Igor Spigelman

GABAA receptor α4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol

The neurotransmitter GABA mediates the majority of rapid inhibition in the CNS. Inhibition can occur via the conventional mechanism, the transient activation of subsynaptic GABAA receptors (GABAA-Rs), or via continuous activation of high-affinity receptors by low concentrations of ambient GABA, leading to “tonic” inhibition that can control levels of excitability and network activity. The GABAA-R α4 subunit is expressed at high levels in the dentate gyrus and thalamus and is suspected to contribute to extrasynaptic GABAA-R-mediated tonic inhibition. Mice were engineered to lack the α4 subunit by targeted disruption of the Gabra4 gene. α4 Subunit knockout mice are viable, breed normally, and are superficially indistinguishable from WT mice. In electrophysiological recordings, these mice show a lack of tonic inhibition in dentate granule cells and thalamic relay neurons. Behaviorally, knockout mice are insensitive to the ataxic, sedative, and analgesic effects of the novel hypnotic drug, gaboxadol. These data demonstrate that tonic inhibition in dentate granule cells and thalamic relay neurons is mediated by extrasynaptic GABAA-Rs containing the α4 subunit and that gaboxadol achieves its effects via the activation of this GABAA-R subtype.

Status epilepticus-induced hilar basal dendrites on rodent granule cells contribute to recurrent excitatory circuitry

Mossy fiber sprouting into the inner molecular layer of the dentate gyrus is an important neuroplastic change found in animal models of temporal lobe epilepsy and in humans with this type of epilepsy. Recently, we reported in the perforant path stimulation model another neuroplastic change for dentate granule cells following seizures: hilar basal dendrites (HBDs). The present study determined whether status epilepticus‐induced HBDs on dentate granule cells occur in the pilocarpine model of temporal lobe epilepsy and whether these dendrites are targeted by mossy fibers. Retrograde transport of biocytin following its ejection into stratum lucidum of CA3 was used to label granule cells for both light and electron microscopy. Granule cells with a heterogeneous morphology, including recurrent basal dendrites, and locations outside the granule cell layer were observed in control preparations. Preparations from both pilocarpine and kainate models of temporal lobe epilepsy also showed granule cells with HBDs. These dendrites branched and extended into the hilus of the dentate gyrus and were shown to be present on 5% of the granule cells in pilocarpine‐treated rats with status epilepticus, whereas control rats had virtually none. Electron microscopy was used to determine whether HBDs were postsynaptic to axon terminals in the hilus, a site where mossy fiber collaterals are prevalent. Labeled granule cell axon terminals were found to form asymmetric synapses with labeled HBDs. Also, unlabeled, large mossy fiber boutons were presynaptic to HBDs of granule cells. These results indicate that HBDs are present in the pilocarpine model of temporal lobe epilepsy, confirm the presence of HBDs in the kainate model, and show that HBDs are postsynaptic to mossy fibers. These new mossy fiber synapses with HBDs may contribute to additional recurrent excitatory circuitry for granule cells. J. Comp. Neurol. 428:240–253, 2000.

Dentate granule cells form novel basal dendrites in a rat model of temporal lobe epilepsy

Mossy fibre sprouting and re-organization in the inner molecular layer of the dentate gyrus is a characteristic of many models of temporal lobe epilepsy including that induced by perforant-path stimulation. However, neuroplastic changes on the dendrites of granule cells have been less-well studied. Basal dendrites are a transient morphological feature of rodent granule cells during development. The goal of the present study was to examine whether granule cell basal dendrites are generated in rats with epilepsy induced by perforant-path stimulation. Adult Wistar rats were stimulated for 24 h at 2 Hz and with intermittent (1/min) trains (10 s duration) of single stimuli at 20 Hz (20 V, 0.1 ms) delivered 1/min via an electrode placed in the angular bundle. The brains of these experimental rats and age- and litter-matched control animals were processed for the rapid Golgi method. All rats with perforant-path stimulation displayed basal dendrites on many Golgi-impregnated granule cells. These basal dendrites mainly originated from their somata at the hilar side and then extended into the hilus. Quantitative analysis of more than 800 granule cells in the experimental and matched control brains showed that 6-15% (mean=8.7%) of the impregnated granule cells have spiny basal dendrites on the stimulated side, as well as the contralateral side (mean=3.1%, range=2.9-3.9%) of experimental rats, whereas no basal dendrites were observed in the dentate gyrus from control animals. The formation of basal dendrites appears to be an adaptive morphological change for granule cells in addition to the previously described mossy fibre sprouting, as well as dendritic and somatic spine formation observed in the dentate gyrus of animal and human epileptic brains. The presence of these dendrites in the subgranular region of the hilus suggests that they may be postsynaptic targets of the mossy fibre collaterals.

Site-specific increases in peripheral cannabinoid receptors and their endogenous ligands in a model of neuropathic pain

&NA; Selective activation of the peripheral cannabinoid receptor 1 (CB1R) has been shown to suppress neuropathic pain symptoms in rodents. However, relatively little is known about changes in CB1R and its endogenous ligands during development or maintenance of neuropathic pain. Using immunohistochemistry, Western blot, real‐time reverse transcription polymerase chain reaction, as well as liquid chromatography/mass spectrometry, we studied the changes in CB1Rs and endocannabinoids N‐arachidonoylethanolamine/anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG) in rat lumbar (L4 and L5) dorsal root ganglia (DRG) after neuropathic pain induction (L5 spinal nerve ligation: SNL). Immunohistochemistry revealed that in control rats, CB1R is expressed in the majority (76–83%) of nociceptive neurons as indicated by co‐labeling with isolectin B4 (IB4) or antibodies recognizing transient receptor potential vanilloid (TRPV1), calcitonin gene related peptide (CGRP), and the NR2C/2D subunits of the N‐methyl‐ d‐aspartate receptor. After L5 SNL, CB1R mRNA and protein increases in the ipsilateral uninjured L4 DRG whereas the percentages of CB1R immunoreactive (CB1R‐ir) neurons remain unchanged in L4 and L5 DRG. However, for these CB1R‐ir neurons, we observe significant increases in percentage of TRPV1‐ir cells in ipsilateral L4 DRG, and decreases in percentage of IB4‐ and CGRP‐co‐labeled cells in ipsilateral L5 DRG. Levels of both AEA and 2‐AG increase significantly only in the ipsilateral L5 DRG. These results are consistent with the preserved analgesic effects of cannabinoids in neuropathic pain and provide a rational framework for the development of peripherally acting endocannabinoid‐based therapeutic interventions for neuropathic pain.

Chronic Intermittent Ethanol-Induced Switch of Ethanol Actions from Extrasynaptic to Synaptic Hippocampal GABAA Receptors

Alcohol withdrawal syndrome (AWS) symptoms include hyperexcitability, anxiety, and sleep disorders. Chronic intermittent ethanol (CIE) treatment of rats with subsequent withdrawal of ethanol (EtOH) reproduced AWS symptoms in behavioral assays, which included tolerance to the sleep-inducing effect of acute EtOH and its maintained anxiolytic effect. Electrophysiological assays demonstrated a CIE-induced long-term loss of extrasynaptic GABAA receptor (GABAAR) responsiveness and a gain of synaptic GABAAR responsiveness of CA1 pyramidal and dentate granule neurons to EtOH that we were able to relate to behavioral effects. After CIE treatment, the α4 subunit-preferring GABAAR ligands 4,5,6,7 tetrahydroisoxazolo[5,4-c]pyridin-3-ol, La3+, and Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5α][1,4]benzodiazepine-3-carboxylate) exerted decreased effects on extrasynaptic currents but had increased effects on synaptic currents. Electron microscopy revealed an increase in central synaptic localization of α4 but not δ subunits within GABAergic synapses on the dentate granule cells of CIE rats. Recordings in dentate granule cells from δ subunit-deficient mice revealed that this subunit is not required for synaptic GABAAR sensitivity to low [EtOH]. The profound alterations in EtOH sensitivity and α4 subunit localization at hippocampal GABAARs of CIE rats suggest that such changes in these and other relevant brain circuits may contribute to the development of tolerance to the sleep-inducing effects and long-term dependence on alcohol.

Mechanisms of Reversible GABAA Receptor Plasticity after Ethanol Intoxication

The time-dependent effects of ethanol (EtOH) intoxication on GABAA receptor (GABAAR) composition and function were studied in rats. A cross-linking assay and Western blot analysis of microdissected CA1 area of hippocampal slices obtained 1 h after EtOH intoxication (5 g/kg, gavage), revealed decreases in the cell-surface fraction of α4 and δ, but not α1, α5, or γ2 GABAAR subunits, without changes in their total content. This was accompanied (in CA1 neuron recordings) by decreased magnitude of the picrotoxin-sensitive tonic current (Itonic), but not miniature IPSCs (mIPSCs), and by reduced enhancement of Itonic by EtOH, but not by diazepam. By 48 h after EtOH dosing, cell-surface α4 (80%) and γ2 (82%) subunit content increased, and cell-surface α1 (−50%) and δ (−79%) and overall content were decreased. This was paralleled by faster decay of mIPSCs, decreased diazepam enhancement of both mIPSCs and Itonic, and paradoxically increased mIPSC responsiveness to EtOH (10–100 mm). Sensitivity to isoflurane- or diazepam-induced loss of righting reflex was decreased at 12 and 24 h after EtOH intoxication, respectively, suggesting functional GABAAR tolerance. The plastic GABAAR changes were gradually and fully reversible by 2 weeks after single EtOH dosing, but unexplainably persisted long after withdrawal from chronic intermittent ethanol treatment, which leads to signs of alcohol dependence. Our data suggest that early tolerance to EtOH may result from excessive activation and subsequent internalization of α4βδ extrasynaptic GABAARs. This leads to transcriptionally regulated increases in α4 and γ2 and decreases in α1 subunits, with preferential insertion of the newly formed α4βγ2 GABAARs at synapses.

Concurrent release of ATP and substance P within guinea pig trigeminal ganglia in vivo.

Neurons within sensory ganglia have been proposed to communicate via non-synaptic release of a diffusible chemical messenger, but the identity of the chemical mediator(s) remains unknown [J. Neurosci. 16 (1996) 4733-4741]. The present study addressed the possibility of co-released ATP and substance P (SP) within sensory ganglia to further advance the hypothesis of non-synaptic communication between sensory neurons. Microdialysis probes inserted into trigeminal ganglia (TRGs) of anesthetized guinea pigs were perfused with artificial cerebrospinal fluid and the collected perfusate analyzed for ATP and SP content using the firefly luciferin-luciferase (L/L) assay and radioimmunoassay, respectively. Significant reversible increases in ATP and SP levels were observed after infusion of 100 mM KCl or 1 mM capsaicin. Ca(2+)-free ACSF produced an eightfold increase in ATP levels, interpreted as a decrease in activity of Ca(2+)-dependent ecto-nucleotidases that degrade ATP. In contrast, KCl-induced release of ATP in the presence of normal Ca(2+) was blocked by Cd(2+), a voltage-gated Ca(2+) channel blocker, illustrating Ca(2+)-dependence of evoked ATP release. Since ganglionic release of ATP could arise from several neuronal and non-neuronal sources we directly tested acutely dissociated TRG neuron somata for ATP release. Neuron-enriched dissociated TRG cells were plated onto glass tubes and tested for ATP release using the L/L assay. Robust ATP release was evoked with 5 microM capsaicin. These data suggest that ATP is released concurrently with SP from the somata of neurons within sensory ganglia.

Behavior and Physiology of Mice Lacking the GABAA-Receptor δ Subunit

Summary:  Purpose: Mice with a targeted disruption of the γ‐aminobutyric acid (A) receptor (GABAAR) δ subunit exhibited spontaneous seizures and a strikingly selective attenuation of responses to neuroactive steroids, but not to other neuromodulators. This study further characterized the behavior and physiology of the δ mutants.

Persistent reduction of GABAA receptor-mediated inhibition in rat hippocampus after chronic intermittent ethanol treatment

GABA(A) receptor-mediated function was studied in rats treated with chronic intermittent ethanol (CIE). Rats were given 60 doses of 6g/kg ethanol every 24 h by gastric intubation, with repeated intoxicating and withdrawal episodes leading to a kindling-like increase in seizure susceptibility (Kokka et al., Alcohol: Clin. Exp. Res., 17 (1993) 525-531). Efflux of 36Cl-, evoked by application of muscimol, a measure of GABA(A) receptor function, was examined in 300 mu m slices obtained from frontal, parietal, and temporal cortex, hippocampus, and inferior colliculus, one day after the last administration of ethanol. Compared to controls, the 36Cl- efflux in hippocampal slices of CIE rats was significantly reduced by 29%, while there were no changes in the other brain regions studied. In hippocampal slices, paired-pulse inhibition in CA1 pyramidal neurons, measured extracellularly using homosynaptic orthodromic stimulation at an interval of 10 ms, was significantly reduced in CIE rats. A significant decrease by 40% both at 2 and 40 days after 60 doses of ethanol was found, implying a persistent decrease in GABA(A) receptor-mediated inhibition in CIE rats. These reductions in paired-pulse inhibition are consistent with the decrease in the pentylenetetrazol (PTZ) seizure threshold which was previously observed in CIE rats. Therefore, we suggest that this reduction of GABA(A) receptor-mediated inhibition contributes to the persistent increase in seizure susceptibility of CIE rats.

Increased peripheral nerve excitability and local NaV1.8 mRNA up-regulation in painful neuropathy

BackgroundNeuropathic pain caused by peripheral nerve injury is a chronic disorder that represents a significant clinical challenge because the pathological mechanisms have not been fully elucidated. Several studies have suggested the involvement of various sodium channels, including tetrodotoxin-resistant NaV1.8, in affected dorsal root ganglion (DRG) neurons. We have hypothesized that altered local expression of NaV1.8 in the peripheral axons of DRG neurons could facilitate nociceptive signal generation and propagation after neuropathic injury.ResultsAfter unilateral sciatic nerve entrapment injury in rats, compound action potential amplitudes were increased in both myelinated and unmyelinated fibers of the ipsilateral sciatic nerve. Tetrodotoxin resistance of both fiber populations and sciatic nerve NaV1.8 immunoreactivity were also increased. Further analysis of NaV1.8 distribution revealed that immunoreactivity and mRNA levels were decreased and unaffected, respectively, in the ipsilateral L4 and L5 DRG; however sciatic nerve NaV1.8 mRNA showed nearly an 11-fold ipsilateral increase. Nav1.8 mRNA observed in the sciatic nerve was likely of axonal origin since it was not detected in non-neuronal cells cultured from nerve tissue. Absence of changes in NaV1.8 mRNA polyadenylation suggests that increased mRNA stability was not responsible for the selective peripheral mRNA increase. Furthermore, mRNA levels of NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 were not significantly different between ipsilateral and contralateral nerves. We therefore propose that selective NaV1.8 mRNA axonal transport and local up-regulation could contribute to the hyperexcitability of peripheral nerves in some neuropathic pain states.ConclusionCuff entrapment injury resulted in significantly elevated axonal excitability and increased NaV1.8 immunoreactivity in rat sciatic nerves. The concomitant axonal accumulation of NaV1.8 mRNA may play a role in the pathogenesis of this model of neuropathic pain.