Regional stimulatory and inhibitory effects of guanine nucleotides on [125I]galanin binding in rat brain: relationship with the rate of occupancy of galanin receptors by endogenous galanin.

Galanin has been shown to stimulate feeding or modulate neuroendocrine secretions when administered centrally. In the present work, using quantitative autoradiography, we documented the existence of [125I]galanin specific binding sites in several hypothalamic nuclei expected to mediate these effects. In standard binding conditions, [125I]galanin specific binding can be visualized in the hypothalamic ventromedial nucleus, stria terminalis, piriform cortex, central amygdaloid nucleus and medial amygdaloid nucleus, while it is almost undetectable in most neuroendocrine or autonomic hypothalamic areas. We hypothesized that high endogenous galanin levels in these regions might mask galanin receptors. We first showed that a high ionic strength/acid wash of brain slices is effective in removing more than 80% of specifically prebound [125I]galanin in all tested regions. After such treatments, specific binding sites could be revealed in the hypothalamus namely in the parvocellular paraventricular nucleus, periventricular nucleus, arcuate nucleus and median eminence. In contrast, regions already labeled in standard conditions exhibited a slight decrease in [125I]galanin binding. Thus, regions were ranked from low to high rate of occupancy of galanin receptors by endogenous galanin, the rate of occupancy of galanin receptors being maximal in median eminence (greater than 90%). We thus studied the regional effect of guanine nucleotides on [125I]galanin specific binding. A high concentration (100 microM) of guanyl 5'-yl imidodiphosphate, a nonhydrolyzable analog of GTP directly added to the incubation medium, inhibited [125I]galanin binding in all telencephalic regions. On the same sections and only in regions of high index of galanin receptor occupancy (arcuate nucleus, median eminence, dorsomedial nucleus, paraventricular nucleus, and periventricular hypothalamic nucleus), guanyl 5'-yl imidodiphosphate paradoxically enhanced [125I]galanin binding. The effects of acid preincubation and guanyl 5'-yl imidodiphosphate incubation on [125I]galanin binding were strongly correlated in these hypothalamic areas (r = 0.97). In all regions, guanyl 5'-yl imidodiphosphate increased the rate of dissociation of [125I]galanin. In competition studies, guanyl 5'-yl imidodiphosphate decreased the IC50 s of unlabeled galanin which were homogenized around 4 nM in most telencephalic and hypothalamic regions. Thus, the guanyl 5'-yl imidodiphosphate-induced stimulation of [125I]galanin specific binding measured in the neuroendocrine and autonomic hypothalamus is linked to an increase in receptor capacity and not to a rise in receptor affinity. Both inhibitory and stimulatory guanyl 5'-yl imidodiphosphate effects observed in [125I]galanin equilibrium binding studies were dose-dependent and guanine nucleotide-specific with guanyl 5'-yl imidodiphosphate more potent than GTP or GDP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Galanin receptor over-expression within the amygdala in early Alzheimer's disease: an in vitro autoradiographic analysis

Galanin receptor (GALR) expression is increased in various areas of the limbic system in end stage Alzheimer's disease (AD). The amygdaloid complex is a key component of the limbic circuit, is involved in homeostatic and cognitive functions, is impacted in AD and contains the peptide and receptor for galanin. Although GALR expression occurs in the amygdala in end stage AD, it remains to be determined whether a plasticity response occurs early or late in the disease. Therefore, we analyzed the distribution and associated changes in GALR binding in the amygdala during the progression of AD using an in vitro receptor autoradiographic method. Human galanin ([125I]hGAL) receptor binding was performed on brain sections from early and late AD cases, as well as normal age-matched control subjects. In aged controls, densities of [125I]hGAL binding sites were found in the central and the corticomedial nuclei. Relative to controls, possible/early AD cases displayed significantly greater numbers of [125I]hGAL binding sites in the central nucleus and cortico-amygdaloid transition area. In contrast, we found a decrease in the number of binding sites for [125I]hGAL in late as compared to early AD cases. The over-expression of GALRs in subfields of the amygdaloid early in AD suggests that galaninergic systems play a key role in limbic related behavioral changes during the disease process.     

Galanin receptors in the post-mortem human brain. Regional distribution of I-galanin binding sites using the method of in vitro receptor autoradiography

The distribution of putative receptors for the peptide galanin was studied in the normal post-mortem human brain by using ¹²⁵I-galanin (0.5 nM) in combination with in vitro receptor autoradiography. Specific binding of ¹²⁵I-galanin was found in a large number of brain areas throughout the neuraxis. Highest binding densities occurred in the basal forebrain and hypothalamus, while the basal ganglia, major parts of the thalamus and the tectum were found to be poor in binding sites. All cortical areas harboured ¹²⁵I-galanin binding, and in the visual cortex a laminated pattern was present. In the hippocampus, ¹²⁵I-galanin binding occurred in layer 2 of the entorhinal cortex, in the uncus and in the hippocampal-amygdala area. In the brain-stem, ¹²⁵I-galanin binding was found in serotoninergic and noradrenergic cellgroups as well as in the reticular formation and in the parabrachial nuclei. Galanin receptors may, thus, mediate the response of galanin in numerous structures in the human brain.     

Distribution of nicotinic cholinergic receptors in the rat tel- and diencephalon: a quantitative receptor autoradiographical study using [3H]-acetylcholine, [α-125l]bungarotoxin and [3H]nicotine

The topographical distribution of [α:-¹²T]bungarotoxin [¹²⁵I]BTX, [³H]nicotine ([³H]Nic), [³H]acetylcholine ([³H]ACh) (in the presence of atropine) binding in rat teland diencephalon was investigated using a quantitative receptor autoradiographical technique. With the [³H|ACh and [³H]Nic radioligands, a strong labelling was observed in various thalamic nuclei, including the medial habenula, a moderate labelling in different areas of the cortex cerebri, the nucleus caudatus putamen, the nucleus accumbens and tuberculum olfactorium and a uniform weak labelling in the hypothalamus. When the binding data for [³H]Nic were plotted against binding data for [³H]ACh in various brain nuclei, a significant correlation was obtained. Considering [¹²⁵I]BTX, the strongest labelling was observed in the lateral mammillary nucleus and the hilus gyrus dentatus of the hippocampal formation. A weak labelling occurred in areas such as the nucleus causatus putamen, the thalamus and the cerebral cortex. No significant correlation was therefore obtained between the degree of [¹²⁵I]BTX binding in various brain nuclei and the degree of binding observed with [³H]Nic or [³H]ACh. The present results underline the view that the high-affinity |³H]Nic and [³H]ACh binding sites label the same cholinergic nicotinic receptor binding site, while [¹²⁵I]BTX labels another subpopulation of nicotinic cholinergic receptors, predominantly found in discrete areas of the hypothalamus and the limbic cortex.     

Autoradiographic mapping of galanin receptors in the monkey brain

The distribution of specific binding sites for the peptide galanin was studied in the monkey brain by using 125I-galanin and the method of quantitative in vitro autoradiography. The binding to tissue sections was found to be rapid, reversible and saturable with a calculated Bmax of around 13 fmol mg-1 tissue in cortical regions and a KD ranging between 0.39 and 0.22 nM in different brain regions. Specifically bound 125I-galanin was detected in a number of brain regions throughout the rostrocaudal axis of the monkey brain. More specifically, the majority of the 125I-galanin binding sites were present in the basal forebrain (e.g. olfactory tubercle, lateral septal nuclei, nucleus accumbens, substantia innominata and the basal nucleus of Meynert), the amygdala (e.g. the lateral, medial accessory and central nuclei), the amygdala-hippocampal area, hypothalamus (e.g. the anterior hypothalamic area, the ventro-medial hypothalamus and the zona incerta), as well as in certain brain stem cell groups such as the substantia nigra (pars compacta), locus coeruleus, central grey substance, parabrachial nucleus, sensory and motor nuclei of the trigeminal nerve and the dorsal vagal complex. In the neocortex, highest binding density occurred in layer 4 of all neocortical fields, except in the visual cortex where the highest densities occurred in layers 4cb, 5a and 6. Major fibre tracts such as the fimbria and the stria terminalis also contained high densities of 125I-galanin binding sites. Areas poor in, or lacking, specific 125I-galanin binding sites included the basal ganglia, the thalamus, the mammillary bodies, the cerebellum, and the reticular formation of the brain stem. The pattern of galanin receptor distribution in the monkey brain reported here closely resembles that observed for galanin receptors and galanin-immunoreactive preterminal processes described previously in the rat brain. An exception, however, was neocortex, where in the rat neither 125I-galanin binding nor galanin-positive fibres have been observed. The present findings may indicate a close association between galanin receptors and nerve terminals containing galanin immunoreactivity in the monkey brain and implies important role(s) for galanin in neurotransmission in the monkey central nervous system.     

Galanin receptors and their second messengers in the lumbar dorsal spinal cord

The ligand binding properties of galanin receptors were examined in crude synaptosomal fraction preparations of lumbar dorsal spinal cord, using chloramin-T mono-iodinated porcine Tyr²⁶ galanin as ligand. The equilibrium binding of [¹²⁵I]galanin showed the presence of a single population of high-affinity binding sites with K_D= 0.6±0.2 nM in a concentration of 55±15 fmol mg^-1 protein (B_max. The N-terminal fragments galanin (1–16) and galanin (1–12) fully displaced specific [¹²⁵1]galanin binding from membranes with IC₅₀ values 6 nM and 4 μM, respectively. The C-terminal fragment galanin (17–29) did not displace [¹²⁵I]galanin when applied in the concentration range 10^-11–10^-4 M. GTP inhibited the specific binding of [¹²⁵I] galanin in a concentration dependent manner, with 54% inhibition at 1 mM, suggesting that the galanin receptor found in lumbar dorsal spinal cord is G-protein coupled. Second messenger systems, through which the galanin receptor in lumbar dorsal spinal cord may exert its effect, were also studied. A galanin (10,μm) produced inhibition (58%) of the depolarization induced cGMP increase was found, whereas galanin (10 μM) did not inhibit the noradrenalin (100 μM) activated CAMP synthesis or phosphoinositide turnover in tissue slices of the spinal cord. Bilateral transection of the sciatic nerve at midthigh level 14 days prior to the binding experiment was performed, a treatment which is known to cause a dramatic increase of galanin-like immunoreactivity in the superficial layers of the dorsal spinal cord, dorsal root ganglion and in galanin mRNA levels, but no significant effect on B_max or K_D of the galanin receptor was found.     

Distribution of nicotinic cholinergic receptors in the rat tel- and diencephalon: a quantitative receptor autoradiographical study using [3H]-acetylcholine, [alpha-125I]bungarotoxin and [3H]nicotine

The topographical distribution of [alpha-125I]bungarotoxin [125I]BTX, [3H]nicotine ([3H]Nic), [3H]acetylcholine ([3H]ACh) (in the presence of atropine) binding in rat tel- and diencephalon was investigated using a quantitative receptor autoradiographical technique. With the [3H]ACh and [3H]Nic radioligands, a strong labelling was observed in various thalamic nuclei, including the medial habenula, a moderate labelling in different areas of the cortex cerebri, the nucleus caudatus putamen, the nucleus accumbens and tuberculum olfactorium and a uniform weak labelling in the hypothalamus. When the binding data for [3H]Nic were plotted against binding data for [3H]ACh in various brain nuclei, a significant correlation was obtained. Considering [125I]BTX, the strongest labelling was observed in the lateral mammillary nucleus and the hilus gyrus dentatus of the hippocampal formation. A weak labelling occurred in areas such as the nucleus causatus putamen, the thalamus and the cerebral cortex. No significant correlation was therefore obtained between the degree of [125I]BTX binding in various brain nuclei and the degree of binding observed with [3H]Nic or [3H]ACh. The present results underline the view that the high-affinity [3H]Nic and [3H]ACh binding sites label the same cholinergic nicotinic receptor binding site, while [125I]BTX labels another subpopulation of nicotinic cholinergic receptors, predominantly found in discrete areas of the hypothalamus and the limbic cortex.     

Distribution of angiotensin IV binding sites (AT4 receptor) in the human forebrain, midbrain and pons as visualised by in vitro receptor autoradiography

Angiotensin IV and other AT₄ receptor agonists, improve memory retention and retrieval in the passive avoidance and swim maze learning paradigms. Angiotensin IV binding sites (also known as the AT₄ receptors) are widely distributed in guinea pig and monkey (Macaca fascicularis) brains where high densities of the binding sites have been detected in the hippocampus, neocortex and motor nuclei. However, the distribution of the binding sites in the human brain is not known. We have recently localised the angiotensin IV binding sites (AT₄ receptors) in post-mortem human brain using iodinated Nle-angiotensin IV, a higher affinity and more stable analogue of angiotensin IV. This radioligand bound with relatively high affinity and specificity to angiotensin IV binding sites. In competition studies on consecutive sections through the prefrontal cortex and claustrum, angiotensin IV, Nle-angiotensin IV and LVV-hemorphin 7 competed for the binding of ¹²⁵I[Nle]-angiotensin IV with nanomolar affinities. Angiotensin II and the AT₁ and AT₂ receptor antagonists were ineffective in competing for the binding at concentrations of up to 10 μM. We found high densities of ¹²⁵I[Nle]-angiotensin IV binding sites throughout the cerebral cortex including the insular, entorhinal, prefrontal and cingulate cortices. Very high densities of the binding sites were observed in the claustrum, choroid plexus, hippocampus and pontine nucleus. Some thalamic nuclei displayed high densities of binding including the anteroprincipal, ventroanterior, anteromedial, medial dorsal and ventrolateral nuclei. The caudate nucleus, putamen, many amygdaloid nuclei and the red nucleus all displayed moderate densities of binding with a higher level detected in the substantia nigra pars compacta. In the hypothalamus, high densities binding sites were found in the ventromedial nucleus with lower levels in the dorsomedial and paraventricular nuclei. The distribution of ¹²⁵I[Nle]-angiotensin IV binding sites in the human brain is similar to that found in other species and supports multiple roles for the binding sites in the central nervous system, including facilitation of memory retention and retrieval.     

Distribution of vasopressin and oxytocin binding sites in the brain and upper spinal cord of the common marmoset

The aim of this study was to label selectively and to map central vasopressin (AVP) and oxytocin (OT) binding sites in the common marmoset. [¹²⁵I]VPA, a compound selective in rodents and human for the AVP V_1a receptor, yielded the same labeling pattern as [³H]AVP, thus suggesting that most AVP receptors present in the marmoset brain are of the V_1a subtype. Numerous areas exhibited AVP binding sites, among which the olfactory bulb, the accumbens nucleus, the bed nucleus of the stria terminalis, the hypothalamic suprachiasmatic, arcuate and ventromedial nuclei, the medial amygdaloid nucleus, the nucleus of the solitary tract and the cerebral cortex. Binding sites for [¹²⁵I]OTA, a selective OT receptor antagonist in rat and human, were markedly less abundant than [¹²⁵I]VPA ones, and, to a few exceptions, expressed in different areas. Neither AVP, nor OT binding sites were detected in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei identified by neurophysin immunoreactivity. Marked species-related differences were observed in the distribution of both AVP and OT binding sites. Altogether, our data provide a morphological basis to investigate the function of central AVP and OT in the marmoset.     

Evidence for the existence of galanin receptors on cultured astrocytes of rat CNS: colocalization with cholinergic receptors

The cellular localization of binding sites for [¹²⁵I]galanin was studied in explant cultures of rat neocortex, cerebellum, locus coeruleus and spinal cord by means of autoradiography. Binding sites for the peptide were observed on a great number of astrocytes in all CNS regions studied. In addition to astrocytes, many neurones were intensely labelled by [¹²⁵I]galanin. Binding of [¹²⁵I]galanin (10⁻⁸ M) to both astrocytes and neurones was markedly reduced or inhibited by the unlabelled peptide at high concentration (10⁻⁶ M), suggesting `specific' binding of the radioligand. Evidence for the colocalization of galanin and cholinergic receptors on astrocytes was provided by combined autoradiographic and immunohistochemical studies. Many astrocytes were labelled by [¹²⁵I]galanin and immunostained with antibodies to either muscarinic or nicotinic receptors. Electrophysiological studies revealed that addition of galanin (10⁻⁹ to 10⁻⁷ M) to the bathing fluid caused a dose-dependent hyperpolarization of the majority of astrocytes studied. When galanin (10⁻⁸ M) and the cholinergic agonists muscarine and nicotine (10⁻⁶ M) were tested on the same astrocyte, all three compounds induced a hyperpolarization, suggesting a colocalization of functional galanin and cholinergic receptors on the glial membrane.     

Autoradiographic visualization of A1-adenosine receptors in brain and peripheral tissues of rat and guinea pig using 125I-HPIA

A1-adenosine receptors were identified in sections of rat brain and guinea pig kidney with the radioiodinated agonist 125I-N6-p-hydroxyphenylisopropyladenosine (125I-HPIA) using in vitro autoradiography. The affinities of adenosine receptor ligands in competing with 125I-HPIA binding to tissue sections were in good agreement with those found in membranes, and indicate that the binding site represents an A1-adenosine receptor. The distribution of 125I-HPIA binding sites in rat brain sections was similar to the pattern of [3H]N6-cyclohexyladenosine ([3H]CHA) binding sites determined previously, with highest densities in the hippocampus and dentate gyrus, the cerebellar cortex, some thalamic nuclei and certain layers of the cerebral cortex. In the guinea pig kidney 125I-HPIA labelled longitudinal structures in the medulla. This study demonstrates that 125I-HPIA allows the autoradiographic detection of A1 adenosine receptors in the brain and peripheral organs and has the advantage of short exposure times.     

Autoradiographic distribution of receptors to FLFQPQRFamide, a morphine-modulating peptide, in rat central nervous system.

The neuropeptide FLFQPQRFamide is a structure related to FMRFamide which is able to inhibit the effects of both endogenous and exogenous opiates. This morphine-modulating activity is mediated via the stimulation of specific FLFQPQRFamide receptors, different from opiate receptors. In vitro quantitative receptor autoradiography was performed on frozen sections of rat central nervous system to characterize binding properties and visualize FLFQPQRFamide receptors using the specific ligand [125I]YLFQPQRFamide, a radio-iodinated analogue of FLFQPQRFamide. [125I]YLFQPQRFamide appeared to interact reversibly with a single class of binding sites (KD = 0.2 nM). The specific binding represented 80% of the total binding at 0.05 nM, the FLFQPQRFamide concentration used in this mapping study. Sites labelled with [125I]YLFQPQRFamide were distributed heterogeneously within the brain and spinal cord. A high density of FLFQPQRFamide binding sites was detected in the most external layers of the dorsal horn of spinal cord and various nuclei of pons and medulla including trigeminal, dorsal tegmental and reticular nuclei. Nucleus of solitary tract, parabrachial, ambiguous and facial nuclei are also intensively labelled. Some structures of mesencephalon and diencephalon exhibited a high density of FLFQPQRFamide binding sites: central gray, raphe nuclei and thalamic nuclei such as parafascicular, laterodorsal, central median, paratenial and paraventricular nuclei. Suprachiasmatic and mammillary nuclei, lateral, posterior and anterior areas of hypothalamus and medial preoptic area exhibited high labelling. FLFQPQRFamide binding sites were also seen in some structures of the dopaminergic meso-cortico-limbic system including ventral tegmental area, cingulate cortex, lateral septum and the head of the caudate-putamen. Dense labelling appeared in the presubiculum of hippocampus. The dissimilar mapping of FLFQPQRFamide and opiate brain receptors confirms our previous pharmacological findings in FLFQPQRFamide binding studies on rat spinal cord membranes, showing that FLFQPQRFamide receptors are different from opiate receptors. There was a good correspondence between localization of binding sites and that of the putative endogenous peptide. Both occur in brain areas previously associated with analgesic action of opiates. However, the mapping of FLFQPQRFamide receptors in the central nervous system suggests that the FLFQPQRFamide system could be implicated in other physiological functions.     

Galanin receptors and their second messengers in the lumbar dorsal spinal cord.

The ligand binding properties of galanin receptors were examined in crude synaptosomal fraction preparations of lumbar dorsal spinal cord, using chloramin-T mono-iodinated porcine Tyr26 galanin as ligand. The equilibrium binding of [125I]galanin showed the presence of a single population of high-affinity binding sites with KD = 0.6 +/- 0.2 nM in a concentration of 55 +/- 15 fmol mg-1 protein (Bmax). The N-terminal fragments galanin (1-16) and galanin (1-12) fully displaced specific [125I]galanin binding from membranes with IC50 values 6 nM and 4 microM, respectively. The C-terminal fragment galanin (17-29) did not displace [125I]galanin when applied in the concentration range 10(-11)-10(-4) M. GTP inhibited the specific binding of [125I] galanin in a concentration dependent manner, with 54% inhibition at 1 mM, suggesting that the galanin receptor found in lumbar dorsal spinal cord is G-protein coupled. Second messenger systems, through which the galanin receptor in lumbar dorsal spinal cord may exert its effect, were also studied. A galanin (10 microM) produced inhibition (58%) of the depolarization induced cGMP increase was found, whereas galanin (10 microM) did not inhibit the noradrenalin (100 microM) activated cAMP synthesis or phosphoinositide turnover in tissue slices of the spinal cord. Bilateral transection of the sciatic nerve at midthigh level 14 days prior to the binding experiment was performed, a treatment which is known to cause a dramatic increase of galanin-like immunoreactivity in the superficial layers of the dorsal spinal cord, dorsal root ganglion and in galanin mRNA levels, but no significant effect on Bmax or KD of the galanin receptor was found.     

Multiple conformations of 5-HT2A and 5-HT2C receptors in rat brain: an autoradiographic study with [125I](±)DOI

Earlier autoradiographic studies with the 5-HT₂ receptor agonist [¹²⁵I](±)DOI in human brain showed unexpected biphasic competition curves for various 5-HT_2A antagonists. We have performed similar studies in rat brain regions with selective 5-HT_2A (M100907) and 5-HT_2C (SB242084) antagonists together with ketanserin and mesulergine. The effect of GTP analogues on antagonist competition was also studied. Increasing concentrations of Gpp(NH)p or GTPγS resulted in a maximal inhibition of [¹²⁵I](±)DOI-specific binding of approximately 50 %. M100907 competed biphasically in all regions. In the presence of 100 μM Gpp(NH)p, M100907 still displaced biphasically the remaining [¹²⁵I](±)DOI binding. Ketanserin showed biphasic curves in some regions and monophasic curves in others. In the latter, Gpp(NH)p evidenced an additional high-affinity site. SB242084 competed biphasically in brainstem nuclei and monophasically in the other regions. In most areas, SB242084 affinities were not notably altered by Gpp(NH)p. Mesulergine competed monophasically in all regions without alteration by Gpp(NH)p. These results conform with the extended ternary complex model of receptor action: receptor exists as an equilibrium of multiple conformations, i.e. ground (R), partly activated (R*) and activated G-protein-coupled (R*G) conformation/s. Thus, [¹²⁵I](±)DOI would label multiple conformations of both 5-HT_2A and 5-HT_2C receptors in rat brain, and M100907 and ketanserin would recognise these conformations with different affinities.     

Benzodiazepine receptor sites in the human brain: autoradiographic mapping

Receptor autoradiography was used to localize and quantify the distribution of benzodiazepine receptor sites in human post mortem materials using [3H]flunitrazepam. The distribution and density of these sites was analysed in the brains of 21 patients dying without reported neurological disease. The distribution of benzodiazepine receptors in the human brain was found to be comparable from case to case although differences in the density occurred among the brains examined. No influence of the post mortem delay, age, gender or pre mortem drug treatment on the distribution and densities was observed in our series. The highest densities of benzodiazepine receptors in human brain were localized in cortical and hippocampal areas, nucleus accumbens, amygdala and mammillary bodies. Intermediate densities were found in the basal ganglia and thalamic and hypothalamic nuclei. [3H]Flunitrazepam binding was low in the brainstem nuclei and very low in white matter. The triazolopyridazine Cl 218872, reported to differentiate between type I and type II benzodiazepine receptor sites, exhibited regional differences in affinity when used to block [3H]flunitrazepam binding. Benzodiazepine receptors in the cerebellar cortex were more sensitive to this compound than those in the dentate gyrus of the hippocampus and the tuberal nuclei of the hypothalamus. An enrichment in the concentration of type I benzodiazepine receptor Cl 218872-sensitive sites was observed in motor areas as compared to structures of the limbic system. The addition of GABA to the incubation medium resulted in an increase of [3H]flunitrazepam binding, suggesting the coupling of these sites to a GABAA receptor. The increase in binding was directly proportional to the density of benzodiazepine receptors but unrelated to the density of high-affinity GABAA sites. The distribution of benzodiazepine receptor sites in the human brain compares well with that previously described in the rat brain. The high densities of receptors localized in the limbic system and in the cortical areas suggest that the effects of benzodiazepines are mediated through an interaction with the sites we have visualized in these anatomical structures. Our results provide a detailed map of the distribution of benzodiazepine receptors and a basis for the understanding of pharmacological effects of these drugs in humans and for future studies of modifications of these receptors in neurological and neuropsychiatric conditions in humans.     

Distribution of galanin-binding sites in the monkey and human telencephalon: preliminary observations

Summary Using X-ray film autoradiography the distribution of ¹²⁵I-galanin binding sites was studied in the forebrain of monkey and man. In the monkey a high density was found in all areas of the neocortex, especially layer 4, and in certain subfields in the hippocampal region. Also in the human brain high activity was seen in neocortex, mainly layer 6 and in hippocampal areas, as well as in amygdala, piriform cortex and hypothalamus. These results suggest that the 29-amino acid peptide galanin may be involved in the regulation of higher cortical functions in primates.     

Tissue-Binding Properties of the Cholera Toxin

[¹²⁵I]choleragen was employed to study further the tissue-binding properties of highly purified choleragen. It was observed that [¹²⁵I]choleragen was bound when combined with mucosal homogenates from all regions of the gastrointestinal tract of adult guinea pigs. Gastric, duodenal, jejunal, and ileal mucosa appeared equally effective in toxin-binding capacity. Preparations of large intestinal mucosa could bind an exceptionally larger amount of toxin. The binding property of small intestinal homogenates could not be attributed to any particular fraction after differential centrifugation; rather, the toxin receptor appeared to be associated with several sizes of particles containing cell membrane components. Although binding to mammalian cells was easily demonstrable, no binding to several types of bacterial cells was observed. The toxin receptor was found to be a “universal component” of many mammalian cell membranes, since specific binding of the toxin to a variety of guinea pig tissues was clearly demonstrated. [¹²⁵I]choleragen binding to all tissues, with the exception of those prepared from brain and large intestinal mucosa, could be inhibited by preincubation of the tissue homogenates with unlabeled choleragen but not with comparable concentrations of normal rabbit serum proteins. The determination of the specificity of [¹²⁵I]choleragen binding to brain and large intestinal mucosal homogenates was hampered by the continual release of soluble receptor from the homogenates, both of which contained the highest concentration of cholera toxin receptor. The data support and extend observations that cholera toxin binding to tissue receptor(s) is a very specific reaction, and further indicate that binding may occur with a variety of tissues to different degrees.     

GABA[A] receptor level changes in female hamster forebrain following in vivo estrogen progesterone and benzodiazepine treatment: a quantitative autoradiography analysis

Summary The levels of gamma amino butyric acid (GABA_A) receptors (i.e. ³H-Muscimol binding sites) were determined by quantitative neurotransmitter receptors autoradiography in ovariectomized (OVX), OVX-estradiolo (E), OVX-progesterone (P), OVX-E+P, diazepam (DZ) and DZ + Ro15-1788 treated female hamsters.The various hormonal treatments altered ³H muscimol binding in many brain areas, whereas DZ and DZ + Ro15-1788 had little influence on GABA_A receptor levels at the onset of the blocked aggressive behavioral activity. For example, E, P and E+P all significantly increased ³H muscimol binding in medial preoptic area, ventromedial hypothalamic nuclei, and vertical diagonal bandmedial septal nucleus, whereas P treatment increased binding in the caudate-putamen and decreased it in reuniens nucleus of the thalamus. E and E+P treatments increased ³H muscimol binding in the corticomedial amygdala nucleus and hippocampus. Diazepam treatment decreased the GABA_A receptor binding in the caudate-putamen and basolateral amygdala, while having no effect in the other brain regions where hormone treatment was effective. In vitro incubation of brain sections with micromolar concentrations of E or P did not change muscimol binding. These results suggest that ovarian steroids, and benzodiazepines to a lesser extent, modulate ³H muscimol binding but do so in different brain regions. The hormone effects on ³H muscimol binding in the critical reproductive centres at a time period that coincides with the onset of its behavioral effect is consistent with a genomic controlled activity.     

Different pharmacological anatomy in the paraventricular hypothalamic nucleus,supraoptic nucleus,and suprachiasmatic nucleus of rats: Quantitative autoradiography on angiotensin II receptor binding sites

Angiotensin II (AII) and vasopressin (VP) play important roles in cardiovascular function. Using ¹²⁵I-[Sar¹, Ile⁸]-angiotensin II (¹²⁵I-SI-AII), a potent AII antagonist, AII receptor binding sites were autoradiographically localized in three VP-producing areas of the hypothalamus and compared in hypertensive and normotensive rats. Within three major VP-producing areas, AII receptor binding was highest in the paraventricular hypothalamic nucleus and lowest in the supraoptic nucleus, suggesting that a differential AII regulation of separate VP systems exists in the brainstem. No statistical difference in ¹²⁵I-SI-AII receptor binding was found between WKY and SHR rats in each of the three major VP-producing nuclei studied. These results are consistent with a role of AII receptors in a subtle and complicated regulation of VP in cardiovascular function.     

Expression of the galanin receptor subtype Gal-R2 mRNA in the rat hypothalamus

The distribution of galanin receptor subtype 2 (Gal-R2) mRNA-expressing cells was examined by in situ hybridization in the rat hypothalamus using a full-length rat ³⁵S-riboprobe. Gal-R2 receptor mRNA-expressing cells were found at moderate to high levels of expression in most nuclei and regions of hypothalamus. The labeling was observed within well-defined anatomical nuclei: preoptic, suprachiasmatic, periventricular, paraventricular, arcuate, dorsomedial, mammillary nuclei. The supraoptic and ventromedial nuclei were almost devoid of labeling. Some scattered labeled cells were also observed in the pituitary. This distribution of Gal-R2 mRNA-expressing cells corresponds well with that of galanin binding sites studies. As compared to the distribution of the galanin receptor subtype 1 (Gal-R1), our results indicate that the Gal-R2 type is differentially distributed, although a significant overlap exists in some regions such the preoptic area, arcuate and dorsomedial nuclei. The functional implications of these results are discussed in light of the role of galanin receptors plays in neuroendocrine regulation and feeding behavior.