Modulation of net outward current in cultured neurons by angiotensin II: involvement of AT1 and AT2 receptors

In this study we have used whole-cell, voltage-clamp procedures to determine the effects of angiotensin II (AII) on net outward current (I_no) in neurons co-cultured from the hypothalamus and brainstem of 1-day-old rats. I_no is the sum of all inward and outward membrane currents (minus Na⁺, which is blocked by tetrodotoxin) which occur during the repolarization phase of the action potential. We have determined that AII elicits two separate effects on I_no in cultured neurons. AII caused a reversible and concentration (0.1 nM–10 μM)-dependent increase in I_no. This effect is inhibited by the AT₂ receptor-selective antagonists, PD123177 and PD123319 (both 100 nM), but not by the AT₁-selective receptor blocker, DuP753 (Losartan; 100 nM), and so it is mediated by AT₂ receptors. In a smaller number of neurons AII induced a reversible and concentration (0.01 nM–10 μM)-dependent decrease in I_no that was blocked by Losartan (100 nM) but not by PD123177 (100 nM). Thus the decrease in I_no is mediated by AT₁ receptors. Additionally, some neurons displayed both AT₁- and AT₂ receptor-mediated effects on I_no. Our results demonstrate two distinct actions of AII on membrane ionic currents in cultured neurons, effects that are mediated by different AII receptor subtypes.     

The AT2 angiotensin receptor subtype predominates in the 18 day gestation fetal rat brain.

The angiotensin II receptor subtype-specific antagonists Dup 753 (AT₁) and PD 123177 (AT₂) were used to characterize the angiotensin II receptor subtypes present in 18 day gestation fetal Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rat brain using in vitro receptor autoradiography. The AT₂ subtype was predominant in the brain of both rat strains, even in areas that display predominantly the AT₁ subtype in the adult rat brain.     

The role of angiotensin, AT1 and AT2 receptors in the pressor, drinking and vasopressin responses to central angiotensin.

Angiotensin II (Ang II) given centrally produces an increase in blood pressure and motivation to drink. The physiological mechanisms that mediate the pressor response include release of vasopressin (AVP) and activation of the sympathetic nervous system. Using 2 new Ang II receptor antagonists, we were able to investigate the role of AT1 or AT2 receptors in mediating these effects. Adult male Sprague-Dawley rats were cannulated in the lateral ventricle and 5 days later catheterized in the carotid artery for blood pressure measurements. All experiments were carried out in conscious rats. Three treatments were given intraventricularly (i.v.t.), in 2 microliters artificial cerebrospinal fluid (ACSF) at 30 min intervals: (1) 50 ng Ang II, (2) 0.7 micrograms AT1 antagonist Losartan or 7.0 micrograms AT2 antagonist PD123177, followed by 50 ng Ang II, and (3) 50 ng Ang II, to test for recovery. Blood pressure and drinking measurements were recorded. Also, blood samples for assay of AVP were drawn at 1 or 3 min post-injection in 2 separate groups of rats. We found that both Losartan and PD123177 significantly reduced release of AVP to Ang II 1 min post-injection. Losartan significantly blocked the pressor response (P less than 0.001), while PD123177 had no significant effect. Drinking was also antagonized by Losartan (P less than 0.05) and reduced (n.s.) by PD123177. The results suggest that the pressor response to Ang II (i.v.t.) is predominantly AT1 mediated, while the drinking and AVP responses may be mediated by both receptor subtypes.     

Cardiovascular effects produced by microinjection of angiotensins and angiotensin antagonists into the ventrolateral medulla of freely moving rats

In this study we determined the cardiovascular effects produced by microinjection of angiotensin peptides [Angiotensin-(1–7) and Angiotensin II] and angiotensin antagonists (losartan, L-158,809, CGP 42112A, Sar¹-Thr⁸-Ang II, A-779) into the rostral ventrolateral medulla of freely moving rats. Microinjection of angiotensins (12.5–50 pmol) produced pressor responses associated to variable changes in heart rate, usually tachycardia. Unexpectedly, microinjection of both AT₁ and AT₂ ligands produced pressor effects at doses that did not change blood pressure in anesthetized rats. Conversely, microinjection of Sar¹-Thr⁸-Ang II and the selective Ang-(1–7) antagonist, A-779, produced a small but significant decrease in MAP an HR. These findings suggest that angiotensins can influence the tonic activity of vasomotor neurons at the RVLM. As previously observed in anesthetized rats, our results further suggest a role for endogenous Ang-(1–7) at the RVLM. The pressor activity of the ligands for AT₁ and AT₂ angiotensin receptor subtypes at the RVLM, remains to be clarified.     

Activation of glycogen phosphorylase by serotonin and 3,4-methylenedioxymethamphetamine in astroglial-rich primary cultures: involvement of the 5-HT2A receptor

Neurotransmitters, neuropeptides, and ions regulate glycogen levels in the brain by modulating the activity of glycogen synthase (GSase) and glycogen phosphorylase (GPase). GPase is co-localized with glial fibrillary acidic protein (GFAP), an astroglia-specific marker, suggesting that glycogen is localized in astroglial cells. Additionally, functional serotonin (5-HT) receptors are found in both neurons and glia, and 5-HT is known to stimulate glycogenolysis. It is reported that 3,4-methylenedioxymethamphetamine (MDMA), a drug of abuse, stimulates the release and inhibits the reuptake of 5-HT, and selectively inhibits the activity of MAO-A. These biochemical consequences of MDMA lead to increased extra-cellular 5-HT levels. This study investigates the effects of MDMA(+) and serotonin (5-HT) on glycogen metabolism in the rat brain. A histochemical method was designed to visualize active glycogen phosphorylase (GPase) in an astroglial-rich primary culture.

Serotonin activated GPase in a concentration-dependent manner (100 nM−100 μM). Maximal activation by 5-HT was achieved by 50 μM and resulted in a 167% increase in the number of reactive sites (P < 0.001). MDMA(+) (500 nM−50 μM) directly stimulated GPase activity with maximal activation induced by 5 μM, which caused a 70% increase in the number of reactive sites (P < 0.001). The 5-HT₂ receptor agonist, 1-(2,5-dimethoxy-4-bromophenyl)-2-aminopropane (DOB), also displayed a concentration-dependent increase in the number of GPase reactive sites. Maximal stimulation by DOB occurred at 100 nM which increased the number of reactive sites by 166% (P < 0.001). These effects of 5-HT and MDMA(+) were significantly attenuated by mianserin (200 nM), a 5-HT₂ receptor antagonist.

An astrocyte-neuron metabolic link may be vital for synaptic homeostasis. By increasing 5-HT levels in the synapse, MDMA(+) may increase GPase activity and promote glycogenolysis via activation of the 5-HT₂ receptor. Prolonged GPase activity may lead to depletion of synaptic energy stores, thereby compromising the energy state of the synapse. The resulting deficiency in synaptic energy may contribute to terminal degeneration induced by substituted amphetamines.     

The antiepileptic drug losigamone decreases the persistent Na current in rat hippocampal neurons

The tetronic acid derivative losigamone is a new anticonvulsant drug with a mechanism of action that was previously unknown. The drug decreases the frequency of spontaneous action potentials and suppresses repetitive firing of neurons. Here we tested the hypothesis that losigamone suppresses the persistent Na⁺ current (I_NaP) in hippocampal neurons of rat brain slices and in cultured hippocampal neurons. Whole-cell voltage clamp recordings from neurons of juvenile rats (P15–P25) were performed with pipettes filled with Cs-gluconate or CsF. After pharmacological block of K⁺ and Ca²⁺ currents I_NaP was revealed by applying slow depolarizing voltage ramps from −70 to 0 mV. Losigamone (100–200 μM) was dissolved in DMSO (0.1%) and was applied by bath application or local pressure application. Losigamone induced a decrease in amplitude of I_NaP at depolarized membrane potentials which was reversible in cultured neurons. When tetrodotoxin (TTX) was added to the bath, I_NaP was blocked and only a residual non-specific outward cation current (I_cat) remained. Losigamone had no obvious effect on responses to voltage ramps under these conditions. Thus, losigamone did not affect I_cat or induce any additional currents. The data suggest that losigamone decreases neuronal excitability via a decrease in I_NaP.     

Dopamine D1 receptors are involved in the modulation of D2 receptors induced by cholecystokinin receptor subtypes in rat neostriatal membranes

The action of cholecystokinin octapeptide (CCK-8) on rat neostriatal dopamine (DA) D₂ receptors was evaluated in membrane binding experiments. 0.1 nM of CCK-8 inreased theK_d value of the D₂ agonist [³H]N-propylnorapomorphine (NPA) binding sites by 42%. The CCK_B antagonist PD134308 blocked this action. Kinetic analysis demostrated that this effect of CCK-8 was related to a reduction by 45% of the association rate constant of [³H]NPA. In contrast, 1 nM of CCK-8 decreased theK_H and theK_L values of DA for the D₂ antagonist [³H]raclopride binding sites by 56% and 50%, respectively. Both the CCK_A antagonist L364718 and the CCK_B antagonist PD134308 blocked this effect. The D₁ antagonist SCH23390 counteracted the CCK-8 induced decrease in theK_H and theK_L values of DA, and allowed 1 nM of CCK-8 to produce a significant increase in the IC₅₀ value of NPA for the [³H]raclopride binding sites. These results indicate that CCK-8 can reduce the affinity of the neostriatal D₂ agonist binding sites, but increase the affinity of D₂ receptors for DA. D₁ receptors may exert a switching role in the modulation of the neostriatal D₂ receptors by the CCK receptors.     

Chronic paroxetine desensitises 5-HT1D but not 5-HT1B autoreceptors in rat lateral geniculate nucleus

The present study examined the effect of chronic paroxetine (10 mg/kg p.o., 21 days) on the 5-HT_1B and 5-HT_1D autoreceptors controlling 5-HT efflux in slices of rat ventrolateral geniculate nucleus. Electrically stimulated 5-HT efflux (10 pulses, 200 Hz, 0.1 ms, 10 mA) was measured using fast cyclic voltammetry. Peak 5-HT efflux was greater (P<0.01) after chronic paroxetine (22.2±1.4 nM, mean±S.E.M.) than water (15.8±1.4 nM). 5-HT efflux was inhibited by CP 93129 (1 nM–10 μM) and sumatriptan (1 nM–1 μM), agonists at 5-HT_1B and 5-HT_1D receptors, respectively. Chronic paroxetine did not affect the sensitivity of the 5-HT_1B autoreceptor but shifted the sumatriptan concentration-response curve to the right (P<0.05). These data suggest that chronic paroxetine increases evoked 5-HT efflux. This may be the result of desensitisation of 5-HT_1D but not 5-HT_1B autoreceptors.     

Rapid changes in hippocampal CA1 pyramidal cell function via pre- as well as postsynaptic membrane mineralocorticoid receptors

Corticosterone (100 n m ) rapidly increases the frequency of miniature excitatory postsynaptic currents in mouse CA1 pyramidal neurons via membrane-located mineralocorticoid receptors (MRs). We now show that a presynaptic ERK1/2 signalling pathway mediates the nongenomic effect, as it was blocked by the MEK inhibitors U0126 (10 µ m ) and PD098059 (40 µ m ) and occluded in H-Ras ^G12V -mutant mice with constitutive activation of the ERK1/2 presynaptic pathway. Notably, the increase in mEPSC frequency was not mediated by retrograde signalling through endocannabinoids or nitric oxide, supporting presynaptic localization of the signalling pathway. Unexpectedly, corticosterone was also found to have a direct postsynaptic effect, rapidly decreasing the peak amplitude of I _A currents. This effect takes place via postsynaptic membrane MRs coupled to a G protein-mediated pathway, as the effect of corticosterone on I _A was effectively blocked by 0.5 m m GDP-β-S administered via the recording pipette into the postsynaptic cell. Taken together, these results indicate that membrane MRs mediate rapid, nongenomic effects via pre- as well as postsynaptic pathways. Through these dual pathways, high corticosterone concentrations such as occur after stress could contribute to enhanced CA1 pyramidal excitability.     

Differential effects of the adenosine A(1) receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes.

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A₁ receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [³H]cyclohexyladenosine ([³H]CHA) to A₁ receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81,723 (0.3–100 μM) produced a concentration-dependent enhancement of [³H]CHA binding to rat brain A₁ receptors. These effects were also species-dependent with larger enhancements (150–200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [³H]CHA binding to A₁ receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 μM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A₁ receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 μM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [³H]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 μM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A₁ receptor in a high-affinity state and that the relative proportions of high-affinity A₁ receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A₁ receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A₁ receptor.     

Lanthanum and zinc sensitivity of GABAA-activated currents in adult medial septum/diagonal band neurons from ethanol dependent rats

The impact of chronic ethanol treatment, sufficient to induce tolerance and physical dependence, on GABA_A receptor function was studied in acutely isolated neurons from the medial septum/nucleus diagonal band (MS/nDB) of adult rats using whole cell, patch-clamp recordings. In ethanol-naive Controls, GABA (0.3–300 μM) induced concentration-dependent increases in Cl⁻ current with a threshold of 0.3–1 μM, a mean maximal current of 7645 ± 2148 pA at 100–300 μM, an EC₅₀ of 11.3 ± 1.3 μM and a slope of 1.53 ±0.07. GABA-activated currents in neurons from animals receiving two weeks of ethanol liquid diet treatment did not differ significantly on any of these measures. The rate of GABA_A receptor desensitization (t_1/2 = 6.49 ± 1.19 s) estimated as the time required for loss of 50% of peak current during sustained application of 10 μM GABA, as well as the residual steady state current remaining following complete desensitization for controls was unchanged by chronic ethanol. The impact of chronic ethanol treatment on the GABA_A receptor modulation by lanthanum and zinc which act as positive and negative allosteric modulators, respectively, was also evaluated. Test pulses of 3 μM GABA in control neurons showed maximal potentiation by 141 ± 30% at ~ 1000 μM lanthanum with an EC₅₀ of 107 ± 34 μM and a slope of ~ 1. Lanthanum potentiation remained the same following chronic ethanol treatment. Initial estimates based on fitted concentration response curves suggested that maximal inhibition of 3 μM GABA responses by zinc at the level of 70.2 ± 8.5% in control cells was significantly increased by chronic ethanol treatment to 95.3 ± 2.5%, although the IC₅₀ of 60.2 ± 25 μM was not changed. However, this difference was not supported by direct tests of maximal 3–10 mM zinc concentrations. These results suggest that chronic ethanol treatment, sufficient to induce tolerance and physical dependence, probably does not lead to readily detectible changes in GABA_A receptor function in MS/nDB neurons.     

Currents evoked by GABA and glycine in acutely dissociated neurons from the rat medial preoptic nucleus

The responses of acutely dissociated medial preoptic neurons to application of GABA and glycine were studied using the perforated-patch whole-cell recording technique under voltage-clamp conditions. GABA, at a concentration of 1 mM, evoked outward currents in all cells (n=33) when studied at potentials positive to −80 mV. The I–V relation was roughly linear. The currents evoked by GABA were partially blocked by 25–75 μM picrotoxin and were also partially or completely blocked by 100–200 μM bicuculline. Glycine, at a concentration of 1 mM, did also evoke outward currents in all cells (n=12) when studied at potentials positive to −75 mV. The I–V relation was roughly linear. The currents evoked by glycine were largely blocked by 1 μM strychnine. In conclusion, the present work demonstrates that neurons from the medial preoptic nucleus of rat directly respond to the inhibitory transmitters GABA and glycine with currents that can be attributed to GABA_A receptors and glycine receptors respectively.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [³H]cyclohexyladenosine (CHA) to the cellular fractions and P₂ subfractions of the goldfish brain was studied. The A₁ receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P₂), 30 mM K⁺ stimulated glutamate, taurine and GABA release in a Ca²⁺-dependent fashion, whereas the aspartate release was Ca²⁺-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 μM) inhibited K⁺-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A₁ receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K⁺ (30 mM) stimulated Ca²⁺ influx (46.8±6.8%) and this increase was completely abolished by pretreatment with 100 nM ω-conotoxin. Pretreatment with 100 μM R-PIA or 100 μM CHA, reduced the evoked increase of intra-synaptosomal Ca²⁺ concentration, respectively by 37.7±4.3% and 39.7±9.0%. A possible correlation between presynaptic A₁ receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

Role of AT1 receptors in area postrema on baroreceptor reflex in spontaneously hypertensive rats

Intravenous injection of angiotensin II type 1 (AT₁) receptor antagonist improves the baroreceptor reflex gain in spontaneously hypertensive rats (SHRs). To investigate the role of area postrema in the modulation of the baroreflex control by AT₁ receptor, the effects of intravenous injection of CV-11974 (AT₁ receptor antagonist) on the baroreflex control of renal sympathetic nerve activity (RSNA) and heart rate (HR) were examined in sham and area postrema-lesioned SHRs. The baseline mean arterial pressure was similar in both groups. However, baseline heart rate was significantly lower (p<0.01) in area postrema-lesioned SHR than in sham-lesioned SHR, 307±11 and 365±10 beats/min (bpm), respectively. Intravenous CV-11974 (0.05 mg/kg) significantly decreased mean arterial pressure; however, it did not change HR and RSNA in either group. Reflex changes in RSNA and HR were elicited by intravenous infusion of either phenylephrine or sodium nitroprusside before and after intravenous injection of CV-11974. Intravenous CV-11974 increased baroreflex control of RSNA (G_max; −1.57±0.08 vs. −1.92±0.12%/mmHg, p<0.05) and HR (G_max; −0.54±0.12 vs. 1.25±0.24 bpm/mmHg, p<0.05) in sham-lesioned SHRs. However, intravenous CV-11974 failed to alter the baroreflex sensitivities in area postrema-lesioned SHRs. These results suggest that the area postrema does not play a crucial role in maintenance of high blood pressure in adult SHRs, and that the improvement of baroreflex control of RSNA and HR by intravenous CV-11974 is mediated via the area postrema in SHRs.     

Dual effects of thrombin and a 14-amino acid peptide agonist of the thrombin receptor on septal cholinergic neurons

We have compared the effects of thrombin and of the 14-amino acid peptide agonist (TRAP-14) of the thrombin protease activated receptor (PAR) on cholinergic neurons in pure cultures of rat septal neurons and in co-cultures of septal neurons and glial cells. In pure septal cultures, low concentrations of thrombin (up to 10 nM) did not affect choline acetyltransferase (ChAT) activity, a marker of cholinergic neurons, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, an index of cell viability. However, 100 nM thrombin decreased ChAT activity and MTT reduction by 44 and 17%, respectively. In co-cultures, a low concentration of thrombin (1 nM) increased ChAT activity (+75%), whereas a high concentration (100 nM) decreased it (−83%). At this high concentration, thrombin was neurotoxic, as indicated by a large decrease in MTT reduction (−80%). Thrombin effects on ChAT activity were mimicked by TRAP-14 both in pure septal cultures (no effect at 0.1 μM and −63% at 100 μM) and in co-cultures (+25% at 0.1 μM and −28% at 100 μ M). In contrast, this peptide did not affect MTT reduction. These dual effects of thrombin and TRAP-14 on ChAT activity in co-cultures, were also observed on pure cultures of septal cells supplied with NGF. The activation and inhibition by TRAP-14 of the expression of ChAT activity in septal neuron/glial cell cultures were inhibited by a 9-amino acid peptide antagonist of thrombin PAR. Thus, the effects of thrombin on cholinergic neurons seem to be mainly mediated by thrombin PAR and glial cells seem to play a major role in these thrombin actions.     

Hypothalamic cocaine-amphetamine regulated transcript (CART) is regulated by glucocorticoids

Nicorandil, a clinically useful drug for the treatment of ischemic heart disease, has an anti-apoptotic effect in cardiomyocytes, and activation of mitochondrial ATP-sensitive potassium (mitoK_ATP) channels underlies this effect. Recently, several studies showed that nicorandil reduced brain injury in animal models of brain ischemia. Based on these facts, we hypothesized that nicorandil may have anti-apoptotic effects in neurons mediated by mitoK_ATP channels. We investigated the effect of nicorandil on apoptosis induced by oxidative stress using cultured cerebellar granule neurons. Nicorandil (100 μmol/l) significantly suppressed the number of cells with TUNEL-positive nuclei and the increase in caspase-3 activity induced by 20 μmol/l H₂O₂. An indicator dye for mitochondrial inner membrane potential (ΔΨ_m) revealed that nicorandil prevented the loss of ΔΨ_m induced by H₂O₂ in a concentration-dependent manner. These effects were abolished by 5-hydroxydecanoate (5HD; 500 μmol/l), a mitoK_ATP channel blocker. The present results showed that nicorandil has anti-apoptotic effects in neurons, at least in part, by preserving ΔΨ_m.     

Presynaptic D2 dopaminergic receptors mediate inhibition of excitatory synaptic transmission in rat neostriatum

The effect of dopamine (DA) on excitatory synaptic transmission was studied in rat neostriatal neurons using intracellular- and whole-cell voltage clamp-recording methods. Depolarizing excitatory postsynaptic potentials (EPSPs) were evoked by cortical stimulation. Superfusion of DA (0.01–10 μM) reversibly decreases EPSP in a concentration-dependent manner and with a estimated IC₅ of 0.3 μM. In addition, the inhibitory effect induced by DA at a low concentratiion (0.1 μM) was antagonized by sulpiride (1–10 nM), a selective D₂ dopaminergic receptor antagonist. However, D₁ dopaminergic receptor antagonist SKF-83566 (1–5 μM) did not affect the blocking effect by DA 0.1 μM. Based on these findings, we conclude that DA at a low concentration ( 0.1 μM) reduced the excitatory response of neostriatal neurons following cortical stimulation via the activation of D₂, but not D₁ dopaminergic receptors, located on the terminals of corticostriatal neurons.     

Ethanol enhances GABAB-mediated inhibitory postsynaptic transmission on rat midbrain dopaminergic neurons by facilitating GIRK currents

It is largely accepted that an activation of the dopaminergic system underlies the recreational and convivial effects of ethanol. However, the mechanisms of action of this drug on the dopaminergic neurons are not fully understood yet. In the present study, we have used intracellular electrophysiological techniques (current and single-electrode voltage-clamp) to investigate the actions of ethanol on the γ-aminobutyric acid (GABA)_B-mediated inhibitory postsynaptic potentials (IPSPs) in rat midbrain dopaminergic neurons. Ethanol (10–200 m m ) augmented, in a concentration-dependent and reversible manner, the amplitude of the GABA_B–IPSP. In addition, the GABA_B agonist baclofen generated G-protein-gated inward rectifying K⁺ channels (GIRK)-related membrane hyperpolarizations/outward currents that were potentiated by ethanol. The potentiating effect of ethanol persisted in tetrodotoxin (TTX)-treated neurons, suggesting a postsynaptic site of action. These effects of ethanol were not changed by manipulating adenyl cyclase, protein kinases and phospholipase C activity, or by chelating intracellular Ca²⁺ with EGTA. Interestingly, the outward current caused by the intracytoplasmatic diffusion of the irreversible G-protein activator GTPγS was transiently enhanced by ethanol. Our observations suggest that the action of ethanol occurs on activated GIRK channels downstream of the GABA_B receptors. These enhancing effects of ethanol on GABA_B-induced synaptic responses could modulate alcohol intake and the altered mental and motor performance of individuals in an acute intoxicative phase.     

Nitric oxide induces an increased Na conductance in identified neurons of Aplysia

The ionic mechanism of the effects of micropressure ejections of hydroxylamine (HOA) and sodium nitroprusside (SNP), nitric oxide (NO) generators, on the membrane of identified neurons (R9–R12) of Aplysia kurodai was investigated with conventional voltage-clamp, micropressure ejection, and ion-substitution techniques. Micropressure ejection of HOA and SNP onto the neurons caused a marked depolarization in the unclamped neurons. Clamping the same neurons at their resting potential level (−60 mV) and reejecting HOA and SNP with the same dose produced a slow inward current (I_i(HOA) and I_i(SNP), 3–7 nA in amplitude, 15–60 s in duration) associated with an increase in input membrane conductance. Bath-applied hemoglobin (50 μM), a nitric oxide scavenger, almost completely blocked I_i(HOA) and I_i(SNP), and 3-isobutyl-1-methylxanthine (IBMX, 50 μM) prolonged and enhanced both I_i(HOA) and I_i(SNP). An intracellular injection of cyclic guanosine 3′,5′-monophosphate (cGMP) into the same neurons produced a slow inward current (I_i(cGMP)) which resembled the responses to HOA and SNP, and this current was enhanced in IBMX. Bath-applied methylene blue (10 μM), an inhibitor of guanylate cyclase, significantly reduced I_i(HOA) and I_i(SNP). The inward currents induced by HOA, SNP and cGMP were sensitive to changes in the external Na⁺ concentration. These results suggest that extracellular NO can induce a slow inward current associated with an increase in Na⁺ conductance, mediated by an increase in intracellular cGMP.     

Development of GABAA receptors on medial septum/diagonal band (MS/DB) neurons after postnatal ethanol exposure

The impact of ‘binge-like' ethanol exposure on postnatal days (PD) 4–9 was examined on development of γ-aminobutyric acid type A receptors (GABA_AR) during the first month of life in the rat. Whole-cell patch-clamp recordings in acutely isolated medial septum/diagonal band (MS/DB) neurons were used to define effects of rapidly applied ethanol and other allosteric modulators on bicuculline-sensitive GABA currents. Three age groups were examined including ‘pups' (PD 4–10), ‘juveniles' (PD 11–16) and ‘young adults' (PD 25–35). In untreated neurons, maximum responses to GABA and the apparent GABA EC₅₀ increased 2-fold during the first month of life. Potentiation of GABA responses by pentobarbital, midazolam, and loreclezole all increased with age, while Zn²⁺ inhibition declined. Initial inhibition by ethanol switched to potentiation of GABA responses during this time. In vivo, binge-like ethanol treatment (4.5 g kg⁻¹ day⁻¹ divided into two doses, 2 h apart on PD 4–9) reduced both the GABA maximal response and GABA EC₅₀ measured on PD 11–16. These measures returned to control levels by PD 25–35. After binge-like postnatal ethanol exposure, age-dependent loss of Zn²⁺ inhibition of GABA responses was increased, while potentiating actions of in vitro ethanol were blocked. GABA_AR modulation by other drugs was unaffected. These data suggest that early postnatal ethanol exposure disrupts the expected developmental pattern of GABA_AR function in MS/DB neurons, an action that could contribute to neurobehavioral deficits associated with the fetal alcohol syndrome. Whether these changes are due to cellular damage, delayed gene expression or post-translational modification needs to be determined.