Biomedical subjects

J L Barker

Publications and source records attributed to J L Barker.

At least 73 records · Page 4Linked to original sources

PubMed1998

Potentiometric study of resting potential, contributing K+ channels and the onset of Na+ channel excitability in embryonic rat cortical cells.

Resting membrane potential (RMP), K+ channel contribution to RMP and the development of excitability were investigated in the entire population of acutely dissociated embryonic (E) rat cortical cells over E11-22 using a voltage-sensitive fluorescent indicator dye and flow cytometry. During the period of intense proliferation (E11-13), two cell subpopulations with distinct estimated RMPs were recorded: one polarized at approximately -70 mV and the other relatively less-polarized at approximately -40 mV. Ca2+o was critical in sustaining the RMP of the majority of less-polarized cells, while the well-polarized cells were characterized by membrane potentials exhibiting a approximately Nernstian relationship between RMP and [K+]o. Analysis of these two subpopulations revealed that > 80% of less-polarized cells were proliferative, while > 90% of well-polarized cells were postmitotic. Throughout embryonic development, the disappearance of Ca2+o-sensitive, less-polarized cells correlated with the disappearance of the proliferating population, while the appearance of the K+o-sensitive, well-polarized population correlated with the appearance of terminally postmitotic neurons, immuno-identified as BrdU-, tetanus toxin+ cells. Differentiating neurons were estimated to contain increased K+i relative to less-polarized cells, coinciding with the developmental expression of Cs+/Ba2+-sensitive and Ca2+-dependent K+ channels. Both K+ channels contributed to the RMP of well-polarized cells, which became more negative toward the end of neurogenesis. Depolarizing effects of veratridine, first observed at E11, progressively changed from Ca2+o-dependent and tetrodotoxin-insensitive to Na+o-dependent and tetrodotoxin-sensitive response by E18. The results reveal a dynamic development of RMP, contributing K+ channels and voltage-dependent Na+ channels in the developing cortex as it transforms from proliferative to primarily differentiating tissue.

Animals↗

PubMed1998

GABAergic cells and signals in CNS development.

GABA is formed primarily from decarboxylation of glutamate by a family of cytosolic and membrane-bound GAD enzymes. In the adult, GAD-derived GABA sustains the vitality of the central nervous system (CNS), since blockage of GAD rapidly leads to convulsions and death. In plants, cytosolic GAD synthesizes GABA in response to hormones and environmental stress. Since decarboxylation involves protonation, secretion of GABA serves to buffer cytosolic pH in plant cells. Families of GAD and GABAA receptor/Cl- channel transcripts and encoded proteins emerge early and seemingly everywhere during CNS development, with their abundance closely paralleling neurogenesis and peaking before birth. Micromolar GABA acts at receptor/Cl-channels to depolarize progenitor cells in the cortical neuroepithelium; it also elevates their cytosolic Ca2+ (Cac2+) levels. In some way, these effects decrease proliferation. GABA directs the migration of postmitotic neuroblasts at femtomolar concentrations and stimulates their random motility at micromolar concentrations via Ca2+ signaling mechanisms. Activation of GABAA receptors by micromolar GABA may limit motility via membrane depolarization and elevated Cac2+. These results indicate that in vitro GABA can affect embryogenesis of the CNS through effects on cell proliferation and migration. As neurons differentiate postnatally, Cl(-)-dependent depolarization disappears together with GABAergic Cac2+ signals. Physiologically occurring GABAergic signals at Cl-channels exist in tonic and transient forms. Since the former are found on progenitor cells while both are present in postmitotic neurons, mechanisms to generate transients differentiate in the latter. Surprisingly, tonic and transient forms of GABAergic signaling at Cl-channels are rapidly and smoothly interconvertible and seem to be derived from online GABA synthesis in a surface-accessible compartment of the membrane.

Animals↗

PubMed1997

Astrocytes regulate amino acid receptor current densities in embryonic rat hippocampal neurons.

Embryonic rat hippocampal neurons were cultured in a serum-free defined medium (MEM/N3) either directly on poly-D-lysine (PDL) or on a confluent monolayer of postnatal cortical astrocytes, C6 glioma cells, or Rat2 fibroblasts. Neurons on PDL were grown in MEM/N3 or in MEM/N3 conditioned for 24 h by astrocytes or C6 cells. Membrane capacitance (Cm) and gamma-aminobutyric acid (GABA)-, glycine-, kainate-, and N-methyl-D-aspartate (NMDA)-induced currents were quantified using whole-cell patch-clamp recordings. Cm as well as the amplitude and the density of these currents in neurons cultured on astrocytes were significantly greater than those in neurons grown on PDL after 24 and 48 h. C6 cells mimicked astrocytes in promoting Cm and GABA-, glycine-, and NMDA-evoked, but not kainate-evoked, currents. Cm and currents in neurons grown on Rat2 cells were comparable to those in neurons on PDL. Astrocytes maintained in culture for 3 months were noticeably less effective than freshly prepared ones just grown to confluence. Suppression of spontaneous cytoplasmic Ca2+ (Ca[c]2+) elevations in astrocytes by 1,2-bis(2-aminophenoxy) ehane-N, N, N, N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) loaded intracellularly blocked the observed modulatory effects. Medium conditioned by either astrocytes or C6 cells mimicked the effects of direct coculture of neurons on these cells in promoting Cm and amino acid-evoked currents. Inclusion of antagonists at GABA and glutamate receptors in coculture experiments blocked the observed effects. Thus, diffusible substances synthesized and/ or secreted by astrocytes in a Ca(c)2+-dependent manner can regulate neuronal growth and aminoacid receptor function, and these effects may involve neuronal GABA and glutamate receptors.

Animals↗

PubMed1997

5-HT1A receptor mRNA expressions differ in the embryonic spinal cord of male and female rats.

During critical periods of development, the effects of testosterone (T) on promoting androgenization of the central nervous system (CNS) are reflected not only by behavior, morphology, and hormone secretion but also by gene expression. The mechanisms involved in sexual differentiation of the CNS, however, remain incompletely defined. The current set of experiments examined with in situ hybridization the dimorphism in 5-HT1A receptor mRNA expression in the embryonic rat spinal cord and the possible role of T in the dimorphism. We found sex-related differences in expression of 5-HT1A mRNA in the spinal cord, which were altered by a single injection of T. The results suggest that this gonadal steroid is responsible for the sexual dimorphism in 5-HT1A mRNA expression occurring during the critical period.

Animals↗

PubMed1997

Proton-induced cation current in embryonic rat spinal cord neurons changes ion dependency over time in vitro.

A rapid increase in proton concentration [H+]0 induces Na+ conductance in a variety of cell types. Here we report that H+ trigger a cation-selective channel whose ion dependency changes over time in culture. Whole-cell recordings of ventral spinal cord neurons dissociated at E15 and cultured for up to 14 days revealed that more than 80% had H(+)-induced inward current responses exhibiting a rapid decay phase. The current response was activated beginning about pH 6.8. Following decay, several minutes were required for complete recovery. More modest decreases in pH, which by themselves failed to activate this current, depressed those triggered by effective changes in pH. The currents recorded from cells in culture for less than 7 days could be abolished completely in the absence of Ca2+ and persisted in Na(+)-free and Ba(2+)-containing solutions. Ensemble analysis of current fluctuations recorded at the peak of the current allowed us to estimate a unitary channel conductance of 7.0 pS and a mean open time of 4.1 ms. In neurons cultured 2 weeks or more, protons induced an inward current response with similar kinetic properties, but with [Na+]0 dependency. Thus, proton-activated cation conductance in embryonic rat spinal cord neurons is self-limiting and involves brief openings of cation-selective channels whose ion dependency changes over time in culture.

Animals↗

PubMed1997

GABA induces GABAergic MSCs in cultured embryonic rat thalamic neurons.

Application of 0.1-10 microM GABA in the vicinity of cultured embryonic rat thalamic neurons recorded with patch pipettes in the presence of 2 microM TTX induced or increased the frequency of miniature synaptic currents (MSCs) that reversed polarity at the Cl- equilibrium potential. These MSCs were blocked by the GABAA receptor antagonist bicuculline and exhibited exponential decay kinetics that closely paralleled those estimated from fluctuation analysis of Cl- channels activated pharmacologically by applying 1-10 microM GABA to the same cells. We conclude that the MSCs are mediated by GABA. Application of the GABAA receptor agonist muscimol activated Cl- current but failed to induce GABAergic MSCs while submicromolar concentrations of GABA evoked GABAergic MSCs but did not activate Cl- channels. The GABAB receptor agonist (-)baclofen did not mimic GABA in inducing MSCs. Induction of GABAergic MSCs by GABA required extracellular Ca2+. Verapamil and Co2+, which block voltage-dependent calcium channels, completely blocked GABA-induced MSCs independent of their effects on the direct activation of a Cl- current response. The results indicate that GABA can trigger GABAergic Cl(-)-dependent MSCs in a Cao(2+)-dependent manner. The mechanism may involve a novel receptor and/or signal transduction pathway.

Animals↗

PubMed1997

Potassium current expression during prenatal corticogenesis in the rat.

Using in situ patch-clamp techniques, we have studied K current expression in rat telencephalon from embryonic day 12 to 21. For cells recorded in the ventricular zone, the K current consisted of a delayed rectifier and a large-conductance calcium-activated component, and displayed little variation from embryonic day 12 to 21. Cells recorded in pial regions could be separated into two classes: radially oriented, putatively migrating cells, and cells tangentially oriented in layer I, which were assumed to be Cajal-Retzius cells. When using a voltage-clamp protocol that included a prepulse to -120 mV, Cajal-Retzius cells displayed a larger density of total K current than radial cells, and both types revealed an inactivating component (IA). The proportion of this component increased from embryonic day 18 to 21 in both cell types, although the amplitude of total K current, in the respective cell type, did not vary. This suggested a concomitant decrease in delayed rectifier current, which was verified directly with an appropriate protocol. The activation rate of the delayed rectifier current was slower for ventricular zone cells than for radial or Cajal-Retzius cells. IA was studied in Cajal-Retzius cells and displayed a strikingly negative (approximately -100 mV) voltage of half-maximal steady-state inactivation. Tetraethylammonium ions only blocked the non-inactivating component(s) of K current whereas 4-aminopyridine appeared to decrease both inactivating and non-inactivating components. The quantitative changes in K current expression are likely to underlie the overall increase in excitability of differentiating cells. On the other hand, the observation of qualitative differences among channel properties opens an interesting area of investigation into their physiological significance.

4-Aminopyridine↗

PubMed1997

Anatomical gradients in proliferation and differentiation of embryonic rat CNS accessed by buoyant density fractionation: alpha 3, beta 3 and gamma 2 GABAA receptor subunit co-expression by post-mitotic neocortical neurons correlates directly with cell buoyancy.

Development of the CNS occurs as a complex cascade of pre-programmed events involving distinct phases of cell proliferation and differentiation. Here we show these phases correlate with cells of specific buoyant densities which can be readily accessed by density gradient fractionation. Sprague-Dawley dams were pulse-labelled with bromodeoxyuridine (BrdU) and selected regions of embryonic (E) CNS tissues at E11-22 dissociated with papain into single-cell suspensions. Proliferative cell populations were assessed by anti-BrdU and propidium iodide staining using flow cytometry. Cell differentiation was evaluated using molecular and immunocytochemical probes against mRNAs and antigens differentiating the neuroepithelial, neuronal and glial cell lineages. The results show the emergence of distinctive spatiotemporal changes in BrdU+ populations throughout the CNS during embryonic development, which were followed by corresponding changes in the cellular distributions of antigens distinguishing specific cell types. Fractionation of neocortical cells using discontinuous Percoll gradients revealed that an increasing number of cells increase their buoyancy during corticogenesis. Immunocytochemical and molecular characterization showed that the proliferative and progenitor cell populations are for the most part associated with lower buoyancy or higher specific buoyant densities (> 1.056 g/ml) whereas the post-mitotic, differentiated neurons generally separated into fractions of higher buoyancy or lower specific buoyant densities (< 1.043 g/ml). Immunostaining with antibodies against several GABAA receptor subunits (alpha 3, beta 3, gamma 2) revealed that the highest percent (70-90%) of immunopositive cells could be identified in the most buoyant, differentiating neurons found in the cortical plate/subplate regions, with the lowest percent of the immunopositive cells found in the least buoyant, proliferative and progenitor cell populations originating from the ventricular/subventricular zones. Taken together, these results indicate that buoyant density is a distinguishing characteristic of embryonic CNS cells transforming from primarily proliferative to mainly differentiating, and that fractionation of these cells according to their buoyant densities provides rapid access to the properties of specific cell lineages during the prenatal period of CNS development.

Animals↗

PubMed1997

Neurotrophins stimulate chemotaxis of embryonic cortical neurons.

During mammalian cortical development, neuronal precursors proliferate within ventricular regions then migrate to their target destinations in the cortical plate, where they organize into layers. In the rat, most cortical neuronal migration occurs during the final week of gestation (Bayer et al, 1991; Jacobson, 1991). At this time (E15-E21), reverse transcriptase-polymerase chain reaction demonstrated that cortical homogenates contain mRNA encoding brain derived neurotrophic factor (BDNF) and the catalytic form of its high-affinity receptor, TrkB. Immunocytochemistry and in situ hybridization of sections revealed that the catalytic TrkB receptors predominantly localize to regions containing migratory cells. Many TrkB+ cells exhibited the classic morphology of migrating neurons, suggesting that TrkB ligands play a role in cortical neuronal migration. We analysed whether TrkB ligands influence the motility of embryonic cortical cells (from E15-E21) using a quantitative in vitro chemotaxis assay. High-affinity TrkB ligands (BDNF and NT4/5) stimulated chemotaxis (directed migration) of embryonic neurons at concentrations ranging from 1 to 100 ng/ml. NT-3, a low-affinity TrkB ligand, only stimulated significant migration at high concentrations (> or =100 ng/ml). Peak migration to BDNF was observed at gestational day 18 (E18). BDNF-induced chemotaxis was blocked by either tyrosine kinase inhibitor, K252a, or the Ca2+-chelator, BAPTA-AM, suggesting that BDNF-induces motility via autophosphorylation of TrkB receptor proteins and involves Ca2+-dependent mechanisms. BDNF-stimulation of increased cytosolic Ca2+ was confirmed with optical recordings of E18 cortical cells loaded with Ca2+ indicator dye. Thus, signal transduction through the TrkB receptor complex directs neuronal migration, suggesting that, in vivo, BDNF exerts chemotropic effects that are critical to morphogenesis of the cortex.

Animals↗

PubMed1996

Upregulation of GABAA current by astrocytes in cultured embryonic rat hippocampal neurons.

Embryonic rat hippocampal neurons were cultured on poly-D-lysine (PDL) or a monolayer of postnatal cortical astrocytes to reveal putative changes in neuronal physiology that involve astrocyte-derived signals during the first 4 d of culture, GABA-induced Cl- current (IGABA) was quantified using outside-out and whole-cell patch-clamp recordings beginning at 30 min, when cells had become adherent. The amplitude and density (current normalized to membrane capacitance) of IGABA in neurons grown on astrocytes became statistically greater than that recorded in neurons grown on PDL after 2 hr in culture (HIC). Although the current density remained unchanged in neurons on astrocytes, that in neurons on PDL decreased and became statistically lower beginning after 2 HIC. The differences in amplitude and density of IGABA in the two groups of neurons were maintained during the 4 d experiment. The upregulation effect of astrocytes on neuronal IGABA required intimate contact between the neuronal cell body and underlying astrocytes. Suppression of spontaneous Cac2+ elevations in astrocytes by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid that was loaded intracellularly decreased their modulatory effects on IGABA. IGABA in all cells was blocked completely by bicuculline and exhibited virtually identical affinity constants, Hill coefficients, and potentiation by diazepam in the two groups. Outside-out patch recordings revealed identical unitary properties of IGABA in the two groups. More channels per unit of membrane area could explain the astrocyte enhancement of IGABA. The results reveal that cortical astrocytes potentiate IGABA in hippocampal neurons in a contact-dependent manner via a mechanism involving astrocyte Cac2+ elevation.

Animals↗

PubMed1996

GABA stimulates chemotaxis and chemokinesis of embryonic cortical neurons via calcium-dependent mechanisms.

During rat cortical development, when neurons migrate from the ventricular zone to the cortical plate, GABA localizes within the target destinations of migratory neurons. At this time, cells in germinal zones and along migratory pathways express GABA receptor subunit transcripts, implying that in vivo, GABA may be a chemoattractant. We used an in vitro strategy to study putative chemotropic effects of GABA on embryonic rat cortical cells. GABA stimulated neuronal migration in vitro at embryonic day 15 (E15). From E16 onward, two concentration ranges (fM and microM) induced motility. Femtomolar GABA primarily stimulated chemotaxis (migration along a chemical gradient), whereas micromolar GABA predominantly initiated chemokinesis (increased random movement). These effects were mimicked by structural analogs of GABA with relative specificity at GABAA (muscimol), GABAB (R-baclofen), and GABAC (trans- or cis-4-aminocrotonic acid) receptors. Antagonists of GABAB (saclofen) and GABAC (picrotoxin) receptors partially inhibited responses to both femto- and micromolar GABA; however, only responses to femtomolar GABA were partially blocked by bicuculline, a well established antagonist of GABA at GABAA receptors. Hence, chemotactic responses to femtomolar GABA seem to involve all three classes of GABA receptor proteins, whereas chemokinetic responses to micromolar GABA involve GABAB and GABAC receptor proteins. GABA-induced motility was blocked by loading the cells with the Ca(2+)-chelating molecule bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid, suggesting that intracellular Ca2+ mediates GABA-induced cell movement. Optical recordings of cells loaded with Ca2+ indicator dye revealed that both femto- and micromolar GABA evoked increases in intracellular Ca2+. Thus, GABA-stimulated increases in intracellular Ca2+ may mediate both chemotactic and chemokinetic responses in embryonic cortical cells.

Animals↗

PubMed1996

Immature properties of large-conductance calcium-activated potassium channels in rat neuroepithelium.

The pharmacological and biophysical properties of large-conductance Ca-activated K (BK) channels from embryonic rat telencephalic neuroepithelium were investigated with in situ patch-clamp techniques. A fraction of these channels exhibited properties characteristic of BK channels recorded in well differentiated cells, including normal gating mode (BKN channels). The vast majority of BK channels expressed distinctive properties, the most conspicuous being their buzz gating mode (BKB channels). BKB channels were insensitive to a concentration of charybdotoxin that completely and reversibly blocked BKN channels. In contrast with the strict dependence of BKN channel activation on cytoplasmic Ca, BKB channels displayed substantially high open probability (Po) after inside-out patch excision in a Ca-free medium. Intracellular trypsin down-regulated the Po of BKB channels, which then exhibited a greater sensitivity to cytoplasmic Ca, mainly in the positive direction (increased Po with increased Ca). This suggested a modulatory role for Ca as opposed to its gating role in BKN channels. Ca ions also reduced current amplitude of both types of channels. BKB channels were less voltage sensitive than BKN channels, but this was not correlated with their lower Ca sensitivity. We speculate that BKB channels may represent immature forms in the developmental expression of BK channels.

Animals↗

PubMed1996

Mechanosensitive properties of BK channels from embryonic rat neuroepithelium.

The mechanosensitive properties of large-conductance Ca2+-activated K+ (BK) channels from embryonic rat neuroepithelium were investigated with the cell-attached and inside-out configurations of the patch-clamp technique. The channels were activated in both recording configurations by negative pressures applied to the patch electrode, but reversal of the effect was total and immediate in inside-out patches whereas it was incomplete and delayed in on-cell patches. This mechanosensitivity was not mediated by Ca2+ ions or fatty acids, suggesting that it is an intrinsic property of these channels. Cytochalasin B did not affect mechanosensitivity in on-cell patches but increased it in inside-out patches. Kinetic studies showed that stretch increased the mean open time of the channels and decreased the slowest time constant of their closed-time distributions. The present as well as previous results suggest complex interactions between embryonic BK channels and their membranous and submembranous environment.

Animals↗

PubMed1996

Early upregulation of medium neurofilament gene expression in developing spinal cord of the wobbler mouse mutant.

Homozygous wobbler mouse mutants develop a progressive paralysis due to spinal motoneuron degeneration. To understand the molecular aspect underlying the genetic defect we have studied the embryonic (from E13) and postnatal expression of the three neurofilament and choline acetyltransferase genes in each member from several wild-type (wt) and wobbler (wr) progenies. There are no variations among wt littermates at all ages studied. In contrast, analyses of neurofilament mRNA reveals a 3-4-fold increase of medium neurofilament (NFM) mRNA in wobbler mice (wr/wr). The pattern of increased NFM mRNA during development, prior to the appearance of the wobbler phenotype, among littermates (from heterozygous carriers) conforms to a mendelian inheritance of a single gene defect 1:2:1 (wr/wr:wr/+:+/+). Light and heavy neurofilament mRNA levels are also increased later in development exclusively in those individuals with high NFM mRNA values indicating that increase of the latter is associated with increase of the light and heavy subunit expression. Also NF proteins are increased. Expression of choline acetyltransferase gene is instead always comparable to normal control. Our study provides novel insights into the nature of the wobbler defect, strengthening the hypothesis that neurofilament accumulation plays a pivotal role in the etiopathogenesis of motoneuron degeneration.

Animals↗

PubMed1996

Gamma Knife treatment of 100 consecutive meningiomas.

Clinical and imaging results of Gamma Knife treatment of 100 consecutive patients with intracranial meningiomas are reported. Only 1 patient refused follow-up imaging and her symptoms remain improved after 1 year. Mean values for the patient and treatment parameters were age 61 years, duration of symptoms 3.6 years, time since diagnosis 3 years, average tumor diameter 2.4 cm, surface radiation dose 15 Gy and number of isocenters 5. Clinical outcomes revealed that 6 were improved, 75 were unchanged and 17 had deteriorated. Of the latter, 8 were operated, 4 were treated medically and 5 died. Imaging follow-up showed no growth in 87 patients. The size of tumors treated ranged from 0.66 to 6.8 cm average diameter. In the 77 patients with tumors with an average diameter of 3 cm or less, only 2 (3%) showed further growth, and none died of tumor-related causes.

Adult↗

PubMed1996

Differential coexpression of genes encoding prothyrotropin-releasing hormone (pro-TRH) and prohormone convertases (PC1 and PC2) in rat brain neurons: implications for differential processing of pro-TRH.

Pro-TRH is cleaved at paired basic residues to yield five copies of TRH and cryptic peptides. Recent studies have shown that the prohormone convertases, PC1 and PC2, can process pro-TRH correctly. To determine whether these two enzymes could play a role in pro-TRH processing in vivo, the regional and cellular colocalization of pro-TRH messenger RNA (mRNA) with the mRNAs encoding the prohormone convertases PC1 and PC2 was examined in rat brain, using in situ hybridization histochemistry. Differential regional distribution of pro-TRH mRNA with PC1 and/or PC2 mRNA was found in several brain regions. For example, in the olfactory regions, there was coexpression of pro-TRH mRNA in the glomerular layer with PC2 mRNA, but not PC1 mRNA, whereas in the tenia tecta, coexpression of pro-TRH and PC1 mRNAs was evident, but PC2 mRNA was absent. Pro-TRH mRNA in the paraventricular nucleus was coexpressed with both PC1 and PC2 mRNAs, whereas the basal lateral hypothalamus showed coexistence of pro-TRH mRNA with PC2 mRNA, but not PC1 mRNA. Interestingly, pro-TRH was expressed in the thalamic reticular nucleus, but neither PC1 nor PC2 was detectable in this region. Cellular colocalization studies using double in situ hybridization histochemistry showed the presence of PC2 mRNA in the pro-TRH neurons of the olfactory glomerular layer and basal lateral hypothalamus, and PC1 mRNA in the pro-TRH neurons in the paraventricular nucleus. These results suggest that PC1 and PC2 are enzyme candidates for the processing of pro-TRH in vivo. Moreover, the differential distribution of PC1 and PC2 mRNAs with pro-TRH mRNA may be responsible for the differential processing of this prohormone in the central nervous system. The absence of PC1 and PC2 mRNAs in certain TRH neurons raises the possibility that prohormone convertases other than PC1 and PC2 may be involved in the processing of brain pro-TRH.

Animals↗

PubMed1995

Depolarizing GABA-activated Cl- channels in embryonic rat spinal and olfactory bulb cells.

1. We have compared the electrical properties of the Cl- channels activated by GABA in cells acutely dissociated from embryonic (E) spinal cord (SC) and olfactory bulb (OB) regions at E15 using different configurations of the patch-recording technique. By in situ analysis these cells express GABAA receptor mRNAs encoding a common set of subunits (alpha 2, beta 2, and beta 3). SC cells also express alpha 3, alpha 5 and gamma 2s transcripts. 2. Whole-cell recordings revealed current responses to GABA (0.5 microM to 1 mM) in 242 out of 294 cells. In both SC and OB cells, currents evoked by 2 microM GABA could be potentiated by diazepam (DZP) in a dose-dependent manner with an EC50 of approximately 50 nM in both SC and OB. The maximal effect was approximately 300%. Both SC and OB cells exhibited GABA-activated currents that were only partially sensitive to zinc even at high micromolar concentrations. The effect of DZP and the relatively modest sensitivity to zinc suggest the presence of gamma subunits in both preparations. 3. Spectral analysis of current responses in twenty-six cells showed that power spectra could be fitted by three exponential components (tau 1-3) in the cells of both areas. The tau of the longest-lasting component (tau 3) was significantly different in the cells of the two areas: approximately 50 ms in OB and 80-100 ms in SC. No statistically significant differences in the average inferred unitary conductance between the two cell types could be resolved. 4. Single-channel properties were examined directly using the cell-attached configuration. GABA-activated channels could be recorded in only 89 out of well-sealed 984 patches and most of them exhibited multiple channel activity. The mean open time in the response to 10 microM GABA was significantly shorter in OB cells (12 ms) compared to SC cells (25 ms) while the average conductance values were not significantly different between the two cell types. 5. On average, Cl- channels reversed polarity when the on-cell patch pipette potential was approximately -30 mV. Thus, in these embryonic neurons, micromolar GABA activates Cl- channels, which, when open, effectively depolarize cells by approximately 30 mV. 6. Cl- channels activated by GABA are open longer in embryonic SC cells than in OB cells. This statistically significant difference in native GABAA receptor Cl- channel properties correlates with, and may be related to differences in subunit mRNA expression.

Action Potentials↗

PubMed1995

GABA-induced motility of spinal neuroblasts develops along a ventrodorsal gradient and can be mimicked by agonists of GABAA and GABAB receptors.

During embryogenesis, neuroblasts proliferate within germinal zones, then migrate to their final positions. Although many neurons migrate along radial glial fibers, evidence suggests that environmental factors, as yet unidentified, also influence neuroblast movement. In vivo, nerve growth factor (NGF) and gamma-aminobutyric acid (GABA) colocalize near target destinations of migratory neuroblasts. In vitro, embryonic spinal neurons migrate towards NGF and GABA (Behar et al.: J Neurosci 14:29-38, 1994), implying that the molecules may act as chemoattractants in vivo. Here, we have used an in vitro assay of migration to show that migratory responses to these attractants develop along a ventrodorsal gradient that parallels terminal mitosis during cord development, and that GABA stimulates chemokinesis (motility without a gradient) via heterogeneous receptors involving separate signalling pathways. Both GABAA (muscimol) and GABAB (baclofen) agonists mimicked the effects of GABA in stimulating chemokinesis. Muscimol-induced motility was only blocked by GABAA antagonists (bicuculline or picrotoxin), whereas migration to baclofen was blocked by antagonists of both GABAA and GABAB (2-hydroxysaclofen) receptors. Migration to baclofen, but not muscimol, was abolished in the presence of 8-bromo cAMP or pertussis toxin, indicating that the former, but not the latter, attractant may stimulate motility via Gi/Go GTP binding proteins, and that PKA may modulate migratory responses to baclofen. Migration to GABA was partially attenuated by each of the GABA receptor antagonists. These results lead us to conclude that the natural ligand stimulates neuroblast motility via heterogeneous receptors coupled to different signalling mechanisms.

Age Factors↗