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Disgust is a prototypical type of negative affect. In animal models of excessive disgust, only a few brain sites are known in which localized dysfunction (lesions or neural inactivations) can induce intense 'disgust reactions' (e.g. gapes) to a normally pleasant sensation such as sweetness. Here, we aimed to map forebrain candidates more precisely, to identify where either local neuronal damage (excitotoxin lesions) or local pharmacological inactivation (muscimol/baclofen microinjections) caused rats to show excessive sensory disgust reactions to sucrose. Our study compared subregions of the nucleus accumbens shell, ventral pallidum, lateral hypothalamus, and adjacent extended amygdala. The results indicated that the posterior half of the ventral pallidum was the only forebrain site where intense sensory disgust gapes in response to sucrose were induced by both lesions and temporary inactivations (this site was previously identified as a hedonic hotspot for enhancements of sweetness 'liking'). By comparison, for the nucleus accumbens, temporary GABA inactivations in the caudal half of the medial shell also generated sensory disgust, but lesions never did at any site. Furthermore, even inactivations failed to induce disgust in the rostral half of the accumbens shell (which also contains a hedonic hotspot). In other structures, neither lesions nor inactivations induced disgust as long as the posterior ventral pallidum remained spared. We conclude that the posterior ventral pallidum is an especially crucial hotspot for producing excessive sensory disgust by local pharmacological/lesion dysfunction. By comparison, the nucleus accumbens appears to segregate sites for pharmacological disgust induction and hedonic enhancement into separate posterior and rostral halves of the medial shell.

Intense feeding can be elicited by injections of the GABA(A) receptor antagonist bicuculline into the medial ventral pallidum (VPm), a basal forebrain structure anatomically interposed between two other feeding-related brain regions, the nucleus accumbens shell and the lateral hypothalamus (LH). To determine whether the VPm effects changes in feeding behavior through actions on the LH, we examined feeding following unilateral injections of bicuculline into the VPm made either ipsilateral or contralateral to a unilateral excitotoxic lesion of the LH in nondeprived rats. We found that lesions of the LH significantly attenuated feeding induced from the ipsilateral VPm, as compared to sham-operated controls. In striking contrast, unilateral LH lesions significantly potentiated the feeding response elicited by injections of bicuculline into the contralateral VPm. The 'ipsilateral-contralateral disruption' design we used makes it extremely unlikely that our findings could have resulted from nonspecific effects of the lesions. These results suggest that the LH is causally involved in mediating the ingestive effects produced by activation of the VPm, and provide an important insight into the functional circuitry by which basal forebrain structures control food intake in mammals.

Behavioral sensitization describes the intensification of motor activity that results from repeated exposure to drugs of misuse, and the underlying neuronal adaptations are hypothesized to model aspects of the brain changes that occur in humans misusing such drugs. The α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor is an ionotropic glutamate receptor involved in the neuroplasticity that accompanies acute and repeated drug administration. Changing surface expression is one means to regulate AMPA receptor function, and the present study tested the hypothesis that behavioral sensitization to the μ-opioid receptor agonist morphine is accompanied by changes in the subcellular distribution of AMPA receptors in limbic brain regions. To test this hypothesis, we used a protein cross-linking assay to assess cell surface and intracellular levels of GluA1 and GluA2 subunits in the nucleus accumbens, medial prefrontal cortex and ventral pallidum. Repeated morphine treatment decreased surface expression of GluA1 in the medial prefrontal cortex without affecting levels of GluA2. In contrast, surface levels of GluA1 or GluA2 were unchanged in the nucleus accumbens and ventral pallidum, demonstrating that although AMPA receptors in accumbal and pallidal regions are critical mediators of behaviors induced by repeated opiate exposure, these effects are not accompanied by changes in surface expression. The findings reveal that the involvement of AMPA receptor trafficking in opiate-induced behavioral sensitization is relegated to selective regions and that AMPA receptors in the medial prefrontal cortex may be particularly sensitive to these actions.

We previously found that the blockade of γ-aminobutyric acid (GABA) A receptors in the ventral pallidum (VP) alters the taste palatability of a conditioned stimulus (CS) from aversive to ingestive after the establishment of conditioned taste aversion (CTA). Because these results suggest that GABAergic transmission in the VP mediates decreased palatability of the taste in CTA, the present study aimed to examine the effects of taste stimulation on the extracellular release of GABA in the VP using in vivo microdialysis. Initially, rats received a paired presentation of 5 m M saccharin or 0.3 m M quinine solution with an intraperitoneal injection of 0.15 M lithium chloride (S-CTA and Q-CTA groups) or saline (S-control and Q-control groups). After conditioning, microdialysis was carried out before, during and after the presentation of the CS via an intra-oral cannula. We measured the latency of the first aversive orofacial responses to the CS as behavioral indices. In the S-CTA group, which rapidly rejected the CS (within 100 s), the GABA efflux was significantly increased (147%) and was maintained for 2 h. On the other hand, the S-control group expressed no aversive responses and showed no significant alterations in GABA efflux. Although the Q-CTA group immediately expressed aversive responses to the CS (within 30 s), GABA release was not changed by presentation of the CS, which was similar in the Q-control group. These findings suggest that the palatability shift from ingestive to aversive in conditioned aversion to saccharin, but not quinine, is mediated by the change in GABAergic transmission in the VP.

The behavioral profile in response to (+/-)-3,4-methylenedioxymethamphetamine (MDMA) is characterized by acute hyperlocomotion that is primarily restricted to the periphery of the open field, whereas behavioral sensitization to MDMA reflects a selective increase in activity in the central zone, suggesting that acute effects and sensitization might rely on neuroadaptations in different systems. This study was thus undertaken to determine whether specific changes in neuronal activation could be correlated with either the acute or sensitized behavioral responses to MDMA. Animals received five daily intraperitoneal (i.p.) injections of saline or MDMA (10 mg/kg). Two days later, animals that received saline were injected with saline or MDMA (5 or 10 mg/kg, i.p.). Animals pretreated with MDMA were injected with saline or MDMA (5 mg/kg, i.p.). Locomotor activity was measured in an open field, and neuronal activation was examined by immunodetection of Fos. Acute MDMA exposure produced a dose-dependent increase in locomotion in the peripheral zone of the open field that was related to an increase in Fos expression in the ventromedial shell of the nucleus accumbens, ventral pallidum, several hypothalamic nuclei and rhomboid thalamic nucleus. Following repeated, intermittent exposure to MDMA, drug-produced hyperactivity became sensitized but, unlike the effect of increasing dose, the increased response was due to increased activity and time spent in the central zone. Furthermore, the sensitized behavioral response was related to changes in Fos expression in the lateral shell of the nucleus accumbens, central nucleus of the amygdala and anteromedial part of the lateral habenula. This study identifies neural substrates that might specifically underlie the sensitized response to MDMA.

The ventral tegmental area (VTA), primary source of the mesocorticolimbic dopaminergic system, is regarded as a critical site for initiation of behavioural sensitization to psychostimulants. The present study was undertaken to identify the neural pathways converging on the VTA that are potentially implicated in this process. Rats were sensitized by a single exposure to amphetamine (5 mg ⁄kg, s.c.). The distribution of VTA-projecting neurons activated by amphetamine was examined by combining retrograde transport of the cholera toxin b subunit (CTb), injected into the VTA, with immunodetection of Fos. The quantitative analysis of CTb– Fos double labelling demonstrates that amphetamine induced a rapid activation of Fos in a large number of brain areas projecting to the VTA. More than half of the CTb–Fos double-labelled neurons were located in the prefrontal cortex, lateral preoptic area–lateral hypothalamus, pontomesencephalic tegmentum, dorsal raphe nucleus, ventral pallidum and nucleus accumbens. In addition, scattered CTb–Fos double-labelled cells were observed in many other VTA afferent structures, such as claustrum, lateral septum, diagonal band–magnocellular preoptic nucleus, deep mesencephalic nucleus, oral part of pontine reticular nucleus and dorsomedial tegmental area. This suggests that systemic amphetamine activates a wide population of neurons projecting to the VTA that may be important for the modulation of neurobehavioural plasticity produced by this psychostimulant.

The dopamine D4 receptor (D4R) has received considerable interest because of its higher affinity for atypical antipsychotics, the extremely polymorphic nature of the human gene and the genetic association with attention deficit and hyperactivity disorder (ADHD). Several efforts have been undertaken to determine the D4R expression pattern in the brain using immunohistochemistry, binding autoradiography and in situ hybridization, but the overall published results present large discrepancies. Here, we have explored an alternative genetic approach by studying bacterial artificial chromosome (BAC) transgenic mice that express enhanced green fluorescent protein (EGFP) under the transcriptional control of the mouse dopamine D4 receptor gene (Drd4). Immunohistochemical analysis performed in brain sections of Drd4-EGFP transgenic mice using an anti-EGFP polyclonal antibody showed that transgenic expression was predominant in deep layer neurons of the prefrontal cortex, particularly in the orbital, prelimbic, cingulate and rostral agranular portions. In addition, discrete groups of Drd4-EGFP labelled neurons were observed in the anterior olfactory nucleus, ventral pallidum, and lateral parabrachial nucleus. EGFP was not detected in the striatum, hippocampus or midbrain as described using other techniques. Given the fine specificity of EGFP expression in BAC transgenic mice and the high sensitivity of the EGFP antibody used in this study, our results indicate that Drd4 expression in the adult mouse brain is limited to a more restricted number of areas than previously reported. Its leading expression in the prefrontal cortex supports the importance of the D4R in complex behaviours depending on cortical dopamine (DA) transmission and its possible role in the etiopathophysiology of ADHD.

The nucleus accumbens and its related circuitry have been shown to play an important role in promoting the intake of hedonically desirable food. A previous report has demonstrated that the blockade of GABA A receptors in the ventral pallidum (VP), a target of GABAergic projection from the nucleus accumbens, greatly increases food, but not water, intake in satiated rats [Stratford et al. (1999) Brain Research, 825, 199-203]. The present study examined which neurotransmission in the VP is specifically involved in the intake of normally preferred taste stimuli. Microinjections of the GABA A antagonist bicuculline selectively increased the intake of saccharin solution but not that of water and quinine solution in water-deprived rats. In contrast, the facilitation of GABA A receptors by microinjections of muscimol in the VP generally suppressed the intake of saccharin, water and quinine. The same injections induced strong aversive taste reactivity responses to oral stimulation with not only quinine but also water and saccharin. The local administration of D-Ala2,N-Me-Phe4,Glyol5-enkephalin, a selective μ-opioid receptor agonist, into the VP had time-dependent effects, decreasing saccharine intake early and increasing intake late. Microinjections of SCH-23390, a dopamine D 1 receptor antagonist, in the VP suppressed the intake of saccharin but not water or quinine. Microinjections of sulpiride, the dopamine D 2 receptor antagonist, and 6-cyano-7-nitroquinoxaline-2,3-dione, the AMPA/kainate glutamate receptor antagonist, had no effect on fluid intake. These results reveal that GABA, opioid and D 1 receptors in the VP are involved in the consumption of hedonically positive taste stimuli.

Neurons in ventral pallidum fire to reward and its predictive cues. We tested mesolimbic activation effects on neural reward coding. Rats learned that a Pavlovian conditioned stimulus (CS+1 tone) predicted a second conditioned stimulus (CS+2 feeder click) followed by an unconditioned stimulus (UCS sucrose reward). Some rats were sensitized to amphetamine after training. Electrophysiological activity of ventral pallidal neurons to stimuli was later recorded under the influence of vehicle or acute amphetamine injection. Both sensitization and acute amphetamine increased ventral pallidum firing at CS+2 (population code and rate code). There were no changes at CS+1 and minimal changes to UCS. With a new 'Profile Analysis', we show that mesolimbic activation by sensitization/amphetamine incrementally shifted neuronal firing profiles away from prediction signal coding (maximal at CS+1) and toward incentive coding (maximal at CS+2), without changing hedonic impact coding (maximal at UCS). This pattern suggests mesolimbic activation specifically amplifies a motivational transform of CS+ predictive information into incentive salience coded by ventral pallidal neurons. Our results support incentive-sensitization predictions and suggest why cues temporally proximal to drug presentation may precipitate cue-triggered relapse in human addicts.

Basal forebrain cholinergic neurons project to diverse cortical and hippocampal areas and receive reciprocal projections therefrom. Maintenance of a fine-tuned synaptic communication between pre- and postsynaptic cells in neuronal circuitries also requires feedback mechanisms to control the probability of neurotransmitter release from the presynaptic terminal. Release of endocannabinoids or glutamate from a postsynaptic neuron has been identified as a means of retrograde synaptic signalling. Presynaptic action of endocannabinoids is largely mediated by type 1 cannabinoid (CB1) receptors, while fatty-acid amide hydrolase (FAAH) is involved in inactivating some endocannabinoids postsynaptically. Alternatively, vesicular glutamate transporter 3 (VGLUT3) controls release of glutamate from postsynaptic cells. Here, we studied the distribution of CB1 receptors, FAAH and VGLUT3 in cholinergic basal forebrain nuclei of mouse and rat. Cholinergic neurons were devoid of CB1 receptor immunoreactivity. A fine CB1 receptor-immunoreactive (ir) fibre meshwork was present in medial septum, diagonal bands and nucleus basalis. In contrast, the ventral pallidum and substantia innominata received dense CB1 receptor-ir innervation and cholinergic neurons received CB1 receptor-ir presumed synaptic contacts. Consistent with CB1 receptor distribution, FAAH-ir somata were abundant in basal forebrain and appeared in contact with CB1 receptor-containing terminals. Virtually all cholinergic neurons were immunoreactive for FAAH. A significant proportion of cholinergic cells exhibited VGLUT3 immunoreactivity in medial septum, diagonal bands and nucleus basalis, and were in close apposition to VGLUT3-ir terminals. VGLUT3 immunoreactivity was largely absent in ventral pallidum and substantia innominata. We propose that specific subsets of cholinergic neurons may utilize endocannabinoids or glutamate for retrograde control of the efficacy of input synapses, and the mutually exclusive complementary distribution pattern of CB1 receptor-ir and VGLUT3-ir fibres in basal forebrain suggests segregated input-specific signalling mechanisms by cholinergic neurons.

The ventral pallidum (VP) is situated at the convergence of midbrain dopamine and accumbal opioid efferent projections. Using in vivo electrophysiological procedures in chloral hydrate-anaesthetized rats, we examined whether discrete application of mu- [D-Ala2,N-Me-Phe4,Gly-ol5 (DAMGO)] or kappa- (U50488) opioid receptor agonists could alter VP responses to electrical stimulation of ventral tegmental area. Rate suppressions occurred frequently following ventral tegmental area stimulation. Consistent with an involvement of dopamine in this effect, none of the 12 spontaneously active ventral pallidal neurons recorded in rats that had monoamines depleted by reserpine responded to electrical stimulation of ventral tegmental area. Moreover, in intact rats, the dopamine antagonist flupenthixol attenuated evoked suppression in 100% of the neurons tested; however, the GABAA antagonist bicuculline was able to slightly attenuate the response in 50% of the neurons tested. These observations concur with our previous studies in indicating that ventral tegmental area stimulation releases dopamine (and sometimes GABA) onto ventral pallidal neurons. Both DAMGO and U50488 decreased the inhibitory effects of ventral tegmental area stimulation. These effects on the endogenously released transmitter differed from those seen with exogenously applied dopamine, for DAMGO did not alter the efficacy or potency of microiontophoretically applied dopamine. Taken together, these observations suggest that the interaction between DAMGO and dopamine does not occur at a site that is immediately postsynaptic to the dopaminergic input within the VP, but rather that opioid modulation involves mechanisms governing presynaptically released dopamine. These modulatory processes would enable ventral pallidal opioids to gate the influence of ventral tegmental area dopamine transmission on limbic system outputs at the level of the VP.

Sonic hedgehog (SHH) is considered to play an important role in tissue induction and patterning during development, particularly in determining neuronal cell fate in the ventral neural tube and in the embryonic forebrain. SHH precursor is autoproteolytically cleaved to an aminoterminal fragment (SHHN) which retains all known SHH biological activities. Here, we demonstrate the expression of a 22-kDa SHHN immunoreactive peptide in developing and adult hamster brain regions using a rabbit antiserum directed against a mouse SHHN fragment. Interestingly, SHHN was developmentally regulated with the highest expression observed in the adult brain, was resistant to Triton X-100 solubilization at 4 degrees C and partitioned with the raft component ganglioside GM1 during density gradient centrifugation. In rat brain, Shh transcripts were identified by double in situ hybridization in GABAergic neurons located in various basal forebrain nuclei including globus pallidus, ventral pallidum, medial septum-diagonal band complex, magnocellular preoptic nucleus and in cerebellar Purkinje cells as well as in motoneurons of several cranial nerve nuclei and of the spinal cord. We show that radiolabelled SHHN peptides are synthesized in the adult hamster retina and are transported axonally along the optic nerve to the superior colliculus in vivo. Our data indicate that SHHN is associated with cholesterol rich raft-like microdomains and anterogradely transported in the adult brain, and suggest that the roles of this extracellular protein are more diverse than originally thought.

Certain neurochemical and connectional characteristics common to extended amygdala and the nucleus accumbens shell suggest that the two represent a single functional-anatomical continuum. If this is so, it follows that the outputs of the two structures should be substantially similar. To address this, projections from the caudomedial shell and central nucleus of the amygdala, a key extended amygdala structure, were demonstrated in Sprague-Dawley rats with different anterograde axonal tracers processed separately to exhibit distinguishable brown and blue-black precipitates. The caudomedial shell projection is strong in the ventral pallidum and along the medial forebrain bundle through the lateral preopticohypothalamic continuum into the ventral tegmental area, distal to which it thins abruptly. The central nucleus projects strongly to the bed nucleus of the stria terminalis and the sublenticular extended amygdala, but substantially to the lateral hypothalamus only at levels behind the rostral part of the entopeduncular nucleus. Innervation of the ventral tegmental area by the central amygdala is minimal, but the lateral one-third of the substantia nigra, pars compacta and an adjacent lateral part of the retrorubral field receive substantial central amygdala input. Central amygdaloid projections are robust in caudal brainstem sites, such as the reticular formation, parabrachial nucleus, nucleus of the solitary tract and dorsal vagal complex, all of which receive little input from the accumbens. The substantial differences in the output systems of the caudomedial shell of accumbens and central amygdala suggest that the two represent distinct functional-anatomical systems.

Microiontophoresis was used to investigate the influence of dopamine on GABA- and glutamate-induced responses from ventral pallidal neurons recorded extracellularly in chloral hydrate-anaesthetized rats. Modulation was determined by comparing dopamine-induced alterations in amino acid-induced activity ('signal') with dopamine-induced effects on spontaneous firing ('noise'). A dopamine ejection current-response curve was generated to determine the current levels that did not alter spontaneous firing ('subthreshold') and those that produced approximately 50% of the maximal dopamine-induced response (ECur50). Co-iontophoresis of dopamine with GABA generally diminished the inhibitory influence of GABA on pallidal neuron firing; 70% of neurons tested with ECur50 dopamine demonstrated a decrease in the signal-to-noise ratio whereas 10% displayed an increase. At subthreshold dopamine ejection currents, 59% of neurons responded with a decrease and 18% responded with an increase in the GABA signal-to-noise ratio. When ECur50 dopamine was co-iontophoresed with glutamate, 84% of the neurons displayed a decrease in the signal-to-noise ratio for glutamate-evoked excitations whereas 11% demonstrated an increase. Subthreshold dopamine ejection currents decreased the signal-to-noise ratio in 62% of the ventral pallidal neurons excited by glutamate and increased the ratio in 23%. These data illustrate that dopamine substantially alters GABA- and glutamate-evoked responses even at ejection currents that are below those necessary to change spontaneous firing. Thus, it appears that neuromodulation is an important means by which dopamine influences ventral pallidal neuronal activity.

The neuropathological hallmark of Parkinson's disease is the loss of dopaminergic neurons in the pars compacta of the substantia nigra (SNc). The degenerative process starts unilaterally and spreads to the dopaminergic system of both hemispheres. However, the complete characterization of the nigra lesion and the subsequent changes in basal ganglia nuclei activity has not yet been achieved invivo. The aim of this study was to characterize the time course of the nigral lesion invivo, using longitudinal T2 relaxometry and diffusion tensor imaging, and the changes in basal ganglia nuclei activity, using manganese-enhanced magnetic resonance imaging, in 6-hydroxydopamine (6-OHDA)-lesioned rats. Our results showed that a unilateral SNc lesion induces bilateral alterations, as indicated by the enhancement of magnetic resonance imaging T2 relaxation times in both the ipsilateral and contralateral SNc. Moreover, axial and radial diffusivities demonstrated bilateral changes at 3 and 14days after 6-OHDA injection in the pars reticulata of the substantia nigra and cortex, respectively, in comparison to the sham group, suggesting bilateral microstructural alterations in these regions. Unexpectedly, manganese-enhanced magnetic resonance imaging showed decreased axonal transport from the ipsilateral subthalamic nucleus to the ventral pallidum in 6-OHDA-lesioned animals compared with the sham group. These findings demonstrate, for the first time invivo, the temporal pattern of bilateral alteration induced by the 6-OHDA model of Parkinson's disease, and indicate decreased axonal transport in the ipsilateral hemisphere. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd., This work was partially supported by a grant from the Fondo de Investigaciones Sanitarias (FIS08/0060 and PS09/00527) from the Ministerio de Sanidad y Consumo and SAF2009-0807 from the Ministerio de Ciencia e Innovacion, Spain. CIBERBBN is an initiative funded by the VI National R&D&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, and CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. G. S. has a JAE-Doc contract from CSIC.

Using an in vitro incubation method, we stimulated cGMP production in rat brain slices by rat ANF-(103 - 126). The localization of the cells responding to this ANF stimulation with an increase in cGMP production was studied by cGMP immunocytochemistry. ANF-responding cells were found in specific loci throughout the central nervous system of the rat. Regions containing the highest number of these cells were: the olfactory bulb, the lateral septum, the bed nucleus of the accessory olfactory tract, the mediobasal amygdala, the central grey area, the medial vestibular nucleus, and the nucleus of the solitary tract. Scattered ANF-responding, cGMP-immunoreactive cells were found in the hippocampus, the cingulate cortex, the ventral pallidum, the medial preoptic area, and the endopeduncular nucleus. ANF-responding cells in these areas had the same morphology, that is, multipolar with numerous processes. The nature of these ANF-responding cells was studied by sequential staining with an antiserum against glial fibrillary acidic protein (GFAP). In the hippocampus it was demonstrated that all ANF-responding cells are astroglial cells. However, not all astroglial cells in this area showed a cGMP response, demonstrating a regional heterogeneity. ANF-responding cells, having the appearance of neuronal cell bodies, could be found in the subfornical organ, and the hypothalamic paraventricular nucleus. Fibres producing cGMP immunoreactivity in response to ANF were found in the median preoptic nucleus, the medial preoptic area, and the dorsal hypothalamus.