Lateral hypothalamic involvement in feeding elicited from the ventral pallidum.

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.

Extreme tolerance to ammonia fumes in African naked mole-rats: animals that naturally lack neuropeptides from trigeminal chemosensory nerve fibers.

Naked mole-rats (Heterocephalus glaber) naturally lack neuropeptides associated with the signaling of chemical irritants from C type trigeminal nerve fibers. The goal of the present study was to assess behavioral responses of these animals to stimulation of the trigeminal chemosensory system, and to determine if stimulation would increase post-synaptic activity in the trigeminal nucleus, as seen in laboratory mice and rats. The results show that naked mole-rats are behaviorally insensitive to capsaicin solution applied to the nostrils and to ammonia fumes in a behavioral avoidance test. Centrally, the number of c Fos labeled cells in the spinal trigeminal nucleus increased from exposure to ammonia although the magnitude of the increase was less than for rats. The increase observed in naked mole-rats likely reflects activity from glutamate release, which appears insufficient to drive pain and aversion behaviors. The results support the idea that neuropeptides in the C fibers of the trigeminal system may be required to signal the aversive quality of specific chemical irritants. The natural lack of neuropeptides in naked mole-rats may be an adaptation to living in a challenging subterranean environment with extremely high levels of ammonia and carbon dioxide, stimuli known to excite trigeminal chemosensory C fibers.

Feeding specific glutamate surge in the rat lateral hypothalamus revealed by low-flow push-pull perfusion.

Substantial evidence implicates the lateral hypothalamus (LH) in the control of ingestive behavior and previous studies have found that glutamate release within the LH increases during meals. It is not known, however, whether this effect is selective for feeding, or whether similar changes are also seen during drinking. In this work, we examined this question using low-flow push-pull perfusion which allows sampling from small tissue volumes. Presentation of highly palatable solid or liquid foods to food-deprived rats resulted in an immediate increase in glutamate output of more than 200% over baseline. The response was maximal immediately after food presentation. In contrast, significant changes in glutamate output were not seen when water was presented to water-deprived animals, despite the occurrence of vigorous drinking. These findings confirm reports of feeding related glutamate release in the LH and demonstrate that this effect is specific to feeding, rather than being a general concomitant of all ingestive behaviors. The push-pull technique described here may allow the relevant region of the LH to be identified with greater precision than other methods.

Rotation and immediate-early gene expression in rats treated with the atypical D1 dopamine agonist SKF 83822.

Classical agonists of the dopamine D1 receptor activate both adenylyl cyclase and phospholipase C (PLC) signaling pathways. As a result, the extent to which these two pathways are essentially involved in various effects produced by D1 receptor agonists is currently uncertain. In the present report we examined the effects of SKF 83822, a dopamine D1 agonist which has been reported to activate adenylyl cyclase, but not PLC, on behavior and immediate early gene (IEG) expression in rats with unilateral 6-hydroxydopamine lesions. SKF 83822 (25-100 microg/kg) induced dose dependent contralateral rotation in these subjects, and, additionally, stimulated strong expression of the IEG products c-Fos, Fra2, Zif/268 and Arc in the deinnervated striatum. All of these effects could be antagonized by pretreatment with the selective D1 dopamine antagonist SCH 23390 (0.5 mg/kg). Although PLC may be involved in many effects mediated through dopamine D1 receptors, these results suggest that direct activation of PLC is not necessary for the induction of either rotation or IEG expression in dopamine depleted rats.

The selective m1 muscarinic antagonist MT-7 blocks pilocarpine-induced striatal Fos expression.

Systemic administration of the nonselective muscarinic agonist pilocarpine induces pronounced striatal Fos expression. Intrastriatal injections of the mamba snake toxin MT-7, a highly selective and irreversible m1 muscarinic antagonist, drastically attenuated this response when given 2, but not 8, days before pilocarpine. In contrast, MT-7 did not alter the response to amphetamine (5 mg/kg). These results suggest that pilocarpine induces Fos expression in the striatum as a result of stimulating m1 muscarinic receptors located within this structure and demonstrate the utility of the MT-7 for in vivo studies of cholinergic function.

Prenatal cocaine and morphine alter brain cyclin-dependent kinase 5 (Cdk5) activity in rat pups.

Pregnant rats received daily injections of saline, cocaine (20 mg/kg), morphine (2 mg/kg), or the combination of both drugs, on days 13-20 of gestation. Cyclin-dependent kinase 5 (Cdk5) activity was then measured in the resulting pups on postnatal days 1, 7, 14 and 28. Cocaine resulted in a time dependent increase in brain Cdk5 activity which peaked on day 14. Morphine, in contrast, induced a decrease in Cdk5 activity which was also maximal on day 14. Combined administration of the two drugs led to smaller increases than those seen after cocaine alone. These findings demonstrate that prenatal drug exposure can modify postnatal activity of Cdk5 in the brain and raise the possibility that alterations in Cdk5 may play a role in some of the neural and behavioral effects produced by these treatments.

Chemogenetic inhibition of cells in the paramedian midbrain tegmentum increases locomotor activity in rats.

Pronounced hyperactivity can be produced by lesions or pharmacological inhibition of cells in the median raphe nucleus (MR) located in the paramedian midbrain tegmentum. In the current study we examined whether a similar effect can be seen after chemogenetic inhibition of cells in this region using the DREADD (Designer Receptors Exclusively Activated by Designer Drugs) approach. We found that the DREADD ligand clozapine-N-oxide (CNO) increased locomotor activity in animals expressing the inhibitory DREADD hM4Di, but not those injected with a control virus in the MR. The effect was of rapid onset and short duration and persisted for at least four months after virus injections. Histological examination of the brains indicated that labeled fibers followed the known projection patterns of the MR to a variety of forebrain and midbrain structures. These findings confirm the role of the MR region in the control of locomotion and suggest that the DREADD technique may be a useful approach to the study of the functional architecture of this complex area. Methodological and interpretive aspects of DREADD studies are discussed.

Behavioral and physiological characterization of PKC-dependent phosphorylation in the Grin2a∆PKC mouse.

Activity-dependent plasticity in NMDA receptor-containing synapses can be regulated by phosphorylation of serines and tyrosines in the C-terminal domain of the receptor subunits by various kinases. We have previously identified S1291/S1312 as important sites for PKC phosphorylation; while Y1292/Y1312 are the sites indirectly phosphorylated by PKC via Src kinase. In the oocyte expression system, mutation of those Serine sites to Alanine (that cannot be phosphorylated) in the GluN2A subunit, resulted in a decreased PKC stimulated current enhancement through the receptors compared to wild-type NMDA receptors. To investigate the behavioral and physiological significance of those PKC-mediated phosphorylation sites in vivo, the Grin2a∆PKC mouse expressing GluN2A with four mutated amino acids: S1291A, S1312A, Y1292F and Y1387F was generated using homologous recombination. The Grin2a∆PKC mice exhibit reduced anxiety in the open field test, light dark emergence test, and elevated plus maze. The mutant mice show reduced alternation in a Y maze spontaneous alternation task and a in a non-reinforced T maze alternation task. Interestingly, when the mutant mice were exposed to novel environments, there was no increase in context-induced Fos levels in hippocampal CA1 and CA3 compared to home-cage Fos levels, while the Fos increased in the WT mice in CA1, CA3 and DG. When the SC-CA1 synapses in slices from mutant mice were stimulated using a theta-burst protocol, there was no impairment in LTP. Overall, these results suggest that at least one of those PKC-mediated phosphorylation sites regulates NMDAR-mediated signaling that modulates anxiety.

Cholinergic involvement in the cortical and hippocampal Fos expression induced in the rat by placement in a novel environment.

Placing rats into a series of novel environments induced vigorous c-fos expression in the infralimbic, anterior cingulate and retrosplenial cortices, and in the hippocampus. Pretreatment with the antimuscarinic drugs scopolamine and atropine was able to greatly suppress novelty-induced Fos expression at these sites. Placement into the novel environments also induced Fos expression in the habenula and the paraventricular thalamic nucleus, but the response at these sites did not appear to be sensitive to cholinergic blockade. These findings suggest that cholinergic mechanisms play an important role in ability of behavioral events to influence cortical and hippocampal immediate-early gene expression and are consistent with the possibility that some of the effects of anticholinergic drugs on placticity and learning may be mediated through alterations in the expression of these genes.

The atypical dopamine D1 receptor agonist SKF 83959 induces striatal Fos expression in rats.

The effects of dopamine D1 receptor agonists are often presumed to result from an activation of adenylyl cyclase, but dopamine D1 receptors may also be linked to other signal transduction cascades and the relative importance of these various pathways is currently unclear. SKF 83959 is an agonist at dopamine D1 receptors linked to phospholipase C, but has been reported to be an antagonist at receptors linked to adenylyl cyclase. The current report demonstrates that SKF 83959 induces pronounced, nonpatchy, expression of the immediate-early gene product Fos in the striatum of intact rats which can be converted to a patchy pattern by pretreatment with the dopamine D2-like receptor agonist quinpirole. In rats with unilateral 6-hydroxydopamine lesions SKF 83959 induces strong behavioral rotation and a greatly potentiated Fos response. All of the responses to SKF 83959, in both intact and dopamine-depleted animals, can be blocked by pretreatment with the dopamine D1 receptor antagonist SCH-23390. In intact subjects, SKF 83959 induced Fos expression less potently than the standard dopamine D1 receptor agonist SKF 82958, but the two drugs were approximately equipotent in deinnervated animals. These results demonstrate for the first time that possession of full efficacy at dopamine D1 receptors linked to adenylyl cyclase is not a necessary requirement for the induction of striatal Fos expression in intact animals and suggest that alternative signal transduction pathways may play a role in dopamine agonist induced Fos expression, especially in dopamine-depleted subjects.

The distribution of m4 muscarinic acetylcholine receptors in the islands of Calleja and striatum of rats and cynomolgus monkeys.

The distribution of m4 muscarinic acetylcholine receptors, and their relation to a number other markers, was examined using immunocytochemical techniques. Staining in the dorsal striatum tended to be more pronounced in the striosomal than the matrix compartment of both rats and cynomolgus monkeys. Within the ventral striatum, immunoreactivity was more pronounced within the olfactory tubercle and the shell region of the nucleus accumbens than in the nucleus accumbens core and was especially marked within the lateral striatal stripe. Modest staining was also seen in the external plexiform layer of the olfactory bulb. By far, the most intense staining in the forebrain of both rats and cynomolgus monkeys was found in islands of Calleja, where it appeared to be a selective marker for the core or hilus regions of the islands, or an analogous region found adjacent to them. The core regions of different islands appear to be continuous with each other so as to form a complex three-dimensional structure, which is largely encased by layers of granule cells. The neuronal elements in the islands of Calleja, which express m4 receptors, remain to be identified, but it is unlikely that cholinergic neurons are a major locus of these receptors. Although there are certain similarities between the islands of Calleja and other components of the striatal complex, the current studies emphasize the extent to which the islands are unique in terms of their architecture and chemical anatomy.

NPY mediates the feeding elicited by muscimol injections into the nucleus accumbens shell.

Injections of muscimol into the nucleus accumbens shell (AcbSh) induce large increases in food intake in satiated rats and also activate neurons in a number of feeding-related brain regions, including NPY-containing neurons in the arcuate hypothalamic nucleus and cells in the paraventricular hypothalamic nucleus. This suggests that the NPY system may participate in the expression of AcbSh-mediated feeding behavior. Therefore, we examined the effects of intraventricular administration of the Y1 receptor antagonist 1229U91 or the Y5 receptor antagonist L-152,804 on AcbSh-mediated food intake. Intra-AcbSh muscimol elicited a large increase in food intake which was potently suppressed by blocking either central Y1 or Y5 receptors. Our results suggest that the AcbSh influences food intake, in part, through the release of NPY.

Effects of haloperidol and clozapine on Fos expression in the primate striatum.

In cynomolgus monkeys, the typical neuroleptic haloperidol induced strong expression of the immediate early gene product Fos in both the nucleus accumbens shell and the dorsal striatum. In the caudate nucleus, haloperidol induced staining was more marked in the striosomes than the matrix. The atypical neuroleptic clozapine also induced Fos expression in the nucleus accumbens, but, in contrast to haloperidol, had only a small effect in the dorsal striatum. Additionally, clozapine was more potent than haloperidol at inducing Fos-like immunoreactivity in the islands of Calleja. These results are similar to those typically obtained in rodents, and suggest that the basic mechanisms underlying the regional specificity of the effects of atypical neuroleptics are likely be conserved between these two mammalian orders.

Role of dopamine D1 receptors in the striatal and cortical fos expression induced by the muscarinic agonist pilocarpine.

Injections of the muscarinic cholinergic receptor agonist pilocarpine (50 mg/kg) induced pronounced expression of the immediate early gene (IEG) product Fos in the striatum and cortex of rats. Pretreatment with the dopamine D1 receptor antagonist 7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-H-3-benzazepine hydrochloride (SCH-23390; 0.2-2.0 mg/kg) drastically attenuated the pilocarpine response in the striatum, but had no effect in the cortex. In contrast, the muscarinic receptor antagonist scopolamine (0.75-3.00 mg/kg) virtually abolished the Fos response at both sites. These results suggest that stimulation of dopamine D1 receptors may mediate the effects of muscarinic agonists on Fos expression in the striatum, but not the cortex.

Role of dopamine D2 receptors in the striatal immediate early gene response to amphetamine in reserpinized rats.

The indirect dopamine agonist amphetamine has been shown to induce a patchy pattern of immediate early gene (IEG) expression in the rostral striatum of both pharmacologically intact and reserpinized rats. The available data suggest that stimulation of D(2) dopamine receptors may play a role in the patterning of amphetamine-induced IEG expression, but direct evidence is lacking. In the current study of reserpinized animals, we found that pretreatment with the selective D(2) dopamine antagonist raclopride did not block the induction of the IEGs Fos and Arc by amphetamine, but greatly reduced the "patchiness" of the induced expression. Raclopride did not induce Fos or Arc expression by itself under the conditions studied here. These findings suggest that although stimulation of D(2) receptors is not necessary for amphetamine to induce IEG expression in reserpinized animals, these receptors do play a critical role in the spatial patterning of the resulting response.

Dopamine is differentially involved in the locomotor hyperactivity produced by manipulations of opioid, GABA and glutamate receptors in the median raphe nucleus.

The median raphe nucleus (MR) has been shown to exert a powerful influence on behavioral arousal and marked locomotor hyperactivity can be produced by intra-MR injections of a variety of drugs including GABAA and GABAB agonists, excitatory amino acid antagonists, and µ- and δ-opioid agonists. Other studies have indicated that the MR exerts an inhibitory influence on ascending dopamine systems, suggesting that MR induced alterations in activity may be mediated through changes in dopaminergic transmission. In the present study, we explored this possibility by examining whether systemic administration of the preferential D2 dopamine antagonist haloperidol is able to antagonize the hyperactivity produced by intra-MR injections of various drugs. We found that haloperidol completely blocked the locomotor response to intra-MR injections of the µ-opioid receptor agonist DAMGO and the δ-opioid receptor agonist DPDPE. In marked contrast, at doses which abolished the locomotor response to systemic amphetamine, haloperidol had no effect on the hyperactivity induced by intra-MR injections of GABAA agonist muscimol, the GABAB agonist baclofen, or the kainate/quisqualate antagonist pBB-PZDA, even though it suppressed baseline activity in these same animals. These results indicate that there must be at least two mechanisms capable of influencing behavioral arousal within the MR region, one of which is dependent on D2 dopamine receptors and the other is not.

Manipulation of GABA in the ventral pallidum, but not the nucleus accumbens, induces intense, preferential, fat consumption in rats.

Injections of the GABAA antagonist bicuculline into the medial ventral pallidum (VPm) induce marked increases in food intake, but nothing is known about the way in which these injections alter the distribution of intake in a macronutrient selection situation. We investigated this topic by adapting rats to a diet containing independent sources of protein, carbohydrate and fat, and then examining the effects of intra-VPm bicuculline on diet selection. Under these conditions, bicuculline produced a massive, preferential increase in fat intake with subjects consuming a mean of 97% of their calories from fat. Furthermore, all treated subjects ate fat before any other macronutrient, suggesting that the animals' behavior was directed selectively toward this dietary component even before consumption had begun. Similar effects were not observed following food deprivation, which exerted its largest effect on carbohydrate intake. To compare the intra-VPm bicuculline response to that seen after activation of GABA receptors in the nucleus accumbens shell (AcbSh), a major source of projections to the VPm, we conducted similar experiments with intra-AcbSh injections of muscimol and baclofen. These injections also enhanced food intake, but did not reproduce the selective preference for fat seen after intra-VPm bicuculline. These experiments provide the first demonstration of preferential enhancement of fat intake following manipulations of a nonpeptide neurotransmitter. Since mean intakes of fat under baseline conditions and after deprivation tended to be lower than those of carbohydrates, it seems unlikely that the effects of intra-VPm bicuculline are related to the intrinsic "rewarding" properties of fat, but might rather reflect the induction of a state of "fat craving."

Injections of muscimol into the paraventricular thalamic nucleus, but not mediodorsal thalamic nuclei, induce feeding in rats.

The paraventricular thalamic nucleus (PVT) is a component of the midline thalamic group that is interconnected with several brain regions known to play important roles in the control of food intake, including the lateral hypothalamus and nucleus accumbens shell, suggesting that the PVT itself may be involved in mediating feeding behavior. In the current study, we examined whether inhibition of cells in the PVT with the GABA(A) agonist muscimol could alter food intake in non-deprived rats. To control for possible spread of the drug, we also observed food intake after injections of muscimol into the overlying ventricle or laterally adjacent mediodorsal thalamic nuclei (MD). We found that muscimol injections into the central PVT dose-dependently increased food intake. In contrast, intra-MD injections of muscimol resulted in a potent dose-dependent suppression of food intake, while those into the overlying ventricle had no effect. These results support the proposal that the PVT is a component of the neural circuitry controlling feeding behavior.

Effects of muscimol, amphetamine, and DAMGO injected into the nucleus accumbens shell on food-reinforced lever pressing by undeprived rats.

Previous studies have shown that large increases in food intake in nondeprived animals can be induced by injections of both the GABA(A) agonist muscimol and the µ-opioid agonist DAMGO into the nucleus accumbens shell (AcbSh), while injections of the catecholamine agonist amphetamine have little effect. In the current study we examined whether injections of these drugs are able to increase food-reinforced lever pressing in nondeprived rats. Twelve subjects were trained to lever press on a continuous reinforcement schedule while food deprived and were then tested after being placed back on ad libitum feeding. Under these conditions, responding was markedly increased by injections of either muscimol or DAMGO, although the onset of the effects of the latter drug was delayed by 30-40 min. In contrast, amphetamine injections failed to increase reinforced lever pressing, although they did enhance responding on a non-reinforced lever, presumably reflecting alterations in behavioral activation. These results demonstrate that stimulation of GABA(A) and µ-opioid receptors within the AcbSh is able to promote not only food intake, but also food-directed operant behavior. In contrast, stimulation of AcbSh dopamine receptors may enhance behavioral arousal, but does not appear to specifically potentiate behaviors directed toward food procurement.

Evidence that the nucleus accumbens shell, ventral pallidum, and lateral hypothalamus are components of a lateralized feeding circuit.

Pronounced feeding can be elicited by injections of the GABA(A) agonist muscimol into the medial shell region of the nucleus accumbens (AcbSh). This region of AcbSh has been shown to project to both the lateral hypothalamus (LH) and the medial ventral pallidum (VPm). The current study examined the effects of unilateral LH or VPm lesions on the ingestive responses induced by injections of muscimol into the AcbSh on either the same or the opposite side of the brain. We found that lesions of either of these structures drastically attenuated feeding induced from the ipsilateral, as compared to the contralateral, AcbSh. The "ipsilateral/contralateral disruption design" employed here virtually rules out the possibility that the suppressive effects of the lesions were nonspecific and suggests that the VPm and LH play essential roles in mediating the ingestive effects of inactivation of the AcbSh.