Thomas R. Stratford

Injections of nociceptin into nucleus accumbens shell or ventromedial hypothalamic nucleus increase food intake

THE novel opioid receptor ORL1 is widely distributed throughout the CNS of the rat, and is present in high densities in several brain regions known to participate in the control of food intake. We injected a recently identified endogenous agonist of this receptor, nociceptin, into two of these feeding-related areas. Microinjections of nociceptin (2.5–25 nmol) into either the ventromedial hypothalamic nucleus or the nucleus accumbens shell significantly increased food intake in rats. We believe this to be the first report of a specific effect of nociceptin on a motivated behavior.

Ascending dopaminergic projections from the dorsal raphe nucleus in the rat

The projections of putative dopamine containing cells within the dorsal raphe nucleus (DR) were studied using a combination of tyrosine hydroxylase (TH) immunocytochemistry and fluorescent retrograde tracing. Substantial numbers of TH-immunoreactive cells in the DR were found to project to the nucleus accumbens. Progressively smaller numbers of cells were found to project to the lateral septum and medial prefrontal cortex. Very few TH-immunoreactive cells projected to the dorsal striatum, and none to the substantia nigra. TH-immunoreactive cells did not display serotonin-like immunoreactivity. These findings indicate that the projection pattern of TH-immunoreactive cells within the dorsal raphe more closely resembles that of dopaminergic cells within the ventral tegmental area (VTA) than that of serotonergic cells within the DR.

Studies on the behavioral activation produced by stimulation of GABAB receptors in the median raphe nucleus

Injections of the GABAB agonist baclofen into the median raphe nucleus (MR) resulted in marked hyperactivity and in increases in food and water intake by non-deprived animals. The locomotor effects of baclofen were stereospecific and could be antagonized by coinjection of the GABAB antagonist 2-hydroxysaclofen. Hyperactivity was produced by lower doses of baclofen, at shorter latencies, when the drug was injected into the MR than when it applied to the dorsal raphe nucleus (DR) or the ventral tegmental area (VTA). The locomotor response to intra-MR baclofen was unaltered in animals pretreated with the serotonin synthesis inhibitor p-chlorophenylalanine. Finally, intra-MR injections of baclofen produced a large increase in dopamine metabolism in the nucleus accumbens and striatum but failed to alter hippocampal or striatal serotonin metabolism. These findings suggest that baclofen may produce increases in activity and ingestive behavior as a result of an action on non-serotonergic cells in the MR.

Activation of feeding-related neural circuitry after unilateral injections of muscimol into the nucleus accumbens shell.

Chemical inhibition of neurons in the nucleus accumbens shell (AcbSh) elicits intense, behaviorally specific, feeding in satiated rats. We have demonstrated previously that this treatment activates a number of brain regions, most significantly the lateral hypothalamus (LH). This activation could be elicited through a direct neural connection with the AcbSh or secondarily through changes in autonomic activity, stress, or circulating levels of orexigenic or satiety factors. In the present study, we used the immunohistochemical localization of Fos protein to map neuronal activation after unilateral muscimol injections into the AcbSh to determine whether AcbSh-mediated Fos expression remains lateralized in the circuit and whether secondary systemic changes in the rat can be excluded as primary factors in the activation of downstream component nuclei. Rats receiving only saline injections exhibited very little Fos immunoreactivity. In contrast, unilateral injections of muscimol into the AcbSh consistently increased Fos expression in several brain regions. Three distinct patterns of expression were observed. Fos synthesis in the LH was increased only on the side of the brain ipsilateral to the muscimol injection. Fos expression remained primarily ipsilateral to the injection site in the septohypothalamic, paraventricular hypothalamic (PVN), paratenial thalamic, and lateral habenular nuclei, and medial substantia nigra, but was increased bilaterally in the piriform cortex, supraoptic nucleus, central nucleus of the amygdala, and nucleus of the solitary tract. Smaller numbers of Fos-immunoreactive cells were seen unilaterally in the bed nucleus of the stria terminalis, medial ventral pallidum, arcuate nucleus, and ventral tegmental area and bilaterally in the supraoptic and tuberomammillary nuclei. The labeling in the LH, PVN, and other unilaterally labeled structures provides evidence that these brain regions are components of an AcbSh-mediated neural circuit and suggests that they may be involved in the expression of AcbSh-mediated feeding behavior.

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.

Evidence that serotonergic projections to the substantia nigra in the rat arise in the dorsal, but not the median, raphe nucleus

Following microinjections of the fluorescent retrograde tracer Fast blue into the substantia nigra, large numbers of retrogradely labeled cells were observed in the dorsal raphe nucleus. Using immunocytochemical techniques it could be demonstrated that the majority of these cells contained serotonin-like-immunoreactivity. In contrast, careful examination of the region of the median raphe nucleus revealed no suggestion of a significant projection from the median raphe to the substantia nigra.

Evidence for motivational effects elicited by activation of GABA-A or dopamine receptors in the nucleus accumbens shell

Microinjections of the inhibitory GABA-A receptor agonist muscimol into the shell region of the nucleus accumbens (AcbSh) have been reported to induce large increases in food intake, but the effect of these injections on motivational processes is less clear. In the current study, bilateral injections of saline, muscimol (50ng/side) or d-amphetamine (10mug/side) were made into the AcbSh of rats trained to lever press on a progressive ratio schedule for food reward. Injections of both muscimol and amphetamine were found to produce a large increase in the breaking point relative to saline injections. This result suggests that inactivation of the AcbSh does not simply drive ingestive behavior, but also affects motivational processes assessed by the progressive ratio schedule. Breaking points were also increased by injections of amphetamine into the AcbSh.

Placement in a novel environment induces Fos-like immunoreactivity in supramammillary cells projecting to the hippocampus and midbrain

Injections of fluorescent retrograde tracers into either the hippocampal formation or the midbrain raphe nuclei resulted in retrograde labeling of many cells in the supramammillary region of the hypothalamus. Double labeling studies indicated that these two projections originate from different populations of supramammillary cells. Expression of the proto-oncoprotein Fos could be induced in some retrogradely labeled cells by placing rats in a novel open field before sacrifice. Although seen in both cell types, Fos-like immunoreactivity was significantly more common in supramammillary cells projecting to the hippocampus than in those projecting to the midbrain. These findings suggest that the supramammillary region may contain several populations of neurons which are differentially responsive to certain behavioral manipulations.

Evidence for GABAergic projections from the tegmental nuclei of Gudden to the mammillary body in the rat

Immunochemical studies have demonstrated the presence of large numbers of cells immunoreactive for glutamic acid decarboxylase (GAD) within the dorsal and ventral tegmental nuclei of Gudden. Following injections of Fluoro-Gold into the medial mammillary nucleus, a substantial proportion of the retrogradely labeled neurons within the ventral tegmental nucleus displayed GAD-like immunoreactivity. Conversely, electrolytic or excitotoxic lesions of the ventral tegmental nucleus produced a large decrease in the number of fibers and terminals immunoreactive for GAD within the medial mammillary nucleus. In contrast, electrolytic lesions of the dorsal tegmental nucleus were found to produce a large decrease in GAD-like immunoreactivity which was restricted to the lateral mammillary nucleus. Control lesions placed caudal to the dorsal tegmental nucleus were without effect. These findings suggest that the dorsal and ventral nuclei send a substantial, topographically organized, GABAeregic input to the mammillary body.

Opposite effects on the ingestion of ethanol and sucrose solutions after injections of muscimol into the nucleus accumbens shell

Injection of the GABA(A) receptor agonist muscimol into the nucleus accumbens shell (AcbSh) elicits robust feeding in satiated rats, but has no effect on water intake. The current study was designed to examine whether intra-AcbSh muscimol injections influence the intake of ethanol solutions in rats trained to drink using a limited access paradigm. We confirmed that bilateral injections of muscimol (100 ng) into the AcbSh produce large increases in the intake of sucrose solutions and of the chow maintenance diet but found in two independent experiments that these injections potently reduce the intake of a 10% ethanol solution. Furthermore, intra-AcbSh muscimol significantly increased intake of an ethanol-sucrose mixture. These results demonstrate that activating GABA(A) receptors in the vicinity of the AcbSh can have opposite effects on the intake of different caloric substances and are consistent with the possibility that GABAergic circuits in the AcbSh may play a role in mediating voluntary ethanol intake.