Jonathan P. Turner

The 'window' component of the low threshold Ca2+ current produces input signal amplification and bistability in cat and rat thalamocortical neurones

1 The mechanism underlying a novel form of input signal amplification and bistability was investigated by intracellular recording in rat and cat thalamocortical (TC) neurones maintained in slices and by computer simulation with a biophysical model of these neurones. 2 In a narrow membrane potential range centred around −60 mV, TC neurones challenged with small (10–50 pA), short (50–200 ms) current steps produced a stereotyped, large amplitude hyperpolarization (> 20 mV) terminated by the burst firing of action potentials, leading to amplification of the duration and amplitude of the input signal, that is hereafter referred to as input signal amplification. 3 In the same voltage range centred around −60 mV, single evoked EPSPs and IPSPs also produced input signal amplification, indicating that this behaviour can be triggered by physiologically relevant stimuli. In addition, a novel, intrinsic, low frequency oscillation, characterized by a peculiar voltage dependence of its frequency and by the presence of plateau potentials on the falling phase of low threshold Ca2+ potentials, was recorded. 4 Blockade of pure Na+ and K+ currents by tetrodotoxin (1 μm) and Ba2+ (0.1–2.0 mm), respectively, did not affect input signal amplification, neither did the presence of excitatory or inhibitory amino acid receptor antagonists in the perfusion medium. 5 A decrease in [Ca2+]0 (from 2 to 1 mm) and an increase in [Mg2+]0 (from 2 to 10 mm), or the addition of Ni2+ (2–3 mm), abolished input signal amplification, while an increase in [Ca2+]0(from 2 to 8 mm) generated this behaviour in neurones where it was absent in control conditions. These results indicate the involvement of the low threshold Ca2+ current (IT) in input signal amplification, since the other Ca2+ currents of TC neurones are activated at potentials more positive than −40 mV. 6 Blockade of the slow inward mixed cationic current (Ih) by 4‐(N‐ethyl‐N‐phenylamino)‐1,2‐dimethyl‐6‐(methylamino)‐pyrimidinium chloride (ZD 7288) (100–300 μm) did not affect the expression of the large amplitude hyperpolarization, but abolished the subsequent repolarization to the original membrane potential. In this condition, therefore, input signal amplification was replaced by bistable membrane behaviour, where two stable membrane potentials separated by 15–30 mV could be switched between by small current steps. 7 Computer simulation with a model of a TC neurone, which contained only IT, Ih, K+ leak current (ILeak) and those currents responsible for action potentials, accurately reproduced the qualitative and quantitative properties of input signal amplification, bistability and low frequency oscillation, and indicated that these phenomena will occur at some value of the injected DC if, and only if, the ‘window’ component of IT (IT window) and the leak conductance (gLeak) satisfy the relation (dITWindow/dV)max > gLeak. 8 The physiological implications of these findings for the electroresponsiveness of TC neurones are discussed, and, as IT is widely expressed in the central nervous system, we suggest that ‘window’IT will markedly affect the integrative properties of many neurones.

CHARACTERIZATION OF SENSORY AND CORTICOTHALAMIC EXCITATORY INPUTS TO RAT THALAMOCORTICAL NEURONES IN VITRO

1 Using an in vitro slice preparation of the rat dorsal lateral geniculate nucleus (dLGN), the properties of retinogeniculate and corticothalamic inputs to thalamocortical (TC) neurones were examined in the absence of GABAergic inhibition. 2 The retinogeniculate EPSP evoked at low frequency (<= 0.1 Hz) consisted of one or two fast‐rising (0.8 ± 0.1 ms), large‐amplitude (10.3 ± 1.6 mV) unitary events, while the corticothalamic EPSP had a graded relationship with stimulus intensity, owing to its slower‐rising (2.9 ± 0.4 ms), smaller‐amplitude (1.3 ± 0.3 mV) estimated unitary components. 3 The retinogeniculate EPSP exhibited a paired‐pulse depression of 60.3 ± 5.6 % at 10 Hz, while the corticothalamic EPSP exhibited a paired‐pulse facilitation of > 150 %. This frequency‐dependent depression of the retinogeniculate EPSP was maximal after the second stimulus, while the frequency‐dependent facilitation of the corticothalamic EPSP was maximal after the fourth or fifth stimulus, at interstimulus frequencies of 1‐10 Hz. 4 There was a short‐term enhancement of the <= 0.1 Hz corticothalamic EPSP (64.6 ± 9.2 %), but not the retinogeniculate EPSP, following trains of stimuli at 50 Hz. 5 The <= 0.1 Hz corticothalamic EPSP was markedly depressed by the non‐NMDA antagonist 1‐(4‐amino‐phenyl)‐4‐methyl‐7,8‐methylene‐dioxy‐5H‐2,3‐benzodiazepine (GYKI 52466), but only modestly by the NMDA antagonist 3‐((RS)‐2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid ((RS)‐CPP), and completely blocked by the co‐application of GYKI 52466, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), (RS)‐CPP and (5R,10S)‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine (MK‐801). Likewise, the corticothalamic responses to trains of stimuli (1‐500 Hz) were greatly reduced by this combination of ionotropic glutamate receptor antagonists. 6 In the presence of GYKI 52466, CNQX, (RS)‐CPP and MK‐801, residual corticothalamic responses and slow EPSPs, with a time to peak of 2‐10 s, could be generated following trains of five to fifty stimuli. Neither of these responses were occluded by 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD), suggesting they are not mediated via group I and II metabotropic glutamate receptors.

Spontaneous paroxysmal activity induced by zero magnesium and bicuculline: suppression by NMDA antagonists and GABA mimetics.

Slices of rat cerebral cortex developed spontaneous paroxysmal discharges when superfused with Krebs medium containing zero Mg2+ or 50 microM bicuculline. In both situations, the N-methyl-D-aspartate (NMDA) antagonists APV, 100 microM, and ketamine, 100 microM substantially reduced the frequency of the paroxysmal events, the reduction being greater in zero Mg2+. gamma-Aminobutyric acid (GABA) 1 mM, the GABA-A agonist muscimol 2 microM and the GABA-B receptor agonist baclofen 10 microM, each reduced the frequency of events in zero Mg2+ while muscimol and GABA also reduced the amplitude of the events. GABA and baclofen were similarly effective against bicuculline-induced events but the muscimol concentration required was 5-10-fold higher. These results suggest that, under our vitro conditions, neocortical cells are normally restrained from paroxysmal discharges by Mg2+. Inhibition by GABA through GABA-A receptors and inhibition by GABA through GABA-B receptors, may also contribute to this restraint.

Gamma-hydroxybutyrate promotes oscillatory activity of rat and cat thalamocortical neurons by a tonic GABAB receptor-mediated hyperpolarization

The actions of gamma-hydroxybutyrate, a drug known to lead to an increase in nocturnal slow wave sleep and induce epileptic states following systemic application, on the membrane properties of thalamocortical neurons from brain slices of the rat and cat dorsal lateral geniculate nucleus were studied using sharp electrode intracellular recordings. Gamma-hydroxybutyrate applied by addition to the perfusion medium led to a concentration-dependent and reversible hyperpolarization of the membrane potential accompanied by a decrease in apparent input resistance (0.1 mM: 2.3 +/- 0.3 mV, 9.5 +/- 1.0%; 10 mM: 11.3 +/- 1.3 mV, 37.5 +/- 10.8%, respectively). In six of seven neurons the iontophoretic or bath (0.1-0.2 mM) application of low concentrations of gamma-hydroxybutyrate led to a hyperpolarization accompanied by the appearance of low-frequency (< 4 Hz) membrane potential oscillations crowned by bursts of action potentials, when the membrane potential of these neurons was initially set depolarized to the range where ongoing oscillatory activity occurred. The gamma-hydroxybutyrate-elicited hyperpolarization was reversibly antagonized by the co-application of the GABAB receptor antagonist CGP 35348 (0.4-1 mM), but was not affected by the putative gamma-hydroxybutyrate receptor antagonist NCS 382 (0.1-5 mM) or tetrodotoxin (1 microM), suggesting that gamma-hydroxybutyrate tonically activates postsynaptic GABAB receptors. The gamma-hydroxybutyrate-induced promotion of oscillatory activity and action potential burst firing of thalamocortical neurons may be one mechanism by which gamma-hydroxybutyrate leads to an increase in the deep stages of sleep and the generation of electroencephalogram and behavioural patterns typical of absence epilepsy.

Modulation of the GABAA receptor complex by steroids in slices of rat cuneate nucleus.

1 Several derivatives of pregnane and androstane that have hypnotic properties have been investigated for their ability to potentiate responses to the GABAA receptor agonist muscimol and to reduce the effect of the non‐competitive GABAA antagonist picrotoxin. 2 Depolarizing responses to muscimol in slices of rat cuneate nucleus were potentiated most potently by 3α‐hydroxy, 5α‐pregnane‐11,20‐dione (alphaxalone), which gave half‐maximal potentiation at 0.15 μm. The 5β isomer of alphaxalone had little effect up to 3 μm but in analogues lacking an 11‐keto substituent (pregnanolones), both the 5α‐ and 5β‐isomers potentiated with potencies 20 and 10 times lower, respectively, than that of alphaxalone. The a configuration of the 3‐hydroxy was essential in alphaxalone, the 3β‐hydroxy isomer being inactive. However, there was little difference between the potencies of the 3α‐ and 3β‐hydroxy configurations in the pregnanolones, although the maximal effects of the 30‐hydroxy isomers were rather lower than those of the 3α‐hydroxy isomers. 3 Reductions in the effect of picrotoxin as an antagonist of muscimol were caused most potently by the 3α‐hydroxy pregnanolones, with a ten fold reduction in picrotoxin potency at 1 μm concentrations of these steroids. Alphaxalone and its 5β‐isomer were about half as potent. Androsterone was about 10 times less potent and the 3β‐hydroxy pregnanolones were ineffective. 4 This difference in the structure‐activity relationships for steroidal potentiation of muscimol and reduction in picrotoxin antagonism of muscimol is reminiscent of an analogous distinction found with the barbiturates.

Sensory input and burst firing output of rat and cat thalamocortical cells: The role of NMDA and non-NMDA receptors

1. Intracellular and patch‐clamp recordings were obtained from thalamocortical (TC) cells in the rat and cat dorsal lateral geniculate nucleus (dLGN) in vitro to study the role of N‐methyl‐D‐aspartate (NMDA) and non‐NMDA receptors in the synaptic potential and burst firing evoked by electrical stimulation of the optic tract. 2. At membrane potentials more positive than ‐65 mV, the sensory synaptic potential consisted of a fast EPSP that was followed by a smaller, slower component. At membrane potentials more negative than ‐65 mV, this slower component became more prominent owing to the presence of a low‐threshold (LT) Ca2+ potential, which in turn evoked a high‐frequency (> 150 Hz) burst of action potentials. The lower, but not the upper limit of the range of membrane potential over which burst firing occurred was dependent on the amplitude of the fast EPSP. 3. The non‐NMDA receptor antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 5‐10 microM) and 1‐(4‐amino‐phenyl)‐4‐methyl‐7,8‐methylene‐dioxy‐5H‐2,3‐ benzodiazepine (GYKI 52466, 100 microM) greatly depressed the fast EPSP, abolished the burst firing generated by the LT Ca2+ potential, and left a relatively small, slow EPSP, which was sensitive to the NMDA antagonist DL‐2‐amino‐5‐phosphonovaleric acid (DL‐AP5, 50‐100 microM). 4. In the absence of CNQX or GYKI 52466, DL‐AP5 depressed the slow but not the fast EPSP. DL‐AP5 also increased the latency of the first action potential evoked by the LT Ca2+ potential or even abolished the LT Ca2+ potential and associated burst firing. The latter effect was only present when this type of firing occurred within a small membrane potential range. 5. DL‐AP5 had no effect on the properties of the LT Ca2+ current IT, indicating that its effect on the burst firing was not mediated by a direct action on IT. 6. The response of TC cells to high‐frequency (100 Hz) stimulation consisted of an initial burst firing response, followed by a sustained depolarization that could reach firing threshold. This sustained depolarization was markedly depressed by DL‐AP5 but not by CNQX. 7. These results demonstrate that with low‐frequency stimulation of the sensory afferents, the generation of TC cell output in the rat and cat dLGN is mainly controlled by non‐NMDA receptors, while the contribution of NMDA receptors is limited to the burst firing generated by the LT Ca2+ potential, and depends on the membrane potential range over which this type of firing occurs.(ABSTRACT TRUNCATED AT 400 WORDS)

Reduction of sensory and metabotropic glutamate receptor responses in the thalamus by the novel metabotropic glutamate receptor-1-selective antagonist S-2-methyl-4-carboxy-phenylglycine

Previous work has shown that responses of thalamic neurons in vivo to the metabotropic glutamate receptor agonists 1S,3R-aminocyclopentane-1,3-dicarboxylate and S-3,5-dihydroxyphenylglycine can be reduced by a variety of phenylglycine antagonists. Responses of thalamic neurons to noxious thermal somatosensory stimuli were reduced in parallel by these antagonists, indicating that these responses are mediated by Group I metabotropic glutamate receptors (i.e. metabotropic glutamate receptor-1 and/or metabotropic glutamate receptor-5), which are known to be linked to phosphoinositol phosphate hydrolysis. The recent development of S-2-methyl-4-carboxyphenylglycine as an antagonist which is highly selective for metabotropic glutamate receptor-1 compared to metabotropic glutamate receptor-5 on human receptors expressed in AV-12 cells, now offers the possibility of discriminating between these two receptor subtypes in order to distinguish which is involved in thalamic responses. We have made recordings from single somatosensory neurons in the thalamus of the rat, and find that S-2-methyl-4-carboxy-phenylglycine is able to reduce responses of neurons to 1S,3R-aminocyclopentane-1,3-dicarboxylate, S-3,5-dihydroxyphenylglycine, and noxious stimuli without significant effect on responses to either N-methyl-D-aspartate or (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. These results suggest that excitatory responses of thalamic neurons to 1S,3R-aminocyclopentane-1,3-dicarboxylate and S-3,5-dihydroxyphenylglycine may be mediated by metabotropic glutamate receptor-1. Furthermore, the reduction of nociceptive responses by S-2-methyl-4-carboxy-phenylglycine indicates that metabotropic glutamate receptor-1 is involved in thalamic nociceptive processing and that such antagonists may have analgesic properties.

Group III metabotropic glutamate receptors control corticothalamic synaptic transmission in the rat thalamus in vitro

1 Corticothalamic (CT) EPSPs evoked at ≤ 0.1 Hz were recorded from thalamocortical neurones in the rat dorsal lateral geniculate nucleus in vitro, with both GABAA and GABAB receptors blocked. 2 The group III metabotropic glutamate (mGlu) receptor agonists L‐2‐amino‐4‐phosphono‐butyric acid (L‐AP4) and O‐phospho‐L‐serine (L‐SOP) both caused a concentration‐dependent depression of the CT EPSP. The maximum depression and EC50 values for these effects were 64.4 ± 3.8 % and 88.0 ± 24.7 μm for L‐AP4, and 42.0 ± 2.5 % and 958 ± 492 μm for L‐SOP, respectively (means ±s.e.m.). Neither agonist had any effect on membrane potential or input resistance. 3 The depression of the CT EPSP caused by L‐AP4 was reversed using the group III antagonist (S)‐2‐amino‐2‐methyl‐4‐phosphonobutanoic acid (MAP4, 1 mM), and the group II/III antagonist LY341495 (3 μm), but not using the group II antagonist (2S)‐α‐ethylglutamic acid (300 μm). The potencies of L‐AP4, L‐SOP and LY341495 indicate that this action of L‐AP4 is mediated via mGlu7 and mGlu8 and not mGlu4 receptors. 4 Neither MAP4 nor LY341495 had any effect on the CT EPSPs evoked by 10 Hz trains of five stimuli, indicating the lack of endogenous activation of group III mGlu receptors in the thalamus during short bursts of cortical input. However, the magnitude of the depression caused by L‐AP4 indicates that any physiological activation of group III mGlu receptors would have a profound effect on the CT input to the thalamus, and hence cortical control of thalamic function.

On the nature of anomalous rectification in thalamocortical neurones of the cat ventrobasal thalamus in vitro

1 Intracellular sharp electrode current clamp and discontinuous single electrode voltage clamp recordings were made from thalamocortical neurones (n= 57) of the cat ventrobasal thalamus in order to investigate the mechanisms underlying anomalous rectification. 2 Under current clamp conditions, voltage‐current (V–I) relationships in a potential range of −55 to −110 mV demonstrated anomalous rectification with two components: fast rectification, which controlled the peak of negative voltage deviations, and time‐dependent rectification. Time‐dependent rectification was apparent as a depolarizing sag generated during the course of negative voltage deviations, was first formed at potentials in the range −60 to −70 mV, and was sensitive to 3 mm Cs+ (n= 6). Similarly, under voltage clamp conditions, instantaneous and steady‐state I–V relationships demonstrated anomalous rectification. A slowly activating inward current with an activation threshold in the range of −65 to −70 mV formed time‐dependent rectification. This current was sensitive to Cs+ (3 mm) (n= 3) and had properties similar to the slow inward mixed cationic current (Ih). 3 4‐(N‐Ethyl‐N‐phenylamino)‐l,2‐dimethyl‐6‐(methylamino)‐pyrimidinium chloride (ZD7288) (100–300 μm) irreversibly blocked time‐dependent rectification mediated by Ih (n= 23 of 25 neurones), and led to a hyperpolarization of the resting membrane potential (6.8 ± 0.5 mV). In the presence of ZD 7288, V–I and I–V relationships exhibited fast anomalous rectification, first activated from potential more negative than −80 mV. 4 Ba2+ (100 μm) (n= 8), in the continuous presence of ZD 7288, reversibly linearized peak V–I and instantaneous I–V relationships over a potential range of −70 to −120 mV, and led to a membrane depolarization (13.3 ± 4.2 mV) or tonic inward current (192 ± 36 pA). 5 The co‐application of ZD 7288 and Ba2+ revealed a depolarizing sag in negative voltage deviations under current clamp conditions, or a large inward current with kinetics two to three times slower than those of Ih under voltage clamp conditions. This novel form of time‐dependent rectification was first apparent at potentials more negative than about −85 mV, was sensitive to 5 mm Cs+ (n= 4), and is termed Ih,slow. Ih,slow tail currents reversed between −65.3 and −56.6 mV (with potassium acetate electrodes, n= 3) or −57.6 and −50.3 mV (with KCl electrodes, n= 3). 6 Computer simulations confirmed that the pattern of anomalous rectification in thalamocortical neurones of the cat ventrobasal thalamus is mediated by the concerted action of Ih and a Ba2+ ‐sensitive current with properties similar to an inwardly rectifying K+ current (IKIR).

Potentiators of responses to activation of gamma-aminobutyric acid (GABAA) receptors.

Quantitative aspects of the potentiation of GABA and muscimol by benzodiazepines and barbiturates are reviewed, taking account of both electrophysiological and receptor binding data. It has been a consistent finding that barbiturates cause a greater maximal potentiation than do benzodiazepines. The steroid anaesthetic alphaxalone and some naturally occurring steroids were compared as potentiators of electrophysiological responses to muscimol. From the relative potencies, important structural features of the steroid molecule for this effect have been identified. The possibility of the barbiturates and the steroids having a common mode of action as potentiators of GABA and muscimol is discussed, together with the idea that this action may involve perturbation of membrane lipids rather than a barbiturate/steroid receptor site. The GABA-potentiating effect of ethanol may also be barbiturate-like but potentiations by chlormethiazole and ketamine appear to involve different mechanisms. It is predicted that any endogenous potentiators of GABA would be unlikely to have more than a modest effect.