Release of Acetylcholine and Noradrenaline from the Cholinergic and Adrenergic Afferents in Rat Hippocampal CA1, CA3 and Dentate Gyrus Regions

An attempt was made to study the release of acetylcholine (ACh) and noradrenaline and their presynaptic modulation in isolated slice preparations dissected from different subfields of the hippocampus: CA1, CA3 and the dentate gyrus. The slices were perfused and loaded with [³H]choline or with [³H]noradrenaline. The release in response to field stimulation was determined radiochemically and the content of transmitters was assayed by a chemiluminescent method or by HPLC combined with electrochemical detection. After 30 min of loading with [³H]choline there were marked subregional differences in the specific activity of [³H]ACh content. The highest concentration was measured in the dentate gyrus and the lowest in CA3. Evidence was obtained that in all three subfields the cholinergic axon terminals are equipped with inhibitory muscarinic autoreceptors and the noradrenergic terminals with α₂-autoreceptors, as indicated by an increase in transmitter release when the tissue was exposed to selective muscarinic or α₂-adrenoceptor antagonists. In contrast, the cholinergic boutons are not equipped with α₂-adrenoceptors, and noradrenergic terminals do not possess inhibitory muscarinic receptors. It is therefore concluded that while the release of both ACh and noradrenaline is controlled by negative feedback modulation, there is no possibility of establishing a presynaptic inhibitory interaction between the two.     

Voltage-dependent calcium channels in ventromedial hypothalamic neurones of postnatal rats: modulation by oestradiol and phenylephrine

Oestradiol actions in the hypothalamus play an important role in reproductive behaviour. Oestradiol treatment in vivo induces α_1b-adrenoceptor mRNA and increases the density of α_1B-adrenoceptor binding in the hypothalamus. Oestradiol is also known to modulate neuronal excitability, in some cases by modulating calcium channels. We assessed the effects of phenylephrine, an α₁-adrenergic agonist, on low-voltage-activated (LVA) and high-voltage-activated (HVA) calcium channels in ventromedial hypothalamic (VMN) neurones from vehicle- and oestradiol-treated female rats. Whole-cell and gramicidin perforated-patch recordings were obtained, with barium as the charge carrier. In the absence of phenylephrine, oestradiol treatment increased the magnitude of LVA currents compared to controls, but had no effect on HVA currents. Phenylephrine enhanced HVA currents in a significantly greater proportion of neurones from oestradiol-treated rats (76%) than from vehicle-treated (41%) rats. The L-channel blocker nifedipine abolished this oestradiol effect on phenylephrine-enhanced HVA currents. Preincubating slices with the N-type channel blocker omega-conotoxin GVIA completely blocked the phenylephrine response, suggesting that the N-type channel is essential. Phenylephrine also stimulated LVA currents in approximately two-thirds of neurones in slices from both vehicle- and oestradiol-treated rats. Our data show that oestradiol increases LVA currents in the VMN. Oestradiol also amplifies α₁-adrenergic signalling by increasing the proportion of neurones showing phenylephrine-stimulated HVA currents mediated by N- and L-type calcium channels. In this way, oestradiol may increase excitatory responses to arousing adrenergic inputs to VMN neurones governing oestradiol-dependent reproductive behaviour.     

Central α2-Adrenergic Control of Colonic Transit in Rats

The effects of central and peripheral administration of α₂-adrenoceptor agonists on colonic propulsion were examined in conscious rats chronically fitted with a catheter inserted in the lumen of the proximal colon and a cannula placed in a cerebral lateral ventricle. The mean retention time of a marker in the colon was determined by administration of a bolus of [⁵¹Cr]sodium chromate into the proximal colon and collection of the feces at hourly intervals. In control studies the colonic mean retention time was 7.5 ± 1.6 hours. Clonidine administered intraperitonedy or intracerebroventricularly produced a dose-dependent increase in mean retention time but was more potent when given intracerebroventricularly. St-91, a quaternary derivative of clonidine which poorly crosses the blood-brain barrier, delayed colonic transit after intracerebroventricular, but not after intraperitoneal, administration at the same dose. The increase in colonic mean retention time induced by intraperitonedy administered clonidine was antagonized by intracerebroventricular administration of yohimhine but not of prazosin, an α₁-adrenoceptor antagonist. Yohimbine intracerebroventricularly or intraperitonedy administered alone at the same dose accelerated the colonic transit. Such data suggest a possible role of central α₂-adrenoceptors in the regulation of colonic motility in rats.     

Functional Properties of Neuronal Nicotinic Acetylcholine Receptor Channels Expressed in Transfected Human Cells

To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)-induced single-channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat α₃β₄, chick α_<3/β₂, α₄β₂, α₄β₄, α₇and α₈, expression levels were high enough to allow measurements of acetylcholine-evoked whole-cell currents and nicotine-elicited Ca²⁺ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine-evoked events of α₈ nAChR had a slope conductance of 23 pS, whereas two conductance classes (19–23 and 32–45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric α₇ and α₈ nAChRs (6–7 ms) than for heteromeric nAChRs (1–3 ms), with the exception of α₃α₄nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (α₃α₄and α₃α₂) exhibited single-channel characteristics similar to those reported for native receptors. The variety of nAChRs channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells.     

Noradrenalin Enhances the Activity of Cochlear Nucleus Neurons in the Rat

The cochlear nucleus of rats is heavily innervated by noradrenergic fibres from the locus coeruleus. The physiological meaning of this innervation is poorly understood. Therefore, iontophoretically applied noradrenalin was tested on single neurons of the cochlear nucleus in urethane-anaesthetized rats. Iontophoresis of noradrenalin had a dual effect. During application noradrenalin led to moderate inhibition of tone-evoked activity in 37% of the tested neurons. In contrast, ∼20-30 s after the onset of iontophoresis a long-lasting increase in discharge activity was found in most neurons. Data from iontophoresis of the α₁-receptor agonist phenylephrine and the α₂-receptor agonist clonidine suggest that the fast moderate inhibition is mediated by α₂-receptors while the pronounced long-lasting elevated neuronal firing is mediated by α₁-receptors. However, these data do not exclude the possibility that part of the response to noradrenalin is also mediated by β-receptors. Electrical stimulation of the locus coeruleus resulted in an increase in discharge activity comparable with iontophoresis of noradrenalin or phenylephrine. Thus, activation of the locus coeruleus predominantly increases spontaneous and tone-evoked neuronal firing in the cochlear nucleus of the rat. This α₁-receptor-mediated enhanced discharge activity may serve to increase the sensitivity of acoustic processing mechanisms or to lower the threshold for short-latency acoustic reflexes.     

Effect of ethacrynic acid on sodium pump alpha isoforms in SH-SY5Y cells

Background:  Ethacrynic acid (ECA), a diuretic that has several cellular actions, increases expression of the sodium and potassium-activated adenosine triphosphatase (Na, K-ATPase or Na pump) in normal lymphocytes, but not in lymphocytes of bipolar patients. While this has been proposed to be important in the pathophysiology of bipolar illness, the response of neural tissues to ECA is unknown.
Methods:  Human neuroblastoma SH−SY5Y cells differentiated with 10-μM retinoic acid were treated with various ECA concentrations for 3 days, and changes in Na-pump α-isoform expression were quantified with densitometric analysis of Western bands.
Results:  Expression of α1 and α3 Na pump isoforms significantly increased with 10⁻⁵ M ECA. Cells treated with 10⁻⁶ or 10⁻⁷ M ECA showed no change in Na-pump expression, while cells treated with 10⁻⁴ M ECA died. The α2 isoform could not be detected in differentiated SH-SY5Y cells.
Conclusions:  The effect of ECA on α₁-isoform in neural tissue is similar to that observed in lymphocytes. As α3 isoform is not expressed in lymphocytes, however, we conclude that lymphocytes are an incomplete model of neural tissue.     

Noradrenaline Modulates GIutamate-mediated Neurotransmission in the Rat Basolateral Amygdala In Vitro

The entorhinal cortex and the amygdala are interconnected structures of the limbic system in which paroxysmal activity occurs during temporal lobe epilepsy. Conflicting evidence shows that noradrenaline (i) inhibits the spreading to other parts of the limbic system of paroxysmal activity generated in the amygdala or the entorhinal cortex, but also (ii) increases glutamatergic transmission in the basolateral amygdala. Given our previous work on the inhibitory effect of noradrenaline on entorhinal cortex neurons, we developed an in vitro slice preparation to study the synaptic transmission in the basolateral amygdala and its modulation by noradrenaline. Noradrenaline reduced the fast excitatory postsynaptic potential (EPSP) by ∼40% at 100 μM and the slow EPSP by ∼50% at 50 μM. A similar effect was obtained with the α₂-agonist UK 14304 at 100 and 50 μM respectively. In contrast, the β-agonist isoproterenol increased the fast EPSP by ∼40% at 100 μM and the slow EPSP by ∼20% at 50 μM. Accordingly, the effect of noradrenaline on the EPSPs was blocked by the α₂-antagonist yohimbine (10 μM) but not by the α₁-antagonist prazosine (10 μM) and the β-antagonist propranolol (10 μ M ). Noradrenaline (50–100 μM) was ineffective on most (14/16) of the isolated inhibitory postsynaptic potentials (IPSPs). These experiments provide evidence that noradrenaline inhibits the excitatory synaptic response of basolateral amygdala neurons. A pharmacological analysis revealed that the noradrenergic modulation of the excitatory transmission in the basolateral amygdaia can be dissected into a predominant α₂-adrenoreceptor-mediated inhibition and a β-adrenoreceptor-mediated excitation.     

HIV-1 Envelope Protein gp120 Potentiates NMDA-evoked Noradrenaline Release by a Direct Action at Rat Hippocampal and Cortical Noradrenergic Nerve Endings

Exposure of rat or human neocortical or hippocampal tissue to glutamate receptor agonists elicits a Ca²⁺-dependent, exocytotic-like release of previously accumulated [³H]noradrenaline through activation of both N -methyl- d -aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors colocalized on the noradrenergic axon terminals. Here we show that the NMDA (100 μM)-evoked release of [³H]noradrenaline from superfused thin layers of isolated rat hippocampal or cortical nerve endings was potentiated when the human immunodeficiency virus type 1 coat protein gp120 was added to the superfusion medium concomitantly with NMDA. The effect of gp120 (10 pM to 3 nM) on the 100 μM NMDA-evoked release of [³H]noradrenaline was concentration-dependent; the maximal effect (-140% potentiation) was reached at 100 pM of gp120. The protein was inactive on its own. The [³H]noradrenaline release evoked by NMDA (100 μM) + gp120 (100 μM) was prevented by classical NMDA receptor antagonists, as well as by 10 μM memantine. Neither the release evoked by NMDA nor that elicited by NMDA + gp120 was sensitive to the nitric oxide synthase inhibitor N ^G -nitro- l -arginine, suggesting no involvement of nitric oxide. The [³H]noradrenaline release elicited by 100 nM AMPA was unaffected by gp120. The protein potentiated the release evoked by 100 nM glutamate; the effect of 100 pM gp120 was quantitatively identical to that of 1 μM glycine, with no apparent additivity between gp120 and glycine. The antagonism by 1 μM 7-chloro-kynurenic acid of the NMDA-induced [³H]noradrenaline release was reversed by glycine or gp120. The data are compatible with gp120 acting directly as a powerful positive allosteric modulator at a neuronal NMDA receptor.     

Stimulatory Effect of Calcitonin Gene-Related Peptide on Adrenocorticotropin Release from Rat Anterior Pituitary Cells

In the present study, we examined the direct regulatory effect of rat calcitonin gene-related peptide (CGRP) on adrenocorticotropin (ACTH) release from rat cultured anterior pituitary cells. CGRP significantly increased ACTH release at concentrations of 10⁻⁸–10⁻¹¹ M. The ACTH release was gradually increased by CGRP concentrations lower than 10⁻¹⁰ M, and was decreased at concentrations higher than 10⁻⁹ M, presenting a bell-shaped dose-response curve. As well as having an additive effect on corticotropin-releasing factor-induced ACTH release, CGRP stimulated the accumulation of intracellular cAMP. The CGRP-induced ACTH release was inhibited by a protein kinase A inhibitor, suggesting that its stimulatory effect on the ACTH release was mediated via an adenylate–cyclase–protein kinase system. CGRP-like immunoreactive nerve fibers have been reported to innervate the anterior pituitary, so that the stimulatory effect of CGRP on the ACTH release suggests that this peptide may be involved in neural regulation of hormone secretion in the anterior pituitary.     

Influence of Selective α- and β-Adrenoceptor Antagonists on the Control of Motor Activity and Transmural Potential Differences in the Rabbit Ileum in Vitro

Although the sympathetic nervous system influences intestinal motility and intestinal fluid and electrolyte transport, the role of sympathetic nerves in the relationship between the two processes has not been characterized. We investigated the effect of sympathetic nerve stimulation of intestinal function by monitoring concurrently predominantly longitudinal smooth muscle activity and transmural potential difference as an on-line marker of intestinal ion transport. In a segment of rabbit small intestine in vitro, perivascular nerve stimulation inhibits spontaneous phasic motor activity and reduces transmural potential difference, which reflects enhanced absorption of fluids and electrolytes. The aim of this study was to investigate the particular adrenoceptors involved in both the smooth muscle and epithelial responses. Using the selective antagonists for the α₁-(prazonsin), α₂-(idazoxan), β₁-(atenolol), and β₂-adrenoceptors (butoxamine), we demonstrated that the enhanced fluid absorption, as shown by a fall in transmural potential difference, is α-mediated with both α₁-and α₂-adrenoceptors being involved. However, there was considerable variation with regard to the type of α-adrenoceptor subtype exerting the dominant effect. The sympathetically induced loss of spontaneous smooth muscle motor activity was found to be predominantly β₁-mediated, with the remainder of the response involving β₂-adrenoceptors.     

Linkage analysis between idiopathic generalized epilepsies and the GABAA receptor α5, β3 and γ3 subunit gene cluster on chromosome 15

Introduction - We tested the hypothesis that genetic variants within the GABA_Aα₅, β₃ and γ₃ subunit gene cluster on chromosome 15q11-q13 confer genetic susceptibility to common subtypes of idiopathic generalized epilepsy (IGE). Material and methods - Ninety-four families were selected from IGE patients with either juvenile myoclonic epilepsy (JME), juvenile (JAE) or childhood absence epilepsy (CAE). Cosegregation was tested between dinucleotide polymorphisms associated with the human GABA_Aα₅, β₃ and γ₃ subunit gene cluster and three different IGE trait models. Results - Evidence against linkage to the GABA_Aα₅, β₃ and γ₃ subunit gene cluster was found in the entire family set and subsets selected from either CAE or JAE. In 61 families of JME patients, a maximum lod score (Z_max=1.40 at θ_max=0.00) was obtained for a broad IGE spectrum ("idiopathic" generalized seizure or generalized spike and wave discharges in the electroencephalogram) assuming genetic heterogeneity (α=0.37; P =0.06) and an autosomal recessive mode of inheritance. Conclusion - The possible hint of linkage in families of JME patients emphasizes the need for further studies to determine whether a recessively inherited gene variant within the GABA_Aα₅, β₃ and γ₃ subunit gene cluster contributes to the pathogenesis of "idiopathic" generalized seizures and associated EEG abnormalities in a proportion of families.     

Neurotensin Excitation of Serotonergic Neurons in the Dorsal Raphe Nucleus of the Rat In Vitro

Neurotensin-containing terminals and radioligand binding sites are present in the dorsal raphe nucleus. The purpose of this study was to test, in brain slices containing this nucleus, the effect of neurotensin on the electrical activity of serotonergic neurons. In extracellular recordings, the cells were identified by the ability of the α₁-adrenoceptor agonist phenylephrine to induce firing, and serotonin to reduce this effect. After washout of phenylephrine, neurotensin (10 nM to 10 μM) induced a concentration-dependent increase in the firing rate of serotonergic neurons (EC₅₀= 142 nM; maximum effect ˜1 μM). The neurotensin excitation, which was mimicked by neurotensin fragments 8–13 but not neurotensin peptide fragment 1–8 and selectively blocked by SR 48692 (100 nM), was observed mainly in the ventral part of the nucleus. Most serotonergic neurons showed marked desensitization to neurotensin, even at low concentrations. The neurotensin response was occluded by supramaximal concentrations of phenylephrine. In intracellular recordings using KCl-containing electrodes, neurotensin induced an inward current associated in some cases with a decrease in apparent input conductance. In conclusion, neurotensin was found to have an excitatory action on serotonergic neurons in the ventral part of the dorsal raphe nucleus, an effect which could be subject to desensitization and was occluded by phenylephrine. This occlusion phenomenon may be important for the physiological role of neurotensin in the dorsal raphe nucleus.     

Hypothalamic corticotrophin-releasing factor and norepinephrine mediate sympathetic and cardiovascular responses to acute intracarotid injection of tumour necrosis factor-alpha in the rat

Systemic administration of tumour necrosis factor (TNF)-α induces the release of norepinephrine in the paraventricular nucleus (PVN) of hypothalamus and an increase in expression of corticotrophin-releasing factor (CRF) and CRF type 1 receptors. We explored the hypothesis that CRF and norepinephrine in PVN mediate the cardiovascular and sympathetic responses to acute systemic administration of TNF-α. In anaesthetised rats, the increases in arterial pressure and heart rate induced by intracarotid artery injection of TNF-α were attenuated by intracerebroventricular (ICV) injection of either the α₁-adrenergic antagonist prazosin or the CRF antagonist α-helical CRF. Prazosin blocked the TNF-α-induced increase in renal sympathetic nerve activity (RSNA), whereas α-helical CRF substantially reduced the RSNA response. Conversely, CRF and the α₁-adrenergic agonist phenylephrine, administered ICV, both elicited increases in PVN neuronal activity, RSNA, arterial pressure and heart rate. Microinjection of CRF and phenylephrine directly into PVN evoked smaller responses. These results are consistent with the hypothesis that norepinephrine and CRF in the PVN mediate the cardiovascular and sympathetic responses to acute systemic administration of TNF-α.     

A Comparative Study of α2- and β-Adrenoceptor Distribution in Pigeon and Chick Brain

The pharmacological properties and anatomical distribution of α₂, β₁ and β₂-adrenoceptors in pigeon and chick brains were studied by both homogenate binding and tissue section autoradiography. [³H]Bromoxidine (α₂-adrenoceptor-), [³H]CGP 12177 (β-adrenoceptor) and [¹²⁵1]cyanopindolol (β-adrenoceptor) were used as radioligands. In both species, [³H]bromoxidine binding to avian brain tissue showed a pharmacological profile similar to that previously reported for α₂-adrenoceptors in mammals. Regarding the anatomical distribution, the areas with the highest densities of α₂-adrenoceptors in the pigeon brain included the hyperstriatum, nuclei septalis, tectum opticum and some brainstem nuclei. Most β-adrenoceptors found in tissue membranes and sections from chick and pigeon brain were of the β₂ subtype, in contrast to what has been reported in the mammalian brain, where the β₁ subtype is predominant. A striking difference was found between the two species regarding the densities of these receptors: while pigeon brain was extremely rich in [¹²⁵1]cyanopindolol binding throughout the brain (mainly cerebellum) in the pigeon, the levels of labelling in the chick brain were much lower; the exception was the cerebellum, which displayed a higher density than other parts of the brain in both species. Overall, our results support the proposed anatomical equivalences between a number of structures in the avian and mammalian encephalon.     

Effect of Melatonin on the Release of Gonadotropin-Releasing Hormone and Cyclic AMP from the Rat Hypothalamus: an in vitro Study

Using an in vitro static incubation system of adult male rat hypothalami, we have studied the effect of melatonin on the release of gonadotropin-releasing hormone (GnRH) and cyclic adenosine monophosphate (cAMP). Mediobasal hypothalamus (MBH) and preoptic area (POA) were incubated separately in Minimum Essential Medium (MEM) for 6  h. The release of GnRH was measured by radioimmunoassay in the incubation medium sampled every 7.5 min. In the MBH and POA incubation medium, the mean amount of GnRH released was 8.9±1.1 and 3.4±0.6  pg GnRH/7.5 min, respectively (P<0.01). The mean number of GnRH pulses under basal conditions was 2±0.3 per 2  h in the MBH and 1.6±0.3 per 2  h in the POA (P>0.05). Melatonin (10⁻⁸  M) did not alter the release of GnRH in the presence or absence of forskolin (10⁻⁴  M). Melatonin, which was without effect on basal cAMP, inhibited forskolin-stimulated cAMP accumulation in the medium by 50% in the MBH and 40% in the POA. These results suggest that in our incubation system, melatonin does not modify GnRH release, but probably acts through the melatonin binding sites located in the hypothalamus to inhibit forskolin-stimulated cAMP.     

Activation of the GABAA Receptor Inhibits the Proliferative Effects of bFGF in Cortical Progenitor Cells

Recent studies have localized γ-aminobutyric acid (GABA)-containing neurons and identified cells that express subunits of the GABA_A receptor in the proliferative zone of the developing cerebral cortex and have demonstrated a role for GABA in cortical neurogenesis. We examined here the interactions between a number of neurotrophic factors, known to be involved in cortical cell proliferation and differentiation, and the GABAergic system (GABA and GABA_A receptors) in the regulation of cell production in dissociated cortical cell cultures. We found that basic fibroblast growth factor (bFGF) increased the number of cells labelled for the α₁ subunit of the GABA_A receptor but not for the α₂, α₃ or α₅ subunits. The α₁ subunit was expressed by the majority of proliferating neuroepithelial cells as well as by differentiated neurons. We also found that activation of the GABA_A receptor by GABA or muscimol inhibited the proliferative effects of bFGF on cortical progenitors, leading to an increased number of differentiated neurons. These results suggest that bFGF stimulates cell proliferation and GABA_A receptor expression in cultured progenitor cells of the developing neocortex, and that GABA regulates cell production by providing a feedback signal that terminates cell division.     

Comparison of Stress-induced Changes in Noradrenergic and Serotonergic Neurons in the Rat Hippocampus Using Microdialysis

The effects of stress on the serotonergic and noradrenergic projection to the hippocampus were compared in freely moving rats using microdialysis. Stress-induced changes in 5-hydroxytryptamine (5-HT), noradrenaline and their metabolites 5-hydroxyindoleacetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured in the presence of their respective uptake blockers. Local infusion of tetrodotoxin and replacement of Ca²⁺ with Cd²⁺ were used to test dependence on impulse traffic. A 5 min tail pinch or 10 min restraint stress increased 5-HT, 5-HIAA, noradrenaline and DOPAC levels. A subcutaneous saline injection produced an increase in 5-HT and DOPAC but not noradrenaline or 5-HIAA. Although α₂ adrenoceptor agonists and antagonists produced changes in the baseline values of noradrenaline and DOPAC, they had little or no effect on stress-induced changes. Both the abolition of impulse traffic and its enhancement by stress had a greater effect on transmitter than on metabolite levels. Although the responses to stress of the noradrenergic and serotonergic pathway showed many similarities, there was evidence for their activation by separate pathways.