Histochemical and electrophysiological evidence for estrogen receptors on cultured astrocytes: colocalization with cholinergic receptors

By means of autoradiographic and immunohistochemical methods it was demonstrated that astrocytes in explant and primary cultures of rat neocortex, hippocampus, preoptic area and spinal cord express estrogen alpha- and beta-receptors. Immunoreactivity was mainly distributed over the soma, the nuclei being more intensely stained. Combined autoradiographic and immunohistochemical studies as well as double-immunostaining revealed a colocalization of estrogen alpha- and beta-receptors on many astrocytes. There was also a coexistence of estrogen receptors and cholinergic muscarinic and nicotinic sites. Electrophysiological investigations have shown that 17beta-estradiol induced hyperpolarizations on the majority of astrocytes in explant cultures of hippocampus and spinal cord, providing evidence for the existence of functional estrogen receptors on these cells. Furthermore, on the same astrocytes, 17beta-estradiol, muscarine and nicotine caused hyperpolarizations, suggesting a coexistence of receptors for estrogen and the cholinergic agonists on glial cells. The presence of glial estrogen receptors and their colocalization with cholinergic receptors is discussed with respect to the effects of these neurotransmitters/neuromodulators in development and maturation of the central nervous system, as well as to neurodegenerative events such as Alzheimer's disease.     

Colocalization of androgen, estrogen and cholinergic receptors on cultured astrocytes of rat central nervous system

By means of immunohistochemical and electrophysiological methods, we have investigated the presence of androgen receptors on astrocytes in explant and primary cultures from various regions of rat central nervous system. Our studies have shown that a great number of astrocytes and neurones express androgen receptors as recognized by a specific monoclonal antibody. Immunoreactivity was mainly distributed over the soma of the astrocytes, the nuclei being intensely stained. In contrast, glial processes were only faintly stained or not stained. Double-immunostaining studies have provided evidence for a colocalization of androgen and estrogen alpha- and beta-receptors on many astrocytes. Furthermore, there was also a coexistence of glial androgen receptors with cholinergic muscarinic and nicotinic sites. Our immunohistochemical findings are supported by electrophysiological investigations demonstrating that 5alpha-androstan, 17beta-estradiol as well as the cholinergic agonists muscarine and nicotine caused hyperpolarizations on the same astrocytes. Our studies suggest that there is a coexistence of functional receptors for androgen, estrogen as well as for the cholinergic agonists on glial cells. Further investigations are needed to elucidate the physiological role of glial androgen, estrogen and cholinergic receptors and to define their function in neurodegenerative diseases.     

Metabolism and binding of androgens in the spinal cord of the rat

The binding of [3H]androgens and estrogens, and the metabolism of [3H]androgens, were studied in the spinal cord of the adult rat. High-affinity, specific binding sites for [3H]testosterone and [3H]estradiol were detected in cytosol fractions from the spinal cords of castrate animals. Equilibrium dissociation constants for reaction of these sites with their respective ligands were similar to those of androgen and estrogen receptors from other regions of the central nervous system. Nuclear binding of [3H]estradiol was observed in the spinal cord 1 h after intravenous administration of the isotope. Likewise, exchange assay demonstrated the presence of high-affinity androgen binding sites in spinal cord nuclei from orchidectomized, testosterone propionate treated animals. 5 alpha-Reductase activity in homogenates of the spinal cord was relatively high, approximately 3 times that in the pooled hypothalamus, preoptic area, septum and amygdala. However, in contrast to the latter brain regions, estrogen formation was not detectable in spinal cord tissue. No sex differences were observed in the metabolism of [3H]testosterone by spinal cord homogenates. These results confirm the presence of androgen and estrogen receptors in the rat spinal cord. The lack of detectable aromatase activity in the spinal cord is consistent with the hypothesis that the effects of circulating testosterone on spinal reflex function are mediated primarily through the androgen receptor system.     

Binding of [H]glycine, [H]β-alanine and [H]strychnine in cultured rat spinal cord and brain stem

Cultures of rat brain stem and spinal cord were used to visualize binding sites for [³H]glycine, [³H]β-alanine and their antagonist [³H]strychnine by light microscopic autoradiography. In spinal cord cultures, all radio-ligands were bound mainly to large neurones, probably motoneurones whereas in brain stem cultures, both medium-sized and large neurones were labelled. In contrast, glial cells did not show binding sites for any of the compounds studied, suggesting that glial elements may not possess receptors for glycine and β-alanine.     

Characterization of nicotinic receptors inducing noradrenaline release and absence of nicotinic autoreceptors in human neocortex

Presynaptic facilitatory nicotinic receptors (nAChRs) on noradrenergic axon terminals were studied in slices of human or rat neocortex and of rat hippocampus preincubated with [3H]noradrenaline ([3H]NA). During superfusion of the slices, stimulation by nicotinic agonists for 2 min only slightly increased [3H]NA outflow in the rat neocortex, but caused a tetrodotoxin-sensitive. Ca(2+)-dependent release of [3H]NA in rat hippocampus and human neocortex. In both tissues a similar rank order of potency of nicotinic agonists was found: epibatidine > DMPP > nicotine approximately cytisine > or = acetylcholine; choline was ineffective. In human neocortex, the effects of nicotine (100 microM) were reduced by mecamylamine, methyllycaconitine, di-hydro-beta-erythroidine (10 microM, each) and the alpha3beta2/alpha6betax-selective alpha-conotoxin MII (100/200 nM). The alpha3beta4 selective alpha-conotoxin AuIB (1 microM), and the alpha7 selective alpha-conotoxin ImI (200 nM) as well as alpha-bungarotoxin (125 nM) were ineffective. Glutamate receptor antagonists (300 microM AP-5, 100 microM DNQX) acted inhibitory, suggesting the participation of nAChRs on glutamatergic neurons. On the other hand, nAChR agonists were unable to evoke exocytotic release of [3H]acetylcholine from human and rat neocortical slices preincubated with [3H]choline. In conclusion: (1) alpha3beta2 and/or alpha6 containing nAChRs are at least partially responsible for presynaptic cholinergic facilitation of noradrenergic transmission in human neocortex; (2) nicotinic autoreceptors were not detectable in rat and human neocortex.     

Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors

The distribution of serotonin-2 (5-HT2) receptors in the rat brain was studied by light microscopic quantitative autoradiography. Receptors were labeled with four ligands: [3H]ketanserin, [3H]mesulergine, [3H]LSD and [3H]spiperone, which are reported to show high affinity for 5-HT2 receptors. Co-incubation with increasing concentrations of several well-known 5-HT2-selective drugs, such as pirenperone, cinanserin and ketanserin, resulted in an inhibition of the binding of the four 3H-labeled ligands to the same areas. However, all of them recognized, in addition to 5-HT2 sites, other populations of binding sites. Receptor densities were quantified by microdensitometry with the aid of a computer-assisted image-analysis system. Our results reveal a heterogeneous distribution of 5-HT2 receptor densities in the rat brain. Very high concentrations were localized in the claustrum, olfactory tubercle and layer IV of the neocortex. The anterior olfactory nucleus, piriform cortex and layer I of neocortex were also rich in 5-HT2 receptors. Intermediate concentrations of receptors were found in caudate putamen, nucleus accumbens, layer V of neocortex, ventral dentate gyrus and mammillary bodies. Areas containing only low concentrations of receptors included the thalamus, hippocampus, brainstem, medulla, cerebellum and spinal cord. The specificity of the different ligands used is discussed in terms of the other populations of sites recognized by them. The distribution of 5-HT2 receptors here reported is discussed in correlation with (a) the known distribution of serotoninergic terminals, (b) the specific anatomical systems and (c) the central effects reported to be mediated by 5-HT2-selective drugs.     

Characterization of benzodiazepine receptors in primary cultures of fetal mouse brain and spinal cord neurons

Primary cultures of fetal mouse brain and spinal cord were examined for the presence of binding sites for [³H]diazepam. Both brain and spinal cord cultures contain high affinity binding sites which resemble benzodiazepine receptors found in mammalian CNS with respect to both pharmacologic profile and response to exogenously applied GABA. These observations, coupled with the electrophysiologic properties of these cells suggest that primary cultures of fetal mouse brain and spinal cord may be valid models for studying the role and regulation of the benzodiazepine receptor.     

Tritium-sensitive film autoradiography of guinea pig brain α2-noradrenergic receptors

Tritium-sensitive film autoradiography was used to determine the distribution of α-noradrenergic receptors (i.e. [³H] p-aminoclonidine binding sites) in guinea pig forebrain. α₂-Receptors are heterogeneously distributed throughout the forebrain. Many limbic system structures, such as bed nucleus of stria terminalis, medial preoptic area, medial amygdaloid nucleus and lacunosum molecular layer in hippocampus were heavily labeled. We did not quantify receptor density in areas containing principally white matter but the optical density in those areas was similar to film background suggesting a very low receptor density. Low receptor concentrations were also found in areas that do not contain a high percentage of white matter, such as lateral septum and ventromedial hypothalamic nucleus.     

A critical evaluation of the use of toxins fromDendro- aspis viridis to block nicotinic responses at central and ganglionic synapses

Previous work by other investigators has shown that toxins prepared fromDendroaspis viridis venom block cholinergic transmission at the neuromuscular junction, as well as nicotinic transmission in frog spinal cord and in snail neurons. This suggested that these ligands may be useful for studying nicotinic receptors in the central nervous system. Thus,Dendroasis viridis venom was fractionated into its toxin components. Only one of the fractions possessed activity as assessed by: (1) inhibition of α-bungarotoxin (α-BGT) binding at the neuromuscular junction (25% at 50 μg toxin/ml) or (2) inhibition of the ventral root-dorsal root potential (VR-DRP), a nicotinic response in frog spinal cord. However, in the spinal cord preparation, in addition to this blockade of the nicotinic pathway, convulsant activity and an increase in the amplitude of other root potentials was observed. Binding experiments using [¹²⁵I]den- drotoxin demonstrated that the labeled compound bound to central nervous tissue such as brain or spinal cord; this was not displaced by nicotine (10⁻⁴ M) ord-tubocurarine (10^-4 M), a nicotinic antagonist, indicating either non-specific binding or binding to a non-nicotinic receptor. These results thus suggest that toxins fromDendroaspis viridis venom are not suitable ligands for central nicotinic receptors. In addition, as experiments also demonstrated that the dendrotoxins did not block cholinergic transmission in frog sympathetic ganglia, it contraindicates their use at ganglionic nicotinic receptors.     

Kappa opioid receptors in human lumbo-sacral spinal cord

[3H]Etorphine and [3H]ethylketocyclazocine bind with high affinity (Kd between 0.25-2.0 nM) to a single class of sites in human lumbo-sacral spinal cord. Other ligands such as [3H]morphine, [3H]dihydromorphine and [3H]D-Ala2, D-Leu5-enkephalin (DADLE) did not bind to significant number of sites under our incubation conditions. Ligand selectivity pattern strongly suggests that [3H]etorphine labels kappa opioid binding sites in the human lumbo-sacral spinal cord since benzomorphans and oripavines are much more potent than mu and delta agonists. Furthermore, [3H]etorphine and [3H]ethylketocyclazocine binding is sensitive to high concentrations of DADLE suggesting that these sites are of the kappa 2 sub-type. Finally, the visualization of these sites by receptor autoradiography demonstrates that they are mainly concentrated in lamina II and III of the dorsal horn. Moderate densities of sites are present around the central canal. Thus, it is possible that kappa opioid binding sites could be involved in the control of sensory and autonomic functions in the human lumbo-sacral spinal cord.     

Regional distribution of cholinergic muscarinic receptors in spinal cord

Quinuclidinylbenzilate ([³H]QNB) binding sites are present in the rat spinal cord. The binding sites are muscarinic in character based on displacement of [³H]QNB by cholinoceptive drugs. They are distributed rather uniformly along the cord, although the receptor density is greater in gray matters than in white matter. Binding to white matter may be associated with glial cells. Within the gray matter, the receptor density is higher in the ventral born than in the dorsal horn. In the thoracic region receptor density is about equal in the intermediate zone and ventral horn. Midthoracic transection of the cord does not change the receptor density or the dissociation constant of [³H]QNB in the lumbar cord. In contrast, treatment with the neurotoxin, 6-aminonicotinamide, which produces lesions of the cord, loss of motor control and paralysis, reduces the receptor density and affinity of [³H]QNB for lumbar gray matter but not white matter. The presence of [³H]QNB binding sites throughout the spinal cord as well as the documented presence of acetylcholine-containing neurons, suggest that muscarinic receptors play a role in all phases of spinal cord physiology.     

Differential modulation of γ-aminobutyric acid receptors by caprolactam derivatives with central nervous system depressant on convulsant activity

The effects of a series of caprolactam derivatives with central depressant, convulsant or muscle relaxant activity were investigated upon γ-aminobutyric acid (GABA) receptor-ionophore binding to rat brain membranes using [³H]GABA, [³H]muscimol and [³⁵S]-tert.-butylbicyclophophorothionate ([³⁵S]TBPS) as ligands, and GABA resonses in mouse spinal cord neurones in dissociated cell culture. Some caprolactams produced a picrotoxin-like chloride-dependent partial inhibition of muscimol binding and were potent inhibitors of TBPS binding. One compound that was further investigated (4,4,6,6,-tetramethylhexahydro-2H-azepin-2-one), inhibited GABA responses and increased the frequency of paroxysmal depolarizations in cultured neurones. Other caprolactams enhanced muscimol binding and were relatively weak inhibitors of TBPS binding, and one (3,3-diallyl-6,6-dimethylhexahydro-2H-azepin-2,4-dione) was shown to enhance GABA responses and produced quiescence of activity in cultured neurones. There was a direct correaltion between caprolactam effects on muscimol binding in the presence of chloride ions and their effects on TBPS binding suggesting a similar site of action for the caprolactams influencing the binding of these two ligands. For the two classes of caprolactams, with respect to inhibition or enhancement of muscimol binding, there appeared to be a relationship between in vitro effects and their convulsant or depressant activity in mice. Caprolactams may be useful low molecular weight probes for the study of GABA receptor-ionophore complexes.     

Regional distribution of cholinergic neurons in human spinal cord transections in the patients with and without motor neurons disease

Regional distribution of enzymic activities in acetylcholine (ACh) metabolism was examined on thinly-sectioned transverse slices of human spinal cords obtained during autopsy of 5 motor neuron disease (MND) and 5 control patients without MND. Choline acetyltransferase (ChAT) activity was highly concentrated in the ventral horn regions (gray and white matters) of cervical, thoracic and lumbar spinal cord of non-MND patients. This enzyme activity was found to be remarkably low in the ventral gray and white matter of MND patients compared with that of the controls. Although the distribution of acetylcholinesterase (AChE) activity was found to be high in both ventral and dorsal gray matter of the spinal cord, little difference was observed between each corresponding region of MND and control patients, except relatively low enzyme activity in the cervical ventral horn region of MND patients. Muscarinic cholinergic receptors, examined as specific [³H]quinuclidinylbenzilate ([³H]QNB) binding, was also highly concentrated in the ventral and dorsal gray matter of the control spinal cord, and was strongly reduced in the ventral horn region of MND patients, indicating a quite similar distribution pattern of ChAT activity. These biochemical changes of cholinergic transmission system may be paralleled to the morphological degeneration of the spinal lower motor neurons in MND patients. Activity of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase), a marker enzyme of central myelin structure, was evenly distributed throughout the whole spinal cord section, without regard to the gray and white matter, of both MND and control patients.     

A comparison of the regional ontogenesis of nicotine- and muscarine-like binding sites in mouse brain

The postnatal development of the cholinergic neurotransmitter system was studied in the cortex, hippocampus, midbrain and cerebellum of 3-, 7-, 12-, 17- and 30-day-old NMRI mice. The concentration of muscarine-like binding sites determined with [³H]quinuclidinyl benzilate as a ligand increased progressively with age. A similar developmental pattern was found for the activity of a presynaptic marker, choline acetyltransferase (ChAT). When expressed as a percentage of the values for 30-day-old mice, however, the muscarine-like receptors were parallel but preceding the development of ChAT in all the brain regions studied. The concentration of nicotine-like binding sites studied with [³H]α-bungaro-toxin as a ligand gradually increased with age in the cortex, hippocampus and midbrain, with a peak between days 7 and 12, followed by a decrease towards day 30. With [³H]tubocurarine as a ligand, on the other hand, the concentration of nicotine-like binding sites was in general high at 3 days and gradually decreased with age, suggesting that different subpopulations of nicotine-like receptors might be determined when the two ligands are used.     

[H]Prazosin binding in the intermediolateral cell column and the effects of iontophoresed methoxamine on sympathetic preganglionic neuronal activity in the anaesthetized cat and rat

The autoradiographic localization of [³H]prazosin (α₁-adrenoceptor ligand) binding sites was determined in cat spinal cord sections. High levels of [³H]prazosin binding were found in the intermediolateral cell column (IML) at thoracic and lumbar levels. The iontophoresis of theα₁-adrenoceptor agonist methoxamine onto sympathetic preganglionic neurones (SPNs) in anaesthetized cats and rats caused excitation of 8 cat SPNs and 13 rat SPNs. These results suggest an excitatory role for some of the catecholaminergic of the IML.     

Opiate receptor binding sites in human spinal cord

Opiate receptor binding sites were analyzed in various regions of human spinal cord and compared to results obtained in spinal cord and brain of certain animals. mu-, delta- and kappa binding sites were individually monitored by the overall labeling of opiate binding sites with [3H]diprenorphine followed by the sequential elimination of binding to particular sites by the use of selective ligands. kappa-Receptors were the predominant type (approximately 50%), followed by mu-receptors (approximately 40%), and, in rather small amounts, delta-receptors. A similar proportion of receptor types was found in the spinal cord of guinea pigs.     

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.     

Cholinergic agonist-induced down regulation of neuronal α-bungarotoxin receptors

The ability of cholinergic ligands to regulate neuronal α-bungarotoxin receptor number was studied in dissociated monolayer cultures of embryonic chick ciliary gangliion neurons. Carbamylcholine and nicotine, but notD-tubocurarine, caused a loss of 25% of the surface [¹²⁵I]α-bungarotoxin receptors within 1 h at 37°C. This receptor loss occured withour change in affinity for [¹²⁵I]α-bungarotoxin, was temperature-sensitive and was prevented by co-incubation withD-tubocurarine.     

Histamine H1 receptors in the brain and spinal cord of the cat

The regional distribution of histamine H₁ receptors in the feline brain and spinal cord was determined in vitro using the radio-actively labeled histamine H₁ antagonist, [³H]pyrilamine. This distribution of H₁ receptors, which was different from that reported for other species, was highest in the hypothalamus and mammillary bodies. Intermediate levels of binding were observed in the cerebral cortical and limbic regions, corpus striatum, colliculi, cerebellum, and medulla. The lowest binding was found in the pons and spinal cord. Binding in the spinal cord was concentrated in the gray matter, but the number of binding sites detected in the dorsal and ventral horns did not differ. The equilibrium dissociation constants for [³H]pyrilamine were similar for the various regions and were in the range of 2–3 nM and the pharmacological characteristics of the feline brain and spinal H₁ receptors were similar to those found in the brains of other mammalian species with the exception of the guinea pig.     

Localization and function of cat carotid body nicotinic receptors

Acetylcholine and nicotinic agents excite cat carotid body chemoreceptors and modify their response to natural stimuli. The present experiments utilized [125I]alpha-bungarotoxin [( 125I]alpha-BGT) to localize within the chemosensory tissue the possible sites of action of exogenous and endogenous nicotinic cholinergic substances. In vitro equilibrium binding studies of intact carotid bodies determined a Kd of 5.57 nM and a Bmax of 9.21 pmol/g of tissue. Chronic section (12-15 days) of the carotid sinus nerve (CSN) did not change the amount of displaceable toxin binding. In contrast, the specific binding was reduced by 46% following removal of the superior cervical ganglion. Light microscope autoradiography of normal, CSN-denervated and sympathectomized carotid bodies revealed displaceable binding sites concentrated in lobules of type I and type II cells. Treatment of carotid bodies with 50 nM alpha-BGT in vitro reduced by 50% the release of [3H]dopamine (synthesized from [3H]tyrosine) caused by hypoxia or nicotine, and also significantly reduced the stimulus-evoked discharges recorded from the CSN. The data suggest an absence of alpha-BGT binding sites on the afferent terminals of the CSN and that nicotinic receptors located with parenchymal cell lobules may modulate the release of catecholamines from these cells.