Ceri H. Davies

A hippocampal GluR5 kainate receptor regulating inhibitory synaptic transmission

The principal excitatory neurotransmitter in the vertebrate central nervous system, L-glutamate, acts on three classes of ionotripic glutamate receptors, named after the agonists AMPA (α-amino-3-hydroxy-5-methyl-4-isoxalole-4-propionic acid), NMDA ( N -methyl-D-aspartate) and kainate. The development of selective pharmacological agents has led to a detailed understanding ofthe physiological and pathological roles of AMPA and NMDA receptors. In contrast, the lack of selective kainate receptor ligands has greatly hindered progress in understanding the rolesof kainate receptors,. Here we describe the effects of a potent and selective agonist, ATPA (( RS)-2-amino-3-(3-hydroxy-5- tert -butylisoxazol-4-yl)propanoic acid) and a selective antagonist, LY294486 ((3SR, 4aRS, 6SR, 8aRS)-6-((((1H-tetrazol-5-yl) methyl)oxy)methyl)-1, 2, 3, 4, 4a, 5, 6, 7, 8, 8a-decahydroisoquinoline-3-carboxylic acid), of the GluR5 subtype of kainate receptor. We have used these agents to show that kainate receptors, comprised of or containing GluR5 subunits, regulate synaptic inhibition in the hippocampus, an action that could contribute to the epileptogenic effects of kainate.

Epileptogenesis and Enhanced Prepulse Inhibition in GABA B1 -Deficient Mice

The recent cloning of two GABA(B) receptor subunits, GABA(B1) and GABA(B2), has raised the possibility that differences in GABA(B) receptor subunit composition may give rise to pharmacologically or functionally distinct receptors. If present, such molecular diversity could permit the selective targeting of GABA(B) receptor subtypes specifically involved in pathologies such as drug addiction, spasticity, pain, and epilepsy. To address these issues we have developed a GABA(B1) subunit knockout mouse using gene targeting techniques. In the brains of GABA(B1) null mice, all pre- and postsynaptic GABA(B) receptor function was absent demonstrating that the GABA(B1) subunit is essential for all GABA(B) receptor-mediated mechanisms. Despite this, GABA(B1) null mice appeared normal at birth, although by postnatal week four their growth was retarded and they developed a generalized epilepsy that resulted in premature death. In addition, GABA(B1) heterozygote animals showed enhanced prepulse inhibition responses compared to littermate controls, suggesting that GABA(B1) deficient mice exhibit increased sensorimotor gating mechanisms. These data suggest that GABA(B) receptor antagonists may be of benefit in the treatment of psychiatric and neurological disorders in which attentional processing is impaired.

Identification and characterisation of SB-366791, a potent and selective vanilloid receptor (VR1/TRPV1) antagonist

Vanilloid receptor-1 (TRPV1) is a non-selective cation channel, predominantly expressed by peripheral sensory neurones, which is known to play a key role in the detection of noxious painful stimuli, such as capsaicin, acid and heat. To date, a number of antagonists have been used to study the physiological role of TRPV1; however, antagonists such as capsazepine are somewhat compromised by non-selective actions at other receptors and apparent modality-specific properties. SB-366791 is a novel, potent, and selective, cinnamide TRPV1 antagonist isolated via high-throughput screening of a large chemical library. In a FLIPR-based Ca(2+)-assay, SB-366791 produced a concentration-dependent inhibition of the response to capsaicin with an apparent pK(b) of 7.74 +/- 0.08. Schild analysis indicated a competitive mechanism of action with a pA2 of 7.71. In electrophysiological experiments, SB-366791 was demonstrated to be an effective antagonist of hTRPV1 when activated by different modalities, such as capsaicin, acid or noxious heat (50 degrees C). Unlike capsazepine, SB-366791 was also an effective antagonist vs. the acid-mediated activation of rTRPV1. With the aim of defining a useful tool compound, we also profiled SB-366791 in a wide range of selectivity assays. SB-366791 had a good selectivity profile exhibiting little or no effect in a panel of 47 binding assays (containing a wide range of G-protein-coupled receptors and ion channels) and a number of electrophysiological assays including hippocampal synaptic transmission and action potential firing of locus coeruleus or dorsal raphe neurones. Furthermore, unlike capsazepine, SB-366791 had no effect on either the hyperpolarisation-activated current (I(h)) or Voltage-gated Ca(2+)-channels (VGCC) in cultured rodent sensory neurones. In summary, SB-366791 is a new TRPV1 antagonist with high potency and an improved selectivity profile with respect to other commonly used TRPV1 antagonists. SB-366791 may therefore prove to be a useful tool to further study the biology of TRPV1.

A model of atropine-resistant theta oscillations in rat hippocampal area CA1.

Theta frequency oscillations are a predominant feature of rhythmic activity in the hippocampus. We demonstrate that hippocampal area CA1 generates atropine‐resistant theta population oscillations in response to metabotropic glutamate receptor activation under conditions of reduced AMPA receptor activation. This activity occurred in the absence of inputs from area CA3 and extra‐ammonic areas. Field theta oscillations were co‐expressed with pyramidal distal apical dendritic burst spiking and were temporally related to trains of IPSPs with slow kinetics. Pyramidal somatic responses showed theta oscillations consisted of compound inhibitory synaptic potentials with initial IPSPs with slow kinetics followed by trains of smaller, faster IPSPs. Pharmacological modulation of IPSPs altered the theta oscillation suggesting an inhibitory network origin. Somatic IPSPs, dendritic burst firing and stratum pyramidale interneuron activity were all temporally correlated with spiking in stratum oriens interneurons demonstrating intrinsic theta‐frequency oscillations. Disruption of spiking in these interneurons was accompanied by a loss of both field theta and theta frequency IPSP trains. We suggest that population theta oscillations can be generated as a consequence of intrinsic theta frequency spiking activity in a subset of stratum oriens interneurons controlling electrogenesis in pyramidal cell apical dendrites.

Region-specific reduction in entorhinal gamma oscillations and parvalbumin-immunoreactive neurons in animal models of psychiatric illness.

Psychiatric illnesses, particularly schizophrenia, are associated with disrupted markers for interneuronal function and interneuron-mediated brain rhythms such as gamma frequency oscillations. Here we investigate a possible link between these two observations in the entorhinal cortex and hippocampus by using a genetic and an acute model of psychiatric illness. Lysophosphatidic acid 1 receptor-deficient (LPA1-deficient) mice show psychomotor-gating deficits and neurochemical changes resembling those seen in postmortem schizophrenia studies. Similar deficits are seen acutely with antagonism of the NMDA subtype of glutamate receptor. Neither model induced any change in power or frequency of gamma rhythms generated by kainate in hippocampal slices. In contrast, a dramatic decrease in the power of gamma oscillations was seen in superficial, but not deep, medial entorhinal cortex layers in both models. Immunolabeling for GABA, parvalbumin, and calretinin in medial entorhinal cortex from LPA1-deficient mice showed an ∼40% reduction in total GABA- and parvalbumin-containing neurons, but no change in the number of calretinin-positive neurons. This deficit was specific for layer II (LII). No change in the number of neurons positive for these markers was seen in the hippocampus. Acute NMDA receptor blockade, which selectively reduces synaptic drive to LII entorhinal interneurons, also disrupted gamma rhythms in a similar manner in superficial entorhinal cortex, but not in hippocampus. These data demonstrate an area-specific deficit in gamma rhythmogenesis in animal models of psychiatric illness and suggest that loss, or reduction in function, of interneurons having a large NMDA receptor expression may underlie the network dysfunction that is seen.

Temporal Interactions between Cortical Rhythms

Multiple local neuronal circuits support different, discrete frequencies of network rhythm in neocortex. Relationships between different frequencies correspond to mechanisms designed to minimise interference, couple activity via stable phase interactions, and control the amplitude of one frequency relative to the phase of another. These mechanisms are proposed to form a framework for spectral information processing. Individual local circuits can also transform their frequency through changes in intrinsic neuronal properties and interactions with other oscillating microcircuits. Here we discuss a frequency transformation in which activity in two co-active local circuits may combine sequentially to generate a third frequency whose period is the concatenation sum of the original two. With such an interaction, the intrinsic periodicity in each component local circuit is preserved – alternate, single periods of each original rhythm form one period of a new frequency – suggesting a robust mechanism for combining information processed on multiple concurrent spatiotemporal scales.

Cholinergic modulation of hippocampal cells and circuits

Septo‐hippocampal cholinergic fibres ramify extensively throughout the hippocampal formation to release acetylcholine upon a diverse range of muscarinic and nicotinic acetylcholine receptors that are differentially expressed by distinct populations of neurones. The resultant modulation of cellular excitability and synaptic transmission within hippocampal circuits underlies the ability of acetylcholine to influence the dynamic properties of the hippocampal network and results in the emergence of a range of stable oscillatory network states. Recent findings suggest a multitude of actions contribute to the oscillogenic properties of acetylcholine which are principally induced by activation of muscarinic receptors but also regulated through activation of nicotinic receptor subtypes.

Regulation of EPSPs by the synaptic activation of GABAB autoreceptors in rat hippocampus.

1. Intracellular recording was used to study the influence of GABAB autoreceptor‐mediated regulation of monosynaptic GABAA and GABAB receptor‐mediated hyperpolarizing inhibitory postsynaptic potentials (IPSPAs and IPSPBs, respectively) on alpha‐amino‐3‐hydroxy‐5‐methyl ‐4‐isoxazolepropionic acid (AMPA) and N‐methyl‐D‐aspartate (NMDA) receptor‐mediated excitatory postsynaptic potentials (EPSPAs and EPSPNs, respectively) in the CA1 region of rat hippocampal slices. To achieve this, synaptic potential were evoked monosynaptically by near stimulation following blockade of either EPSPNs, by the NMDA receptor antagonist (R)‐2‐amino‐5‐phosphonopentanoate (AP5; 0.05 mM), or EPSPAs, by the AMPA/kainate receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 0.01 mM). 2. Paired‐pulse stimulation at 3‐50 Hz caused an increase in the duration (paired‐pulse widening) of EPSPAs, which paralleled the time course of paired‐pulse depression of monosynaptic IPSCs, and a potentiation of the amplitude (paired‐pulse potentiation) of EPSPAs, which did not. Paired‐pulse stimulation also caused frequency‐dependent changes in EPSPNs. At frequencies > 40 Hz it produced paired‐pulse depression of EPSPNs, along with marked summation of IPSPS, and at frequencies < 40 Hz it caused paired‐pulsed enlargement of EPSPNs, concomitant with a reduction in IPSPS. 3. Paired‐pulse potentiation of EPSPAs at 50 Hz was enhanced by picrotoxin (0.1 mM) but was not significantly affected by 3‐amino‐propyl(diethoxymethyl)phosphinic acid (CGP 35348; 1 mM). Paired‐pulse depression of EPSPNs at 50 Hz was converted to paired‐pulse enlargement by picrotoxin but was unaffected by CGP 35348. These effects can be explained by block of IPSPAs by picrotoxin. 4. Paired‐pulsed widening of EPSPAs at 5 Hz was occluded by picrotoxin and abolished by CGP 35348. Similarly, paired‐pulsed enlargement of EPSPNs at 5 Hz was occluded, and in some cases converted to paired‐pulse depression, by picrotoxin. The effects of CGP 35348 were more complex in that this antagonist reduced paired‐pulse enlargement of EPSPNs in control medium whereas it eliminated paired‐pulsed depression of EPSPNs in the presence of picrotoxin, effects consistent with its block of GABAB autoreceptors and IPSPBS, respectively. 5. ‘Priming’ using a ‘priming stimulation protocol’ (a single ‘priming stimulus’ followed at 1‐50 Hz (‘priming frequency’) by a ‘primed burst’ of four shocks at 20‐100 Hz (‘burst frequency’)) caused an increase in both ‘primed’ EPSPAs and EPSPNs compared with ‘unprimed’ EPSPAs and EPSPNs. This effect was optimal when the respective priming and burst frequencies were 5 and 100 Hz. 6. In the presence of either picrotoxin or CGP 35348 the primed EPSPAs and EPSPNs resembled unprimed EPSPAs and EPSPNs, respectively. This was because picrotoxin occluded whereas CGP 35348 blocked the effect of priming on EPSPS. 7. CGP 35348 had only modest effects on EPSPAs but enhanced EPSPNs evoked by a tetanus (20 stimuli at 100 Hz), in either the presence or absence of picrotoxin. In the absence of picrotoxin, CGP 35348 also promoted depolarization by enhancing a depolarizing GABAA receptor‐mediated component (IPSPD). These effects can all be attributed to block of IPSPBS by CGP 35348. 8. CGP 35348 blocked the induction of long‐term potentiation (LTP) of extracellularly recorded field EPSPs elicited by a priming stimulation protocol in control medium but was ineffective in the presence of picrotoxin. CGP 35348 was also ineffective at preventing tetanus‐induced LTP (100 Hz, 1 s) in both the absence and presence of picrotoxin. 9. These data demonstrate the complex regulation of AMPA and NMDA receptor‐mediated EPSPs during various patterns of synaptic activation caused by the dynamic changes in GABA‐mediated synaptic inhibition, which are orchestrated by GABAA autoreceptors in a frequency‐dependent

CGP 55845A: a potent antagonist of GABAB receptors in the CA1 region of rat hippocampus.

The new GABAB receptor antagonist CGP 55845A was tested on pre- and post-synaptic GABAB receptors in the hippocampus. CGP 55845A (1 microM) blocked (-)-baclofen (5-10 microM)-induced postsynaptic hyperpolarization and depression of evoked IPSPs and EPSPs. It also blocked three physiological consequences of GABAB receptor activation: the late IPSP, paired-pulse depression of IPSCs, and heterosynaptic depression of EPSPs. Therefore, CGP 55845A is an antagonist at pre- and post-synaptic GABAB receptors in the hippocampus and is approximately three orders of magnitude more potent than previously described GABAB receptor antagonists.

Gabapentin is not a GABAB receptor agonist.

Recent experiments have demonstrated that formation of functional type B gamma-aminobutyric acid (GABA(B)) receptors requires co-expression of two receptor subunits, GABA(B1) and GABA(B2). Despite the identification of these subunits and a number of associated splice variants, there has been little convincing evidence of pharmacological diversity between GABA(B) receptors comprising different subunit combinations. However, Ng et al. [Mol. Pharmacol., 59 (2000) 144] have recently suggested a novel and important pharmacological difference between GABA(B) receptor heterodimers expressing the GABA(B1a) and GABA(B1b) receptor subunits. This study suggested that the antiepileptic GABA analogue gabapentin (Neurontin) is an agonist at GABA(B) receptors expressing the GABA(B1a) but not the GABA(B1b) receptor subunit. The importance of this finding with respect to identifying novel GABA(B) receptor subunit specific agonists prompted us to repeat these experiments in our own [35S]-GTPgammaS binding and second messenger assay systems. Here we report that gabapentin was completely inactive at recombinant GABA(B) heterodimers expressing either GABA(B1a) or GABA(B1b) receptor subunits in combination with GABA(B2) receptor subunits. In addition, in both CA1 and CA3 pyramidal neurones from rodent hippocampal slices we were unable to demonstrate any agonist-like effects of gabapentin at either pre- or post-synaptic GABA(B) receptors. In contrast, gabapentin activated a GABA(A) receptor mediated chloride conductance. Our data suggest that gabapentin is not a GABA(B)-receptor agonist let alone a GABA(B) receptor subunit selective agonist.