T.E. Salt

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.

Contributions of mGlu1 and mGlu5 receptors to interactions with N-methyl-d-aspartate receptor-mediated responses and nociceptive sensory responses of rat thalamic neurons

The nociceptive responses of rat ventrobasal thalamus neurons can be reduced by N-methyl-D-aspartate antagonists and by selective metabotropic glutamate receptor mGlu1 antagonists. The recent development of the mGlu5-selective antagonist 6-methyl-2-(phenylethynyl)-pyridine now allows the direct probing of the possible involvement of mGlu5 receptors in thalamic nociceptive responses. Extracellular recordings were made from single neurons in the ventrobasal thalamus and immediately overlying dorsal thalamic nuclei of adult urethane-anaesthetized rats using multi-barrel electrodes. Responses of neurons to iontophoretic applications of the mGlu5-selective agonist (R,S)-2-chloro-5-hydroxyphenylglycine were selectively reduced during continuous iontophoretic applications of 6-methyl-2-(phenylethynyl)-pyridine. Similar applications of 6-methyl-2-(phenylethynyl)-pyridine reduced neuronal responses to noxious thermal stimuli to 53+/-9.5% of control responses. Co- application by iontophoresis of N-methyl-D-aspartate and metabotropic glutamate receptor agonists resulted in a mutual potentiation of excitatory responses. This effect could be reduced by either 6-methyl-2-(phenylethynyl)-pyridine or the mGlu1 antagonist LY367385. These results, taken together with previous data, suggest that acute thalamic nociceptive responses are mediated by a combination of mGlu1, mGlu5 and N-methyl-D-aspartate receptor activation, and that co-activation of these receptors produces a synergistic excitatory effect. Thus blockade of any of these receptor types would have a profound effect on the overall nociceptive response.

Different roles for GABAA and GABAB receptors in visual processing in the rat superior colliculus

1 The superficial grey layer of the superior colliculus (SGS) contains a high proportion of GABAergic inhibitory neurones. We have investigated the role of GABA receptors in synaptic transmission of aspects of visual activity in the SGS that may be driven by inhibitory mechanisms, such as surround inhibition and response habituation. 2 Multi‐barrel glass iontophoretic pipettes were used to record single neuronal activity in the SGS of urethane‐anaesthetized rats. Visual stimulation was provided by the display of moving bars and stationary spots of light on a monitor placed in the receptive field. 3 Both ejection of GABA and the GABAB agonist baclofen reduced responses to moving bars (interstimulus intervals ≥ 8 s). The effects of GABA were reversed by the GABAA antagonist bicuculline, and the effects of baclofen were antagonized by the GABAB antagonist CGP 35348. 4 Surround inhibition was estimated by plotting the response to flashed spots of increasing diameter. In controls, expanding the spot diameter beyond the excitatory receptive field caused a decrease in the response. This inhibitory surround was reversibly reduced by bicuculline, but CGP 35348 had no effect. 5 Response habituation is the progressive reduction in the visual response during repetitive stimulus presentation. In controls, the visual response was reduced to 44 ± 3% of its initial level when a stimulus (moving bar) was presented 5 times with an interstimulus interval of 0.5 s. During CGP 35348 ejection, response habituation was reversibly reduced. Bicuculline had no effect on response habituation. 6 The effects of bicuculline on surround inhibition in the superior colliculus are consistent with similar studies in the lateral geniculate nucleus which indicate that GABAA receptors mediate this effect. The function of GABAB receptors in the visual system is less well researched. The reduction of response habituation with CGP 35348 demonstrates that, at least in the SGS, GABAB receptors have an important role in visual transmission which is distinct from that of GABAA receptors.

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.

Role of N-methyl-d-aspartate and metabotropic glutamate receptors in corticothalamic excitatory postsynaptic potentials In vivo

The ventrobasal thalamus is the principal somatosensory thalamic relay nucleus, and it receives two major sources of excitatory input: firstly an input from ascending sensory afferents, and secondly a descending projection from the primary somatosensory cortex. There is considerable anatomical evidence to suggest that both of these projections utilise the excitatory amino acid L-glutamate as their neurotransmitter. Previous work from this laboratory has shown that the sensory input to the rat ventrobasal thalamus in vivo is mediated by ionotropic excitatory amino acid receptors of both the N-methyl-D-aspartate and non-N-methyl-D-aspartate type. These findings are consistent with data from other studies in various thalamic relay nuclei. In contrast, there are considerably less data available concerning the synaptic pharmacology of the corticothalamic projection although there have been both speculation and studies concerning the functional significance of this pathway. There is some evidence to suggest an involvement of N-methyl-D-aspartate receptors and metabotropic glutamate receptors. The aim of this study was to determine which excitatory amino acid receptors might mediate cortically-elicited excitatory postsynaptic potential in the ventrobasal thalamus in vivo. Intracellular recordings were made, and neurotransmitter antagonists were applied on to rat ventrobasal thalamus neurons by microiontophoresis. Cortically-elicited excitatory postsynaptic potentials were reduced by the N-methyl-D-aspartate antagonist 3-[(+/-)-2-carboxy-piperazin-4-yl]-propyl-1-phosphonate, or the Group I metabotropic antagonist (S)-4-carboxyphenylglycine. These data indicate that both N-methyl-D-aspartate receptors and Group I (possibly metabotropic glutamate receptors type I) metabotropic receptors are involved in the mediation of corticothalamic transmission. Such a transmitter mechanism would allow a modulatory system that could selectively enhance other excitatory inputs. Some of these data have been reported in abstract form.

Synaptic activation of the group I metabotropic glutamate receptor mGlu1 on the thalamocortical neurons of the rat dorsal lateral geniculate nucleus in vitro

Intracellular recordings were made from thalamocortical neurons in slices of rat dorsal lateral geniculate nucleus in vitro, where ionotropic glutamate receptors and ionotropic and metabotropic GABA receptors had been blocked. The activation of specific metabotropic glutamate receptors by exogenous agonists and by the electrical stimulation of the corticothalamic pathway was then assessed using selective antagonists. The specific group I agonist (S)-3, 5-dihydoxyphenylglycine and the non-selective agonist (1S, 3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid both caused a concentration-dependent depolarization of membrane potential. These effects were associated with an increase in the apparent input resistance, and a more robust expression of both the depolarizing sag of the voltage response and the low-threshold Ca(2+) potential and an increase in thalamocortical neuron excitability. However, group I agonists selective for the mGlu5 receptor and agonists selective for group II and III receptors did not have these effects. Consequently, these data suggested that these actions were mediated specifically by the group I mGlu1 receptor. The activation of cortical fibres, with trains of 50 stimuli at 50Hz, resulted in a two-component depolarizing response. The first part of this synaptic response and the agonist-induced depolarization of membrane potential were depressed by the novel group I receptor antagonists LY367366 and LY367385, which are active at mGlu1 receptors. However, they were not blocked by 6-methyl-2-(phenylethyl)-pyridine, a highly selective mGlu5 receptor antagonist.Thus, the membrane potential depolarization of thalamocortical neurons caused either by exogenous agonists or by the stimulation of cortical fibres resulted from the specific activation of mGlu1 but not mGlu5 receptors. This result is consistent with the location of this receptor type on the distal dendrites of thalamocortical neurons in the dorsal lateral geniculate nucleus of the thalamus.

Modulation of sensory inhibition in the ventrobasal thalamus via activation of group II metabotropic glutamate receptors by 2R,4R-aminopyrrolidine-2,4-dicarboxylate

Abstractu2002Recordings were made from single neurones responsive to somatosensory input in the ventrobasal thalamus of the anaesthetised rat. GABAergic afferent inhibition arising from the thalamic reticular nucleus was evoked using a condition-test vibrissal stimulation paradigm. Local iontophoretic application of the group II metabotropic glutamate receptor (mGluR) agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC) in the vicinity of the recorded neurones produced a reduction of the afferent inhibition (from 78±3.0% to 25±5.3%), presumably via a presynaptic mechanism. This effect could be antagonised by LY307452, a known group II mGluR antagonist. In contrast, two selective group I mGluR agonists, (S)-3,5-dihydroxyphenylglycine (DHPG) and trans-azetidine-2,4-dicarboxylate (tADA), were without effect on the GABAergic inhibition. These data show that group II but not group I mGluRs can have a significant role in the modulation of GABAergic afferent inhibition in the ventrobasal thalamus. This could be of importance in the control of sensory discriminative processes and functions of sleep, arousal and seizure generation.

CHARACTERIZATION OF THE METABOTROPIC GLUTAMATE RECEPTORS (mGluRs) WHICH MODULATE GABA-MEDIATED INHIBITION IN THE VENTROBASAL THALAMUS

The ventrobasal thalamus (VB) relays and processes somatosensory information ascending to the cerebral cortex. Several types of mGluR are known to be present in VB, and we have previously shown that Group II and Group III mGluR agonists can reduce inhibitory synaptic transmission by acting at presynaptic receptors on GABAergic terminals in this structure. We have tested the action of several antagonists against the disinhibitory action of the Group II agonist CCG-I [(2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine] and the Group III agonist L-AP4 [L-2-amino-4-phosphonobutyrate] in the VB of anaesthetized rats using extracellular single-neurone recording techniques and iontophoretic applications of mGluR antagonists and agonists. The antagonists MAP4 [alpha-methyl-L-AP4] and MPPG [(+/-)-alpha-methyl-4-phosphonophenylglycine] reduced the disinhibitory actions of L-AP4 whilst having little effect on the disinhibitory action of CCG-I. In contrast, MCCG [alpha-methyl-CCG-I] and MCPG [(+)-alpha-methyl-4-carboxyphenylglycine] antagonized CCG-I, whilst having less effect against L-AP4 responses. These results support the hypothesis that GABAergic inhibitory transmission in VB can be modulated by at least two types of mGluR, belonging to Group II and Group III. Furthermore, the novel antagonists appear to be useful tools for the future study of the physiological role of these receptors in thalamic sensory processing.

Evaluation of agonists and antagonists acting at Group I metabotropic glutamate receptors in the thalamus in vivo.

Recordings were made from single neurones in the ventrobasal thalamus of anaesthetised rats in order to evaluate the properties of several agonists and antagonists of Group I mGlu receptors. The selective mGlu1 receptor antagonist LY367385 was found to reduce excitatory responses to iontophoretically applied ACPD and DHPG whereas the mGlu5 agonist CHPG was resistant to antagonism. The antagonists LY367366 and LY393053 reduced responses to all three agonists, but without reducing responses to NMDA or AMPA. Although AIDA was also found to reduce mGlu agonist-evoked responses, this antagonist also produced significant reductions in responses to NMDA and AMPA. These data suggest that there are functional mGlu1 and mGlu5 receptors in the thalamus. Furthermore, LY367385 is a useful tool for investigating mGlu1 functions whereas LY367366 and LY393053 have a broader spectrum of action. The usefulness of AIDA as an antagonist in physiological experiments would appear to be limited by its effects against NMDA and AMPA.

Developmental changes in NMDA receptor-mediated visual activity in the rat superior colliculus, and the effect of dark rearing.

Abstractu2002N-methyl-D-aspartate (NMDA) receptor-mediated activity is considered important for experience-dependent plasticity in the developing visual system. We investigated the influence of age and experience on the role of NMDA receptors in the visual transmission in the superficial grey layer of the superior colliculus (SGS) of the superior colliculus, where, in the adult, NMDA receptors mediate a substantial part of the visual response. In normally reared (postnatal day 14, P14, to adult) rats, visual responses were challenged with NMDA receptor-selective iontophoretic applications of the antagonist D-2-amino-5-phosphonovalerate (AP5). After eye opening (at P14), there was a significant increase in the number of neurones whose visual responses were reduced during AP5 ejection, which peaked at P22 (85%; n = 21), and then declined to adult levels (66%; n = 47) at P25. The mean reduction of the response (from control levels) by AP5 was similar at all ages (approximately 40%). Dark rearing had striking effects on the role of NMDA receptors in visual transmission, especially when comparisons were made between age-matched subjects greater than P25. In these subjects, AP5 ejection reduced the visual responses of all neurones studied. In addition, AP5 ejection caused a significantly larger reduction of visual responses in dark-reared rats (mean reduction 62 ± 4; n = 29) compared with age-matched controls (mean reduction 44 ± 8; n = 23). The D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reduced the visual responses of every neurone studied and there were no age- or experience-dependent effects. We conclude that NMDA receptors, but not AMPA receptors, assume greater importance for visual transmission in the SGS of dark-reared rats.