Biomedical subjects

J L Barker

Publications and source records attributed to J L Barker.

At least 55 records · Page 3Linked to original sources

PubMed1999

A role for inwardly rectifying K+ channels in differentiation of NG108-15 neuroblastoma x glioma cells.

The whole-cell patch-clamp technique was used to assess the current carried by inwardly rectifying K+ channels (K(ir)) and the resting membrane potential (RMP) during long-term culture of NG108-15 cells. Culture of this cell line in serum-free medium triggers differentiation of a type I, neuron-like cell type followed by an eventual predominance of a type II, proliferative cell type. NG108-15 K(ir) currents, which strongly resemble currents carried by human ether-a-go-go related gene (HERG) K+ channels, exhibited significantly smaller current density for the more depolarized undifferentiated cells in growth media (GM) and type II cells compared to the neuron-like type I cells. Detailed examination of the transition from undifferentiated GM cells to type I cells revealed a shift in the voltage dependence of K(ir) activation which paralleled the more hyperpolarized RMP, neurite outgrowth, and biochemical differentiation characteristic of type I cells. Reverse-transcription polymerase chain reaction experiments using primers for the rat variant of HERG, RERG, revealed a a nearly twofold increase in RERG mRNA as cells differentiate from GM to type I, a finding entirely consistent with the increased K(ir) current density derived from patch-clamp recordings. Administration of CsCl(5 mM) blocked K(ir) currents and depolarized the RMP of type I cells. Furthermore, culture of NG108-15 cells in serum-free medium but with CsCl added significantly prevented neurite extension, an effect which was entirely reversible upon subsequent removal of CsCl. In contrast, other K+ channel inhibitors (4-aminopyridine and tetraethylammonium), at concentrations without marked effects on K(ir), failed to affect neurite extension. These results suggest an important role of the K(ir) channels in determining the RMP and triggering morphological differentiation of the cell line.

Animals↗

PubMed1999

Inhibition of spontaneous GABAergic transmission by trimethylolpropane phosphate.

Trimethylolpropane phosphate (TMPP) is a neuroactive organophosphate generated during partial pyrolysis of a synthetic ester turbine engine lubricant. While TMPP had been shown to have little affinity for acetylcholinesterase, previous binding studies and 6Cl- flux measurements have implicated TMPP as an antagonist of GABA, receptor/Cl- channels. Using the whole-cell patch clamp method, spontaneous inhibitory postsynaptic currents (sIPSCs) mediated by bicuculline-sensitive GABA(A) receptors were measured in neurons cultured from the rat embryonic hippocampus for 13-21 days. Experiments were conducted in the presence of tetrodotoxin and 6-cyano-7-nitroquinoxaline to inhibit spontaneous presynaptic action potentials and glutamate transmission, respectively, thus isolating GABAergic sIPSCs for study. TMPP induced a concentration-dependent inhibition of sIPSC amplitude and frequency suggesting both postsynaptic and presynaptic actions. Administration of 5 microM TMPP reversibly diminished sIPSC amplitude by 23 +/- 8% (mean SEM, n=5 cells) while markedly decreasing the mean sIPSC frequency by 40 +/- 2% (n=5). The mean time constant of sIPSC decay was reversibly decreased by 20 +/- 4% (n=3) in the presence of 20 microM TMPP, suggesting an increase in the rate of inactivation. To directly verify the blockade of ionotropic GABA receptors by TMPP, the effects of TMPP were examined on whole-cell Cl- current responses activated by exogenous GABA. Administration of TMPP (5 microM) depressed peak whole-cell GABA-induced currents to 73 1% (n=4) of control levels, consistent with the results on sIPSC amplitude. Our data directly demonstrate that TMPP directly inhibits GABA(A) receptor function, as indicated by the blockade of whole-cell GABA-mediated Cl- current and the reduction in sIPSC amplitude. Furthermore, TMPP exerts a presynaptic effect on GABAergic transmission, as evidenced by the reduction in sIPSC frequency, which may be independent of a GABA(A) receptor. The molecular basis for the presynaptic action of TMPP remains to be elucidated.

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PubMed1998

Astrocyte-conditioned saline supports embryonic rat hippocampal neuron differentiation in short-term cultures.

Embryonic rat hippocampal neurons were cultured for 1-2 days in serum-free, HEPES-buffered Tyrode's solution. The effects of cortical astrocytes and astrocyte-conditioned saline on neuron survival, membrane surface area and the expression of functional amino acid neurotransmitter receptors were studied. Neurons grown in Tyrode's solution alone survived well for 1 day but deteriorated thereafter both in terms of percent neurons surviving and the amplitudes and densities of GABA-, glycine-, kainate-and NMDA-induced currents. Neurons grown in Tyrode's previously conditioned by astrocytes for 24 h had significantly larger apparent plasma membrane surface area, as indexed by whole-cell membrane capacitance, and larger amplitudes and densities of the amino acid-induced currents after both 1 and 2 days. The survival rate and neurite outgrowth were also greater in the astrocyte-conditioned saline group after 2 days in culture. Similarly, neurons cultured on glass cover-slips facing a confluent monolayer of astrocyte were larger in apparent plasma membrane area and amino acid-induced currents than neurons cultured in Tyrode's alone. Neurons cultured in saline conditioned by astrocytes provide a strategy to study the physiological basis of astrocyte-directed neuronal differentiation in the absence of ambiguities arising from the inclusion of sera and other additives often used in vitro.

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PubMed1998

Central neuronal synapse formation on micropatterned surfaces.

Controlling synapse formation is a key to patterning of neurons into functional circuits and networks in vitro. However, the process of synapse formation among neurons grown on artificial surfaces is relatively unstudied. We cultured embryonic hippocampal cells on trimethoxysilylpropyl-diethylenetriamine (DETA) and tridecafluoro-1, 1,2,2-tetrahydrooctyl-1-dimethylchlorosilane (13F), and on patterns composed of DETA lines separated by 13F spaces. For comparison, neurons were concurrently plated on surfaces coated with uniform poly-d-lysine (PDL). Pre- and postsynaptic specializations were identified by immunostaining for synapsin I and microtubule-associated protein-2 (MAP-2). Spontaneous (SPCs) and evoked (EPCs) postsynaptic currents were recorded using dual patch-clamp techniques. We found that DETA promoted synapse formation, whereas evidence for synapse formation on 13F was barely detected. MAP-2+ neuronal soma and rapidly growing dendrites were co-localized with synapsin I puncta faithfully along DETA lines. The expression of synapsin I puncta, and MAP-2+ soma and dendrites correlated well with the appearance of SPCs. Synapsin I, MAP-2 and SPCs emerged together at days 3-4 and increased at day 7, when EPCs appeared. Synaptic signals occurring during 4-7 days in culture were all GABAergic. These results indicate that fully functional synapses are formed on silane surfaces, demonstrating the suitability of patterned silane surfaces for organizing synapse formation in vitro.

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PubMed1998

Amyloid beta-protein impairs astrocyte-mediated differentiation of hippocampal neurons.

Embryonic rat hippocampal neurons were cultured on poly-D-lysine (PDL) or on cortical astrocytes, some of which had been pretreated for 24 h with amyloid beta-protein (beta-AP). Amino acid-induced currents were quantified. Membrane capacitance (Cm), as well as the amplitude and density of amino acid-evoked currents recorded in neurons cultured on untreated astrocytes were all statistically greater than those recorded in neurons grown on PDL. However, compared to untreated astrocytes, those treated with beta-AP led to significantly lower values in neurons for Cm and GABA, kainate- and NMDA-induced currents, while glycine-activated current values were not significantly different. Furthermore, beta-AP treatment abolished spontaneous Cac2+ fluctuations in astrocytes, which may account for their impaired ability to promote the expression of functional transmitter receptors in neurons.

Amyloid beta-Peptides↗

PubMed1998

Platelet-derived growth factor induces chemotaxis of neuroepithelial stem cells.

The ability of differentiating cells to migrate within the developing central nervous system (CNS) depends on extrinsic guidance signals, some of which are growth factors. In this study we have investigated the chemotactic response of cultured stem cells from the embryonic rat cortex to platelet-derived growth factor (PDGF). Nestin-positive stem cells from the developing CNS can be maintained and expanded in vitro under serum-free conditions in the presence of basic fibroblast growth factor (bFGF). Northern blot analysis of PDGF receptor expression revealed both alpha- and beta-receptors on bFGF-treated neural stem cells. Both PDGF-AA and PDGF-BB readily induced directed migration of cultured neuroepithelial cells as measured in a microchemotaxis assay. Blocking of the migratory response was achieved by incubation with PDGF isoform-specific antibodies. More than 90% of the migrating cells were nestin-positive and incorporation of BrdU was also seen suggesting the cells to be immature and not yet committed to a specific cell lineage. These findings suggest a role for PDGF in cell migration in the developing cortex.

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PubMed1998

Differential response of cortical plate and ventricular zone cells to GABA as a migration stimulus.

A microdissection technique was used to separate differentiated cortical plate (cp) cells from immature ventricular zone cells (vz) in the rat embryonic cortex. The cp population contained >85% neurons (TUJ1(+)), whereas the vz population contained approximately 60% precursors (nestin+ only). The chemotropic response of each population was analyzed in vitro, using an established microchemotaxis assay. Micromolar GABA (1-5 microM) stimulated the motility of cp neurons expressing glutamic acid decarboxylase (GAD), the rate-limiting enzyme in GABA synthesis. In contrast, femtomolar GABA (500 fM) directed a subset of GAD- vz neurons to migrate. Thus, the two GABA concentrations evoked the motility of phenotypically distinct populations derived from different anatomical regions. Pertussis toxin (PTX) blocked GABA-induced migration, indicating that chemotropic signals involve G-protein activation. Depolarization by micromolar muscimol, elevated [K+]o, or micromolar glutamate arrested migration to GABA or GABA mimetics, indicating that migration is inhibited in the presence of excitatory stimuli. These results suggest that GABA, a single ligand, can promote motility via G-protein activation and arrest attractant-induced migration via GABAA receptor-mediated depolarization.

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PubMed1998

Embryonic rat hippocampal neurons and GABAA receptor subunit-transfected non-neuronal cells release GABA tonically.

We used patch-clamp recording techniques to investigate the contribution of GABA to baseline membrane properties in cultured embryonic rat hippocampal neurons. Almost all of the neurons recorded with Cl--filled pipettes and clamped at negative potentials exhibited baselines that were noticeably noisy, with microscopic fluctuations superimposed on the macroscopic holding current. A gentle steam of saline applied to the neuronal surface rapidly and reversibly reduced the baseline current and fluctuations, both of which were completely eliminated by bicuculline. Fluctuation analysis showed that the variance in the baseline current signal was exponentially distributed with estimated kinetics comparable to those activated by submicromolar concentrations of exogenous GABA. The kinetics of Cl- channels activated by endogenous GABA displayed a potential sensitivity comparable to those activated by exogenous GABA. Non-neuronal cells stably transfected with alpha1 and gamma2 GABAA receptor subunits exhibited little baseline current variance when recorded with Cl--filled pipettes. Addition of micromolar GABA to the extracellular saline or to the pipette solution induced a saline- and bicuculline-sensitive baseline current signal comparable to that recorded in hippocampal neurons. Thus, both intra- and extracellular sources of GABA could contribute to the baseline properties recorded in these cultured neurons.

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PubMed1998

Kainate induces an intracellular Na+-activated current in cultured embryonic rat hippocampal neurones.

1. In embryonic rat hippocampal neurones cultured for < 3 days, kainate induced an inward current at negative potentials that recovered to baseline levels immediately upon termination of agonist application. However, in neurones cultured for longer, the kainate-induced current was often followed by a long-lasting inward current that slowly recovered to baseline levels. The amplitude of the delayed current (Idelay) triggered by kainate was positively related both to the duration of application at constant agonist concentration and to concentration at constant application duration. 2. Idelay could last for several minutes and was accompanied by a conductance increase, which closely paralleled current amplitude. Depression of the kainate-induced current response at receptor level with CNQX or at ionic level with Na+-free solution eliminated Idelay. However, when applied during Idelay neither CNQX nor Na+-free solution had any effect on Idelay. Li+ effected the same response as Na+ in mediating kainate-induced Idelay. 3. GABA-activated Cl- current, which was associated with the same amount of inwardly directed charge flow at the same potential as that induced by kainate, did not trigger a long-lasting delayed current. 4. Idelay depended on the existence of extracellular K+ and its amplitude increased with the increase in K+ concentration. Neither applying Cl-- or Ca2+-free solutions nor increasing intracellular Ca2+ buffering speed and capacity altered Idelay. Exposure to the specific KCa channel blockers apamin and charybdotoxin also failed to alter Idelay. However, Idelay could be blocked by Cs+, Ba2+ and high concentrations of 4-aminopyridine (4-AP) and TEA. 5. Inside-out excised patch-clamp recordings revealed that low density or highly clustered Na+-activated K+ channels were expressed in the cell bodies of cultured embryonic rat hippocampal neurones. These could be the elementary channels underlying Idelay.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1998

GABA receptor subunit mRNA expression in brain of conflict, yoked control and control rats.

Animal conflict models have been used for years as a preclinical screen for predicting anxiolytic therapeutic efficacy. Anxiolytics, including benzodiazepines, increase punished responding. This suggests that the punished behavior may be mediated by the GABA receptor. To evaluate this hypothesis, we performed in situ hybridization histochemistry studies of GABA receptor subunits (alpha1-alpha4) and synthetic enzymes glutamic acid decarboxylase (GAD65 and GAD67) in four groups of rats: conflict (punishment), yoked controls (rats shocked without conflict training history), fixed interval only controls (rats that worked for food but were not shocked) and untreated controls. With conflict behavioral training, bilateral reduction of mRNA for the GABAA alpha1 subunit was seen relative to controls in the cortex, thalamus and hippocampus. In contrast, alteration of alpha2 mRNA levels appeared only in the yoked control group, with increased levels seen in the thalamus and cortex and decreased levels in the hippocampus. There were no differences in the alpha2 mRNA level between the control and the conflict behavioral trained animals. Further, no significant differences were found between groups in the mRNA levels for the alpha3 subunit, alpha4 subunit, GAD65, and GAD67. These results suggest that the behaviors related to conflict and uncontrollable aversive stimuli (yoked control group) are accompanied and perhaps mediated by selective changes in the GABAA alpha1 or alpha2 subunits, respectively. These findings highlight the potential usefulness of the conflict model as a means of elucidating the biological underpinnings of anxiety disorder. Published by Elsevier Science B.V. All rights reserved.

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PubMed1998

Astrocytes regulate developmental changes in the chloride ion gradient of embryonic rat ventral spinal cord neurons in culture.

1. Embryonic rat ventral spinal cord neurons were dissociated at day 15 and grown on: (i) poly-D-lysine (PDL); (ii) a confluent monolayer of type I astrocytes; or (iii) PDL in astrocyte-conditioned medium (ACM) to examine the influence of astroglia on the regulation of GABAA receptor/Cl- channel properties. 2. Potentiometric oxonol dye recordings of intact cells indicated that embryonic neurons were uniformly depolarized by muscimol. The depolarizing effects disappeared in cells dissociated during the early postnatal period and recovered in culture for 24 h. Similar recordings using the calcium-imaging dye fura-2 AM revealed that GABA or muscimol triggered a sustained rise in cytosolic Ca2+ (Ca2+c ) in embryonic neurons that was dependent on extracellular Ca2+, blocked by bicuculline and nifedipine and sensitive to changes in extracellular chloride. The incidence and amplitude of the Ca2+ response decreased with time in vitro and was accelerated in neurons cultured on astrocytes compared with those on PDL. 3. Perforated patch-clamp recordings revealed that GABA depolarized neurons in a Cl--dependent and bicuculline-sensitive manner. Both the resting membrane potential and the GABA equilibrium potential became more hyperpolarized with time in vitro. 4. Astrocytes and ACM accelerated the transformation of GABAergic potential responses from depolarizing to hyperpolarizing. The change occurred over the first 4 days in co-culture or in ACM but took more than 2 weeks in neurons cultured on PDL alone. 5. The intrinsic, elementary properties of GABAA receptor/Cl- channels including open time and unitary conductance changed independently of the presence of astrocytes or ACM. Mean open time of the dominant kinetic component decreased and conductance increased with time in vitro. 6. In sum, astrocytes accelerate the developmental change in the Cl- ion gradient extrinsic to GABAA receptor/Cl- channels, which is critical for triggering Ca2+ entry, without influencing parallel changes in the intrinsic properties of the channels.

Animals↗

PubMed1998

Neuronal and glial epitopes and transmitter-synthesizing enzymes appear in parallel with membrane excitability during neuroblastoma x glioma hybrid differentiation.

The membrane excitability and the presence of neural proteins, including neuronal and glial markers and neurotransmitter-synthesizing enzymes, were examined in parallel while the NG108-15 cell line was maintained in a serum-free medium. Whole-cell recordings in voltage-clamp or current-clamp configurations were used to evaluate the membrane excitability, and immunostaining was done with a panel of well-characterized antibodies against NSE, NF150, S-100 beta, GFAP, ChAT and TH. Culture for 4 to 10 days led to a striking rise in neurite outgrowth, electrical excitability and expression of neural proteins in type I neuron-like cells, which were of both neuronal and glial character, and expressed both cholinergic and adrenergic traits. After about 2 weeks, type II cells which lack neurite processes began to emerge. The type II cells proliferated, as revealed by BrdU uptake, and gradually overgrew differentiated cell types. They exhibited little or no membrane excitability and absence of immunoreactivity for the neuronal and glial specific proteins tested. These measurements indicate that the presence of these neural proteins at crucial stages of membrane excitability development is an important characteristics of NG108-15 cell differentiation, providing insights into the neural development and the reversible nature of neoplasia in the nervous system.

Animals↗

PubMed1998

GABA, GAD, and GABA(A) receptor alpha4, beta1, and gamma1 subunits are expressed in the late embryonic and early postnatal neocortical germinal matrix and coincide with gliogenesis.

Increasing evidence indicates that the classical, fast-acting neurotransmitter gamma-amino butyric acid (GABA) may initially act as morphogen in cell proliferation and differentiation via specific receptors. In view of the potential roles for GABA in central nervous system development, we examined the expression of GABA, GABA(A) receptor beta1 and gamma1 subunits by immunocytochemistry and the expression of transcripts for two GABA-synthesizing enzymes, glutamate decarboxylase (GAD65, GAD67 mRNAs), and for alpha4, beta1, and gamma1 subunits of GABA(A) receptor by in situ hybridization in the developing neocortex. Tissue sections were taken from embryonic days (E) 17 and E20 embryos and newborn rats (P0). The embryos' mothers and newborn rats had been injected with 5-bromo-2'-deoxyuridine (BrdU) and had survived for 2 hours. At E17, BrdU-positive cells were largely restricted in the synthetic zone at the ventricular margin when cortical neurogenesis was still active. GAD mRNAs and GABA immunoreactivity were detected in the subventricular zone, while alpha4, beta1, and gamma1 subunits were abundant in the ventricular zone. At E20 and P0, when neurogenesis had largely ceased and gliogenesis had commenced, BrdU-positive cells were found throughout the ventricular zone with GABA, GAD mRNAs, and alpha4, beta1, and gamma1 subunits. GABA, GAD mRNAs and alpha4, beta1, and gamma1 subunit signals intensified in the ventricular zone from E17 to P0 as gliogenesis proceeded. Thus, specific components of a putative GABAergic circuit are expressed in cells of the ventricular zone during the late embryonic/early postnatal period coincident with gliogenesis, suggesting a role for GABA in glial cell proliferation.

Animals↗

PubMed1998

Large-scale temporal gene expression mapping of central nervous system development.

We used reverse transcription-coupled PCR to produce a high-resolution temporal map of fluctuations in mRNA expression of 112 genes during rat central nervous system development, focusing on the cervical spinal cord. The data provide a temporal gene expression "fingerprint" of spinal cord development based on major families of inter- and intracellular signaling genes. By using distance matrices for the pair-wise comparison of these 112 temporal gene expression patterns as the basis for a cluster analysis, we found five basic "waves" of expression that characterize distinct phases of development. The results suggest functional relationships among the genes fluctuating in parallel. We found that genes belonging to distinct functional classes and gene families clearly map to particular expression profiles. The concepts and data analysis discussed herein may be useful in objectively identifying coherent patterns and sequences of events in the complex genetic signaling network of development. Functional genomics approaches such as this may have applications in the elucidation of complex developmental and degenerative disorders.

Animals↗

PubMed1998

Basic FGF-responsive telencephalic precursor cells express functional GABA(A) receptor/Cl-channels in vitro.

We have previously described the expression of specific gamma-aminobutyric acid (GABA)A receptor subunits and their transcripts in the cortical neuroepithelium (Ma and Barker, 1995, 1998). However, it is not clear whether neural precursor cells exposed to basic fibroblast growth factor (bFGF) in vitro reproduce the biological properties of neuroepithelial cells in vivo within the embryonic ventricular zone. In the present study, neural precursor cells were isolated from the telencephalic neuroepithelium of embryonic day 13-13.5 rats and exposed to bFGF in serum-free medium. Basic FGF-responsive cells were capable of dividing and differentiating into neurons and astrocytes. The rapidly dividing cells formed multicellular spheres and then a rosette-like formation in which a majority of cells expressed GABA(A) receptor alpha4, beta1, or gamma1 subunit proteins. We found in perforated patch-clamp recordings that GABA depolarized bromodeoxyundine (BrdU)+ precursor cells, and under voltage-clamp induced a bicuculline-sensitive current that reversed at the Cl- equilibrium potential. GABA also increased cytoplasmic Ca2+ in a significant number of BrdU+ cells that was blocked by bicuculline. The bicuculline sensitivity of these pharmacological effects implicates GABA(A) receptor/Cl- channels, since bicuculline is a competitive GABA(A) antagonist at these channels in well-differentiated cells. It is possible that the three GABA(A) receptor subunits (alpha4, beta1, and gamma1) found in precursor cells form the Cl- channels detected electrophysiologically. The functional GABA(A) receptor/Cl- channels and associated regulation of their cytoplasmic Ca2+ levels via bicuculline-sensitive mechanisms may play significant roles in the regulation of neural cell proliferation in this model neuroepithelium.

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PubMed1998

Buoyant density gradient fractionation and flow cytometric analysis of embryonic rat cortical neurons and progenitor cells.

We have used the property of natural cell buoyant density to selectively fractionate embryonic rat neocortical cells into 20 subpopulations ranging in phenotype from proliferatively active progenitors to terminally postmitotic neurons. Immunocytochemical and cell cycle analysis of the cellular fractions with flow cytometry revealed an inverse relationship between cell buoyant density and neuronal differentiation. The most buoyant fractions contained predominantly terminally postmitotic, tubulin betaIII-positive, tetanus toxin-positive, and nestin-negative differentiating neurons, while immature, bromodeoxyuridine-positive and nestin-positive proliferating cells were more prevalent in less buoyant fractions. Double loading of isolated cells with voltage- and Ca2+-sensitive fluorescent indicator dyes followed by simultaneous recordings of membrane potential and cytoplasmic [Ca2+] ([Ca2+]c]) using flow cytometry revealed that >50% of the least buoyant cells produced functional responses to veratridine, a Na+ channel agonist, and muscimol, a GABA(A) receptor agonist, but <10% responded to kainic acid, an agonist of a subset of glutamate receptors. As cells became more buoyant the percentage of cells that depolarized and produced a rise in [Ca2+]c to each ligand increased, particularly in response to kainic acid. Short-term culture of select fractions revealed a marked enrichment for cells with morphologies and epitopes characteristic of neuronal and progenitor cell subpopulations. The results show that embryonic cortical cells exhibit a range of naturally occurring buoyant densities that can be used to expeditiously fractionate cortical cells according to their pre- or postmitotic status, thus providing ready access for cellular and molecular studies of proliferation and differentiation.

Animals↗

PubMed1998

Dominant GABA(A) receptor/Cl- channel kinetics correlate with the relative expressions of alpha2, alpha3, alpha5 and beta3 subunits in embryonic rat neurones.

The embryonic appearance of GABAergic cells and signals in the rat nervous system coincides with the appearance of transcripts encoding some but not all of the subunits forming GABA(A) receptor/Cl- channels. Quantitative in situ hybridization studies reveal higher variabilities in alpha2 and alpha3 subunit transcripts relative to others examined (alpha5, beta2, beta3 and gamma2) in six spinal and supraspinal regions. Immunocytochemistry of cells dissociated from the embryonic CNS shows that alpha2 and alpha3 subunits are detectable in differentiating neurones. FACS analyses of dissociated cells immunostained with alpha2- or alpha3- antibodies reveal immunopositive subpopulations of variable size in each region. Whole-cell recordings of acutely adherent neurones show that GABA activates Cl- currents whose fluctuations characteristically vary depending on a neurone's region of origin. Spectral analyses indicate a predominance of the low frequency (< 5 Hz) components, which vary regionally. Regression analyses reveal that (i) channel properties correlate with subunit transcript levels and (ii) dominant channel kinetics correlate with alpha2 and alpha3 subunit transcripts indexed as a ratio and with coexpressions of alpha5 and beta3. The correlations strongly suggest that alpha3 subunits in embryonic neurones are expressed in native receptor/channel complexes with slower kinetics than those containing alpha2 without alpha3 subunits. Thus, GABA(A) receptor/Cl- channels in these embryonic neurones may be encoded by the six transcripts (alpha2, alpha3, alpha5, and beta2, beta3, and gamma2) with proportions of alpha2, alpha3, alpha5, and beta3 subunits critical in determining their dominant kinetics.

Animals↗

PubMed1998

Initially expressed early rat embryonic GABA(A) receptor Cl- ion channels exhibit heterogeneous channel properties.

We have studied the earliest expression of GABA-induced CI- channels in the rat embryonic dorsal spinal cord (DSC) using in situ hybridization, immunocytochemistry, flow cytometry and electrophysiology. At embryonic day 13 (E13) cells in the dorsal region are still proliferating. In situ hybridization consistently showed transcripts encoding only three GABAA receptor subunits (alpha4, beta1 and gammal); immunocytochemistry both in tissue sections and in acutely isolated cells in suspension demonstrated the expression of the corresponding proteins and also revealed staining for other subunits (alpha2, alpha3, beta3, gamma2). In patch-recordings performed in cells acutely isolated from the dorsal cord, responses to GABA were detected in 356 out of 889 cells. GABA-evoked responses, which often displayed the opening of a few channels, were mediated by CI- ions, were inhibited by bicuculline and picrotoxin, and potentiated by benzodiazepines. Taken together, these observations indicate that CI- channels likely involve GABAA type receptors. Fluctuation analysis revealed channel kinetics consisting of three exponential components (Ts: approximately 1,9 and 90 ms) and a wide variety of inferred unitary conductance values, ranging between 4 and 40 pS. A comparison of these results with observations in other, later embryonic cell types and recombinant receptors suggests that most of the earliest E13 DSC GABAA receptors may include alpha3 subunit. These GABAA receptor Cl- channels may be activated physiologically as both GABA synthesizing enzymes and GABA are present in the E13 dorsal cord.

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