Rewarding brain stimulation induces only sparse Fos-like immunoreactivity in dopaminergic neurons

Pergamon

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Neuroscience Vol. 83, No. 2, pp. 501–515, 1998 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00409-0

REWARDING BRAIN STIMULATION INDUCES ONLY SPARSE FOS-LIKE IMMUNOREACTIVITY IN DOPAMINERGIC NEURONS G. E. HUNT*‡ and I. S. MGREGOR† *Department of Psychological Medicine, University of Sydney Clinical Sciences Building, Concord Hospital, Concord, N.S.W., Australia 2139 †Department of Psychology, University of Sydney, Sydney, Australia 2006 Abstract––In this study, c-fos immunohistochemistry was used to identify the brain regions activated by rewarding brain stimulation in rats. Rats had monopolar electrodes implanted in the medial forebrain bundle and were allocated to either a self-stimulation (n=4), yoked stimulation (n=4) or no stimulation (n=6) group. In a single 1 h test session, each rat in the self-stimulation group made 1000 nose poke responses with each response followed by a 0.5 s train of brain stimulation. Rats in the yoked-stimulation group were paired with a partner in the self-stimulation group and received brain stimulation whenever their partner did. However, their nose poke responses did not trigger stimulation. This yoked procedure was thus used to identify any Fos-like immunoreactivity due to operant responding. Rats in the no stimulation group were placed in the same apparatus as the other rats but received no brain stimulation and were thus used to assess baseline Fos-like immunoreactivity. Results showed that stimulation increased Fos-like immunoreactivity in many areas of the brain in both the self-stimulation and yoked groups. The areas with the highest Fos-like immunoreactivity were ipsilateral to the electrode site and included the medial prefrontal cortex, lateral septum, nucleus accumbens (shell), the medial and lateral preoptic areas, bed nucleus of the stria terminalis, central amygdala, lateral habenula, dorsomedial hypothalamus, lateral hypothalamus and the anterior ventral tegmental area. Bilateral Fos-like immunoreactivity was evident in the nucleus accumbens core, paraventricular nucleus of the hypothalamus, the retrorubral fields and the locus coeruleus. A double-labelling procedure identifying both Fos and tyrosine hydroxylase was used to show that very few (<5%) of the A10 dopamine cell bodies in the ventral tegmental area expressed Fos following brain stimulation. In contrast, most of the noradrenergic neurons of the locus coeruleus (A6), rubrospinal tract (A5) and pontine tegmental area (A7) were Fos positive. Overall, the results show that rewarding brain stimulation induces Fos-like immunoreactivity in many forebrain regions but only sparsely in mesolimbic and mesocortical dopamine neurons. The similar patterns of Fos-like immunoreactivity seen in the self-stimulation and yoked-stimulation groups suggests that the operant responding for brain stimulation causes minimal Fos expression in itself. ? 1998 IBRO. Published by Elsevier Science Ltd. Key words: c-Fos, immediate early genes, lateral hypothalamus, self-stimulation, reward, dopamine.

The self-stimulation phenomenon, in which animals work to administer trains of electrical stimulation to their own brains, has long fascinated psychologists and neuroscientists alike. A large body of work has attempted to identify the neuroanatomical basis of self-stimulation, with the promise that this may in turn shed light on the neural processes underlying the reinforcing properties of natural rewards and drugs of abuse.20,65 Most of these studies have focused on medial forebrain bundle (MFB) self-stimulation which arguably gives rise to the most vigorous self-stimulation of any brain region.40,70 Numerous lesion studies have been performed in an attempt to find the critical brain region that ‡To whom correspondence should be addressed. Abbreviations: 2-DG, 2-deoxyglucose; Fos-LI, Fos-like immunoreactivity; LH, lateral hypothalamus; MFB, medial forebrain bundle; PBS, phosphate-buffered saline; TH, tyrosine hydroxylase; VTA, ventral tegmental area.

subserves MFB self-stimulation. The greatest inhibition of self-stimulation was seen with destruction or isolation of the lateral preoptic area or with knife cuts immediately caudal to the site of stimulation.19,31,32,44 However, lesion studies bring inevitable problems of interpretation and this has led to a search for alternative means of verifying the key neural substrates for self-stimulation. The use of 2-deoxyglucose (2-DG) or cytochrome oxidase has allowed visualization of areas activated by brain stimulation5,16,18,58,59 and has indicated that MFB or ventral tegmental area (VTA) selfstimulation increases metabolic activity in many projection areas of the mesolimbic and mesocortical dopamine pathways including the medial prefrontal cortex, nucleus accumbens, olfactory tubercle and lateral septum.16,58,59 These results, coupled with pharmacological studies showing inhibition of selfstimulation by dopamine antagonists,30,40,79,80 have

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suggested a central role for the mesolimbic and mesocortical dopamine pathways in mediating self-stimulation. More recent studies of the neuroanatomical basis of self-stimulation have employed the technique of c-Fos immunohistochemistry, which offers superior spatial resolution to the 2-DG technique and superior temporal resolution to the cytochrome oxidase method.43,61 An initial study reported increased Fos expression all along the MFB, nucleus accumbens, vertical limb of the diagonal band and medial septum in two rats after experimenter-delivered stimulation of the VTA.62 A further study showed that electrical stimulation of the MFB in anaesthetized rats increased Fos expression in the nucleus accumbens shell, major islands of Calleja, lateral septum, but not in the motor related structures of the medial or lateral striatum or nucleus accumbens core.9 Two more recent studies reported increased Fos expression after multiple self-stimulation training sessions in several forebrain structures implicated in rewarding brain stimulation1,52 and two other brief reports describe similar findings.8,17 The present study was designed to further identify the neurons activated by MFB self-stimulation with the use of three innovations compared to previous studies. Firstly, the effects of stimulation were assessed in rats on their first exposure to brain stimulation. Previous studies have largely assessed Fos immunoreactivity following multiple selfstimulation sessions.1,17,52 However, this may lead to less Fos being expressed8 similar to the reduced Fos immunoreactivity seen following repeated drug injections.10,15,27,60 To produce rapid acquisition of self-stimulation, a nose-poke response was used because rats learn this more quickly than leverpressing.21 In addition, stimulation parameters were selected from previous studies that produced a substantial rewarding effect without eliciting unwanted motor movements or seizures.4,29 The second innovation in the present study was to use an antibody directed against tyrosine hydroxylase (TH) to identify which catecholaminergic neurons express Fos following self-stimulation.25 If the dopaminergic theory of self-stimulation is correct,40,70,78–80 then it might be reasonably inferred that Fos expression will be seen in the somata of mesolimbic and mesocortical neurons located in the VTA as well as in the projection areas of these neurons. The present study also quantified Fos expression in non-dopaminergic brain regions, either because they have been implicated in rewarding brain stimulation (such as the medial and lateral preoptic areas)25,65,75 or because they have been shown to be responsive to stressful or aversive events (such as the paraventricular nucleus of the hypothalamus and locus coeruleus).13,14,57,63 A final feature of the present study was the inclusion of a control group that received yoked (non-contingent) stimulation. Previous studies have

not usually included such a control making it uncertain the extent to which Fos expression seen with self-stimulation reflects the simple act of responding for stimulation. The design involved allowing one rat to self-stimulate while another rat in an adjacent chamber received yoked stimulation independently of its behaviour. A third group consisted of rats placed in the chamber with the stimulator switched off in order to quantify baseline Fos expression for rats exposed to a novel environment.23,53 EXPERIMENTAL PROCEDURES

Subjects The subjects were male Wistar rats (SPF Breeding Facility, Sydney) weighing 350–425 g at the time of surgery. They were housed individually in a temperature controlled room (20&2)C) and had free access to food and water. All testing occurred during the light hours of a 12 h light/dark cycle. The study was approved by the Animal Welfare Committee of the Central Sydney Area Health Service and carried out in accordance with the guidelines of the National Health and Medical Research Council of Australia. Surgery and testing The rats were anaesthetized with an intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (2 mg/kg). They were then positioned in a stereotaxic apparatus and implanted with monopolar electrodes as described previously.29 The electrode tip was aimed at the lateral hypothalamic component of the MFB using level-skull coordinates of 2.8 to 3.3 mm posterior to Bregma, 1.6 mm lateral to the midline and 8.7 mm ventral from the superior surface of the skull. An uninsulated stainless steel wire wrapped around two of the four screws retaining the acrylic skull cap served as the indifferent wire. The rats were handled three times a week during a four week postsurgical period and were monitored for weight gain. Prior to their first and only 1 h test session, the rats were randomly allocated into three groups: self-stimulation (n=4), yoked stimulation (n=4) and no stimulation (n=6). The self-stimulation apparatus consisted of two identical testing chambers (Coulbourn Instruments). Noses pokes into a 28 mm diameter hole in the front panel on the chamber were detected by a photobeam, and triggered the delivery of brain stimulation to rats in the self-stimulation group and to their yoked partner in the adjacent chamber. Nose pokes by rats in the yoked-stimulation or no stimulation groups were without consequence. The stimulation consisted of 0.5 s trains of 100 Hz cathodal rectangular pulses, of 0.2 ms pulse duration and 0.2 mA amplitude. A constant-current stimulator (A-310 Accupulser and A-365 stimulus isolators, World Precision Instruments) delivered the brain stimulation through mercury-filled commutators via connecting leads. The stimulation was continuously monitored on an oscilloscope as a voltage drop across a 1 KÙ resistor. A personal computer controlled the experimental process and recorded all events and event times. A maximum of 1000 stimulation trains were available to rats in the self-stimulation group during the 1 h test session. The stimulation was delivered under a fixed-interval 1 s schedule of reinforcement in order to avoid extreme density of stimulation that might lead to seizures. Rats in the no stimulation group were placed in the operant chambers for 1 h but did not receive any stimulation during their test session. On completion of the test session, the rats were returned to their home cages.

Fos-like immunoreactivity and rewarding brain stimulation Immunohistochemistry Rats remained in their home cages for a period of 60 or 90 min after the self-stimulation session, and were then deeply anaesthetized with pentobarbitone (120 mg/kg, i.p.). They were then perfused transcardially with 100 ml of 0.1 M phosphate-buffered saline (PBS) followed by 250 ml of 4% paraformaldehyde in PBS (pH 7.3). The brains were removed and a small sagittal incision made on the left side of the brain to allow subsequent identification of the hemisphere containing the electrode. The brains were placed in paraformaldehyde overnight at 4)C, then blocked in the coronal plane and stored in cold 30% sucrose for 72 h. Tissue blocks were then placed on microtome stages, frozen to "17)C, sliced at 40 µm and serial sections were then placed in PBS. Free-floating sections were incubated for 30 min in 1% hydrogen peroxide in PBS and then for 30 min in 3% normal horse serum in PBS. They were incubated in the primary Fos antibody (sheep polyclonal; Cambridge Research Biochemicals, OA-11-823, directed against residues 2–16 of the N-terminal region of the Fos molecule) diluted 1:2000 in phosphate-buffered horse serum (0.1% bovine serum albumin, 0.2% Triton X-100, 2% normal horse serum in PBS) for 72 h at 4)C. Sections were washed for 30 min in PBS at room temperature and then incubated for 1 h in biotinylated anti-sheep IgG (Vector Laboratories; diluted 1:500) in phosphate-buffered horse serum. They were then washed in PBS for a further 30 min and then incubated for 2 h in ExtrAvidin–horseradish peroxidase (Sigma; diluted 1:1000 in phosphate-buffered horse serum). After three 30 min washes in PBS, horseradish peroxidase activity was visualized with the nickel diaminobenzidine and glucose oxidase reaction as described previously.66 This reaction was terminated after approximately 10 min by washing in PBS. For double labelling of cells, tyrosine hydroxylase (TH) (mouse monoclonal; IncStar; diluted 1:10,000) was added at the same stage as the primary Fos antibody. Following completion of the Fos processing described above, the tissue was further processed using the same procedure described for Fos except that anti-mouse IgG was used for the secondary antibody and nickel ammonium chloride was not included in the visualization process.68 This produced TH immunoreactivity in catecholamine-containing neurons, visualized as an amber immunoprecipitate.25 The sections were then mounted onto subbed slides, dehydrated, xylene-cleared and coverslipped. Some sections were Nissl stained with Toludine Blue O or Cresyl Fast Violet to help identify electrode tip placements.

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contralateral side and this group would also have more Fos-LI nuclei compared to rats in the no-stimulation or yoked-stimulation groups. The four planned contrasts were made for numbers of Fos-LI nuclei in the (i) ipsilateral side of the self-stimulation group versus the no stimulation group, (ii) contralateral side of the self-stimulation group versus the no stimulation group, (iii) ipsilateral versus contralateral sides of the self-stimulation group, and (iv) ipsilateral side of the self-stimulation group versus the yoked-stimulation group. Where homogeneity of variance between groups was not evident using the Levine test, the probability of rejecting the null hypothesis (P<0.05) was based on the separate variance estimate. RESULTS

Behavioural testing On being placed in the chamber, most rats explored the novel environment with vigorous sniffing and occasionally placed their nose in the nose-poke recess. For the self-stimulation group, the nose poke initiated the electrical brain stimulation and within the first 10 min of the test session all four rats in this group began self-stimulating (Fig. 2). By 20 min all four rats were vigorously nose-poking and did little else until the 1000 reinforcement criterion was met after approximately 40 min. In the adjacent chamber, the yoked-stimulation rats showed continuous locomotion, sniffing, rearing, licking and occasionally showing short bouts of grooming and nose pokes (the mean number of nose pokes over the session was 9). In contrast, the control rats that did not receive any stimulation, moved around the chamber for the first 10–20 min, placing their snout in the nose-poke recess a few times (mean of 7), and then rested or groomed for the remainder of the 60 min session. Electrode locations Histological analysis revealed that all 14 of the electrode tips were located in the MFB within the boundaries of the lateral hypothalamus (LH) (Fig. 3).

Counting of labelled cells The Fos antibody used produces a black oval-shaped immunoprecipitate confined to the nucleus of neurons. Since the antibody used recognizes Fos as well as other Fos-related antigens, the term Fos-like immunoreactivity (Fos-LI) is used to describe this staining. In control procedures, no Fos-LI was evident after omission of the primary antibody. Fos-LI labelling was quantified microscopically at 21 brain sites in each hemisphere (Fig. 1). At 200# magnification, a 0.5 mm square graticule was placed over the region of interest using a brain atlas for guidance.54 Counting of Fos-LI positive nuclei within the graticule was then performed by an observer who was blind to group assignment. Only darkly-labelled oval-shaped nuclei that fell within the grid were counted. One-way ANOVA with planned comparisons (SPSS for Windows, Release 6.1) was used to compare the number of Fos-LI nuclei in different brain sites across the three experimental groups. It was hypothesized that the self-stimulators would have greater amounts of Fos-LI nuclei on the ipsilateral side to the stimulating electrode compared to the

Fos-like immunoreactivity in the no stimulation group Very little Fos-LI labelling was seen in the brains of the six unstimulated control rats in any of the 21 regions of interest (Table 1). The highest Fos-LI cell counts (10–25 cells) were seen in the medial prefrontal cortex, orbital cortex, lateral septum, lateral habenula and dorsomedial hypothalamus. Examples of sparse Fos-LI labelling for one representative rat are shown in Figs 4–7. Fos-like immunoreactivity in the self-stimulation group There were no significant differences in Fos-LI between rats perfused 60 or 90 min after the test session within each group, so the data were pooled. Compared to control rats, prominent Fos-LI was found in most of the 21 regions of interest in the

Fig. 1. Schematic diagrams of the 21 areas that were used to count Fos-like immunoreactivity adapted from the atlas of Paxinos and Watson.54 Open squares indicate the placement of the 0.5 mm2 grid from which the counts were made. For explanation of abbreviations see Table 1.

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Fig. 2. Cumulative graphs of the four rats in the selfstimulation group during their first and only 1 h exposure in an operant chamber. Placing their snout in a nose-poke recess was reinforced with a 0.5 s train of electrical stimulation. Response rates were low at the beginning of the session while the rats explored the novel environment. High rates were uniformly achieved for the group after 10 min elapsed illustrated by the sharp rise in the cumulative records. Vigorous responding continued uninterupted until the 1000 reinforcement criterion was attained (dotted line) except for one rat that temporarily stopped responding for 5 min toward the end of the interval because he was grooming. Each rat from this group had a yoked-partner in an adjacent chamber that received the same electrical stimulus passively without the need to respond.

self-stimulation group (Table 1). Only two ipsilateral areas did not show significantly elevated Fos-LI after self-stimulation relative to unstimulated controls (Table 1, planned comparisons marked with *), namely the lateral aspect of the caudate–putamen and the olfactory tubercle. The highest densities of Fos-LI nuclei (>100 cells/ 0.5 mm2) were observed on the side of the brain ipsilateral to the stimulating electrode within the dorsomedial nucleus of the hypothalamus, lateral septum, nucleus accumbens shell, the paraventricular nucleus of the hypothalamus and the orbital cortex. Moderate densities of Fos-LI cells (50–100 cells/ 0.5 mm2) were observed within the LH, medial and lateral preoptic areas, the bed nucleus of the stria terminalis, the medial prefrontal cortex, the anterior VTA and the locus coeruleus. Other areas with significantly more Fos-LI nuclei (15–50 cells/ 0.5 mm2) included the nucleus accumbens core, medial caudate–putamen, central amygdala, lateral habenula, the ventromedial nucleus of the hypothalamus, the retrorubral fields and the VTA. Examples of Fos-LI labelling for one representative rat from the self-stimulation group are shown in Figs 4–7. Fos-LI was also present in many brain regions contralateral to the stimulating electrode. Compared to no stimulation control rats, there were eight brain regions that had significantly more Fos-LI nuclei on the contralateral side after self-stimulation (Table 1, comparisons marked with †). These areas included the nucleus accumbens (core and shell), the lateral septum, the paraventricular nucleus of the hypotha-

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lamus, the dorsomedial nucleus of the hypothalamus, the anterior VTA, the retrorubral fields and the locus coeruleus. An asymmetric distribution of Fos-LI neurons was evident in several brain regions when comparing the ipsilateral and contralateral sides (Table 1, comparisons marked with ‡). The structures with more Fos-LI on the ipsilateral side (ratios>3:1) were the LH, bed nucleus of the stria terminalis, nucleus accumbens shell, the medial and lateral preoptic areas, the central amygdala and the lateral habenula. Several other structures had ratios between 2:1 and 3:1 favouring the ipsilateral side including the orbital cortex, lateral septum, the dorsomedial and ventromedial hypothalamus, and the VTA. Fos-like immunoreactivity in the yoked-stimulation group Most of the areas that had high densities of Fos-LI nuclei in the self-stimulation group also had a high number of Fos-positive nuclei in the yokedstimulation group. There were only five areas in which the self-stimulators had significantly more Fos-LI on the ipsilateral side than the yoked group (Table 1, comparisons marked with §). These were the lateral preoptic area, the LH, the lateral habenula, the dorsomedial hypothalamus, and the anterior VTA. In addition, many of the structures that showed greater labelling on the ipsilateral side in the self-stimulation group also showed this asymmetric distribution in the yoked-stimulation group. Fos labelling in tyrosine hydroxylase-positive neurons Co-staining of Fos-LI and TH was seen in a small proportion of A10 dopamine neurons in the VTA (Fig. 8A, B). Fos-LI nuclei were present in numerous neurons in the retrorubral fields but these neurons were not co-stained with TH. However, many of the Fos-LI nuclei were closely co-distributed with the A8 dopamine neurons (Fig. 8C, D). In contrast, all of the Fos-LI nuclei located in the locus coeruleus were co-labelled with TH (Fig. 8E, F). Other TH-positive neurons that expressed Fos-LI included many of the A5 and A7 noradrenergic neurons located in the brain stem and a few A12 dopamine neurons in the arcuate nuclei (data not shown). Double labelling was not evident in A11, A13 or A14 dopaminergic cells in and around the hypothalamus. DISCUSSION

The present experiment demonstrates that a single session of MFB self-stimulation in rats induces a distinctive pattern of Fos-LI. Since there was only sparse Fos-LI in the brains of unstimulated control rats, it is clear that electrode implantation or exposure to a novel environment are not responsible for the pattern of Fos-LI seen with self-stimulation.

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amygdala, lateral habenula, and VTA following rewarding brain stimulation.1,8,17,52 Taken together, these studies show a consistent pattern of Fos-LI after rewarding brain stimulation despite the use of different tasks, training procedures, sampling times, stimulation parameters and electrode sites. They are also in general agreement with studies based on the 2-DG method which show that many structures along the path of the MFB50 are activated by self-stimulation.18,59 There are, however, some differences between the present results and previous studies. Most notable was the failure of the present study to find increased Fos-LI in the olfactory tubercle of stimulated rats, a result that contrasts with previous studies showing increased Fos expression in the olfactory tubercle after VTA self-stimulation8,17 or increased 2-DG utilization in the olfactory tubercle after LH self-stimulation.37,59 Differences between self-stimulation and yoked stimulation

Fig. 3. Schematic diagrams of the electrode placements for the 14 rats used in this experiment. Filled stars indicate placements for the rats in the self-stimulation group, filled circles for the yoked-stimulation group and open circles for the no stimulation group. All of the placements for the rats that received electrical stimulation were within the boundaries of the medial forebrain bundle.

The highest densities of Fos-LI was observed in regions on the same side of the brain as the stimulating electrode. These areas included the medial prefrontal cortex, orbital cortex, lateral septum, nucleus accumbens (shell), the medial and lateral preoptic areas, bed nucleus of the stria terminalis, central amygdala, lateral habenula, dorsomedial hypothalamus, ventromedial hypothalamus, LH and the anterior VTA. Bilateral Fos-LI was evident in the nucleus accumbens core, paraventricular nucleus of the hypothalamus, the retrorubral fields and the locus coeruleus. These findings confirm and extend previous studies reporting increased Fos expression in the medial prefrontal cortex, lateral septum, nucleus accumbens, bed nucleus of the stria terminalis, parts of the

The present study shows differences in Fos-LI between the self-stimulation and yoked-stimulation groups in five areas ipsilateral to the stimulating electrode, namely the lateral preoptic area, lateral habenula, LH, dorsomedial hypothalamus, and anterior VTA. In each case, more Fos-LI was seen in the self-stimulation rather than the yokedstimulation group. Since the major behavioural difference between these group was the high levels of operant responding seen in the self-stimulation group, it seems logical to conclude that these five regions may be involved in the initiation and maintenance of such responding. However, it may also be the case that differences in Fos-LI between these two groups may in some way relate to the rewarding impact of the stimulation. Previous studies suggest that rats prefer to self-administer brain stimulation rather than have it delivered passively.71 At least one other recent study has also shown differences in Fos expression in self-stimulating compared to passively stimulated rats, although this study involved self-stimulation of the ventral pallidum and did not involve a strict yoking procedure.52 However, an earlier 2-DG study found no difference in 2-DG utilization between self-stimulators and their yoked partners,18 while another such study reported increased 2-DG uptake in the medial prefrontal cortex and nucleus accumbens of self-stimulating rats in comparison to yoked controls.58 These results using 2-DG are both discrepant with the present findings. Whether this discrepancy reflects different electrode placements, training procedures, stimulation configurations or differences between the metabolic maps produced by 2-DG autoradiography and Fos immunohistochemistry13,64 is unknown. Large discrepancies between Fos expression and the 2-DG

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Table 1. Fos-like immunoreactive cells per 0.5 mm2 (Mean&S.E.M.) within different brain areas ipsilateral and contralateral to the electrode for the three groups of rats No stimulation (n=6) Area 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

MPC Orbital NAC CPu-L CPu-M LS NAC Tu BST LPO MPA PVN Amg LH LHb DMH VMH VTA VTA RRF LC

Intracranial self-stimulation (n=4)

Yoked stimulation (n=4)

Bregma

Ipsilateral

Ipsilateral

Contralateral

Ipsilateral

Contralateral

+3.7 +3.7 +2.7 +0.7 +0.7 +0.7 +0.7 "0.3 "0.3 "0.3 "0.3 "1.8 "2.3 "2.8 "2.8 "2.8 "2.8 "4.8 "5.3 "6.7 "9.8

20&4 23&3 8&2 0&0 5&1 17&7 6&1 2&0 6&2 3&2 9&2 6&1 3&2 7&3 13&3 12&4 4&1 9&3 1&0 2&1 1&0

69&20* 112&37* 29&6* 1&0 43&17* 164&20* 141&30* 6&2 75&13* 65&7* 75&30* 130&30* 46&19* 93&27* 41&13* 190&10* 32&5* 67&12* 16&6* 26&11* 71&5*

31&9 50&16‡ 22&5† 1&1 7&3 58&6†‡ 43&4†‡ 4&2 11&2‡ 11&5‡ 22&8‡ 79&22† 7&2‡ 13&4‡ 12&2‡ 95&16†‡ 13&4‡ 35&2†‡ 7&1‡ 23&10† 63&8†

66&15 57&12 23&5 2&1 33&19 167&56 110&37 4&2 74&20 27&8§ 59&12 120&16 42&25 32&5§ 18&5§ 110&26§ 43&25 30&4§ 12&3 17&5 50&16

37&10 35&17 21&6 0&0 22&17 40&9 39&9 2&2 14&4 12&4 17&4 39&6 6&3 6&2 7&1 65&10 10&2 11&1 7&2 13&5 38&8

One-way ANOVA, d.f.(4,17), planned comparisons: *P<0.05 no stimulation vs self-stimulation group, ipsilateral hemisphere; †P<0.05 no stimulation vs self-stimulation group, contralateral hemisphere; ‡P<0.05 ipsilateral vs contralateral side within the self-stimulation group; §P<0.05 ipsilateral sides self-stimulation vs yoked-stimulation group MPC, medial prefrontal cortex; orbital, orbital cortex; NAC, nucleus accumbens-core; CPu-L, caudate–putamen-lateral; CPu-M, caudate–putamen-medial; LS, lateral septum; NAC, nucleus accumbens-shell; Tu, olfactory tubercle; BST, bed nucleus of the stria terminalis; LPO, lateral preoptic area; MPA, medial preoptic area; PVN, paraventricular nucleus of the hypothalamus; Amg, central nucleus of the amygdala; LH, lateral hypothalamus; LHb, lateral habenula; DMH, dorsomedial hypothalamus; VMH, ventromedial hypothalamus; VTA, ventral tegmental area-anterior; VTA, ventral tegmental area; RRF, retrorubral fields; LC, locus coeruleus.

method can be explained by the different cellular compartments visualized by the two techniques.64 Fos labelling is confined to the cell nucleus close to the region that gives rise to the nerve impulse and is induced by specific signal transduction mechanisms like increased intracellular levels of calcium,24 whereas 2-DG images reflect metabolically-activated nerve terminals, glial elements and other neuronal processes that constitute neuropil. Moreover, 2-DG has the capacity for labelling all brain regions involved in a given behaviour, whereas Fos labelling may only be induced in a few subsets of neurons in a given pathway.61,64 Fos-like immunoreactivity in catecholamine-containing neurons The high density of Fos-LI just posterior to the electrode dissipated rapidly from the anterior VTA to more posterior levels of the VTA (Fig. 7). Therefore in the medial portion of the VTA ("5.2 to "6.3 mm bregma), where most of the A10 dopamine neurons are located,25 there were very few Fos-LI cells (mean=16 cells/0.5 mm2, range 5–35) and many of these Fos-positive cells were not co-labelled with TH (Fig. 8B). In addition, none of the A8 or A9 dopamine neurons expressed Fos-LI after selfstimulation even though there were many Fos-

positive cells co-distributed within the TH-rich areas of the retrorubral fields (Fig. 8D) and the lateral aspects of the substantia nigra. In contrast, most of the A6 noradrenaline neurons in the locus coeruleus (Figs 6F, 8F) and many of the A5 and A7 neurons in the brainstem expressed Fos-LI following brain stimulation. TH labelling was much darker in the groups that received electrical stimulation making it difficult at times to distinguish double labelling in the densely packed cells of the VTA, especially if the Fos-like protein was not stained black. Previous investigators3 have noted the same difficulty and reported that few of the A10 dopamine cells expressed Fos following male sexual activity in hamsters. Even though VTA neurons are activated during rewarding selfstimulation and sexual activity as shown by increased dopamine release in the striatum,48,76 this activation may not be sufficient to cause Fos-LI. It is unlikely that these dopamine neurons lack the phenotype necessary to synthesize Fos12 since several studies have shown Fos-LI in TH-labelled cells of the VTA following aversive procedures such as formalin injection into the forepaw,41 electroacupuncture of the hindlimb42 and restraint stress.11 Many pharmacological studies have suggested that the ascending dopaminergic system is critically involved with self-stimulation since dopamine

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Fig. 4. Low magnification (100#) photomicrographs illustrating the distribution of Fos-LI in the nucleus accumbens (NAC) shell (A, B), the bed nucleus of the stria terminalis (BST) medial division (C, D) and the medial preoptic area (MPA) (E, F) from a rat that received no stimulation (A, C, E) and from the ipsilateral side of a rat after self-stimulation (B, D, F). LV, lateral ventricle; aca, anterior commissure, anterior; ac, anterior commissure; 3V, 3rd ventricle. Scale bar=200 µm.

antagonists attenuate the rewarding efficacy of brain stimulation.30,40,79,80 Thus, it has been proposed that the directly-stimulated neurons responsible for the primary rewarding effect of intracranial selfstimulation integrate with the ascending dopaminergic projections at some stage to form a final common pathway for all rewards.35,36,77,80 If this hypothesis is correct then various stimuli that are positively reinforcing should activate this common system. The present data partly support this

hypothesis since many of the areas that express Fos after rewarding brain stimulation also display Fos-LI after acute cocaine or amphetamine injections including the nucleus accumbens, lateral septum, dorsomedial striatum and amygdala.7,22,27,49,51,55 However, there are important differences in the distribution of Fos expression induced by electrical brain stimulation and psychomotor stimulants. For example, sensory-motor areas such as the lateral aspect of the caudate–putamen express high amounts

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Fig. 5. Photomicrographs illustrating the distribution of Fos-LI in the lateral habenula (LHb) (A, B), the paraventricular nucleus of the hypothalamus (PVN) (C, D) and the central nucleus of the amygdala (Amg) (E, F) from a rat that received no stimulation (A, C, E) and from the ipsilateral side of a rat after self-stimulation (B, D, F). D3V, dorsal 3rd ventricle; 3V, 3rd ventricle; opt, optic tract. Scale bar=200 µm.

of Fos after a single exposure to cocaine22 but the present study did not see Fos-LI in such regions even in yoked-stimulation rats that displayed high levels of locomotor activity during the entire test session. A lack of Fos expression in the dorsolateral striatum has also been observed following ventral pallidum self-stimulation.52 This suggests that the locomotor activating effects of intracranial stimulation and psychomotor stimulants may be subserved by different neuronal mechanisms.72

Identification stimulation

of

the

neurons

subserving

self-

It should be noted that regions expressing Fos-LI after MFB self-stimulation are not necessarily involved with the rewarding aspects of the stimulation, and indeed many of these regions also show Fos-LI following aversive events such as stress, anxiety, unconditioned footshock and aversive brain stimulation.14,26,28,38,56,57,62,63,67 Examples include

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Fig. 6. Photomicrographs illustrating the distribution of Fos-LI in the medial amygdala (A, D), the dorsomedial and ventromedial nuclei of the hypothalamus (DMH, VMH) (B, E) and the locus coeruleus (LC) (C, F) from a rat after no stimulation (A–C) and from the ipsilateral side of a rat after self stimulation (D–F). opt, optic tract; MePD, medial posterodorsal nucleus of the amygdala; f, fornix; 4V, 4th ventricle. Scale bar=100 µm (C, F) and 200 µm for the others.

the paraventricular nucleus of the hypothalamus, dorsomedial and ventromedial hypothalamus, the locus coeruleus and the amygdala. This is not surprising since stimulation of the LH has many emotional and behavioural consequences, and elicits changes in a wide range of cardiovascular, endocrine and thermoregulatory functions.70 Therefore, the presence of Fos-LI following electrical stimulation could be attributed to other processes not relating to the rewarding aspects of the stimulation. However, some regions may conceivably be engaged by both aversive and rewarding events. For example, Fos is induced in A10 dopamine neurons by stressful stimuli11,41,42 but numerous studies also indicate the primary importance of the VTA in drug reward.35,36 It should also

be noted that the lack of Fos-LI staining cannot be used to unequivocally exclude a brain region from involvement in a process because not all stimuli induce Fos-LI in activated neurons.12,28,61 Studies of the electrophysiological properties of the neurons subserving self-stimulation indicate the critical involvement of descending myelinated fibres originating in forebrain nuclei.20,45,46,65,81 It is notable in the present study that many of these forebrain nuclei contained dense Fos-LI. However, destruction or severing connections to many of these structures does not always affect self-stimulation. Thus, while lesions in the septal area34 decrease current thresholds for LH self-stimulation, lesions of the amygdala,74 diagonal band of Broca, medial

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Fig. 7. Photomicrographs illustrating the distribution of Fos-LI in the lateral hypothalamus (LH) at the level of the electrode tip (filled stars) approximately 2.8 mm posterior from bregma (A, B), the LH approximately "4.8 mm from bregma (C, D) and the ventral tegmental area (VTA) "5.2 mm from bregma. Panels on the left (A, C, E) were taken from a rat after no stimulation and panels on the right (B, D, F) were taken from the ipsilateral side of a rat after self stimulation. f, fornix; mp, mammillary peduncle; MT, medial terminal nucleus of the accessory optic tract. Scale bar=200 µm.

septum, medial preoptic area,75 dorsomedial nucleus of the hypothalamus,73 nucleus accumbens or ventral pallidum33 have little or only temporary effects. Even excitotoxic lesions within the LH itself do not always produce lasting decrements in self-stimulation.69,72 To date, unilateral lesions in the lateral preoptic area,2,31,44,75 anterolateral LH47 or knife cuts between the LH and the VTA19,32 have been shown to produce long-lasting inhibition of MFB self-stimulation. Interestingly, both the lateral preoptic area and the

anterior VTA show increased Fos expression and metabolic activation after LH self-stimulation1,18 and are two of the five areas in the present study with higher densities of Fos-LI in self-stimulators compared to the yoked-stimulation group. Taken together, these data indicate that the lateral preoptic area and the anterior VTA may be likely candidate areas for comprising an essential part of the neural substrate for rewarding brain stimulation and be important in mediating operant responding for reward.

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Fig. 8. Low-power (400#, A, B, E) and higher-power (1000#, B, D and F) photomicrographs illustrating the distribution of Fos-LI and tyrosine hydroxylase (TH)-positive neurons in the A10 dopamine neurons of the VTA (A, B), the A8 dopamine neurons in the retrorubral fields (C, D) and the A6 noradrenergic neurons of the locus coeruleus (E, F) from the ipsilateral side of a rat after self stimulation. Filled straight arrows indicate examples of Fos-LI nuclei, filled curved arrows indicate examples of TH-positive neurons and open arrows indicate doublely-labelled neurons. Very few of the A10 dopamine neurons expressed Fos-LI after self-stimulation (B), none of the A8 dopamine neurons were double labelled (D), whereas almost all of the A6 noradrenaline neurons were double labelled. 3N, oculomotor nerve; IP, interpeduncular nucleus; 4V, 4th ventricle. Scale bar=62.5 µm (A, C and E) and 25 µm (B, D and F).

It is also worth noting that two recent studies have suggested that the reward-relevant neurons linking the LH and VTA may extend caudally into the posterior mesencephalon, and that the retrorubral fields and pedunculopontine tegmental nucleus may also be involved in positive reinforcement.6,39

CONCLUSIONS

The present study has shown that many structures along the path of the MFB express Fos-LI following rewarding brain stimulation, especially on the ipsilateral side to the stimulating electrode. These data are in general agreement with studies based on the

Fos-like immunoreactivity and rewarding brain stimulation

2-DG method showing that many neurons that link midbrain and forebrain regions are activated during LH or VTA self-stimulation. Unfortunately, due to the widespread area of activation, neither Fos nor 2-DG studies reveal the exact anatomical locus of the neurons responsible for positive reinforcement. Unlike the dramatic increases in Fos-LI seen in dopaminergic projection areas, neither rewarding brain stimulation nor psychomotor stimulants have been reported to produce similar increases of Fos-LI in A10 dopamine neurons. The low number of Fosand TH-positive neurons in the VTA observed in the present study does not necessarily mean that mesolimbic or mesocortical dopamine neurons lack involvement in LH self-stimulation. There are a number of reasons mentioned above why more of the A10 dopamine neurons did not show Fos-LI. Although not quantified in this study, there was evidence of increased TH immunoreactivity in the A10 neurons of stimulated rats, suggesting that stimulation may increase metabolic demand despite the absence of Fos induction. It could be argued that Fos-LI may have been more pronounced in the dopamine cells bodies if electrodes were placed within the VTA, or if higher currents,52 or wider pulse widths were used. The use of wider pulses (2–5 ms) is thought to recruit fibres of

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a smaller diameter and may be more likely to affect the small unmyelinated dopamine fibres.20,65 None the less, even if these procedures were shown to induce widespread Fos expression in VTA neurons, the present study demonstrates that this is not a necessary prerequisite for self-stimulation to occur. In order to further study the neurons involved with rewarding brain stimulation, subsequent studies might examine a wide range of stimulation frequencies to ascertain the impact of decreasing stimulation charge on Fos expression,52 and test the effects of varying pulse widths5 and reward blocking doses of dopamine antagonists.9,30 In addition, changes in Fos-LI following lesions to areas that produce longterm decrements to self-stimulation such as the lateral preoptic area and anterior VTA may also help further identify the anatomical substrate of rewarding brain stimulation.

Acknowledgements—This research was funded by the National Health and Medical Research Council (NH and MRC) of Australia (project grant 960911). We thank Professor Gordon Johnson for helpful discussions relating to aspects of the article. Part of this work was presented at the Collegium Internationale Neuro-Psychopharmacologium (CINP) meeting in Melbourne, July 1996 and the Society for Neuroscience, November 1996.

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