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Fos-like immunoreactivity in the caudal diencephalon and brainstem following lateral hypothalamic self-stimulation
Behavioural Brain Research 88 (1997) 275 – 279
Short communication
Fos-like immunoreactivity in the caudal diencephalon and brainstem following lateral hypothalamic self-stimulation Andreas Arvanitogiannis, Cecilia Flores, Peter Shizgal * Center for Studies in Beha6ioral Neurobiology, Department of Psychology, Concordia Uni6ersity, 1455 de Maisonneu6e Bl6d. West Montre´al, Que´. H3G 1M8, Canada Received 10 February 1997; received in revised form 7 April 1997; accepted 7 April 1997
Abstract Fos immunohistochemistry was used to stain neurons in the caudal diencephalon, midbrain and hindbrain driven by rewarding stimulation of the lateral hypothalamus (LH). Increases in Fos-like immunoreactivity were most pronounced ipsilateral to the site of stimulation and tended to be confined within discrete structures such as the posterior LH, arcuate nucleus, ventral tegmental area (VTA), central gray, dorsal raphe´, pedunculopontine area (PPTg), parabrachial nucleus, and locus coeruleus. At least two of these structures, the VTA and PPTg, have been implicated in medial forebrain bundle self-stimulation. © 1997 Elsevier Science B.V. Keywords: Brain stimulation reward; Medial forebrain bundle; Immediate-early genes; Midbrain; Hindbrain; Ventral tegmental area; Pedunculopontine nucleus
1. Introduction
Abbre6iations: 3V, third ventricle; Aq, cerebral aqueduct; Arc, arcuate hypothalamic nucleus; CG, central grey; cp, cerebral peduncle; DM, dorsomedial hypothalamic nucleus; DR, dorsal raphe´ nucleus; f, fornix; ic, internal capsule; LC, locus coeruleus; LDTg, laterodorsal tegmental nucleus; LG, Lateral geniculate nucleus; LH, lateral hypothalamic area; LHb, lateral habenular nucleus; me5, mesencephalic trigeminal tract; ml, medial lemniscus; mlf, medial longitudinal fasciculus; MnR, median raphe´ nucleus; MTu, medial tuberal nucleus; PBN, parabrachial nucleus; PH, posterior hypothalamic nucleus; Pn, pontine nuclei; PPTg, pedunculopontine tegmental nucleus; PrC, precommissural nucleus; PVP, paraventricular thalamic nucleus, posterior; pv, periventricular fiber system; RLi, rostral linear nucleus of the raphe´; RRF, retrorubral field; scp, superior cerebellar peduncle; SNC, substantia nigra, compact; Sol, nucleus of the solitary tract; SuG, superficial gray layer of the superior colliculus; SuM, supramammillary nucleus; VMH, ventromedial hypothalamic nucleus; VTA, ventral tegmental area; xscp, decussation of the superior cerebellar peduncle. * Corresponding author. Tel.: +1 514 8482191; fax: + 1 514 8482817; e-mail: [email protected]. 0166-4328/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 4 3 2 8 ( 9 7 ) 0 0 0 6 5 - X
Despite recent advances in clarifying the relationship between brain stimulation reward (BSR) and natural reinforcers [28], the identification of the neural circuitry subserving BSR remains to be established. Two key questions must be answered to bring the analysis of BSR to the cellular level. First, what is the origin of the directly stimulated (‘first-stage’) neurons responsible for self-stimulation and what are their morphological and neurochemical characteristics? Psychophysical [5–7, 19,38], electrophysiological [20,25,29], and lesion [2,14,21] studies of the medial forebrain bundle (MFB) reward substrate have led to the proposal that at least some of the first stage neurons have myelinated axons, somata in the basal forebrain and synaptic terminals in, or caudal to, the VTA. Although there is general agreement about the electrophysiological properties of the first stage neurons [30], the location of their somata and their axonal morphology remain matters of contro-
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versy [12,16]. The second question concerns postsynaptic elements of the circuitry subserving the rewarding effect. A preponderance of evidence indicates that MFB self-stimulation is dependent on the mesolimbic dopamine system [32,35,36] as well as the cholinergic projection from the pedunculopontine nucleus to the VTA [39,40]. What place do these systems occupy in the neural substrate giving rise to the rewarding effect? A first step toward answering these questions is to employ a high-resolution anatomical method to visualize neurons activated by rewarding stimulation. It has been suggested that Fos expression is triggered by neuronal activation, at least in some neurons [8,15,18,26]. If so, mapping the distribution of the Fos protein is a means of visualizing the neural elements that respond to natural and artificial stimuli [8,26]. We have previously shown that rewarding electrical stimulation of the LH and VTA increased ipsilateral Fos expression in discrete regions of the basal forebrain and diencephalon [1,10]. We now report the effect of rewarding LH stimulation on Fos-like immunoreactivity (FLIR) in the caudal diencephalon, midbrain and hindbrain. Staining of cell nuclei ipsilateral to the stimulation electrode was compared to staining in homologous contralateral structures. Under pentobarbital anesthesia (65 mg/kg), monopolar electrodes were aimed stereotaxically at the LH (AP, 2.8 mm behind bregma; ML, 1.7 mm lateral to the mid-sagittal sinus; DV, 7.8 mm below dura) in nine male, Long-Evans rats (350 – 400 g). Subjects learned to lever press for 0.5-s stimulation trains composed of 0.1 ms, 1000 mA rectangular cathodal pulses. Rate-frequency curves were collected, and the minimum stimulation frequency that supported asymptotic responding (the ‘shoulder’ frequency) was determined for each subject. During the final 1-h test session, conducted 3 days after the previous session, the experimental subjects self-stimulated at the shoulder frequency. For two control subjects, the stimulator was disconnected during the final 1-h test. Fifteen minutes after the end of the final session, the rats were deeply anesthetized with sodium pentobarbital (120 mg/kg) and intracardially perfused with 200 ml of cold physiological saline followed by 400 ml of cold 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were removed and stored overnight in 4% paraformaldehyde at 4°C. Coronal, vibratome (50 mm) sections were cut and then processed for FLIR as previously described [1,10], using a mouse monoclonal antibody raised against N-terminal residues 4 – 17 of the human Fos protein (NCI/BCB Repository, Quality Biotech, Camden, NJ). FLIR was detected with a Vectastain Elite ABC Kit (Dimension Labs) using diaminobenzidine as the chromogen. An image-analysis program (NIH Image) was used to count the cells within a circumscribed region and to
Fig. 1. Mean ( 9S.E.M) FLIR density ipsilaterally and contralaterally to the stimulating electrode in caudal diencephalic, midbrain, and hindbrain regions where asymmetric activation was observed and analyzed. Densities were calculated from mean numbers obtained using the three sections per region exhibiting maximum FLIR for each animal (ns =P \0.05, t-test).
convert the cell counts to densities [10]; the borders of the region to be analyzed were defined manually to correspond to structure boundaries in the Paxinos and Watson [23] atlas. The density of Fos-positive cells was taken as the mean for a given structure in the three sections exhibiting maximum FLIR for the structure in question. Substantial staining, heaviest ipsilateral to the stimulating electrode, was seen in the seven stimulated brains. In the caudal diencephalon, midbrain, and hindbrain of the unstimulated subjects, labeling was absent Table 1 Relative and absolute FLIR density in the stimulated versus the unstimulated hemisphere across brain regions Structure
Ratio
Density
n
LH MTu Arc DM VMH PH PVP LHb PrC pv SuM SNC VTA RRF CG DR PPTg Pn LDTg PBN LC
7.4:1 4.6:1 2.4:1 2.2:1 3.5:1 3.0:1 2.0:1 1.7:1 3.0:1 2.6:1 1.9:1 3.4:1 2.0:1 2.2:1 2.2:1 1.7:1 2.1:1 1.7:1 1.6:1 4.2:1 2.6:1
++++ +++ ++++ ++++ ++ ++ +++ +++ +++ ++ ++++ + ++ ++ ++ ++ ++ ++ ++ ++ +++
7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 6 5 5 5
Column 2 contains the ratios of FLIR densities in homologous structures in the two hemispheres (stimulated/unstimulated). Column 3: 0 – 100 cells/mm2 =‘+’; 101 – 250 cells/mm2 =‘++’; 251–400 cells/mm2 =‘+++’; and \400 cells/mm2 =‘++++’).
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Fig. 2. Representative digitized images showing substantially more FLIR in the stimulated (left) as opposed to the unstimulated (right) hemisphere. Structures pictured: LH, MTu, Arc, DM (A: bregma, − 3.6 mm); SuM, SNC, VTA (B: bregma, − 4.8 mm); CG (C: bregma, − 5.8 mm). Scale bars: A – C, 250 mm.
or very sparse ( 5 13 Fos-positive cells/mm2); what little labeling was seen was bilaterally symmetrical. Estimates of the density of Fos-positive cells in the stimulated subjects are shown in Fig. 1. FLIR tended to be the highest in the caudal diencephalon and to fall off in the brainstem. Nonetheless, intense staining was seen in the LC. Although a predominance of labeled cells ipsilateral to the stimulation electrode was found in the SNC, the difference between FLIR in the ipsilateral and contralateral sides was not significant. Bilateral staining was seen in the LG, RLi, MnR, and SuG (not shown in Fig. 1). In one subject, the anterior part of the Sol was examined and moderate FLIR was observed, predominantly ipsilateral to the stimulating electrode. Bilateral comparisons of the relative density in homologous structures are shown in Table 1, along with ordinally scaled values indicating absolute density. Fig. 2 and Fig. 3 illustrate the asymmetrical distribution of FLIR in some of the structures analyzed.
The present study provides new information concerning the discrete distribution of the FLIR induced by LH stimulation in regions caudal to the stimulation electrode. To our knowledge, this is the first study to provide a detailed description of the midbrain and hindbrain structures that are activated by rewarding stimulation of the LH. Gallistel et al. [11] and Porrino et al., [24] using 14C-2-deoxyglucose autoradiography, as well as Bielajew [4], using cytochrome oxidase histochemistry, failed to detect activation posterior to the VTA after LH self-stimulation. Esposito et al. [9] reported increases in glucose utilization caudal to the VTA after rewarding stimulation of the VTA, but only in a subset of the structures that showed activation in the present study. Thus, apart from offering cellular resolution, Fos immunostaining appears to be a highly sensitive method for visualizing neurons activated by rewarding stimulation. We should note, that the LH stimulation current used in this study is at the upper end of the range typically self-administered in BSR
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Fig. 3. Representative digitized images showing substantially more FLIR in the stimulated (left) as opposed to the unstimulated (right) hemisphere. Structures pictured: PPTg (A: bregma, − 7.8 mm); DR, LDTg, PBN (B: bregma, −8.7 mm); LC (C: bregma, −9.8 mm). Scale bars: A, B, 250 mm; C, 100 mm.
experiments. Thus, it is not surprising that a substantial density of Fos-positive cells was found in many nuclei that give rise to or receive synaptic input from fibers passing through the LH [3,13,22,27,31,33,34]. It would be interesting to determine whether substantially lower currents produce a similar pattern of Fos staining. Cells in some of the regions showing increased Fos expression, such as the VTA and PPTg, have been implicated as postsynaptic elements in the circuitry subserving the rewarding effect of LH stimulation [35,36,39,40]. In future studies, Fos immunostaining can be combined with anterograde and retrograde tracers and with specific stains for dopaminergic and cholinergic neurons to determine the neurochemistry of Fos-positive neurons as well as their afferent and efferent connections. McGregor and Hunt [17] have already taken a first step in that direction by combining Fos and tyrosine hydroxylase labeling. Combining methods could also serve to test specific hypotheses about the identity of the first stage neurons. For example, Gallis-
tel et al. [12] recently proposed that these neurons are bipolar, extensively collateralized, cells with both their somata and the majority of their terminals located caudal to the diencephalic MFB. Do any of the Fospositive neurons in the midbrain or hindbrain have these morphological characteristics? To find out, Fos immunostaining could, for example, be combined with the anti-200-kDa neurofilament immunostaining method that permits the visualization of myelinated axons [37]. Thus, if neurons responsible for BSR do indeed express Fos, the combination of morphological, neurochemical and tracing methods with Fos immunohistochemistry should make possible a detailed cellular analysis of the reward-related circuitry.
Acknowledgements A.A. was supported by a scholarship from the ‘Fonds pour la Formation de Chercheurs et l’Aide a` la
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Recherche du Que´bec’ (FCAR). This work was supported by the Medical Research Council of Canada ( c MT-8037, P.S., p.i.). References [1] A. Arvanitogiannis, C. Flores, J.G. Pfaus, P. Shizgal, Increased ipsilateral expression of Fos following lateral hypothalamic selfstimulation, Brain Res. 720 (1996) 148–154. [2] A. Arvanitogiannis, M. Waraczynski, P. Shizgal, Effects of excitotoxic lesions of the basal forebrain on MFB Self-stimulation, Physiol. Behav. 59 (1996) 795–806. [3] F.C. Barone, M.J. Wayner, S.L. Scharoun, R. Guevara-Aguilar, H.U. Aguilar-Baturoni, Afferent connections to the lateral hypothalamus: a horseradish peroxidase study in the rat, Brain Res. Bull. 7 (1981) 75 – 88. [4] C. Bielajew, Distribution of cytochrome oxidase in response to rewarding brain stimulation: effect of different pulse durations, Brain Res. Bull. 26 (1991) 379–384. [5] C. Bielajew, P. Shizgal, Behaviorally derived measures of conduction velocity in the substrate for rewarding medial forebrain bundle stimulation, Brain Res. 237 (1982) 107–119. [6] C. Bielajew, P. Shizgal, Evidence implicating descending fibers in self-stimulation of the medial forebrain bundle, J. Neurosci. 6 (1986) 919 – 929. [7] S.M. Boye, P.-P. Rompre´, Mesencephalic substrate of reward: axonal connections, J. Neurosci. 16 (1996) 3511–3520. [8] M. Dragunow, R. Faull, The use of c-fos as a metabolic marker in neuronal pathway tracing, J. Neurosci. Methods 29 (1989) 261 – 265. [9] R.U. Esposito, L.J. Porrino, T.F. Seeger, A.M. Crane, H.D. Everist, A. Pert, Changes in local cerebral glucose utilization during rewarding brain stimulation, Proc. Natl. Acad. Sci. USA 81 (1984) 635 – 639. [10] C. Flores, A. Arvanitogiannis, P. Shizgal, Fos-like immunoreactivity in forebrain regions following self-stimulation of the lateral hypothalamus and the ventral tegmental area, Behav. Brain Res., in press. [11] C.R. Gallistel, Y. Gomita, E. Yadin, K.A. Campbell, Forebrain origins and terminations of the medial forebrain bundle metabolically activated by rewarding stimulation or by reward-blocking doses of pimozide, J. Neurosci. 5 (1985) 1246–1261. [12] C.R. Gallistel, M. Leon, B.T. Lim, J.C. Sim, M. Waraczynski, Destruction of the medial forebrain bundle caudal to the site of stimulation reduces rewarding efficacy but destruction rostrally does not, Behav. Neurosci. 110 (1996) 766–790. [13] A.E. Hallanger, B.H. Wainer, Ascending projections from the pedunculopontine tegmental nucleus and the adjacent mesopontine tegmentum in the rat, J. Comp. Neurol. 274 (1988) 483 – 515. [14] J.D. Janas, J.R. Stellar, Effects of knife cut-lesions of the medial forebrain bundle in self-stimulating rats, Behav. Neurosci. 101 (1987) 832 – 845. [15] T.L. Krukoff, Expression of c-fos in studies of central, autonomic and sensory systems, Mol. Neurobiol. 7 (1993) 247–263. [16] J. Malette, E. Miliaressis, Interhemispheric links in brain stimulation reward, Behav. Brain Res. 68 (1995) 117–137. [17] I.S. McGregor, G.E. Hunt, Rewarding brain stimulation induces only sparse c-fos immunoreactivity in dopaminergic neurons, Soc. Neurosci. Abstr. 22 (1996) 686. [18] J.I. Morgan, T. Curran, Stimulus-transcription coupling in the nervous system: Involvement of the inducible protooncogenes fos and jun, Annu. Rev. Neurosci. 14 (1991) 421–451. [19] B. Murray, P. Shizgal, Behavioral measures of conduction velocity and refractory period for reward-relevant axons in the anterior LH and VTA, Physiol. Behav. 59 (1996) 643–652.
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[20] B. Murray, P. Shizgal, Physiological measures of conduction velocity and refractory period for putative reward-relevant MFB axons arising in the rostral MFB, Physiol. Behav. 59 (1996) 427 – 437. [21] B. Murray, P. Shizgal, Anterolateral lesions of the medial forebrain bundle increase the frequency threshold for self-stimulation of the lateral hypothalamus and ventral tegmental area in the rat, Psychobiology 19 (1991) 135 – 146. [22] R. Nieuwenhuys, L.M.G. Geeraeddts, J.G. Veening, The medial forebrain bundle of the rat. I. General introduction, J. Comp. Neurol. 206 (1982) 49 – 81. [23] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 2nd ed., Academic Press, Sydney, 1986. [24] L.J. Porrino, D. Huston-Lyons, G. Bain, L. Sokoloff, C. Kornetsky, The distribution of changes in local cerebral energy metabolism associated with brain stimulation reward to the medial forebrain bundle of the rat, Brain Res. 511 (1990) 1–6. [25] P.-P. Rompre´, P. Shizgal, Electrophysiological characteristics of neurons in forebrain regions implicated in self-stimulation of the medial forebrain bundle in the rat, Brain Res. 364 (1986) 338– 349. [26] S.M. Sagar, F.R. Sharp, T. Curran, Expression of c-fos protein in the brain: metabolic mapping at the cellular level, Science 244 (1988) 1328 – 1331. [27] C.B. Saper, L.W. Swanson, W.M. Cowan, An autoradiographic study of the efferent connections of the lateral hypothalamic area in the rat, J. Comp. Neurol. 183 (1979) 689 – 706. [28] P. Shizgal, K. Conover, On the neural computation of utility, Curr. Dir. Psychol. Sci. 5 (1996) 37 – 43. [29] P. Shizgal, D. Schindler, P.-P. Rompre´, Forebrain neurons driven by rewarding stimulation of the medial forebrain bundle in the rat: comparison of psychophysical and electrophysiological estimates of refractory periods, Brain Res. 499 (1989) 234–248. [30] P. Shizgal, B. Murray, Neuronal basis of intracranial self-stimulation, in: J.M. Liebman, S.J. Cooper, (Eds.), The Neuropharmacological Basis of Reward, Oxford University Press, Oxford, 1989, pp. 106 – 163. [31] T.L. Steininger, D.B. Rye, B.H. Wainer, Afferent projections to the cholinergic pedunculopontine tegmental nucleus and adjacent midbrain extrapyramidal area in the albino rat. I. Retrograde tracing studies, J. Comp. Neurol. 321 (1992) 515 – 543. [32] J.R. Stellar, D. Corbett, Regional neuroleptic microinjections indicate a role for nucleus accumbens in lateral hypothalamic self-stimulation reward, Brain Res. 477 (1989) 126 – 143. [33] J.G. Veening, L.W. Swanson, W.M. Cowan, R. Nieuwenhuys, L.M.G. Geeraedts, The medial forebrain bundle of the rat. II. An autoradiographic study of the topography of the major descending and ascending components, J. Comp. Neurol. 206 (1982) 82 – 108. [34] J. Villalobos, A. Ferssiwi, The differential descending projections from the anterior, central and posterior regions of the lateral hypothalamic area: an autoradiographic study, Neurosci. Lett. 81 (1987) 95 – 99. [35] R.A. Wise, Addictive drugs and brain stimulation reward, Ann. Rev. Neurosci. 19 (1996) 319 – 340. [36] R.A. Wise, P.-P. Rompre´, Brain dopamine and reward, Ann. Rev. Psychol. 40 (1989) 191 – 225. [37] D.-L. Yao, S. Komoly, Q.-L. Zhang, H. Webster deF, Myelinated axons demonstrated in the CNS and PNS by anti-neurofilament immunoreactivity and luxol fast blue counterstaining, Brain Pathol. 4 (1994) 97 – 100. [38] J.S. Yeomans, The absolute refractory periods of self-stimulation neurons, Physiol. Behav. 22 (1979) 911 – 919. [39] J.S. Yeomans, M. Baptista, Both nicotinic and muscarinic receptors in ventral tegmental area contribute to brain-stimulation reward, Pharmacol. Biochem. Behav., in press. [40] J.S. Yeomans, A. Mathur, M. Tampakeras, Rewarding brain stimulation: role of tegmental cholinergic neurons that activate dopamine neurons, Behav. Neurosci. 107 (1993) 1077 –1087.
Short communication
Fos-like immunoreactivity in the caudal diencephalon and brainstem following lateral hypothalamic self-stimulation Andreas Arvanitogiannis, Cecilia Flores, Peter Shizgal * Center for Studies in Beha6ioral Neurobiology, Department of Psychology, Concordia Uni6ersity, 1455 de Maisonneu6e Bl6d. West Montre´al, Que´. H3G 1M8, Canada Received 10 February 1997; received in revised form 7 April 1997; accepted 7 April 1997
Abstract Fos immunohistochemistry was used to stain neurons in the caudal diencephalon, midbrain and hindbrain driven by rewarding stimulation of the lateral hypothalamus (LH). Increases in Fos-like immunoreactivity were most pronounced ipsilateral to the site of stimulation and tended to be confined within discrete structures such as the posterior LH, arcuate nucleus, ventral tegmental area (VTA), central gray, dorsal raphe´, pedunculopontine area (PPTg), parabrachial nucleus, and locus coeruleus. At least two of these structures, the VTA and PPTg, have been implicated in medial forebrain bundle self-stimulation. © 1997 Elsevier Science B.V. Keywords: Brain stimulation reward; Medial forebrain bundle; Immediate-early genes; Midbrain; Hindbrain; Ventral tegmental area; Pedunculopontine nucleus
1. Introduction
Abbre6iations: 3V, third ventricle; Aq, cerebral aqueduct; Arc, arcuate hypothalamic nucleus; CG, central grey; cp, cerebral peduncle; DM, dorsomedial hypothalamic nucleus; DR, dorsal raphe´ nucleus; f, fornix; ic, internal capsule; LC, locus coeruleus; LDTg, laterodorsal tegmental nucleus; LG, Lateral geniculate nucleus; LH, lateral hypothalamic area; LHb, lateral habenular nucleus; me5, mesencephalic trigeminal tract; ml, medial lemniscus; mlf, medial longitudinal fasciculus; MnR, median raphe´ nucleus; MTu, medial tuberal nucleus; PBN, parabrachial nucleus; PH, posterior hypothalamic nucleus; Pn, pontine nuclei; PPTg, pedunculopontine tegmental nucleus; PrC, precommissural nucleus; PVP, paraventricular thalamic nucleus, posterior; pv, periventricular fiber system; RLi, rostral linear nucleus of the raphe´; RRF, retrorubral field; scp, superior cerebellar peduncle; SNC, substantia nigra, compact; Sol, nucleus of the solitary tract; SuG, superficial gray layer of the superior colliculus; SuM, supramammillary nucleus; VMH, ventromedial hypothalamic nucleus; VTA, ventral tegmental area; xscp, decussation of the superior cerebellar peduncle. * Corresponding author. Tel.: +1 514 8482191; fax: + 1 514 8482817; e-mail: [email protected]. 0166-4328/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 4 3 2 8 ( 9 7 ) 0 0 0 6 5 - X
Despite recent advances in clarifying the relationship between brain stimulation reward (BSR) and natural reinforcers [28], the identification of the neural circuitry subserving BSR remains to be established. Two key questions must be answered to bring the analysis of BSR to the cellular level. First, what is the origin of the directly stimulated (‘first-stage’) neurons responsible for self-stimulation and what are their morphological and neurochemical characteristics? Psychophysical [5–7, 19,38], electrophysiological [20,25,29], and lesion [2,14,21] studies of the medial forebrain bundle (MFB) reward substrate have led to the proposal that at least some of the first stage neurons have myelinated axons, somata in the basal forebrain and synaptic terminals in, or caudal to, the VTA. Although there is general agreement about the electrophysiological properties of the first stage neurons [30], the location of their somata and their axonal morphology remain matters of contro-
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versy [12,16]. The second question concerns postsynaptic elements of the circuitry subserving the rewarding effect. A preponderance of evidence indicates that MFB self-stimulation is dependent on the mesolimbic dopamine system [32,35,36] as well as the cholinergic projection from the pedunculopontine nucleus to the VTA [39,40]. What place do these systems occupy in the neural substrate giving rise to the rewarding effect? A first step toward answering these questions is to employ a high-resolution anatomical method to visualize neurons activated by rewarding stimulation. It has been suggested that Fos expression is triggered by neuronal activation, at least in some neurons [8,15,18,26]. If so, mapping the distribution of the Fos protein is a means of visualizing the neural elements that respond to natural and artificial stimuli [8,26]. We have previously shown that rewarding electrical stimulation of the LH and VTA increased ipsilateral Fos expression in discrete regions of the basal forebrain and diencephalon [1,10]. We now report the effect of rewarding LH stimulation on Fos-like immunoreactivity (FLIR) in the caudal diencephalon, midbrain and hindbrain. Staining of cell nuclei ipsilateral to the stimulation electrode was compared to staining in homologous contralateral structures. Under pentobarbital anesthesia (65 mg/kg), monopolar electrodes were aimed stereotaxically at the LH (AP, 2.8 mm behind bregma; ML, 1.7 mm lateral to the mid-sagittal sinus; DV, 7.8 mm below dura) in nine male, Long-Evans rats (350 – 400 g). Subjects learned to lever press for 0.5-s stimulation trains composed of 0.1 ms, 1000 mA rectangular cathodal pulses. Rate-frequency curves were collected, and the minimum stimulation frequency that supported asymptotic responding (the ‘shoulder’ frequency) was determined for each subject. During the final 1-h test session, conducted 3 days after the previous session, the experimental subjects self-stimulated at the shoulder frequency. For two control subjects, the stimulator was disconnected during the final 1-h test. Fifteen minutes after the end of the final session, the rats were deeply anesthetized with sodium pentobarbital (120 mg/kg) and intracardially perfused with 200 ml of cold physiological saline followed by 400 ml of cold 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were removed and stored overnight in 4% paraformaldehyde at 4°C. Coronal, vibratome (50 mm) sections were cut and then processed for FLIR as previously described [1,10], using a mouse monoclonal antibody raised against N-terminal residues 4 – 17 of the human Fos protein (NCI/BCB Repository, Quality Biotech, Camden, NJ). FLIR was detected with a Vectastain Elite ABC Kit (Dimension Labs) using diaminobenzidine as the chromogen. An image-analysis program (NIH Image) was used to count the cells within a circumscribed region and to
Fig. 1. Mean ( 9S.E.M) FLIR density ipsilaterally and contralaterally to the stimulating electrode in caudal diencephalic, midbrain, and hindbrain regions where asymmetric activation was observed and analyzed. Densities were calculated from mean numbers obtained using the three sections per region exhibiting maximum FLIR for each animal (ns =P \0.05, t-test).
convert the cell counts to densities [10]; the borders of the region to be analyzed were defined manually to correspond to structure boundaries in the Paxinos and Watson [23] atlas. The density of Fos-positive cells was taken as the mean for a given structure in the three sections exhibiting maximum FLIR for the structure in question. Substantial staining, heaviest ipsilateral to the stimulating electrode, was seen in the seven stimulated brains. In the caudal diencephalon, midbrain, and hindbrain of the unstimulated subjects, labeling was absent Table 1 Relative and absolute FLIR density in the stimulated versus the unstimulated hemisphere across brain regions Structure
Ratio
Density
n
LH MTu Arc DM VMH PH PVP LHb PrC pv SuM SNC VTA RRF CG DR PPTg Pn LDTg PBN LC
7.4:1 4.6:1 2.4:1 2.2:1 3.5:1 3.0:1 2.0:1 1.7:1 3.0:1 2.6:1 1.9:1 3.4:1 2.0:1 2.2:1 2.2:1 1.7:1 2.1:1 1.7:1 1.6:1 4.2:1 2.6:1
++++ +++ ++++ ++++ ++ ++ +++ +++ +++ ++ ++++ + ++ ++ ++ ++ ++ ++ ++ ++ +++
7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 6 5 5 5
Column 2 contains the ratios of FLIR densities in homologous structures in the two hemispheres (stimulated/unstimulated). Column 3: 0 – 100 cells/mm2 =‘+’; 101 – 250 cells/mm2 =‘++’; 251–400 cells/mm2 =‘+++’; and \400 cells/mm2 =‘++++’).
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Fig. 2. Representative digitized images showing substantially more FLIR in the stimulated (left) as opposed to the unstimulated (right) hemisphere. Structures pictured: LH, MTu, Arc, DM (A: bregma, − 3.6 mm); SuM, SNC, VTA (B: bregma, − 4.8 mm); CG (C: bregma, − 5.8 mm). Scale bars: A – C, 250 mm.
or very sparse ( 5 13 Fos-positive cells/mm2); what little labeling was seen was bilaterally symmetrical. Estimates of the density of Fos-positive cells in the stimulated subjects are shown in Fig. 1. FLIR tended to be the highest in the caudal diencephalon and to fall off in the brainstem. Nonetheless, intense staining was seen in the LC. Although a predominance of labeled cells ipsilateral to the stimulation electrode was found in the SNC, the difference between FLIR in the ipsilateral and contralateral sides was not significant. Bilateral staining was seen in the LG, RLi, MnR, and SuG (not shown in Fig. 1). In one subject, the anterior part of the Sol was examined and moderate FLIR was observed, predominantly ipsilateral to the stimulating electrode. Bilateral comparisons of the relative density in homologous structures are shown in Table 1, along with ordinally scaled values indicating absolute density. Fig. 2 and Fig. 3 illustrate the asymmetrical distribution of FLIR in some of the structures analyzed.
The present study provides new information concerning the discrete distribution of the FLIR induced by LH stimulation in regions caudal to the stimulation electrode. To our knowledge, this is the first study to provide a detailed description of the midbrain and hindbrain structures that are activated by rewarding stimulation of the LH. Gallistel et al. [11] and Porrino et al., [24] using 14C-2-deoxyglucose autoradiography, as well as Bielajew [4], using cytochrome oxidase histochemistry, failed to detect activation posterior to the VTA after LH self-stimulation. Esposito et al. [9] reported increases in glucose utilization caudal to the VTA after rewarding stimulation of the VTA, but only in a subset of the structures that showed activation in the present study. Thus, apart from offering cellular resolution, Fos immunostaining appears to be a highly sensitive method for visualizing neurons activated by rewarding stimulation. We should note, that the LH stimulation current used in this study is at the upper end of the range typically self-administered in BSR
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Fig. 3. Representative digitized images showing substantially more FLIR in the stimulated (left) as opposed to the unstimulated (right) hemisphere. Structures pictured: PPTg (A: bregma, − 7.8 mm); DR, LDTg, PBN (B: bregma, −8.7 mm); LC (C: bregma, −9.8 mm). Scale bars: A, B, 250 mm; C, 100 mm.
experiments. Thus, it is not surprising that a substantial density of Fos-positive cells was found in many nuclei that give rise to or receive synaptic input from fibers passing through the LH [3,13,22,27,31,33,34]. It would be interesting to determine whether substantially lower currents produce a similar pattern of Fos staining. Cells in some of the regions showing increased Fos expression, such as the VTA and PPTg, have been implicated as postsynaptic elements in the circuitry subserving the rewarding effect of LH stimulation [35,36,39,40]. In future studies, Fos immunostaining can be combined with anterograde and retrograde tracers and with specific stains for dopaminergic and cholinergic neurons to determine the neurochemistry of Fos-positive neurons as well as their afferent and efferent connections. McGregor and Hunt [17] have already taken a first step in that direction by combining Fos and tyrosine hydroxylase labeling. Combining methods could also serve to test specific hypotheses about the identity of the first stage neurons. For example, Gallis-
tel et al. [12] recently proposed that these neurons are bipolar, extensively collateralized, cells with both their somata and the majority of their terminals located caudal to the diencephalic MFB. Do any of the Fospositive neurons in the midbrain or hindbrain have these morphological characteristics? To find out, Fos immunostaining could, for example, be combined with the anti-200-kDa neurofilament immunostaining method that permits the visualization of myelinated axons [37]. Thus, if neurons responsible for BSR do indeed express Fos, the combination of morphological, neurochemical and tracing methods with Fos immunohistochemistry should make possible a detailed cellular analysis of the reward-related circuitry.
Acknowledgements A.A. was supported by a scholarship from the ‘Fonds pour la Formation de Chercheurs et l’Aide a` la
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