Role of gap junctions on synchronization in human neocortical networks.

Gap junctions (GJ) have been implicated in the synchronization of epileptiform activities induced by 4-aminopyrine (4AP) in slices from human epileptogenic cortex. Previous evidence implicated glial GJ to govern the frequency of these epileptiform events. The synchrony of these events (evaluated by the phase unlocking index, PUI) in adjacent areas however was attributed to neuronal GJ. In the present study, we have investigated the effects of GAP-134, a recently developed specific activator of glial GJ, on both the PUI and the frequency of the 4AP-induced epileptiform activities in human neocortical slices of temporal lobe epilepsy tissue. To delineate the impact of GJ on spatial spread of synchronous activity we evaluated the effects of carbenoxolone (CBX, a non-selective GJ blocker) on the spread in three axes 1. vertically in a given cortical column, 2. laterally within the deep cortical layers and 3. laterally within the upper cortical layers. GAP-134 slightly increased the frequency of the 4AP-induced spontaneous epileptiform activities while leaving the PUI unaffected. CBX had no effect on the PUI within a cortical column or on the PUI in the deep cortical layers. CBX increased the PUI for long interelectrodes distances in the upper cortical layers. In conclusion we provide new arguments toward the role played by glial GJ to maintain the frequency of spontaneous activities. We show that neuronal GJ control the PUI only in upper cortical layers.

Different pharmacology of N-desmethylclozapine at human and rat M2 and M 4 mAChRs in neocortex.

Cholinergic transmission plays a pivotal role in learning, memory and cognition, and disturbances of cholinergic transmission have been implicated in neurological disorders including Alzheimer's disease, epilepsy and schizophrenia. Pharmacological alleviation of these diseases by drugs including N-desmethylclozapine (NDMC), promising in animal models, often fails in patients. We therefore compared the effects of NDMC on glutamatergic and GABAergic transmission in slices from rat and human neocortex. We used carbachol (CCh; an established agonist at metabotropic muscarinic acetylcholine (ACh) receptors (mAChRs)) as a reference. Standard electrophysiological methods including intracellular and field potential recordings were used. In the rat neocortex, NDMC prevented the CCh-induced decrease of GABAA and GABAB receptor-mediated responses but not the CCh-induced increase of the paired-pulse depression. NDMC reduced neither the amplitude of the excitatory postsynaptic potentials (EPSP) nor antagonized the CCh-induced depression of EPSP. In the human neocortex, however, NDMC failed to prevent CCh-induced decrease of the GABAB responses and directly reduced the amplitude of EPSP. These data suggest distinct effects of NDMC in rat and human at M2 and M4 mAChRs underlying presynaptic modulation of GABA and glutamate release, respectively. In particular, NDMC might be a M2 mAChR antagonist in the rat but has no activity at this receptor in human neocortex. However, NDMC has an agonistic effect at M4 mAChR in the human but no such effect in the rat neocortex. The present study confirms that pharmacology at mAChRs can differ between species and emphasizes the need of studies in human tissue.

Effects of VU0240551, a novel KCC2 antagonist, and DIDS on chloride homeostasis of neocortical neurons from rats and humans.

The normal function of GABAA receptor-mediated inhibition is governed by several factors, including release of GABA, subunit composition and density of the receptors and in particular by the appropriate ionic gradient. In the human epileptogenic neocortex an impaired chloride (Cl(-)) gradient has been proposed, due to decreases of potassium-coupled chloride transport (KCC2) and voltage-gated Cl(-) channels (ClC). Regarding sodium- and potassium-coupled Cl(-) transport (NKCC1) both up- and downregulations have been proposed. We investigated changes of Cl(-) homeostasis of human and rat neocortical neurons (layer 2/3) with intracellular recordings and iontophoretic Cl(-) loading employing selective compounds. After cessation of iontophoresis, the IPSPA amplitudes of rat neurons recovered with a time constant (τrec) of 6.5s (n=21). In human neurons, τrec averaged 17.8s (n=36; 23 resections). Application of the novel KCC2 blocker VU0240551 (1 µM) caused in rat neurons a reversible prolongation of τrec from 5.7 to 8.1s (n=11), corresponding to a VU0240551-sensitive Cl(-) transport rate (1/Δτrec) of 0.0504s(-1). In human neurons, τrec increased on application of 1µM VU0240551, on average from 15.1 to 20.3s (n=17). The human neurons comprised two subgroups with different τrec when segregated according to a border given by the mean+2s.d. of rat neurons. In one group, τrec averaged 8.7s (n=6) and reversibly increased to 14.6s in the presence of 1µM VU0240551, corresponding to a Cl(-) transport rate of 0.0504s(-1). The other group had an average τrec of 18.5s which increased in the presence of 1µM VU0240551 to 23.3s (n=11), indicating a much smaller rate (0.0151s(-1)). Addition of DIDS, a presumed blocker of anion exchanger (AE), increased the τrec of rat neurons from 7.5 to 8.8s (n=6) corresponding to a DIDS-sensitive rate of 0.0185s(-1). In human neurons, DIDS increased τrec from 23.3 to 50.7s (n=7), corresponding to a DIDS-sensitive rate of 0.0200s(-1). These data suggest a greatly reduced KCC2-mediated transport rate in most of the human neurons. The two subgroups observed in human tissue indicate a considerable variability of Cl(-) transport within a given tissue from almost normal to greatly impeded, predominated by a decline of KCC2 whereas AE is unaltered.

Muscarinic acetylcholine receptor-mediated effects in slices from human epileptogenic cortex.

Acetylcholine has been implicated in higher cortical functions such as learning, memory and cognition, yet the cellular effects of muscarinic acetylcholine receptor (mAChR) activation are poorly understood in the human cortex. Here we investigated the effect of the mAChR agonist carbachol (CCh) and various mAChR antagonists in human cortical slices (from tissue removed during neurosurgical treatment of epilepsy) by intracellular and extracellular recordings. CCh increased neuronal firing, which was antagonised by atropine (non-selective mAChR antagonist) and pirenzepine (M(1)/M(4) mAChRs antagonist) when applied before or after CCh application. AF-DX 116 (M(2)/M(4) mAChRs antagonist) had no effect on CCh-induced increase of firing. CCh also reduced evoked excitatory postsynaptic potentials (EPSP), and the CCh-induced depression of EPSP was fully reversed by atropine. Pirenzepine reversed the depression of CCh on EPSP, but failed to prevent the depression when applied before CCh. AF-DX 116 prevented the CCh-induced depression of evoked EPSP when applied before CCh. CCh also depressed GABAergic transmission and this effect was antagonised by AF-DX 116. Xanomeline (M(1)/M(4) mAChR agonist) increased neuronal firing and decreased EPSP, but had no effect on GABAergic transmission. Reduction (with linopirdine) and enhancement (with retigabine) of the M-current (mediated by K(V)7 channels), increased and decreased neuronal firing, respectively, but had marginal effects on the evoked EPSP. Our results indicate that three pharmacologically distinct mAChRs modulate neuronal firing, glutamatergic and GABAergic transmissions in the human epileptogenic neocortex. The data are discussed towards possible implications of altered mAChR signalling in hyperexcitability and cognitive functions in the human neocortex.

Pathological cholesterol metabolism fails to modify electrophysiological properties of afflicted neurones in Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is a recessive inherited neurovisceral lipid storage disease characterized by progressive motor impairment and a loss of neurones including those integrated into the motor system. One of the key neuropathological findings is the intracellular accumulation of lysosomes enriched with free cholesterol. This accumulation is due to impaired transport proteins named NPC1 (approx. 95% of the cases) or NPC2 (approx. 5%) responsible for the transport of endocytosed cholesterol from lysomes to plasma membranes. The perturbed lipid-transport in NPC cells leads to an altered lipid composition of the plasma membrane. Available evidence suggests that the lipid matrix influences the electrophysical properties of ion channels in membranes. We therefore evaluated whether electrophysiological properties of NPC neurones differ from healthy neurones. Both, acute brain slices and primary neuronal cell cultures from wildtype and NPC mice, a well-established mouse model for the Niemann-Pick type C disease, were used for a comparison of electrophysiological properties like resting membrane potential, input resistance, action potential amplitudes and synaptic properties of the neurones. In addition we optically recorded the changes of intraneuronal calcium levels elicited by depolarization. Our results show that the characteristics of ion channels in NPC neurones do not differ significantly from wildtype neurones. We therefore conclude that gross alterations of the electrophysiological properties of neurones will probably not initiate or substantially contribute to the development of the motor impairment or other neurological signs of NPC.

Cellular mechanisms of pharmacoresistance in slices from epilepsy surgery.

Slices of human cortical tissue from epilepsy surgery were investigated with intracellular recordings to elucidate the mechanisms contributing to augmented synaptic excitation and to repetitive activity. The analysis of single synaptic potentials revealed, amongst other differences to rodent cortex, a disturbance of GABAA inhibition, namely depolarizing responses. A tentative ionic mechanism, impaired KCl outward-transport (KCC2), was evaluated in a rat model (0-Mg hyperexcitability). The observed down-regulation of KCC2 mRNA after 0-Mg-ACSF exposure of slices may contribute to the depolarizations by GABA. The factors enabling repetitive activity were addressed with a paired-pulse paradigm. In slices from epilepsy surgery, synaptic responses were virtually constant with interstimulus intervals between 100 and 1000 ms. Tiagabine markedly prolonged the effects of released GABA at GABAA receptors, but paired-pulse behaviour was only slightly affected. We demonstrate that bicuculline-induced paroxysmal activity of rat cortex is frequency-limited (to about <1 Hz) by presynaptic GABAB receptors. The lack of frequency limitation of synaptic events suggests an impaired GABAB receptor function in the human epileptogenic cortex. The data are discussed regarding the pivotal role of KCl transport in epileptic disorders of various origins and the role of GABAB receptors in the frequency limitation of paroxysmal activity.

Molecular and functional analysis of hyperpolarization-activated pacemaker channels in the hippocampus after entorhinal cortex lesion.

Differential display of hippocampal tissue after entorhinal cortex lesion (ECL) revealed decreases in mRNA encoding the neuronal hyperpolarization-activated, cyclic nucleotide-gated channel HCN1. In situ hybridization confirmed that hippocampal transcripts of HCN1, but not HCN2/3/4, are down-regulated after ECL. Expression recovered at approximately 21 days after lesion (dal). Immunohistochemistry demonstrated a corresponding regulation of HCN1 protein expression in CA1-CA3 dendrites, hilar mossy cells and interneurons, and granule cells. Patch-clamp recordings in the early phase after lesion from mossy cells and hilar interneurons revealed an increase in the fast time constant of current activation and a profound negative shift in voltage activation of Ih. Whereas current activation recovered at 30 dal, the voltage activation remained hyperpolarized in mossy cells and hilar interneurons. Granule cells, however, were devoid of any detectable somatic Ih currents. Hence, denervation of the hippocampus decreases HCN1 and concomitantly the Ih activity in hilar neurons, and the recovery of h-current activation kinetics occurs parallel to postlesion sprouting.

Human brain-cell death induced by tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL)

Cell death induced by tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) was believed to occur exclusively in tumour cells, suggesting that this drug is safe to use as an antitumour therapy. Concerns were raised, however, when cultured normal human hepatocytes were shown to be susceptible to TRAIL. Here we report that TRAIL induces apoptosis in the human brain. Our finding therefore argues against the use of TRAIL for therapy of human brain tumours. However, neuroinflammatory T cells that express TRAIL might induce apoptosis of brain tissue, indicating a potential target for treatment of multiple sclerosis.

GABA(B) receptor-mediated effects in human and rat neocortical neurones in vitro.

GABA(B) receptor-mediated responses were investigated in human and rat neocortical neurones in vitro by using intracellular recording. Human epileptogenic tissue and cortex from rats were compared for differences related to the cellular mechanisms of hyperexcitability. In both tissues, single stimuli of various intensities were used to compare basic properties of excitatory and inhibitory postsynaptic potentials (EPSP, IPSP). Paired stimuli, causing a decrease of a second IPSP, were used as an index of presynaptic activation of GABA(B) receptors. In neocortical neurones of rats, increasing intensities of stimulation elicited at low intensities (6-8 V) a fairly pure EPSP which was curtailed at higher stimulus intensities (10-14 V) by a GABA(A) receptor mediated IPSP (IPSP(A)). In all rat neocortical neurones the IPSP(A) was followed by a late inhibitory component (IPSP(B), time to peak about 150 ms) which was eliminated by the GABA(B) receptor antagonists CGP 35348 or CGP 55845A. On average, paired stimuli reduced the amplitude of a second IPSP(A) to 57% of the first (in the presence of 10 microM CNQX and 20 microM D-APV). Paired-pulse depression was only antagonized by CGP 55845A, but not by CGP 35348. The magnitude and time course of paired-pulse depression was markedly enhanced at lower temperatures. In human cortical neurones obtained following epilepsy surgery only low intensity stimuli (4 V) elicited EPSPs. Intermediate to higher stimulus intensities (8-10 V) elicited often all-or-none depolarization shifts or prolonged and increased EPSPs. Few neurones exhibited a sequence of EPSP and IPSPs comparable to that observed in rat neurones. Application of CGP 55845A caused little change in excitability near 150 ms, indicating that the IPSP(B) is weak. Paired-pulse depression of inhibition was small in most neurones, the second IPSPA was reduced to 82.8% of the first at a 500 ms interval (n = 6). Only two neurones exhibited a paired-pulse depression comparable to rat neurones. The consequences of GABA receptor-mediated paired-pulse depression on neuronal synchronisation are discussed towards the different cellular mechanisms of focal and bilateral synchronous epilepsies.

The GABA(B) receptor antagonist CGP 55845A reduces presynaptic GABA(B) actions in neocortical neurons of the rat in vitro.

Use-dependent depression of inhibitory postsynaptic potentials was investigated with intracellular recordings and the paired-pulse paradigm in rat neocortical neurons in vitro. Pairs of stimuli invariably reduced the second inhibitory postsynaptic potential-A (GABA(A) receptor-mediated inhibitory postsynaptic potential) of a pair; at interstimulus intervals of 500 ms, the amplitude of the second inhibitory postsynaptic potential-A was considerably smaller than the first (36.2 +/- 6.2%, n= 17). Decreasing the interstimulus interval reduced the second inhibitory postsynaptic potential-A further and with interstimulus intervals shorter than 330 ms the compound excitatory postsynaptic potential-inhibitory postsynaptic potential response reversed from a hyperpolarizing to a depolarizing response. The depression of the inhibitory postsynaptic potential-A exhibited a maximum at interstimulus intervals near 150 ms and recovered with a time constant of 282 +/- 96.2 ms. Elimination of excitatory transmission by the application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(-)-2-amino-5-phosphonovaleric acid yielded an essentially unaltered time-course of paired-pulse depression (maximum depression near 150 ms, time constant of recovery 232 +/- 98 ms). The polarity change of the compound excitatory postsynaptic potential response at shorter interstimulus intervals was abolished in the presence of CNQX and D(- )-2-amino-5-phosphonovaleric acid. CNQX and D(-)-2-amino-5-phosphonovaleric acid also reduced the apparent depolarizing shift of the reversal potential between the first and second inhibitory postsynaptic potential-A from about 6 mV to less than 2 mV. Application of the GABA(B) receptor antagonist CGP 55845A in the presence of CNQX and (-)-2-amino-5-phosphonovaleric acid abolished the inhibitory postsynaptic potential-B and paired-pulse depression. Under these conditions, the amplitude of the second inhibitory postsynaptic potential was, on average, about 90% of the first, i.e. reduced by about 10%. The second inhibitory postsynaptic potential-A was approximately constant at interstimulus intervals between 100 and 500 ms. It is concluded that paired-pulse depression of cortical inhibition is predominantly mediated by presynaptic GABA(B) receptors of GABAergic interneurons. The abolition of net inhibition at interstimulus intervals near 330 ms may facilitate spread of excitation and neuronal synchrony during repetitive cortical activation near 3 Hz. This use-dependent depression of inhibition may contribute to highly synchronized slow electroencephalogram activity during spike-and-wave or delta activity.

Bidirectional effects of the neuroactive steroid tetrahydrodeoxycorticosterone on GABA-activated Cl- currents in cultured rat hypothalamic neurons.

1. The non-genomic effects of tetrahydrodeoxycorticosterone (THDOC; 5-alpha-pregnane-3-alpha, 21-diol-20-one) were studied in cultured hypothalamic neurons of the rat. 2. The effects of THDOC (10 nM - 1 microM) on responses to different concentrations of exogenously applied GABA and on spontaneous inhibitory postsynaptic currents (IPSCs) were measured with whole-cell voltage clamp recordings. 3. Application of GABA induced inward currents with dose-dependently increasing amplitudes (up to 3.9 nA at a holding potential of -20 mV). High doses of THDOC (100 nM-1 microM) induced small inward currents on its own (14+/-3 and 24+/-3 pA, respectively). 4. Simultaneous application of 10 microM GABA with 100 nM or 1 microM THDOC increased current amplitudes by 125 and 128%, respectively. At 10 nM THDOC exerted no consistent effects on GABA currents. 5. Responses to 1 microM of GABA were modulated in a bidirectional manner by different doses of THDOC: 10 nM THDOC reduced the amplitude of GABA responses to 80% (P=0.018, n=15), whereas 100 nM and 1 microM THDOC enhanced the GABA response to 115 and 180% (P=0.0007, n = 15), respectively. 6. The time constant of decay of spontaneous inhibitory postsynaptic currents (IPSCs) was reversibly increased from 91+/-10 to 314+/-34 ms (n=3) by the application of THDOC (1 microM). The amplitudes of the IPSCs were not affected by THDOC. 7. These data indicate that THDOC modulates GABA responses of hypothalamic neurons in a bidirectional manner, resulting in a complex tuning of neuronal excitability in the hypothalamus.

Effect of the GABA uptake inhibitor tiagabine on sleep and EEG power spectra in the rat.

1. The sleep profiles induced by agonists and agonistic modulators of gamma-aminobutyric acidA (GABA[A]) receptors differ markedly. With regard to GABA(A) agonists, the effects may be due to the fact that these agents are poor substrates for uptake and are therefore likely to activate GABA(A) receptors tonically. To investigate this possibility, we assessed the sleep effects of two doses (2 and 10 mg kg[-1]) of the GABA re-uptake inhibitor tiagabine, administered intraperitoneally at light onset in 8 rats. Electroencephalogram (EEG) and electromyogram were recorded during the first 8 h after the injection. 2. Compared with vehicle, tiagabine had minimal effects on the temporal pattern of non-rapid eye movement sleep (non-REMS) and on the total time spent therein. However, tiagabine dose-dependently elevated EEG activity during non-REMs, most prominently in the lower frequencies (1-8 Hz) and least pronounced in the frequencies between 11 and 16 Hz. During the first 2 h after the injection, 10 mg kg(-1) tiagabine elicited repetitive episodes of hypersynchronous EEG waves during wakefulness and slightly suppressed REMS. Except for these effects, tiagabine hardly influenced the time spent in and EEG activity during wakefulness and REMS. 3. The effects of tiagabine on state-specific EEG activity were qualitatively very similar to those elicited by GABA(A) agonists. These findings support the hypothesis that the influence of GABA(A) agonists on EEG signals may be caused by tonic stimulation of GABA(A) receptors.

Muscimol and midazolam do not potentiate each other's effects on sleep EEG in the rat.

The interaction of a gamma-aminobutyric acid-A (GABAA) receptor agonist and a benzodiazepine-type modulator of GABAA receptors on sleep was investigated. Low doses of muscimol (0.3 mg/kg) and the benzodiazepine midazolam (1.5 mg/kg) were administered alone and in combination, in random order, to eight rats. All injections were given intraperitoneally at light onset. Electroencephalogram (EEG) and electromyogram were recorded during the first 6 h post injection. Compared with vehicle, muscimol hardly affected the time spent in non-rapid eye movement sleep (non-REMS) and REMS, but significantly enhanced EEG activity in the frequency range between 2 and 6 Hz during non-REMS. Midazolam significantly increased the time spent in non-REMS, reduced EEG activity at frequencies < 12 Hz, and elevated EEG activity in most higher frequencies during this state. The combined administration of muscimol and midazolam affected non-REMS-specific EEG activity in an unexpected fashion: the effects were intermediate between those of muscimol and midazolam. These results indicate that muscimol and midazolam have dissimilar effects on EEG within non-REMS and demonstrate that midazolam does not augment but attenuates the muscimol-induced changes in sleep EEG. Our data are at variance with established mechanisms, according to which agonistic modulators would have similar effects and should potentiate the effects of GABAA agonists. The present data suggest that application of agonists and agonistic modulators of GABAA receptors causes differential net effects on sleep parameters.

Presynaptic and postsynaptic GABAB receptors of neocortical neurons of the rat in vitro: differences in pharmacology and ionic mechanisms.

The properties of pre- and postsynaptic GABAB receptors were investigated with intracellular recordings from rat neocortical neurons in vitro. An antagonist of the GABAB receptor (CGP 35348) and ions or drugs interfering with GABAB receptor-mediated K+ conductance (Ba2+, QX 314) were employed to delineate possible differences. CGP 35348 reduced the conductance of the late inhibitory postsynaptic potential (IPSPB) in a dose-dependent manner. Neither the early GABAA receptor-mediated inhibitory postsynaptic potential (IPSPA), nor resting membrane potential or direct excitability, were consistently affected by CGP 35348. Bath application of 100 mumol/l Ba2+ decreased IPSPB conductance to about 40% and increased IPSPA conductance to 130% of control. The depression of a second IPSP by a pair of stimuli (paired pulse depression, or PPD) was used as an index for presynaptic GABAB receptor activation. Neither CGP 35348 nor Ba2+ exerted significant effects on the PPD at intervals of 400 msec. The dependence of PPD on the latency of the interval of the stimulus pair was investigated after intracellular applicatio of QX 314 had virtually abolished the IPSPB. Decreasing the stimulus interval from 500 msec to 100 msec revealed a stronger depression of the second IPSPA. Application of CGP 35348 alleviated PPD for stimulus intervals below 300 msec. The data indicate a distinct pharmacological difference between pre- and postsynaptic GABAB receptors. Moreover, we suggest that two temporally distinct presynaptic GABAB receptor effects contribute to PPD: a short-lasting effect, sensitive to CGP 35348, and a long-lasting effect, insensitive to CGP 35348. The latter is insensitive to Ba2+, implying that this component is not associated with a K+ conductance mechanism.

A tetrodotoxin-insensitive [corrected] sodium current initiates burst firing of neocortical neurons.

The ability to generate burst discharges is a widespread phenomenon in central neurons and often has been attributed to Ca2+ currents. The diversity of burst patterns seems to be at variance with a single mechanism. Selected neocortical bursting neurons were examined by intracellular recordings. Selective blockers and pulse protocols were applied to characterize the crucial components of bursting activity. In the presence of tetrodotoxin and Ca2+ channel antagonists a transient depolarization persists, which shares the activation and deinactivation properties with the burst. The sensitivity to Na+ removal and resistance to tetrodotoxin suggests a tetrodotoxin-insensitive Na+ current as the crucial component in neocortical bursting neurons. A tetrodotoxin-insensitive Na+ current has been isolated in neocortical bursting neurons. The biophysical properties of this current allow for burst firing at frequencies up to about 12 Hz. This tetrodotoxin-insensitive Na+ may generate the alpha-rhythm of the electroencephalogram by effectively synchronizing arrays of follower cells. The implications of the intrinsic currents of bursting neurons for the initiation of epileptic discharges are discussed.

The role of bicarbonate in GABAA receptor-mediated IPSPs of rat neocortical neurones.

1. The ionic mechanism underlying the fast, GABAA receptor-mediated inhibitory postsynaptic potential (IPSPA) was examined in rat neocortical neurones using intracellular recording techniques. Synaptic responses were evoked by orthodromic stimulation applied to the subcortical white matter or to the pial surface. All experiments were carried out at a constant extracellular Cl- concentration. 2. The resting membrane potential was -76.2 +/- 1.0 mV (mean +/- S.E.M., n = 32) and in most cells IPSPA was depolarizing. The reversal potential of IPSPA (EIPSP-A) was -70.2 +/- 0.9 mV (n = 32) and that of a more slowly developing hyperpolarizing response (IPSPB) was -91.4 +/- 1.3 mV (n = 28). 3. An examination of the temporal relationships between excitatory postsynaptic potentials (EPSPs) and IPSPAs in different cells suggested that, despite partial overlap of these responses, EPSPs had little influence on the measured values of EIPSP-A. 4. Application of 20 mM trimethylamine (TriMA), a membrane-permeant weak base which is expected to produce a rise in pHi (and hence in intracellular HCO3-), induced a reversible positive shift in EIPSP-A of up to +9.0 mV (mean + 4.2 mV) at an extracellular pH (pHo) of 7.4. In some experiments, the shift in reversal potential was associated with a change in the polarity of IPSPA from hyperpolarizing to depolarizing. 5. Application of 20 mM lactate (a membrane-permeant weak acid which is expected to produce a fall in pHi and hence in intracellular HCO3-) at pHo 7.0 produced a hyperpolarizing shift in EIPS-A of up to -7.5 mV (mean -5.6 mV). In some experiments, exposure to lactate changed the polarity of IPSPA from depolarizing to hyperpolarizing. 6. Changes in pHo from 7.4 to 7.0 reduced the effect of TriMA and augmented that of lactate on EIPSP-A, as could be expected on the basis of the pHo-dependent change in the fraction of membrane permeable non-charged weak base or acid. 7. Under control conditions, a change in pHo from 7.4 to 7.0 produced a slight positive shift (< +2 mV) in EIPSP-A. In the presence of TriMA, a similar change in pHo gave rise to a negative shift (-1.8 to -2.7 mV). 8. The results obtained indicate that HCO3- ions contribute significantly to the IPSPA, thereby making EIPSP-A more positive than the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Pre- and postsynaptic GABAB receptors of rat neocortical neurons differ in their pharmacological properties.

GABA (gamma-amino butyric acid)B receptors have been proposed to play a dual role in synaptic transmission in the mammalian central nervous system (CNS): they participate in a late inhibitory postsynaptic potential (1-IPSP) and reduce the release of GABA by a presynaptic action. To further characterize these mechanisms, two established GABAB receptor antagonists were applied to intracellularly recorded rat neocortical neurons in vitro. The depression of the 1-IPSP by the GABAB receptor antagonists phaclofen and 2-OH-saclofen averaged 30% and 50%, respectively. Phaclofen had no effect on direct excitability or excitatory synaptic transmission. The depression of a second IPSP evoked by paired-pulse stimulation was used as an index for presynaptic GABAB receptor activation. Neither antagonist exerted significant effects on this transient depression of GABAergic inhibition. The present results suggest that the pre- and postsynaptic GABAB receptors involved in GABAergic transmission of neocortical neurons differ in their pharmacological properties.

Voltage dependence of excitatory postsynaptic potentials of rat neocortical neurons.

1. The properties of excitatory postsynaptic potentials (EPSPs) of rat neocortical neurons were investigated with a fast single-electrode current-voltage clamp in vitro. Typically, apparently pure EPSPs were obtained by selection of electric stimuli of low intensity. 2. The amplitude and time integral of the EPSP increased when the neuron was depolarized. At threshold for generation of action potentials, the amplitude of EPSPs was increased by approximately 30% [from 5.0 +/- 2.1 to 6.3 +/- 1.0 (SD) mV, n = 12]. The integral of EPSPs was maximally about fourfold (3.7 +/- 1.5, n = 16) larger than at resting membrane potential (Em). The mechanisms involved in this augmentation of EPSPs were further investigated. 3. The amplitude and the time integral of excitatory postsynaptic currents (EPSCs) decreased linearly with shifts in command potential from -100 to -60 mV. The decrease of the EPSC integral with depolarization indicates that the enhancement of the EPSP may be brought about by recruitment of a voltage-dependent inward current. 4. Evoking EPSPs at various delays after the onset of small depolarizing current pulses (0.3-0.6 nA, 600 ms) revealed that augmentation decays with time. The integral of EPSPs evoked approximately 80 ms after the onset of the current pulse was 3.7 (+/- 1.5, n = 16) times larger than at Em. The integral of EPSPs evoked at 480 ms. however, were only twofold (+/- 0.7, n = 16) larger. Hence EPSPs evoked after a delay of 80 ms were 1.7-fold (+/- 0.4, n = 24) larger than EPSPs evoked after 480 ms. EPSCs were independent of the delay of stimulation at all potentials. 5. Intracellular application of the lidocaine derivative N-(2,6-dimethyl-phenylcarbamoylmethyl) triethylammonium bromide (QX 314) at 100 mM from pipettes rapidly abolished fast action potentials and inward rectification. During comparable depolarizations the increase in EPSP integrals was much smaller in QX 314-treated neurons than in controls. On average, the integral of EPSPs evoked at 70-90 ms was 1.7 times (+/- 1.0) larger than at Em, and the integral of EPSPs evoked with larger delays was close to the value obtained at resting Em (0.9 +/- 0.3, n = 8). The ratio of EPSP integrals early versus late (1.8 +/- 0.5) is comparable to controls, suggesting that QX 314-sensitive currents are unlikely to be involved in the time-dependent enhancement. 6. Mimicking EPSPs by brief depolarizations atop long depolarizations revealed a time- and voltage-dependent enhancement comparable to that of EPSPs.(ABSTRACT TRUNCATED AT 400 WORDS)

Frequency-dependent depression of inhibition in guinea-pig neocortex in vitro by GABAB receptor feed-back on GABA release.

1. The mechanisms involved in the lability of inhibition at higher frequencies of stimulation were investigated in the guinea-pig in vitro neocortical slice preparation by intracellular recording techniques. We attempted to test the possibility of a feedback depression of GABA on subsequent release. 2. At resting membrane potential (Em, -75.8 +/- 5.2 mV) stimulation of either the pial surface or subcortical white matter evoked a sequence of depolarizing and hyperpolarizing synaptic components in most neurones. An early hyperpolarizing component (IPSPA) was usually only obvious as a pronounced termination of the EPSP, followed by a later hyperpolarizing event (IPSPB). Current-voltage relationships revealed two different conductances of about 200 and 20 nS and reversal potentials of -73.0 +/- 4.4 and -88.6 +/- 6.1 mV for the early and late component, respectively. 3. The conductances of IPSPA and IPSPB were fairly stable at a stimulus frequency of 0.1 Hz. At frequencies between 0.5 and 2 Hz both IPSPs were attenuated with the second stimulus and after about five stimuli a steady state was reached. Concomitantly IPSPs were shortened. The average decrease in synaptic conductance between 0.1 and 1 Hz was 80% for the IPSPA and 60% for the IPSPB. At these frequencies the reversal potentials decreased by 5 and 2 mV, respectively; Em and input resistance (Rin) were not consistently affected. 4. The amplitudes of field potentials, action potentials and EPSPs of pyramidal cells were attenuated less than 10% at stimulus frequencies up to 1 Hz, suggesting that alterations in local circuits between the stimulation site and excitatory input onto inhibitory interneurones may play only a minor role in the frequency-dependent decay of IPSPs. 5. Localized application of GABA produced multiphasic responses. With low concentrations and application near the soma an early hyperpolarization prevailed followed by a depolarizing late component. Brief application of GABA at low frequencies induced constant responses; at higher frequencies, the responses sometimes declined. The current-voltage relationships of the two GABA responses were similar to each other and to the early IPSP. An apparently fivefold higher conductance was estimated at lower Ems, suggesting that the GABA response had a voltage sensitivity. The slope conductance of IPSPs was decreased by up to 50% for tens of seconds after postsynaptically detectable effects of GABA had dissipated. 6. Application of the GABA uptake inhibitor nipecotic acid (50-500 microM) reduced the conductance of both components of orthodromically evoked inhibition and shortened the IPSP at low frequencies, but had no additional effects at higher stimulation rates.(ABSTRACT TRUNCATED AT 400 WORDS)