitivity and responsiveness to phasic D A release. The mesolimbic D A system has also been implicated in drug addiction (Koob, 1999). Intravenous self administration of cocaine, a D A transporter blocker, caused an increase in D A efflux in both the core and the shell of the N A c of rats (Ito et al., 2000). Cocaine-associated conditioned stimulus also increased D A efflux in the core of the N A c . Kalivas et al. (1999) proposed that motivation was linked to the motor system by a 'motive circuit ' , which in turn had three functional components, namely the afferent component, the connecting component, and the efferent component. The afferent component involves the generation of stimuli within the limbic subcircuit. The connecting component refers to the thalamocortical pathway facilitating the intercommunication between the 2 afferent component and the efferent component. The efferent component involves the production of adaptive behaviors through the motor subcircuit in response to afferent stimuli. One of the major structures in the limbic subcircuit is the N A c . The N A c is part of the ventral striatum consisting of ventral parts of the caudate-putamen, and extensive parts of the olfactory tubercle. The N A c and the ventral striatum have no distinct anatomical boundaries with the dorsal aspects of the striatal complex (De Olmos and Heimer, 1999). They are composed mainly (90-95%) of G A B A e r g i c (gamma amino butyric acid) medium-sized spiny interneurons (Chang et al., 1982; Chang and K i t a i , 1985). The N A c can be separated into a rostral pole, which occupies the rostral quarter of the N A c , and a caudal pole, located in the caudal three fourths of the N A c (Heimer et al., 1995). The caudal part of the N A c can in turn be divided into two sub-regions, namely N A c core and N A c shell. The boundary between the N A c core and the N A c shell is not very distinct, but generally the N A c shell surrounds the N A c core medially, ventrally and laterally. The calbindin D28K staining is strong in the N A c core but weak in the N A c shell (Groenewegen et al., 1996). Injection of an anterograde tracer biotinylated dextranamine ( B D A ) to the ventral subiculum (vSub) region of the ventral hippocampus revealed that neurons in the vSub send projections mainly to the caudal shell of the N A c (Groenewegen et al. , 1996). Semi-quantitative analysis showed that more than 85% of hippocampal projections, as identified by anterograde transport of wheat germ agglutinin-conjugated horseradish peroxidase ( W G A -HRP) and anterograde degeneration, synapse with medium spiny neurons in the medial N A c (Sesack and Pickel , 1990). The N A c also receives dopaminergic inputs from the ventral • tegmental area ( V T A ) (Fallon and Moore, 1978). The dynorphin-containing G A B A e r g i c neurons in the N A c send projections back to dopaminergic neurons in the V T A to form a 3 negative feedback loop (Fallon et al., 1985). A t least 25% of the hippocampal terminals synapse with spiny dendrites in the N A c that are connected to tyrosine hydroxylase-labeled terminals (Sesack and Pickel , 1990). These tyrosine hydroxylase-labeled terminals are most likely dopaminergic projections from the V T A . Another important structure in the limbic subcircuit is the ventral pallidum (VP), part of the anterior or subcommissural substantia innominata, which is continuous with the dorsal part of the pallidal complex (De Olmos and Heimer, 1999). The V P can be further divided into the medial V P (VPm), which has an important role in the limbic subcircuit, as distinct from the lateral V P (VP1) which plays a role in the motor circuit. Most (95%) of the medium spiny neurons in the shell of the N A c send G A B A e r g i c projections to the G A B A e r g i c interneurons in the V P m (Groenewegen et al., 1991; Heimer et al. , 1991). This ventral striopallidal G A B A pathway is tonically modulated by mesolimbic D A neurons. Infusion of the D l antagonist SCH23390 and the D2 antagonist raclopride ( l O p M , 150min.) into the N A c increased G A B A efflux in the ipsilateral V P (O'Connor, 2001). The G A B A e r g i c interneurons in the V P m , in turn, send projections to the mediodorsal thalamus ( M D ) (O'Donnell etal., 1997; Fig . 1). 1.1.1 The Anatomy of the Thalamocortical Pathway Glutamatergic neurons in the M D communicate with the prefrontal cortex (PFC) via the thalamocortical pathway (Krettek and Price, 1977). Injections of W G A - H R P and Lucifer Yel low to the M D showed that this projection terminated in layer I V and deep layer III of the cortex (McFarland and Haber, 2002). These terminal sites were analogous to Brodmann's areas 9 and 46 and extended into the orbitofrontal areas 12 and 13. Some fibers were also 4 Fig . 1. The neurocircuitry of the mesocorticolimbic system. Double headed arrows represent reciprocal connections and single headed arrows represent unidirectional nonreciprocal connections. G A B A , G A B A e r g i c neurons; G l u , glutamatergic neurons; M D thalamus, mediodorsal thalamus; N A c core, core of the nucleus accumbens; N A c shell, shell of the nucleus accumbens; P F C , prefrontal cortex; S N , substantia nigra; VP1, lateral ventral pallidam; V P m , medial ventral pallidam; vSub, ventral subiculum; V T A , ventral tegmental area. Adapted from Kalivas et al. (1999). 5 found in medial regions of prefrontal cortex, areas 32 and 25. The flow of information in the thalamocortical pathway might be inferred from pharmacological experiments in which inhibition of the M D by the G A B A B agonist baclofen or the mu agonist D A M G O changed the efflux of D A in the P F C and the core of the N A c (Klitenick and Kalivas, 1994). This suggests that M D is "upstream" of P F C and the core of the N A c in the thalamocortical pathway. 1.1.2 The Anatomy of the Motor Circuit in the Basal Ganglia The motor circuit in the basal ganglia has two main pathways, namely the direct striatonigral and the indirect trans-subthalamic pathways. Both the direct and the indirect pathways originate in the postcommissural region of the putamen in the striatum which receives somatosensory, motor and premotor cortical inputs (Smith et al. , 1998). These corticostriatal projections are excitatory and are most likely glutamatergic (Spencer, 1976). The subthalamic nucleus receives inputs from the motor, premotor and prefrontal cortices (Monakow et al., 1978). The direct and the indirect pathways have some convergent inputs from different cortical areas, but they also have divergent cortical inputs. Stimulation of the indirect trans-subthalamic pathway via the motor cortex excited neurons in the substantia nigra pars reticulata (SNr) (Kolomiets et al., 2003). Approximately half of these excited cells were inhibited by stimulation of the direct striatonigral pathway via the auditory cortex. Stimulation of the indirect trans-subthalamic pathway via the motor cortex excited neurons in the SNr, but only a few of these excited cells were inhibited by stimulation of the P F C . Cor t ica l glutamatergic projections synapse wi th and modulate dopaminergic projections from the substantia nigra pars compacta (SNc) to the striatum. In the direct 7 pathway, G A B A e r g i c neurons containing D A Dl - type receptors are colocalized with substance P and dynorphin (Kiernan, 1998; Fig. 2). These neurons send projections from the striatum to the G A B A e r g i c neurons in the internal segment of the globus pallidus (GPi), also known as the entopeduncular nucleus of rats, and in turn project to the thalamus (Groenewegen et al, 1996). Glutaminergic neurons in the thalamus send projections to the cerebral cortex. Stimulation of striatal neurons in the direct pathway disinhibits G A B A e r g i c projections from the G P i to the thalamus and in turn increases stimulation of the cortex resulting in the initiation of movements. In the indirect pathway, G A B A e r g i c striatal neurons expressing D A D2-receptors also contain enkephalin (Kiernan, 1998). They send projections from the striatum to the G A B A e r g i c neurons in the external segment of the globus pallidus (GPe). G A B A e r g i c efferents in the GPe innervate the subthalamic nucleus (Bevan et al., 1997) and also the G P i (Bolam and Smith, 1992). Glutamatergic neurons in the subthalamic nucleus send projections to the G P i where the direct and the indirect pathways merge. Neurons in the subthalamic nucleus project to both the GPe and the G P i (Parent and Hazrati, 1995). Stimulation of the indirect pathway in the striatum disinhibits the G A B A e r g i c projects from the GPe to the subthalamic nucleus which activates inhibitory projections from the G P i to the thalamus. Stimulation of the indirect pathway decreases stimulation of the cortex and therefore can inhibit movements initiated by stimulating the direct pathway. 1.2 The Role of Mesolimbic D A System in Locomotion Ikemoto (2002) observed hyperactivity after injecting D A or amphetamine unilaterally into either the core of the N A c or the shell of the N A c . Similarly, microinjection 8 Fig . 2. The circuitry of the basal ganglia. Cortical glutamatergic projections synapse with and modulate dopaminergic projections from the substantia nigra pars compacta (SNc) to the striatum. In the direct pathway, G A B A e r g i c neurons send projections from the striatum to the G A B A e r g i c neurons in the internal segment of the globus pallidus and substantia nigra pars reticulata (GPi / SNr), and in turn project to the thalamus (Thai). Glutaminergic neurons in the thalamus send projections to the cerebral cortex. In the indirect pathway, G A B A e r g i c neurons send projections from the striatum to the G A B A e r g i c neurons in the external segment o f the globus pallidus (GPe). G A B A e r g i c efferents in the GPe innervate the subthalamic nucleus (STN) and also the G P i . Glutamatergic neurons in the S T N send projections to the G P i where the direct and the indirect pathways merge. Double lines represent excitatory projections whereas solid lines represent inhibitory projections. Adapted from Smith et al. (1998). 9 Cerebral Cortex Indirect Pathway Direct Pathway G P i / SNr 1 0 of cocaine (300 nmol) into the medial shell of the N A c produced an immediate increase in locomotor activity and rearing within 5 minutes. However, this enhancement of locomotor activity was delayed when cocaine was administrated in the core of the N A c . This delay may be due to diffusion of cocaine from the core to the medial shell of the N A c . The difference between the core and the shell of the N A c in affecting locomotor activity may reflect the difference between the two regions in response to different chemical compounds. Lesions of the N A c shell by quinolinic acid produced hypoactivity whereas core lesions produced hyperactivity (Parkinson et al, 1999). However, in the presence of either lesion, infusions of D-amphetamine stimulated the locomotor activity suggesting a primary role for D A in the initiation of motor behavior. 1.2.1 The Role of Glutamatergic Efferents from the Ventral Hippocampus in Locomotion Locomotor activity was increased by microinjecting N-methyl-D-aspartate ( N M D A ) , an agonist of glutaminergic N M D A receptors, or _-amino-3-hydroxy-5-methylisoxazole-4-propionate ( A M P A ) , an agonist of glutaminergic n o n - N M D A receptors, into the N A c (Wu et al . , 1993). Locomotor activity of rats with bilateral excitotoxic vSub lesions was significantly decreased compared to the sham controls (Burns et al. , 1996). Locomotor activity was increased by microinjecting N M D A to the vSub, a major output region of the hippocampus (Wu and Brudzynski, 1995). Unilateral stimulation of the shell of the N A c in rats by A M P A , but not N M D A , produced contraversive pivoting which was a tight head-to-tail chasing movement in the contralateral direction to stimulation (Ikeda et al., 2003). The AMPA- induced pivoting movement was attenuated in a dose-dependent fashion by injection of N B Q X being an A M P A receptor antagonist, MK-801 being a N M D A receptor antagonist, 11 or cis-(Z)-flupentixol being a D A D1/D2 receptor antagonist into the shell of the N A c . These findings suggest that A M P A and D A D i / D 2 receptors in the shell of the N A c are involved in generating this pivoting movement and N M D A receptors may play a modulatory role. 1.2.2 The Role of D A in Modulating the Electrophysiological State of Medium-sized Spiny Neurons It is hypothesized that D A acts as a gate for glutamatergic inputs from the cortex and the hippocampus to the N A c by modulating the electrophysiological state of medium-sized spiny neurons (Horvitz, 2002; N i c o l a et al., 2000). The electrophysiological state of medium-sized spiny neurons determines the success of generating action potentials. This explains how D A exerts dual effects, namely excitatory and inhibitory effects, on other neurons. A t rest, these neurons in the N A c are in a "down-state" with a membrane potential of about - 8 5 m V maintained by the inward rectifying channels. Activation of afferents from the cortex can bring these hyperpolarized neurons from the "down-state" to an "up-state" with a membrane potential of approximately - 6 0 m V near the threshold of depolarization. The activation of D I receptors in medium spiny neurons stimulates voltage-gated L -type calcium channels which have high threshold for activation (Zigmond et al., 1999). This high threshold prevents weak excitatory signals from depolarizing the N A c neurons in a "down-state". When activated, L-type calcium channels promote long-lasting ionic currents. Thus, L-type calcium channels facilitate depolarization of "up-state" neurons and evoke more robust spikes (Nicola et al., 2000). These findings may explain the excitatory and inhibitory roles of D A in the N A c . The result of D A modulation is that strong glutamatergic stimuli are passed to medium spiny neurons whereas weak signals are blocked. 12 1.2.3 Functional Interaction between Glutamatergic Efferents from the Ventral Hippocampus and the Mesolimbic D A System Excitatory glutaminergic projections to the N A c from the medial prefrontal cortex (mPFC) (Sesack et al., 1989), ventral hippocampus and amygdala (DeFrance et al., 1980; Kelley et al., 1982) interact with dopaminergic inputs from the ventral tegmental area ( V T A ) (Fallon and Moore, 1978). Approximately 17% of hippocampal projections form direct axo-axonal contacts with tyrosine hydroxylase-labeled terminals (Sesack and Pickel, 1990). This finding suggests that glutamatergic projections from the hippocampus.may modulate dopaminergic terminals in the N A c presynaptically. The application of quinpirole, a D A D2 agonist, into the nAc but not S K F 38393, a D l agonist, diminished the hyperkinetic effect of glutamate agonists in a dose-dependent fashion by D A autoreceptor mediated presynaptic inhibit ion (Wu et al . , 1993). Lesions of dopaminergic neurons in the V T A by 6-hydroxydopamine attenuated the hyperkinetic effect triggered by applying N M D A to the vSub (Wu et al., 1993). This latter finding suggests that the vSub activates the N A c via the V T A . Legault and Wise (1999) observed an ipsilateral increase in the amount of extracellular D A in the V T A and the N A c following the N M D A - i n d u c e d stimulation of the unilateral vSub. Effects of vSub stimulation on D A efflux in the N A c and the V T A were reduced by bilateral infusion of 4% lidocaine to either the P F C or the V T A (Taepavarapruk and Phillips, 2000). However, after infusion of lidocaine into the P F C , extracellular D A levels remained significantly higher in the stimulation group than the control group. These results suggest that the V T A plays an important role in modulating D A release in the N A c triggered by vSub stimulation. 13 Intravenous injection of D i receptor antagonist SCH23390 blocked the potentiation of fimbria-induced spiking activity in the N A c (Floresco et al . , 2001). Similar ly, the potentiation of hippocampal-induced spiking activity in the N A c was inhibited by injecting N M D A receptor antagonist 3-2-carboxypiperazin-4-yl-propyl-l-phosphonic acid (CPP) either before or after tetanic electrical stimulation of the fimbria. Electrical stimulation [200 pulses, 300 u A , delivered at 20 H z for 10s] of the vSub caused a significant increase in D A efflux in the rat N A c in the first 10-minute sampling period (Taepavarapruk and Phillips, 2000). Using similar stimulation parameters, Taepavarapruk et al. (2000) found both a significant 30% and prolonged (>30 min.) increase in the D A level in the N A c compared to pre-stimulation D A levels, along with a significant increase in locomotor activity. The increase in extracellular D A level in the N A c normally induced by vSub stimulation was blocked by reverse dialysis of DL-2-amino-5-phosphopentanoic acid ( A P - V ) , a N M D A antagonist, or 6,7-dinitroquinoxaline-2,3-dione ( D N Q X ) , an AMPA/ka ina te antagonist, into the N A c (Taepavarapruk et al., 2000). These results confirmed the importance of ionotropic glutamate receptors in vSub stimulation effects on D A efflux in the N A c . Intraperitoneal injections of the D I antagonist SCH23390, but not D2 antagonist sulpiride, caused a dose-related inhibition of vSub stimulation-evoked locomotion. These findings indicated that exploratory locomotion might be caused by glutamatergic facilitation of mesoaccumbal D A efflux acting via D I receptors in the N A c . 1.2.4 The Role of Dopamine in Long-term Potentiation Gurden et al. (2000) found a significant increase in the hippocampal-PFC long-term potentiation (LTP) after infusing the D I agonistSKF81297 into the P F C . In contrast, 14 infusion of the D l antagonist SCH23390 into the P F C caused a dose-related reduction in the hippocampal-PFC L T P . Furthermore, tetantic stimulation (250 H z , 200 ms) of the C A 1 / subicular region of the hippocampus caused a significant increase in the N M D A receptor-dependent L T P in the P F C . Taepavarapruk et al . (2000) reported both short-term potentiation and long-term potentiation of basal D A levels and vSub stimulation-evoked D A efflux in the N A c . Stimulation of the vSub by 1-second stimulation train (20Hz, 300 p A ) increased both basal D A levels and stimulation-evoked D A efflux into the N A c within 1 hour after a 10-second vSub stimulation. Repeated vSub stimulation (20Hz, 10s) once per day for 4 days increased and potentiated both basal D A levels and stimulation-evoked D A efflux (Taepavarapruk and Phillips; Society of Neuroscience's abstract, 2000). Locomotor activity was also increased and potentiated. 1.3 The Role of Dopamine and Presynaptic Proteins in Learning and Memory Dopamine has an important role in memory function. Bilateral microinjections of D l antagonist SCH-23390, but not D2 anatagonist sulpiride, into the prelimbic region of the P F C produced a dose-dependent impairment on a delayed-foraging task (Seamans et al., 1998). This delayed task was disrupted by a combination of unilateral injection of D l antagonist SCH-23390 into the P F C and contralateral microinjection of lidocaine into the hippocampus. Presynaptic proteins within the pathway have also been implicated in learning and memory. Davis et al. (1996) showed that the m R N A of syntaxin I B was increased in the dentate gyrus, the C A 3 region and the C A 1 region of the rat hippocampus after learning a reference memory task on a radial arm maze. The m R N A of syntaxin I B was also increased in the P F C and the shell of the n A c during working memory phase of the same task. These results 15 suggest that syntaxin plays a role in learning and memory. Changes in the level of syntaxin IB m R N A is region specific in response to specific type of memory task. The level of syntaxin I B m R N A in parietal cortex, motor cortex and the core of the n A c has no significant difference among the two memory task groups and the motor control group. 1.4 The Role of Presynaptic Proteins in Neurotransmission It is wel l established that neurons communicate by a process in which action potentials in axons trigger the release of diffusible chemical neurotransmitters into the synaptic cleft. Neurotransmitters are released into the synaptic cleft in a controlled manner when synaptic vesicles fuse with the active zone of the plasma membrane of axonal terminals also known as the presynaptic membrane. According to the soluble N-efhylmaleimide-sensitive factor attachment receptor ( S N A R E ) hypothesis, presynaptic proteins play an important role in neurotransmission (Rothman, 1994). 1.4.1 The Role of Presynaptic Proteins in Docking and Priming of Synaptic Vesicles Evolutionarily conserved S N A R E proteins belong to a family of presynaptic proteins involved in membrane fusion and they are found in both axonal terminals and intracellular membranes of eukaryotic cells (Antonin et al., 2002). The two groups of S N A R E s involved in neuronal exocytosis are target S N A R E s ( t -SNAREs) and vesicular S N A R E s (v-SNAREs) . The two t - S N A R E s , namely syntaxin 1 and SNAP-25 located on the presynaptic membrane, bind to each other to form a heterodimer. The main v - S N A R E is synaptobrevin 2 (vesicle-associated membrane protein; V A M P ) which is located on the synaptic vesicular membrane. 16 Syntaxin 1 is believed to be negatively regulated by munc-18, also known as nSecl or rbSec l , which is regulated by protein kinase C dependent phosphorylation. V A M P 2 is negatively regulated by synaptophysin, a synaptic vesicular protein with four transmembrane domains co-immunoprecipitated with V A M P 2. Syntaxin 1 and V A M P 2 each contain one helix whereas S N A P - 2 5 contains two helices. These three S N A R E proteins bind together to form a core complex by coiled-coil interaction of the four parallel a-helices (Mochida, 2000; F i g . 3). The t - S N A R E s contributing three glutamine residues in the hydrophilic layer (0-layer) within the four-helical bundle are also known as the Q - S N A R E s , whereas the v - S N A R E having one arginine residue in the bundle is also called the R - S N A R E (Antonin et al. , 2002). A t this stage, the heterotrimeric S N A R E core complex, which is resistant to sodium dodecyl sulphate (SDS) denaturation, is known as the 7S intermediate complex in the priming step of the synaptic vesicle cycle (Bennett and Scheller, 1994). In the synaptic vesicle cycle, the priming step, a biochemical docking involving S N A R E s , follows the docking step which does not require S N A R E s . The docking step also known as morphological docking is initiated when neurotransmitter filled synaptic vesicles are located at the closest distance of the active zone, as revealed by electron microscopy (Lin and Scheller, 2000). Docked vesicles in the docking step can still tether and have not fully committed to exocytosis. Docking or priming of synaptic vesicles is not affected by clostridal neurotoxins which cleaves SNAP-25 or V A M P (Mochida, 2000). However, docking is required for fusion of vesicles with the presynaptic membrane in exocytosis. Drosophila mutants with gene deletion of syntaxin, or V A M P exhibited a complete loss of neurotransmission (Schulze et a l , 1995; Deitcher et a l , 1998). 17 Fig . 3. The structure of S N A R E core complex. The S N A R E core complex consists of three S N A R E presynaptic proteins, namely syntaxin, synaptosome-associated protein of 25,000 Daltons (SNAP-25) and vesicle-associated membrane protein ( V A M P ) . Syntaxin and V A M P are integral proteins and each of them contains one helix. SNAP-25 is a peripheral protein linked to the plasma membrane by palmitylation and it contains two helices. These three S N A R E proteins bind together to form a core complex by interaction of the four parallel oc-helices. Complexin helix binds to the groove between syntaxin and V A M P in an antiparallel orientation to the surface of a parallel S N A R E complex bundle. N represents the amino terminus and C represents the carboxyl terminus. Adapted from Schiavo and Stenbeck(1998). 18 Lumen of synaptic vesicle Presynaptic vesicular membrane Cytosol of Synaptic terminal Plasma membrane of synaptic terminal Synaptic cleft The structure of S N A R E core complex V A M P N C N N Complexin ' N (Z_ SNAP-25 Syntaxin Hydrophobic Region Each core complex increases the binding affinity o f three a-soluble N -ethylmaleimide-sensitive factor attachment protein (oc-SNAP) molecules, which bind to syntaxin (Woodman, 1997). This complex facilitates binding of one molecule of trimeric N -ethylmaleimide-sensitive factor (NSF), which is a homooligomer of three hydrophilic 76kDa proteins. A t this stage a new complex is formed referred to as the 20S complex. Ca channels bind to syntaxin and maximize local C a 2 + concentration at the active zone (Schiavo and Stenbeck, 1998). The activity of P/Q-type C a 2 + channels is negatively regulated by SNAP-25 until synaptic vesicles are docked onto the presynaptic membrane (Zhong et al., 1999). Syntaxin in the S N A R E core complex and synaptotagmin in the synaptic vesicle reactivate P/Q-type C a 2 + channels when synaptic vesicles are docked. Synaptotagmin, a 65 kDa glycoprotein on synaptic vesicular membrane, can inhibit spontaneous fusion between the synaptic vesicle and the plasma membrane until the influx of calcium ions occurs (Woodman, 1997). Littleton et al. (2001) demonstrated that synaptotagmin is a C a 2 + sensor for fast exocytosis. The AD3 mutation disrupts Ca 2 + -induced conformational changes in the C2B domain of synaptotagmin and in turn inhibits synaptic vesicle exocytosis by preventing S N A R E complexes to assemble in vivo. 1.4.2 The Role of Presynaptic Proteins in Synaptic Vesicle Fusion and Exocytosis Vesicle fusion requires A T P . N S F hydrolyzes A T P and in turn dissociates the core complex. The synaptic vesicle membrane and the presynaptic plasma membrane form a hemifusion by fusing only one l ipid layer. Complete fusion is believed to occur immediately and irreversibly after hemifusion. The exact process involved in the complete fusion of the two lipid bilayers is still unknown. However, recent studies suggest that complexins, also 20 known as synaphins, may play a role in modulating this fusion step in neurotransmitter release. Tokumaru et al. (2001) postulated that complexins promote S N A R E complex oligomerization by stabilizing S N A R E complexes and this is an essential step in fast synaptic vesicle exocytosis. Furthermore, complexins bind to syntaxin 1 that in turn is bound in a S N A R E complex there by freeing one coiled-coil domain of SNAP-25 for interacting with other S N A R E complexes. The S N A R E complexes oligomerize when the two free termini of S N A P - 2 5 in one complex interact with those of S N A P - 2 5 in other complexes. The oligomerized t r a n s - S N A R E complexes become a ring o f S N A R E proteins which may synchronize the process of hemifusion to produce a complete membrane fusion. However, Chen et al. (1999) argued that P C 12 cells did not require S N A P - 2 5 S N A R E motif for mediating oligomerization of core complexes. Pabst et al. (2002) also demonstrated by in vitro fluorescence resonance energy transfer (FRET) measurements that the complexin helix bound in a 1:1 stoichiometry and in an antiparallel orientation to the groove between syntaxin and V A M P on the surface of a parallel S N A R E complex bundle. This binding of complexin to the core complex may stabilize the repulsive forces exerted by the two fusing membranes. Thus, complexins can bind to S N A R E complexes in a C a 2 + -independent manner, however, fast, C a 2 + - dependent exocytosis is decreased by knockout of the complexin gene (Reim et al., 2001). Once the synaptic vesicular membrane fuses completely with the presynaptic plasma membrane, exocytosis occurs instantaneously allowing efflux of neurotransmitters via a fusion pore into the synaptic cleft, by simple diffusion. A t any given time, only one vesicle undergoes exocytosis triggered by calcium influx because of the low release probability 21 (Sudhof, 2000). On average, five to ten calc ium signals are needed to trigger neurotransmitter release during an action potential. 1.5 The Role of Presynaptic Proteins in Plasticity of the Brain Syntaxin is a presynaptic plasma membrane protein with a single transmembrane region exposing the N-terminus to the cytosol (Schiavo and Stenbeck, 1998). A s mentioned above, syntaxin binds to SNAP-25 and V A M P via coiled-coil interactions during priming, which is a precursor step of synaptic vesicle exocytosis (Mochida, 2000). Davis et al. (1996) have shown that the m R N A of syntaxin I B is increased in rat hippocampus during a radial arm maze learning using in situ hybridization technique. This suggests that syntaxin may play a role in learning and memory. The t - S N A R E synaptosome-associated protein o f 25,000 Daltons (SNAP-25) presynaptic protein links to the plasma membrane via the palmitoylation of cysteine residues in the middle of the polypeptide chain which creates a U-shape exposing the two termini in the cytosol (Schiavo and Stenbeck, 1998). Hess et al. (1996) demonstrated that coloboma mice with hemizygous gene deletion at chromosome 2 including SNAP-25 gene exhibited hyperactivity. Insertion of a transgene encoding SNAP-25 in the mutant mice corrected the hyperactivity, but not the head-bobbing or ophthalmic responses (Barr et al., 2000). This suggests that S N A P - 2 5 may be responsible for the hyperactivity but not other symptoms related to coloboma. Unfortunately, the mechanism by which decreased SNAP-25 may cause hyperactivity has not been deduced. Synaptophysin, which is located on the synaptic vesicular membrane, is a regulatory protein that binds to synaptobrevins, also known as vesicle-associated membrane protein 22 ( V A M P ) (Sudhof, 1995). Changes on the amount of synaptophysin provide a general measure of changes in the number of synapses. Therefore, Bozdagi et al. (2000) using synaptophysin as an indicator found that the number of synaptic puncta was increased in C A 1 region of the hippocampus after the induction of hippocampal late-phase long term potentiation (LTP) . However, these changes may indicate changes in the protein level within each synaptic vesicle. Short-term synaptic plasticity was impaired in synaptophysin I and synaptogyrin I double knockout mice. Both paired-pulse facilitation (PPF) and posttetanic potentiation (PTP) were decreased in the double knockout mice compared to the control group and they were assessed by measuring EPSP from the C A 1 region of the hippocampus (Janz et al., 1999). Long-term potentiation in the hippocampus was also decreased in the double knockout mice compared to that of the control group. These results demonstrate that presynaptic proteins involve in synaptic plasticity. Complexins are cytosolic regulatory presynaptic proteins that bind to the groove between synaptobrevin and syntaxin in an antiparallel fashion (Yamada et al., 1999; Chen et al., 2002). Complexin I has been identified in inhibitory axonal terminals at axosomatic synapses and complexin II has been localized in excitatory axonal terminals at axodendritic synapses (Takahashi et al . , 1995). The exact function of complexins has not been fully characterized. Complexins have been hypothesized to modulate neurotransmitter release negatively by competing with a-soluble N-ethylmaleimide-sensitive factor attachment protein ( a - S N A P ) (Ono et al., 1998). However, recent studies suggest that complexins play a modulatory role in facilitating neurotransmitter release by stablizing S N A R E complexes as mentioned before (Chen et al., 2002, Pabst et al., 2002). Complexin II is not essential for all neurotransmitter release because homozygous mutant mice lacking the complexin II gene 23 still survive and are fertile, however this protein appears to be important in establishing hippocampal L T P (Takahashi et a l , 1999). In both C A 1 and C A 2 regions, L T P induced by tetantic stimulation was decreased in hippocampal slices of complexin II knockout mice compared to the wild-type control mice. This further suggests complexin II facilitates neurotransmission in excitatory neurons. 1.6 Hypotheses and Objectives of This Study The present thesis tested the hypothesis repeated electrical stimulation of the vSub at parameters shown previously to potentiate locomotor activity and D A efflux in the N A c and the P F C w i l l produce significant changes in specific presynaptic proteins. These would include synaptophysin, S N A P - 2 5 , syntaxin, complexin I and complexin II within brain regions receiving dopaminergic projections from the V T A and glutamatergic projections from the vSub. Specific regions examined include the N A c core, the N A c shell, the P F C and the dorsal striatum as a control structure. This project is divided into two parts, namely behavioral assessments and biochemical assessments. Behavioral assessments focused on the measurement of motor activity scores. Locomotor activity was expected to increase shortly after vSub stimulation in the stimulation group as previously seen in Taepavarapruk et al. (2000). The locomotor activity was also expected to be potentiated after repeated vSub stimulation. In biochemical assessments, the amount of presynaptic proteins in the stimulation group was compared to the implanted control group. Synaptophysin, a general presynaptic vesicular protein, served as a reference presynaptic protein marker. The question of whether or not the level of synaptophysin would be changed within each synaptic vesicle in response to electrical stimulation is beyond the scope of the present project. Changes in 24 the expression of t - S N A R E s were compared to changes in the expression of synaptophysin. It was hypothesized that the level of synaptophysin would be increased in the P F C and the N A c in the stimulation group because this general presynaptic vesicular protein may reflect synaptic plasticity. Furthermore, an increased expression of syntaxin and SNAP-25 in the P F C and the N A c was postulated because these two t - S N A R E s are actively involved in neurotransmission. I also hypothesized that complexin II level would be enhanced while complexin I level would be decreased in the P F C and the N A c in the stimulation group. This hypothesis assumes that complexin I is found mainly in inhibitory axosomatic synapses and complexin II is found mainly in excitatory axodendritic synapses. Furthermore, this hypothesis implies that both excitatory and inhibitory neurons may be involved in modulating synaptic efficacy to create hyperlocomotion in response to vSub stimulation. Therefore, changes in the expression of complexin I and II may indicate the roles of inhibitory and excitatory neurons in potentiating D A release in N A c . Finally, the present thesis examined whether a correlation exists between locomotor activity and the amount of presynaptic proteins. This correlation may provide further support for a modulatory role of presynaptic proteins in the initiation of locomotor activity. 25 C H A P T E R II M A T E R I A L S A N D M E T H O D S 2.1 Animals Nineteen Long-Evans rats (Charles River, St. Constant, Quebec, Canada) weighing between 320 g and 350 g at the time of surgery were used in this experiment. They were housed individually in plastic cages in a temperature-controlled colony room at about 25°C with a 12-hour light/dark cycle (12 h; lights on at 0700 h and off at 1900 h). Surgeries and test sessions were performed during the light phase. Food (Purina Rat Chow) and water were available ad libitum. A l l experiments were conducted in accordance with standards of the Canadian Council on Animal Care. 2.2 Surgical Procedures A l l nine rats in the control group and ten rats in the testing group had stereotaxic surgery. They were anesthetized by ketamine hydrochloride (100 mg/kg, i.p.; M T C Pharmaceuticals) and zylazine (10 mg/kg, i.p.; Rompun) and mounted in a stereotaxic apparatus. Holes were drilled into the dorsal skull surface, and then a concentric bipolar stimulating electrode was implanted in the v S u b / C A l region [anterioposterior (A.P.) = -5.8mm from bregma, mediolateral (M.L . ) = +5.5 mm from midline, and dorsoventral (D.V.) = -6.0 mm from dura; according to the atlas of Paxinos and Watson 1986] of the hippocampus of anesthetized rats (Taepavarapruk et al., 2000). The stimulating electrode was secured to the skull by screws and dental acrylic. A l l rats in the testing group were tested after at least 5 days of recovery from surgery. 26 2.3 Measurement of Locomotor Activity Locomotor activity was measured by placing each rat in the stimulation group in a Plexiglas activity chamber (length 32cm x width 32cm x height 41cm) for at least 24 h prior to testing to habituate spontaneous activity (Taepavarapruk et al., 2000). Rats were divided into groups of 3 for testing. Testing periods started at 1000 h. Locomotor activity was recorded by four pairs of infra-red beams that were 10 cm apart and were located at a height of 2.5 cm above the metal grid base of the activity chambers. A computer-control system ( M A N X ) interfaced with sensors was used to measure the locomotor activity of individual rats simultaneously in 10-min. intervals throughout the testing period. Four consecutive points that were lower than 100 scores of the motor activity were collected to establish a baseline. Four more data points were collected afterward. 2.4 Application of Electrical Stimulation After establishing a baseline, in-vivo electrical stimulation was applied to the testing group by delivering a train of currents (200 pulses at 300 p A , at 20 H z for 10 seconds) to the vSub (Taepavarapruk et al., 2000). A Master-8 stimulator (an eight-channel programmable pulse generator, A . M . P . I . , Israel) delivered cathodal constant current pulses via an isolator (Iso-flex, A .M.P . I . ) to the vSub region. This stimulation was repeated once per day for four days in the stimulation group. In order to confirm that vSub stimulation had a potentiating effect on these subjects, prior to determining the effect on the expression of presynaptic proteins, one data point (1 interval of 10 min.) was collected in day 4 before sacrificing these rats. 27 2.5 Verification of Electrode Placements by Histology The correct placement of each stimulating electrode was determined by histology. Following removal of the brain, the posterior portion of the brain containing the vSub region was fixed in 10% sucrose in 10% formaldehyde solution for at least 2 days before cryostat sectioning. Serial 35-jxm coronal sections were cut on a freezing microtome. The placement of the electrode was assessed by comparing the tip of the electrode tract to the vSub region illustrated in the atlas of Paxinos and Watson (1986). 2.6 Homogenization and Detergent Compatible (DC) Assay Technique The anterior part of the brain containing the four target regions was not fixed, in order to preserve the integrity of proteins. Three brain regions, namely the P F C , the core of the N A c , and the dorsal striatum, in the left and the right hemispheres of nineteen rats and the shell of the N A c in the left and the right hemispheres of six pairs of rats were dissected out and frozen at - 8 6 ° C . Samples were homogenized separately. Homogenates were suspended in the Tris-buffered saline ( T B S : 10 m M T i r s - H C l , 140 m M N a C l , p H 7.4) by a sonic dismembrator (Fisher Scientific). To determine the concentration of homogenates, a detergent compatible (DC) assay was performed. Bovine serum albumin ( B S A , R I A grade; Sigma, U . S . A . ) following serial dilution from 2 u.g / ( i L to 0.125 | i g / u X was used as a concentration standard. Five u,L of diluted (1:10) homogenate samples were mixed with 25 (xL of Reagent A (BioRad, C A , U S A ) and 200 u L of Reagent B (BioRad, C A , U S A ) in 96-well uncoated plates (Sarstedt, Canada). The colormetric product formed from the reaction was quantified by means of optical density measured at 650 nm using a microplate reader (Bio-Tek Instruments, Winooski , Vermont, U . S . A . ) 15 minutes later. The total protein 28 concentration of each sample was obtained from the linear regression plot of the protein standard's reactivity. 2.7 Enzyme-Linked Immunoabsorbent Assay (ELISA) Technique After determining the exact amount of protein present, homogenates were diluted to 1.5 p,g protein / 50 p X using distilled water. Duplicate aliquots were dried onto 96-well E L I S A plates for coating after six steps 1:1 serial dilution in distilled water over a 64-fold range. They were incubated at room temperature in a 5% milk (non-fat instant milk powder; Carnation, Nestle, Canada) in Tris-buffered saline (TBS) for an hour to block non-specific binding of the monoclonal primary antibody. Monoclonal antibodies against specific presynaptic-like proteins (1:10 in 5% mi lk -TBS) were applied to the wells for binding to presynaptic proteins after washing 5 times with 0.02% Tween-TBS. These primary antibodies include S p l 5 against synaptophysin, S p l 2 against S N A P - 2 5 , Sp6 against syntaxin, Sp33 against complexin I, and Lp27 against complexin II. Supernatant from the parental, non-secreting myeloma (NSO) cel l line was used to determine background immunoreactivity. Peroxidase-conjugated goat anti-mouse secondary antibodies ( p - G A M IgG + I g M , 1:1000 in 5% m i l k - T B S ) were applied to wells and incubated for one hour followed by five Tween-TBS washes. Bound antibodies were detected by color development following the addition of 2,2'-azino-di,3-ethylbenzthiazoline sulphonate (BioRad) substrate to each well . The optical density (OD) was measured at 405 nm using a microplate reader (Bio-Tek Instruments, Winooski, Vermont, U .S .A. ) 30 minutes later. The O D values of the N S O background immunoreactivity were subtracted from those of different samples. These O D values were used to establish a serial dilution curve using linear regression. The log-log 29 Fig . 4. Indirect log-log analysis. Serial dilution curves were established by quantifying the immunoreactivity in terms of optical density (OD) values. A fixed half-maximal O D (OD max) value was used to calculate the amount of protein (|ig protein / 50 u X ) in different samples by interpolation. Sample A requires less amount of protein than sample B to produce the same optical density. Therefore, the concentration of antigen is higher in sample A than B . 30 indirect analysis of the immunoreactivity was used in synaptophysin, syntaxin and SNAP-25 stainings. A fixed half-maximal O D value in the serial dilution curve was used to calculate the amount of protein (jig protein / 50 u,L) in different samples by interpolation (Fig. 4). These protein concentrations were used for comparing the stimulation group to the control group. The A v g - N S O direct analysis of the immunoreactivity was used in complexin I and II stainings. The averaged O D values of an 8-fold linear range (first four steps) were used for comparisons between the stimulation group and the control group. The E L I S A experiment was conducted twice, on different days, to ensure reproducibility. The batch-to-batch correlation was assessed by rho (r) values in which 0.80 or above were acceptable. 2.8 Statistical Methods One-way analysis of variance ( A N O V A ) was used to analyze data of the locomotor activity in the stimulation group by comparing different data points to. the baseline. One-way A N O V A was used to assess the effect of repeated vSub stimulation on locomotor activity potentiation. The E L I S A results were analyzed by unpaired t-tests for assessing the difference in the expression of presynaptic proteins between the stimulation group and the control group. A s a control for multiple comparisons, the acceptable p value was lowered to 0.05 / n, where n is the number of unpaired t-tests performed. Paired t-tests were used to evaluate differences in the expression of presynaptic proteins between the ipsilateral hemisphere and the contralateral hemisphere in response to vSub stimulation. Changes in the expression of S N A R E presynaptic proteins in relation to vesicular presynaptic proteins in response to vSub stimulation were assessed by two-way A N O V A (group x region). The 31 Spearman rank correlation analysis was used to review the association between the immunoreactivity of presynaptic proteins and locomotor activity. 32 C H A P T E R III R E S U L T S 3.1 Effects of Repeated vSub Stimulation on Locomotor Activity Each of the four daily train of electrical stimulation of the vSub evoked a significant (p < 0.0001) increase in locomotor activity which peaked in the first 10-minute bin post stimulation (Fig. 5). Locomotor activity remained elevated for between 20-40 minutes. Importantly, the magnitude of peak locomotor activity on Day 3 (mean = 398) and Day 4 (mean = 336) was potentiated relative to Day 1 (mean = 260). These effects were statistically significant for both Day 3 (p = 0.004) and Day 4 (p = 0.0401) relative to Day 1. Histological analysis showed that stimulating electrode placements were all located in the ventral C A 1 or vSub / C A 1 regions of the hippocampus in both the stimulation group and the sham control group (Fig. 6). 3.2 Effects of Repeated vSub Stimulation on Presynaptic Proteins Differences in the expression of each presynaptic protein in stimulation and control groups were analyzed via A N O V A (Table 1). These analyses revealed a highly significant difference (p < 0.0001) between the stimulation group and the control group for most presynaptic proteins including synaptophysin, S N A P - 2 5 , complexin I and complexin II, except for complexin II/I ratios (p = 0.0917 in the ipsilateral hemisphere and p = 0.0366 in the contralateral hemisphere). A significant group difference (p = 0.0012) was also seen in syntaxin expression in the contralateral hemisphere. Regional differences in the expression of most presynaptic proteins including synaptophysin, complexin I, complexin II and complexin II/I ratio were highly significant (p < 0.0001) except for syntaxin. 33 Fig. 5. Change in locomotor activity following single trains of electrical stimulation of the vSub, given daily for four days. In-vivo electrical stimulation was applied to the stimulation group by delivering a train of currents (200 pulses at 300 A , at 20 H z for 10 seconds) to the vSub after establishing a stable baseline with three consecutive points that were lower than 50 motor activity scores. Each data point represents the mean motor activity scores of the ten rats in the stimulation group. The error bar represents the standard error of means. Solid arrows represent vSub stimulation. * and ' represent data points that are significantly different (p < 0.0001 and p < 0.05, respectively) from the baseline in day 1. A represents data points that are significantly different (p < 0.05) from the first data point after the vSub stimulation on day 1. 34 35 Fig . 6. Histology of stimulating electrode placements. Schematic drawings of coronal sections at -5.8 millimeters from bregma show that tips of stimulating electrodes (solid circles) are mainly located in the ipsilateral subicular/CAl region of the hippocampus in the control group (A) and in the stimulation group (B). Plates are modified from Paxinos and Watson (1986). 36 37 a o o Q. O) CO c 1* E o ca ^ CD QC JO h-+- ( / ) °? W (0 O ( j .<2 LL co * a _r £ a> o ^ U W < ir > % O z (/) z o w CO ^ c (0 ~ o '5> CO o .52 ro c "co c W 0) rt ® 3 O CO E E £ o O — c o o 3 E c Q) a) +J ro i : c h a B Q . o C D — > c a. o . E CD O 3 O 75 > L L CD _=! CO — > £ Q -X _CD Q . P, * o ^ > Li-CD _3 CO > .= a. x CO c C O 9> To > CD CO > L O Q . a. < co f °-Q . CO c > . CD C O 3 CO > L L !•>. l - h-r O CO O) O T -o o o o d d o> . oo « s « ,_ ,_ CO o o •T— o o CO o o 00 d d d co co CO co O T - i - m O O -3-© O h~ O O T-d d d i - CM co co m 00 o 00 o 00 05 o 00 05 d d d in co CM CO i -o o LO 05 o o o o CM d d d T - T -O O o o o o r*. d d d -tf en co in co co c o CD DC CO CD r-•55 2 g» 2 P - O L t (J CO T - 1-<o o r» co o o o o o o d d r-m co co 00 T - r -o o o o o c o o o •* o d d S £ « O i -co i -i - l - CO O O O O CD © © O o d d 1 - O r-. o d in CM CM OO T — CO o 00 o o CD d d d 0 ) 00 o CM CO CM O 00 i n O CM •<* o CO o o o d d d co CO CM m oo , i n i — oo CM CM d co CM ,_ ,_ c o O o 00 O o CD O o CO d d d oo r» O ) 03 CO oo o CO B CO o c o CD QC CL CD ¥ o o cc a CO CO c c CD 0 E E o o o o co ca CO CO a) o o o c c CD CD -C ^ O O O ^ o co 2 1 o x : o co > o o CD < < * z z rte E o CJ CO To c t o o To CO CD ^ o Q . ~u II II o cc 1-a. co 38 Regional differences in SNAP-25 expression were also observed but were not as robust (p < 0.0495 in the ipsilateral hemisphere and p < 0.0328 in the contralateral hemisphere). The E L I S A results summarized in tables 2 and 3 showed a general trend of an increase in the expression of specific presynaptic proteins (i.e. S N A R E proteins, vesicular proteins and complexin proteins) in all four brain regions, in response to vSub stimulation. In the ipsilateral core of the N A c , only synaptophysin expression was increased significantly (Fig. 7). In the contralateral shell of the N A c , the expression of complexin I was increased significantly in the vSub stimulation group (Fig. 8). In the P F C (Fig. 9), the expression of a majority of presynaptic proteins following vSub stimulation was increased significantly relative to control values, except for complexin II in the ipsilateral hemisphere of the brain. In the dorsal striatum, only SNAP-25 expression was increased significantly in the ipsilateral hemisphere, whereas syntaxin, synaptophysin and complexin I expression were increased significantly in the contralateral hemisphere (Fig. 10). 3.3 The Relationship between the Expression of Syntaxin and Synaptophysin A ratio of the immunoreactivity of syntaxin to synaptophysin was used to assess relative changes between a vesicular presynaptic protein as a general marker of synaptic terminal density and a t - S N A R E protein involved in exocytosis. A significant group difference (p < 0.0086) in the syntaxin to synaptophysin ratio was observed in the ipsilateral hemisphere (Fig. 11). This indicated that vSub stimulation affected these two proteins differently. However, the syntaxin to synaptophysin ratio in the contralateral hemisphere, did not differ between the stimulation group and the control group (Fig. 12). Significant 39 Table 2. Quantification and percent differences of the presynaptic protein concentration at a fixed half-maximal optical density in different brain regions between the stimulation and the control groups Protein type Reqion SNARE presynaptic protein Statistical Significant: p < 0.003125 Vesicular presynaptic protein p < 0.00625 SNAP-25 (Loq-Loq); OD = 0.4 syntaxin (Log-Log); OD = 0.4 synaptophysin (Loa-Locn: 0D = 0.45 Ipsil. PFC control: mean+-SD stim. :mean+-SD % change Contra. PFC control: mean+-SD stim. :mean+-SD % change .612 +- .105 .411 +- .104 + 49% (.0006) .583 +- .116 .387 +- .105 + 51% (.0012) .511 +- .033 .429 +- .057 + 19% (.0015) .531 +- .066 .420 +- .061 + 26% (.0014) .290 +- .052 .215 +- .030 + 35% (.0011) .277 +- .037 .207 +- .042 + 34% (.0014) Ipsi. STR control: mean+-SD stim. :mean+-SD % change Contra. STR control: mean+-SD stim. :mean+-SD % change .504 +- .067 .386 +- .077 + 31% (.0024) .487 +- .078 .394 +- .074 + 24% (.0161) .546 +- .063 .460 +- .070 + 19% (.0121) .554 +- .055 .460 +- .063 + 20% (.0031) .386 +- .079 .289 +- .069 + 34% (.0113) .379 +- .051 .298 +- .048 + 27% (.0024) Ipsi. NAc core control: mean+-SD stim. :mean+-SD % change Contra. NAc core control: mean+-SD stim. :mean+-SD % change .529 +- .137 .439 +- .051 + 21% (.0686) .534 +- .097 .477 +- .086 + 12% (.1974) .545 +- .100 .456 +- .128 + 20% (.108) .569 +- .084 .536 +- .191 + 6% (.6382) .209 +- .044 .133 +- .021 + 57% (.0001) .211 +- .033 .163 +- .048 + 29% (.0222) Ipsi. NAc shell control: mean+-SD stim. :mean+-SD % change Contra. NAc shell control: mean+-SD stim. :mean+-SD % change .464 +- .108 .386 +- .074 + 20% (.1726) .446 +- .094 .382 +- .094 + 17% (.2645) .559 +- .037 .469 +- .049 + 19% (.0048) .567 +- .047 .492 +- .053 + 15% (.026) .219 +- .067 .163 +- .037 + 34% (.1022) .239 +- .059 .183 +- .033 + 31% (.0728) Key: PFC = prefrontal cortex NAc core = core of the nucleus accumbens STR = dorsal striatum NAc shell = shell of the nucleus accumbens Log-Log = Indirect analysis of the ELISA data OD = fixed half-maximal optical density Ipsi. = Ipsilateral hemisphere Contra. = Contralateral hemisphere SD = Standard deviation ( ) = p value from unpaired t-tests control = sham control group stim. = stimulation group mean = mean of presynaptic protein concentration as a fixed half maximal OD Note: For PFC, STR & NAc core, 19 male Long-Evans rats were used (stim. n = 10, control n = 9) For NAc shell, 6 pairs of male Long-Evans rats were used (stim. n = 6, control n = 6) 40 Table 3. Quantification and percentage differences between the average optical density of complexin presynaptic proteins in different regions of the brain of the stimulation and the control groups Protein type Reqion Complexin presynaptic protein Statistical Significant: p < 0.0015625 Complexin I (Avg-NSO) Complexin II (Avg-NSO) Cpx II/I (Avg-NSO) Ipsil. PFC control: mean+-SD stim. :mean+-SD % change Contra. PFC control: mean+-SD stim. :mean+-SD % change .128 +- .020 .173 +- .024 + 35% (.0005) .125 +- .021 .174 +- .022 + 39% (.0002) .075 +- .008 .133 +- .049 + 77% (.0027) .070 +- .011 .129 +- .037 + 84% (.0003) .592 +- .081 .757 +- .214 + 28% (.0442) .565 +- .076 .743 +- .175 + 32% (.0121) Ipsi. STR control: mean+-SD stim. :mean+-SD % change Contra. STR control: mean+-SD stim. :mean+-SD % change .136 +- .028 .196 +- .049 + 44% (.0045) .126 +- .022 .185 +- .042 + 47% (.0015) .071 +- .017 .132 +- .051 + 86% (.0034) .068 +- .019 .120 +- .039 + 76% (.002) .520 +- .054 .655 +- .120 + 26% (.0069) .531 +- .082 .638 +- .102 + 20% (.0228) Ipsi. NAc core control: mean+-SD stim. :mean+-SD % change Contra. NAc core control: mean+-SD stim. :mean+-SD % change .060 +- .016 .083 +- .013 + 38% (.0031) .054 +- .016 .075 +- .022 + 39% (.0282) .118 +- .025 .144 +- .031 + 22% (.0623) .106 +- .025 .132 +- .053 + 25% (.2042) 2.047 +- .846 1.757 +- .385 - 14% (.1953) 2.141 +- .846 1.712 +- .509 - 20% (.1933) Ipsi. NAc shell control: mean+-SD stim. :mean+-SD % change Contra. NAc shell control: mean+-SD stim. :mean+-SD % change .057 +- .020 .092 +- .011 + 61% (.004) .051 +- .004 .087 +- .007 + 71% (<.0001) .138 +- .029 .182 +- .016 + 32% (.0083) .138 +- .017 .179 +- .031 + 30% (.017) 2.610 +- .886 1.977 +- .213 - 24% (.1197) 2.727 +- .340 2.063 +- .274 - 24% (.004) Key: PFC = prefrontal cortex NAc core = core of the nucleus accumbens STR = dorsal striatum NAc shell = shell of the nucleus accumbens Avg-NSO = Direct analysis of the ELISA data Ipsi. = Ipsilateral hemisphere Contra. = Contralateral hemisphere SD = Standard deviation ( ) = p value from unpaired t-tests control = sham control group stim. = stimulated group mean = mean of presynaptic protein concentration as a fixed half maximal OD Note: For PFC, STR & NAc core, 19 male Long-Evans rats were used (stim. n = 10, control n = 9) For NAc shell, 6 pairs of male, Long-Evans rats were used (stim. n = 6, control n = 6) 41 Fig . 7. Immunoreactivity (IR) of presynaptic proteins in the core of the nucleus accumbens in the stimulation group relative to control values (100%; no difference). In-vivo electrical stimulation was applied to the stimulation group by delivering a train of currents (200 pulses at 300 _ A , at 20 H z for 10 seconds) to the vSub once per day for four days. A concentric bipolar stimulating electrode was implanted in the v S u b / C A l region in one hemisphere of the control group. Each bar represents the percentage change in immunoreactivity of presynaptic proteins in the stimulation group compared to the control group (+S.E.M.) using E L I S A techniques. Sol id bars represent the ipsilateral hemisphere of the brain and open bars represent the contralateral hemisphere of the brain. * represents p < 0.003125 in S N A R E presynaptic proteins including SNAP-25 and syntaxin, p < 0.00625 in vesicular presynaptic proteins namely synaptophysin, and p < 0.0015625 in complexin presynaptic proteins including complexin I and complexin II; the stimulation group is significantly different from the sham control group using unpaired t-test 42 Core of the nucleus accumbens C O O 0) > 0) 190 180 170 160 150 140 130 120 110 100 • ipsilateral • Contralateral V) > . . E a o a. ra c >. m CM CL < z X 03 c X o Q. E o O c "x a) a. E o o (Vesicular) ( SNARE ) ( Complexins ) 43 Fig. 8. Immunoreactivity (IR) of presynaptic proteins in the shell o f the nucleus accumbens in the stimulation group relative to control values (100%; no difference). In-vivo electrical stimulation was applied to the stimulation group by delivering a train of currents (200 pulses at 300 _ A , at 20 H z for 10 seconds) to the vSub once per day for four days. A concentric bipolar stimulating electrode was implanted in the v S u b / C A l region in one hemisphere of the control group. Each bar represents the percentage change in immunoreactivity of presynaptic proteins in the stimulation group compared to the control group (+S.E.M.) using E L I S A techniques. Sol id bars represent the ipsilateral hemisphere of the brain and open bars represent the contralateral hemisphere of the brain. * represents p < 0.003125 in S N A R E presynaptic proteins including SNAP-25 and syntaxin, p < 0.00625 in vesicular presynaptic proteins namely synaptophysin, and p < 0.0015625 in complexin presynaptic proteins including complexin I and complexin II; the stimulation group is significantly different from the sham control group using unpaired t-test 44 Shell of the nucleus accumbens 45 Fig . 9. Immunoreactivity (IR) of presynaptic proteins in the prefrontal cortex in the stimulation group relative to control values (100%; no difference). In-vivo electrical stimulation was applied to the stimulation group by delivering a train of currents (200 pulses at 300 _ A , at 20 H z for 10 seconds) to the vSub once per day for four days. A concentric bipolar stimulating electrode was implanted in the v S u b / C A l region in one hemisphere of the control group. Each bar represents the percentage change in immunoreactivity of presynaptic proteins in the stimulation group compared to the control group (+S.E.M.) using E L I S A techniques. Sol id bars represent the ipsilateral hemisphere of the brain and open bars represent the contralateral hemisphere of the brain. * represents p < 0.003125 in S N A R E presynaptic proteins including SNAP-25 and syntaxin, p < 0.00625 in vesicular presynaptic proteins namely synaptophysin, and p < 0.0015625 in complexin presynaptic proteins including complexin I and complexin II; the stimulation group is significantly different from the sham control group using unpaired t-test 46 Prefrontal Cortex 220 i (Vesicular) ( SNARE ) ( Complexins ) Fig . 10. Immunoreactivity (IR) o f presynaptic proteins in the dorsal striatum in the stimulation group relative to control values (100%; no difference). In-vivo electrical stimulation was applied to the stimulation group by delivering a train of currents (200 pulses at 300 _ A , at 20 H z for 10 seconds) to the vSub once per day for four days. A concentric bipolar stimulating electrode was implanted in the v S u b / C A l region in one hemisphere of the control group. Each bar represents the percentage change in immunoreactivity of presynaptic proteins in the stimulation group compared to the control group (+S.E.M.) using E L I S A techniques. Sol id bars represent the ipsilateral hemisphere of the brain and open bars represent the contralateral hemisphere of the brain. * represents p < 0.003125 in S N A R E presynaptic proteins including SNAP-25 and syntaxin, p < 0.00625 in vesicular presynaptic proteins namely synaptophysin, and p < 0.0015625 in complexin presynaptic proteins including complexin I and complexin II; the stimulation group is significantly different from the sham control group using unpaired t-test 48 Dorsal Striatum 240 100 • ipsilateral • Contralateral >. Q. O CL ro c >. w CM • Q. < Z CO c X (0 +-> c • > N CO X o a E o o x o Q. E o o (Vesicular) ( SNARE ) ( Complexins ) 49 regional differences (p < 0.0001) in the syntaxin to synaptophysin ratios were observed in both hemispheres suggesting that the increased expression of syntaxin was greater relative to general changes in synaptic terminals in some brain regions. However, group x region interactions indicated that effects of vSub stimulation on the relative expression of syntaxin and synaptophysin did not differ significantly in different brain regions (Fig. 11 and 12). 3.4 The Relationship between the Expression of Complexin I and Complexin II The group x region interaction for complexin II/I ratios was significant in the contralateral hemisphere (p < 0.0074; F ig . 13). This interaction indicated that vSub stimulation was accompanied by regional differences in the relative expression of the two complexin proteins and this effect was confirmed by percent changes in the amount of the complexin proteins (Table 3). Increments in the percent change of complexin II/I ratios between the stimulation group and the control group indicated that the magnitude of enhanced expression of complexin II was greater than that of complexin I in the P F C and the dorsal striatum. On the other hand, percent changes of complexin II/I ratios in the core and the shell of the N A c were negative. These results suggest that the expression of complexin I increased more than complexin II in the core and the shell of the N A c in the stimulation group compared to the control group. 3.5 Correlation between Locomotor Act ivi ty and the Expression of Presynaptic Proteins Induced by Electrical Stimulation of the vSub A s already noted, data obtained from the behavioral testing showed both acute increase in locomotor activity on each test day and also a potentiation of locomotor activity 50 Fig. 11. Comparisons among syntaxin to synaptophysin ratios in different brain regions in the ipsilateral hemisphere of the stimulation group and the control group. Brain regions include the prefrontal cortex (PFC), the core of the nucleus accumbens ( N A c core), the shell of the nucleus accumbens ( N A c shell), and the dorsal striatum (STR). Error bars represent standard error of means. Open circles represent the control group and solid squares represent the stimulation group. 51 Syntaxin to synaptophysin ratios in the ipsilateral hemisphere .25 J 1 1 1 1— P F C N A c core N A c shell S T R Regions 52 Fig . 12. Comparisons among syntaxin to synaptophysin ratios in different brain regions in the contralateral hemisphere of the stimulation group and the control group. Brain regions include the prefrontal cortex (PFC), the core of the nucleus accumbens ( N A c core), the shell of the nucleus accumbens ( N A c shell), and the dorsal striatum (STR). Error bars represent standard error of means. Open circles represent the control group and solid squares represent the stimulation group. 53 Syntaxin to synaptophysin ratios in the contralateral hemisphere 25 J 1 1 1 1— P F C N A c core N A c shell S T R Regions Fig. 13. Comparisons among complexin II to complexin I (Cx II /1) ratios in different brain regions in the contralateral hemisphere of the stimulation group and the control group. Brain regions include the prefrontal cortex (PFC), the core of the nucleus accumbens ( N A c core), the shell of the nucleus accumbens ( N A c shell), and the dorsal striatum (STR). Error bars represent standard error of means. Open circles represent the control group and solid squares represent the stimulation group. 55 Complexin II/I ratios in the contralateral hemisphere PFC NAc core NAc shell STR Regions 56 on test day 3 and day 4 relative to day 1. To minimize variation, the average of peak locomotor activity scores on day 3 and day 4 was used to represent the maximum effect of vSub stimulation on locomotor activity. The Spearman rank correlation analysis revealed that a significant correlation between the immunoreactivity of syntaxin in many brain regions and locomotor activity (Table 4). Significant correlation coefficients for locomotion and immunoreactivity of syntaxin were observed in the ipsilateral N A c (r = -0.709; p < 0.0334), the contralateral N A c (r = -0.794; p < 0.0172), and the ipsilateral dorsal striatum (r = -0.685; p < 0.0399). A significantly high correlation coefficient for locomotor activity and the immunoreactivity of syntaxin was observed in the contralateral P F C (r = -0.879; p < 0.0084). Other presynaptic proteins showing significant correlation with locomotor activity were complexin I in the ipsilateral N A c (r = -0.709; p < 0.0334), complexin II in the ipsilateral P F C (r = -0.685; p < 0.0399) and complexin II in the contralateral N A c (r = -0.661; p < 0.0475). 57 c CD T3 C c o ro c Q) O c o o c "55 o a o a ro c CO c > o > +3 O 0) ro l _ L_ o o o o rank com c o ro c c > ro CO CD c Q . o CO "5) i_ CD _c . 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Results from behavioral assessments confirm that the vSub plays an important role in regulating locomotor activity (Mogenson et al., 1980). A s expected, the acute effect of vSub stimulation was an increase in motor activity scores in the stimulation group compared to the control group. Motor activity was significantly increased at the first 10-min. sampling period after vSub stimulation in each o f the four days. This result confirms a report by Taepavarapruk et al. (2000) and is similar to previous findings of increased locomotor activity fo l lowing unilateral vSub st imulat ion by N M D A microinjections (Wu and Brudzynski, 1995; Legault and Wise, 1999). The chronic treatment with vSub stimulation over four days resulted in potentiation of motor activity scores at 10 min. after electrical stimulation of vSub on day 3 (mean = 398) and day 4 (mean = 336) compared to day 1 (mean = 260). This finding replicates a recent report on the effect of repeated electrical stimulation of the vSub on locomotor activities (Taepavarapruk and Phillips, 2000). Previous studies have demonstrated that vSub stimulation increased locomotor activity and also D A efflux in the N A c (Legault and Wise, 1999; Taepavarapruk et al., 2000). These findings are supported by anatomical evidence. The N A c receives glutamatergic projections from the ventral hippocampus and dopaminergic projections from the V T A 59 (DeFrance et al , 1980; Fal lon and Moore, 1978). Furthermore, hippocampal projections synapse with tyrosine-labeled terminals in the N A c (Sesack and P icke l , 1990). The potentiation of fimbria-induced spiking activity in the N A c was impaired by intravenous injection of D i receptor antagonist SCH23390 (Floresco et al., 2001). The involvement of D A in locomotor activity was clearly demonstrated when depletion of D A in the N A c by 6-O H D A lesions of the V T A decreased vSub stimulation-induced locomotor activity (Wu and Brudzynski, 1995). Intraperitoneal injections of the D l antagonist SCH23390 caused a dose-related inhibition o f vSub stimulation-evoked locomotion (Taepavarapruk et al., 2000). Taepavarapruk and Phi l l ips (2003) also demonstrated that vSub stimulation caused reinstatement of D-amphetamine self-administration during abstinence. Together these findings suggest that the effect of vSub stimulation on locomotor activity seen in the present findings has biological significance. 4.2 Effects of vSub Stimulation on the Expression of Presynaptic Proteins Significant group differences in the expression of presynaptic proteins were observed following vSub stimulation. This finding suggests that potentiation of vSub stimulation-induced hyperactivity may involve presynaptic changes, in addition to the postsynaptic component. The vSub stimulation-induced changes in presynaptic protein expressions were observed in specific brain regions and were discussed below. In the core o f the N A c ipsilateral to the stimulating electrode, synaptophysin expression was increased (157%) significantly in the stimulation group compared to the control group (100%). This result confirmed the hypothesis described in section 1.6 that vSub stimulation would evoke the increase of synaptophysin level in the N A c . This result is 60 consistent with the involvement of the N A c core in initiation of locomotor activity. Unilateral injection of D A or amphetamine into the core of the N A c has been shown to induce hyperactivity (Ikemoto, 2002). The specific increase of synaptophysin expression in the ipsilateral N A c core of the stimulation group may reflect a primary effect of vSub stimulation on the N A c core. Expression of complexin I in the contralateral shell of the N A c was increased significantly in the vSub stimulation group. This result supports the involvement of the N A c shell in initiation of locomotor activity. Microinjection of cocaine (300 nmol) into the medial shell of the N A c produced an immediate increase in locomotor activity and rearing within 5 minutes (Ikemoto, 2002). Lesions of the N A c shell by quinolinic acid produced hypoactivity (Parkinson et al, 1999). Anatomical evidence suggests that glutamatergic neurons in the vSub send projections mainly to the caudal N A c shell (Groenewegen et al., 1996). Complexin I has been identified in inhibitory axonal terminals (Takahashi, 1995). Therefore, changes in the amount of complexin I may reflect changes in the release of G A B A . The vSub stimulation evoked general increase in the expression of presynaptic proteins in the P F C in both hemispheres, and this may reflect the fact that the P F C receives inputs from various regions including the vSub, the V T A and the M D of thalamus. Lesions of the V T A lowered the level of D A in the P F C and decreased the hippocampal-PFC L T P measured by postsynaptic potentials in the P F C (Gurden et al., 1999). This shows that L T P in the hippocampal-PFC pathway is dependent on mesocorfical dopaminergic projections from the V T A . Therefore, changes in the expression of presynaptic proteins following daily stimulation of the vSub may be related to plasticity of hippocampal-PFC synapses. Given the well documented role of the P F C in executive function and cognition (Floresco et al., 61 1999), vSub stimulation-induced plasticity in the P F C may reflect changes in cognitive function and not in locomotor activity. There are no direct projections from the vSub to the dorsal striatum. Therefore, changes in the expression of presynaptic proteins in the dorsal striatum after vSub stimulation may be secondary to the increased activity in other brain structures including the cerebral cortex. Somatosensory, motor and premotor cortices send glutamatergic projections to the dorsal striatum (Spencer, 1976). Dopamine facilitates strong glutamatergic inputs from the cortex to the striatum and blocks weak signals (Horvitz, 2002). The dorsal striatum is involved in habit formation as microinjection of either NMDA-receptor or D2-dopamine-receptor antagonists in this structure impairs memory for habitual behaviors (Packard and Knowlton, 2002). The role of the dorsal striatum in motor learning raises the possibility that changes in presynaptic protein levels observed in this project are secondary consequences of behavioral activation induced by vSub stimulation. The level of S N A P - 2 5 was increased in the dorsal striatum (131% in the ipsilateral hemisphere) and the P F C (149% in the ipsilateral hemisphere and 151% in the contralateral hemisphere) in the stimulation group compared to the control group (100%). This finding confirms the hypothesis that vSub stimulation induces the increase of SNAP-25 expression in the P F C . Hess et al. (1996) showed that the deletion of S N A P - 2 5 gene in transgenic mice caused hyperactivity. However, results of the present project cannot be compared to findings in Hess et al. (1996). The reason is that the mutant mice lacking SNAP-25 gene affect all brain regions rather than specific brain regions as in the case of the present project. The amount of syntaxin was increased significantly in the P F C in the both hemispheres (119% in the ipsilateral hemisphere and 126% in the contralateral hemisphere) 62 in the stimulation group compared to the control group (100%). This result was consistent with the hypothesis that syntaxin expression in the P F C would be increased by vSub stimulation. Syntaxin has been localized in terminals of excitatory neurons (Sesack and Snyder, 1995). Changes in the syntaxin level may be due to the initial increase of glutamate efflux in the P F C synaptic terminals of the projections from the vSub and M D of thalamus. The significant increase in the expression of syntaxin in the contralateral dorsal striatum is most likely a secondary effect of changes in the expression of presynaptic proteins in the P F C . The vSub stimulation-evoked increase in syntaxin level complements previous findings on the increased m R N A level of syntaxin in response to radial arm maze learning task (Davis etal., 1996). The expression o f synaptophysin was increased significantly in the P F C of both hemispheres (135% in the ipsilateral hemisphere and 134% in the contralateral hemisphere) in the stimulation group compared to the control group (100%). This finding is more likely to reflect the plasticity of synaptic terminals in the P F C than vSub stimulation-evoked enhanced locomotion. Changes in the expression of synaptophysin have been related to Alzheimer's Disease (AD) , a neurodegenerative disease that affects memory and cognition. The amount of synaptophysin and V A M P was decreased by about 30% in the hippocampus of A D patients' brains compared to age-matched subjects by Western blotting (Shimohama et al., 1997), an effect that may be attributed to the loss of neurons. Using immunoblot analyses, Shimohama et al. (1998) has shown that the amount of synaptophysin and V A M P was increased with age from birth (5%) to 24 weeks (100%) in the hippocampus of rats and then showed a decline until 96 weeks (75%) which was the end point of the study. The initial increase in the expression of synaptophysin in young rats may be due to a direct 63 increase in synaptic density, with the ensuring decrease related to a decline in synaptic density with age. A significant group difference in the syntaxin to synaptophysin ratio was observed in the ipsilateral hemisphere. This finding indicates that vSub stimulation affected these two proteins differently. Furthermore, the initiation of locomotor activity may require changes in both t - S N A R E s and vesicular presynaptic proteins. Significant regional differences were observed in the syntaxin to synaptophysin ratios. This finding suggests that the expression of syntaxin is lowered relative to synaptophysin in different brain regions in response to vSub stimulation, although syntaxin itself is higher in stimulated animals. This result is consistent with the involvement of different brain regions in the initiation of locomotor activity. The significant difference in the group x region interaction of complexin II/I ratios in the contralateral hemisphere between the stimulation group and the control group suggests that vSub stimulation has different effects on the relative expression of the two complexin proteins in different brain regions. Enhanced expression of complexin II was higher than that of complexin I in the P F C and the dorsal striatum. This finding confirms the hypothesis that vSub stimulation increases the complexin II level in the P F C . On the other hand, the increment of the expression of complexin I was higher than that of complexin II in the core and the shell of the N A c . These findings may be related to clinical studies. Using the ratio of complexin II/I (Cx II/Cx I), the balance between the amount of the two complexins was assessed in patients with severe mental illness. A significant increase of Cx II/Cx I ratio in the anterior frontal cortex (anterolateral inferior or middle frontal gyrus) of suicidal patients with schizophrenia (43%) and non-suicidal patients with schizophrenia (26%) or depression (32%) was found using E L I S A (Sawada et al., 2002). To show that 64 these increases were not due to pharmacological therapies, the amount of presynaptic proteins in the frontal cortex of rats treated with chlorpromazine, trifluoperazine, or haloperidol was measured. N o significant changes were found in drug-treated subjects compared to control rats treated with vehicle. On the other hand, the level of m R N A for both complexin I and II were reduced significantly in the C A 4 region of the hippocampus of schizophrenics compared to controls using in situ hybridization techniques (Eastwood and Harrison, 2000). Furthermore, the amount of m R N A for complexin II was reduced significantly in the dentate gyrus of the hippocampus of schizophrenia patients. In the case of bipolar disorders, the expression of complexin I and II m R N A was decreased in C A 4 , subiculum and parahippocampal gyrus (Eastwood and Harrison, 2000). These data show that abnormalities in the expression of presynaptic proteins are region specific. Synaptophysin and SNAP-25 levels were not correlated with locomotor activity. In contrast, syntaxin and complexin II have been located mainly in excitatory neurons whereas complexin I has been identified in inhibitory neurons (Sesack and Snyder, 1995; Takahashi, 1995). Therefore, the expression of these specific presynaptic proteins may be related to changes in specific group o f neurons, thereby increasing the possibility of detecting a significant correlation with the locomotor activity. Syntaxin has a high and significantly negative correlation with locomotor activity in the N A c core in both hemispheres. The negative correlation between the syntaxin level and average motor scores following vSub stimulation requires special comment because although levels of the protein are increased overall, within this group, rats with higher protein levels had lower motor activity scores. Therefore, enhanced locomotor activity may be related to modest increases in syntaxin levels in the N A c core. Previous pharmacological studies have already indicated that D A and 65 glutamate play an important role in generating locomotor activity (Taepavarapruk et al., 2000; Floresco et al., 2001). Repeated vSub stimulation can cause not only an increase of but also potentiation in the release of D A in the N A c (Taepavarapruk and Phillips, Society of Neuroscience's Abstract 2000). Presynaptic proteins are important in neurotransmission and these are the most likely mechanisms by which they could influence locomotor activity. 4.3 Limitations The present project was based on the hypothesis that vSub stimulation increases D A efflux in the N A c as seen in previous studies (Legault and Wise, 1999; Taepavarapruk et al., 2000). These findings were supported by the neuroanatomy o f glutamatergic and dopaminergic interaction in the N A c and the biological significance of D A in adaptive locomotor activity (Groenewegen et al. , 1996; Mogenson et al . , 1980). Glutamatergic afferents from the hippocampus to the N A c may play a presynaptic role in modulating dopaminergic varicosities in the N A c . This leads to the hypothesis that changes in the expression of presynaptic proteins in glutamatergic and/or dopaminergic neurons may facilitate the efflux o f neurotransmitters in response to vSub stimulation causing hyperlocomotion. It is important to note that this hypothesis does not exclude the possibility that dopaminergic varicosities may also act as presynaptic modulators on other efferents. The N A c also contains interneurons which may contribute to the level of presynaptic protein measured. Therefore we cannot specify with certainty which type of synaptic connection is primary responsible for the increased levels of synaptophysin in the ipsilateral core of the N A c following vSub stimulation. 66 Changes in presynaptic protein expressions observed in the present project could reflect the effect of repeated vSub stimulation or an acute effect of vSub stimulation on day 4. The difference between chronic and acute effect of vSub stimulation on presynaptic protein expressions must be resolved by additional experiments. A s noted above, in the P F C , vSub stimulation induced significant increases bilaterally in expressions of all presynaptic proteins except complexin II. These changes may be related to vSub stimulation-induced plasticity of the P F C , an effect independent of vSub stimulation-induced hyperactivity. In view of the fact that L T P can be induced by tetanic stimulation of the glutamatergic projection from the vSub to the P F C (Gurden et a l , 1999; Gurden et a l , 2000), it w i l l be of interest to determine whether L T P is associated with changes in any or all of the presynaptic proteins measured in the present study. Syntaxin has been localized in terminals of excitatory neurons (Sesack and Snyder, 1995). Takahashi (1995) has described complexin I (Cx I) was localized in inhibitory axonal terminals whereas complexin II (Cx II) was localized in excitatory axonal terminals. Despite this level of specificity, no other relationship between presynaptic proteins and specific cell type has been established to dose. Therefore, the present findings on presynaptic protein levels following vSub stimulation cannot be related to a particular class of neuron (i.e. dopaminergic or glutamatergic). Synaptophysin has been used as a synaptic marker for synaptic terminal density (Bozdagi et al., 2000), however this may be limited in situation in which changes of this protein occurred within each synaptic terminal. Therefore, the question of whether subicular stimulation affects the expression of presynaptic proteins at individual synapses as distinct from synaptogenesis, cannot be answer in the present findings. 67 Although the amounts of syntaxin, complexin I or complexin II in specific brain regions showed significant correlations with locomotor activity, a cause and effect relationship has not been proven in the present findings. Exercise increases the m R N A level of brain-derived neurotrophic factor (BDNF) in the hippocampus and the cortex of rats which in turn enhances plasticity of the brain (Neeper et al., 1995; Neeper et al., 1996). Expression of presynaptic proteins may be related to neuroplasticity (Davis et al., 1996) and this raised the possibility that vSub-induced locomotor activity may cause an increase in the expression of presynaptic proteins. However, this is highly unlikely because the increased locomotor activity lasted for less than one hour, whereas the effect of exercise on neurotrophin release was observed after several days of voluntary wheel-running (Neeper et al., 1995). 4.4 Future directions Changes in the expression of presynaptic proteins in response to vSub stimulation seen in the present project w i l l only be understood in light of future experiments. First it w i l l be important to investigate the difference between acute and chronic effect of vSub stimulation on presynaptic protein expression. This w i l l involve application of a single train of vSub stimulation to one group of rats and repeated daily vSub stimulation to another group of rats. The acute effect of vSub stimulation may cause changes in a more specific type of presynaptic protein expression in a more specific brain region. This experiment w i l l also establish a dose response of presynaptic protein expressions to vSub stimulation. Another experiment could involve characterizing the expression of different presynaptic proteins that have responded to vSub stimulation by Western blotting technique. This immunoblot study w i l l not only confirm the specificity of the monoclonal antibodies 68 against individual presynaptic proteins, but w i l l also reveal whether or not vSub stimulation has an effect on the function of presynaptic S N A R E proteins. Differences in the position of immunoreactive bands on an immunoblot w i l l indicate differences in the molecular weight of presynaptic proteins in different group. H igh molecular weight bands may represent complexes of different presynaptic proteins. Non-denatured samples w i l l detect complexes of different presynaptic proteins whereas denatured samples w i l l indicate the native state of individual presynaptic proteins. A slight shift of the molecular weight of a band representing a specific presynaptic protein may detect small changes o f that protein, for example phosphorylation. Takahashi (1995) has described complexin I (Cx I) in inhibitory axonal terminals and complexin II (Cx II) in excitatory axonal terminals. However, since their functions in neurotransmission are very similar, it w i l l be interesting to find out whether C x I is present in excitatory glutamatergic axonal terminals and whether C x II is localized in inhibitory G A B A e r g i c terminals. To test this hypothesis, Cx I w i l l be colocalized with glutamic acid decarboxylase, which is a glutaminergic marker. Similarly, C x II w i l l be colocalized with calretinin, which is a calcium-binding protein found in G A B A e r g i c interneurons. The overlapping of two antibodies would signify the presence of complexins at a specific type of neurons. The same colocalization technique can be applied to locating other presynaptic proteins such as syntaxin. Another study can investigate whether subicular stimulation affects the expression of presynaptic proteins at individual synapses as distinct from synaptogenesis. To answer this question, a quantitative analysis on the average number of synapses in a given structure of the stimulation group and the control group can be performed. If the level of a S N A R E has a 69 simple linear relationship (slope > 0) with the number of synapses, this w i l l mean no change in the amount of that S N A R E at individual synapses. However, i f the amount of a S N A R E protein increases exponentially with the amount of the number of synapses, this w i l l show that both the number of synapses and the amount of that S N A R E have increased. 4.5 Conclusion Although the exact mechanism involved in generating adaptive voluntary movements, reinitiating drug seeking behavior during abstinence or those related to the etiology of psychiatric disorders involve complex neural systems, each o f their functions may be mediated in part by the mesolimbic D A system. The present project has addressed the role of presynaptic proteins as a possible subcellular mechanism for regulating locomotor activity via the release of D A as reported in previous studies (Taepavarapruk et al., 2000; Floresco et al., 2001). Results in the present project raise the possibility that changes in the expression of presynaptic proteins may be involved in the mesolimbic D A system by modulating the D A efflux. 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