Reevaluation of the Anhedonia Hypothesis of Dopamine Blocking

國立中正大學心理學研究所博士論文

Haloperidol 干擾正向與負向味覺刺激的反應：多巴胺阻斷

劑的”Anhedonia”假設之檢視

Haloperidol interferes with the impact of rewarding and

aversive stimuli: Re-appraisal of the anhedonia

hypothesis of dopamine blocking

研究生黃智偉撰

指導教授蕭世朗博士

中華民國九十二年七月

Haloperidol Interferes with the impact of Rewarding and aversive Stimuli: Reappraisal of the Anhedonia

Hypothesis of Dopamine Blocking

Andrew Chih Wei Huang

Department of Psychology

National Chung Cheng University

160 San-Hsing, Min-Hsiung,

Chia-Yi 621, Taiwan, R.O.C.

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy

July, 2003

ii Abstract

The present study examined that a non-selective D2 receptor antagonist, haloperidol, attenuated the US regardless of rewarding and aversive stimuli on the conditioned taste aversion and taste solution intake paradigms. It was shown that the brain DA system mediated the impact of aversive or rewarding US, and revised the preceding anhedonia hypothesis of DA blocking in general without the valence of stimulus. Besides, the salient stimuli hypothesis of brain DA is to substitute the anhedonia hypothesis. However, the data indicated that so-called salient stimuli did not mean the taste unfamiliarity (novelty). Rather, it is probable the reinforcing effect of stimuli. Taken together, brain DA system probably plays a crucial role to implicate the reinforcing effect of stimuli. However, whether a stimulus has a reinforcing value seems to depend on the animals’ condition, except few primary reinforcers. After the investigation, some new issues emerge, and need to investigate in the future.

iii 摘要

本研究使用味覺嫌惡制約及味覺溶液攝食的動物行為模式，檢驗

多巴胺阻斷劑 haloperidol 是否能削弱正向及負向未制約刺激的效

果。結果顯示腦內多巴胺系統不僅牽涉正向未制約刺激也影響負向未

制約刺激，此結果修正早期多巴胺 anhedonia 的假設，支持最近的

腦內多巴胺牽涉 salient stimuli 的新假設，並且對該假設提出更

進一步的釐清，認為所謂的 salient stimuli很可能指的不是刺激的

新奇性(novelty)，而是刺激的酬賞特性，而腦內多巴胺系統可能負

責此一功能。然而刺激是否具有酬賞效果，也可能會隨動物的內外環

境的狀態而改變。許多關於腦內多巴胺系統功能的新問題及新議題在

本研究裏發現，仍待之後的研究釐清。

iv Table of Contents

Abstract ------ii

Chapter 1. ------1.

Introduction ------1.

The discovery of the neural substrate of positive reinforcement ------1.

The anhedonia hypothesis of dopamine (DA) ------1.

Challenges to the anhedonia hypothesis of DA blocking ------5.

Critique 1. ------5.

Critique 2. ------6.

Critique 3. ------7.

Opposite evidence ------10.

Sensory processing function of brain DA neurons ------12.

The involvement of amygdala to aversive and rewarding stimulus

associations ------13.

Neuroanatomical and behavioral data ------15.

The present investigation ------17.

Chapter 2. ------19.

General Method ------19.

Chapter 3. ------22.

v Experiment 1A ------22.

Experiment 1B ------27.

Experiment 2A ------30.

Experiment 2B ------33.

Discussion ------35.

Chapter 4. ------39.

Experiment 3 ------40.

Discussion ------51.

Chapter 5. ------52.

Experiment 4 ------53.

Discussion ------60.

Chapter 6. ------63.

Experiment 5 ------63.

Experiment 6 ------66.

Discussion ------69.

Chapter 7. ------71.

Experiment 7 ------74.

Experiment 8 ------83.

Discussion ------92.

vi Chapter 8. ------96.

General Discussion ------96.

Against the viewpoint of motor impairment ------98.

What is a salient stimulus? ------98.

Arguments and Issues ------101.

1. Confirmation of HAL Effect ------101.

2. High sucrose is not attenuated by HAL ------102.

3. Relativity of Reinforcement ------103.

4. A Probable Cause Regards to Generate Animals’ Approaching and

Avoiding Behaviors ------105.

Conclusion ------106.

References ------107.

vii List of Figures

1 ------26.

2 ------29.

3 ------32.

4 ------34.

5 ------42.

6 ------44.

7 ------46.

8 ------48.

9 ------50.

10 ------56.

11 ------57.

12 ------58.

13 ------59.

14 ------65.

15 ------68.

16 ------75.

17 ------76.

18 ------78.

viii 19 ------80.

20 ------82.

21 ------84.

22 ------85.

23 ------87.

24 ------89.

25 ------91.

ix Appendix 1. Histological Verification: Central nucleus of amygdala

ID No target Unilateral target Bilateral target Left Right 1g1 ＊ 2g1 ＊ 3g1 ＊ 4g1 ＊ 5g1 ＊ 6g1 ＊ 7g1 ＊ 8g1 ＊ 1g2 ＊ 2g2 ＊ 3g2 ＊ 4g2 ＊ 5g2 ＊ 6g2 ＊ 7g2 ＊ 8g2 ＊

Note: g1 represents the group that was given vehicle prior to NaCl, whereas g2 was injected HAL before NaCl. Besides, one week later, the same rats received the identical drug treatment and then LiCl test. 「＊」 indicates the condition of injected location.

x Appendix 2. Histological Verification: Central nucleus of amygdala

ID No target Unilateral target Bilateral target Left Right 1g3 ＊ 2g3 ＊ 3g3 ＊ 4g3 ＊ 5g3 ＊ 6g3 ＊ 7g3 ＊ 8g3 ＊ 1g4 ＊ 2g4 ＊ 3g4 ＊ 4g4 ＊ 5g4 ＊ 6g4 ＊ 7g4 ＊ 8g4 ＊

Note: g 3 and g4 was respectively injected HAL and vehicle before sucrose. 「＊」 indicates the condition of injected location.

xi Appendix 3. Histological Verification: Basolateral nuclei of amygdala

ID No target Unilateral target Bilateral target Left Right 1g1 ＊ 2g1 ＊ 3g1 ＊ 4g1 ＊ 5g1 ＊ 6g1 ＊ 7g1 ＊ 8g1 ＊ 9g1 ＊ 1g2 ＊ 2g2 ＊ 3g2 ＊ 4g2 ＊ 5g2 ＊ 6g2 ＊ 7g2 ＊ 8g2 ＊ 9g2 ＊ 10g2 ＊

xii Appendix 4. Histological Verification: Basolateral nuclei of amygdala

ID No target Unilateral target Bilateral target Left Right 1g3 ＊ 2g3 ＊ 3g3 ＊ 4g3 ＊ 5g3 ＊ 6g3 ＊ 7g3 ＊ 8g3 ＊ 9g3 ＊ 1g4 ＊ 2g4 ＊ 3g4 ＊ 4g4 ＊ 5g4 ＊ 6g4 ＊ 7g4 ＊ 8g4 ＊ 9g4 ＊ 10g4 ＊

Note: g 3 and g4 was respectively injected vehicle and HAL before sucrose. 「＊」 indicates the condition of injected location.

xiii Chapter 1. Introduction

The discovery of the neural substrate of positive reinforcement

The discovery that reward learning has distinctive neural substrates at the forebrain was by Olds and Milner (1954) in an accidental event. They stimulated the forebrain, thalamus, and midbrain of rats, expecting aversive responding, but, instead, they found that the stimulation of septal area was able to support vigorous bar-press responses. This effect, known as intracranial self-stimulation, was thought to indicate that positive reinforcement was mediated by forebrain activation. During that time electrical brain stimulation was regarded to evoke pain-related reflexes such as vocalization, defensive and offensive responses, like those induced by peripheral noxious stimuli and the stimulation of mesencephalic areas (Delgado, 1955b; Delgado,

1955a). Their finding connected the brain directly to reinforcement and opened a new era of research on the rewarding brain mechanisms.

The anhedonia hypothesis of dopamine (DA) blockade

The effect of a neuroleptic, chlorpromazine, to interfere with learned responses was reported by Wilson and Schuster (1972). Yet this drug antagonizes DA as well as noradrenergic receptors, so the effect was not specific (Wise, 1994). In 1970s Wise and associates used a DA-selective blocker, pimozide, to show that operant

1 responding to food was decreased and they suggested that this effect was due to attenuation of food’s reinforcement effects (Wise, Spindler, deWit, & Gerber, 1978).

The drugged animals initially showed normal responding of bar pressing, but then progressively decreased, and eventually ceased, their responses. This pattern of responding resembled that during extinction when food was no longer available. This pattern of declination suggested that the effect was due to “blunting” of the hedonic effect, as if the reward was withdrawn, rather than motor deficits induced by the DA blocker. They showed further that DA blockade attenuated intravenous self-administration of amphetamine (Yokel & Wise, 1975) or cocaine (de Wit & Wise,

1977), as well as lateral hypothalamic brain stimulation (Fouriezos, Hansson, & Wise,

1978; Fouriezos & Wise, 1976). Thus, the anhedonia hypothesis of DA blocking was born (Wise, 1982).

Another line of evidence supporting this hypothesis came from the studies of

“reward summation function”. In a series of experiments Gallistel and associates

(Edmonds & Gallistel, 1974; Edmonds, Stellar, & Gallistel, 1974; Gallistel, Stellar, &

Bubis, 1974) showed that the running speed in a runway could be plotted as a function of the parameters of the stimulating electricity (rate, intensity and pulse number) and called this relationship the reward summation function. Various factors, such as task difficulty, degree of training, and pharmacological state of the rat, have been shown to

2 affect the height (asymptote) or the slope of the reward summation curve. The factors related to each reinforcement, such as current intensity, the number of electrical pulses, and the interval of pulses induced a shift of the curve without affecting the asymptotic value (Edmonds et al., 1974). Subsequently, the reward summation function was used to examine the relationship between the operant response and electrical brain stimulation to regarding to the effect of central DA blockade. The results indicated that when low doses of neuroleptics were injected, the curve shifted toward right, indicating that each reinforcement was devalued and a higher stimulation was needed to attain the same response level, but the asymptote was not altered. Therefore, it was concluded that the neuroleptics caused the reward reduction. In contrast, at a high dose, neuroleptics reduced the asymptote as well, indicating that motor ability, as well as the rewarding effect, was impaired (Franklin, 1978; Gallistel & Freyd, 1987;

Miliaressis, Rompre, Laviolette, Philippe, & Coulombe, 1986; Stellar, Kelley, &

Corbett, 1983).

Several other studies using diverse approaches supported the view that neuroleptics altered the hedonic effect. First, Ettenberg and associates have demonstrated that the reinstatement effect of food, heroin, amphetamine, following extinction, was eliminated by haloperidol (HAL) (Ettenberg, 1990; Horvitz &

Ettenberg, 1989; McFarland & Ettenberg, 1995; McFarland & Ettenberg, 1998).

3 Second, the antagonists of DA receptor subtypes, such as D1 blocker SCH23390 and

D2 blocker pimozide, decreased the lever pressing for food reward in a way similar to that observed in the operant response during extinction (Beninger et al., 1987). Third, intermittent injections of HAL during the reinforcement phase produced the partial reinforcement effect that was indistinguishable to the real partial reinforcement effect with periodic reward omission (Ettenberg & Camp, 1986b; Ettenberg & Camp,

1986a). Fourth, the reward summation function between the magnitude of lever pressing and numerous concentrations of sucrose solution showed a right-ward shift when pimozide was given, as well as when quinine was used to adulterate the reinforcing sucrose solutions (Bailey, Hsiao, & King, 1986). Fifth, licking responses to a concentrated sucrose solution (10%) resembled that to a less concentrated one

(5%) when raclopride was given to rats (Hsiao & Smith, 1995; Schneider, Davis,

Watson, & Smith, 1990). Sixth, the best latency scores to access to the first pellet under the pretreatment of DA antagonist equaled those under the vehicle condition on the free feeding task, and the effect was claimed as due to the reinforcement deficits

(Koechling, Colle, & Wise, 1988; Wise & Colle, 1984). Finally, the reinstatement effect of lever pressing for the brain stimulation could be generated by light-on regardless of following truncation of the electrical brain stimulation and the pretreatment of pimozide conditions (Franklin & McCoy, 1979).

4 Challenges to the anhedonia hypothesis of DA blockade

Critique 1. The assumption of functional equivalence

One challenge of the anhedonia hypothesis was regarding to the functional equivalence between reduction of operant responding without the food reward

(extinction) and that under the neuroleptics pretreatment. Wise and associates (1978) showed that operant responding in the absence of reward could be transferred to a condition in which rats received neuroleptics pretreatment with food reward, thus it was regarded to be functionally equivalent. However, the study of Tombaugh and colleagues (1979) failed to observe such functional equivalence, no matter whether the shift is from the pimozide condition to extinction or the other way around. Phillips and Fibiger (1979) and Gerber et al. (1981) ascribed the interfering effect generated by the neuroleptic injection preceding the reward to a simple motor deficit. The failure to demonstrate functional equivalence between the pimozide pretreatment and extinction situation was also reported with the partial reinforcement schedules

(Tombaugh, Anisman, & Tombaugh, 1980; Gray & Wise, 1980), a fixed-ratio reinforcement schedule (Faustman & Fowler, 1981), and a fixed-interval reinforcement schedule (Salamone, 1986). All of data showed that the effect of extinction-like decrements could be generated by the neuroleptic pretreatment in

5 subsequent trials. However, it was not the case if the shift was from the pimozide to the extinction conditions.

Critique 2. The assumption that neroleptics takes away the “goodness” of the primary reward

Wise and associates found that the neuroleptics only interfered with the lever pressing for the primary reward in their initial investigation, and suggested that the effect was due to blunting of the impact of primary reward. However, they did not mention whether the DA antagonist also blocked the effect of a conditioned reinforcer, which has been associated with a primary reward. Tombaugh et al. (1982) replicated a similar procedure of Wise et al. (1978) where they compared the lever responses for food reward with treatments of pimozide/reinforcement, vehicle/extinction, and pimozide/extinction groups on several trials per session over three separated test sessions. They observed not only the pimozide/reinforcement group has a greater number of responses than the vehicle/extinction group, but also the pimozide/extinction group emitted a significantly lesser number of responses than the vehicle/extinction group. It was suggested that the cause to a stronger suppression of the lever pressing on the pimozide/extinction than that of the vehicle/extinction was probably due to that pimozide not only blocked the hedonic effect of the primary reinforcing stimulus but also interfered with the secondary reinforcement associated

6 with primary reward (see also Gray and Wise, 1980) Thus, an incentive-motivation explanation of the anhedonia hypothesis was favored.

Beninger and associates (1980) used a clever design to clarify whether environmental secondary reinforcement was subjected to the effects of the neuroleptic effect: During the pre-exposure phase rats were trained with two levers, one at a time, and one of them was followed by a 3s tone. During the conditioning phase, rats were given the tone and then a food pellet. The experimental group received a pimozide injection prior to conditioning session whereas the control group received vehicle.

Finally, during the test session, the two levers were presented. The results showed that during the test phase, the group treated with pimozide had a decreased lever-pressing rate than the control group (Beninger & Phillips, 1980). Moreover, they found the magnitude of the conditioned reinforcement induced by the primary reward was proportioned to the antagonizing effect of pimozide (Hoffman & Beninger, 1985).

Their findings indicated that the brain DA system was related to the secondary reinforcement and put forward a hypothesis of the brain DA and incentive motivation.

Critique 3. Is the activation of brain DA system also related to aversive events?

One line of vigorous investigation is an assessment of whether blockade of DA affected acquisition of responses to avoid a foot shock. The responses observed included lever pressing (White, Ciancone, Haracz, & Rebec, 1992) or running to the

7 safe side of compartment to avoid a foot shock on an active avoidance task

(Blackburn & Phillips, 1989; Hillegaart, Ahlenius, Magnusson, & Fowler, 1987;

Sanger, 1985; Sanger, 1987). In a typical case of an active avoidance task, rats were injected neuroleptics prior to being placed into one side of the compartment in a shuttle box, a neutral stimulus (tone) was present for 10 s, and then the tone was turned off, followed by a foot shock lasting for 10 s. Rats without the neuroleptics treatment could avoid the shock during the presence of the tone before the onset of shock by moving to the other side of compartment after several trainings. However, if rats received neuroleptics, the active avoidance responses would vanish (Beninger,

Mason, Phillips, & Fibiger, 1980a; Fibiger, Zis, & Phillips, 1975; Anisman, Irwin,

Zacharko, & Tombaugh, 1982; Beninger, Mason, Phillips, & Fibiger, 1980b; Sanger,

1986). Thus, the brain DA seemed to be also involved in aversive learning (Salamone,

1994; Salamone, Cousins, & Snyder, 1997).

To account for these results, the group of investigators explained the effect of neuroleptics by the motor deficits based on the following reasons. First, the data showed that rats with neuroleptics increased the latency to initiate a movement; however, did not impair the escape responding to the shock during the period of shock

(Fibiger et al., 1975). This effect is seen as similar to a symptom of Parkinsonism

(Blaszczyk, 1998). Consequently, it was suggested that the effect of neuroleptics to

8 the active avoidance response was attributed to the motor impairment (Beninger et al.,

1980a; Beninger et al., 1980b; Fibiger et al., 1975). Second, rats were trained with the pimozide pretreatment to learn the active avoidance response to avoid a foot shock on the early sessions, and then withdrew the pretreatment of pimozide on the later sessions. Rats showed a fewer numbers of the avoidance response in the initial sessions, but then increased in the later no-drug-treatment sessions. It was asserted that the effect of neuroleptics on the initial drug treatment session was not caused by the impairment of the avoidance learning, instead, it was due to motor impairment

(Beninger et al., 1980b). Finally, the neurotoxic lesion sites that impaired avoidance responses to a foot shock (Beer & Lenard, 1975; Cooper, Breese, Grant, & Howard,

1973; Cooper, Breese, & Howard, 1972; Delacour, Echavarria, Senault, & Houcine,

1977; Fibiger, Phillips, & Zis, 1974; Lenard & Beer, 1975; Neill, Boggan, &

Grossman, 1974; Zis, Fibiger, & Phillips, 1974) paralleled those due to the sensorimotor deficits related to adipsia and aphagia induced by lesioning of the nigrostriatal DA system (Marshall, Levitan, & Stricker, 1976; Marshall & Richardson,

1974; Ungerstedt, 1971).

9 Evidence counters the viewpoint of the motor impairment interpretation on the aversive events and DA

Delacour and associates (1977) microinjected 6-hydroxydopamine (6-OHDA) into the substantia nigra and caudate nucleus, and reported a decreased active avoidance response without influencing locomotor behavior. Wadenberg and associates (1990) reported that the local injection of a D2 antagonist into the ventral striatum (such as nucleus accumbens and amygdala) decreased avoidance behavior, but injections into the dorsal striatum (such as the dorsolateral neostriatum and the posterior neostriatum) did not. The dorsal striatum is crucial to the motor activity

(Blaszczyk, 1998) whereas the ventral striatum, mainly comprising nucleus accumbens and olfactory tubercle and receiving DA innervation from the ventral tegmental area (Fallon & Moore, 1978; Lindvall, 1979; Lindvall & Bjorklund, 1983;

Swanson, 1982), is implicated in the reward learning (Wise & Rompre, 1989). Thus, the above result suggested that the specific function of ventral striatum was to mediate the positive reinforcing learning, but not motor impairments.

Another line of the passive avoidance studies also showed the results that were difficult to explain with the view of motor deficit. The passive avoidance paradigm requires that rats learn to inhibit to enter the preferred dark compartment because the foot shock is present. However, when the DA antagonist was injected, rats decreased

10 the latency to enter the dark compartment (Castellano, Battaglia, & Sansone, 1992).

Moreover, it has been shown that 6-OHDA injected into the ventral striatum (nucleus accumbens and olfactory tubercle), interfered with its acquisition (Schwarting &

Carey, 1985), and manifested as reduction of the time to enter the dark place. Thus, this inhibitory response cannot be due to motor impairment.

Similarly, conflicting evidence has been demonstrated on the microdialysis experiments. Concentrations of DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), increased by aversive events, that included foot-shock (Deutch, Tam, & Roth, 1985; Fadda et al., 1978; Thierry, Tassin,

Blanc, & Glowinski, 1976), tail-shock (Abercrombie, Keefe, DiFrischia, & Zigmond,

1989), tail-pinch (Pleim, Matochik, Barfield, & Auerbach, 1990; Taber & Fibiger,

1997), restraint (Imperato, Angelucci, Casolini, Zocchi, & Puglisi, 1992; Puglisi,

Imperato, Angelucci, & Cabib, 1991), cold restraint (Dunn & File, 1983), and even saline injection (Jaskiw, Karoum, & Weinberger, 1990). The areas involved included three major DA terminal areas (the prefrontal cortex of the mesocortical system, nucleus accumbens and olfactory tubercle of the mesolimbic system, and the striatum of the nigrostriatal DA system) and its origin of projections (the ventral tegmental area of the mesocortical and mesolimbic systems, and the substantia nigra of the nigrostriatal system). Unfortunately, the results remain uncertain for the diverse

11 category of aversive agents. However, most researchers agree on two points: (a). The increase in concentrations of DA or/and its metabolites generated by aversive stimuli was linked to the activation of the innervation from the ventral tegmental area to the prefrontal cortex (i.e. the mesocortical system) (Dunn, 1988) as well as those from the ventral tegmental area to the nucleus accumbens (i.e. the mesolimbic system)

(Imperato et al., 1992; Thierry et al., 1976). (b). Activation of the DA mechanism related to aversive stimuli excluded the nigrostriatal system (Deutch et al., 1985;

Thierry et al., 1976). Apparently, the data from in vivo microdialysis studies did not support the motor impairment hypothesis.

Sensory processing function of brain DA neurons: From rewarding and aversive properties to unexpectedness and saliency of stimuli

Recent studies provide alternative explanations aside from the motor impairment or anhedonia explanation of DA blocking. One such explanation is related to the salient-stimulus processing function. First, Schultz and his co-workers measured the activity of DA neuron activity in the midbrain during reward learning (Mirenowicz &

Schultz, 1994). They found that when an unpredicted reward was presented (US) the neuron responded with a vigorous activity (UR). However, as the CS became associated to the US, the responding shifted to the CS, rather than to the US. Thus, the saliency of stimuli might be the stimulus to invoke DA release. The unexpected US

12 was salient but expected US was not, and the CS was salient. It was suggested that the midbrain DA neurons mediated the signaled changes in the saliency of stimuli

(Schultz, 1997). Similar finding of DA mediating the salient stimuli was reported

(Redgrave, Prescott, & Gurney, 1999; Spanagel & Weiss, 1999; Wickelgren, 1997).

Although Schultz and associates have even attempted to use the identical technique that recorded vigorous activation of the midbrain DA neuron by aversive stimuli, and hoped to confirm the identical result just as that in positive rewarding events, yet they found that it had a weaker response of the brain DA neurons to aversive events than to rewarding events (Mirenowicz & Schultz, 1996). Nevertheless, this inconsistency is resolved by the literature review of Horvitz (Horvitz, 2000) and the study of Young and associates (Young, Ahier, Upton, Joseph, & Gray, 1998).

These researchers suggested that non-conditioned neutral stimuli could activate midbrain DA neurons if the intensity of stimuli is high enough regardless of the valence of the stimuli (Horvitz, Stewart, & Jacobs, 1997; Horvitz, 2000).

The involvement of the amygdala to aversive and rewarding stimulus associations

The amygdala is thought to be a crucially neural substrate mediating emotional behaviors for a long period since it was found that monkey with damages in the temporal lobe (mainly in the amygdala) would reveal psychic blindness, hypersexuality, alternation of the dietary habits, increasing tendency to examine every

13 objects, and becoming more tameness; that is termed as Klüver-Bucy syndrome

(Klüver & Bucy, 1939). Furthermore, a body of studies have shown that these emotional behaviors related to the amygdala not only has a close relationship with aversive stimulus associations such as fear conditioning and anxiety (Davis, 1992;

LeDoux, 2000), but also with stimulus-reward learning (Everitt et al., 1999). To illustrate, rats with the amygdala lesions interfered with acquisition of cued and contextual fear conditioning which appeared as decrement of freezing behavior in footshock task (Phillips & LeDoux, 1992) whereas rats with the amygdala subjected to bilateral quisqualate-induced lesions (Everitt, Morris, Brien, & Robbins, 1991) or lidocaine inactivation (Schroeder & Packard, 2000) would also be impaired in conditioned association on an environmental cue and a rewarding nature stimulus.

Second, lesions of the amygdala, particularly to the central nucleus, could reflect a reduction in the heart rate changes on the aversively acoustic startle testing (Young &

Leaton, 1996). In addition, effects of amygdala blockade has also been exhibited in the rewarding drug-seeking behavior when the sodium channel blocke-- tetrodotoxin-- was microinjected into the amygdala (Kruzich & See, 2001). Third, electrolytic lesions of the amygdala could retard the suppression of a flavor paired with lithium chloride (LiCl) on the taste aversive learning (Dunn & Everitt, 1988; Grupp,

Linseman, & Cappell, 1976), and the identical lesion effect have also been

14 demonstrated on the runway task in which rats with electrolytic lesions of the amygdala revealed an increase in response latency for food pellets (Kemble &

Beckman, 1970). Moreover, electrolytic lesions of the amygdala would not only decrease the reactivity to the rewarding effects of sucrose solution (Kemble, Levine,

Gregoire, Koepp, & Thomas, 1972; Kemble & Schwartzbaum, 1969; Schwartzbaum,

1960) but also produce an increment for LiCl intake, particularly when the basolateral amygdala was damaged in the latter case (Rolls & Rolls, 1973). Consequently, it is suggested that the amygdala perhaps has a crucial role in learning that involves both aversive and rewarding stimuli.

Neuroanatomical and behavioral data of the mediation of the amygdaloid central and basolateral D2 receptors in aversive and rewarding events

According to neuroanatomical and behavioral data, DA terminals located within various subnuclei of the amygdala are proposed to involve in the aversive and rewarding events. For example, a pathway projects from the basolateral nuclei of the amygdala to the ventral striatum (Kelley, Domesick, & Nauta, 1982) has been shown to implicate the reward-related stimuli association (Cador, Robbins, & Everitt, 1989;

Everitt, Cador, & Robbins, 1989; Everitt et al., 1991). Second, one of the anatomical projections within the mesolimbic DA system terminates in the amygdala nucleus

(Alheid & Heimer, 1988; Lindvall & Bjorklund, 1978) is suggested to involve in the

15 positively rewarding learning (Wise, 1982). Third, the ventral taste pathway from the parabrachial nucleus through the lateral hypothalamus into the central nucleus of the amygdala is thought to modulate the reinforcing function of gustatory stimuli

(Pfaffmann, Norgren, & Grill, 1977). Fourth, the fear or anxiety could be attenuated by a D2 receptor antagonist, eticlopride or raclopride, when this drug was infused into the amygdala prior to the conditioning on the aversive paradigms of fear-potentiated startle or freezing (Greba, Gifkins, & Kokkinidis, 2001; Guarraci, Frohardt, Falls, &

Kapp, 2000). Finally, it has been demonstrated that there are numerous D1 and D2 receptors in the subnuclei of the amygdala complex though their distributions are unequal (Scibilia, Lachowicz, & Kilts, 1992). Thus, it is plausible that the DA projections within different subnuclei of the amygdala, particularly those in the central and basolateral components, are very likely to be differentially involved in the reward and aversive stimuli events.

16 The Present Investigation

As above mentioned, investigators have found that DA antagonism affects conditioning in both aversive and appetitive learning tasks. However, critical evaluation of the anhedonia hypothesis has not been carried out on the taste aversion-learning task (Acquas, Carboni, Leone, & Di Chiara, 1989; Caulliez, Meile,

& Nicolaidis, 1996; Di Scala & Sandner, 1989; Fenu, Bassareo, & Di Chiara, 2001;

Hoffman & Beninger, 1989; Mark, Blander, & Hoebel, 1991; Wagner, Foltin, Seiden,

& Schuster, 1981). Second, several recent literatures suggested that the brain DA system is involved in the saliency and unexpectedness of stimuli no matter what a valence of them, and termed as the saliency hypothesis of DA function (Horvitz, 2000;

Huang & Hsiao, 2002; Redgrave et al., 1999; Schultz, Dayan, & Montague, 1997;

Wickelgren, 1997). However, what qualify a saliency of stimulus remains ambiguous.

Besides, a gustatory stimulus initially has no reinforcing effect. However, after conditioned with an aversive agent, whether the “assumed reinforcement” of this gustatory solution is blocked by the pretreatment of DA antagonists. Neuroanatomical and behavioral data indicated that the subnuclei of the amygdala play a differential role in aversive and appetitive events. However, these data are always collected with a more rough approach (such as electrolytic or excitotoxic injury), and less known which category of neurotransmitter of receptors involve in this function.

17 Consequently, the present project examined the role of D2 receptors in gustatory solution intake with the peripheral intraperitoneal administration of a nonselective D2 antagonist, HAL, in a taste aversion-learning and an innate gustatory solution intake tasks or microinjection of this drug into the central and the basolateral amygdaloid nuclei in an innate gustatory solution intake task. The study intended to focus on the following issues: (a). Whether the brain DA system mediates aversive as well as rewarding learning. (b). To determine a strongest HAL dose which does not affect rats’ licking responses to water intake, and then injecting this dose of HAL prior to each of gustatory solutions intake trials to examine the influence of HAL on the reinforcing effect of stimuli. (This part is designed to verify the saliency of stimuli of

DA hypotheis.) (c). To examine whether a neutral gustatory solution after conditioning with a aversive agent LiCl can elicit a reduction and this effect is blocked by the pretreatment of HAL. (d). To evaluate whether D2 receptor of the amygdaloid subnuclei, particularly those in the central nucleus and basolateral nuclei, mediates the sucrose-generated rewarding or the LiCl-induced aversive effects, or both.

18 Chapter 2. General Method

Subjects

Male Wistar rats were purchased from the Laboratory Animal Center, National

Taiwan University College of Medicine, Taipei, Taiwan and National Laboratory for

Animal Breeding and Research Center, Taipei, Taiwan. At the beginning of experiments, rats weighed 220-450 g. They were individually housed in stainless hanging cages in a colony room that was maintained at 20±2 oC with 12-12 hr light-dark cycles (0600-1800). During the experiments food was available but water was controlled.

Apparatus

Intake volume was measured in lickometer cage with single-bottle procedure.

The drinking tube was a 25ml-burette with 0.1 ml graduation fitted with metal spout that was connected with the white panel in front of the test cage. When rats’ tongue made a contact with the tube, a circuit closed and a current of no more than 60 nA passed through the body. The electrical signal was received by a personal computer programmed to record the time of each lick to the nearest 1 ms (Davis & Smith, 1992;

Hsiao & Fan, 1993).

19 Surgical procedure

All rats were injected with atropine and, 20-min later, anesthetized with sodium pentobarbital 30-min prior to the surgery by an intraperitoneal injection. Two stainless steel guide cannulas (0.63 mm outside diameter, 0.33 mm inside diameter, 15 mm long) were bilaterally implanted in the central nucleus or basolateral nuclei of the amygdala, and were chronically fixed to the skull with jeweler’s screws and acrylic dental cement. After more than 7 days of postoperative recovery, rats were subjected to behavioral procedure.

Cannulae

The used cannulae include the guide cannulae and the inner cannulae. Both are made from stainless steel. The format of guide cannulae is 0.63 mm outside diameter,

0.33 mm inside diameter, and 15 mm long, whereas those of inner cannulae are respectively 0.3 mm, 0.08 mm, and 50 mm long. As the animals are received the bilateral microinfusions of HAL or its vehicle, one side of inner cannulae is attached to a Hamilton 2-µl syringe with PE-50 tubing and the other side is inserted the guide cannulae into its target site. Then, the drug is injected through the CAM/100 microinjection pump with a volume of 0.2-µl over 1-mins. After that, the cannulae remain in place for three minutes, and then inserted stylets into guide cannulas to avoid the contamination of the microsyringe.

20 Behavioral procedure

Conditioned taste aversion task

Animals were given a tastant (such as saccharin or sodium chloride solutions) as the conditioned stimulus (CS). This was followed by the treatment of LiCl as the unconditioned stimulus (US). Subsequent intake of the CS solution would be suppressed if the conditioned taste aversion (CTA) occurred. In the series of experiments, the Exp 1 and Exp 2 (in Chapter 3), and Exp 5 and Exp 6 (in Chapter 6) utilized this CTA paradigm. Because each experiment manipulated distinct parameters the detailed procedure was described separately.

Histology

After the completion of the behavioral testing, all rats were anesthetized with an overdose of chloralhydrate and perfuse with 0.9 % normal saline followed by a 10 % formalin-saline solution. Next, brains were removed and stored in a 15 % formalin-sucrose solution until the brain sank. After that, brains were sectioned

(40-µm) on a freezing microtome, mounted on glass slides, and then stained with thionin. The slides were assessed by the light microscopy for verification of the location of an available injection and drawings were made by the atlas of Paxinos and

Watson (1986).

21 Chapter 3. Haloperidol Attenuates Rewarding and Aversively

Conditioned Suppression of Saccharin Solution Intake

The present study used the conditioned taste aversion (CTA) paradigm to test the hypothesis that DA blockade attenuates the impact of the unconditioned stimulus

(US), thereby weakening the conditioning of intake suppression induced by aversive stimulation of LiCl as well as by a rewarding drug, AMPH. Experiment 1 studied whether the injection of HAL prior to the AMPH US, but after the saccharin conditioned stimulus (CS), would attenuate the conditioned taste suppression induced by its rewarding effect. Experiment 2 studied whether HAL injected prior to the LiCl

US, but after the CS, would attenuate the CTA. In each experiment, various treatment groups were adopted in an attempt to narrow down the effect of DA blockade to the impact of the US.

Experiment 1A

AMPH’s rewarding effect is manifested in a conditioned place preference (Carr

& White, 1983) and in the operant response (Hoebel et al., 1983). However, AMPH also can induce a suppression of tastant ingestion, and be claimed it might also possess an aversive effect on CTA (Cappell & LeBlanc, 1971; D'Mello & Stolerman,

1978). This coexistence of reward and aversive effects for a drug of abuse is a

22 “paradox” (Schultz, 1997; Hunt & Amit, 1987). Recently, it is suggested that the suppressive effect of abused drugs on the intake of saccharin resulted from drugs’ higher rewarding effect than the preferable saccharin solution (Grigson, 1997;

Grigson & Freet, 2000). Accordingly, it is served as a rewarding agent on this experiment, and examined whether HAL injection between saccharin and AMPH could attenuate the reduction of saccharin intake.

Method

Drugs. All drugs were obtained from Sigma Company. Sodium saccharin, sodium chloride (NaCl), and tartaric acid were dissolved in distilled water with the following concentrations: 0.1 % saccharin solution (w/v), 0.15 M NaCl (normal saline), and 2 % tartaric acid solution (w/v). AMPH and HAL were respectively prepared in normal saline and 2 % tartaric acid solutions. Because a dose of 0.2 mg/kg

HAL could effectively block AMPH-induced conditioned place preference and locomotor activity (Mithani, Martin-Iverson, Phillips, & Fibiger, 1986), the following doses were used: AMPH, 2 mg/kg; HAL, 0.2 mg/kg. All injections were intraperitoneal at the following volume: AMPH and NaCl were 4 ml/kg; HAL and tartaric acid were 1 ml/kg.

Procedure. All subjects were accustomed to a water deprivation regimen (23.5 hrs/day) in home cage for about a week followed by training to drink in the

23 lickometer cage for about 3 days. They were assigned to the following treatment groups (n = 8 per group): VH-AMPH, HAL-AMPH, and HAL-NaCl. During the conditioning regimen, rats were given a CS fluid (0.1 % saccharin solution) for

15-min in lickometer cage, immediately injected a treatment drug (HAL or 2 % tartaric acid solution), and 45-min later, administered the US agent (AMPH or NaCl) in home cage. The treatment was given for 5 consecutive days. The treatment was completed before noon, and thereafter water was given for 30-min in late afternoon

(1800-1900).

Results

Mean (± SEM) suppression scores for each treatment group is shown in Figure 1.

A 3×4 factorial ANOVA with repeated sessions indicated that the group effect, F(2,

21) = 30.36, session effect, F(3, 63) = 28.52, and interaction, F(6, 63) = 18.33, were significant (all p < .01). Further analysis indicated that the HAL-NaCl group did not have the suppression effect, (F(3, 21) = 2.95, p > .05). This is consistent with reports that HAL, by itself, does not induce a CTA (Asin & Montana, 1989; Risinger, Brown,

Oakes, & Love, 1999). The comparison between the VH-AMPH group and the

HAL-AMPH group indicated a significant group difference, (F(1, 14) = 9.84, p < .01).

Thus, HAL at the 0.2 mg/kg dose, by itself, did not suppress the saccharin solution intake, but it reduced the conditioned suppression induced by AMPH (2

24 mg/kg) when it was injected between the CS and the rewarding US agent. This appears to support the anhedonia hypothesis.

Figure 1. Effect of HAL before AMPH (or NaCl) on the suppression score of saccharin intake. Mean (± SEM) suppression score (in milliliters) for the VH-AMPH, the HAL-AMPH, and the HAL-NaCl groups (n = 8, per group) in four conditioning sessions. HAL (or VH) was given prior to unconditioned stimulus, AMPH, or NaCl.

VH: vehicle, HAL: haloperidol, AMPH: amphetamine, NaCl: sodium chloride.

Experiment 1B

A question is addressed: Whether the effect of HAL is a combined effect of

AMPH and HAL rather than this drug acting specifically to the US. Accordingly, give a group the 0.2 mg/kg HAL treatment after the 2 mg/kg AMPH presentation to probe the temporal properties of the attenuation effect of HAL. If HAL is unable to attenuate the reduction of saccharin intake, it would further suggest that, at the specific HAL dose and the single AMPH dose treatments, HAL probably affected the impact of the perception of the US itself.

Method

Procedure. The sequence of drug treatments was different with that in Exp 1A as the following: AMPH-VH (n = 8), AMPH-HAL (n = 8). Rats were given a CS fluid (0.1

% saccharin solution) for 15-min, immediately administered US agent (AMPH) and,

20-min later, injected a treatment drug (HAL or 2 % tartaric acid). The conditioning was repeated 4 times and thus, there were 3 sessions of the suppression scores in this experiment.

Results

A 2×3 factorial ANOVA was used to compare the suppression scores of the two groups, the AMPH-VH group and the AMPH-HAL group (see Figure. 2). The results

27 revealed that there was no effect of HAL, F(1, 14) = 0.16, p > .05; however, the session effect was significant, F(2, 28) = 41.60, p < .01. Thus, the HAL treatment did not affect the conditioned suppression of AMPH when it was injected following the

CS-US pairing. Taken together, the results of the two experiments indicate that the effect of HAL is probably to modulate the conditioned suppression through inhibiting the impact of the US, while used the 0.2 mg/kg dose of HAL and a single dose of 2 mg/kg AMPH as US agent.

Figure 2. Effect of HAL after AMPH on the suppression score of saccharin intake.

Mean (+ SEM) suppression score (in milliliters) for the AMPH-VH and the

AMPH-HAL groups (n = 8, per group) in three conditioning sessions. HAL (or VH) was given after unconditioned stimulus, AMPH. VH: vehicle, HAL: haloperidol,

AMPH: amphetamine.

29 Experiment 2

Two experiments were run for testing the effect of HAL on a CTA that was based on an aversive drug, LiCl. The experimental paradigm replicated that of Exp 1.

Experiment 2A examined whether the HAL injection would attenuate the conditioned suppression induced by LiCl’s aversive effect when it was injected between a saccharin solution and LiCl. Experiment 2B tested whether the HAL treatment would affect the conditioned taste aversion when HAL injections followed the CS-US pairing.

Experiment 2A

Method

Drugs. LiCl was dissolved in distilled water (dH2O) at a concentration of 0.15 M

(isotonic solution) and intraperitoneally injected at 4 ml/kg volume. Other drugs were identical to that described in Exp 1A.

Procedure. Same experimental procedure as that described in Exp 1A was used, except that AMPH was replaced by LiCl. The treatments were as following (n = 8 per group): VH-LiCl, HAL-LiCl, and HAL-NaCl. The analysis method was the same as that in Exp 1A.

Results

A 3×4 factorial ANOVA with repeated sessions revealed the significant difference (all p < .01) for the group effect, F(2, 21) = 8.43, session effect, F(3, 63) =

23.02, and the interaction, F(6, 63) = 3.57 (see Figure 3). Further analysis indicated that the HAL-NaCl group did not have the suppression, (F(3, 21) = 0.98, p > .05). The

VH-LiCl group showed the strongest suppression effect with repeated sessions (F(3,

21) = 19.39, p < .01). However, when the DA antagonist HAL was injected between the CS and LiCl, HAL significantly attenuated the suppression effect, (F(1, 14) = 4.83, p < .05). Thus, as with AMPH, HAL, by itself, did not induce a conditioned suppression, but it was able to attenuate the conditioned suppression induced by

LiCl’s aversive effect.

Figure 3. Effect of HAL before LiCl (or NaCl) on the suppression score of saccharin intake. Mean (+ SEM) suppression score (in milliliters) for the VH-LiCl, the

HAL-LiCl, and the HAL-NaCl groups (n = 8, per group) in four conditioning sessions.

HAL (or VH) was given prior to unconditioned stimulus, LiCl, or NaCl. VH: vehicle,

HAL: haloperidol, LiCl: lithium chloride, NaCl: sodium chloride.

Experiment 2B

Method

Procedure. The same experimental procedure as that described in Exp 1B was used, except that AMPH was replaced by LiCl. The treatment groups included LiCl-VH (n

= 8) and LiCl-HAL (n = 8), the same as that in Exp 1B. All drugs were the same as those in Exp 2A. The analysis method was the same as that in Exp 1B.

Results

A 2×3 factorial ANOVA with repeated sessions indicated that the HAL effect was not significant, F(1, 14) = 0.16, p > .05 (see Figure 4). Thus, HAL could not affect the conditioned suppression by LiCl when it followed the CS-US pairing in a

CTA.

Figure 4. Effect of HAL after LiCl on the suppression score of saccharin intake. Mean

(+ SEM) suppression score (in milliliters) for the LiCl-VH and the LiCl-HAL groups

(n = 8, per group) in three conditioning sessions. HAL (or vehicle) was given after unconditioned stimulus, LiCl. VH: vehicle, HAL: haloperidol, LiCl: lithium chloride.

Discussion

Four experiments examined the effect of HAL on the conditioned suppression of a CS fluid intake induced by AMPH or LiCl. The study demonstrated that (a) HAL, by itself, was not effective in reducing the intake of the saccharin solution. (b) When

HAL was injected between the CS and US, the suppression was attenuated, whereas this attenuation did not occur when the HAL was injected after the US indicating that

HAL probably affected the perception of US. (c) This attenuation was found with both 2 mg/kg AMPH and 4 ml/kg of 0.15 M LiCl treatments indicating that the valence of US is probably unimportant. In sum, the results call into question one of the main aspects of anhedonia hypothesis, that DA is involved only in a reward stimulus. The results implied that DA antagonism impairs probably not only the hedonic impact of stimuli but also the aversive effect of stimuli. It is easily reasoned that the “anhedonia response” may be due to weakening of the US effect in general.

In considering previous studies, several questions should be addressed. The first question is whether the attenuation of the conditioned suppression is specific to D1 or

D2 receptor blockers or both. Caulliez, et al (1996) and Fenu et al (2001) used a specific D1 receptor antagonist, SCH23390 or SCH39166, and a specific D2 receptor antagonist, sulpiride or raclopride, to impair the acquisition of CTA induced by LiCl.

35 Their results indicated that only the specific D1 receptor antagonist impaired the acquisition of CTA, but the specific D2 receptor antagonist did not. However, the present results show that nonselective D2 receptor antagonist HAL, which is predominantly binding to the D2 receptor and has partial affinity for the D1 receptor, is able to attenuate the conditioned suppression. Second, which stage of the process of

CTA is impaired by DA blocker? Caulliez and associates (1996) manipulated DA blockers (included SCH 23390 and sulpiride) relative to the timing of the US or the

CS. However, their experiments failed to clearly show the effect on the processing of the CS or the US information. Recently, Fenu and associates (2001) found that the D1 receptor was important for the short-term memory trace of the gustatory CS during the process of CTA. The present results showed that HAL, by itself, did not suppress the conditioning of the saccharin solution intake. Besides, HAL was able to attenuate the conditioned suppression when it was injected before the US, but not after the US.

At the first glance, it was concluded that the attenuated effect of HAL was on the perception of the US. However, this conclusion should be taken with reservation because there are few probabilities that the effect of HAL partially affects the consolidation of saccharin. Thus, on the study it needs to add the saccharin-VH-NaCl group to compare with saccharin-HAL-NaCl group, and examines whether the effect of saccharin can be reduced by HAL. Otherwise, the conclusion cannot exclude the

36 probable one of opportunities that the effect of HAL partially affects the consolidation of saccharin solution, even though HAL, itself, did not elicit conditioned suppression of saccharin on the present study.

Third, what may be an appropriate hypothesis to account for the phenomenon that DA antagonism attenuates both the conditioned aversive as well as appetitive learning?

Redgrave and associates (1999) reviewed the studies of Schultz and colleagues on the function of DA system (Mirenowicz et al., 1996; Schultz, 1997; Schultz et al.,

1997). They suggest that “…we will, however, argue that the ‘short-latency dopamine response’ could have a rather different functional role… we suggest that this response could represent an important component of the processes that are responsible for reallocating attentional and behavioural resources in favour of unexpected salient events…”(p.146). Accordingly, it is suggested that the DA activation is probably to mediate unexpected and salient stimuli, and that is not limited to reward stimuli.

Horvitz et al (1997) recorded the burst of activity on the ventral tegmental DA neuron in freely moving cats during the presentation of non-reward sensory stimuli (auditory and visual stimuli). They found that the sensory-induced DA activation was disproportionally large relative to the basal discharge frequency. Horvitz (2000) then reviewed a great deal of single-unit, microdialysis and voltammetry studies and

37 suggested that the DA system, including mesolimbocortical and nigrostriatal DA neurons, would respond to a large category of salient events including appetitive, aversive, high intensity, and novel stimuli. Thus, the HAL treatment that partially attenuates the conditioned suppression induced by aversive LiCl as well as by rewarding AMPH is probably due to the decrease of the US’s salience or the capacity of unexpectedness of the US without regard to their valence.

There are questions that remain to be solved regarding the role of DA in learning and behavior. Nevertheless, “the salient stimuli hypothesis” of brain DA extends the anhedonia hypothesis to appropriately explain the role of the DA system in the conditioned suppression induced by LiCl’s aversive effects as well as those rewarding effects induced by AMPH. The anhedonia hypothesis may be viewed as a subset to a more broadly based hypothesis related to the stimulus saliency.

38 Chapter 4. Determine the Highest Haloperidol Dose That Does Not

Affect Licking Responses

The brain DA systems control multiple functions including the reward effect

(mesolimbic pathway) (Wise et al., 1989), the motor effect (nigrostriatal pathway)

(Blaszczyk, 1998; Hornykiewicz, 1975) and the cognitive effect (mesocortical pathway) (Epstein, Stern, & Silbersweig, 1999; Snyder, Banerjee, Yamamura, &

Greenberg, 1974). The D2 receptor appears to be involved in those functions.

Therefore, a reduction in licking responses and CTA induced by HAL might be attributed to any of the above functions and the motor effect and reward effect often confound to confuse the interpretation as mentioned earlier. Yet it has been shown that at low doses HAL seems to interfere with the reward effect without much effect on the motor functioning (see Introduction). The aim of this study was to obtain a wide-ranged dose-effect relation between HAL and licking of water to determine the dose of HAL that did not interfere with the motor aspect. Water is the neutral and most familiar liquid and it should be able to serve as the basal level to determine the motoric effect of HAL. The dose that did not affect the licking of water was then used to study its effect on the intake of various tastants to investigate involvement of the

DA system in intake of tastants with and without reinforcing effect in the further

39 experiment.

Experiment 3

Method

Drug administration. HAL was dissolved into vehicle (VEH) of 2 % (w/v) tartaric acid. The doses used were 0 (VEH), 0.025, 0.05, 0.1, and 0.2 mg/kg HAL.

The volume of the drug injected was 1 ml/kg.

Procedure. After preliminary training rats were randomly assigned to each dose group (n = 5 for all, except 0 mg/kg HAL was 4 subjects). Rats were intraperitoneally injected with an appropriate dose 45-min prior to water intake for 15 minutes for 5 consecutive daily sessions. They were given water for 30-min in the afternoon (6:00 p.m.-7:00 p.m.) in home cages.

Statistics. Data were analyzed by a 5 x 5 mixed analysis of variance (ANOVA) for the factors of dose condition and sessions with repeated measures. When appropriate, post hoc tests were conducted using Tukey’s honestly significant difference (HSD) test. Standard error of the difference of the means (SED) represents polled standard errors from all groups of each session.

Result

Five behavioral indices were used to show the intake behavior: total lick numbers, total intake volume, lick duration, interlick interval, and lick numbers

40 during each 3-min period (Minutes 0-3, Minutes 3-6, Minutes 6-9, Minutes 9-12, and

Minutes 12-15) (see Figure 5 to Figure 9).

Figure 5 depicts the mean numbers of licking for 15 minutes by each group during sessions 1-5 on lickometer test. It revealed that different doses of HAL had differential effects on suppressing licking numbers to water (F(4, 19) = 4.97, p < .01).

A statistical significant influence occurred just at the 0.2 mg/kg HAL group on the session 3 and 4 when it was compared with 0 mg/kg HAL group (all, p < .05).

Figure 6 indicates that mean intake volume of water for each group during sessions 1-5. There was a significant dose effect (F(4, 19) = 4.72, p < .01). Moreover, the effect of 0.2 mg/kg HAL was significant only at session 2 and session 3 after comparing all does (p < .05).

Figure 7 depicts that the mean lick duration by various groups (except the 0.2 mg/kg HAL group) during sessions 1-5. Because 0.2 mg/kg HAL on session1-5 and

0.1 mg/kg HAL on session 5 at least one rat completely ceased to lick the groups were not included in the analysis.

Figure 8 indicates that the mean interlick interval by various groups (except the

0.2 mg/kg HAL group) during sessions1-5 on lickometer test. To avoid the bias of default data, omits data of interlick intervals from groups of 0.2 mg/kg HAL (session

1-5) and 0.1 mg/kg HAL (session 5). It appeared that no group on any sessions

41 showed its difference than 0 mg/kg HAL control group (p > .05). Thus, the interlick interval between all groups (except the 0.2 mg/kg HAL) was not affected by HAL injection on sessions 1-4.

Figure 9 depicts the mean lick numbers to water during each 3-mins period

(Minutes 0-3, Minutes 3-6, Minutes 6-9, Minutes 9-12, and Minutes 12-15) on session

1-5 under the various doses (0, 0.025, 0.05, 0.1, and 0.2 mg/kg) of HAL injections.

It showed that only the 0.2 mg/kg HAL group has a significant reduction of water intake than 0 mg/kg HAL control group on the first 3-mins period no matter what any sessions (from 1 to 5; all p < .05). Besides, the licking pattern of 0.2 mg/kg

HAL group is unlikely to other groups. Instead, 0.2 mg/kg HAL group almost go down to the bottom of y-axis at the initial periods. It means that this high dose of

HAL would generate a strong motor impairment to access to water intake.

Figure 5. Effect of HAL under various doses on the licks of water intake during

15-mins for five sessions. Mean lick numbers to water by water-deprived rats during fifteen minutes under pretreatment of various doses of HAL (0, 0.025, 0.05, 0.1, 0.2 mg/k; n = 5 per group except 0 mg/kg HAL n = 4). **p < .01, *p < .05, different from

0 mg/kg HAL group for each session. HAL: haloperidol.

Figure 6. Effect of HAL under various doses on the intake volume of water during

15-mins for five sessions. Mean intake volume (in milliliters) to water by water-deprived rats during fifteen minutes under pretreatment of various doses of

HAL (0, 0.025, 0.05, 0.1, 0.2 mg/kg; n = 5 per group except 0 mg/kg HAL n = 4). **p

< .01, *p < .05, different from 0 mg/kg HAL group for each session. HAL: haloperidol.

Figure 7. Effect of HAL under various doses on the lick duration of water during

15-mins for five sessions. Mean lick duration (in milliseconds) to water by water-deprived rats during fifteen minutes under pretreatment of various doses of

HAL (0, 0.025, 0.05, 0.1 mg/kg; n = 5 per group except 0 mg/kg HAL n = 4). HAL: haloperidol. Note: Because one rat of 0.1 mg/kg HAL group on session 5 completely ceased to lick, data of this group were not included in session 5.

Figure 8. Effect of HAL under various doses on the interlick interval of water intake during 15-mins for five sessions. Mean interlick interval (in milliseconds) to water by water-deprived rats during fifteen minutes under pretreatment of various doses (0,

0.025, 0.05, 0.1 mg/kg; n = 5 per group except 0 mg/kg HAL n = 4). HAL: haloperidol. Note. Because one rat of 0.1 mg/kg HAL group on session 5 completely ceased to lick, data of the group were not included in session 5.

Discussion

The results indicate that HAL had a dose-dependent effect on mean lick numbers, mean intake volume, and lick numbers during each 3-mins period on session 1-5.

Only at the dose of 0.2 mg/kg did HAL affect licking. The data suggest that doses higher than 0.2 mg/kg would impair the motoric licking responses. Hence, it seems that the 0.1 mg/kg HAL is the strongest dose that it does not influence rats’ licking responses before the first three sessions. Thereby, this data can provide bases for further investigation of studying brain DA functioning relative to the stimulus.

48 Chapter 5. Haloperidol Attenuates the Reinforcing Effect of

Gustatory Stimuli: Re-verification of the Unexpectedness and

Saliency of Hypothesis of DA Function

Numerous literatures have asserted that the brain DA system mediates the stimulus processing in the conditioned learning regardless the stimulus valence

(Horvitz, 2000; Huang et al., 2002; Redgrave et al., 1999; Spanagel et al., 1999;

Wickelgren, 1997). For instance, single unit recording has shown that an initially vigorous activity of midbrain DA neurons for the positive primary reward (food or preferable juice) becomes silent after pairing with a neutral stimulus and the activity shifts to the stimulus that predicts it (Mirenowicz et al., 1996; Mirenowicz et al., 1994;

Schultz et al., 1997). Moreover, the ventral tegmental DA neurons could give a burst of activities to a brief presentation of non-conditioned auditory or visual stimulus in a freely moving cat (Horvitz et al., 1997). The dialysis experiments with an aversively sensory preconditioning paradigm show that the DA concentration in the nucleus accumbens increases at the presence of tone during a footshock conditioning and also on the pairing of flashing light and the tone (Young et al., 1998). All data mentioned above do not support the viewpoint of anhedonia of DA blocking, and thereby the brain DA neurons probably play a crucial role on mediating the unexpectedness and

49 saliency of stimuli processing. This view is termed the salient stimuli hypothesis of

DA function (Huang & Hsiao, 2002). However, a question arises: What kind of stimulus or what a property of stimulus qualifies as “salient”?

Almost thirty years ago Kalat (1974) has manipulated two properties of gustatory stimulus (taste strength and taste unfamiliarity) to examine this issue, and assumed that a salient stimulus is that one able to elicit a stronger CTA. He shows that the taste unfamiliarity (i.e. novelty), not taste strength, is related to CTA. Depended on this operational definition, it was expected that any concentration of gustatory solution used in the experiment should be affected by HAL injection. Various tastants were used in this experiment to study whether the saliency aspect of stimuli was mediated by the brain DA system. The tastant used included various taste qualifies and intensities: I started with an absolute threshold level of 0.34 % sucrose and 0.0005 % quinine solutions (Koh & Teitelbaum, 1961), and 0.15 M NaCl and LiCl (isotonic)

(Nachman & Ashe, 1973). It was expected that HAL was capable of altering the intake volume of any of those salient solutions.

Experiment 4

Method

Procedure. Rats received the identical experimental procedure as described in experiment 3. The different part was that water was replaced by a tastant and 0.1

50 mg/kg HAL was injected.

Rats were injected with HAL or its vehicle (2 % tartaric acid solution), and

45-min later, given an appropriate gustatory solution for 15-min. The groups were:

VH-Sucrose and HAL-Sucrose for a 0.34 % low sucrose solution (n = 7, for each group), VH-Sucrose and HAL-Sucrose for a 10 % high sucrose solution (n = 4, for each group), VH-Quinine and HAL-Quinine for a 0.0005 % low quinine solution (n =

7, for each group), VH-Quinine and HAL-Quinine for a 0.005 % high quinine solution (n = 4, for each group), VH-NaCl and HAL-NaCl for isotonic NaCl solution

(n = 8, for each group), and VH-LiCl, HAL-LiCl for isotonic LiCl (n = 8, for each group). Additionally, the behaviorally suppressive effect of LiCl must be measured on the next day, so rats treated with LiCl had one more testing trial that was given to access to an isotonic NaCl solution for 15-mins on the testing session next to the HAL treatment.

Statistics. All available data were analyzed with one-way analysis of variance method to compare the mean intake volume of a gustatory solution (sucrose, quinine, NaCl, or

LiCl) between vehicle and HAL groups.

Results

On the innate taste intake task, the 0.1 mg/kg dose of HAL injection prior to numerous gustatory solutions indicated that (a) Sucrose: A significant suppression

51 effect of HAL on sucrose intake was occurred at the 0.34 % concentration low solution groups (F(1, 12) = 9.86, p < .01), but not at the 10 % high solution groups

(F(1, 6) = 0.05; see Figure 10). (b) Quinine: HAL pretreatment did not affect their intake volume to the low quinine (F(1, 12) = 0.02) as well as to the high quinine (F(1,

6) = 0.12) groups, when it was compared to its control group (see Figure 11). (c) NaCl:

HAL-NaCl group did not have a significant difference of the intake volume of the isotonic NaCl solution from the VH-NaCl group (F(1, 14) = 0.21; see Figure 12). It indicated that HAL pretreatment is not effective. (d) LiCl: The effect of HAL was not significant between the VH-LiCl and the HAL-LiCl groups during drug treatment session (F(1, 14) = 3.14, p > .05; see left panel of Figure 13). However, rats with the

HAL-LiCl treatment increased the intake volume of NaCl solution on the testing session than that of the LiCl solution on the drug treatment session (no CTA effect on the HAL-LiCl group), whereas rats with the VH-LiCl treatment decreased the NaCl intake on the testing session than the LiCl on the drug treatment session (CTA effect on the VH-LiCl group). Thereby, the results were shown that the VH-LiCl and the

HAL-LiCl groups had an inverse direction on the mean difference scores on Figure 13.

Moreover, the effect of HAL is approaching significance on testing session (F(1, 14)

= 4.11, p = .06; see right panel of Figure 13).

Figure 10. Effect of HAL on the intake volume of low and high sucrose solutions

during the drug treatment session. Mean (+ SEM) intake volume (in milliliters) of

sucrose for the VH-Sucrose and the HAL-Sucrose groups on the drug treatment

session (15 min). Left panel: Low sucrose is a solution of 0.34 % (n = 7, per group).

Right panel: High sucrose is a solution of 10 % (n = 4, per group). Stars indicate a significant difference relative to its control group (VH-Sucrose), **p < .01. VH: vehicle, HAL: haloperidol. Note: The y-axis of left and right panels is on different scales.

Figure 11. Effect of HAL on the intake volume of low and high quinine solutions during the drug treatment session. Mean (+ SEM) intake volume (in milliliters) of

quinine for the VH-Quinine and the HAL-Quinine groups on the drug treatment

session (15 min). Left panel: Low quinine is a solution of 0.0005 % (n = 7, per group).

Right panel: High quinine is a solution of 0.005 % (n = 4, per group). VH: vehicle,

HAL: haloperidol.

Figure 12. Effect of HAL on the intake volume of NaCl solution during the drug treatment session. Mean (+ SEM) intake volume (in milliliters) of isotonic sodium chloride for the VH-NaCl and the HAL-NaCl groups (n = 8, per group) on the drug treatment session (15 min). VH: vehicle, HAL: haloperidol, 0.15 M NaCl: isotonic sodium chloride.

Figure 13. Effect of HAL on the intake volume of LiCl solution during the drug treatment session and on the difference score of NaCl intake during the testing session.

Left panel: Mean (+ SEM) intake volume (in milliliters) of isotonic LiCl for the

VH-LiCl and the HAL-LiCl groups (n = 8, per group) on the drug treatment session

(15 min). Right panel: Mean (+ SEM) difference score (in milliliters) of isotonic NaCl intake for the VH-LiCl and the HAL-LiCl groups (n = 8, per group) on the testing session (15 min). VH: vehicle, HAL: haloperidol, NaCl: sodium chloride, LiCl: lithium chloride.

56 Discussion

The results indicated that: First, the intake volume of low sucrose and of LiCl was altered by the HAL pretreatment. It could be seen that the HAL-sucrose group had lesser amount of intake than VH-sucrose on the drug treatment session, and the

HAL-LiCl group had an increased amount of intake of NaCl than the VH-LiCl group on the testing session. However, any used concentration of quinine or isotonic NaCl was not affected by the HAL injection. The novelty hypothesis of DA function was taken to explain the attenuated effect of HAL to the gustatory intake was seemly not appropriate because the HAL did not affect all gustatory solutions intake as rats encountered these solutions at their first time of life.

Thus, a question emerges: What kind of an effect is affected by HAL, if it is not accounted by the novelty of stimuli (i.e. Kalat named as taste unfamiliarity)?

Conventionally, NaCl is seen as a “neutral” gustatory solution and quinine is an

“aversive” solution though rats show different volume of intake of either solution than water at the behavioral expression. To illustrate, rats would drink a little more of NaCl or a significant less of quinine as compared with water on a two-bottle taste preference test (Yamada, Wada, Imaki, Ohki, & Wada, 1999). Moreover, quinine produced rejection responses (Grill & Norgren, 1978) as well as those induced by

LiCl after conditioning on the taste reactivity test (Berridge & Robinson, 1998). In

57 spite of that, these two solutions were generally taken to be a CS rather a US on the taste-aversive learning, and both had a reduction intake once they were respectively paired with an aversive US agent (e.g. LiCl) (Bevins, Jensen, Hinze, & Besheer, 1999;

Nowlis, Frank, & Pfaffmann, 1980; Scott & Giza, 1987; Yasoshima, Shimura, &

Yamamoto, 1995).

In contrast, sucrose and LiCl solutions are used to be as US agents (though sometimes sucrose can be used as a CS solution) on conditioning, and further, the greatest difference between US and CS is that US has the capacity of strengthening learned stimulus-response association (White, 1989) or stimulus-stimulus association

(Harre & Lamb, 1983), which is an operationally defined properties of the US reinforcing effect. (Note: In the classical conditioning, the reinforcing event is thought as the US, however, in the operant conditioning it is a consequence of stimuli

(Berlyne, 1969)).

Thus, it would be surmised that the HAL effect on low concentration of sucrose in the drug treatment session and on LiCl in the testing session probably resulted from attenuating the reinforcing effect of these two gustatory solutions. It seems to imply the saliency of stimuli should be defined as the “reinforcing effect” of stimuli rather the “taste unfamiliarity”. Related to high concentration of sucrose, why is it not affected by HAL? It is likely that the concentration of sucrose is too high to be

58 blocked by the relatively low dose of 0.1 mg/kg HAL (see General Discussion).

59 Chapter 6. Haloperidol does not Affect the suppression of NaCl after

paired with LiCl on taste-aversive learning

A gustatory solution (NaCl) initially does not have the reinforcing effect.

However, after pairing with an US (LiCl), it is conventionally thought to acquire a secondary reinforcing effect. Is the acquired effect mediated by the DA system? This experiment addressed the issue of DA involvement in the acquired reinforcement effect. If this effect is involved in the DA system, it is expected that HAL blocks the reinforcing effect of NaCl acquired and increases the amount of NaCl intake.

Experiment 5

Method

Procedure. Following adaptation rats were randomly assigned to the following conditions: NaCl-LiCl or NaCl-NaCl. During the conditioning session, they were exposed to 0.15 M NaCl for 15-mins and then injected with 4 ml/kg of 0.15 M LiCl or the control solution (0.15 M NaCl, 4 ml/kg). Next day (the testing session) half of

NaCl-LiCl and NaCl-NaCl rats were given the injection of HAL (0.1 mg/kg) and the other half received its vehicle (2 % tartaric acid solution) before they were given 0.15

M NaCl solution for 15-mins. The four groups were indicated as NaCl-LiCl/VH,

NaCl-LiCl/HAL, NaCl-NaCl/VH, and NaCl-LiCl/HAL (n = 7, per group).

60 Results

As shown in Figure 14, it appeared that the NaCl/LiCl treatment decreased the amount of NaCl intake than the NaCl/NaCl treatment (F(1, 24) = 5.32, p < .05). It represented that the LiCl induced the suppression of NaCl intake. However, the HAL injection after conditioning of NaCl and LiCl did not block the intake of NaCl (F(1,

24) = 0.00). Thus, the result was inconsistent with our expectation.

Figure 14. Effect of HAL on the intake volume of NaCl solution after conditioning of

NaCl and LiCl during the testing session. Mean (+ SEM) intake volume (in milliliters) of isotonic NaCl for the NaCl-LiCl/VH, the NaCl-LiCl/HAL, the NaCl-NaCl/VH, and the NaCl-NaCl/HAL groups (n = 7, per group) on the testing session (15 min). A star indicates a significant difference between the NaCl-LiCl and the NaCl-NaCl treatments, *p < .05. VH: vehicle, HAL: haloperidol, NaCl: sodium chloride, LiCl: lithium chloride.

62 Experiment 6

The experiment concentrates the next question: When does the HAL can alter the

NaCl intake? It was designed to treat the HAL prior to the LiCl and posterior to the presence of NaCl to examine whether HAL could attenuate the reinforcing effect of

NaCl acquired and increases the amount of NaCl intake during the conditioning sessions.

Method

Procedure. Rats were randomly assigned to each of four groups: NaCl-VH-LiCl;

NaCl-HAL(0.1 mg/kg)-LiCl; NaCl-HAL(0.2 mg/kg)-LiCl, and NaCl-VH-NaCl. Rats were given access to 0.15 M NaCl for 15-mins, immediately injected HAL (0.1 or 0.2 mg/kg) or its vehicle, and 45-mins later treated with LiCl (4 ml/kg of 0.15 M) or

NaCL (4 ml/kg of 0.15 M). The intake of the NaCl solution for 15 min was measured for four sessions.

Results

Mean intake volume of isotonic NaCl solution was measured for each of four groups such as NaCl-VH-LiCl, NaCl-HAL(0.1 mg/kg)-LiCl, NaCl-HAL(0.2 mg/kg)-LiCl, and NaCl-VH-NaCl when the HAL or its vehicle injected prior to the

LiCl or NaCl and posterior to the NaCl. Figure 18 indicated that (a) all groups had a significant session effect from the session 1 to the session 2 (F(1, 24) = 112.33, p

63 < .01); that is due to the different regimen of water-deprived treatment. (b) The

VH-LiCl group significantly reduced the intake volume of NaCl rather than the

VH-NaCl group from the session 2 to session 4 (F(1, 12) = 9.95, p < .01); it represented to produce a CTA when rats received the LiCl treatment posterior to the

NaCl solution and the vehicle injection. Moreover, (c) analyze the mean intake volume of NaCl solution for all groups from the session 2 to the session 4 with a 4 by

3 two-way ANOVA with repeated session design. It showed that the significant interaction between groups and sessions (F(6, 48) = 5.39, p < .01). That is, the suppressive effect was attenuated by the HAL injection. (d) The further post hoc tests were conducted by using Tukey’s honestly significant difference (HSD) test to compare all groups for session 2-4. The results showed that the attenuated effect only yield at the high dose of HAL (0.2 mg/kg) but not the low dose of HAL (0.1 mg/kg) because the high dose of HAL did not produce a significant difference with VH-LiCl

(p > .05) and with the control VH-NaCl (p > .05) on the session 3. Thus, HAL could attenuate the reinforcing effect of NaCl acquired and increases the amount of NaCl intake. Besides, it occurs only during the conditioning sessions.

Figure 15. Effect of HAL on the intake volume of NaCl solution during conditioning of NaCl and LiCl for four sessions. Mean intake volume (in milliliters) for the

VH-LiCl, the HAL(0.1 mg/kg)-LiCl, the HAL(0.2 mg/kg)-LiCl, and the VH-NaCl groups (n = 7, per group) on four acquisition sessions. NaCl: sodium chloride, LiCl: lithium chloride, VH: vehicle, HAL: haloperidol.

65 Discussion

The results of Exp 5 and Exp 6 have demonstrated that the HAL did not affect the reduction of NaCl solution after paired with LiCl. However, when HAL was injected prior to LiCl and posterior to NaCl, the emetic effect could be attenuated by

0.2 mg/kg HAL as it was seen to produce less suppression for NaCl intake than the control group.

A question is noteworthy that why the NaCl after a paired with LiCl does not affect by the HAL injection? In this case, two probable reasons are provided here to explain the data. First, the NaCl after LiCl substitutes the reinforcing effect of LiCl, but the conditioned effect is not mediated by the DA system. That is because according to Pavlov’s stimulus substitution theory, a neutral stimulus (CS) can elicit the responses that initially induced only by the US by repeated pairings with CS and

US, and supposed that CS has substituted all capacities (of course, including the reinforcing effect) of US. However, the HAL given cannot affect decrease of NaCl intake for rats. Therefore, the DA system probably does not mediate the effect after conditioning on CTA. Rather, other brain neurotransmitters implicate this function.

The second probable reason is that the reduction of NaCl merely represents a signal to predict the being presenting US, but not the reinforcing effect. Hence, the decrement of NaCl intake was not altered by the 0.2 mg/kg HAL injection. In order to resolve the

66 issue, the further studies maybe use the sensory preconditioning paradigm by means of pairing NaCl and another gustatory stimulus (CS2) before the NaCl and LiCl conditioning, and then test the CS2. It will be able to examine the reinforcing effect whether is mediated by the DA system.

67 Chapter 7. Microinjection of Haloperidol into the Amygdaloid

Central and Basolateral Nuclei Does not Interfere With Rewarding

and Aversive US Impact As Well As the CS

According to previous electrolytic and excitatory lesion data, it is well known that injury of amygdaloid subnuclei, particularly the central and basolateral regions, can interfere with the conditioned learning about aversive and rewarding stimuli (see

Introduction). However, whether D2 receptors in the amygdaloid central nucleus and basolateral nuclei regulate the reinforcing effect of aversive and rewarding gustatory stimuli remains unclear. Thus, the present study is designed to use HAL microinfusion into the amygdaloid central nucleus (in Exp 7) and basolateral nuclei (in Exp8) prior to the intake of 0.34 % sucrose, 0.15 M NaCl, and 0.15 M LiCl solutions in order to test the issue.

Method

Subjects. Seventy Wistar male rats were given an appropriate drug infusion (VH or

HAL) into amygdaloid subnuclei. Each group had eight subjects and given an injection of intra-central nucleus in Exp 7. For Exp 8, each vehicle group had nine subjects and each HAL group had ten subjects for all taste solutions, whereas all were injected with suitable drugs into the basolateral nuclei of the amygdala.

68 Surgery. All rats were injected with atropine (0.3 mg/kg) 20-mins and anesthetized with sodium pentobarbital (50 mg/kg, i.p.) 30-mins prior to the surgery. Stainless steel guide cannulas (0.63 mm outside diameter, 0.33 mm inside diameter, 15 mm long) were bilaterally implanted into the amygdaloid central (in Exp 7) and basolateral nuclei (in Exp 8); thereafter, fixed to the skull with two jeweler’s screws and acrylic dental cement. Since completing the surgery, stylets that cut flush with the guide cannula tips were inserted into the guide cannulas. The stereotaxic coordinates used were: the central nucleus, AP –2.3 mm from bregma, L + 4.2 mm from the midline,

V –7.9 mm from the skull surface, whereas the basolateral nuclei, AP –2.8 mm from bregma, L +5.0 mm from the midline, V –8.4 mm from the skull surface (Ploeger,

Spruijt, & Cools, 1994). After more than 7 days of postoperative recovery, rats received the behavioral procedure.

Behavioral Procedure. After preliminary training, rats were randomly assigned into each of groups with a drug-tastant treatment: VH-NaCl and HAL-NaCl; VH-Sucrose and HAL-Sucrose. All rats were injected HAL or its vehicle into the target site,

15-mins later to access to a gustatory solution (NaCl or sucrose) for 15-mins, and then compared the change of the intake volume between HAL and VH groups on the drug treatment session. One week later, rats of NaCl groups was replaced NaCl with LiCl, and followed the identical experimental procedure as described as NaCl and sucrose

69 groups; indicated as VH-LiCl and HAL-LiCl. However, VH-LiCl and HAL-LiCl groups had additionally a NaCl solution test on the testing session in which HAL- and

VH- LiCl rats received a NaCl solution for 15-mins. The time between drug infusion withdrawn and a taste solution given was determined by the experimental procedure of Ploeger and associates (1994).

Drugs and Injection Procedure

HAL (0.5 µg) or its vehicle infusion (0.2μl , over 1 min) into the amygdaloid subnuclei ( the central nucleus or the basolateral nuclei) was employed by an infusion pump through a Hamilton 2.0-μl syringe which was attached to the infusion cannulae

(0.3 mm outside diameter, 0.08 mm inside diameter) by means of polyethylene tubing

(PE-50). After injection, the cannulae remained in place for 3-mins, and then inserted stylets into guide cannulas to avoid the contamination of the microsyringe. The dose of 0.5 µg HAL was selected because it could effectively attenuate the acquisition of spatial learning in the Morris water maze under intra-accumbens injection (Ploeger et al., 1994).

The used solutions comprised 0.15 M NaCl, 0.15 M LiCl, and 0.34 % sucrose as identical as employed in the previous studies.

Histology. The behavioral testing later, all rats were anesthetized with an overdose of chloralhydrate and perfuse with 0.9 % normal saline followed by a 10 %

70 formalin-saline solution. Brains were removed and stored in a 15 % formalin-sucrose solution until the brain sank. After that, brains were sectioned (40 µm) on a freezing microtome, mounted on glass slides, and then stained with thionin. The accuracy of cannulae placements and the effect of microinfusion were then assessed.

Statistical analysis. Data were analyzed by one-way analysis of variance. Note that the difference score was subtracted the intake volume on the testing session from that on the drug treatment session.

Experiment 7

Results

Histology

Histological verification found that each groups (included the HAL- and VH- sucrose and NaCl groups) had three subjects with cannulas missing the target location, so these data were not included for analysis. Figure 16 shows the histological verification to the placement of cannulae injections for the amygdaloid central nucleus in which was infused HAL or VH.

Figure 16. Placement of cannulae aiming at the central amygdaloid nucleus (VH-NaCl and VH-LiCl, open circles; HAL-NaCl and HAL-LiCl, filled circles; VH-Sucrose, filled squares; HAL-Sucrose, open squares). Numbers adjacent to each section indicate distances from Bregma (mm) in the anterior-posterior axis. (The plates are from The Rat Brain in Seterotaxic Coordinates by G. Paxinos & C. Watson, 1986. San

Diego, CA: Academic Press. Copyright (1986), With Permission from Elsevier.)

Figure 17. A photomicrograph (at × 40 magnification) represents the location of the injection cannula with a 40-μm section at approximate 2.3 mm posterior to bregma

(Paxinos & Watson, 1986). Ce: Central amygdaloid nuclus. BLA: Basolateral amygdaloid nuclei. ec: external capsule. opt: optic tract. ic: internal capsule.

73 Mean intake volume of each one of numerous gustatory solutions (NaCl, sucrose, and LiCl) between VH and HAL was shown in Figure 18 to Figure 20. It indicated the effect of bilateral HAL infusion into the central nucleus of the amygdala on the behavioral measures.

As shown in Figure 18, no significant difference was detected in the intake volume of NaCl solution between VH and HAL (F(1, 8) = 0.28, p > .05). For the

HAL-Sucrose group, which received HAL infusion into the central nucleus of the amygdala, it did not has a significant reduction on the intake volume in comparison with that of VH-Sucrose control group (F(1, 8) = 1.07, p > .05; see Figure 19). The intra-central nucleus HAL injection for the HAL-LiCl did not significantly affect their intake than the VH-LiCl group on the drug treatment session (F(1, 8) = 1.36, p > .05; see left panel in Figure 20). On the testing session, the mean difference score of NaCl intake on the HAL-LiCl group was not significant than the VH-LiCl group (F(1, 8) =

2.78, p > .05; see right panel in Figure 20).

Figure 18. Effect of bilateral HAL infusion into the central nucleus of the amygdala on the intake volume of NaCl solution during the drug treatment session. Mean (+

SEM) intake volume (in milliliters) of isotonic NaCl on the drug treatment session (15 min) in rats bilaterally infused with HAL or VH into the central amygdala. An available subject number is respectively five for the VH-NaCl and the HAL-NaCl.

VH: vehicle, HAL: haloperidol, 0.15 M NaCl: isotonic sodium chloride.

Figure 19. Effect of bilateral HAL infusion into the central nucleus of the amygdala on the intake volume of sucrose solution during the drug treatment session. Mean (+

SEM) intake volume (in milliliters) of 0.34 % sucrose solution on the drug treatment session (15 min) in rats bilaterally infused with HAL or VH into the central amygdala.

An available subject number is respectively five for the VH-Sucrose and the

HAL-Sucrose. VH: vehicle, HAL: haloperidol.

Figure 20. Effect of bilateral HAL infusion into the central nucleus of the amygdala on the intake volume of LiCl solution during the drug treatment session and on the difference score of NaCl intake during the testing session. Left panel: Mean (+ SEM) intake volume (in milliliters) of isotonic LiCl for the VH-LiCl and the HAL-LiCl groups (n = 5, per group) on the drug treatment session (15 min). Right panel: Mean

(+ SEM) difference score (in milliliters) in isotonic NaCl intake for the VH-LiCl and the HAL-LiCl (n = 5, per group) on the testing session (15 min). HAL or VH was bilaterally infused into the central amygdala. VH: vehicle, HAL: haloperidol, 0.15 M

NaCl: isotonic sodium chloride, 0.15 M LiCl: isotonic lithium chloride.

77 Experiment 8

Results

Histology

Figure 21 shows the histological verification to the placement of all injections for the basolateral amygdaloid nuclei in which was infused by HAL or VH. Note:

Sucrose and NaCl (i.e. LiCl’s subject number) groups respectively have five and four rats without an available injection at the target site, so these data were excluded for analysis.

Figure 21. Placement of cannulae aiming at the basolateral amygdaloid nuclei

(VH-NaCl and VH-LiCl, open circles; HAL-NaCl and HAL-LiCl, filled circles;

VH-Sucrose, open squares; HAL-Sucrose, filled squares). Numbers adjacent to each section indicate distances from Bregma (mm) in the anterior-posterior axis. (The plates are from The Rat Brain in Seterotaxic Coordinates by G. Paxinos & C. Watson,

1986. San Diego, CA: Academic Press. Copyright (1986), With Permission from

Elsevier.)

Figure 22. A photomicrograph (at × 40 magnification) represents the location of the injection cannula with a 40-μm section at approximate 2.8 mm posterior to bregma

(Paxinos & Watson, 1986). Ce: Central amygdaloid nucleus. BAL: Basolateral amygdaloid nuclei. opt: optic tract. ic: internal capsule.

80 Each group of Exp 8 received HAL or VH microinfusions into the basolateral nuclei of amygdala prior to freely access to a specific one of gustatory solutions

(NaCl, sucrose, and LiCl) as shown on the mean intake volume between HAL and VH groups.

As shown on Figure 23, for the intake of isotonic NaCl, the HAL-NaCl group did not have a significantly difference on the intake volume than that of the VH-NaCl group (F(1, 13) = 0.25, p > .05; see Figure 23). As can be seen in Figure 24, HAL did not significantly reduce to sucrose solution intake (F(1, 12) = 3.15, p > .05). A group received the HAL-LiCl treatment did not drink significantly different amount of LiCl solution than the VH-LiCl group on the drug treatment session (F(1, 13) = 0.00, p

> .05; see left panel in Figure 25), and the mean difference score of NaCl intake in the

HAL-LiCl group was not different with the VH-LiCl group on the testing session (F(1,

13) = 0.08, p > .05; see right panel in Figure 25).

Figure 23. Effect of bilateral HAL infusion into the basolateral nuclei of the amygdala on the intake volume of NaCl solution during the drug treatment session. Mean (+

SEM) intake volume (in milliliters) of isotonic NaCl on the drug treatment session (15 min) in rats bilaterally infused with HAL or VH into the basolateral amygdala.

VH-NaCl (n = 8), HAL-NaCl (n = 7). VH: vehicle, HAL: haloperidol, 0.15 M NaCl: isotonic sodium chloride.

Figure 24. Effect of bilateral HAL infusion into the basolateral nuclei of the amygdala on the intake volume of sucrose solution during the drug treatment session. Mean (+

SEM) intake volume (in milliliters) of 0.34 % sucrose solution on the drug treatment session (15 min) in rats bilaterally infused with HAL or VH into the basolateral amygdala. VH-NaCl (n = 6) and HAL-NaCl (n = 8). VH: vehicle, HAL: haloperidol,

0.15 M NaCl: isotonic sodium chloride.

Figure 25. Effect of bilateral HAL infusion into the basolateral nuclei of the amygdala on the intake volume of LiCl solution during the drug treatment session and on the difference score of NaCl intake during the testing session. Left panel: Mean (+ SEM) intake volume (in milliliters) of isotonic LiCl for the VH-LiCl (n = 8) and the

HAL-LiCl (n = 7) on the drug treatment session (15 min). Right panel: Mean (+ SEM) difference score (in milliliters) in isotonic NaCl intake for the VH-LiCl (n = 8) and the HAL-LiCl (n = 7) on the testing session (15 min). HAL or VH was bilaterally infused into the basolateral amygdala. VH: vehicle, HAL: haloperidol, 0.15 M NaCl: isotonic sodium chloride, 0.15 M LiCl: isotonic lithium chloride.

84 Discussion

The present results indicated that microinjection of HAL into the amygdaloid central and basolateral nuclei failed to interfere with the intake of NaCl, sucrose, and

LiCl solutions. Thus, it is not consistent my previous findings that the intake of those solutions was disrupted with a peripheral administration of HAL.

Accordingly, several plausible causes are discussed as following. First, D2 receptors in the amygdala did not mediate the reinforcing effect of rewarding or aversive stimuli on the CTA. Instead, other subtype of DA receptors controls this category of function. For instance, Phillips and his co-workers suggested that D3 receptor seemly played a crucial role on the rewarding learning (Hitchcott & Phillips,

1998; Hitchcott, Bonardi, & Phillips, 1997; Phillips, Harmer, & Hitchcott, 2002a;

Phillips, Harmer, & Hitchcott, 2002b). They used the post-session intra-amygdala infusion procedure in which rats learned to associate one neutral stimulus followed sucrose reward and another neutral stimuli paired with no reward, and rats following learning showed discriminative approach behavior. After training, subjects were bilaterally given three subtypes of DA agonists (D1, SKF-38393; D2, quinpirole; D3,

7-OH-DPAT) into the intra-amygdala. They found that only D3 agonist increased the acquisition of discriminative approach for the neutral stimuli with sucrose reward

(Hitchcott et al., 1997). In a follow-up study, they manipulated rats to pre-expose

85 d-amphetamine which enhanced subjects’ learning of Pavlovian appetitive conditioning. After the learning sessions, they bilaterally infused a D3 receptor antagonist, nafadotride, into the amygdala. It appeared that rats infused with nafadotride showed an attenuated pre-exposure sensitization effect in the acquiring

Pavlovian conditioning, which was otherwise intact in the VH treated group (Phillips et al., 2002a). Besides, another line of studies have shown that bilateral injection of

D1 receptor antagonist, SCH-23390, into the amygdala indicated a decrement of response rate for intravenous cocaine reinforcement on a self-administration paradigm

(Caine, Heinrichs, Coffin, & Koob, 1995; McGregor & Roberts, 1993). However, this line of research was mostly restricted to the role of D1 receptors, never examined whether the amygdaloid D2 or other subtype of amygdala DA receptors modulated this behavior. Thus, amygdaloid D1 (but not D2) receptor is a candidate to play a role for mediating the intravenously administrated drugs of abuse by itself.

Second, other neural sites other than the amygdala may probably be involved in this function of D2 receptor related to the aversive and rewarding events. For instance, according to existing neuroanatomical data, it was well known that the ventral striatum has mainly received the afferent projection from the basolateral amygdaloid nuclei (DeFrance, Marchand, Stanley, Sikes, & Chronister, 1980; Kelley et al., 1982;

Price, Russchen, & Amaral, 1987) and also from the subiculum and the frontal cortex

86 in parallel (DeFrance et al., 1980; Kelley & Domesick, 1982; Price et al., 1987).

Everitt, Robbins, and their colleagues used the method of excitotoxic lesions to explore involvement of the pathway from the basolateral amygdaloid nuclei to the ventral striatum related to the signal process of reward. In their series of stimulus-reward associations studies, they found that rats with basolateral amygdala damages reduced their learned responses for the conditioned reinforcement which represented the primary reward, no matter what an amphetamine intra-accumbens infusion (Cador et al., 1989), a second-order sexual reinforcement (Everitt et al.,

1989), and a conditioned place (Everitt et al., 1991). Besides, either bilateral lesions of the ventral striatum or ventromedial caudate-putamen also attenuate the place preference effect (Everitt et al., 1991). Followed up the line of research, they further extend their viewpoint and reported that not only the amygdala-ventral striatum system but the projection from the ventral subiculum to the ventral striatum also mediated the control over behavior by the conditioned reinforcement (Burns, Robbins,

& Everitt, 1993). There are several subsequent studies that employed a selected D2 antagonist and agonist to show that D2 receptors of the nucleus accumbens has a crucial role to mediate the aversive and rewarding effects (Bassareo, De Luca, & Di

Chiara, 2002; Wan & Swerdlow, 1993; Wolterink et al., 1993). Intra-accumbenal infusion of D2 receptor antagonist, raclopride would abolish the conditioned response

87 that was induced by the amphetamine infusion into nucleus accumbens (Wolterink et al., 1993). In addition, infusion of the D2 agonist quinpirole into the nucleus accumbens impaired the prepulse inhibition of the startle reflex (Wan et al., 1993).

Moreover, a recent microdialysis study has demonstrated that intraoral infusion of rewarding sucrose solution could elevate DA levels in the prefrontal cortex, whereas application of aversive taste stimuli generated vigorously releasing in the prefrontal cortex and the core of nucleus accumbens (Bassareo et al., 2002).

Third, an inadequacy of manipulation such as the accuracy of implantation or inadequate drug dosage may result in a non-significant effect of HAL. To address the anatomical question, all brains were cut to a piece of slices, and verified the location of injection for each rat as seen in Figure 16 and Figure 21. Besides, the condition of implanted cannulas for all rats on the central nucleus and basolateral nuclei was shown on appendix 1 to appendix 4. Only data from rats with both cannulas in the target were analyzed in the present study. However, the present study can not rule out the possibility that an effect may be detected with other doses of the drug.

Take together, whether the amygdaloid D2 receptor is involved in the reinforcing effect of both rewarding and aversive stimuli remains to be resolved by future studies.

88 Chapter 8. General Discussion

The present investigation indicated that 0.2 mg/kg HAL could partially attenuate the reduction of saccharin intake while it was given prior to (but not posterior to) the rewarding AMPH or aversive LiCl US on a CTA paradigm. It was proposed that brain

DA system is involved in the US impact whatever the US valence might be. Such findings are consistent with the saliency hypothesis of DA. However, the further data does not fully support the viewpoint, and thus we went forward to access the concept of DA involvement in saliency and unexpectedness of stimuli. The data showed that

HAL (0.1 mg/kg) merely reduced the intake of low sucrose (not high sucrose) on the drug treatment session and blocked the deleterious effect of LiCl on the subsequent intake of NaCl solution on the testing session (the last drug treatment session rats were given HAL before an aversive agent LiCl test), but not affect the intake of low and high quinine and isotonic NaCl solutions even though all used gustatory solutions were exposed to rats for the first time. Thus, it seems to imply that a so-called saliency of stimuli may not be equivalent to the novelty of these stimuli but instead represents the reinforcing potency of US. Moreover, the brain DA system mediates this action. This study further tested the suppression of NaCl after paired with LiCl, and found that HAL could not alter the reduction effect of NaCl intake. It was

89 suspected that the NaCl probably failed to obtain the reinforcing potency of LiCl but simply served as a signal to predict the US’s. To test this conjecture, a sensory preconditioning experiment was proposed. On the study in which HAL was administered centrally, the intra-central and intra-basolateral nuclei HAL microinfusion did not affect the intake to any solutions tested (contained neutral NaCl, rewarding sucrose, and aversive LiCl). The result may be due to several reasons included involvement of other DA receptor subtype within amygdala, the mediation of other brain sites, the inadequacy of manipulation, etc. The issue of the relationship between amygadaloid D2 receptor and aversive as well as appetitive learning remains to be clarified in the future.

Nevertheless, the present data are thought to be consistent with recent studies showing that the brain DA system participates in mediating the stimulus-reward association such as the conditioned place preference (Hoffman et al., 1989; Acquas et al., 1989), and generating instrumental responses to obtain drug administration (Yokel et al., 1975) or for nature reward (Wise et al., 1978) as well as the aversive stimulus learning such as drug-induced place aversion (Di Scala et al., 1989; Acquas et al.,

1989), or taste aversion learning (Caulliez et al., 1996; Fenu et al., 2001), and producing an instrumental response to avoid an aversive consequence (Blackburn &

Phillips, 1990; Sanger, 1986; White et al., 1992).

90 Against the viewpoint of motor impairment

The present results cannot be explained by motor impairment ensuing from DA blockade. That is because if HAL affected the motor ability, the sucrose and LiCl should be both reduced to intake. Instead, the attenuation of HAL on aversive and rewarding US results in responses of opposite direction: HAL-Sucrose decreased the intake of the rewarding sucrose on the drug treatment session and yet HAL-LiCl increased the NaCl intake on the testing session. Such results argue against the notion that HAL could generate the effect by impeding motor performance, but instead suggest that the reinforcing values of US are obliterated. Besides, the dose of 0.1 mg/kg HAL has been demonstrated that this dose did not influence the licking responses to water. Therefore, these results consistently indicate that the attenuated effect of HAL is caused by the blockade of US impact but not of the motor ability.

Hence, the present data in Exp 4 was suggested that DA blocker interfered with the reinforcing effect of US, rather the explanation of motor impairment.

What is a salient stimulus?

Several recent studies examined the function of brain DA neuron related to the neutral stimulus (CS) signaled the further consequence (US) reported that the DA activation is seemly to implicate unexpected and salient stimuli, not restrictedly to the rewarding stimulus (Horvitz, 2000; Redgrave et al., 1999; Schultz et al., 1997).

91 Horvitz and colleagues (1997) suggested that if the magnitude of the stimulus was

great enough, it could elicit burst of activity on the ventral tegmental DA neuron even

that was a neutral stimulus (Horvitz et al., 1997). It was therefore termed as a saliency

hypothesis of DA function to explain the aversive and the strong neutral cases (Huang

& Hsiao, 2002).

However, my data was not fully congruent with the view. Because HAL only

affect the intake of low sucrose and LiCl, but had little influence on quinine and NaCl

even though all taste solutions were the first time given for rats.

To explain this inconsistency, it is probably crucial to compare the characteristics

of gustatory stimuli. Sucrose and LiCl are suggested to have two different properties

(Pfaffmann et al., 1977; Niijima & Yamamoto, 1994): The first is the sensation

property, and the other is the reinforcing effect. The sweet-tasting quality is used as a

CS to generate the conditioned reduction after paired with an aversive US agent

(Ramirez, 1994; Smith & Theodore, 1984). In addition, it is also utilized as a US to

reinforce a relationship with a preceding flavor solution after the taste preference

conditioning (Bolles, Hayward, & Crandall, 1981; Messier & White, 1984). Likewise,

LiCl also has two taste properties. One is the salty-tasting property from the taste buds, and it cannot be discriminated with NaCl (Nachman, 1963). The other is the ailing sense derived from the visceral organ, and it can be used as an aversive US to

92 reinforce the association with CS, and elicit the suppression on CTA (Garcia &

Koelling, 1966).

In contrast, the aversion induced by quinine and LiCl appears to be different though these two gustatory solutions can both generate the rejection responses on the taste reactivity test and decrease the intake volume on the taste preference task.

However, no literatures related to CTA reported that quinine was served as a US rather a CS though it can decrease the lick responses for rats just the same as

LiCl-induced taste aversion (Jones, 1980). Accordingly, quinine and NaCl probably have the only one property of taste quality from the taste buds, but sucrose and LiCl have a reinforcing effect besides a sweet and a salty sensations. In addition, the reinforcing effect is served as not only a US to reinforce the relation with a CS on

Pavlovian conditioning, but also a consequence to reinforce between a stimulus and a response to reflect on approaching and avoiding behaviors on instrumental conditioning (Hergenhahn & Olson, 2001).

Hence, the attenuated effect of HAL is probably due to the blockade of the reinforcing effect of a rewarding sucrose and an aversive LiCl US. The salient stimulus probably implies the reinforcing stimulus, but not the novelty one

(unfamiliarity).

93 Arguments and Issues

The present investigation raises several arguments and issues to be resolved. They are described as below.

1. Confirmation of HAL Effect

In Exp 1, AMPH was used as a US agent, and found that when HAL was injected prior to AMPH, the reduction of saccharin intake was attenuated. Thereby, it was concluded that the brain DA system probably interfered, in part, with the AMPH impact. In light of the result, the suggestion is likely to be argued that attenuated effect may be caused by involvement of other monoamine systems. As demonstrated in previous literatures, AMPH is an indirect agonist. Its action is mainly due to blockade of reuptake in the presynaptic endings of catecholamine neurons, and elicits vigorous releases of DA in the corpus striatum and norepinephrine in the cerebral cortex (Azzaro, Ziance, & Rutledge, 1974; Harris & Baldessarini, 1973). Furthermore, serotonin (5-HT) reuptake is also inhibited under a high concentration of AMPH

(Taylor & Ho, 1978). Accordingly, an experiment can be designed as follows to verify which monoamines mediate the HAL effect. For example, it can be replaced HAL with a monoamine (5-HT, norepinephrine, or epinephrine but not DA) antagonist, and then given prior to the AMPH. If antagonists to other monoamine system cannot attenuate the reduction of saccharin intake by AMPH, the effect of AMPH can be

94 entirely ascribed to the brain DA system. Another method is given a specific DA receptor agonist, which is not involved in other monoamine functions, served as the

US agent (a premise is that the DA agonist is also capable of inducing a suppression to saccharin like as AMPH). HAL is given before the agonist, and then test the saccharin intake. If the suppression of saccharin intake is attenuated, it can be suggested that the HAL effect actually influences the brain DA system.

The two methods as described above can resolve the argument that which kind of neurotransmitter actually mediates the HAL effect.

2. High sucrose is not attenuated by HAL

Results of Exp 4 showed that 0.1 mg/kg HAL could affect low sucrose and LiCl, but did not low and high quinine, NaCl, and high sucrose. It was suggested that the attenuating effect of HAL was probably attributed to the blockade of the reinforcing effect of sucrose and LiCl. It seems plausible because quinine and NaCl under normal conditions was dissimilar to sucrose and LiCl, which were able to be a US and possessed a reinforcing effect. However, yet it seems contradictory to observe that high concentration of sucrose solution.

In a review literature of Hunt and Amit (1987), it was stated that ”…Quantitative

CS variables such as the amount consumed, duration of CS exposure, and concentration of CS solution have been reported to enhance CTA learning…The

95 term ’salience’ was introduced by Kalat and Rozin to describe the finding that taste aversions were more readily formed with certain tastes than others” (p.110.) It seems to imply that taste novelty has a greater potency than taste intensity to influencing

CTA learning. However, taste intensity is also one of factors to influence the magnitude of CTA.

Thus, this conclusion can be applied to explain why the 0.1 mg/kg HAL reduced the low sucrose (0.34 %) but not the high sucrose (10 %). That is because the latter sucrose solution is relatively strong 0.1 mg/kg HAL is not high enough to attenuate the reinforcing effect of high sucrose. Thereby, if it can be given another kind of high dose of DA antagonist, which does not affect subjects’ motor movement, it will be expected to reduce the intake of high sucrose as well as those of low sucrose. Hence, the intake of high sucrose was not affected under the 0.1 mg/kg HAL treatment does not imply that the high sucrose had no reinforcing effect.

3. Relativity of Reinforcement

The present data indicated that HAL seems only to attenuate reinforcement in a typical case of US agent such as sucrose, AMPH, and LiCl. Conversely, it did not alter the intake of CS like as quinine and NaCl in normal. However, some questions should be considered: What qualify as a reinforcer? Whether is the CS (NaCl) able to have the reinforcing effect in some special condition? That is, is the reinforcement a

96 relative or absolute effect? For the typically operational definition of reinforcement, most literatures always stated that it was a tendency of certain stimuli to strength a relation between a stimulus and a correspondent response or a stimulus and another stimulus, and produced a relatively durable change in behavior (Clayton, 1969;

Hergenhahn et al., 2001; White, 1989). Yet, this definition of reinforcer is in question under David Premack’s concept of reinforcement. He suggested that all behaviors could be ranked by its frequency in a hierarchical order. The opportunity to engage in more frequent behavior can reinforce the less frequent behavior, which has been denoted as the termed Premack principle (Hundt & Premack, 1963; Premack, 1959;

Premack, 1962; Premack, 1971). Thus, according to the Premack principle, reinforcement is relative rather than absolute.

Although the Premack principle focus on the responses, the similar concept can be seen on the data that stimulus severed as reinforcement. For example, in a normal condition, NaCl is not thought to have a capacity of reinforcement. However, if an animal is in a sodium-deprived or an adrenalectomized conditions, NaCl can reinforce a correspondent response, and change its behavior (Lewis, 1960; Wagman, 1963).

Thus, the reinforcement is context-dependent, but not absolute. Only few stimuli, that maybe critical to animals’ survive on ontogeny or phylogeny, possess a reinforcing value in origin. Others have got the reinforcing effect later.

97 Accordingly, if HAL is injected prior to NaCl for a sodium-deprived rat, perhaps the intake of NaCl solution will decrease.

4. A Probable Cause Regards to Generate Animals’ Approaching and Avoiding

Behaviors

If the brain DA system to mediate the reinforcing effect of both aversive and rewarding stimuli to associate the relation between a stimulus and a response or a stimulus and another, what does something regulate animal’s approaching and avoiding responses?

Recently, Horvitz (2002) has reviewed a lot of single-unit and brain image data.

He found that after animals exposed a salient stimulus, it would activate the DA neurons of substantia nigra (SN) and ventral tegmental area (VTA). The activation of

DA neuron (on SN and VTA) not only mediated the sensory, motor, and incentive signals to dorsal and ventral striatum, but also simultaneously modulated the glutamate sensory and motor information from the orbitofrontal cortex and basolateral amygdala to striatum. He suggested that the latter pathway is involved in a normal response execution. Accordingly, it can be known that if loss the nigrostriatal and mesolimbic DA neuron, it would result in an abnormal processing of sensory, motor, and incentive information to striatum (included the glutamate functioning of orbitofrontal cortex and basolateral amygdala to striatum), and produced animals

98 cannot execute a correct response for its relative stimulus (i.e. approaching to food or avoiding to a footshock).

Thus, according to this view, I speculate that the glutamate system may probably be involved in the function of an appropriate motor execution.

Conclusion

The present series of studies have shown that the brain DA system mediated the impact of US regardless of its aversive or a rewarding valence, and thus revised the previous anhedonia hypothesis of DA blocking in general without the valence of stimulus. Besides, the salient stimuli hypothesis of brain DA is to substitute the anhedonia hypothesis. However, the data indicated that so-called salient stimuli did not mean the taste unfamiliarity (novelty). Rather, it is probably the reinforcing potency of stimuli. Taken together, brain DA system probably plays a crucial role in the reinforcing effect of stimuli. However, whether a stimulus has a reinforcing value seems to depend on the animals’ condition, except for a few primary reinforcers.

Some new issues emerge from our study and await further investigation.

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131 Appendix 1. Histological verification: The central nucleus of the amygdala

ID No target Unilateral target location Bilateral target location Left Right location 1g1 ＊ 2g1 ＊ 3g1 ＊ 4g1 ＊ 5g1 ＊ 6g1 ＊ 7g1 ＊ 8g1 ＊ 1g2 ＊ 2g2 ＊ 3g2 ＊ 4g2 ＊ 5g2 ＊ 6g2 ＊ 7g2 ＊ 8g2 ＊

Note: g 1 represents the group that was given vehicle prior to NaCl, whereas g 2 was injected HAL before NaCl. Besides, one week later, the same rats received the identical drug treatment and then LiCl test. 「＊」 indicates the condition of injected location.

132 Appendix 2. Histological verification: The central nucleus of the amygdala

ID No target Unilateral target location Bilateral target location Left Right location 1g3 ＊ 2g3 ＊ 3g3 ＊ 4g3 ＊ 5g3 ＊ 6g3 ＊ 7g3 ＊ 8g3 ＊ 1g4 ＊ 2g4 ＊ 3g4 ＊ 4g4 ＊ 5g4 ＊ 6g4 ＊ 7g4 ＊ 8g4 ＊

Note: g 3 and g 4 was respectively injected HAL and vehicle before sucrose. 「＊」 indicates the condition of injected location.

133 Appendix 3. Histological verification: The basolateral nuclei of the amygdala

ID No target Unilateral target location Bilateral target location Left Right location 1g1 ＊ 2g1 ＊ 3g1 ＊ 4g1 ＊ 5g1 ＊ 6g1 ＊ 7g1 ＊ 8g1 ＊ 9g1 ＊ 1g2 ＊ 2g2 ＊ 3g2 ＊ 4g2 ＊ 5g2 ＊ 6g2 ＊ 7g2 ＊ 8g2 ＊ 9g2 ＊ 10g2 ＊

Note: g 1 represents the group that was given vehicle prior to NaCl, whereas g 2 was injected HAL before NaCl. Besides, one week later, the same rats received the identical drug treatment and then LiCl test. 「＊」 indicates the condition of injected location.

134 Appendix 4. Histological verification: The basolateral nuclei of the amygdala

ID No target Unilateral target location Bilateral target location Left Right location 1g3 ＊ 2g3 ＊ 3g3 ＊ 4g3 ＊ 5g3 ＊ 6g3 ＊ 7g3 ＊ 8g3 ＊ 9g3 ＊ 1g4 ＊ 2g4 ＊ 3g4 ＊ 4g4 ＊ 5g4 ＊ 6g4 ＊ 7g4 ＊ 8g4 ＊ 9g4 ＊ 10g4 ＊

Note: g 3 and g 4 was respectively injected vehicle and HAL before sucrose. 「＊」 indicates the condition of injected location.

135