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ABSTRACT: To investigate whether epileptiform activity in the immature brain is modulated by dopamine, we examined the effects of dopaminergic agonists and antagonists in an intact in vitro preparation of the isolated corticohippocampal formation of immature (postnatal days 3 and 4) C57/Bl6 mice using field potential recordings from CA3. Epileptiform discharges were induced by a reduction of the extracellular Mg(2+) concentration to 0.2 mM. These experiments revealed that low concentrations of dopamine (<0.3 μM) attenuated epileptiform activity, whereas >3 μM dopamine enhanced epileptiform activity. The D1-agonist SKF38393 (10 μM) had a strong proconvulsive effect, and the D2-like agonist quinpirole (10 μM) mediated a weak anticonvulsive effect. The proconvulsive effect of 10 μM dopamine was completely abolished by the D1-like receptor antagonist SCH39166 (2 μM) or the D2-like antagonist sulpiride (10 μM), whereas the D2 antagonist L-741626 (50 nM) and the D3 antagonist SB-277011-A (0.1 μM) were without effect. The anticonvulsive effect of 0.1 μM dopamine could be suppressed by D1-like, D2, or D3 receptor antagonists. A proconvulsive effect of 10 μM dopamine was also observed when AMPA, NMDA, or GABA(A) receptors were blocked. In summary, these results suggest that 1) dopamine influences epileptiform activity already at early developmental stages; 2) dopamine can bidirectionally influence the excitability; 3) D1-like receptors mediate the proconvulsive effect of high dopamine concentrations, although the pharmacology of the anticonvulsive effect is less clear; and 4) dopamine-induced alterations in GABAergic and glutamatergic systems may contribute to this effect.
Journal of Neuroscience Research 06/2012; 90(10):2020-33. · 2.74 Impact Factor
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ABSTRACT: 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.
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.
The GABAergic antagonists gabazine (3 μm) and picrotoxin (100 μm) induced epileptiform discharges, whereas activation of GABA(A) receptors attenuated epileptiform discharges. Under low-Mg(2+) 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.
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.
Epilepsia 03/2012; 53(5):888-96. · 3.96 Impact Factor
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ABSTRACT: In immature neurons activation of ionotropic GABA receptors induces depolarizing membrane responses due to a high intracellular Cl(-) concentration ([Cl(-)](i)). However, it is difficult to draw conclusions about the functional consequences of subthreshold GABAergic depolarizations, since GABAergic membrane shunting and additional effects on voltage-dependent ion channels or action potential threshold must be considered. To systematically investigate factors that determine the GABAergic effect on neuronal excitability we performed whole cell patch-clamp recordings from Cajal-Retzius cells in immature rat neocortex, using [Cl(-)](i) between 10 and 50 mM. The effect of focal GABA application was quantified by measuring various parameters of GABAergic responses including the shift in minimal threshold current (rheobase). The rheobase shift was correlated with other parameters of the GABAergic responses by multiple linear regression analyses with a set of simple mathematical models. Our experiments demonstrate that focal GABA application induces heterogeneous rheobase shifts in Cajal-Retzius cells that could not be predicted reliably from [Cl(-)](i) or the GABAergic membrane depolarization. Implementation of a linear mathematical model, which takes the GABAergic membrane conductance and the difference between action potential threshold and GABA reversal potential into account, resulted in a close correlation between calculated and experimentally obtained rheobase shifts. Addition of a linear term proportional to the GABAergic membrane depolarization improved the accuracy of correlation. The main advantage of using multiple linear regression with simple models is that direction and strength of GABAergic excitability shifts can be analyzed by using only measured parameters of GABAergic responses and with minimal a priori information about cellular parameters.
Journal of Neurophysiology 07/2011; 106(4):2034-44. · 3.32 Impact Factor
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ABSTRACT: To unravel the functional implications of activity-dependent Cl- changes during early stages of neuronal development, we determined which changes in the GABA reversal potential (E (GABA)) and GABAergic rheobase shifts were induced by episodes of GABA(A) receptor activation using gramicidin-perforated patch-clamp recordings from Cajal-Retzius cells in tangential cortical slices of newborn mice. Under this condition, focal application of the GABA(A) agonist muscimol (10 μM) depolarized the membrane by 15 ± 0.8 mV (n = 35). Such subthreshold GABAergic depolarizations considerably reduced the rheobase, corresponding to an excitatory action. After repetitive focal muscimol applications (50 pulses at 0.5 Hz) a significant reduction of E (GABA) and an attenuation of the excitatory GABAergic rheobase shift were observed, while the GABAergic membrane conductance and the absolute value of the rheobase were unaltered after the muscimol pulses. Bath application of 100 μM carbachol induced bursts of spontaneous GABAergic postsynaptic potentials. Both, E (GABA) and the excitatory GABAergic rheobase shift was significantly reduced after such barrage of carbachol-induced GABAergic postsynaptic potentials, while neither the GABAergic membrane conductance nor the absolute value of the rheobase was affected under this condition. Both results indicate that GABAergic activity itself can limit the excitatory effects of GABA(A) receptor activation, which supports the hypothesis that the low capacity of the Cl- homeostasis in immature neurons could be a substrate for synaptic scaling and homeostatic plasticity.
Pflügers Archiv - European Journal of Physiology 02/2011; 461(5):557-65. · 4.46 Impact Factor
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ABSTRACT: The action of diuretic furosemide on the GABA(A) receptor was studied in acutely isolated Purkinje cells using the whole-cell recording and fast application system. Furosemide blocked stationary component of GABA-activated currents in a concentration-dependent manner with IC(50) value > 5 mM at -70 mV. The inhibition was rapid in the onset, fully reversible and did not require drug pre-perfusion. The termination of GABA and furosemide co-application was followed by transient increase in the inward current 'tail' current, which was not observed when furosemide was continuously present in the solution. The degree of furosemide block did not depend on GABA concentration. Furosemide block increased with membrane depolarization. Five millimolar furosemide depressed GABA currents by 32.4+/-1.3% at -70 mV and by 76.7+/-5.0% at +70 mV. Analysis of the voltage dependence of the block suggests that furosemide binds at the site located within GABA(A) channel pore with a dissociation constant of 5.3+/-0.5 mM at 0 mV and electric distance of 0.27. Our results provide evidence that furosemide interacts with Purkinje cell GABA(A) receptors (most probably composed of alpha1beta2/3gamma2 subunits) through a low affinity site located in channel pore and suggest that furosemide acts as a sequential open channel blocker, which prevents the dissociation of agonist while the channel is blocked.
Neuropharmacology 06/2002; 42(7):913-21. · 4.81 Impact Factor
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ABSTRACT: The action of diuretic furosemide on the GABAA receptor was studied in acutely isolated Purkinje cells using the whole-cell recording and fast application system. Furosemide blocked stationary component of GABA-activated currents in a concentration-dependent manner with IC50 value > 5 mM at −70 mV. The inhibition was rapid in the onset, fully reversible and did not require drug pre-perfusion. The termination of GABA and furosemide co-application was followed by transient increase in the inward current ‘tail’ current, which was not observed when furosemide was continuously present in the solution. The degree of furosemide block did not depend on GABA concentration. Furosemide block increased with membrane depolarization. Five millimolar furosemide depressed GABA currents by 32.4±1.3% at –70 mV and by 76.7±5.0% at +70 mV. Analysis of the voltage dependence of the block suggests that furosemide binds at the site located within GABAA channel pore with a dissociation constant of 5.3±0.5 mM at 0 mV and electric distance of 0.27. Our results provide evidence that furosemide interacts with Purkinje cell GABAA receptors (most probably composed of α1β2/3γ2 subunits) through a low affinity site located in channel pore and suggest that furosemide acts as a sequential open channel blocker, which prevents the dissociation of agonist while the channel is blocked.
Neuropharmacology.
