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WOS: 000310414300006, PubMed ID: 22960117, Cocaine and amphetamine regulated transcript (CART) mRNA and peptides are highly expressed in the paraventricular (PVN), dorsomedial (DMH) and arcuate (ARC) nuclei of the hypothalamus. It has been suggested that these nuclei regulate the hypothalamic-pituitary-adrenal (HPA) axis, autonomic nervous system activity, and feeding behavior. Our previous studies showed that forced swim stress augmented CART peptide expression significantly in whole hypothalamus of male rats. In another study, forced swim stress increased the number of CART-immunoreactive cells in female PVN, whereas no effect was observed in male PVN or in the ARC nucleus of either sex. In the present study, we evaluated the effect of forced swim stress on CART mRNA expression in PVN, DMH and ARC nuclei in both male and female rats. Twelve male (stressed and controls, n=6 each) and 12 female (stressed and controls, n=6 each) Sprague-Dawley rats were used. Control animals were only handled, whereas forced swim stress procedure was applied to the stressed groups. Brains were dissected and brain sections containing PVN, DMH and ARC nuclei were prepared. CART mRNA levels were determined by in situ hybridization. In male rats, forced swim stress upregulated CART mRNA expression in DMH and downregulated it in the ARC. In female rats, forced swim stress increased CART mRNA expression in PVN and DMH, whereas a decrease was observed in the ARC nucleus. Our results show that forced swim stress elicits region- and sex-specific changes in CART mRNA expression in rat hypothalamus that may help in explaining some of the effects of stress. (C) 2012 Elsevier B.V. All rights reserved., NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [3 R01 DA010732-05S1]; National Center for Research ResourcesUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Center for Research Resources (NCRR) [P51RR165]; Office of Research Infrastructure Programs/OD [P51OD11132], This study was supported by NIH Grant no. 3 R01 DA010732-05S1 and by the National Center for Research Resources (P51RR165) and is currently supported by the Office of Research Infrastructure Programs/OD (P51OD11132).

CART mRNA and peptides are highly expressed in the anatomical structures composing the hypothalamo-pituitary-adrenal (HPA) axis and sympatho-adrenal system. Anatomical and functional studies suggest that CART peptides may have a role in the regulation of the neuroendocrine and autonomic responses during stress. Our previous study showed that CART peptides increased significantly in the male hypothalamus and amygdala 10 min after the forced swim stress. The present study aimed to examine the effect of forced swim stress on CART peptide expression in selected brain regions, including those where CART peptide expression has not been reported before (frontal cortex, pons, medulla oblongata), as well as in endocrine glands related to stress in male Sprague Dawley rats. A total of 16 (n = 8) animals were used, including control groups. Rats were subjected to forced swim on two consecutive days, and sacrificed on the second day, 2 h after the termination of the stress procedure. Frontal cortex, pons, medulla oblongata, hypothalamus, pituitary and adrenal glands were dissected and homogenized. CART peptide expression in these tissues was measured by Western Blotting and six different CART peptide fragments were identified. Our results showed that forced swim stress elicited region-specific changes in CART peptide expression. CART was upregulated in the frontal cortex, hypothalamus, medulla oblongata and adrenal gland while there was no change in the pons and pituitary. Enhanced CART peptide fragments in these brain regions and adrenal glands may have a role in the regulation of the HPA and sympatho-adrenal axis activity during stress response.

Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action.

Production of mRNA from the cocaine- and amphetamine-regulated transcript (CART) gene is regulated by cocaine and other drugs of abuse in the nucleus accumbens (NAc), a brain reward region. Current hypotheses postulate that CART peptides there oppose the rewarding actions of cocaine by opposing the effects of dopaminergic transmission. Since over expression of CREB was shown to decrease cocaine-mediated reward, we hypothesized that CART could be a target gene for CREB in the NAc and that over expression of CREB would increase CART peptide levels. Transcription factor (TF) binding to DNA is influenced by sequences adjacent to consensus TF binding sites and other factors. We thus examined CREB binding to a 27mer oligonucleotide containing the CRE sequence from the CART gene proximal promoter. Using electrophoretic mobility shift assays and TF-antibody super shift assays, CREB was found to bind to the CRE sequence from the CART promoter. To test if over expression of CREB in the NAc affected CART peptide levels, Herpes simplex virus-1 vectors over expressing CREB (HSV-CREB), or a vector that expressed LacZ (HSV-LacZ) as a control, were injected into the NAc of rats. Western blotting and in situ hybridization showed that HSV-CREB injections increased CART mRNA and peptide levels. Injections of a dominant negative CREB mutant (HSV-mCREB) did not alter either CART mRNA or peptide levels. The finding that CREB can regulate the levels of CART mRNA and peptides in vivo in the NAc supports a role for CART peptides in psychostimulant-induced reward and reinforcement.

We have recently shown that CART peptides exhibit a diurnal rhythm in blood that is affected by food intake and glucocorticoids. In the present study, we extend our observations by demonstrating that CART peptides also exhibit a diurnal rhythm in several brain regions, notably the nucleus accumbens, hypothalamus and amygdala, but not in the midbrain. To examine whether the CART peptide rhythm was dependent on food intake, animals were food-deprived for 24 h. In regular-fed animals, CART peptide levels were lower in the morning compared to evening hours. However, this diurnal variation of CART peptide was not apparent in fasted animals, and CART peptide levels were reduced. The diurnal variation of CART mRNA in the nucleus accumbens paralleled the variation of CART peptide in this region. Similar to the peptide, the mRNA did not change in midbrain. These results show that CART peptide levels and gene expression undergo a diurnal variation in some brain regions, and the variation is altered by fasting. These findings suggest a variety of regulatory mechanisms for CART and additional considerations for CART's role in brain.

CART peptide is a neurotransmitter involved in various physiological processes including feeding, sensory processing, development, addiction, and stress. Substantial amounts of CART mRNA and CART peptide expression have been demonstrated in the hypothalamic periventricular area, the paraventricular nucleus of hypothalamus, the anterior lobe of the pituitary gland and the adrenal gland in addition to many other brain areas. This localization defines the HPA axis, responsible for the stress response. The aim of the present study was to assess the possible mediation of the CART peptides in the stress response by testing for changes in CART in adrenalectomized animals. Three groups of male Sprague–Dawley rats were used for the study: sham operated, adrenalectomized (ADX), and ADX+hormone replacement (corticosterone, 30 μg/ml in drinking water/5 days). All rats were perfused 7 days after the surgery, brains were removed and serial coronal sections were prepared. Immunohistochemistry was used to assess CART peptide expression in paraventricular and supraoptic cells. ADX lowered both the number and percentage of CART-positive cells compared to the sham-operated group, and hormone replacement partially restored the decrease in the CART cell numbers in ADX animals. There were no significant changes in the supraoptic nucleus. Our results suggest a role for CART peptides in the stress response.

An earlier study in our laboratory showed that 24 h after intracerebroventricular administration of the irreversible dopamine transporter inhibitor RTI-76, [3H]GBR12935 binding to the dopamine transporter (DAT) protein was inhibited in both the striatum and nucleus accumbens of the rat in a dose-dependent fashion (0.05-5.0 micromol). The rate of return of binding to control levels was used to calculate the half-life of DAT. Since changes in behavior could conceivably influence the half-life, the effects of various doses of RTI-76 on locomotor activity 1 and 3 days after RTI-76 administration were examined. During the first day after i.c.v. administration, 1.25 micromol RTI-76 had no effect on locomotor activity, but 2.5 micromol RTI-76 significantly increased locomotor activity in rats, a time at which this dose inhibited 41 and 42% of [3H]GBR12935 binding in the striatum and in the nucleus accumbens, respectively. These results agree with earlier reports showing that significant blockade of the dopamine transporter protein in the striatum is required for increases in motor activity in rodents. However, 5.0 micromol RTI-76 did not increase locomotor activity, even though binding was inhibited to 38 and 37% of control levels in the striatum and nucleus accumbens, respectively. Furthermore, our present results suggest that locomotor activity does not continue to increase as the blockade of DAT increases. Notably, there were no increases in locomotor activity at the dose of RTI-76 (100 nmol) used to measure DAT half-life.

Because of the reported presence of both CART peptide and NOS activity in the same hypothalamic nuclei, their colocalization was examined. Eighteen percent of the neurons in the supraoptic nuclei, and 16% of the neurons in the paraventricular nucleus contained both CART immunoreactivity and NOS activity. Many other neurons in these regions stained for only one marker although they were often close by. Thus, CART peptides and NO may interact in these regions.

CART was identified as a novel mRNA regulated by psychostimulant drugs. CART peptides appear to be neurotransmitters involved in a variety of functions such as feeding. The mouse gene has been characterized and localized to Chromosome 13. The processing of CART peptides is evident in Western blotting studies.

In order to examine the effect of cocaine and amphetamine regulated transcript (CART) peptide depletion in adult rats, CART shRNAs or scrambled control shRNAs were administered bilaterally into the nucleus accumbens (NAc). There was an increase in body weight of the shRNA injected rats compared with the rats injected with the scrambled RNA. This is compatible with the data showing a role for the peptide in body weight and food intake. Also at this time, there was about a two-and-a-half fold increase in cocaine-mediated locomotion in the shRNA injected rats compared to the control rats. This finding is critical support for the hypothesis that endogenous CART peptides in the NAc inhibit the actions of cocaine and other psychostimulants. In immunohistochemical experiments on these same animals, there was a decrease in the staining density of CART peptide in the NAc of the shRNA injected rats. These data show that shRNA can reduce CART peptides in the NAc and that endogenous CART peptides influence body weight and cocaine-induced locomotor activity (LMA).

Previously we have shown that the [ 125 I]DEEP-labeled dopamine transporter from the rat nucleus accumbens has a higher apparent molecular weight than that from striatum. The present study confirms and extends these observations 24 . Experiments with nucleus accumbens showed [ 125 I]-DEEP to specifically bind to a protein with an apparent molecular weight of 76 kDa and with the pharmacological properties of the dopamine transporter. In exoglycosidase studies, treatment with neuraminidase, but not alpha-mannosidase, reduced the apparent molecular weight of the dopamine transporter from both the striatum and nucleus accumbens; however, a difference in the apparent molecular weight was still observed. N -Glycanase treatment, on the other hand, did reduce the apparent molecular weight of the dopamine transporters from the two regions to a similar value, approximately 56 kDa. In radioligand binding studies examining the effect of partial deglycosylation on striatal dopamine transporters, neuraminidase did not affect specific [ 3 H]WIN 35, 428 binding at 4 and 40 nM concentrations. In conclusion, the present study demonstrates that the difference in the apparent molecular weight of the dopamine transporter from these two regions is due to a difference in glycosylation and that the dopamine transporter from both regions contains similar amounts of sialic acid in their carbohydrate structure. Furthermore, the present data also indicate that the polypeptide portion of the dopamine transporter from both regions could be the same gene product.

CART (cocaine and amphetamine regulated transcript) is a neuropeptide involved in the control of several physiological processes, such as response to psychostimulants, food intake, depressive diseases and neuroprotection. It is robustly expressed in the brain, mainly in regions that control emotional and stress responses and it is regulated by estrogen in the hypothalamus. There is a distinct population of CART neurons located in the vicinity of the Edinger-Westphal nucleus of the midbrain that also colocalize urocortin-1. The aims of this study were 1) to determine the distribution of CART immunoreactive neurons in the monkey midbrain, 2) to examine the effects of estrogen (E) and progesterone (P) on midbrain CART mRNA and peptide expression and 3) to determine whether midbrain CART neurons contain steroid receptors. Adult female rhesus monkeys (Macaca mulatta) were spayed and either treated with placebo (OVX), estrogen alone (E), progesterone alone (P) or E+P. Animals were prepared (a) for RNA extraction followed by microarray analysis and quantitative (q) RT-PCR (n=3/group); (b) for immunohistochemical analysis of CART and CART+tryptophan hydroxylase (TPH), CART+estrogen receptors (ER) or CART+progesterone receptors (n=5/group) and (c) for Western blots (n=3/group). Both E- and E+P-administration decreased CART gene expression on the microarray and with qRT-PCR. Stereological analysis of CART immunostaining at five levels of the Edinger-Westphal nucleus indicated little effect of E or E+P administration on the area of CART immunostaining. However, P administration increased CART-immunopositive area in comparison to the OVX control group with Student's t-test, but not with ANOVA. CART 55-102 detection on Western blot was unchanged by hormone administration. ERbeta and PR were detected in CART neurons and CART fibers appeared to innervate TPH-positive serotonin neurons in the dorsal raphe. In summary, E decreased CART mRNA, but this effect did not translate to the protein level. Moreover, P administration alone had a variable effect on CART mRNA, but it caused an increase in CART immunostaining. Together, the data suggest that CART neurons in the midbrain have a unique steroid response, which may be mediated by nuclear receptors, neuroactive steroids or interneurons.

CART peptide is a novel neurotransmitter that, due to its distribution in the brain and its modulation of dopamine systems, may be involved in aspects of reward and drug abuse. In the nucleus accumbens (NAcc), CART peptide immunoreactivity (IR) is colocalized with substance P-IR in neurons. Approximately 86% of CART-IR cells colocalize with substance P, while only 19% of substance P-IR neurons contain CART. CART peptide does not colocalize with enkephalin-IR in this region. The substance P-CART colocalization exists in a rostro-caudal gradient with more colocalization in rostral regions. The presence of CART in substance P NAcc neurons suggests that CART neurons may be a subset of the basal ganglia direct pathway or that CART neurons are involved in limbic projections of the NAcc, such as to the ventral pallidum.

Cocaine- and amphetamine-regulated transcript (CART) peptides are found in brain and spinal cord areas involved in pain transmission. In the present study, we investigated the role of rat CART (55-102) in the modulation of an acute pain model after intrathecal administration. The results show that CART (55-102) was without effect on the tail-flick test after i.t. injection in mice. Interestingly, i.t. administration of CART (55-102) significantly enhanced the antinociceptive effect of morphine in the tail-flick test. These results suggest that CART (55-102) potentiates the effects of opioids to inhibit the nociceptive information transmission.

We investigated the effects of rat cocaine- and amphetamine-regulated transcript CART (55-102) after i.c.v. administration in mouse models of acute and persistent pain. CART was not active in the tail-flick or PPQ tests. It increased the latency to paw licking in the hot-plate test but only at doses that impaired motor function. CART produces antinociception in the formalin test in both phases. Our results suggest that CART is involved in supraspinal pain transmission.

The objective of this study was to examine the turnover of the serotonin transporter (SERT) by determining its production rate (r), degradation rate constant (k) and half-life of recovery (t1/2). The turnover of SERT was determined from the rate of recovery of binding after administration of RTI-76, an irreversible inhibitor of ligand binding. In preliminary studies, in vitro incubation of rat cerebral cortex with RTI-76 produced a wash and temperature resistant inhibition of SERT binding densities (Bmax). Citalopram protected against the RTI-76-induced inhibition of SERT binding. Following 6 h of in vivo intracerebroventricular injections of 100 nmol of RTI-76, there was a dose- and time-dependent reduction (- 60%) of SERT binding in hippocampus and striatum, without a change in the Kd. SERT binding densities recovered over several days, reaching control levels by day 14. The recovery curve fit the standard model of protein synthesis and degradation. The turnover parameters of SERT were determined in hippocampus and striatum, regions that receive serotonergic innervation from the dorsal and median midbrain raphe nuclei, respectively. In the hippocampus, the production rate constant was 2.36 fmol mg protein (-1)h(-1); the degradation rate constant was 0.0077 h(-1); and the half-life of the SERT recovery was 3.4 days. The values in the striatum were similar. The decrease and recovery of [3H]-5-HT uptake correlated highly (r = 0.93) with the recovery of SERT binding.

Sodium nitroprusside (SNP), a generator of nitric oxide (NO), decreased [3H]dopamine uptake into rat striatal synaptosomal preparations in a dose-, time- and temperature-dependent fashion. Reduced hemoglobin, a substance that binds NO, prevented the SNP-induced decrease in uptake. Potassium ferri- and ferrocyanide, compounds similar to SNP that do not produce NO, were without effect on uptake. SNP inhibited [3H]dopamine uptake in synaptosomes from nucleus accumbens and olfactory tubercle as well but with a lower potency. SNP inhibited [3H]serotonin and [3H]glutamate uptake but had no effect on [3H]norepinephrine uptake. S-Nitroso-N-acetylpenicillamine (SNAP), another generator of NO, had effects similar to those of SNP. The SNP-induced decrease in [3H]dopamine uptake was due to a Vmax decrease at 100 μM SNP and to both a Vmax and Km change at 300 μM SNP. Depletion of calcium by omission of calcium from buffers and addition of EGTA increased the potency of SNP in inhibiting uptake. There was no change in [3H]WIN 35428 binding to the dopamine transporter with doses of SNP that inhibited uptake. These data indicate that NO can decrease [3H]dopamine transporter function.

Because some evidence suggests that cocaine and GBR12935 bind to different sites, we utilized photoaffinity probes from both classes of compounds to see if they label the same protein. [125I]RTI-82 a cocaine analog, and [125I]DEEP, a GBR analog, labeled protein(s) showing the same molecular weight, a similar pharmacological profile and a similar sensitivity to neuraminidase.

Neurotensin receptor autoradiography reveals dense binding in normal human substantia nigra. In nigra from patients with Parkinson's disease, nigral receptor binding is only about one-third of control values. ‘Saturation’ analysis suggests that these changes result from receptor loss. These results support a neurotensin-domapine interaction in human nigro-striatal circuits.

Muscarinic cholinergic receptors build up on the proximal side of a ligature placed on the rat sciatic and vagus nerves. These receptors appear to be flowing down the axons of at least a portion of the nerve fibers present in sciatic and vagus nerves. Most of the anterograde flowing muscarinic binding sites are displaceable with 10−4 M carbachol indicating that many are high affinity agonist binding sites. These receptors can also be localized in the ganglionic nerve cell bodies in the dorsal root ganglia which contribute fibers to the sciatic nerve. We hypothesize that muscarinic cholinergic receptors are synthesized in the ganglion cell bodies and transported distally in sensory nerve axons in the sciatic nerve.

The ontogeny of muscarinic acetylcholine receptors in the rat brain has been examined using the radioligands, [3H]N-methylscopolamine, [3H]propylbenzilylcholine and [3H]oxotremorine-M. In the 3 regions of the brain selected for study, the cerebral cortex, the diencephalon and the medulla-pons, the receptors develop at different rates. The most rapid development takes place in the medulla with considerably slower maturation in the diencephalon and cerebral cortex. In the cortex, the agonist binding properties of the muscarinic receptors vary during development. There appears to be a 6--7 day lag in the appearance of high affinity sites following formation of low affinity sites.

The effects of adrenalectomy on benzodiazepine receptors in discrete regions of rat brain were examined using [3H]flunitrazepam as binding ligand. The concentration of benzodiazepine receptors was significantly increased by 25, 50 and 71% in hippocampus, striatum and hypothalamus, respectively, after adrenalectomy. In contrast, adrenalectomy did not affect the concentration of benzodiazepine receptors in cerebral cortex, olfactory bulb and cerebellum. No significant differences in the apparent binding affinity (Kd) values were seen following adrenalectomy in any brain region examined. The adrenalectomy-induced increases in [3H]flunitrazepam binding sites were completely reversed by glucocorticoid replacement with dexamethasone. These results demonstrate that adrenalectomy is capable of selectively modulating benzodiazepine receptors in brain regions presumably involved with glucocorticoid negative feedback. The data further suggest additional mechanisms by which endogenous hypothalamic-pituitary-adrenocortical hormones may affect 'anxiety' levels.

Opiate receptor distribution, determined by the autoradiographic localization of stereospecific [3Hdiprenorphine binding sites, was examined in the telencephalon. Areas showing very dense or dense localization of receptors included parts of the presubiculum and amygdala, patchy areas in the caudate-putamen and accumbens, the subfornical organ, the interstriatal nucleus of the striate terminalis and the anterior olfactory nucleus, pars externa. Lower densities were found in other parts of the hippocampal formation, the deeper part of the cerebral cortex, the entopenduncular nucleus, globus pallidus, nucleus triangularis septi and nucleus paratenialis. The significance of these findings is discussed in terms of the biochemical and physiological actions of opiates.

Neurotensin is a putative peptide neurotransmitter in the mammalian brain. The light microscopic localization of the neurotensin receptor has been carried out by the autoradiographic localization of [3H]neurotensin binding sites in slide-mounted tissue sections. Large variations in receptor distribution were found, even in small areas. In many regions, the distribution of the receptor paralleled that of the immunoreactive neurotensin found in other studies. The results suggest sites of neurotensin's action in producing various physiological effects such as hypothermia and analgesia.

[3H]Spiperone ([3H]SP) binding sites were localized by light microscopic autoradiography, after in vitro labeling. The kinetic and pharmacological characteristics of these binding sites were studied in slide-mounted sections of rat forebrain, and optimal labeling conditions were defined. Autoradiograms were obtained by apposing emulsion-coated coverslips to labeled sections. Differential drug sensitivity allowed the selective displacement of [3H]SP from dopamine receptors by ADTN, from serotonin receptors by cinanserin, from both by haloperidol and from unique spiperone sites by unlabeled spiperone. The various sites presented a differential anatomical localization. For example, only dopaminergic sites were found in the glomerular layer of the olfactory bulb; only serotonergic sites were found in lamina IV of the neocortex, and a high concentration of unique spiperone sites was found in parts of the hippocampus.

Literature findings indicated that injection of low doses of [ 3 H] spiperone results in a labelling of dopamine receptors in rat brain, but also in a labelling of serotonin receptors. Administration of pipamperone, a drug with serotonergic properties, to animals treated with [ 3 H] spiperone reduced the serotonergic component of the binding and permitted an easier identification of dopamine receptor binding. At the level of Forceps Minor, there were elevated levels of receptors in the deeper layers of the cingulate cortex, in the region above the rhinal sulcus and in an area dorsal to the accumbens. This distribution is in agreement with the results of other biochemical, histochemical and electrophysiological studies.

Enkephalin immunoreactive perikarya, fibers and nerve terminals, visualized by the indirect immunohistofluorescent method in colchicine-pretreated animals, are localized in many discrete regions of the rat brain stem. These specific immunohistofluorescent patterns are similar after staining with selective primary antisera directed against either methionine-enkephalin or leucine-enkephalin. Cell bodies are found in the substantia gelatinosa and interpolaris zones of the trigeminal nuclear complex, the nucleus of the solitary tract, in the vicinity of the nucleus raphé magnus, in the dorsal cochlear, medial vestibular, and paraolivary nuclei and, dorsal to this last region, in the parabrachial nuclei and the dorsal tegmental nucleus of Gudden, in the periaqueductal gray matter and interpeduncular nucleus and along the borders of the lateral lemniscus and medial geniculate. In some areas, such as the parabrachial region, nucleus of the solitary tract and substantia gelatinosa of the trigeminal nucleus, these perikarya are associated with densities of fibers and terminals. Other regions, such as the dorsal cochlear nucleus and the vicinity of the nucleus raphé magnus, contain cell bodies associated with low densities of processes and terminals. In still other nuclei, such as the nucleus of the facial nerve and the locus coeruleus, fiber and terminal densities without associated cell bodies are evident. Many of these enkephalin localizations can be rationalized on the basis of known actions of opiate drugs and the brain stem distribution of opiate receptors.

Corticotropin-releasing factor (CRF) receptors were localized in rat pituitary gland by an in vitro labeling light microscopic autoradiographic technique using the radioiodinate ovine CRF analog, [Nle21, 125I-Tyr32]-CRF. Our data confirms the existence of CRF receptors in anterior pituitary and demonstrates the presence of specific CRF binding sites in the intermediate lobe of rat pituitary. The distribution of CRF binding sites closely resembles the clustering of corticotrophs in anterior lobe and the uniform distribution of opiomelanocortin-producing cells in the intermediate pituitary. These results suggest that endogenous CRF plays a physiological role in regulating secretion of pro-opiomelanocortin-derived peptides from both anterior and intermediate lobes of rat pituitary.

Autoradiographic techniques were used to identify serotonergic binding sites in human and canine basilar artery segments. 5-HT1, but not 5-HT2, receptors could be localized to the medial layer of the vascular wall using both [3H]LSD and [3H]5-HT. The identification of 5-HT1 binding sites in human and canine basilar arteries provides anatomical evidence that the 5-HT1 receptor mediates vasoconstriction in these major intracranial vessels.

The vast majority of morphine-sensitive single units in the area examined were localized to the locus coeruleus. This corresponds well with the known distribution of the highest densities of opiate receptor sites in this region of the midbrain. The effect of iontophoretically applied morphine was a marked and prolonged depression of spontaneous activity. Levorphanol, an opiate agonist, produced an effect similar to that of morphine while comparable doses of dextrorphan, it's clinically inactive stereoisomer, did not. Naloxone and levallorphan prevented as well as reversed the depression due to application of agonists. While the units were depressed following the application of opiate agonists, the cells were still excited by the neurotransmitter acetylcholine. We conclude that (1) neuronal sensitivity to opiates has a high positive correlation with autoradiographically determined opiate receptor sites, and (2) this sensitivity to opiates is blocked by opiate antagonists and is stereospecific in nature.

This investigation is an electron microscopic, histochemical study of the localization of acetylcholinesterase on membranes in partially fractionated sucrose homogenates of the hippocampal formation. A large number of stained membranes were observed; staining was lost after addition of1 μM paraoxon, placement of lesions in the septum and omission of acetylthiocholine from the incubation media. Addition of iso-OMPA (10−4 M) to the media did not alter the staining. Thus, the staining appeared to be due to the presence of specific acetylcholinesterase localized to afferent neurons. Only a minority of the stained membranes were synaptosomal membranes. Most of them appeared to be broken unmyelinated axons, postsynaptic processes, and mitochondria. However, a measurable portion of synaptosomal profiles were completely stained and comprised approximately 6% of the total number of profiles. On the basis of existing evidence, these were tentatively assumed to be cholinergic. After calculation of the volumes of these presumed cholinergic synaptosomes, it was estimated that the concentration of free or ‘labile bound’ acetylcholine within these terminals was about2mM. However, this is probably an underestimation.

Summary We have found a selective reduction in forebrain tryptophan hydroxylase activity afer placement of electrolytic lesions in the midbrain raphe nuclei in rats. Animals with lesions of the lateral midbrain did not show any reduction in enzyme activity. Forebrain serotonin levels were also reduced after placement of lesions, but at first the levels fell more slowly than did the enzyme activity. There were significant reductions in tryptophan hydroxylase activity as soon as one day post-lesion, while serotonin levels were not reduced until after two days post-lesion. By 8 days, both the enzyme and serotonin reduction were maximal and similar in extent. Tyrosine hydroxylase activity was not significantly reduced, and aromatic amino acid decarboxylase activity showed only a small decrease, when measured with either 5-HTP or DOPA as substrate. These results indicate that projections of the midbrain raphe neurons, which are believed to account for the major portion of forebraibn serotonin, also contain the bulk of the enzyme tryptophan hydroxylase in the forebrain.

Previous studies have shown that p-cholorophenylalanine (PCPA), an irreversible inhibitor of serotonin (5-HT) synthesis at the tryptophan hydroxylase step, fails to prevent the l-tryptophan-induced increase in the fluorescence of 5-HT-containing neuronal perikarya of the brain stem raphe nuclei, although there is a marked depletion in whole brain 5-HT concentration. In an effort to explain this apparent discrepancy, the effect of PCPA upon the histofluorescence of raphe cell terminals in the midbrain and forebrain of rats was examined. Demonstration of the fluorescence of raphe cell terminals was aided in part by pretreatment with l-tryptophan in the presence of a monoamine oxidase inhibitor. PCPA was found to produce a marked decrease in the fluorescence of raphe terminals but, in agreement with previous results, not in the perikarya. Parallel biochemical studies showed that 5-HT was depleted by PCPA (with or without l-tryptophan loading) to a much greater extent in the forebrain (a region rich in raphe terminals), than in a block of tissue containing the raphe cell perikarya. These results indicate that the synthesis of 5-HT is more susceptible to inhibition by PCPA in the terminals than in the perikarya of raphe neurons, perhaps due to a continued synthesis of new tryptophan hydroxylase enzyme in the latter site.

Placement of high frequency lesions in the medial habenular area results in a large depletion of acetylcholine (ACh) levels, choline acetyltransferase (ChAc) activity, and high affinity choline uptake in the interpeduncular nucleus area (IPN) at 1, 3 and 7 days post-lesion. Areas adjacent to the IPN did not have a reduction in ChAc activity. The reduction in high affinity choline uptake was selective in that there was no change in the uptake of L-[3H]tyrosine or L-[3H]glutamic acid. Unlike the cholinergic septal-hippocampal neurons, there was no rise in ACh levels in the IPN 1 h after placement of lesion, or 30 min after administration of pentobarbital. While the IPN probably has a much denser cholinergic innervation than the hippocampus (as evidenced by much higher ACh levels, ChAc activity and choline uptake levels), it has only one-fourth as many [3H]QNB binding sites (a measure of cholinergic muscarinic receptors). This, some of the habenulo-interpeduncular neurons are probably cholinergic, and they may have pharmacologic and functional differences compared to the septal-hippocampal neurons.

Using [ 3 H]para-aminoclonidine, α 2 adrenergic binding sites have been mapped in the rat and human CNS using in vitro labeling autoradiographic techniques. In both the rat and human thoracic spinal cord, high densities of α 2 binding sites were associated with the substantia gelatinosa and the intermediolateral cell column. In the rat medulla, high binding site density was observed in the medial nucleus of the solitary tract, dorsal motor nucleus of the vagus, raphe pallidus and the substantia gelatinosa of the trigeminal nucleus, while lower levels of specific binding were found in the lateral and ventrolateral medulla. In the human, a similar distribution was observed. However, significantly lower levels of specific binding were seen in the medial nts as opposed to the dmv. In the rat, high levels of specific binding were seen at pontine and midbrain levels in the locus coeruleus, parabrachial nucleus and periaqueductal gray. In the forebrain, several hypothalmic and limbic regions, including the paraventricular and arcuate nuclei of the hypothalamus, the central, medial and basal nuclei of the amygdala, lateral septum and bed nucleus of the stria terminalis and pyriform, entorhinal and insular cortex were labeled. Each of these regions are involved in either modulating autonomic functions directly or integrating somatosensory and/or affective function with autonomic mechanisms. Further, these regions are interrelated by reciprocal connections, and neurons that utilize nor-adrenaline or adrenaline as their neurotransmitter form a vital part of these connections. Thus, these functional, anatomical and neurochemical correlates of the α 2 binding site distribution establish a neurological basis for the complex pharmacological effects of centrally acting α 2 agonists.

The distribution of the high affinity gamma-aminobutyric acid (GABA) receptor labeled by [3H]muscimol, has been studied in the rat brain by light microscopic autoradiography. Receptors in slide-mounted tissue sections were labeled in vitro with [3H]muscimol. Most of the gray matter areas presented grain densities significantly higher than background or white matter areas. Wide variations in receptor densities were found between different brain areas and nuclei. Areas with very high grain densities are the granule cell layer of the cerebellum, external plexiform layer of the olfactory bulb and nuclei of the thalamus, such as the ventral nucleus, lateral nucleus and dorsal geniculate body. The molecular layer of the hippocampus and the external (I–IV) layers of the cortex are also rich in GABA receptors. The basal ganglia have moderate concentrations of receptors, while the pons, medulla and brainstem have only low concentrations of autoradiographic grains. These distributions are discussed in correlation with the known distributions of GABAergic terminals and the presence of inhibitory GABAergic mechanisms.

High affinity gamma-aminobutyric acid, benzodiazepine, strychnine (glycine), dopamine, spirodecanone, alpha 1-adrenergic, alpha 2-adrenergic, beta-adrenergic and muscarinic cholinergic binding sites were localized by semiquantitative autoradiography in rat and, in some instances, in monkey and human retinae using [3H]muscimol, [3H]flunitrazepam, [3H]strychnine, [3H]spiperone, [3H]prazosin, [3H]para-aminoclonidine, [3H]dihydroalprenolol and [3H]quinuclidinyl benzylate, respectively. In nearly every case, the inner plexiform layer (IP) contained a high receptor density. The distribution of alpha 1 sites was unusual in that binding was concentrated in the outer plexiform layer (OP). Dopaminergic and, to a lesser extent, beta-adrenergic binding was diffusely distributed in the outer nuclear layer, the OP, the inner nuclear layer and the IP. The ganglion cell layer displayed significant benzodiazepine binding. The intraretinal distribution of pre- and postsynaptic markers of these neurotransmitters is discussed.

Summary The distribution of opiate receptors, determined by the autoradiographic localization of stereospecific [ 3 H]diprenorphine binding sites, was studied in the brain stem. Areas showing very dense or dense localizations of receptors included the parabrachial nuclei, the superior colliculus, the ventral median raphe nucleus, components of the accessory optic system, portions of the habenulo-interpenduncular complex, the pretectal nuclei and the ventral lateral geniculate, the infundibulum and the medial thalamus. Moderate grain densities were found in broad areas of the upper medulla, midbrain and diencephalon. The significance of these findings are discussed in terms of pain mechanisms, association of some receptors with small diameter axons, pupillary reflexes, hormonal control, and effects of opiates on neurotransmitter systems.

Rats were administered [3H]spiperone (SP: spiroperidol) by tail vein injection and 2 h later the brain was processed for light microscopic autoradiography. High densities of autoradiographic grains were found in all areas known to have a dopaminergic innervation, including the olfactory tubercles, nucleus accumbens, nucleus caudate-putamen, lateral septum, zona incerta, nucleus subthalamicus, arcuate nucleus, nucleus of the central amygdala, areas in the ventral tegmentum and the claustrum. There were also increased autoradiographic grain densities in other areas such as the midbrain and the frontal cortex indicating that binding occurred to other neurotransmitter receptors besides dopamine receptors. These studies delineate with a high resolution at an anatomical level the major binding sites for neuroleptic drugs in the forebrain. They suggest which areas of the brain are the most involved in neuroleptic drug action and they add further evidence that important regions are those receiving a dense dopaminergic innervation.

Discrete localization of enkephalin immunoreactive sites in the rat forebrain was undertaken using animals with and without colchicine pretreatment. Both the immunofluorescence and unlabeled PAP antibody techniques were employed using antisera directed against methionine-enkephalin. We have found several areas of enkephalin fiber and cell localizations not previously described, as well as confirming the results of other researchers. Thus, we found large amounts of perikaryal enkephalin immunoreactivity in the lateral septal nucleus, bed nucleus of the stria terminalis, the preoptic nuclei, the ventral premammillary nucleus, prelateral mammillary nucleus, the medial mammillary nucleus, paraventricular nucleus, the periventricular, ventromedial, dorsomedial and posterior nuclei of the hypothalamus, the arcuate nucleus, and the ventral nucleus of the lateral geniculate body. High concentrations of immunoreactive neuronal fibers not immediately associated with enkephalin perikarya were seen in the nucleus accumbens, medial forebrain bundle, globus pallidus, mammillothalamic tract, the subthalamic nucleus, and the caudal portion of the zona incerta. Many of the areas presented in this study correspond with areas known to exert some regulation over neuroendocrine function as well as various motor and sensory functions. Nuclei with high immunoreactive fiber content also correspond in many instances with areas reported to have high concentrations of opiate receptors. Possible enkephalin-containing tracts and the relationships of enkephalin-containing cells in fiber bundles are also pointed out.

We examined the distribution of dopamine and serotonin receptors in human pituitary gland by light microscopic autoradiography using [3H]spiperone as a binding ligand. Clusters of specific binding sites for [3H]spiperone were observed in the anterior pituitary. This clustering is similar to the clustering of lactotrophs observed in immunohistochemical studies. No specific binding sites for [3H]spiperone were present in the posterior lobe of the pituitary gland. We found no evidence for the presence of serotonin-2 receptors (i.e. cinanserin-displaceable [3H]spiperone binding) in human pituitary.

[125I]alpha-Bungarotoxin (alpha-BuTX) binding sites accumulate both proximal and distal to a ligature positioned around the sciatic nerve of rats. [125I]alpha-BuTX binding sites, localized using quantitative receptor autoradiography, were found to accumulate at nerve ligatures at a relatively constant rate which suggests that they undergo both anterograde and retrograde axonal transport. [125I]alpha-BuTX binding to sections of ligated sciatic nerve was saturable with apparent dissociation constants of 0.97 nM proximal and 0.53 nM distal to the ligature. D-Tubocurarine, nicotine, decamethonium and atropine displaced [125]alpha-BuTX from sciatic nerve sections with affinities comparable to those previously reported for the toxin binding component of rat brain. These data indicate that [125I]alpha-BuTX binding sites pharmacologically similar to those of rat brain are transported in sciatic nerve. Axonally transported toxin binding sites may correspond to those previously localized to the plasma membrane of peripheral nerve axons and on the terminals of motor neurons.

By using kainic acid (KA) to perform chemical lesions in the guinea pig cerebellum, we have caused degeneration of Purkinje cells without affecting granule cell morphology. Near the injection site we found a large decrease in autoradiographically labeled histamine-H 1 and benzodiazepine receptors of the molecular layer while those receptors distant from the injection site were unaffected. GABA receptors in the granule cell layer remained uniformly constant even immediately adjacent to the lesion site. This evidence suggests that histamine-H 1 and benzodiazepine receptors are present on neuronal elements (possibly on Purkinje cell dendrites) in the molecular layer of the cerebellum and that GABA receptors are associated with the KA-resistant granule cells.