Beta-alanine potentiation of [3H]flunitrazepam binding to rat spinal cord homogenates

The effect of beta-alanine, gamma-aminobutyric acid (GABA), and other functionally related amino acids on [3H]flunitrazepam binding to rat spinal cord homogenates was studied. beta-Alanine potentiated [3H]flunitrazepam binding by 40% and GABA by 88%. Taurine increased the binding by 19%. Hypotaurine produced an 11% increase. No significant effect was seen in glycine, alanine, serine, valine or the dipeptide carnosine. The beta-alanine increase in [3H]flunitrazepam binding was completely inhibited by 10 microM strychnine, whereas the GABA increase required 0.1 mM strychnine to be fully suppressed. Results suggest that beta-alanine specifically potentiates binding of [3H]flunitrazepam in rat spinal cord homogenates.     

Postnatal development of [3H]flunitrazepam and [3H]strychnine binding sites in rat spinal cord localized by quantitative autoradiography

The development of inhibitory receptors in rat spinal cord was investigated by autoradiography using [3H]flunitrazepam as a ligand for benzodiazepine receptors and [3H]strychnine as a ligand for glycine receptors. The development of benzodiazepine receptors follows a similar pattern at all levels of the spinal cord. The density of [3H]flunitrazepam binding sites is already high at birth, increases 2-fold by days 3-7 and thereafter declines to levels already present at birth. In contrast, [3H]strychnine binding sites are weakly expressed at birth and increase up to 7-fold between days 4 and 21. A craniocaudal gradient in the development of glycine receptors is not apparent. However, maturation of [3H]strychnine binding in the ventral horn precedes that in the dorsal horn for 3-4 days. In summary, the developmental expression of these two inhibitory receptors in the spinal cord appears to be regulated differently.     

Cholecystokinin releases [3H]GABA from the perfused subarachnoid space of the anaesthetized rat spinal cord

The neuropeptide cholecystokinin(26-33) (CCK) is widely distributed in the mammalian central nervous system, including the spinal cord. We have studied the possible interaction of CCK with GABA release mechanisms. Low doses of CCK-8 (1 nM) have been found to evoke calcium-dependent [3H]GABA release from an in vivo perfused spinal cord preparation in the anaesthetized rat. Tachyphylaxis was seen to the [3H]GABA releasing action of CCK-8. The injection of proglumide (150 mg/kg i.p.) totally blocked the [3H]GABA release produced by CCK-8 or by a medium containing 50 mM potassium. Substance P (10 microM) did not produce release of [3H]GABA, although in the same animals 50 mM potassium containing solutions could be shown to evoke release of [3H]GABA.     

Binding sites for [3H]gamma-hydroxybutyrate on cultured neurones of rat cerebellum and spinal cord

By means of light microscopic autoradiography, binding sites for the GABA catabolite [3H]gamma-hydroxybutyrate (GHB) were observed on cultured cerebellar and spinal neurones but not on glial cells. [3H]GHB was bound to similar types of neurones as [3H]GABA. However, the number of neurones labelled by [3H]GHB was considerably smaller than that by [3H]GABA, and the intensity of labelling by [3H]GHB was usually weaker. It is suggested that GHB might interact with neurotransmission mediated by GABA or play a role as neuromodulator or neurotransmitter in the mammalian central nervous system.     

Autoradiographic localization of binding sites for [3H]gamma-aminobutyrate, [3H]muscimol,(+)[3H]bicuculline methiodide and [3H] flunitrazepam in cultures of rat cerebellum and spinal cord

Cultures of rat cerebellum and spinal cord were used to visualize binding sites for [³Hγaminobutyrate, [³Hmuscimol, [³Hbicuculline methiodide and [³Hflunitrazepam by autoradiography. In cerebellar cultures, many large neurons (presumably Purkinje cells) and interneurons were labelled. In spinal cord cultures, these compounds were mainly bound to small and medium-sized neurons, whereas the majority of large neurons were unlabelled. No binding sites for these radioligands were found on glial cells. Binding of [³Hγ-aminobutyrate, [³Hmuscimol and [³Hbicuculline methiodide was markedly reduced or inhibited by adding unlabelled γ-aminobutyrate, muscimol and bicuculline (10^?3m) respectively to the incubation medium. Addition of a thienobenzazepine markedly reduced binding with [³Hflunitrazepam.It is concluded that tissue cultures are an excellent tool to visualize the cellular localization of binding sites for neurotransmitters and drugs using autoradiography.     

Glycine enhances [3H]noradrenaline release from slices of rat hippocampus

The effects of glycine on the release of [3H]noradrenaline ([3H]NA) have been investigated using rat hippocampus slices previously labeled with the radioactive catecholamine. Glycine (30-1000 microM) produced a potent and concentration-dependent increase of the release of [3H]NA evoked by 12 mM KCl. The effect of 1000 microM glycine was totally antagonized by 3 microM strychnine suggesting that, in rat hippocampus, glycine may regulate the release of NA by activating a receptor of the strychnine-sensitive subtype.     

Inhibition of [3H] glutamate release from rat hippocampal slices by L-phenylisopropyladenosine

Hippocampal slices were labelled with [3H]glutamate. Evoked release, which was calcium-dependent and inhibited by tetrodotoxin, could be reduced concentration-dependently by L-PIA. The effect of L-PIA was blocked by 10 microM 8-phenyltheophylline. The results indicate that there are adenosine receptors modulating the efflux of glutamate in field stimulated hippocampal slices.     

Stereospecific interaction of bicuculline with specific [3H]strychnine binding to rat spinal cord synaptosomal membranes

The gamma-aminobutyric acid (GABA) antagonist (+/-)-bicuculline inhibits specific [3H]strychnine binding to postsynaptic glycine receptor sites in rat spinal cord synaptosomal membranes with an inhibition constant of about 5 microM, which is fairly similar to its inhibition constant reported for the GABA receptor. This effect is highly stereospecific, since the affinity of (-)-bicuculline for the specific [3H]strychnine binding sites is more than ten times less than that of the pharmacologically active (+)-bicuculline. Besides an unspecific effect at the glycine receptor, the results could suggest that the glycine and the GABA receptors are located close together in spinal cord membranes, so that the antagonist states of both receptors may be able to interfere with each other in some mechanistic way.     

Synergism between A(2A)-adenosine receptor activation and vasoactive intestinal peptide to facilitate [3H]-acetylcholine release from the rat motor nerve terminals

We report positron emission tomographic measurements of regional cerebral blood flow (rCBF) in a male patient with war and torture related post-traumatic stress disorder (PTSD) during symptom provocation. The subject was exposed to war related sounds before and after treatment with a selective serotonin reuptake inhibitor (SSRI; Fluoxetine; Fontex((R))). Therapy reduced PTSD symptoms, provoked anxiety and heart rate. Before treatment trauma reminders resulted in decreased rCBF in the insula, prefrontal, and inferior frontal cortices. Increased activity was evident in the cerebellum, precuneus and supplementary motor cortex. This was normalized after SSRI administration. Prefrontal and cingulate rCBF correlated with heart rate. Hence, the anxiolytic effect of SSRI for PTSD could be mediated by prefrontal and paralimbic cortices. Data suggest that SSRI treatment normalize provocation induced rCBF alterations in areas involved in memory, emotion, attention and motor-control.     

Paleocerebellar stimulation induces in vivo release of endogenously synthesized [3H]dopamine and [3H]norepinephrine from rat caudal dorsomedial nucleus accumbens

The influence of cerebellar vermis stimulation on noradrenergic and dopaminergic activity in the nucleus accumbens was investigated in anesthetised rat. Artificial cerebral spinal fluid containing [3H]tyrosine was continuously circulated through a unilateral push-pull cannula implanted in the nucleus accumbens. Fifteen-minute perfusate samples were collected serially for three consecutive 1-h periods designated pre-, during-, and post-stimulation. The stimulation was applied through a bilateral electrode located subdurally over the fifth vermal lobe. The [3H]norepinephrine and [3H]dopamine components in each sample were isolated by alumina extraction and high-pressure liquid chromatographic fractionation, and then quantified by liquid scintillation counting. For cannula locations in the caudal dorsomedial nucleus accumbens, levels of both [3H]catecholamines were found to be significantly higher during stimulation compared to the prestimulation baselines, and [3H]norepinephrine remained significantly elevated through the post-stimulation period. The relative increase during stimulation for [3H]norepinephrine (130%) was nearly twice that for [3H]dopamine (70%). These results indicate that vermal activation can significantly raise both noradrenergic and dopaminergic in vivo activity in the caudal dorsomedial nucleus accumbens, and provide a possible mechanism for explaining previously demonstrated influences of paleocerebellum upon affective components of behavior.     

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.     

The influence of felodipine and diethyl-stilboestrol on [3H]noradrenaline release in the rat trachea

The fractional release of [3H]noradrenaline was measured in rings from the rat trachea. Transmitter release was induced either by isotonic KCl (124 mM) or by 2.5 mM Ca added to a Ca-free depolarizing solution. Felodipine, a Ca antagonist (10(-4)M), had no effect on the basal release of [3H]NA whereas it reduced the overflow of transmitter caused by KCl or Ca. Diethyl-stilboestrol (25 microM), which also inhibits Ca influx in smooth muscle, had a stronger inhibitory effect than felodipine on [3H]NA release induced by high K and addition of Ca. Basal efflux of [3H]NA was increased by diethyl-stilboestrol. This increase also occurred in the absence of extracellular Ca.     

Uptake and release of [3H]GABA by growth cones isolated from neonatal rat brain

A subcellular fraction highly enriched in neuronal growth cones was isolated from 5-day-old rat forebrain by a recently described method. The growth cone fraction was shown to have a sodium- and temperature-dependent, high-affinity (Km = 4.4 microM) uptake system for [3H]GABA. Electron microscopic autoradiography confirmed that this uptake was into growth cones since only these structures were heavily labelled with silver grains. High potassium induced the release of newly accumulated [3H]GABA from the growth cone fraction, about half of which was Ca2+-dependent. The presence of uptake and release systems for GABA in growth cones may simply reflect the development of growth cones into nerve terminals. Alternatively, these observations may indicate a role for neurotransmitter release in synaptogenesis.     

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.     

Retrograde labeling of neurons in the brain stem following injections of [3H]choline into the rat spinal cord

In an attempt to identify cholinergic neurons in the brain stem which project to the spinal cord, [3H]choline (100, 20, 10, 5 or 1 microCi) was injected into the upper cervical spinal cord in 55 rats. The animals were killed 20 h later and the brains processed for autoradiography of diffusible substances. At all doses of [3H]choline, cells were consistently, retrogradely labeled in the medical medullary reticular formation, the lateral vestibular nucleus, the dorsolateral pontine tegmentum and the red nucleus. The retrogradely labeled cells were found to be moderately to darkly stained for acetylcholinesterase. Injection of [3H]noradrenaline (50 microCi) into the upper cervical spinal cord resulted in retrograde labeling of cells in the locus coeruleus, subcoeruleus and the ventrolateral pontine tegmentum, that correspond in position to the neurons of the A6, A7 and A5 catecholamine cell groups, respectively. Injection of [3H]serotonin (20 microCi) into the upper cervical spinal cord was associated with retrograde labeling of cells in the raphe pallidus, obscurus and magnus nuclei that correspond in position to those of the B1, B2 and B3 serotonin cell groups, respectively. Injection of True Blue into the upper cervical spinal cord was followed by retrograde labeling of a large number of cells located in the areas where cells were retrogradely labeled by [3H]choline, [3H]noradrenaline and [3H]serotonin, and additionally, in the solitary tract nucleus, the lateral, parvicellular medullary reticular formation, the caudal and oral pontine reticular formation, the mesencephalic reticular formation and the superior colliculus. These results indicate that from the cervical spinal cord, [3H]choline selectively retrogradely labels a certain population of non-monaminergic, acetylcholinesterase-positive cells localized in the medial medullary, and secondarily the dorsolateral pontine, reticular formation, the lateral vestibular nucleus, and the red nucleus.     

Immunoreactive somatostatin release from rat spinal cord in vitro.

A calcium-dependent release of immunoreactive somatostatin from rat spinal cord in vitro in response to two depolarising stimuli (60 mM KCl and 75 micrometer veratrine) has been demonstrated. Released somatostatin immunoreactivity comprised 0.53% of total tissue content, showed parallelism when serial dilutions were compared to the immunoassay dose-response curve and eluted similarly to synthetic somatostatin on Sephadex G-25 (f) chromatography. These results provide further evidence for a neurotransmitter or neuromodulator role for somatostatin in mammalian spinal cord.     

Cholecystokinin octapeptide-evoked [3H]acetylcholine release from guinea pig gallbladder

Cholecystokinin-octapeptide (CCK-OP) produced a contractile response in isolated guinea pig gallbladder; the response consisted of scopolamine-sensitive and scopolamine-insensitive components, neither of which were affected by tetrodotoxin or hexamethonium. In the presence of tetrodotoxin, CCK-OP evoked [3H]acetylcholine (ACh) release from strips of gallbladder in a concentration-dependent manner. These results suggest that CCK-OP acts at two sites in the guinea pig gallbladder, viz. the smooth muscle cell and the postganglionic cholinergic nerve terminal.