Intrinsic activation of GABAA receptors suppresses epileptiform activity in the cerebral cortex of immature mice

Purpose: Activation of ionotropic γ‐aminobutyric acid type A (GABA_A) receptors induces in immature neocortical neurons a membrane depolarization that may contribute to the higher epilepsy susceptibility in newborns. To elucidate whether depolarizing GABAergic responses enhance or attenuate epileptiform activity in the immature neocortex, we investigated the effect of agonists, antagonists, and positive modulators of GABA_A receptors on epileptiform activity. Methods: We performed in vitro field potential recordings on isolated whole neocortex preparations and whole cell recordings of identified pyramidal neurons in 400‐μm slices of immature (postnatal day 1–7) mice. Epileptiform activity was induced by low Mg²⁺ solutions with or without 50–100 μm 4‐aminopyridine. Results: Bath application of GABA (3–100 μm , in the presence of tiagabine) attenuated epileptiform activity. The GABA transporter isoform 1 (GAT‐1) inhibitor tiagabine (30 μm ) and the GAT‐2/3 specific inhibitor SNAP 5114 (40 μm ) reduced the frequency of epileptiform activity. The benzodiazepines midazolam (0.2 μm ) and zolpidem (0.5 μm ) as well as the barbiturate phenobarbital (30 μm ) slightly attenuated epileptiform activity. Continuous bath application of the GABAergic antagonist gabazine (SR‐95531, 2–3 μm ) or picrotoxin (15 μm ) induced epileptiform discharges. Discussion: These results demonstrate, that (1) the activation or positive modulation of GABA_A receptors attenuates epileptiform activity, (2) GABA_A antagonists mediate a disinhibition, and (3) GABA uptake contributes to the regulation of extracellular GABA in immature neocortex. We conclude from these findings that a constant inhibition via GABA_A receptors is required to suppress epileptiform activity already in the immature neocortex.     

Phasic GABAA -receptor activation is required to suppress epileptiform activity in the CA3 region of the immature rat hippocampus

Purpose: Despite the consistent observation that γ‐aminobutyric acid A (GABA_A) receptors mediate excitatory responses at perinatal stages, the role of the GABAergic system in the generation of neonatal epileptiform activity remains controversial. Therefore, we analyzed whether tonic and phasic GABAergic transmission had differential effects on neuronal excitability during early development. Methods: We performed whole cell patch‐clamp and field potential recordings in the CA3 region of hippocampal slices from immature (postnatal day 4–7) rats to analyze the effect of specific antagonists and modulators of tonic and phasic GABAergic components on neuronal excitability. Key Findings: The GABAergic antagonists gabazine (3 μm ) and picrotoxin (100 μm ) induced epileptiform discharges, whereas activation of GABA_A receptors attenuated epileptiform discharges. Under low‐Mg²⁺ conditions, 100 nm gabazine and 1 μm picrotoxin were sufficient to provoke epileptiform activity in 63.2% (n = 19) and 53.8% (n = 26) of the slices, respectively. Whole‐cell patch‐clamp experiments revealed that these concentrations significantly reduced the amplitude of phasic GABAergic postsynaptic currents but had no effect on tonic currents. In contrast, 1‐μm 4,5,6,7‐tetrahydroisoxaz‐olo[5,4‐c]‐pyridin‐3‐ol (THIP) induced a tonic current of ?12 ± 2.5 pA (n = 6) and provoked epileptiform discharges in 57% (n = 21) of the slices. Significance: We conclude from these results that in the early postnatal rat hippocampus a constant phasic synaptic activity is required to control excitability and prevent epileptiform activity, whereas tonic GABAergic currents can mediate excitatory responses. Pharmacologic intervention at comparable human developmental stages should consider these ambivalent GABAergic actions.     

Mechanisms underlying the neuropathological consequences of epileptic activity in the rat hippocampus in vitro

Blockade of γ-aminobutyric acid (GABA)ergic synaptic transmission in mature hippocampal slice cultures for a period of 3 days with convulsants was shown previously to induce chronic epileptiform activity and to mimic many of the degenerative changes observed in the hippocampi of epileptic humans. The cellular mechanisms underlying the induction of this degeneration were examined in the present study by comparing the effects of GABA blockers with the effects produced by the K⁺ channel blocker tetraethylammonium (2 mM). Both types of convulsant caused a comparable decrease in the number of Nissl-stained pyramidal cells in areas CA1 and CA3. No significant cell loss was induced by tetraethylammonium when epileptiform discharge was reduced by simultaneous exposure of cultures to tetrodotoxin (0.5 μM) or to the anticonvulsants pentobarbital (50 μM) or tiagabine (50 μM). We conclude that this degeneration was mediated by convulsant-induced epileptiform discharge itself. The hypothesis that N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity underlies cell death in this model was tested by applying convulsants together with specific antagonists of glutamate receptors. Whereas coapplication of antagonists of both non-NMDA and NMDA receptors strongly reduced the degeneration induced by the convulsants, application of either class of antagonist alone did not. Application of exogenous NMDA produced potent cell death, and this degeneration was blocked by the NMDA receptor antagonist methyl-10,11-dihydro-5-H-dibenzocyclohepten-5,10-imine (MK-801). Convulsants also induced a loss of dendritic spines that could be partially prevented by NMDA or non-NMDA receptor antagonists. We conclude that NMDA receptor activation is not solely responsible for the neuronal pathology resulting as a consequence of epileptiform discharge. © 1996 Wiley-Liss, Inc.     

Dopaminergic modulation of the caudal photoreceptor in crayfish

In our study we investigated the influence of dopamine (DA) on the caudal photoreceptor (CPR) in crayfish. Here we report the following: (a) the chromatographic determination of DA in the sixth abdominal ganglion (6th AG) shows a variation in the content during a 24‐h cycle with the maximum value at dawn. (b) There are possibly dopaminergic neurons in the 6th AG with antityrosine hydroxylase antibodies. Immunopositive neurons (164) were located in the anterior and posterior regions of the 6th AG with the mean (± SE) diameter of their somata 23 ± 1 μm. In addition, there is immunopositive staining in axons, neuropilar fibers, and varicosities. (c) We also identified, using immunohistochemistry, 108 neurons in the sixth AG that contain dopamine D1‐like receptors, with the mean (±SE) diameter of their somata 18 ± 1 μm. (d) We examined the exogenous action of DA on the electrical activity of the CPR in the isolated sixth AG by conventional extracellular‐recording methods. This CPR displays spontaneous activity and phasic‐tonic responses to light pulses. Topical application of dopamine to ganglia kept in the dark increased the spontaneous firing rate of the CPR, whereas the photoresponse of the CPR remained unchanged. The effect on the spontaneous activity is dose‐dependent with an ED₅₀ of 33 μM, and is blocked by the dopamine D1‐like antagonist SCH23390. These observations suggested that the DA is playing the role of a neurotransmitter or a neuromodulator of the CPR in the 6th AG in both species of crayfish, Procambarus clarkii and Cherax quadricarinatus. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

Stimulation of glutamine synthetase activity by excitatory amino acids in astrocyte cultures derived from aged mouse cerebral hemispheres may be associated with non-n-methyl-d-aspartate receptor activation

We have been using glial cells derived from aged mouse cerebral hemispheres (MACH) at several passages to study the responsiveness of astrocytes to microenvironmental signals in culture. In the present study, we examined the effects of excitatory amino acids on the activity of glutamine synthetase, a marker for astrocytes. MACH glia cell passages 25 to 29 were used. Culture groups were Dulbecco's modified Eagle's medium +10% fetal bovine serum (control); glutamate 100 μM; γ-amino-3-hydroxy-5-methyl isoxazole-4-propionic acid (AMPA) 50 μM; kainic acid 10 μM; N-methyl-d-aspartate (NMDA) 10 μM. In all treated groups glutamine synthetase activity was significantly higher than in controls. We speculate that this increase represents an enhanced differentiation of immature astrocytes. In a second series, we examined the effects of glutamate receptor antagonists on glutamine synthetase activity as follows. MACH cultures were treated with glutamate 100 μM in combination with either L(+)-2-amino-3-phosphonopropionic acid (L-AP3; 50 μM); D(−)-2-amino-5-phosphonopentanoic acid (D-AP5; 50 μM) or 6,7-dinitroquinoxaline-2,3-dione (DNQX; 50 μM). The increase in GS activity produced by glutamate was exhibited by the non-selective NMDA receptor antagonist, DNQX, but not by the metabotropic receptor antagonist, L-AP3 or a selective NMDA receptor antagonist, D-AP5. We also found that in cultures treated with glutamate, a number of astrocytes resembled “reactive astrocytes” morphologically. These astrocytes were absent in cultures treated with glutamate + DNQX. The findings provide supportive evidence that astrocytes from aged mouse cerebral hemispheres respond to excitatory amino acids and that this response is mediated by non-NMDA receptor activation.     

Involvement of GABAB receptors in convulsant‐induced epileptiform activity in rat neocortex in vitro

The role of γ-aminobutyric acid B (GABA_B) receptors in the generation and maintenance of bicuculline-induced epileptiform activity in rat neocortical slices was studied using electrophysiological methods. A block of GABA_B receptors in the presence of functional GABA_A receptor-mediated inhibition was not sufficient to induce epileptiform activity. In the presence of the GABA_A receptor antagonist bicuculline (10 μm ) and at suprathreshold stimulation, the GABA_B receptor antagonist CGP 35348 (10–300 μm ) significantly potentiated epileptiform activity. With stimulation at threshold intensity, low concentrations of CGP 35348 (10–30 μm ) potentiated bicuculline-induced activity, whereas higher concentrations (100–300 μm ) invariably led to a reversible suppression of stimulus-evoked epileptiform discharges. CGP 35348 also enhanced picrotoxin-induced epileptiform activity, but at higher concentrations it was considerably less effective in suppressing such epileptiform discharges. The GABA uptake inhibitor nipecotic acid partially mimicked the actions of CGP 35348: with stimulation at threshold intensity, it reversibly suppressed bicuculline-induced epileptiform field potentials, but it did not influence epileptiform activity induced by picrotoxin. We conclude that a postsynaptic blockade of GABA_B receptors induces an amplification of epileptiform activity in neocortical slices disinhibited by GABA_A receptor antagonists. An additional blockade of presynaptic GABA_B receptors, especially under conditions of weak stimulation of the neurons, reduces the inhibitory auto-feedback control of GABA release, leading to a displacement of competitive antagonists from the postsynaptic GABA_A receptor and hence, to a suppression of epileptiform activity induced by competitive GABA_A receptor antagonists.     

Dopamine D2-like receptor agonists induce penile erection in male rats: differential role of D2, D3 and D4 receptors in the paraventricular nucleus of the hypothalamus

Pramipexole, a dopamine D3/D2 receptor agonist, induces penile erection when administered subcutaneously (s.c.) or into the paraventricular nucleus of the hypothalamus of male rats, like apomorphine, a mixed D1/D2 receptor agonist, and PD 168,077, a D4 receptor agonist. A U-inverted dose-response curve was found with pramipexole and apomorphine, but not with PD 168,077 (0.025-0.5 mg/kg s.c.). Pramipexole effect was abolished by L-741,626, a D2 receptor antagonist (2.5 and 5 mg/kg s.c.) and raclopride, a D2/D3 receptor antagonist (0.025 and 0.1 mg/kg s.c.), but not by SB277011A (2.5 and 10 mg/kg s.c.) or FAUC 365 (1 and 2 mg/kg s.c.), two D3 receptor antagonists, or L-745,870 (1 and 5 mg/kg i.p.), a D4 receptor antagonist. Similar results were found with apomorphine (0.08 mg/kg s.c.), although its effect was also partially reduced by L-745,870. In contrast, PD 168,077 effect was abolished by L-745,870, but not L-741,626, SB277011A, FAUC 365 or raclopride. Similar results were found when dopamine agonists (5-200 ng/rat) and antagonists (1-5 μg/rat) were injected into the paraventricular nucleus. However, no U-inverted dose-response curve was found with any of the three dopamine agonists injected into this nucleus. As pramipexole- and apomorphine-induced penile erection was reduced mainly by D2, but not D3 or D4 antagonists, D2 receptors are those that mediate the pro-erectile effect of these dopamine agonists. Although the selective stimulation of paraventricular D4 receptors induces penile erection, D4 receptors seem to play only a modest role in the pro-erectile effect of the above dopamine agonists.     

Evidence for dopaminergic innervation on kitten retinal ganglion cells

Effects of iontophoretically applied dopamine and its receptor antagonists on physiologically identified retinal ganglion cells were studied in the optically intact eye of pentobarbitone anaesthetised kittens (7-9 weeks of age) and the results were compared with the effects obtained from adult cats (18-22 weeks of age). In both the adult and the kitten, dopamine had an inhibitory effect on visually evoked and spontaneous activity of the retinal ganglion cells, irrespective of cell type. However, unlike in the adult, the effects of dopamine in kittens were variable and not dependent on retinal eccentricity. In adult cells, only L-sulpiride (a potent D2 receptor antagonist) reduced the inhibitory effect of exogenous dopamine, whereas in kitten cells, both alpha-flupenthixol (a potent D1 receptor antagonist) and L-sulpiride did so. When applied alone, neither alpha-flupenthixol nor affected the activity of ganglion cells in adults, but in kittens both antagonists produced an excitatory effect in some cells. Physiologically active dopaminergic innervation seemed, therefore, to be present on the immature ganglion cells, but was subsequently 'eliminated' during the course of postnatal development. Furthermore, in immature cells, both D1 and D2 type receptors are present but only D2 receptors remain in adult. Therefore, there is a mismatch between dopamine receptors and dopamine in the adult retina and this mismatch may be the consequence of a developmental event.     

Study of auto- and heteroreceptor components of the presynaptic dopamine reuptake modulation in the mechanism of the in vitro action of the novel antiparkinsonian drug hemantane

The effect of Hemantane, a new 2-aminoadamantane derivative (N-adamant-2-ylhexamethylenimine hydrochloride) with antiparkinsonian activity on [³H]-dopamine ([³H]-DA) uptake and binding by D1, D2, and D3 dopamine and NMDA glutamate receptors was studied in comparison with the clinically used drug Amantadine. The method of radioligand binding to rat striatal membrane preparations was used. Both drugs, when used within a concentration range of 10^?11 to 10^?3 M did not affect the[G-³H]-SCH23390 and [G-³H]-Spiperone binding by D1 and D2 receptors. However, at micromolar concentrations (>10^?5 M), Hemantane and Amantadine inhibited the binding of the D3 receptor ligand 7-OH-[G-³H]-DPAT with IC₅₀ values of 39 and 360 μM, respectively; i.e., Hemantane is almost one order of magnitude more efficient. Both preparations exhibited a similar effect on NMDA receptors: the semiinhibition constants IC₅₀ were 5.5 μM for Hemantane and 4 μM for Amantadine. Hemantane and Amantadine were shown to reproducibly inhibit the reuptake of [³H]-dopamine at concentrations of 100–500 μM. The study of inhibition kinetics demonstrated the noncompetitive character of the action: Hemantane decreased the B _max value from 9.0 (control) to 5.1 pmol of [³H]-DA per min/mg of protein (p < 0.05), whereas K _m value remained constant (0.5 μM), which is characteristic of the noncompetitive type of inhibition. The (±)CPP and MK-801 antagonists of NMDA receptors inhibited the reuptake of [³H]-DA with IC₅₀ of 6 and 38 μM, respectively; NMDA (1, 10, and 100 μM) had no effect; and quisqualate, an agonist of nonNMDA receptors, moderately (?37%, p < 0.05) inhibited dopamine transport at 100 μM. These data seem to indicate that the mechanism of increase of dopaminergic transfer under the action of adamantane derivatives could involve noncompetitive inhibition of dopamine transport.     

The effects of intracerebroventricular AM-251, a CB1-receptor antagonist, and ACEA, a CB1-receptor agonist, on penicillin-induced epileptiform activity in rats

Purpose: Several results support the conclusion that the cannabinoid system has a role in generation and cessation of epileptic seizures. The aim of this study was to evaluate the effects of intracerebroventricular AM‐251 [N‐(piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide], a CB1‐receptor antagonist, and ACEA (arachidonyl‐2‐chloroethylamide), a CB1‐receptor agonist, on penicillin‐induced epileptiform activity in rats. Methods: In the first set of experiments, 30 min after penicillin injection, AM‐251, at doses of 0.125, 0.25, 0.5, and 1 μg, was administered intracerebroventricularly (i.c.v.). In the second set of experiments, 30 min after penicillin injection, ACEA, at doses of 2.5, 5, 7.5, and 15 μg (i.c.v.), was administered. In the third set of experiments, AM‐251, at doses of 0.125 and 0.25 μg (i.c.v.), was administered 10 min before ACEA (7.5 μg, i.c.v.) injection. Results: ACEA, at a dose of 7.5 μg, significantly decreased the frequency of penicillin‐induced epileptiform activity without changing the amplitude. ACEA, at doses of 2.5, 5, and 15 μg, had no impact on either frequency or amplitude of epileptiform activity. AM‐251, at doses of 0.25 and 0.50 μg, significantly increased the frequency of epileptiform activity. AM‐251, at a dose of 0.25 μg (i.c.v.), was the most effective in changing the frequency of penicillin‐induced epileptiform activity, and it also caused status epilepticus–like activity. AM‐251, at doses of 0.125 and 0.25 μg, 10 min before ACEA (7.5 μg), reversed the anticonvulsant action of ACEA. Discussion: The results of the present study provide electrophysiologic evidence for the role of CB1 receptors in regulating the frequency of epileptiform activity in the model of penicillin‐induced epilepsy. To elucidate the precise mechanism of cannabinoid action in the brain during seizure, more advanced electrophysiologic and neurochemical studies are required.     

Interactions of Dopamine with Glutamate- and GABA-mediated Synaptic Transmission in the Rat Entorhinal Cortex In Vitro

We have studied the interactions between dopamine and glutamate-mediated transmission in the entorhinal cortex using intracellular recording in a slice preparation from the rat brain. High concentrations (0.1 – 1 mM) of dopamine had weak, direct effects on the membrane potential with predominantly hyperpolarizing responses in layer II neurons and depolarizing responses in layer V. Studies with the dopamine antagonists sulpiride (D2 antagonist, 10 – 50 μM) and SCH-23390 (D1 antagonist, 50 μM) indicated that the hyperpolarization by dopamine could be mediated by D2 receptors, although the pharmacology was not clear-cut. The depolarizing response was not affected by either D1 or D2 antagonists. Synaptic responses of layer II and layer V cells were complex, consisting of both inhibitory and excitatory potentials. In untreated slices, dopamine reduced all components of the synaptic responses. However, when components of the responses were pharmacologically isolated, only the excitatory, glutamate-mediated potentials were consistently affected and the GABAergic inhibitory potentials were more resistant to reduction by dopamine. Excitatory potentials mediated by both N -methyl- d -aspartate and α-amino-3-hydroxy-5-methyl-isoxazolepropionic acid receptors were reduced by dopamine, but the former were more strongly affected. Studies with antagonists suggested that the D1 receptor is more likely to be involved in the decrement of glutamate transmission. Thus, dopamine appears to modulate glutamate-mediated synaptic transmission in the entorhinal cortex, and it is conceivable that a disturbance in this interaction could be involved in the aetiology of schizophrenia.     

Modulation of muscarinic facilitation of epileptiform discharges in immature rat neocortex

We examined the cholinergic effects on epileptiform discharge generation in immature (postnatal days 10-20) rat neocortex. Evoked and spontaneous field potentials were recorded from the deep layers of neocortical slices during GABA(A) receptor blockade by bicuculline methiodide (BMI, 50 microM). The anticholinesterase eserine (10 microM) as well as the ACh-analog carbamylcholine chloride (CCh, 25 microM) decreased the amplitude and duration of evoked field potentials and in parallel, increased significantly the rate of occurrence of spontaneous discharges. These effects were reversed by the muscarinic antagonist atropine (2.5 microM, n = 20), but not by the nicotinic receptor antagonist hexamethonium (50 microM, n = 3). The M1 subtype-selective muscarinic antagonist pirenzepine (1 microM, n = 12) blocked spontaneous discharges in 8/12 slices, while muscarinic antagonists of the M2 (AFDX 116 n = 4), M3 (4-DAMP n = 4) and M4 (gallamine n = 5, tropicamide n = 6) type, all at 1 microM, only reduced their frequency. CCh-induced spontaneous discharges were blocked by the combination of the glutamate receptor antagonists AP5 and CNQX (both at 10 microM; n = 11). Gap junction blockers abolished them (halothane, n = 7) or reduced their frequency by 65% (carbenoxolone, n = 8). Inhibiting Ca2+ release from intracellular stores by dantrolene (100 microM, n = 5) or thapsigargin (1 microM, n = 5) also depressed their frequencies by 55-65%. By contrast, their rates were not altered by perfusion with high Ca2+ (7 mM; n = 6) medium, a manipulation suppressing polysynaptic connections. These findings demonstrate that activation of muscarinic receptors, notably of the M1 type, in immature rat neocortex facilitates the generation of glutamatergic epileptiform discharges. These discharges are strongly inhibited by gap junction blockers, and are also partly mediated by the, presumably muscarinic receptor-dependent, mobilization of intracellular calcium.     

Dopamine-nociceptin/orphanin FQ interactions in the substantia nigra reticulata of hemiparkinsonian rats: involvement of D2/D3 receptors and impact on nigro-thalamic neurons and motor activity

Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor antagonists proved to be effective in alleviating experimental parkinsonism. Nonetheless, loss of effectiveness or even worsening of parkinsonian symptoms have been observed at high doses. With the aim of clarifying the circuitry underlying the dual action of NOP receptor antagonists and the role of endogenous dopamine, the NOP receptor antagonist 1-benzyl-N-[3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl]pyrrolidine-2-carboxamide (Compound 24) and the D₂/D₃ receptor antagonist raclopride were used in 6-hydroxydopamine hemilesioned rats. Systemically administered Compound 24 improved motor activity in the 0.1–10 mg/kg dose range being ineffective at 30 mg/kg. To confirm NOP selectivity, Compound 24 improved motor performance in wild-type mice at 1 and 10 mg/kg and inhibited it at 60 mg/kg, being ineffective in NOP receptor knockout mice. To prove that the bell-shaped profile was mediated by nigral NOP receptors, reverse dialysis of Compound 24 (0.03 μM) in substantia nigra reticulata ameliorated akinesia whereas Compound 24 (3 μM) was ineffective. To demonstrate that motor responses were mediated by tuning inhibitory and excitatory inputs to nigro-thalamic neurons, the low concentration elevated GABA and reduced glutamate in substantia nigra, simultaneously reducing GABA levels in ventro-medial thalamus. Conversely, the higher concentration reduced nigral and elevated thalamic GABA, without affecting nigral glutamate levels. Co-perfusion with raclopride (1 μM) abolished the antiakinetic action of Compound 24 (0.03 μM) and turned the ineffectiveness of Compound 24 (3 μM) into an antiakinetic effect. The low concentration reduced nigral but did not affect thalamic GABA whereas the higher concentration elevated nigral and reduced thalamic GABA. Neither concentration affected nigral glutamate. We conclude that dual motor effects of Compound 24 in hemiparkinsonian rats are accomplished through blockade of nigral NOP receptors resulting in opposite modulation of nigro-thalamic neurons. Endogenous dopamine contributes to these responses affecting the level of GABAergic inhibition of the nigral output via D₂/D₃ receptors.     

The effects of ethanol intake and withdrawal on penicillin-induced epileptiform activity in rats

Previous experiments have shown that ethanol may have either pro-convulsive or anti-convulsive effects on epileptic activity in different experimental epilepsy models. In this study, the effect of low dose ethanol and its withdrawal on penicillin-induced epileptiform activity in rat was investigated. Eight groups of adult, male Wistar rats were studied: (1) control, (2) penicillin pretreated (500 units), (3) alpha-tocopherol (500 mg/kg, i.m.), (4) penicillin pretreated+alpha-tocopherol, (5) ethanol-treated (3g/kg, per day, for 15 days, intragastrically)+penicillin, (6) ethanol-treated+penicillin+alpha-tocopherol, (7) ethanol withdrawal+penicillin, (8) ethanol withdrawal+penicillin+alpha-tocopherol. Each animal group was composed of seven rats. The epileptiform activity was verified by electrocorticographic (ECoG) recordings. The epileptiform activity was induced by microinjection of penicillin into the left sensorimotor cortex. Administration of ethanol (3g/kg, per day, for 15 days, intragastrically) did not change either frequency or amplitude of penicillin-induced epileptiform activity. The frequency and amplitude of epileptiform activity were evaluated 40 h after the last ethanol administration in withdrawal groups. There was no significant change in the mean frequency and amplitude of epileptiform activity compared with penicillin pretreated and ethanol-treated groups. The effective dose of alpha-tocopherol (500 mg/kg, i.m.) significantly decreased the mean frequency of epileptiform activity in the 60, 70, and 120 min after alpha-tocopherol injection in penicillin pretreated, ethanol-treated, ethanol withdrawal groups, respectively. However, alpha-tocopherol did not affect the amplitude of epileptiform activity in all groups. In conclusion, the present results indicate that low dose of ethanol does not have either anticonvulsive or proconvulsive effect on penicillin-induced epileptiform activity. alpha-Tocopherol has anti-convulsive effect on penicillin-induced epileptiform activity in effective dose.     

NK3 receptors mediate an increase in firing rate of midbrain dopamine neurons of the rat and the guinea pig

This in vitro study investigates and compares the effects of NK3 receptor ligands on the firing rate of rat and guinea pig midbrain dopamine neurons. The findings are discussed in the light of choosing suitable animal models for investigating pharmacological properties of NK3 receptor antagonists, which have been proposed to possess therapeutic activity in neuropsychiatric diseases like e.g. schizophrenia. In vitro midbrain slice preparations of both species were used to record (extracellularly) the firing rates of dopamine neurons located in the substantia nigra (SN) and ventral tegmental area (VTA). Furthermore, the effect of the D2 receptor agonist quinpirole on guinea pig SN and VTA dopamine neurons was investigated. The efficacy of quinpirole in inhibiting guinea pig dopamine neuron firing activity was much less as compared to that of rat dopamine neurons, suggesting a lower dopamine D2 autoreceptor density on the guinea pig neurons. The NK3 receptor agonist senktide induced in subpopulations of rat SN (55%) and VTA (79%) and guinea pig SN (50%) and VTA (21%) dopamine neurons an increase in firing rate. In responsive neurons this effect was concentration‐dependent with EC₅₀ values of 3–5 nM (for both species). The selective NK3 receptor antagonist osanetant (100 nM) was able to partly block the senktide‐induced increase in firing rates of dopamine neurons and shifted the concentration‐response relation curves for senktide to the right (pA₂ values were ～7.5). The fractional block of the senktide responses by osanetant appeared to be larger in guinea pig dopamine neurons, indicating that osanetant is a more potent blocker of NK3 receptor‐mediated responses with noncompetitive properties in the guinea pig. Synapse 2011. © 2011 Wiley‐Liss, Inc.     

Antiepileptic drugs abolish ictal but not interictal epileptiform discharges in vitro

Purpose: We established the effects of the antiepileptic drugs (AEDs) carbamazepine (CBZ), topiramate (TPM), and valproic acid (VPA) on the epileptiform activity induced by 4‐aminopyridine (4AP) in the rat entorhinal cortex (EC) in an in vitro brain slice preparation. Methods: Brain slices were obtained from Sprague‐Dawley rats (200–250 g). Field and intracellular recordings were made from the EC during bath application of 4AP (50 μm ). AEDs, and in some experiments, picrotoxin were bath applied concomitantly. Results: Prolonged (>3 s), ictal‐like epileptiform events were abolished by CBZ (50 μm ), TPM (50 μm ), and VPA (1 mm ), whereas shorter (<3 s) interictal‐like discharges continued to occur, even at concentrations up to 4‐fold as high. γ‐Aminobutyric acid (GABA)_A–receptor antagonism changed the 4AP‐induced activity into recurrent interictal‐like events that were not affected by CBZ or TPM, even at the highest concentrations. To establish whether these findings reflected the temporal features of the epileptiform discharges, we tested CBZ and TPM on 4AP‐induced epileptiform activity driven by stimuli delivered at 100‐, 10‐, and 5‐s intervals; these AEDs reduced ictal‐like responses to stimuli at 100‐s intervals at nearly therapeutic concentrations, but did not influence shorter interictal‐like events elicited by stimuli delivered every 10 or 5 s. Conclusions: We conclude that the AED ability to control epileptiform synchronization in vitro depends mainly on activity‐dependent characteristics such as discharge duration. Our data are in keeping with clinical evidence indicating that interictal activity is unaffected by AED levels that are effective to stop seizures.     

Dopamine D2High receptors stimulated by phencyclidines,lysergic acid diethylamide,salvinorin A,and modafinil

Although it is commonly stated that phencyclidine is an antagonist at ionotropic glutamate receptors, there has been little measure of its potency on other receptors in brain tissue. Although we previously reported that phencyclidine stimulated cloned‐dopamine D2Long and D2Short receptors, others reported that phencyclidine did not stimulate D2 receptors in homogenates of rat brain striatum. This study, therefore, examined whether phencyclidine and other hallucinogens and psychostimulants could stimulate the incorporation of [³⁵S]GTP‐γ‐S into D2 receptors in homogenates of rat brain striatum, using the same conditions as previously used to study the cloned D2 receptors. Using 10 μM dopamine to define 100% stimulation, phencyclidine elicited a maximum incorporation of 46% in rat striata, with a half‐maximum concentration of 70 nM for phencyclidine, when compared with 80 nM for dopamine, 89 nM for salvinorin A (48 nM for D2Long), 105 nM for lysergic acid diethylamide (LSD), 120 nM for R‐modafinil, 710 nM for dizocilpine, 1030 nM for ketamine, and >10,000 nM for S‐modafinil. These compounds also inhibited the binding of the D2‐selective ligand [³H]domperidone. The incorporation was inhibited by the presence of 200 μM guanylylimidodiphosphate and also by D2 blockade, using 10 μM S‐sulpiride, but not by D1 blockade with 10 μM SCH23390. Hypertonic buffer containing 150 mM NaCl inhibited the stimulation by phencyclidine, which may explain negative results by others. It is concluded that phencyclidine and other psychostimulants and hallucinogens can stimulate dopamine D2 receptors at concentrations related to their behavioral actions. Synapse 63:698–704, 2009. © 2009 Wiley‐Liss, Inc.     

Muscarinic receptor blockade in the ventral tegmental area attenuates cocaine enhancement of laterodorsal tegmentum stimulation‐evoked accumbens dopamine efflux in the mouse

The reinforcing properties of cocaine have been related to increased extracellular concentrations of dopamine in the nucleus accumbens (NAc). M5 muscarinic acetylcholine receptors (mAChRs) on dopamine cells in the ventral tegmental area (VTA) facilitate mesoaccumbens dopamine transmission and are critically involved in mediating natural and drug reinforcement. We investigated the effects of pharmacological blockade of mAChRs in the VTA on cocaine's ability to enhance electrically evoked NAc dopamine efflux. Using fixed potential amperometry together with carbon fiber recording microelectrodes positioned in the NAc core, we quantified dopamine oxidation currents (dopamine efflux) evoked by brief stimulation (15 monophasic pulses at 50 Hz every 30 s) of the laterodorsal tegmentum (LDT) in urethane (1.5 g/kg, i.p.) anesthetized mice. Compared to predrug baseline responses, cocaine (5 or 10 mg/kg, i.p.) dose‐dependently enhanced LDT stimulation‐evoked NAc dopamine efflux, whereas the nonsubtype selective mAChR antagonist scopolamine (10 μg/0.5 μl) microinfused into the VTA diminished LDT‐evoked NAc dopamine efflux. Preinfusion of scopolamine into the VTA diminished the facilitatory actions of cocaine on LDT stimulation‐evoked NAc dopamine efflux, and when infused at the peak effect of cocaine attenuated LDT‐evoked dopamine efflux to below predrug baseline levels. These findings suggest that LDT cholinergic inputs to dopamine neurons in the VTA, via activation of mAChRs (probably of the M5 subtype), are involved in modulating the facilitatory effects of cocaine on NAc dopamine neurotransmission. They also suggest that the development of antagonists aimed at selectively disrupting M5 receptor function may be valuable in reducing abuse liability of psychostimulants. Synapse 64:216–223, 2010. © 2009 Wiley‐Liss, Inc.     

Involvement of D1 and D2 Dopamine Receptors in the Control of Horizontal Cell Electrical Coupling in the Turtle Retina

We studied the actions of D1 and D2 dopamine agonists and antagonists on the coupling of horizontal cell axons in the turtle retina by a combination of pharmacological and electrophysiological methods. Both D1 and D2 receptors were identified in membrane fractions by radioligand binding using [3H]-SCH 23390 and [3H]-spiperone, respectively. The KD of both receptor classes were identical (0.21 nM) but D1 receptor density exceeded that of D2 receptors by more than four-fold. D1 agonists increased the activity of adenylate cyclase in a dose-dependent manner, whereas D2 agonists were without significant effect by themselves, nor did D2 antagonists block the D1-mediated increase in adenylate cyclase activity. Intracellular recordings and Lucifer Yellow dye injections were used to characterize the modifications of the receptive field profile of horizontal cell axons (H1AT) exposed to different pharmacological agents. Dopamine or D1 agonists (0.05 - 10 microM) induced a marked constriction of the H1AT receptive field, whereas D2 agonists elicited a small expansion of the receptive field. However, in the presence of a D1 antagonist, as well as IBMX to inhibit phosphodiesterase, D2 agonists (10 - 70 microM) induced a marked increase in the receptive field profile. These results indicate that both D1 and D2 dopamine receptors play a role in shaping the receptive field profile of the horizontal cell axon terminal in the turtle retina.     

Inhibition of neuronal activity in rat hippocampal slices by Aconitum alkaloids

The structurally related Aconitum alkaloids aconitine, lappaconitine, and 6-benzoylheteratisine inhibited the orthodromic and antidromic population spike in hippocampal CA1 area in a frequency-dependent manner. Aconitine (1 μM) completely suppressed epileptiform activity induced by omission of Mg²⁺ as well as normal neuronal activity, whereas lappaconitine (10 μM) and 6-benzoylheteratisine (10 μM) diminished epileptiform activity by sparing normal neuronal activity.