The anorectic effect of CNTF does not require action in leptin-responsive neurons.

Leptin resistance is a feature of obesity that poses a significant therapeutic challenge. Any treatment that is effective to reduce body weight in obese patients must overcome or circumvent leptin resistance, which promotes the maintenance of excess body fat in obese individuals. Ciliary neurotrophic factor (CNTF) is unique in its ability to reduce food intake and body weight in obese, leptin-resistant humans and rodents. Although attempts to use CNTF as an obesity therapy failed due to the development of neutralizing antibodies to the drug, efforts to understand mechanisms for CNTF's anorectic effects provide an opportunity to develop new drugs for leptin-resistant individuals. CNTF and leptin share several structural, anatomic, and signaling properties, but it is not understood whether or how the two cytokines might interact to affect energy balance. Here, we conditionally deleted the CNTF receptor (CNTFR) subunit, CNTFRα, in cells expressing leptin receptors. We found that CNTFR signaling in leptin-responsive neurons is not required for endogenous maintenance of energy balance and is not required for the anorectic response to exogenous administration of a CNTF agonist. These results indicate that despite anatomical overlap for CNTF and leptin action, CNTF appears to act within a distinct neuronal population to elicit its potent anorectic effect.

CNTFRalpha and CNTF expressions in the auditory brainstem: light and electron microscopy study.

CNTF receptor alpha (CNTFRalpha) is involved in the development, the maintenance and the regeneration of a variety of brain structures. However, its in vivo distribution has not been determined in the auditory system. CNTFRalpha expression was studied in developing and adult rat brainstem auditory nuclei using immunohistochemistry. At birth, the CNTFRalpha immunolabeling was clearly present in somata of the external nucleus of the inferior colliculus but was diffuse throughout brainstem auditory nuclei. The labeling was present in most brainstem auditory nuclei by post-natal day (PND) 6. The intensity of the staining subsequently increased to its highest level at PND21 and decreased to an adult-like appearance by the fourth post-natal week. In adult, CNTFRalpha labeling occurred in most neurons of the cochlear nucleus (CN), the lateral superior olive (LSO), the medial superior olive (MSO), and the medial nucleus of the trapezoid body (MNTB). CNTFRalpha labeling first appeared in the central nucleus of the inferior colliculus (IC) by the end of the fourth week. There was a general increase in the expression of CNTFRalpha that begins prior to the onset of hearing and reaches its highest level after this important developmental stage. Ultrastructural analysis in the adult ventral CN revealed the presence of CNTFR in post-synaptic sites. The presence of CNTF has been investigated in the adult using both Western blot and immunohistochemistry. Western blot showed the presence of CNTF in both peripheral and central auditory structures. The CNTF label was generally localized to the somatic compartment, in axons and as puncta surrounding neuronal cell bodies and dendrites. Differential CNTF labeling was observed between the different auditory nuclei. CNTF staining is present in neurons of the CN, the MNTB and the LSO, while it is restricted to axons and puncta surrounding neuronal somata in the IC. The clear presence of CNTFRalpha at post-synaptic terminals and that of its ligand the CNTF in axons and puncta surrounding neuronal cell bodies suggest an anterograde mode of action for CNTF in the central auditory system.

An essential role for the H218/AGR16/Edg-5/LP(B2) sphingosine 1-phosphate receptor in neuronal excitability.

A wealth of indirect data suggest that the H218/AGR16/Edg-5/LP(B2) sphingosine 1-phosphate (S1P) receptor plays important roles in development. In vitro, it activates several forms of development-related signal transduction and regulates cellular proliferation, differentiation and survival. It is expressed during embryogenesis, and mutation of an H218-like gene in zebrafish leads to profound defects in embryonic development. Nevertheless, the in vivo functions served by H218 signalling have not been directly investigated. We report here that mice in which the H218 gene has been disrupted are unexpectedly born with no apparent anatomical or physiological defects. In addition, no abnormalities were observed in general neurological development, peripheral axon growth or brain structure. However, between 3 and 7 weeks of age, H218(-/-) mice have seizures which are spontaneous, sporadic and occasionally lethal. Electroencephalographic abnormalities were identified both during and between the seizures. At a cellular level, whole-cell patch-clamp recordings revealed that the loss of H218 leads to a large increase in the excitability of neocortical pyramidal neurons. Therefore, H218 plays an essential, unanticipated and functionally important role in the proper development and/or mediation of neuronal excitability.

Selective introduction of antisense oligonucleotides into single adult CNS progenitor cells using electroporation demonstrates the requirement of STAT3 activation for CNTF-induced gliogenesis.

We have developed a novel method in which antisense DNA is selectively electroporated into individual adult neural progenitor cells. By electroporation of antisense oligonucleotides against signal transducer and activator of transcription 3 (STAT3) we demonstrate that ciliary neurotrophic factor (CNTF) is an instructive signal for astroglial type 2 cell fate specifically mediated via activation of STAT3. Activation of the mitogen-activated protein kinase (MAPK) signaling pathway induced only a transient increase in glial fibrillary acidic protein (GFAP) expression, and inhibition of this signaling pathway did not block the induction by CNTF of glial differentiation in progenitor cells. In addition we show that microelectroporation is a new powerful method for introducing antisense agents into single cells in complex cellular networks.

Additive effects of ciliary neurotrophic factor and testosterone on motoneuron survival; differential effects on motoneuron size and muscle morphology.

Testosterone and ciliary neurotrophic factor (CNTF) each enhance motoneuron survival in the spinal nucleus of the bulbocavernosus (SNB) of newborn rats. Here we directly compared the effects of CNTF and testosterone, alone and in combination, on SNB motoneuron number, SNB cell size, and morphology of the levator ani (LA) target muscle. Female rat pups were treated daily from postnatal day 1 through 6 (P1-P6) with recombinant human CNTF (hCNTF), testosterone propionate (TP), both hCNTF and TP, or neither. Effects of treatment were assessed on P7. TP and hCNTF each increased the number of SNB motoneurons and did so to a similar degree. Females treated with both hCNTF and TP had significantly more SNB cells than those receiving either hCNTF or TP alone. TP administered from P1 to P6 also increased SNB motoneuron size on P7. In contrast, hCNTF alone did not significantly affect SNB cell size, and hCNTF in combination with TP antagonized the effect of TP on motoneuron size. TP also increased LA muscle fiber number and LA fiber size, whereas hCNTF did not significantly influence LA muscle morphology in this study. Immunohistochemistry established that virtually all SNB motoneurons of both males and females express the CNTF alpha receptor (CNTFRalpha) between embryonic day 20 and postnatal day 6. Thus, effects of TP and hCNTF on SNB motoneuron survival were additive, and increases in motoneuron survival were dissociated from changes in target muscle morphology in hCNTF-treated animals. SNB motoneurons express CNTFRalpha perinatally and are therefore potential direct sites of hCNTF action.

In vivo localization and characterization of functional ciliary neurotrophic factor receptors which utilize JAK-STAT signaling.

The ciliary neurotrophic factor receptor is critically involved in embryonic motor neuron development. Postnatally, it may contribute to neuronal maintenance and regeneration. In addition, pharmacological stimulation of the receptor may slow the progression of several neurodegenerative disorders. The widespread nervous system expression of ciliary neurotrophic factor receptor components and the effects of low ciliary neurotrophic factor concentrations on a wide variety of cells in culture combine to suggest that functional ciliary neurotrophic factor receptors are expressed by many classes of neurons in vivo. However, the in vivo signaling properties and distribution of functional ciliary neurotrophic factor receptors have not been directly determined. We developed a novel in vivo assay of functional ciliary neurotrophic factor receptors which revealed that, in the adult nervous system, cranial and spinal motor neurons are very sensitive to ciliary neurotrophic factor and display a rapid, robust increase in phospho-STAT3 in their dendrites, cell bodies and nuclei, which is specifically blocked by the ciliary neurotrophic factor receptor antagonist, AADH-CNTF. In distinct contrast, several other classes of ciliary neurotrophic factor receptor expressing neurons fail to increase phospho-STAT3 levels following ciliary neurotrophic factor treatment, even when ciliary neurotrophic factor is applied at high concentrations. Leukemia inhibitory factor and epidermal growth factor elicit the same cell-type-dependent pattern of phospho-STAT3 increases. Responsive and non-responsive neurons express comparable levels of STAT3.Therefore, in vivo ciliary neurotrophic factor receptor-initiated STAT3 signal transduction is regulated in a very cell-type-dependent manner. The present data suggest that at least some of this regulation occurs at the STAT3 tyrosine phosphorylation step. These unexpected results also suggest that other forms of receptor-initiated STAT3 signal transduction may be similarly regulated.

Antisense studies in PC12 cells suggest a role for H218, a sphingosine 1-phosphate receptor, in growth-factor-induced cell-cell interaction and neurite outgrowth.

Our previous studies of H218, a sphingosine 1-phosphate (S1P) receptor and a member of the G-protein-coupled receptor superfamily, suggest that it may participate in mammalian nervous system development. Thus, brain levels of H218 mRNA are higher during early neurogenesis than postnatally. In addition, embryonic H218 immunoreactivity is preferentially localized in young neuronal cell bodies during their early stages of differentiation and in axons during their extension. This report describes the morphological effects of reducing native H218 levels in PC12 cells. Western blot analyses demonstrated that PC12 cells stably transfected with an expression vector carrying an antisense-oriented H218 cDNA contain less H218 protein than vector-transfected control cells. When differentiated with growth factors, the antisense-H218 cells display more neurite production and form less cell-cell contacts than the control cells. Therefore, these data, along with our previous H218 expression studies and a recent, independent study of H218 overexpression, support the possibility that H218 contributes to developmental processes regulating neuronal interaction and axon growth. The data are also consistent with reports that H218 is a S1P receptor, that S1P is present in serum, like that used in our PC12 cell cultures, and that it causes PC12 cell neurite retraction. Finally, and in agreement with a S1P receptor role for H218, we find that the antisense-H218 cells display less S1P-induced neurite retraction than control cells.

Regulation of ciliary neurotrophic factor receptor alpha in sciatic motor neurons following axotomy.

Spinal motor neurons are one of the few classes of neurons capable of regenerating axons following axotomy. Injury-induced expression of neurotrophic factors and corresponding receptors may play an important role in this rare ability. A wide variety of indirect data suggests that ciliary neurotrophic factor receptor alpha may critically contribute to the regeneration of injured spinal motor neurons. We used immunohistochemistry, in situ hybridization and retrograde tracing techniques to study the regulation of ciliary neurotrophic factor receptor alpha in axotomized sciatic motor neurons. Ciliary neurotrophic factor receptor alpha immunoreactivity, detected with two independent antisera, is increased in a subpopulation of caudal sciatic motor neuron soma one, two and six weeks after sciatic nerve transection and reattachment, while no changes are detected at one day and 15 weeks post-lesion. Ciliary neurotrophic factor receptor alpha messenger RNA levels are augmented in the same classes of neurons following an identical lesion, suggesting that increased synthesis contributes, at least in part, to the additional ciliary neurotrophic factor receptor alpha protein. Separating the proximal and distal nerve stumps with a plastic barrier does not noticeably affect the injury-induced change in ciliary neurotrophic factor receptor alpha regulation, thereby indicating that this injury response is not dependent on signals distal to the lesion traveling retrogradely through the nerve or signals generated by axonal growth through the distal nerve. The prolonged increases in ciliary neurotrophic factor receptor alpha protein and messenger RNA found in regenerating sciatic motor neurons contrast with the responses of non-regenerating central neurons, which are reported to display, at most, a short-lived increase in ciliary neurotrophic factor receptor alpha messenger RNA expression following injury. The present data are the first to demonstrate, in vivo, neuronal regulation of ciliary neurotrophic factor receptor alpha protein in response to injury. Moreover, they suggest that the ability of a subpopulation of spinal motor neurons to regulate ciliary neurotrophic factor receptor alpha levels in response to injury may play a role in their survival and axonal regeneration. Consistent with such a role, we also find relatively high, and probably elevated, levels of ciliary neurotrophic factor receptor alpha immunoreactivity in regenerating axons.

Identification of intestinal-type Barrett's metaplasia by using the intestine-specific protein villin and esophageal brush cytology.

Villin is an actin-binding cytoskeletal protein required for brush-border formation in the normal small intestinal and renal proximal tubule epithelium. Villin is a marker of cell differentiation in small intestinal and renal cell lineages, and recent studies have shown villin to be highly expressed in 100% of intestinal-type Barrett's metaplasias. This epithelium is the single greatest risk factor for developing esophageal adenocarcinoma and arises when the normal esophageal squamous epithelium is replaced by a small intestine-like columnar epithelium after damage by chronic gastroesophageal reflux. In intestinal-type Barrett's metaplasia, the villin protein exhibits a highly characteristic staining pattern in which strong apical, brush-border staining of columnar epithelial cells is observed. In this study, the ability to identify intestinal metaplastic cells by using this distinct villin staining pattern was examined in endoscopic esophageal brushings from patients with confirmed Barrett's metaplasia. Esophageal brushings from 81% (17 of 21) of patients with Barrett's metaplasia demonstrated individual columnar cells with the characteristic villin staining pattern, whereas all normal esophageal squamous cells, blood cells, and gastric columnar cells were negative for villin expression. Northern blot analysis demonstrated villin mRNA expression in Barrett's metaplasia but not in the normal squamous esophagus or gastric mucosa from the same patients. The combined use of villin immunohistochemical analysis and esophageal brush cytology may provide a simple and effective method of detecting intestinal-type Barrett's metaplasia in patients at higher risk for developing this epithelium, such as those experiencing chronic gastroesophageal reflux symptoms.

Ciliary neurotrophic factor receptor alpha in spinal motoneurons is regulated by gonadal hormones.

Ciliary neurotrophic factor receptor alpha (CNTFRalpha) is the ligand-binding component of the CNTF receptor. CNTFRalpha expression is essential for the normal development of spinal motoneurons and is required for the development of a sex difference in motoneuron number in androgen-sensitive perineal motoneurons. We used immunocytochemistry to examine the expression and hormone regulation of CNTFRalpha protein in the spinal nucleus of the bulbocavernosus (SNB), dorsolateral nucleus and retrodorsolateral nucleus of the lower lumbar spinal cord of adult rats. CNTFRalpha immunoreactivity (CNTFRalpha-IR) was observed in the somata and dendrites of virtually all motoneurons. In all three motor pools, the intensity of motoneuron soma labeling was greatest among gonadally intact males and was reduced in females and gonadectomized males. The density of CNTFRalpha-IR in neuropil also tended to be highest in intact males. Short-term (2 d) testosterone propionate treatment reversed the decline in the density of soma labeling in the SNB of castrated males but did not reverse any other effects of castration. Long-term hormone treatment, achieved by implanting males with testosterone capsules at the time of gonadectomy, prevented the decline in soma labeling in all motor pools and partially prevented the decline in neuropil label caused by castration. We conclude that expression of CNTFRalpha protein is androgen-regulated in spinal motoneurons.

Promotion of the development of enteric neurons and glia by neuropoietic cytokines: interactions with neurotrophin-3.

Neurotrophin-3 (NT-3) is known to promote enteric neuronal and glial development. Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) were investigated to test the hypothesis that the development of subsets of enteric neurons and/or glia is also affected by a neuropoietic cytokine, by itself, or together with NT-3. Crest-derived cells, immunoselected from the fetal rat gut (E14) with antibodies to p75NTR, were found by RT-PCR and immunocytochemistry (after culture) to express both alpha (CNTER alpha) and beta components (gp130 and LIFR beta) of the tripartite CNTF receptor. In situ, myenteric ganglia below the esophagus were CNTFR alpha-immunoreactive by E16-E18. In vitro, CNTF and LIF induced in crest-derived cells nuclear translocation of STAT3 (signal transducer and activator of transcription 3), a concentration-dependent increase in expression of neuronal or glial markers, and a decrease in expression of the precursor marker, nestin. LIFR beta was expressed by more cells than CNTFR alpha; therefore, although the factors were equipotent, the maximal effect of LIF > CNTF. The cytokines and NT-3 were additive in promoting neuronal but not glial development. Specifically, the development of neurons expressing NADPH-diaphorase activity (an early marker found in inhibitory motor neurons) was promoted by CNTF and NT-3. These observations support the idea that a ligand for the tripartite CNTF receptor complex plays a role in ENS development.

A putative G-protein-coupled receptor, H218, is down-regulated during the retinoic acid-induced differentiation of F9 embryonal carcinoma cells.

We have previously cloned a novel guanine nucleotide-binding protein (G-protein)-coupled receptor, H218, that has sequence similarity to a lysophosphatidic acid receptor, edg2. We present here Northern analysis indicating that the H218 mRNA is expressed in undifferentiated F9 embryonal carcinoma cells. The H218 message is down-regulated and its stability is decreased during retinoic acid- and dibutyryl cAMP-induced differentiation. Treatment by various receptor-selective retinoids indicated that retinoic acid receptor beta or gamma signaling, but not retinoid X receptor activation, is required for the down-regulation of H218 mRNA. Activation of the H218 receptor may contribute to the phenotype of undifferentiated F9 embryonal carcinoma cells.

Immunohistochemical localization of netrin-1 in the embryonic chick nervous system.

Netrin-1 has profound in vitro effects on the growth properties of vertebrate embryonic axons. In addition, netrin-1 mRNA is found in the floor plate of the embryonic nervous system, an intermediate target of many axons, including commissural axons that are affected by netrin-1 in vitro. Moreover, genetic studies of netrin-1 homologs in Caenorhabditis elegans and Drosophila implicate these proteins in commissure formation. We raised polyclonal antisera that recognize chick netrin-1 in fixed tissue sections. The antisera were used to immunohistochemically map netrin-1 in the embryonic spinal cord, brain, and retina. The relationship between netrin-1 localization and the growth of pioneering axons suggests roles for netrin-1 in the regulation of circumferential, commissural, and longitudinal axon growth in the spinal cord and brain. The data also suggest that the primary or sole effect of netrin-1 on pioneering spinal cord commissural axons is haptotactic. Furthermore, the pattern of netrin-1 localization raises the possibility that this protein helps mediate neuronal migration in the spinal cord, brain, and retina.

Embryonic expression pattern of H218, a G-protein coupled receptor homolog, suggests roles in early mammalian nervous system development.

Heterologous expression studies employing mammalian cell tissue culture techniques and in vivo studies of lower eukaryotes suggest that G-protein coupled receptors may play critical roles in regulating early stages of vertebrate nervous system development. Previous work suggests that H218, a rat G-protein coupled receptor homolog, could serve such a role. Most importantly, northern blot data indicate that whole brain H218 mRNA levels are highest during embryogenesis. In the present studies we raised, affinity-purified and characterized several anti-H218, polyclonal antisera and immunohistochemically mapped the expression of H218 during the early stages of rat embryonic nervous system development. The resulting data indicate that H218 is preferentially expressed in young, differentiating neuronal cell bodies and axons. Moreover, the expression is temporally regulated such that highest H218 levels are found in neuronal cell bodies during their early stages of differentiation and in axons during their outgrowth. Therefore, we propose that H218 signal transduction may widely participate in the regulation of some of the first steps in neuronal differentiation including axon outgrowth.

Ciliary neurotrophic factor receptor alpha-immunoreactivity in the monkey central nervous system.

Ciliary neurotrophic factor (CNTF) sustains the viability and phenotypic expression of a variety of neuronal populations in the central nervous system. Cranial and spinal motor neurons are particularly sensitive to the trophic effects of CNTF, and clinical trials are underway testing the potential therapeutic value of this trophic factor in patients with amyotrophic lateral sclerosis. Yet, the distribution of the alpha subunit of the receptor for ciliary neurotrophic factor (CNTFR alpha), which is essential for the trophic effects of CNTF to occur, is unknown in any primate species. Towards this end, the present study used a polyclonal antibody directed against CNTFR alpha to evaluate the distribution of CNTFR alpha-immunoreactive (-ir) cells within the brain and spinal cord of Cebus apella monkeys. CNTFR alpha-ir was found exclusively within neurons. In the anterior horn of the spinal cord, virtually all motor neurons were darkly immunoreactive for CNTFR alpha. A similar pattern of CNTFR alpha-ir was seen within all cranial motor nuclei with general somatic efferent function (III, IV, motor V, VI, VII, and XII cranial nerves). CNTFR alpha-ir was also seen in other regions involved with motor function including the Purkinje cells of the cerebellum, the substantia nigra pars compacta, red nucleus, dorsal motor nucleus of X cranial nerve, and giant neurons of sensory motor neocortex. A few CNTFR alpha-ir neurons were seen within the globus pallidus with concomitant terminal-like staining within the subthalamic nucleus. Autonomic regions such as the mesencephalic nucleus of the trigeminal nerve and the interomedial lateral cell column of the thoracic spinal cord also contained CNTFR alpha-ir neurons. Finally, the hippocampus displayed dense CNTFR alpha-ir within the pyramidal cell layer of the hippocampal formation and the granule cell layer of the dentate gyrus. The dense expression of this CNTFR alpha protein within regions subserving motor, autonomic, and sensory functions suggests that CNTFR alpha supports many central nervous system regions with diverse functions.

Immunohistochemical localization of ciliary neurotrophic factor receptor alpha expression in the rat nervous system.

Ciliary neurotrophic factor receptor alpha (CNTFR alpha) is essential for normal embryonic development and may be involved in postnatal and adult neuronal maintenance. In addition, a rapidly growing body of evidence suggests that CNTFR alpha serves as a site of action for future growth factor therapeutics capable of treating a wide variety of disorders resulting from neuronal loss. We raised two polyclonal, anti-CNTFR alpha antisera against synthetic peptides corresponding to independent regions of rat CNTFR alpha. Western blot and immunohistochemical analyses indicated that affinity-purified preparations of both antisera specifically recognize CNTFR alpha. In the adult brain, the highest levels of CNTFR alpha immunoreactivity were found in the perikarya, dendrites and, occasionally, the axons of several distinct classes of neurons including hippocampal formation neurons, some sensory neurons, and many neurons involved in motor control. CNTFR alpha immunoreactivity also was concentrated in the following: perikarya, dendrites, and axons of ventral horn motor neurons in adult spinal cord; perikarya and axons of adult dorsal root ganglion neurons; and axons in adult peripheral nerve. In embryonic tissue, the highest levels of CNTFR alpha immunoreactivity were observed in differentiating neurons and their processes. Therefore, the present data suggest that CNTFR alpha serves several diverse functions in adulthood and during development.

Ciliary neurotrophic factor receptor alpha mRNA in NB41A3 neuroblastoma cells: regulation by cAMP.

Ciliary neurotrophic factor (CNTF) affects the in vitro and in vivo survival and differentiation of several classes of neurons by binding to the CNTF receptor alpha. We examined the possibility that intracellular cAMP can regulate CNTF receptor alpha mRNA levels in two neuronal cell lines that display cAMP-dependent process outgrowth. Dibutyryl cAMP did not affect CNTF receptor alpha mRNA levels in PC12 cells but elicited a dose- and time-dependent increase in NB41A3 cell CNTF receptor alpha mRNA levels. Forskolin similarly increased CNTF receptor alpha mRNA levels in NB41A3 cells. The data suggest that signal transduction mechanisms involving cAMP may 'crosstalk' with CNTF-initiated signal transduction in a cell type-specific manner and that CNTF receptor alpha expression is not generally linked to neuronal process outgrowth.

Chronic ethanol administration decreases brain-derived neurotrophic factor gene expression in the rat hippocampus.

We have previously demonstrated that chronic ethanol consumption decreases neurotrophic activity in hippocampal extracts, as assessed by a chick dorsal root ganglia bioassay, but has no effect on hippocampal NGF mRNA or NGF protein levels. We presently report that hippocampal mRNAs encoding neurotrophin-3 and basic fibroblast growth factor are also unaffected. However, in contrast, brain-derived neurotrophic factor mRNA is reliably decreased, thereby suggesting that ethanol-induced damage of the septohippocampal system may at least partially result from an ethanol-induced decrease in hippocampal brain-derived neurotrophic factor expression.

Cloning of the rat edg-1 immediate-early gene: expression pattern suggests diverse functions.

The edg-1 immediate-early gene encodes a G-protein-coupled receptor homolog implicated in endothelial cell differentiation. We report the cloning of the rat edg-1 gene. Our Northern analyses indicate that edg-1 is much more widely expressed than previously thought. edg-1 mRNA was found in many organs at several stages of development with relatively high levels present in adult brain. edg-1 transcripts were also detected in several cell lines. Expression of edg-1 mRNA in the PC12 cell model of neuronal differentiation was unaffected by agents that cause PC12 cells to differentiate or proliferate. Therefore, edg-1 may play a cell-type-specific role in differentiation and also participate in neurotransmission.

D2 dopamine receptor mRNA distribution in cholinergic and somatostatinergic cells of the rat caudate-putamen and nucleus accumbens.

An in situ hybridization procedure that identifies cells expressing D2 dopamine receptor mRNA was combined in double-labelling studies with immunohistochemical procedures that identify cells expressing either choline acetyltransferase (ChAT) or somatostatin. D2 receptor mRNA was detected in almost all of the ChAT positive caudate-putamen cells, approximately half of the ChAT positive nucleus accumbens cells and none of the somatostatin-positive cells in either brain region.