Effect of interleukin-6 and tumor necrosis factor-alpha on GABA release from mediobasal hypothalamus and posterior pituitary

The release of cytokines during infection, inflammation and stress induces brain-mediated responses, including alterations of neuroendocrine functions. We examined the effect of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on release of gamma-aminobutyric acid (GABA) from mediobasal hypothalamic (MBH) explants and posterior pituitaries (PP) of male rats. IL-6 (10 ng/ml) did not modify basal GABA release from MBH and PP, but significantly increased GABA release under depolarizing conditions (40 mM K(+)). This effect was abolished by incubation of the tissue with indomethacin, an inhibitor of cyclooxygenase activity, indicating that prostaglandins could mediate the stimulation of GABA release induced by IL-6. On the contrary, TNF-alpha (50 ng/ml) significantly decreased K(+)-evoked GABA release from both MBH and PP. This inhibitory effect was not modified by indomethacin. Neither IL-6 nor TNF-alpha affected nitric oxide synthesis, as measured by [(14)C]citrulline production. The current results indicate that IL-6 stimulates GABA release from both hypothalamus and posterior pituitary by a mechanism mediated by prostaglandins. On the contrary, TNF-alpha inhibits GABA release from both tissues. These results suggest the possibility that GABAergic activity in the hypothalamic-pituitary axis could be involved in neuroendocrine responses to cytokines.     

Luteinizing hormone-releasing hormone and oxytocin response to hyperosmotic stimulation: in vitro study

It was shown previously that luteinizing hormone-releasing hormone (LHRH) affects the neurohypophysial oxytocin release in water-deprived rats. However, the detailed mechanisms by which LHRH modifies the oxytocin response to hyperosmotic stimulation have not been explained so far. Using the isolated hypothalamo-neurohypophysial explants obtained from euhydrated rats, the effect of LHRH on the oxytocin secretion was studied under conditions of direct osmotic (i.e., Na(+)- evoked) as well as nonosmotic (i.e., K(+)-evoked) stimulation. Additionally, the oxytocin response to LHRH was investigated using the explants obtained from animals drinking 2% saline for eight days (systemic, i. e., both direct and indirect, osmotic stimulation). LHRH significantly enhanced Na(+)- and K(+)-evoked oxytocin release from explants taken from rats drinking tap water, indicating that LHRH could affect the Na(+)/K(+)-dependent depolarization of perikarya of oxytocin neurones. In contrast, LHRH significantly diminished the K(+)-stimulated hormone release when the neurohypophysial complex was obtained from previously salt-loaded rats, suggesting that peripheral osmotic stimulation somehow modifies the sensitivity of oxytocinergic neurones to LHRH (possible mechanisms are discussed). It is concluded that LHRH may participate in the regulation of oxytocin secretion via both direct and indirect impact on magnocellular oxytocinergic neurones depending on the current functional status of the hypothalamo-neurohypophysial complex.     

Effects of ions and ionic channel activators or blockers on release of alpha-MSH from perifused rat hypothalamic slices.

The involvement of sodium and chloride ions in the process of alpha-melanocyte-stimulating hormone (a-MSH) release from hypothalamic neurons was investigated using perifused rat hypothalamic slices. Three different stimuli were found to increase a-MSH release from hypothalamic slices: high K+ concentration (50 mM), veratridine (50 microM), and the Na+/K(+)-ATPase inhibitor ouabain (1 mM). Spontaneous or K(+)-evoked a-MSH release was insensitive to the specific Na+ channel blocker tetrodotoxin (TTX; 1.5 microM) and to the blocker of K+ channels tetraethylammonium (TEA; 30 mM) or 4-aminopyridine (4-AP; 4 mM). In contrast, blockage of ouabain-sensitive Na+/K(+)-ATPase increased the resting level of a-MSH and caused a dramatic potentiation of K(+)-evoked a-MSH release. The Na+ channel activator veratridine (50 microM) triggered a-MSH release. This stimulatory effect was blocked by TTX and prolonged by TEA application, indicating the occurrence of voltage-sensitive Na+ and K+ channels on a-MSH neurons. Replacement of Na+ by impermeant choline ions from 95 to 60 mM did not alter K(+)-evoked a-MSH release. Conversely, dramatic reduction of the external Na+ concentration to 16 mM caused a robust increase of a-MSH secretion from hypothalamic neurons, likely through activation of the Na+/Ca2+ exchange system. These data indicate that the depolarizing effect of K+ results from direct activation of voltage-operated Ca2+ channels. The lack of effect of TEA on basal a-MSH release prompted us to investigate the possible involvement of chloride ions in the regulation of the spontaneous activity of a-MSH neurons. Substitution of Cl- for impermeant acetate ions did not affect basal or K(+)-evoked a-MSH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melatonin inhibits the substance P-induced secretion of vasopressin and oxytocin from the rat hypothalamo-neurohypophysial system: in vitro studies

The aim of the present investigations was to study the influence of substance P (a member of a family of peptides known as tachykinins) on basal and K(+)-evoked vasopressin (AVP) and oxytocin (OT) release from rat hypothalamo-neurohypophysial system in vitro as well as to determine whether this effect of substance P is sensitive to melatonin.The present results show that substance P stimulates basal AVP and OT release from isolated hypothalamo-neurohypophysial system, when used at the concentrations of 10(-6) and 10(-7)M/l. At the concentration of 10(-9)M/l, however, substance P was found to stimulate the in vitro secretion of AVP, but not that of OT. Melatonin diminished basal release of AVP; it also significantly inhibited the substance P-stimulated secretion of AVP and OT. K(+)-evoked release of the neurohypophysial hormones was not further modified by either substance P or melatonin.The present results show that the stimulatory effect of substance P on basal release of AVP and OT from rat hypothalamo-neurohypophysial system in vitro is sensitive to inhibitory influence of melatonin.     

NO and angiotensin II effects on blood pressure and fluid homeostasis

Angiotensin II (50 ng/5 microl) and L-NAME (250 microg/5 microl), an inhibitor of NO synthase (NOS), were administered intracerebroventricularly alone or in combination to conscious rats. Mean arterial blood pressure (MABP) increased reaching a peak within 5 min in all groups compared to controls treated with the vehicle, artificial CSF (5 microl). MABP returned to basal levels at 30 min after angiotensin II and remained stable for the following 90 min. In animals treated with L-NAME alone, after the initial pressor response, MABP declined but began to increase progressively from 30 min until the end of the experiment at 120 min. When administered with angiotensin II, however, the initial pressor response was prolonged. Angiotensin II-induced drinking was significantly attenuated by L-NAME. In control rats, inhibiting NOS elevated plasma levels of oxytocin and vasopressin but in angiotensin II-stimulated animals, only oxytocin was further elevated after L-NAME. Thus, NO formed centrally inhibits basal secretion of oxytocin and vasopressin as well as the resting blood pressure. During stimulation with angiotensin II, NO facilitates drinking, limits the pressor response and selectively inhibits oxytocin release.     

Nitric oxide in the potassium-induced response of the rat middle cerebral artery: a possible permissive role

In the middle cerebral artery (MCA), the presence of nitric oxide (NO) is responsible for maintaining a more dilated state than in its absence during increases in extracellular K(+) and osmolality. The purpose of the present study was to determine whether the involvement of NO was due to (a) a direct effect of the K(+)/osmolality (K(hyper)) on the endothelium or (b) a 'permissive' role of NO. MCAs (approximately 210 microm o.d.) were isolated, cannulated with glass micropipettes, and pressurized to 85 mmHg. When K(+) (KCl) in the extraluminal bath was increased to 21 mM, the diameter increased by 15-20% with the magnitude of dilation diminishing with further increases in K(hyper). The addition of N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-5) mM), an inhibitor of nitric oxide synthase, had no significant effect on dilations at lower K(hyper) concentrations but constricted the arteries relative to the control at 51, 66, and 81 mM K(hyper). In the presence of L-NAME, the addition of an exogenous NO donor, S-nitroso-N-acetylpenicillamine (SNAP, 10(-8) M) or an analog of cGMP, 8-bromo-cGMP (6x10(-5) M), tended to restore the response of K(hyper)to near the original response. We conclude that the basal release of NO from the endothelium plays a permissive role in the K(hyper)-induced response.     

Clonidine inhibition of potassium-evoked release of glutamate and aspartate from rat cortical synaptosomes

Release of endogenous glutamic acid (Glu), aspartic acid (Asp) and gamma-aminobutyric acid (GABA) has been investigated using synaptosomes prepared from rat cerebral cortex. Exposure in superfusion to a depolarizing concentration of KCl (30 mM) evoked 3-, 2- and 2-fold increases in Glu, Asp and GABA release, respectively. More than 70% of Glu and Asp overflow were calcium-dependent, although 67% of the GABA overflow was calcium-independent. Clonidine inhibited the K(+)-evoked overflow of Glu and Asp in a concentration-dependent manner, but the GABA overflow was not inhibited. Clonidine inhibited K(+)-evoked Glu and Asp overflow to 40 and 30% of the control with a potency (IC50) of 11 and 36 nM, respectively. Similarly, norepinephrine inhibited the K(+)-evoked overflow of Glu and Asp, although phenylephrine and isoproterenol showed no effect. Rauwolscine, yohimbine and idazoxan counteracted the effects of clonidine on Glu and Asp overflow. The data suggest that the depolarization-evoked overflow of excitatory amino acids is regulated in an inhibitory fashion by alpha 2 adrenoceptors, which are located on the nerve terminals of Glu and Asp neurons in rat cortex.     

Modulation of prodynorphin peptides release from the rat spinal cord in vitro

The release of immunoreactive (ir-) dynorphin (DYN) and alpha-neoendorphin (alpha-NEO) from spinal cord slices was investigated in rats. A stable, spontaneous, in vitro release of these peptides (6.7 +/- 0.3 of ir-DYN and 15.5 +/- 0.3 fmol/min/g wet tissue of ir-alpha-NEO) was measured in superfusates using highly sensitive radioimmunoassays. The exposure of the slices to the superfusion medium containing 57 mM K+ or 50 microM veratridine increased circa three times the basal release of the peptides. The K(+)-evoked release of ir-alpha-NEO was Ca2(+)-dependent, and the veratridine stimulation was abolished by 1 microM tetrodotoxin. Modulation of the alpha-neoendorphin release from the lumbar enlargement of the rat spinal cord by various neuroactive compounds was studied in vitro. Noradrenaline (1 microM) slightly enhanced the K(+)-induced release of ir-alpha-NEO, but was without effect on the basal release. On the other hand, GABA (10 microM) and muscimol (1 microM) inhibited the K(+)-stimulated release of the peptide. The effect of muscimol was attenuated by bicuculline (10 microM). Other compounds, such as serotonin (1 microM), naloxone (1 microM), U-50, 488H and bicuculline, altered neither the basal nor the K(+)-induced release. These data indicate that both ir-DYN and ir-alpha-NEO are stored in a releasable pool in the spinal cord, which supports the concept that prodynorphin peptides can serve as neurotransmitters in this structure. Furthermore, this study suggests that the spinal cord prodynorphin system may be under an inhibitory gabaergic and an excitatory catecholaminergic control.     

Effects of zonisamide on neurotransmitter exocytosis associated with ryanodine receptors

To clarify the antiepileptic and neuroprotective actions of zonisamide (ZNS), we determined acute effects of ZNS on exocytosis of GABA and glutamate associated with ryanodine-receptor (Ryr) in rat hippocampus using microdialysis. ZNS increased basal GABA release concentration-dependently without affecting basal glutamate release; however, K(+)-evoked glutamate and GABA releases were reduced by ZNS concentration-dependently. Inhibition of Ryr reduced K(+)-evoked GABA and glutamate releases without affecting their basal releases. Ryanodine affected GABA and glutamate releases biphasic concentration-dependently: lower concentration of ryanodine increased both basal and K(+)-evoked releases of GABA and glutamate, whereas higher concentration reduced them. The therapeutically relevant concentration of ZNS inhibited ryanodine-induced GABA and glutamate releases, and abolished the inflection point in concentration-response curve for ryanodine on neurotransmitter exocytosis. These data suggest that ZNS elevates seizure threshold via enhancement of GABAergic transmission during resting stage. ZNS inhibits propagation of epileptic hyperexcitability and Ryr-associated neuronal damage during neuronal hyperexcitable stage. These demonstrations indicate that the indirect inhibition of Ryr activities by ZNS during neuronal hyperexcitability appear to be involved in the mechanisms of action of antiepileptic and neuroprotective actions of ZNS.     

Interaction between norepinephrine, oxytocin, and nitric oxide in the stimulation of gonadotropin-releasing hormone release from proestrous rat basal hypothalamus explants

In the proestrous female rat, norepinephrine, oxytocin and nitric oxide (NO) all participate in the regulation of the preovulatory gonadotropin-releasing hormone (GnRH) surge. Recent studies from our laboratory have demonstrated that oxytocin induces dose-dependent release of GnRH from proestrous basal hypothalamus explants. The present studies were undertaken to determine whether norepinephrine could also stimulate GnRH release from similar explants, to identify the receptors responsible for this effect and to investigate interactions between norepinephrine, oxytocin and NO. Norepinephrine significantly stimulated GnRH release from proestrous basal hypothalamus explants, and coadministration of the alpha(1)-adrenergic antagonist prazosin blocked this effect. Combined administration of oxytocin and norepinephrine stimulated significantly more GnRH release than either drug alone, and this stimulation was blocked by inhibition of NO synthase, or by an oxytocin receptor antagonist. NO production was measured from the same samples using a modified Griess reaction. Oxytocin, but not norepinephrine, significantly increased NO production, as did norepinephrine and oxytocin in combination. Oxytocin receptor antagonist administration attenuated the stimulation of NO production by norepinephrine/oxytocin. These results demonstrate for the first time that oxytocin and norepinephrine dramatically stimulate GnRH release from basal hypothalamus explants harvested on the afternoon of proestrus, and indicate that this involves oxytocin receptor and NO-dependent mechanisms.     

gamma-Aminobutyric acid, through GABAA receptors, inhibits the potassium-stimulated release of calcitonin gene-related peptide- but not that of substance P-like material from rat spinal cord slices.

Superfusion of slices of the dorsal zone of the lumbar enlargement with an artificial cerebrospinal fluid was used to investigate the possible modulation by GABA receptor ligands of the in vitro release of calcitonin gene-related peptide- and substance P-like materials (CGRPLM and SPLM) from the rat spinal cord. Whereas the spontaneous outflow of both peptides remained unaffected, the K+ (30 mM)-evoked overflow of CGRPLM could be partially inhibited (approx. -30%) by GABA (1 microM-0.1 mM) and muscimol (10 microM-0.1 mM) but not by baclofen (1-10 microM). Bicuculline methiodide (1 microM) completely prevented the inhibition by GABA (1 microM) and muscimol (10 microM) as expected from an action through GABAA receptors. By contrast, the K(+)-evoked SPLM overflow was altered neither by GABA nor muscimol and baclofen. These data further support that GABA exerts a presynaptic inhibitory control of (CGRP-containing) primary afferent fibres within the rat dorsal horn.     

Histamine H1 receptor-mediated inhibition of potassium-evoked release of 5-hydroxytryptamine from mouse forebrains.

The release of endogenous serotonin and dopamine from slices of mouse forebrains induced by high extracellular K(+) was examined in histamine H1 receptor knockout mice. The release of 5-hydroxytryptamine (5-HT) evoked by 30 mM K(+) significantly decreased in the presence of 10-50 microM histamine in wild-type mice, but was not inhibited in the mutant mice. Histamine H1 receptor-mediated inhibition of serotonin release in wild-type mice was also observed in the presence of thioperamide, an H3 antagonist. From these data, we postulate that endogenous histamine indirectly inhibits the release of 5-HT through H1 receptors in addition to H3 receptors. The treatment of 2 microM tetrodotoxin could partly abolish the effects of histamine on K(+)-evoked 5-HT release. Bicuculline, a GABA(A) antagonist, could reverse the histamine-induced inhibition of 5-HT release in wild-type mice, suggesting that H1 receptors facilitate the release of GABA, which in turn inhibits 5-HT release through GABA(A) receptors. The difference in the effects of d- and l-chlorpheniramine on K(+)-evoked 5-HT release in wild-type mice further supports the evidence of the function of H1 receptor modulating 5-HT release.     

Role of Nitric Oxide/Cyclic GMP Pathway in the Inhibitory Effect of GABA and Dopamine on Prolactin Release

The anterior pituitary gland is a site of nitric oxide (NO) production and action, suggesting a local regulatory function. We recently reported that NO inhibits in vitro prolactin release. The aim of the present study was to establish the mechanism of action of NO on prolactin release and to determine whether NO is involved in the inhibitory effect of GABA on prolactin release. Since NO exerts its action through cGMP by activating guanylate cyclase in different tissues, we examined the effect of sodium nitroprusside (NP), a NO releaser, on intrapituitary cGMP levels. Incubation of anterior pituitary glands with 0.5 mM NP 4-fold increased intrapituitary cGMP content, but decreased intrapituitary cAMP levels. In addition, we studied the effect of NP on prolactin release in the presence of LY 83583, an inhibitor of guanylate cyclase activity and 3-lsobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase activity. 10μM LY 83583 and 0.5 mM IBMX blocked the inhibitory effect of NP on prolactin release. (10^-3 M) 8Br-cGMP, an analogue of cGMP, mimicked the effect of NP on prolactin release. On the other hand, NO seems to be involved in the inhibitory effect of GABA on prolactin release since hemoglobin, a scavenger of NO, and N^w-nitro-L-arginine methyl ester, an inhibitor of NO synthase (NOS), blocked the pituitary response to GABA. Moreover, GABA (10 ⁶ M) stimulated NOS activity by almost 50%. GABA increased intrapituitary cGMP levels and decreased cAMP. Dopamine stimulated NOS activity weakly.
These observations suggest that NO, acting through the guanylate cyclase-cGMP pathway, inhibits prolactin secretion. In addition, NO may be involved in the inhibitory effect of GABA and dopamine on prolactin release.     

Brain-derived neurotrophic factor facilitates glutamate and inhibits GABA release from hippocampal synaptosomes through different mechanisms

Brain-derived neurotrophic factor (BDNF) has an acute excitatory effect on rat hippocampal synaptic transmission. To compare the action of BDNF upon the release of excitatory and inhibitory neurotransmitters in the hippocampus, we studied the effect of acutely applied BDNF on the K+-evoked glutamate and on the K+-evoked gamma-aminobutyric acid (GABA) release from rat hippocampal nerve terminals (synaptosomes). The acute application of BDNF (30-100 ng/ml) enhanced the K+-evoked [3H]glutamate release. This effect involved tyrosine-kinase B (TrkB) receptor phosphorylation and Ca2+ entry into synaptosomes through voltage-sensitive calcium channels, since it was abolished by K252a (200 nM), which prevents TrkB-mediated phosphorylation, and by CdCl2 (0.2 mM), a blocker of voltage-sensitive calcium channels. In contrast, BDNF (3-100 ng/ml) inhibited K+-evoked [3H]GABA release from hippocampal synaptosomes. This action was also mediated by phosphorylation of the TrkB receptor, but was independent of Ca2+ entry into synaptosomes through voltage-sensitive calcium channels. Blockade of transport of GABA with SKF 89976a (20 microM) prevented the inhibitory action of BDNF upon GABA release, indicating that BDNF influences the activity of GABA transporters. It is concluded that BDNF influences in an opposite way, through distinct mechanisms, the release of glutamate and the release of GABA from hippocampal synaptosomes.     

NMDA receptor-mediated control of GABA release from neurointermediate lobes of female and male rats

The effect of glutamate (GLUT) and its ionotropic receptor agonists on K(+)-evoked GABA release from the neurointermediate lobe (NIL) was investigated in diestrus, ovariectomized, ovariectomized-estrogenized female rats and intact male rats. GLUT and N-methyl-D-aspartate (NMDA) increased K(+)-evoked GABA release from the NIL in all the experimental groups. This stimulatory effect of NMDA was blocked by specific NMDA receptor antagonists but not by non-NMDA receptor antagonists. However, kainate did not modify evoked GABA release from the NIL in any of these groups. Neither GLUT nor NMDA modified nitric oxide synthase activity. These results indicate that GLUT, acting through NMDA receptors, stimulates evoked GABA release from the NIL of female and male rats. This effect is not influenced by gonadal status and does not appear to be mediated by nitric oxide production.     

Nitric oxide enhances MPP(+)-induced hydroxyl radical generation via depolarization activated nitric oxide synthase in rat striatum

We examined the effect of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, on extracellular potassium ion concentration ([K(+)](o))-enhanced hydroxyl radical (.OH) generation due to 1-methyl-4-phenylpyridinium ion (MPP(+)) was examined in the rat striatum. Rats were anesthetized, and sodium salicylate in Ringer's solution (0.5 nmol/microl per min) was infused through a microdialysis probe to detect the generation of.OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Induction of KCl (20, 70 and 140 mM) increased MPP(+)-induced.OH formation trapped as 2,3-dihydroxybenzoic acid (DHBA) in a concentration dependent manner. However, the application of L-NAME (5 mg/kg i.v.) abolished the [K(+)](o) depolarization-induced.OH formation with MPP(+). Dopamine (DA; 10 microM) also increased the levels of DHBA due to MPP(+). However, the effect of DA after application of L-NAME did not change the levels of DHBA. On the other hand, the application of allopurinol (20 mg/kg i.v., 30 min prior to study), a xanthine oxidase (XO) inhibitor was abolished the both [K(+)](o)- and DA-induced.OH generation. Moreover, when iron(II) was administered to MPP(+) then [K(+)](o) (70 mM)-pretreated animals, a marked increase in the level of DHBA. However, when corresponding experiments were performed with L-NAME-pretreated animals, the same results were obtained. Therefore, NOS activation may be no relation to Fenton-type reaction via [K(+)](o) depolarization-induced.OH generation. The present results suggest that [K(+)](o)-induced depolarization augmented MPP(+)-induced.OH formation by enhancing NO synthesis.     

Selective cross-activation/inhibition of second messenger systems and the reduction of age-related deficits in the muscarinic control of dopamine release from perifused rat striata

Possible alterations in muscarinic cholinergic (mACh) signal transduction in senescence were studied in rat neostriata. Acetylcholine (ACh) activation of striatal muscarinic heteroreceptors by carbachol or oxotremorine enhances K(+)-evoked release of dopamine from perifused striata of 6- but not 24-month-old rats. Present experiments determined the effects of simultaneous activation or activation/inhibition of more than one second messenger on K(+)-evoked release of DA from perifused striatal slices from these age groups. Combinations of carbachol (500 microns), which stimulates inositol-1,4,5-bisphosphate (IP3) production and inhibits cyclic AMP production, with oxotremorine (500 microns), which inhibits cyclic AMP production, in the presence of 30 mM KCl (in a modified Krebs-Ringer medium) reduced the age-related reduction in mAChR enhancement of DA release (analyzed by HPLC coupled to electrochemical detection; 5 min fractions were collected on ice in perchloric acid; flow rate 120 microliters/min). Combinations of these agonists with the putative second messenger arachidonic acid (10 microM), also enhanced K(+)-evoked release of DA in the striatal tissue from the 24-month group. IP3 activation was lower in the striatal tissue from old animals than those from young under all conditions, but cross-activation/inhibition actually may have lowered the IP3 threshold necessary for enhanced DA release to occur. In a subsequent experiment, pre-loading striatal tissue from young animals with either carbachol or oxotremorine under basal release conditions reduced the responding when the basal release medium was switched to one containing 30 mM KCl and combinations of the agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between carbamazepine, zonisamide and voltage-sensitive Ca2+ channel on acetylcholine release in rat frontal cortex

To clarify the mechanisms of action of antiepileptic drugs (AEDs), carbamazepine (CBZ) and zonisamide (ZNS), on exocytosis mechanisms, the present study determined the concentration-dependent action of CBZ and ZNS, as well as the interaction between these AEDs and voltage-sensitive Ca(2+) channel (VSCC) activity on basal, Ca(2+)- and K(+)-evoked acetylcholine (ACh) release in frontal cortex of freely moving rat using in vivo microdialysis. Perfusion with therapeutic-relevant concentrations of CBZ and ZNS increased basal ACh release, which was regulated by N-type VSCC predominantly and P-type VSCC weakly, whereas supratherapeutic-relevant concentrations of these AEDs reduced this release. The 3.4 mM Ca(2+)-evoked release, which was regulated by N-type VSCC selectively, but not by P-type VSCC, was increased by therapeutic-relevant concentrations of CBZ and ZNS, whereas this release was reduced by supratherapeutic-relevant concentrations of them. The 50 mM K(+)-evoked release, which was regulated by P-type VSCC predominantly and N-type VSCC weakly, was decreased by CBZ and ZNS, in a concentration-dependent manner. These findings indicate that the interplay between enhancement of basal ACh release and reduction of depolarization-related ACh release in the frontal cortex are at least partially involved in a common mechanism of antiepileptic action between CBZ and ZNS.     

Immunocytochemical and neurochemical evidence for age-related loss of GABA in the inferior colliculus: implications for neural presbycusis

The present study describes substantial, selective, age-related loss of the putative inhibitory neurotransmitter GABA in the central nucleus of the inferior colliculus (CIC) of rat based on immunocytochemical and neurochemical data. For immunocytochemistry, neurons in the CIC were immunolabeled using an antibody against a GABA conjugate in young adult (2- to 7-month-old) and aged (18- to 29-month-old) Fischer-344 rats. Computer-assisted morphometry was then used to generate maps of GABA-immunoreactive neurons in the CIC. The number of GABA-positive neurons was reduced 36% in the ventrolateral portion of the CIC of aged animals (93 neurons/mm2) compared to their matched young adult cohorts (145 neurons/mm2; p less than 0.01). For neurochemistry, basal and K(+)-evoked release of the endogenous amino acids GABA, glutamate (Glu), aspartate (Asp), and tyrosine (Tyr) from micropunches of the CIC were measured in 8 age-paired animals from the 2 age groups using high-performance liquid chromatography. Overflow of radiolabeled acetylcholine (3H-ACh) was also determined. In both age groups, K(+)-evoked release of GABA, Glu, Asp, and 3H-ACh from CIC punches was significantly enhanced above basal efflux (+200, +215, +163, and +309%, respectively), while Tyr release was unchanged. Evoked release of 3H-ACh and all amino acids except Tyr showed substantial Ca2+ dependence. A significant (p less than 0.05) age-related reduction in both basal (-35%) and K(+)-stimulated (-42%) efflux of GABA from the CIC was observed. A corresponding decrease in postrelease tissue content of GABA in CIC of aged rats was observed (-30%, p less than 0.05). In contrast, tissue content as well as basal and evoked release of Glu, Asp, Tyr, and 3H-ACh was similar between the 2 age groups. Age-related GABA neurochemical changes described in the CIC were not observed in the release of the other amino acids or 3H-ACh from either the rostral ventrolateral medulla or the somatosensory cortex, 2 brain regions involved in processing non-auditory sensory input. These data support previous findings that GABA, Glu, Asp, and ACh may subserve neurotransmission in the CIC. Additionally, these data provide clear evidence for a pronounced, region- and neurotransmitter-selective, age-related reduction of GABA in the CIC. These findings support the hypothesis that impairment of inhibitory GABAergic neurotransmission in the CIC may contribute to abnormal auditory perception and processing seen in neural presbycusis.     

Facilitation of GABA release by arachidonic acid in rat hippocampal synaptosomes.

Arachidonic acid (AA) is proposed to be a facilitatory retrograde messenger in hippocampal glutamatergic synapses. In this study, we found that AA (10 microM) increased the basal outflow (19 +/- 4%) and the K+-evoked release of [3H]GABA (38 +/- 3%) from rat hippocampal synaptosomes. This effect is likely to be a direct action of AA, as it was not mimicked by arachidic acid (10 microM) and was not modified by inhibition of either lipooxygenase with nordihydroguaiaretic acid (50 microM) or cyclooxygenase with indomethacin (100 microM). Activation of protein kinase C may be involved, as chelerythrine (6 microM), a protein kinase C inhibitor, attenuated the AA (10 microM)-facilitation of K+-evoked [3H]GABA release by 58 +/- 5%. Phospholipase A2 (2 U/mL), an enzyme that releases AA, and melittin (1 microM), a phospholipase A2 activator, mimicked the AA-facilitation of evoked [3H]GABA release (70 +/- 6% and 76 +/- 7% facilitation, respectively). These results show that exogenously added and endogenously produced AA increased basal outflow and K+-evoked release of [3H]GABA from rat hippocampal synaptosomes. Thus, AA can no longer be considered solely a facilitatory neuromodulator in the hippocampus, as this AA-facilitation of the release of the main inhibitory neurotransmitter may predominate under certain circumstances.