Alteration of interneuron migration in a ferret model of cortical dysplasia

During cerebral cortical development, gamma-aminobutyric acidergic (GABAergic) interneurons arise from a different site than projection neurons. GABAergic cells are generated in the subpallial ganglionic eminence (GE), while excitatory projection neurons arise from the neocortical ventricular zone. Our laboratory studies a model of cortical dysplasia that displays specific disruption of GABAergic mechanisms and an alteration in the overall balance of excitation in the neocortex. To produce this model, the birth of neurons on a specific gestational day in ferrets (embryonic day 33 [E33]) is interrupted by injection of the antimitotic methylazoxymethanol (MAM). We hypothesized that migration of interneurons might be disrupted in this cortical dysplasia paradigm. We observed that although interneurons migrate into the neocortex in both normal and dysplastic cortex, the migrating cells become disoriented over time after E33 MAM treatment. Coculture experiments using normal GE and MAM-treated cortex (and vice versa) demonstrate that cues dictating proper orientation of migrating interneurons arise from the cortex and are not intrinsic to the migrating cells. As a consequence, interneurons in mature brains of MAM-treated animals are abnormally distributed. We report that GABA(A) receptor activation is crucial to the proper positioning of interneurons migrating into the cortex from the GE in normal and MAM-treated animals.     

Receptor-activated calcium signals in tangentially migrating cortical cells

Recent studies have shown that the two main types of cortical neurons, pyramidal and nonpyramidal, have different origins and use different migratory routes--radial and tangential respectively. The role of neurotransmitters in radial migration is well known; however, there are no data about their effect on intracellular calcium--[Ca(2+)](i)--in tangentially migrating cells. We have performed ratiometric and confocal calcium imaging of 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine labelled tangentially migrating neurons in the intermediate zone cells of fetal rat coronal slices. Superfusion with N-methyl-D-aspartic acid (NMDA) leads to an increase in [Ca(2+)](i), which is blocked by the antagonist APV or the presence of Mg(2+) in the medium. Kainate produced an increase in [Ca(2+)](i) that could be blocked by the non-NMDA antagonist CNQX. Muscimol, an agonist of GABAa-receptors, produced a transitory increase in [Ca(2+)](i) that was blocked by the specific antagonist bicuculline or the presence of tetrodotoxin in the medium. We conclude that tangentially migrating cells display consistent [Ca(2+)](i) changes in response to agonists of NMDA, non-NMDA and GABAa receptors, suggesting that these cells are quite mature and homogeneous. The endogenous activation of these receptors may have either a direct effect on tangential migration or modulate the response of migrating cells to external cues.     

Blockade of GABA(B) receptors alters the tangential migration of cortical neurons

To better understand the role of neurotransmitter receptors in neuronal differentiation and maturation a detailed knowledge of their identity, location and function in the plasma membrane of specific neuronal populations during development is required. Combining pre-embedding immunocytochemistry with cell tracking in embryonic brain slice cultures we show that virtually all neurons (approximately 98%) migrating through the lower intermediate zone (LIZ) on their way from the medial ganglionic eminence to the cerebral cortex, express GABA(B)R1. Blockade of GABA(B)Rs with a specific antagonist, CGP52432, resulted in a concentration-dependent accumulation of these tangentially migrating neurons in the ventricular/sub-ventricular zones (VZ/SVZ) of the cortex and fewer cells were observed in the cortical plate/marginal zone (CP/MZ) and LIZ. Moreover, they had significantly shorter leading processes compared with similar migrating cells in control slices. Electrophysiological recording in LIZ and CP cells revealed no direct effect of either CGP52432 or the GABA(B)R agonist, baclofen, on resting membrane properties suggesting that the effect of CGP52432 on migration might be mediated through a metabotropic action or the regulation of release of factors controlling migration. These results suggest that GABA(B)Rs have an important modulatory role in the migration of cortical interneurons.     

GABAergic mechanism of propofol toxicity in immature neurons

Certain anesthetics exhibit neurotoxicity in the brains of immature but not mature animals. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, is excitatory on immature neurons via its action at the GABAA receptor, due to a reversed transmembrane chloride gradient. GABAA receptor activation in immature neurons is sufficient to open L-type voltage-gated calcium channels. As propofol is a GABAA agonist, we hypothesized that it and more specific GABAA modulators would increase intracellular free calcium ([Ca2+]i), resulting in the death of neonatal rat hippocampal neurons. Neuronal [Ca2+]i was monitored using Fura2-AM fluorescence imaging. Cell death was assessed by double staining with propidium iodide and Hoechst 33258 at 1 hour (acute) and 48 hours (delayed) after 5 hours exposure of neurons to propofol or the GABAA receptor agonist, muscimol, in the presence and absence of the GABA receptor antagonist, bicuculline, or the L-type Ca2+ channel blocker, nifedipine. Fluorescent measurements of caspase-3,-7 activities were performed at 1 hour after exposure. Both muscimol and propofol induced a rapid increase in [Ca2+]i in days in vitro (DIV) 4, but not in DIV 8 neurons, that was inhibited by nifedipine and bicuculline. Caspase-3,-7 activities and cell death increased significantly in DIV 4 but not DIV 8 hippocampal neuronal cultures 1 hour after 5 hours exposure to propofol, but not muscimol, and were inhibited by the presence of bicuculline or nifedipine. We conclude that an increase in [Ca2+]i, due to activation of GABAA receptors and opening of L-type calcium channels, is necessary for propofol-induced death of immature rat hippocampal neurons but that additional mechanisms not elicited by GABAA activation alone also contribute to cell death.     

GABA receptor antagonists modulate postmitotic cell migration in slice cultures of embryonic rat cortex

Recent studies indicate that GABA acts as a chemoattractant during rat cortical histogenesis. In vivo, GABA localizes in appropriate locations for a chemoattractant, along migratory routes and near target destinations for migrating cortical neurons. In vitro, GABA induces dissociated embryonic cortical neurons to migrate. Here, embryonic rat cortical slices were cultured in the presence or absence of GABA receptor (GABA-R) antagonists to assess GABA's effects on neuronal migration in situ. Gestational day 18 (E18) cortical slices were incubated overnight in bromodeoxyuridine (BrdU)-containing medium to label ventricular zone (vz) cells as they underwent terminal mitosis. The slices were then cultured in BrdU-free medium with or without GABA-R antagonists. In control slices, most BrdU(+) cells were observed in the cortical plate (cp) after 48 h. In contrast, cultures maintained in either saclofen (a GABA(B)-R antagonist) or picrotoxin (a GABA(A/C)-R antagonist) had few BrdU-labeled cp cells. However, the effects of the two antagonists were distinct. In the picrotoxin-treated slices, nearly half of all BrdU(+) cells remained in the vz and subventricular zone (svz), whereas saclofen treatment resulted in an accumulation of BrdU(+) cells in the intermediate zone (iz). Bicuculline, a GABA(A)-R antagonist, did not block, but rather enhanced migration of BrdU(+) cells into the cp. These results provide evidence that picrotoxin-sensitive receptors promote the migration of vz/svz cells into the iz, while saclofen-sensitive receptors signal cells to migrate into the cp. Thus, as cortical cells differentiate, changing receptor expression appears to modulate migratory responses to GABA.     

Electrophysiologic Evidence for Increased Endogenous GABAergic but Not Glycinergic Inhibitory Tone in the Rat Spinal Nerve Ligation Model of Neuropathy

Background: Changes in the inhibitory activity mediated by [gamma]-aminobutyric acid (GABA) and glycine, acting at spinal GABAA receptors and strychnine-sensitive glycine receptors, are of interest in the development of neuropathic pain. There is anatomic evidence for changes in these transmitter systems after nerve injuries, and blocking either GABAA or glycine receptors has been shown to produce allodynia-like behavior in awake normal animals.

Methods: In this study, the possible changes in GABAergic and glycinergic inhibitory activity in the spinal nerve ligation model of neuropathic pain were studied by comparing the effects of the GABAA-receptor antagonist bicuculline and the glycine-receptor antagonist strychnine in neuropathic rats to their effects in sham-operated and nonoperated control rats.

Results: Bicuculline produced a dose-related facilitation of the A[delta]-fiber-evoked activity in all study groups and increased C-fiber-mediated activity in the spinal nerve ligation group but not in either of the control groups. There were no differences in the effect of bicuculline on low threshold responses between the study groups. The glycine receptor antagonist strychnine did not have a statistically significant effect on any of the parameters studied in any of the control groups.     

gamma-Aminobutyric acid enhances the tone of human internal anal sphincter.

The effect of gamma-aminobutyric acid (GABA) on the human internal anal sphincter was investigated. Cumulative applications of GABA produced concentration-dependent contractions (10(-8)-10(-5) M) of the isolated human sphincter. Pretreatment with bicuculline (GABAA antagonist) turned them to relaxation. Muscimol, a GABAA agonist, induced concentration-dependent contractions (10(-8)-10(-5) M); however, baclofen (GABAB agonist, 10(-8)-10(-5) M) promoted concentration-dependent relaxation of the strips. These results suggested that both excitatory GABAA receptors and inhibitory GABAB receptors exist in the internal anal sphincter. Oral administration of sodium valproate (1600 mg/day), a GABA transaminase inhibitor, enhanced the anal canal resting pressure in 10 normal volunteers. Anal manometry showed a significant elevation in tonus without affecting amplitudes or frequencies. These results indicated that endogenous GABA, which was increased by sodium valproate, produced elevations in the anal canal resting pressure through its specific receptors in the human internal anal sphincter.     

Gamma-aminobutyric acidA receptors do not mediate the immobility produced by isoflurane

Many inhaled anesthetics enhance the effect of the inhibitory neurotransmitter gamma aminobutyric acid (GABA), supporting the view that the GABAA receptor could mediate the capacity of inhaled anesthetics to produce immobility in the face of noxious stimulation (i.e., MAC, the minimum alveolar concentration required to suppress movement in response to a noxious stimulus in 50% of subjects). However, only limited in vivo data support the relevance of the GABAA receptor to MAC. In the present study we used two findings to test for the relevance of this receptor to immobilization for isoflurane: 1) differences among anesthetics in their capacity to enhance the response of receptor expression systems to GABA: isoflurane (considerable enhancement), xenon (minimal enhancement), and cyclopropane (minimal enhancement); and 2) studies showing that the spinal cord mediates MAC for isoflurane. If GABAA receptors mediate isoflurane MAC, then their blockade in the spinal cord should increase isoflurane MAC more than cyclopropane or xenon MAC and the MAC increase should be proportional to the in vitro enhancement of the GABAA receptor. To test this thesis, isoflurane, cyclopropane, or xenon MAC was determined in rats during intrathecal infusion of artificial cerebrospinal fluid (aCSF) via chronically implanted catheters. Then MAC was redetermined during infusion of 1 microL/min aCSF containing either 0.6 or 2.4 mg/mL picrotoxin, which noncompetitively blocks GABAA receptors. There was no consistent increase in MAC consequent to increasing the picrotoxin dose from 0.6 to 2.4 microg/min, which suggests that maximal blockade of GABAA receptors in the spinal cord had been achieved. Picrotoxin infusion increased MAC approximately 40% with all anesthetics. This indicates that GABA release in the spinal cord influences anesthetic requirement. However, the increase did not consistently differ among anesthetics and did not correlate with in vitro enhancement of GABAA receptors by these anesthetics. This supports the view that GABAA receptors do not mediate immobilization for isoflurane.     

Antagonism of the Antinocifensive Action of Halothane by Intrathecal Administration of GABA sub A Receptor Antagonists

Background: The hind brain and the spinal cord, regions that contain high concentrations of gamma-aminobutyric acid (GABA) and GABA receptors, have been implicated as sites of action of inhalational anesthetics. Previous studies have established that general anesthetics potentiate the effects of gamma-aminobutyric acid at the GABAA receptor. It was therefore hypothesized that the suppression of nocifensive movements during anesthesia is due to an enhancement of GABAA receptor-mediated transmission within the spinal cord.

Methods: Rats in which an intrathecal catheter had been implanted 1 week earlier were anesthetized with halothane. Core temperature was maintained at a steady level. After MAC determination, the concentration of halothane was adjusted to that at which the rats last moved in response to tail clamping. Saline, a GABAA, a GABAB, or a glycine receptor antagonist was then injected intrathecally. The latency to move in response to application of the tail clamp was redetermined 5 min later, after which the halothane concentration was increased by 0.2%. Response latencies to application of the noxious stimulus were measured at 7-min intervals during the subsequent 35 min. To determine whether these antagonists altered baseline response latencies by themselves, another experiment was conducted in which the concentration of halothane was not increased after intrathecal administration of GABAA receptor antagonists.

Results: Intrathecal administration of the GABAA receptor antagonists bicuculline (0.3 micro gram) or picrotoxin (0.3, 1.0 micro gram) antagonized the suppression of nocifensive movement produced by the small increase in halothane concentration. In contrast, the antinocifensive effect of the increase in halothane concentration was not attenuated by the GABAB receptor antagonist CGP 35348 or the glycine receptor antagonist strychnine. By themselves, the GABAA receptor antagonists did not alter response latency in rats anesthetized with sub-MAC concentrations of halothane.     

Inhibitory role of gamma-amino-butyric acid in the rabbit urinary bladder.

Gamma-amino-butyric acid (GABA) is an established inhibitory neurotransmitter in the central nervous system (CNS) and it has also been identified in the bladder. We have investigated in the rabbit the effect of GABA on detrusor activity. Rabbit detrusor muscle strips were made to contract by electrical stimulation of their autonomic nerves or by the addition of carbachol. The addition of GABA caused substantial inhibition of muscle contraction. GABA acts on 2 classes of receptors-GABAA and GABAB. The inhibition was mediated via the GABAB receptors as its effect was mimicked by baclofen (a GABAB agonist) and inhibited by 2-hydroxysaclofen (a GABAB receptor antagonist). Inhibition was not prevented by bicuculline (a GABAA receptor antagonist). This inhibition may be due to a direct muscle effect since the inhibition, which occurred with carbachol-induced contraction, was not abolished by the addition of tetrodotoxin. GABA, acting via the GABAB receptor, produces substantial inhibition of muscle contraction in the rabbit urinary bladder. This raises the possibility of using GABAB analogues in the treatment of detrusor instability.     

Gabapentin increases a tonic inhibitory conductance in hippocampal pyramidal neurons

BACKGROUND: The mechanisms underlying the therapeutic actions of gabapentin remain poorly understood. The chemical structure and behavioral properties of gabapentin strongly suggest actions on inhibitory neurotransmission mediated by gamma-aminobutyric acid (GABA); however, gabapentin does not directly modulate GABAA or GABAB receptors. Two distinct forms of GABAergic inhibition occur in the brain: postsynaptic conductance and a persistent tonic inhibitory conductance primarily generated by extrasynaptic GABAA receptors. The aim of this study was to determine whether gabapentin increased the tonic conductance in hippocampal neurons in vitro. As a positive control, the effects of vigabatrin, which irreversibly inhibits GABA transaminase, were also examined. METHODS: GABAA receptors in hippocampal neurons from embryonic mice were studied using whole cell patch clamp recordings. Miniature inhibitory postsynaptic currents and the tonic current were recorded from cultured neurons that were treated for 36-48 h with gabapentin, vigabatrin, or gabapentin and vigabatrin. To determine whether gabapentin increased the expression of GABAA receptors, Western blots were stained with antibodies selective for alpha1, alpha2, and alpha5 subunits. RESULTS: GABAA receptors were insensitive to the acute application of gabapentin, whereas chronic treatment increased the amplitude of the tonic current threefold (EC50 = 209 microm) but did not influence miniature inhibitory postsynaptic currents. Vigabatrin increased the tonic conductance, and the maximally effective concentration did not occlude the actions of gabapentin, which suggests that these compounds act by different mechanisms. Neither gabapentin nor vigabatrin increased the expression of GABAA receptors in the neurons. CONCLUSIONS: Gabapentin increases a tonic inhibitory conductance in mammalian neurons. High-affinity GABAA receptors that generate the tonic conductance may detect small increases in the ambient concentration of neurotransmitter caused by gabapentin.     

Electrophysiologic evidence for increased endogenous gabaergic but not glycinergic inhibitory tone in the rat spinal nerve ligation model of neuropathy

BACKGROUND: Changes in the inhibitory activity mediated by gamma-aminobutyric acid (GABA) and glycine, acting at spinal GABAA receptors and strychnine-sensitive glycine receptors, are of interest in the development of neuropathic pain. There is anatomic evidence for changes in these transmitter systems after nerve injuries, and blocking either GABAA or glycine receptors has been shown to produce allodynia-like behavior in awake normal animals. METHODS: In this study, the possible changes in GABAergic and glycinergic inhibitory activity in the spinal nerve ligation model of neuropathic pain were studied by comparing the effects of the GABAA-receptor antagonist bicuculline and the glycine-receptor antagonist strychnine in neuropathic rats to their effects in sham-operated and nonoperated control rats. RESULTS: Bicuculline produced a dose-related facilitation of the Adelta-fiber-evoked activity in all study groups and increased C-fiber-mediated activity in the spinal nerve ligation group but not in either of the control groups. There were no differences in the effect of bicuculline on low threshold responses between the study groups. The glycine receptor antagonist strychnine did not have a statistically significant effect on any of the parameters studied in any of the control groups. CONCLUSIONS: These results support the idea of an increased GABAergic inhibitory tone in the spinal cord of neuropathic rats, possibly as compensation for increased excitability after nerve injury.     

Neural development of the neuregulin receptor ErbB4 in the cerebral cortex and the hippocampus: preferential expression by interneurons tangentially migrating from the ganglionic eminences

The receptor tyrosine kinases represent an important class of signal transduction molecules that have been shown to play critical roles in neural development. We report in the present study that the neuregulin receptor ErbB4 is preferentially expressed by interneurons that are migrating tangentially from the ventral to the dorsal rat telencephalon. ErbB4 immunoreactivity was detected in the medial ganglionic eminence as early as embryonic day (E) 13 at the inception of tangential migration. Prominent ErbB4-positive migratory streams consisting of cells double-labeled with ErbB4 and Dlx, a marker of tangentially migrating cells, were found to advance along the lower intermediate zone and the marginal zone from the ventrolateral to the dorsomedial cortex at E16-E18. After E20, the ErbB4-positive stream in the lower intermediate zone shifted towards the germinal zone and further extended via the cortex into the hippocampal primordium. ErbB4 was not expressed by Tbr1-positive glutamatergic projection neurons during development. ErbB4 was preferentially expressed by the majority of parvalbumin-positive interneurons and subsets of other GABAergic interneurons in the cerebral cortex and the hippocampus in adulthood. The early onset and preferential expression of ErbB4 in tangentially migrating interneurons suggests that neuregulin/ErbB4 signaling may regulate the development and function of telencephalic interneurons.     

Inhibitory effect of propofol on ketamine-induced c-Fos expression in the rat posterior cingulate and retrosplenial cortices is mediated by GABAA receptor activation

BACKGROUND: Non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, including ketamine, have psychotomimetic activities and cause neuronal damage in the posterior cingulate and retrosplenial cortices (PC/RS), which are suggested to be the brain regions responsible for their psychotomimetic activities. We previously demonstrated that ketamine induced marked c-Fos (c-fos protein) expression in the rat PC/RS, which was inhibited by propofol, and the expression was closely related to ketamine-induced abnormal behavior. In the present study, we investigated whether the inhibition by propofol was mediated by GABAA receptor receptor activation. METHODS: Using Wistar rats, propofol alone, propofol with bicuculline or propofol with flumazenil was injected intravenously and then continuously infused. Fifteen minutes later, 100 mg kg-1 of ketamine or normal saline was injected intraperitoneally. Two hours after the ketamine or saline injection, the brain was extracted and brain sections were prepared, and c-Fos expression was detected using immunohistochemical methods. RESULTS: Ketamine induced marked c-Fos expression in the PC/RS (171 +/- 9/0.4 mm2), which was significantly inhibited by propofol (5 +/- 5/0.4 mm2). The inhibition by propofol was disinhibited dose-dependently by both bicuculline (0.5 and 1.0 mg kg-1 bicuculline groups: 46 +/- 15 and 143 +/- 16, respectively) and flumazenil (0.1 and 1.0 mg kg-1 flumazenil groups: 79 +/- 6 and 130 +/- 15, respectively). CONCLUSION: These results demonstrate that the inhibitory effect of propofol on ketamine-induced c-Fos expression in the PC/RS is mediated by GABAA receptor activation, and suggests that ketamine-induced psychoneuronal adverse effects may be suppressed by propofol via the activation of GABAA receptors.     

Effects of Small Concentrations of Volatile Anesthetics on Action Potential Firing of Neocortical Neurons In Vitro

Background: Volatile general anesthetics depress neuronal activity in the mammalian central nervous system and enhance inhibitory Cl- currents flowing across the [Greek small letter gamma]-aminobutyric acidA (GABA (A)) receptor - ion channel complex. The extent to which an increase in GABAA-mediated synaptic inhibition contributes to the decrease in neuronal firing must be determined, because many further effects of these agents have been reported on the molecular level.

Methods: The actions of halothane, isoflurane, and enflurane on the firing patterns of single neurons were investigated by extracellular recordings in organotypic slice cultures derived from the rat neocortex.

Results: Volatile anesthetics depressed spontaneous action potential firing of neocortical neurons in a concentration-dependent manner. The estimated median effective concentration (EC50) values were about one half the EC50 values for general anesthesia. In the presence of the GABA (A) antagonist bicuculline (20 [micro sign]M), the effectiveness of halothane, isoflurane, and enflurane in reducing the discharge rates were diminished by 48 - 65%, indicating that these drugs act via the GABAA receptor.     

Additive Effects of Sevoflurane and Propofol on γ-Aminobutyric Acid Receptor Function

Background: Previous studies have shown that propofol and sevoflurane enhance the function of [gamma]-aminobutyric acid type A (GABAA) receptors. However, it is not known whether these two drugs modulate the same molecular pathways. In addition, little is known about receptor function in the presence of both propofol and sevoflurane. The aim of this study was to better understand the interactions of propofol and sevoflurane with the GABAA receptor.

Methods: Wild-type [alpha]1, [beta]2, [gamma]2s GABAA receptor subunit complementary DNAs were transfected into human embryonic kidney cells grown on glass coverslips using a calcium phosphate transfection method. After transfection (36-72 h), cells were whole cell patch clamped and exposed to combinations of the following: 0.3-1,000 [mu]m [gamma]-aminobutyric acid (GABA), 0-10 [mu]m propofol, and 0-1,650 [mu]m sevoflurane. Chemicals were delivered to the cells using two 10-channel infusion pumps and a rapid solution exchanger.

Results: Both propofol and sevoflurane alone enhanced the amplitude of GABAA receptor responses to submaximal concentrations of GABA in a dose-dependent manner. The enhancement was underpinned by an increase in the apparent affinity of the receptor for GABA. Coapplication of both anesthetics further enhanced the apparent affinity of the receptor for GABA.     

Different Actions of General Anesthetics on the Firing Patterns of Neocortical Neurons Mediated by the GABAA Receptor

Background: In cultured slice preparations of rat neocortical tissue, clinically relevant concentrations of volatile anesthetics mainly decreased action potential firing of neurons by enhancing [Greek small letter gamma]-aminobutyric acid (GABAA) receptor-mediated synaptic inhibition. The author's aim was to determine if other anesthetic agents are similarly effective in this model system and act via the same molecular mechanism.

Methods: The actions of various general anesthetics on the firing patterns of neocortical neurons were investigated by extracellular single-unit recordings.

Results: Pentobarbital, propofol, ketamine, and ethanol inhibited spontaneous action potential firing in a concentration-dependent manner. The estimated median effective concentration (EC50) values were close to or below the EC50 values for general anesthesia. Bath application of the GABAA antagonist bicuculline (100 [micro sign]M) decreased the effectiveness of propofol, ethanol, halothane, isoflurane, enflurane, and diazepam by more than 90%, indicating that these agents acted predominantly via the GABAA receptor. The depressant effects of pentobarbital and ketamine were not significantly reduced by bicuculline treatment. Drugs acting mainly via the GABAA receptor altered the firing patterns of neocortical cells in different manners. Diazepam reduced the discharge rates by decreasing the number of action potentials per burst, leaving the burst rate unaffected. In contrast, muscimol, GABA, propofol, and volatile anesthetics decreased the burst rate.