David A. Mathers

Convulsant doses of penicillin shorten the lifetime of GABA‐induced channels in cultured central neurones

1 The influence of sodium benzylpenicillin (PCN) on membrane channels activated by γ‐aminobutyric acid (GABA) was studied in cultured spinal neurones of the mouse by the extracellular patch clamp technique. 2 In whole‐cell, current clamp recordings, concentrations of PCN above 0.2 mM significantly reduced the amplitude of the GABA response. 3 Single channel currents activated by GABA were studied in outside‐out patches of neuronal membrane. In both the absence and presence of PCN, cumulative open time distributions for GABA‐activated channels were well fitted by the sum of two exponential terms, characterized by fast (τf) and slow time constants (τs). 4 PCN (2 mM) reduced the mean value of τs from 4.29 ± 0.56 ms (mean ± s.e.mean) to 1.12 ± 0.09 ms but had no significant effect on τf. 5 The mean open time of GABA‐activated channels, calculated from the double exponential fits, decreased from 1.39 ± 0.35 ms to 0.53 ± 0.02 ms in the presence of 2 mM PCN. 6 The reduced mean open time of GABA‐sensitive channels seen in the presence of PCN may contribute to the convulsant action of the drug in vivo.

Pentobarbital modulates intrinsic and GABA-receptor conductances in thalamocortical inhibition.

We investigated interactions of an anesthetic barbiturate, pentobarbital, with non-ligand gated channels and identified inhibitory synaptic transmission in thalamic neurons. Using whole cell voltage-clamp, current-clamp and single channel recording techniques in rat ventrobasal neurons of slices and dispersed preparations, we determined the mechanisms of pentobarbital actions on ionic currents and inhibitory postsynaptic currents (IPSCs), mediated by aminobutyric acid (GABA). We investigated pentobarbital effects on intrinsic currents using hyperpolarizing voltage commands from rest and tetrodotoxin blockade of action potentials. At concentrations near 8 microM, pentobarbital increased input conductance and induced net outward current, I(PB), at potentials near action potential threshold. The reversal potential of I(PB) was -75 mV, implicating K+ and other ions. Cs+ (3 mM) which inhibits both K+ currents and inward rectifier (Ih), completely blocked IPB, whereas the selective Ih blocker, ZD-7288 (25 microM), or Ba2+ (2 mM) which suppresses only K+ currents, reduced IPB. Pentobarbital inhibited the Ih, consistent with a ZD-7288-induced shift in reversal potential for IPB toward K+ equilibrium potential. Pentobarbital increased the inward K+ rectifier, IKir, and leak current, Ileak. We determined the susceptibility of IPSCs, evoked by reticular stimulation, to antagonism by bicuculline, picrotoxinin and 2-hydroxysaclofen and identified their receptor subclass components. At EC50 = 53 microM, pentobarbital increased the duration of IPSCs. The prolonged IPSC duration during pentobarbital was attributable to enhanced open probability of GABAA channels, because combined with GABA, pentobarbital application increased mean channel open time without affecting channel conductance. At concentrations up to 100 microM, pentobarbital did not directly activate GABAA receptors. The concentration-response relationships for pentobarbital effects on the intrinsic currents and IPSCs overlapped, implying multiple sites of action and possible redundancy in anesthetic mechanisms. This is the first study to show that an i.v. anesthetic modulates the intrinsic currents, Ih, IKir, and Ileak, as well as IPSC time course in the same neurons. These effects likely underlie inhibition in thalamocortical neurons during pentobarbital anesthesia.

Barbiturate activation and modulation of GABAA receptors in neocortex

We determined if anesthetic and anti-epileptic barbiturates inhibit neurons by different mechanisms. Current- and voltage-clamp recordings were made from somatosensory neurons of neocortex and some thalamocortical neurons in coronal brain slices of rats. We compared effects of pentobarbital, amobarbital, and phenobarbital on inhibitory postsynaptic currents (IPSCs) mediated by gamma-aminobutyric acid (GABA), input conductance, and evoked action potential firing. In neocortex, pentobarbital (EC(50)=41 microM) and amobarbital (EC(50)=103 microM) increased the decay time constant of GABA(A)ergic IPSCs. At higher concentrations, pentobarbital and amobarbital shunted firing by increasing input conductance through agonism at GABA(A) receptors. At anti-epileptic concentrations, phenobarbital increased the IPSC decay time constant (EC(50)=144 microM), and shunted firing by agonism at GABA(A) receptors (EC(50)=133 microM). In thalamocortical neurons, similar concentrations of phenobarbital had negligible effects on GABA(A)ergic IPSCs, conductance, and firing. In contrast to their thalamic actions, barbiturates inhibit neocortical neurons mostly through GABA receptors. Neocortical enhancement of inhibition by pentobarbital and amobarbital, combined with actions on thalamocortical neurons, may contribute to redundant mechanisms of anesthesia. The ability of phenobarbital at anti-epileptic concentrations to inhibit neocortical firing by direct activation and modulation of GABA(A) receptors relates to its specialized therapeutic effects.

Glycinergic inhibition in thalamus revealed by synaptic receptor blockade.

Using juvenile rat brain slices, we examined the possibility that strychnine-sensitive receptors for glycine-like amino acids contributed to synaptic inhibition in ventrobasal thalamus, where gamma-aminobutyrate (GABA) is the prevalent inhibitory transmitter. Ventrobasal nuclei showed staining for antibodies against alpha1 and alpha2 subunits of the glycine receptor. Exogenously applied glycine, taurine and beta-alanine increased membrane conductance, effects antagonized by strychnine, indicative of functional glycine receptors. Using glutamate receptor antagonists, we isolated inhibitory postsynaptic potentials and currents (IPSPs and IPSCs) evoked by high-threshold stimulation of medial lemniscus. Like the responses to glycine agonists, these synaptic responses reversed near E(Cl). In comparative tests with GABA receptor antagonists, strychnine attenuated inhibition in a majority of neurons, but did not alter slow, GABA(B) inhibition. For complete blockade, the majority of fast IPSPs required co-application of strychnine with bicuculline or gabazine, GABA(A) receptor antagonists. Strychnine acting with an IC50 approximately = 33 nM, eliminated residual fast inhibition during selective GABA(A) receptor blockade with gabazine. The latency of onset for IPSPs was compatible with polysynaptic pathways or prolonged axonal propagation time. Strychnine lacked effects on monosynaptic, GABAergic IPSPs from zona incerta. The specific actions of strychnine implicated a glycine receptor contribution to fast inhibition in somatosensory thalamus.

Pentobarbital promotes bursts of γ-aminobutyric acid-activated single channel currents in cultured mouse central neurons

The extracellular patch clamp method was used to study the influence of pentobarbital (PB) on membrane channels induced by gamma-aminobutyric acid (GABA) in cultured mouse spinal neurons. PB (200 microM) increased the probability of finding GABA-sensitive channels in the open state; it also increased the average frequency at which GABA-induced single channel currents occur, without decreasing the number of data sweeps which showed no single channel activity. These results indicate that PB potentiates GABA responses by promoting burst-like openings of the GABA-sensitive channel.

Internally applied endotoxin and the activation of BK channels in cerebral artery smooth muscle via a nitric oxide‐like pathway

In this study the role of nitric oxide synthase (NOS) in the acute activation of large conductance, Ca2+‐activated K+ channels (BK channels) by internally applied E. coli lipopolysaccharide (LPS, endotoxin) was examined in vascular smooth muscle cells. Cerebrovascular smooth muscle cells (CVSMCs) were enzymatically dispersed from the middle, posterior communicating and posterior cerebral arteries of adult Wistar rats and maintained at 4°C for 2–4 days before recording with standard patch‐clamp techniques. Acute application of LPS (100 μg ml−1) to inside‐out patches of CVSMC membrane isolated in a cell‐free environment rapidly and reversibly increased the open probability, Po of BK channels in these patches by 3.3±0.30 fold. Acute application of the nitric oxide (NO) donor sodium nitroprusside (SNP, 100 μM) to inside‐out patches of CVSMC membrane, studied in the presence of intact cells, also reversibly increased Po, by some 1.8±0.2 fold over control. Kinetic analysis showed that both LPS and SNP increased Po by accelerating the rate of BK channel reopening, rather than by retarding the closure of open channels. Neither LPS nor SNP altered the reversal potential or conductance of BK channels. The NOS substrate L‐arginine (1 μM) potentiated the acute activation of BK channels by LPS, while the synthetic enantiomer D‐arginine (1 μM) inhibited the action of LPS on BK channels. The acute activation of BK channels by LPS was suppressed by pre‐incubation of cells with Nω‐nitro‐L‐arginine (50 μM) or Nω‐nitro‐L‐arginine methyl ester (1  mM), two competitive antagonists of nitric oxide synthases. Nω‐nitro‐D‐arginine (50 μM), a poor inhibitor of NOS in in vitro assays, had no effect on BK channel activation by LPS. These results indicate that excised, inside‐out patches of CVSMC membrane exhibit a NOS‐like activity which is acutely activated when LPS is present at the cytoplasmic membrane surface. Possible relationships between this novel mechanism and the properties of known isoforms of nitric oxide synthase are discussed.

Effects of Intracellular Mg2+ on the Properties of Large-Conductance, Ca2+-Dependent K+ Channels in Rat Cerebrovascular Smooth Muscle Cells

The effects of intracellular magnesium ions, Mg2+i on large-conductance, Ca2+-dependent K+ channels (BK channels) of adult rat cerebrovascular smooth muscle cells (CVSMCs) were studied using patch clamp techniques and cells enzymatically dispersed from basilar, middle, and posterior cerebral arteries. Recordings used inside-out membrane patches and took place at 20–24°C. One millimeter [Mg2+]i produced a fast block of BK channel currents, as well described by the Woodhull model of channel occlusion by a charged species. However, the affinity and voltage-sensitivity of Mg2+i block were dependent on the concentration of free intracellular calcium ions, [Ca2+]i. Calcium ions may stabilize a channel conformation in which Mg2+i binding sites are relocated closer to the inner membrane surface. In the presence of 1 μM [Ca2+]i, 0.5 mM [Mg2+]i shifted the Boltzmann curve relating BK channel open probability, Po, to membrane voltage leftward on the voltage axis, without any change in its slope. The enhancing effect of Mg2+i on Po was, therefore, insensitive to membrane potential. Quantitative considerations suggest that physiological levels of Mg2+i tonically facilitate BK channel activation. Alterations of [Mg2+]i during hyper- or hypomagnesemia may contribute to the dilation or contraction of cerebral vessels seen under these two conditions.

Effect of halothane on large-conductance calcium-dependent potassium channels in cerebrovascular smooth muscle cells of the rat.

Background:Patch-clamp methods were used to examine the effects of the volatile general anesthetic halothane on large-conductance calcium-dependent potassium channels (BK channels) in dispersed cerebrovascular smooth muscle cells of adult rats. Methods:Inside-out membrane patches were used for recordings at 21-23°C. Halothane was administered at aqueous concentrations of 0.5-2.8 mm in conjunction with free cytoplasmic calcium concentrations of 1 or 100 µm and at a membrane potential of -60 mV or +60 mV. Results:At a free cytoplasmic calcium concentration of 1 µm, the clinically relevant dose of 0.5 mM (2 MAC) halothane reduced the open probability of large-conductance calcium-dependent potassium channels without altering the single-channel conductance. This effect was blocked by increasing the concentration of cytoplasmic free calcium to 100 µm, but was not intrinsically voltage dependent. Conclusions:The marked dilation of cerebral vessels seen during surgical anesthesia with halothane cannot be attributed to direct effects of the drug on large-conductance calcium-dependent potassium channels. The protective effect of calcium suggests that halothane exerts its effects at channel sites located within the cell membrane.

Membrane channels activated by taurine in cultured mouse spinal cord neurons

Patch-clamp methods were used to compare biophysical properties of anion channels activated by taurine and gamma-aminobutyric acid in the membrane of cultured mouse spinal neurons. Outside-out patches were voltage clamped at -80 mV at a temperature of 21-23 degrees C. Bath application of GABA (1.5-2 microM) or taurine (5-40 microM) induced chloride-dependent single-channel currents in 14/20 patches tested. Amplitude distributions of these currents showed peaks corresponding to conductance levels of 8, 16, 27 and 46 pS. Only a few percent of GABA-induced events reached the 46 pS level, while 30% of taurine-induced currents were of this size. The average lifetime of taurine-activated channels in the open state was 1.0 +/- 0.07 ms, significantly shorter than the corresponding value for GABA (1.6 +/- 0.08 ms). Taurine-induced currents were abolished by 10 microM strychnine, but persisted in the presence of 50 microM bicuculline.

The benzodiazepine triazolam: direct and GABA depressant effects on cultured mouse spinal cord neurons

Whole-cell and outside-out patch clamp recordings were used to study the effects of the benzodiazepine triazolam at the membrane of mouse spinal cord neurons in cell culture. At 1 microM, triazolam reversibly increased the membrane conductance of about half of the spinal cord cells tested, the average increase being 24 +/- 4%. Depolarizations evoked by successive applications of gamma-aminobutyric acid (GABA) to the spinal cord cell membrane were attenuated by 1 microM triazolam in about half of the neurons tested. At 1-10 microM, triazolam also reduced the charge transfer triggered by GABA in outside-out patches of spinal cord cell membrane. In contrast, 10 microM diazepam potentiated charge transfer by the GABA receptor complexes. Triazolam apparently acts as an inverse agonist at benzodiazepine receptors expressed on spinal cord cells in culture. The well known anxiolytic effects of this drug are presumably mediated by benzodiazepine receptor types not assayed in the present experiments.