Whole brain and regional [125I]-α-bungarotoxin binding in developing rat

These experiments were conducted in order to determine if the total number of binding sites for [¹²⁵I]-α-bungarotoxin ([¹²⁵I]-α-BGT) in rat brain increases and then decreases during postnatal development as predicted by comparison with skeletal muscle, and, if so, to determine at approximately what age the peak in binding occurs in the brain as a whole. A further purpose was to investigate the time-course of development of the [¹²⁵I]-α-BGT binding sites in several brain regions.Specific binding for [¹²⁵I]-α-BGT was studied using the pellets from a 20 min, 14,000 × g centrifugation of rat brain homogenates from 4 or 5 postnatal ages. At least three binding assays were done per region and per age, on cerebral cortex, cerebellum, caudate-putamen, posterior hippocampus, pons-medulla and whole brain. In most regions, the [¹²⁵I]-α-BGT specific binding is measurable, but is low at day one, peaks at about 12–20 days and declines by adulthood. With a few exceptions, these data hold true whether binding is expressed as specific binding per mg protein, specific binding per gram wet tissue, or total specific binding per brain region. The absolute number of specifically bound [¹²⁵I]-α-BGT molecules is undistorted by simultaneous or non-linear growth of cells uninvolved with α-BGT binding and, thus, is the measurement most useful in determining developmental changes. Whole brain has the same age-related pattern as in the majority of the brain regions, i.e., compared to 19–20 days, the adult brain actually has fewer total binding sites.     

Contrasting effect of a brain supernatant extract on neuronal vs neuromuscular α-bungarotoxin receptors

The effects of a rat brain supernatant extract and a partially purified supernatant preparation from bovine brain were determined on the binding of [¹²⁵I]α-bungarotoxin (α-BGT) to muscle membranes, as well as to membranes prepared from brain. In agreement with previous work, the supernatant preparations inhibited α-BGT binding to brain membranes in a dose-dependent fashion, (Brain Research, 245 (1982) 57–67); however, no significant effect of either of the preparations was observed on the binding of the toxin to muscle membranes. As well, the supernatant preparations did not affect binding of radiolabelled α-BGT to muscle cells in culture in competition binding experiments. The effect of long-term incubation of cells in culture with the supernatant preparations was subsequently determined. These studies showed that the binding of [¹²⁵I]α-BGT increased markedly (300%) in the presence of a crude rat brain supernatant preparation, while incubation of the muscle cells in the presence of the partially purified bovine supernatant extract had no significant effect on radiolabelled toxin binding. In contrast, both the rat and bovine supernatant preparations significantly decreased (up to 65%) radiolabelled toxin binding to a cultured neuronal cell population, adrenal medullary chromaffin cells. These results suggest that an endogenous factor(s), present in brain extracts, differentially regulates the neuronal as compared to the neuromuscular nicotinic α-bungarotoxin binding sites.     

Ontogeny of sexual difference in α-bungarotoxin binding capacity in the mouse amygdala

We have shown a sex difference in the nucleus amygdaloideus medialis posterior (NAMP) of the mouse with respect to the binding capacity for α-bungarotoxin (α-BGT) under various steroid-hormonal environments. The present study describes histochemically the postnatal development of α-BGT binding capacity in the NAMP of the intact male and female mouse, and characterizes biochemically the toxin binding component at different developmental stages. Light microscopic autoradiography using radio-iodinated α-BGT revealed characteristic patterns of development of α-BGT binding capacity in the NAMP of both sexes. On the day of birth, the autoradiographic grain density for [¹²⁵I]α-BGT binding sites was low in the NAMP and no sex difference was detected. During the next 4 days the grain density increased in each sex, but the density in the female increased to a lesser extent than in the male, resulting in a marked sex difference at 4 days after birth. The grain density in each sex was maximal at 7–10 days and then declined toward the adult level by 28 days of age. The density in the male exceeded that in the female throughout postnatal life except for the day of birth. The biochemical filtration assay on the tissue homogenates provided evidence suggesting that α-BGT binding sites in the posterior corticomedial amygdaloid region including the NAMP are sexually different in number, but not in the binding affinity, at an early postnatal age as well as in adulthood. These results indicate the importance of the early postnatal days for the sexual differentiation of α-BGT binding sites in the NAMP.     

Distribution of an α-bungarotoxin-binding cholinergic nicotinic receptor in rat brain

Cholinergic nicotinic receptors in rat brain were demonstrated by the use of the potent nicotinic antagonist [¹²⁵I]α-bungarotoxin ([¹²⁵I]α-Btx). Biochemical studies on binding of [¹²⁵I]α-Btx to rat hippocampal homogenates revealed saturable binding sites which are protected by nicotine, d-tubocurarine and acetylcholine but not by atropine or oxotremorine. The hippocampus and hypothalamus displayed relatively high [¹²⁵I]α-Btx specific binding whereas the cerebellum was devoid of specific binding. Other regions displayed intermediate binding levels. Analysis of the regional distribution of [¹²⁵I]α-Btx binding by autoradiography of frontal brain sections revealed high labeling in the hippocampus, hypothalamic supraoptic, suprachiasmatic and periventricular nuclei, ventral lateral geniculate and the mesencephalic dorsal tegmental nucleus. It is suggested that the limbic forebrain and midbrain structures as well as sensory nuclei are the main nicotinic cholinoceptive structures in the brain.     

Heterogeneity of brain melanocortin receptors suggested by differential ligand binding in situ

The existence of multiple brain melanocortin receptor types has been postulated, based on the complex pharmacology of intracerebrally administered melanocortin (melanocyte-stimulating hormone-related) peptides. In this study, this hypothesis was tested by determining whether different brain melanocortin receptor populations can be discriminated on a pharmacologic or neuroanatomic basis. The abilities of various pharmacologically active native melanocortins and structural analogs, as well as other test substances, to compete with biologically active [¹²⁵I]Nle⁴,d-Phe⁷-α-MSH([¹²⁵I]NDP-MSH) for binding to melanocortin receptors was determined, by in vitro binding and autoradiography in frozen rat brain tissue sections. We have previously shown that native melanocortins including α-MSH, γ-MSH and ACTH_1–39 compete with [¹²⁵I]NDP-MSH for binding to brain tissue sites. In the present studies, each of the melanocortin peptides α-MSH, des-acetyl-α-MSH, β-MSH and ACTH_1–24 when present at 1 μM virtually eliminated [¹²⁵I]NDP-MSH binding in each of a series of brain structures, including medial preoptic area, caudate putamen, olfactory tubercle, bed nucleus of the stria terminalis, ventral part of the lateral septal nucleus, hypothalamic periventricular and paraventricular nuclei, dorsal anterior amygdaloid area, substantia innominata and thalamic paraventricular nucleus; as well as in extraorbital lacrimal gland, a peripheral melanocortin target. In contrast, the behaviorally and neurotrophically active melanocortin analogs Met(O₂),d-Lys,Phe⁹-α-MSH_4–9 (Org2766), ACTH_4–9 and the antipyretic peptide α-MSH_11–13 did not affect [¹²⁵I]NDP-MSH binding at concentrations up to 100 μM, implying that the receptors or receptor binding sites which mediate the actions of these analogs must comprise additional types, distinct from those which bind [¹²⁵I]NDP-MSH. Binding of [¹²⁵I]NDP-MSH was also unaffected by the nonmelanotropic peptides ACTH_1–4, ACTH_34–39 and vasoactive intestinal polypeptide (VIP) and by the antipyretic drugs acetaminophen and lysine-salicylate. Although some of the brain structures are known to express mRNA encoding a γ-MSH-preferring melanocortin receptor type known as MC3, the relative order of binding affinities of melanocortins, determined in concentration-response studies, wasNdp-MSH≥ACTH_1–24α-MSH>γ-MSH>ACTH_4–10 probably account for most of the [¹²⁵I]NDP-MSH binding detectable in the brain. Furthermore, the potency relationships between these respective peptides and the binding activity ofD-Trp⁷,D-Phe^{10-α-MSH_6–11} amide, a synthetic α-MSH antagonist, varied considerably among the brain sites and peripheral (lacrimal and melanoma) tissues studied, suggesting some degree of heterogeneity of ligand binding properties among [¹²⁵I]NDP-MSH-binding melanocortin receptor populations in different regions of the brain. Considered together with the available data on the pharmacologic actions of melanocortins and the molecular biology of melanocortin receptors, the present results provide evidence for the existence of multiple, pharmacologically distinct classes of melanocortin receptors in the brain, potentially providing a basis for the pharmacological targeting of specific populations of central melanocortin receptors.     

Axonal transport of α-bungarotoxin binding sites in rat sciatic nerve

[¹²⁵I]α-Bungarotoxin (α-BuTX) binding sites accumulate both proximal and distal to a ligature positioned around the sciatic nerve of rats. [¹²⁵I]α-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]α-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 [¹²⁵I]α-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]α-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.     

α1-Adrenergic receptors and stimulation of [H]inositol metabolism in rat brain: Regional distribution and parallel inactivation

The relationship between norepinephrine-stimulated phosphatidyl-inositol metabolism andα₁-adrenergic receptor density was examined in rat brain. Increases in phosphatidyl-inositol metabolism were determined by accumulation of [³H]inositol phosphates in the presence of lithium in brain slices, while receptor density was determined by specific binding of¹²⁵I-BE 2254 (¹²⁵IBE) in membrane fractions. Treatment of slices of cerebral cortex with increasing concentrations of the irreversibleα₁-adrenergic receptor antagonist phenoxybenzamine caused a parallel inactivation of specific¹²⁵IBE binding sites and norepinephrine-induced [³H]inositol phosphate accumulation, although approximately 20% of the binding sites remained after abolition of the inositol response. Comparison of the density of¹²⁵IBE binding sites and the magnitude of norepinephrine-stimulated [³H]inositol phosphate accumulation in 8 different brain regions did not show a particularly good correlation. The thalamus had the highest density of binding sites and an intermediate inositol response, while the hippocampus had the highest inositol response but an intermediate density of binding sites. However, the cerebellum had the lowest density of binding sites and no measurable inositol response. Treatment of slices of each region with 300 nM phenoxybenzamine abolished the inositol response and caused a 59–73% decrease in the density of¹²⁵IBE binding sites. The lack of correlation between receptor density and inositol response between brain regions could not be explained on the basis of receptor affinity, spare receptors, protein content, nor differences in slice size. These results suggest thatα₁-adrenergic receptors labeled by¹²⁵IBE are coupled to [³H]inositol metabolism in rat brain, but the receptor density is not the sole determinant of the magnitude of norepinephrine-induced increases in [^3H]inositol metabolism.     

α-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with α7 subunits and up-regulation by chronic nicotine treatment

High density neuronal cultures from rat E18 hippocampus and cortex have been characterised with respect to cholinergic binding sites. No specific binding of [³H]nicotine or [³H]cyttine to live cells in situ was detected, although the limit for detection was estimated to be 30 fmol/mg protein. Muscarinic binding sites labelled with [³H]QNB were present at a density of 0.75 pmol/mg protein. [¹²⁵I]α-Bungarotoxin (αBgt) bound to hippocampal cultures with a B_max of 128 fmol/mg protein and a K_d of 0.6 nM; cortical cultures expressed five times fewer [¹²⁵I]α-Bgt binding sites. Fluorescence cytochemistry with rhodamine-α-Bgt indicated that 95% of hippocampal neurons were labelled, compared with only 36% of cortical neurons. Average densities of 4 × 10⁴ and 2 × 10⁴ binding sites/cell were calculated for hippocampal and cortical cultures, respectively. Double labelling experiments with mAb307 (which recognises the rat α7 nicotinic receptor subunit) and rhodamine-α-Bgt gave coincident labelling patterns, supporting the correlation between the α7 subunit and Bgt-sensitive neuronal nicotinic receptor. Treatment of hippocampal cultures with 10 μM nicotine for 14 days elicited a 40% increase in the numbers of [¹²⁵I]α-Bgt binding sites, mimicking the up-regulation observed in vivo studies. Primary cultures offer a useful in in vitro system for investigating the expression and regulation of brain α-Bgt-sensitive receptors.     

Characterization of [I]HEAT binding to α1-receptors in human brain: assessment in aging and Alzheimer's disease

The binding of [¹²⁵I]2-(β-4-hydroxyphenylethylamino-ethyltetralone ([¹²⁵I]HEAT), an α₁-adrenergic receptor antagonist, to human brain membranes was characterized and the binding assessed in tissue from subjects with Alzheimer's disease (AD) and aging controls. Under Na⁺-K⁺ phosphate buffer conditions, [¹²⁵I]HEAT bound to a single class of binding sites in prefrontal cortex (Brodmann area 10) with a K_d of about 120 pM. High binding capacities of [¹²⁵I]HEAT were evident in the hippocampus and neocortex but were low in subcortical areas and cerebral microvessels comparable to the regional distribution of [³H]prazosin binding reported previously. Displacement of [¹²⁵I]HEAT by various adrenergic drugs was consistent with its binding to α₁-adrenergic receptors. The specific binding was not affected by postmortem delay between death and freezing of tissue at autopsy. There was no correlation of [¹²⁵I]HEAT binding with age of subjects. In AD subjects, the binding was significantly decreased in prefrontal cortex by about 25% but not changed in hippocampus, putamen or cerebellum compared to age-matched controls. The reduced binding of [¹²⁵I]HEAT in prefrontal cortex may reflect a region-specific change in α₁-adrenergic receptors associated with neuronal loss in AD.     

A radioiodinated 7 α -O-iodoallyl diprenorphine for mapping opioid receptors

The aim of the current research has been to validate an original radioiodinated diprenorphine (DPN) derivative suitable for imaging studies of opioid receptors. [¹²⁵I]7α-O-iodoallyl diprenorphine (7α-O-IA-DPN) was prepared by radioiododestannylation and in vitro and in vivo opioid receptor binding assays were performed with CDF1 mouse brains.In vitro binding studies showed high affinity (K_i= 0.4 ± 0.2 nM) for mouse brain membranes. In vivo studies showed 63% specific binding. Ex vivo autoradiography of brain sections confirmed high uptake and retention of [¹²⁵I]7α- O -IA-DPN in regions rich in opioid receptors. This new radioiodinated DPN analogue appears to be a potential radioprobe for in vivo visualization of human cerebral opioid receptors with single photon emission computed tomography (SPECT).     

α-Bungarotoxin binding to a high molecular weight component from lower vertebrate brain identified on dodecyl sulfate protein-blots

The binding of [¹²⁵I]iodo-α-bungarotoxin ([¹²⁵]α-BuTX) to the dissociated α-subunit of Torpedo acetylcholine receptor (AChR) can be readily demonstrated in a modified ‘protein-blot’ analysis utilizing electrophoretically transferred, dissociated subunits immobilized onto positively charged nylon membranes which are then incubated directly with [¹²⁵I]α-BuTX. We report here the use of the protein-blotting technique to detect the α-BuTX binding site present in the central nervous system of lower vertebrates and to characterize some of the physicochemical properties of the toxin binding site. High molecular weight (M200,000 and 120,000) α-BuTX-binding components can be readily demonstrated in avian and fish brain extracts upon protein-blotting with [¹²⁵I]α-BuTX following lithium dodecyl sulfate PAGE. Neither extensive reduction with dithiothreitol nor prior reduction followed by alkylation with iodoacetamide alter the mobility of the CNS-derived BuTX-binding sites. In contrast to our findings with Torpedo AChR or muscle AChR derived from a number of different species, no binding is observed in the molecular weight range of the α-subunit (M_r= 40,000) nor is any binding at any molecular weight observed in similar fractions prepared from adult, mammalian (rat, guinea pig) brain using this technique. These results demonstrated the existence in lower vertebrate brain of a BuTX binding site comparable in size to the AChR oligomeric complex of electric organ and muscle. They also suggest, however, striking structural differences between muscle AChR and the central neuronal BuTX-binding complex as well as a considerable difference between the neuronal BuTX-binding sites derived from lower and higher vertebrate brain.     

Changes in γ-aminobutyric acid, μ-opioid and neurotensin receptors in the accumbens-pallidal projection after discrete quinolinic acid lesions in the nucleus accumbens

Discrete quinolinic acid lesions in the nucleus accumbens altered [³H]muscimol binding to γ-aminobutyric acid receptors, [¹²⁵I]neurotensin binding to neurotensin receptors, [¹²⁵I]Tyr-d-Ala-Gly-NMePhe-Gly-OH binding to μ-opioid receptors, and [³H]quinuclidinyl benzilate binding to muscarinic receptors. Within lesions of the lateral accumbens core, [³H]muscimol binding increased and [¹²⁵I]Tyr-d-Ala-Gly-NMePhe-Gly-OH, [¹²⁵I]eurotensin and [³H]quinuclidinyl benzilate binding decreased. Lesions of the medial nucleus accumbens resulted in decreased [¹²⁵I]Tyr-d-Ala-Gly-NMePhe-Gly-OH and [³H]quinuclidinyl benzilate binding while no alterations were observed for [³H]muscimol or [¹²⁵I]neurotensin binding. These data support anatomical distinctions between medial and lateral nucleus accumbens. Destruction of intrinsic neurons in the dorsomedial nucleus accumbens core increased [³H]muscimol binding in the dorsal rim of the ventral pallidum and the rostral globus pallidus without altering [¹²⁵I]Tyr-d-Ala-Gly-NMePhe-Gly-OH binding. Destruction of neurons in the lateral nucleus accumbens core or medial shell did not alter [³H]muscimol binding in the ventral pallidum. The lack of upregulation in γ-aminobutyric acid receptors suggests that the γ-aminobutyric acid-containing projection from the dorsomedial core to the dorsal rim of the ventral pallidum differs from the projection from the lateral accumbens core and medial shell to the more ventral regions of the pallidum. Fluoro-gold retrogade tracer histochemistry confirmed the specific projection from the dorsomedial core to the dorsal ventral pallidum; and from the shell of the nucleus accumbens to more ventral regions of the ventral pallidum.     

Effects of gonadal steroids on the in vivo binding of [I]α-bungarotoxin to the suprachiasmatic nucleus

The radioligand [¹²⁵I]α-bungarotoxin (α-BTX) has been used to test receptor binding to putative nicotinic cholinergic receptors in the hypothalamus. Using light microscopic autoradiography following third ventricular infusion of the radioligand we have previously demonstrated that in normally cycling rats and in normal males, the suprachiasmatic nucleus (SCN) consistently binds the α-neurotoxin. In chronically (5 weeks) oophorectomized female, binding of [¹²⁵I]α-BTX to the SCN is markedly diminished. The present series of experiments were designed to test the effects of gonadal steroids on the binding of [¹²⁵I]α-BTX to the SCN. We first tested whether or not estradiol administered to ovariectomized females could duplicate the presence of the ovary. In females ovariectomized and immediately provided with a constant dose of estradiol-17β (E₂)—1.0 cm silastic capsules for 5 weeks, (n= 4), the binding of the neurotoxin to the SCN was maintained. In females ovariectomized for 3 weeks and replaced with E₂ for 2 weeks (n= 4), the binding of [¹²⁵I]α-BTX to the SCN was restored. In chronically (4 weeks) ovariectomized females receivingf E₂ for 6 dyas (n= 2), the binding of the neurotoxin was partially restored. We next tested the effect of chronic (5 weeks) castration (n= 100) and observed that binding of [¹²⁵I]α-BTX to the SCN of castrated males was like that of intact male controls (n= 6). We also tested the effect of neonatal adrogenization (1.25 mg testosterone proprionate, neonatal days 2 and 5) with subsequent adult oophorectomy (day 83) and observed that neonatally androgenized females with intact ovaries (n= 4) demonstrated [¹²⁵I]α-BTX binding to the SCN like that of non-adrogenized females, while neonatally androgenized females without ovaries (n= 4) had decreased binding of the neurotoxin to the SCN like that of oophorectomized adult females. These data demonstrate that: (1) the administration of estradiol can maintain and restore [¹²⁵I]α-BTX labeling of the SCN in ovariectomized rats; (2) binding of [¹²⁵I]α-BTX to the SCN is different in castrated males and ovariectomized females; and (3) the difference in α-BTX binding to the SCN receptor in adult castrated male and female rats may be dependent upon the presence of gonadal steroids at times other than the ‘critical’ postnatal period.     

Modulation of Interleukin-1 Receptors in the Brain-Endocrine-Immune Axis by Stress and Infection

We summarize data from some of our recent studies on in vitro and in vivo modulation of interleukin-1 (IL-1) receptors in the mouse brain-endocrine-immune axis by stress and infection. Ether-laparotomy stress in mice resulted in a selective increase in pituitary IL-I receptors and a significant decrease in pituitary receptors for corticotropin-releasing factor (CRF), a major regulator of the endocrine response to stress. Intraperitoneal injection of rat/human CRF mimicked the effects of stress and resulted in a dramatic increase in [¹²⁵I]IL-1α binding in the pituitary; [¹²⁵I]IL-1α binding in the hippocampus, spleen, and testis was unaffected by stress or CRF treatment. Glucocorticoid treatment with dexamethasone alone did not alter [¹²⁵I]IL-1α, binding but significantly inhibited CRF-induced upregulation of IL-1 receptors in the pituitary. The intracellular mechanism(s) involved in stress and CRF-induced upregulation of IL-1 receptors in the pituitary gland were examined by evaluating the effects of treatment of AtT-20 mouse pituitary corticotroph cells with a variety of neuroendocrine mediators of stress. CRF, forskolin, and isoproterenol (β₂ adrenergic receptor agonist) produced dose-dependent increases in cAMP production and [¹²⁵I]IL-1α binding. In contrast, somatostatin and dexamethasone significantly inhibited CRF-stimulated increase of cAMP production and [¹²⁵I]IL-1α binding, suggesting a primary role for cAMP in the regulation of pituitary IL-1 receptors. Next, we investigated the modulation of LL-1 β levels and IL-1 receptors following infection of mice with the endotoxin, lipopolysaccharide (LPS). Acute administration of low doses of endotoxin (30 μg LPS/mouse) dramatically increased IL-1 β levels and reciprocally decreased [¹²⁵I]IL-1α binding in peripheral tissues (pituitary, testis, liver, and spleen) but not in brain (hippocampus). This effect appeared to be dose related since higher doses of endotoxin (300 μg LPS/mouse) significantly decreased [¹²⁵I]IL-1α binding in both peripheral tissues and brain. Endotoxin induced modulation of the IL-1 system was also dependent on the treatment regimen since two low-dose LPS injections (at 0 and 12 h) increased IL-1 β concentrations and decreased [¹²⁵I]IL-1α binding in both central and peripheral tissues. These data provide further support for a role for IL-1 in coordinating brain-endocrine-immunoresponses to stress and infection.     

Autoradiographic localization of the binding of calcium channel antagonist, [I]ω-agatoxin IIIA, in rat brain

The calcium channel antagonists ω-agatoxin IIIA (ω-Aga-IIIA) and ω-conotoxin GVIA (ω-CgTx) were radioiodinated and used to locate binding sites in the rat brain by receptor autoradiography. While patterns of regional binding to sagittal sections of rat brain were generally similar for the 2 toxins, notable differences in the cerebellum and hippocampus were observed. Specific [¹²⁵I]ω-Aga-IIIA binding was greatest in the granule cell layers of the cerebellum and of the dentate gyrus. In contrast, binding of [¹²⁵I]ω-CgTx was most intense in the molecular layers of these structures. Less than one-third of [¹²⁵I]ω-Aga-IIIA binding in rat brain slices was inhibited by pre-exposure to 250 nM ω-CgTx, while 40 nM ω-Aga-IIIA virtually eliminated the binding of [¹²⁵I]ω-CgTx under the same conditions. The P-type calcium channel antagonist ω-Aga-IVA blocked only a small fraction of [¹²⁵I]ω-Aga-IIIA and [¹²⁵I]ω-CgTx binding. These autoradiographic data are consistent with membrane binding experiments and indicate that the combined use of agatoxins and conotoxins may be useful in the characterization of separate types of neuronal calcium channels.     

Developments of α-bungarotoxin receptors in cultured chick ciliary ganglion neurons

We have maintained embryonic chick ciliary ganglion neurons in dissociated cell culture and studied the progressive appearance of surface receptors for [¹²⁵I]α-bungarotoxin. Cultures were established from 8-day-old embryos and fed a medium supplemented with 180 μg/ml of a soluble protein extract prepared from the eye, the target organ for the ciliary ganglion. Approximately 8064 neurons survived per ganglion and there was no evident loss of neurons through two weeks in culture. Binding of [¹²⁵I]α-bungarotoxin was determined at room temperature on intact cells still attached to their coverslips. Non-specific binding was less than 2% of the total. Specific binding of [¹²⁵I]α-bungarotoxin was saturable with respect to both time of incubation (20–30 min) and concentration of toxin (5–10 nM), with an apparent K_d = 1.0 nM. Binding sites for [¹²⁵I]α-bungarotoxin increased during the first week in culture from 1.8 fmol per 10⁴ neurons at 1 day in vitro (DIV) to 8.6 fmol per 10⁴ neurons at 7 DIV, after which the number of sites seemed to plateau. Light microscopic autoradiography was performed on cultures at 4 DIV and showed most of the grains associated with the surfaces of neuronal cell bodies, while scattered grains occurred over neuronal processes. When compared with previous reports on the in vivo development of α-bungarotoxin receptors in chick ciliary ganglia, the appearance of receptors in these cultured neurons followed a time course similar to, but at lower levels than, their in vivo counterparts. Nevertheless, this culture system should prove useful for the study of questions concerning the regulation, surface distribution and intracellular pathways of neuronal α-bungarotoxin receptors.     

Effect of repeated novel stressors on depressive behavior and brain norepinephrine receptor system in Sprague-Dawley and Wistar Kyoto (WKY) rats

This study compared the effects of repeated novel stressors on ‘depressive behaviors’, defined by the forced-swim and open-field tests, in Sprague-Dawley (S-D) and Wistar Kyoto (WKY) rats. Since stress appears to alter brain norepinephrine (NE) activity, this study also investigated the effects of the stressors on β-adrenoceptors (β-ARs),α₂-adrenoceptors (α₂-ARs) and NE transporter (NET) sites in S-D and WKY rats. Stress did not alter¹²⁵I-iodopindolol (¹²⁵I-PIN) binding to β-ARs, nor [³H]idazoxan ([³H]IDAZ) binding toα₂-ARs in S-D rats, compared to non-stressed controls. However, WKY-stressed rats showed a significant reduction in¹²⁵I-IPIN binding to β-ARs in the cortex, hippocampus, amygdala and hypothalamus, and a reduction in [³H]IDAZ binding toα₂-ARs in the amygdala. [³H]nisoxetine ([³H]NIS) binding to NET sites in WKY-stressed rats was also reduced in the cortex, hippocampus and amygdala. When both strains were compared, the most surprising finding was a significantly higher density of NET sites in the hippocampus and amygdala in WKY rats compared to S-D rats. The results of this study indicate that stress, not only exacerbates depressive behavior in WKY rats, but also selectively alters β-ARs,α₂-ARs and NET sites in limbic brain regions. Thus, the WKY strain may serve as a useful animal model for depressive behavior and for the investigation of novel antidepressant drugs.     

Autoradiographic localization of α5 subunit-containing GABAA receptors in rat brain

Multiple subtypes of GABA_A receptors are expressed in the rat central nervous system (CNS). To determine the distribution and proportion of α5 subunit containing receptors, quantitative autoradiographic analyses were performed with both [³H]L-655,708 and [³H]Ro15-1788, an α5 selective and a non selective benzodiazepine binding site ligand, respectively. High densities of [³H]L-655,708 binding sites were observed in hippocampus and olfactory bulb, where α5 receptors accounted for 20–35% of total [³H]Ro15-1788 binding sites. Low levels of [³H]L-655,708 sites were associated with the cortex as well as amygdala, thalamic, hypothalamic and midbrain nuclei. These observations indicate that although [³H]L-655,708 binding sites have an overall low expression in rat CNS, they may contribute significantly to GABAergic inhibition in specific brain regions.     

Postnatal changes of nicotinic acetylcholine receptor 2, α3, α4, α7 and β2 subunits genes expression in rat brain

Postnatal changes of nicotinic acetylcholine receptor (nAChR) α2, α3, α4, α7 and β2 subunits mRNAs were investigated in rat brain using ribonuclease protection assay. Multiple developmental patterns were observed: (1) transient expression during the first few postnatal weeks; α2 in the hippocampus and brain stem, α3 in the striatum, cerebellum and cortex, α4 in the hippocampus, striatum and cerebellum, α7 in the cerebellum and β2 in the striatum. (2) Constant expression across development; α2 and α3 in the thalamus, α4 in the cortex, thalamus and brain stem, α7 in the thalamus and brain stem and β2 in all brain regions except striatum. (3) Non-detection across development; α2 in the cortex, striatum and cerebellum. (4) Increase with age; α7 in the cortex and hippocampus. (5) Bell-shaped development; α7 in the striatum. Postnatal changes of nAChR isoforms in different brain regions of rat were investigated by receptor binding assays. The developmental patterns of [³H]epibatidine and (−)-[³H]nicotine binding sites were similar to each other in each brain region, but different from that of [³H]α-bungarotoxin binding sites. No obvious correlation was observed between the developmental patterns of [³H]α-bungarotoxin, [³H]epibatidine and (−)-[³H]nicotine binding sites and corresponding subunits mRNAs. These results indicate that multiple mechanisms are involved in changes of gene expression of nAChRs subunits in the brain of developing rats.     

α1-Adrenergic receptors in the brain: characterization in astrocytic glial cultures and comparison with neuronal cultures

Binding of [¹²⁵I]HEAT to membranes prepared from primary cultures of astrocytic glial cells was time-dependent and 70–85% specific. Various adrenergic agonists and antagonists competed for [¹²⁵I]HEAT binding to the potencies of prazosin >, yohimbine , clonidine, norepinephrine (NE), and propranolol. Scatchard analysis showedB_max of 209 fmol/mg protein and a K_d of 184 pM for [¹²⁵I]HEAT binding by astrocytic glial membranes. Pretreatment of astrocytes with NE resulted in a dose-dependent downregulation of [¹²⁵I]HEAT binding sites with a maximal response observed after 8 h at 100 μM NE. Removal of NE from cultures after pretreatment resulted in a time- and protein synthesis-dependent recovery of binding sites to control levels within 120 h. Incubation of astrocytic glial cultures with NE stimulated phosphoinositide (PI) hydrolysis in a time- and dose-dependent manner with a maximal stimulation of 2-fold observed in 60 min by 100 μM NE.Clonidine expressed differential effects on α₁-adrenergic receptors of the neuronal and astrocytic glial cultures. Pretreatment with 10 μM clonidine caused a 40% decrease in the B_max of [¹²⁵I]HEAT binding without influencing the K_d value in neuronal cultures. This downregulatory effect of clonidine was associated with a reduction in the ability of NE to stimulate PI hydrolysis in clonidine pretreated cells. In contrast to neuronal cultures, clonidine neither downregulated [¹²⁵I]HEAT binding sites nor stimulated PI hydrolysis in glial cultures.