Biomedical subjects

J L Barker

Publications and source records attributed to J L Barker.

At least 109 records · Page 6Linked to original sources

PubMed1993

Ontogeny of GABAA receptor subunit mRNAs in rat spinal cord and dorsal root ganglia.

Relatively little is known about the development of GABAA receptor subunits and their gene expression in mammalian spinal cord. The expression of mRNAs encoding 13 GABAA receptor subunits (alpha 1-6, beta 1-3, gamma 1-3, and delta) in embryonic, postnatal, and adult rat spinal cord and dorsal root ganglia (DRG) cells were studied by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Both techniques revealed the presence of all subunit mRNAs originally found in the rat brain, except for alpha 6, which was not detectable, and delta, which was weakly detected only by RT-PCR. Two anatomically distinctive sets of subunit mRNAs were found by in situ hybridization within the ventricular zone (VZ) and mantle zone (MZ). The trio of alpha 4, beta 1, and gamma 1 subunit mRNAs emerged exclusively in neuroepithelial cells at embryonic day 13 (E13) and remained detectable in the VZ until E17. In the MZ, beta 3 subunit mRNA was first detected at E12, while alpha 2, alpha 3, alpha 5, beta 2, gamma 2, and gamma 3 transcripts appeared at E13. Expressions of the subunit mRNAs in the MZ rapidly increased and expanded in a ventrodorsal sequence from motoneurons to dorsal horn neurons before reaching a peak in the late embryonic/early postnatal period. The mRNA expressions declined during postnatal development, by region-selective depletion, with alpha 4, alpha 5, beta 1, beta 2, gamma 1, and gamma 3 subunit mRNAs becoming barely detectable. In contrast, alpha 2, alpha 3, beta 3, and gamma 2 transcripts persisted into adulthood with distinct anatomical distributions. RT-PCR analysis revealed unique developmental patterns in the intensities of PCR products, most of which were in good agreement with developmental changes in the densities of hybridized mRNA signals. However, RT-PCR amplified minute amounts of mRNAs for alpha 1, alpha 4, alpha 5, beta 1, beta 2, gamma 1, gamma 3, and delta subunits in adults, which were not found in film autoradiograms, but could be detected in a few grain-positive cells in emulsion-dipped sections. DRG cells expressed alpha 2, alpha 3, alpha 5, beta 2, beta 3, and gamma 2 subunit mRNAs during embryogenesis but only alpha 2, beta 3, and gamma 2 subunit mRNAs were reliably detected in the adult.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Tonic GABA secretion of cultured rat hippocampal neurons rapidly transformed by Zn2+ into quantal release.

Different mechanisms of neurotransmitter secretion at synapses have been ascribed to quantal, transient signals [4-9] and to continuous, tonic activity [11, 21, 25]. Quantal transmission is imputed to be the release of transmitter packets, or quanta [6], assembled and stored in vesicles present in presynaptic terminals [2,8]. We report that 300 microM Zn2+ rapidly transformed tonic Cl- conductance mediated by the transmitter gamma-aminobutyric acid (GABA) into transient synaptic-like signals. After addition of Zn2+, the size of the elementary fluctuations in the Cl- current progressively increased while randomly activated Cl- channel kinetics remained unchanged and accounted for the bi-exponential time course of average transient decay. The results suggest that Zn2+ rapidly transforms tonic GABA secretion into quantal-like transient release.

Animals↗

PubMed1993

Cl- channels are randomly activated by continuous GABA secretion in cultured embryonic rat hippocampal neurons.

Throughout the adult vertebrate central nervous system (CNS) gamma-aminobutyric acid (GABA) mediates transient Cl- conductances commonly identified as fast, Cl(-)-dependent inhibitory synaptic signals [Prog. Neurobiol., 36 (1991) 35-92]. In the rat hippocampus Cl(-)-dependent excitatory transients mediated by GABA emerge during the first postnatal week superimposed on a steady-state baseline that is also Cl(-)- and GABA-dependent [Int. J. Dev. Neurosci., 8 (1990) 481-490]. Here we report that many embryonic rat hippocampal neurons cultured for hours to days exhibit random fluctuations in Cl- channel activity that are mediated by continuous secretion of GABA in the absence of transients. Thus, GABA is broadcast tonically before it is released transiently.

Action Potentials↗

PubMed1993

GABAergic cells and signals appear together in the early post-mitotic period of telencephalic and striatal development.

Single cell suspensions derived from embryonic telencephala taken from embryos of gestational day 13 (E13) as well as rat striatal tissue from E14, 15 and 17 were prepared by tissue digestion with papain. Cell suspensions were analyzed by flow cytometry or plated onto poly-D-lysine-coated culture dishes for either nuclear staining or immunocytochemistry. Experiments on functional Na+ channels and GABAA receptor expression were carried out using a fluorescence-activated cell sorter (FACS) and a negatively charged fluorescent indicator dye (oxonol). FACS analysis of embryonic cell suspensions at E13-17 consistently revealed one major subpopulation accounting for 85-90% of the events and one minor subpopulation (10-15% of the total). When sorted, the major subpopulation consisted of phase-bright cells of 5-7 microns diameter some of which had neurites. The minor population consisted of phase-dark cells and resealed membranes of 0.5-4 microns diameter as well as debris. Almost all the cells obtained in the high FALS (forward-angle light scatter) subpopulation at E17 expressed 200-kDa neurofilament and tetanus toxin antigens while the small diameter cells seldom expressed tetanus toxin and particles never did. A small number of GABA-containing neurons were detected in the telencephalon at E13 (3%) and in the developing striatum at E14 (6%). All of the GABA-containing neurons expressed neurofilament. In the embryonic rat striatum, nanomolar concentrations of muscimol (GABAA agonist) induced depolarizing responses. A small number of cells in the high FALS subpopulation were responsive to muscimol starting at embryonic day 14, and the number of responsive cells increased at E15.(ABSTRACT TRUNCATED AT 250 WORDS)

Acridine Orange↗

PubMed1993

Quantitative analysis of transient GABA expression in embryonic and early postnatal rat spinal cord neurons.

GABA expression was investigated using biochemical analysis of spinal cord homogenates and immunocytochemical analysis of cells acutely dissociated from the embryonic and postnatal rat spinal cord. gamma-Aminobutyric acid (GABA) was detected by both methods as early as embryonic day 13 (E13). At E13, the percentage of neurons that were GABA+ was 0.5%. This value increased during embryogenesis, peaked during the first two postnatal weeks to just over 50%, and declined to approximately 20% by the third postnatal week emphasizing the transient nature of GABA expression. At E17 there was a pronounced, positive ventro-dorsal and rostro-caudal gradient of GABA+ cells that persisted until just before birth. At this time the gradients reversed in cervical and lumbosacral regions indicating that GABA immunoreactivity in discrete anatomical regions is also a transient phenomenon. During the embryonic period GABA immunoreactivity was diffusely distributed throughout cell bodies and proximal processes. At E21, both GABA and synaptophysin were present in the same cells. However the two antigens did not co-localize point for point. By postnatal day 21 GABA immunoreactivity appeared in puncta that co-localized entirely with puncta of synaptophysin immunoreactivity. The sizable percentage of neurons that transiently express GABA during development, and the fact that it can be detected prior to the synaptic form of glutamic acid decarboxylase (GAD65), suggest that the amino acid may play a significant role during differentiation before it functions as an inhibitory neurotransmitter.

Animals↗

PubMed1993

Quantal and subquantal GABAergic transmissions in cultured rat hippocampal neurons.

At the neuromuscular junction, spontaneous miniature excitatory synaptic currents mediated by acetylcholine are considered elementary, "quantal" transmissions. These miniature conductances can be quantitatively dichotomized into a large-mode class whose mode is the mean of a normal, bell-shaped distribution and a small-mode class whose distribution is skewed to lower values with its mode being a fraction of the large-mode class. The large-mode class constitutes the population of synaptic signals originally utilized to formulate tenets of "quantal" transmission, which have been tacitly adopted in more recent studies of fast transmission at central synapses. Large- and small-mode conductance classes of inhibitory synaptic elementary conductances mediated by GABA have now been recorded in cultured hippocampal neurons (Vautrin J, Schaffner AE, Barker JL, 1991, Neurosci Lett 138:67). Pairs of hippocampal neurons were patch-recorded at optimal signal-to-noise and, using time course analysis, two elementary fluctuations (0.1-0.3 nS and 1-2 nS) were found within synaptic conductances evoked either by presynaptic action potentials or by presynaptic terminal stimulation. These results were interpreted with a simple model that shows how different frequencies of unitary GABA release can generate either small-mode, skew-distributed conductance (0.5-3 kHz) or large-mode, normally-distributed conductances (> or = 10 kHz). Only the latter satisfies the original tenets of the classic quantal theory.

Action Potentials↗

PubMed1993

Two functionally different glutamate receptors of the kainate subtype in embryonic rat mesencephalic cells.

The amino acid glutamate is a widespread excitatory neurotransmitter in the brain. It activates cation-selective channels expressed by nearly every neuron and by glial cells; also various second messenger cascades. Little is known about the ontogeny of glutamate neurotransmission during neurogenesis. We have analyzed the development and differentiation of excitatory amino acid responses and Na+ channels in cells dissociated from embryonic rat ventral mesencephalon and striatum as well as cortex and cerebellum using fluorescent voltage-sensitive oxonol dyes and flow cytometry. Analysis of fluorescence distribution revealed complex profiles under resting conditions which changed in a characteristic manner over the period studied (Embryonic (E) Days 12-20). The response to the Na+ channel agonist veratridine appeared at E12/13 in the mesencephalon. At E13 L-glutamate and kainate evoked changes in membrane potential interpreted as cellular hyperpolarization. At E15 some cells still responded by hyperpolarizing but an equal number began to depolarize. By E18 most cells depolarized. Both hyper- and depolarizations were eliminated by a specific antagonist at kainate receptors (6-cyano-7-nitroquinoxyline-2,3-dione) and by resuspending the cells in Na(+)-free medium. Both responses exhibited a concentration dependency with higher doses evoking stronger effects. In contrast, there was little effect of veratridine in the striatum at E15-E16, and the response to kainate or L-glutamate was predominantly depolarizing during the same embryonic period, with little or no effect until E18. These data show that in the developing CNS, sodium channel responses as well as excitatory aminoacid neurotransmitter responses first become functional in the mesencephalon and subsequently in the striatum, thus suggesting an anatomical gradient of expression. Our results also show that glutamate receptor-coupled functions vary with embryonic age and with regional distribution, suggesting possible roles of glutamate in early CNS embryogenesis, as morphogens or modulators of synaptic plasticity.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1993

Fast pre-potential generation in rat hippocampal CA1 pyramidal neurons.

Small all-or-none pre-potentials have been shown under some conditions to underlie antidromic and orthodromic spike discharge in somatic recordings of hippocampal pyramidal neurons [Andersen P. and Lomo T. (1966) Expl Brain Res. 2, 247-260; Kandel E. R. et al. (1961) J. Neurophysiol. 24, 225-242; Schwartzkroin P. A. (1977) Brain Res. 128, 53-68; Spencer W. A. and Kandel E. R. (1961) J. Neurophysiol. 24, 272-285]. These potentials are taken to reflect spike discharge in distant regions of the cell (axonal or dendritic) [Andersen P. and Lomo T. (1966) Expl Brain Res. 2, 247-260; Kandel E. R. and Spencer W. A. (1961) Ann. N. Y. Acad. Sci. 94, 570-603; Schwartzkroin P. A. (1977) Brain Res. 128, 53-68] or electronic spike conduction across a gap junction between neighboring pyramidal cells [Dudek F. E. et al. (1983) In Basic Mechanisms of Neural Hyperexcitability, pp. 31-73]. The present study compared pre-potentials recorded at the somatic and dendritic levels and used restricted applications of tetrodotoxin to examine the relationship between pre-potentials and Na+ spike discharge. Intrasomatic and intradendritic recordings were obtained from CA1 pyramidal neurons of rat hippocampal slices maintained in vitro. Orthodromic and antidromic spike discharge was evoked by stimulation of afferent fibers in stratum radiatum and pyramidal cell axons in the alveus, respectively. Focal pressure application of tetrodotoxin in the immediate vicinity of somatic or dendritic recordings uncovered pre-potentials following blockade of antidromic spike discharge. Blockade of these pre-potentials required the diffusion of tetrodotoxin to a location remote from the recording site. Focal application of tetrodotoxin in the cell body layer reliably uncovered orthodromic pre-potentials at the soma only when stimulus intensity was raised beyond threshold for somatic spike discharge; e.g. to intensities shown to initiate spike discharge in apical dendritic locations [Turner R. W. et al. (1991) J. Neurosci. 11, 2270-2280]. These data provide evidence that propagation of a Na+ spike over the pyramidal cell axis is preceded by a depolarization in the form of a pre-potential. The uncovering of orthodromic somatic pre-potentials by tetrodotoxin during suprathreshold activation further supports the proposal [Spencer W. A. and Kandel E. R. (1961) J. Neurophysiol. 24, 272-285] that dendritic spike discharge [Turner R. W. et al. (1991) J. Neurosci. 11, 2270-2280] can underlie fast pre-potential generation in pyramidal cell somata.

Animals↗

PubMed1993

Co-existent expression of GABAA receptor beta 2, beta 3 and gamma 2 subunit messenger RNAs during embryogenesis and early postnatal development of the rat central nervous system.

The expression of beta 1, beta 2, beta 3, gamma 2 and delta subunit messenger RNAs of the GABAA receptor was followed by in situ hybridization histochemistry using radiolabeled oligodeoxynucleotide probes in sections of embryonic (E12-21) and early postnatal (P1-5) rat. beta 2, beta 3 and gamma 2 subunit messenger RNAs were first detectable at E15 in the spinal cord (ventral > dorsal) and lower central nervous system regions (e.g. pons, medulla and thalamus). beta 3 subunit messenger RNA was abundantly expressed in olfactory bulb neurons at E15. At E17, the expression pattern of these subunit messenger RNAs continued in the lower central nervous system. In the upper central nervous system, beta 2, beta 3, and gamma 2 subunit messenger RNAs were first detectable in the outer layer of the hippocampal and entire cortical neuroepithelium. The expression for both beta 3 and gamma 2 subunit messenger RNAs increased significantly over that observed at E15, whereas beta 2 subunit messenger RNA increased to a lesser extent and was more discretely expressed in inferior colliculus, cerebellar neuroepithelium and spinal cord (ventral = dorsal). By E19, messenger RNAs for beta 2, beta 3 and gamma 2 subunits a widespread and abundant co-existent distribution throughout the central nervous system. Exceptions to this co-expression were the absence of beta 2 messenger RNA in the dentate gyrus and beta 3 messenger RNA in entorhinal cortex, areas in which they are present in adult. There was also a differential distribution of subunit messenger RNAs in developing olfactory bulb at E19-20: the glomerular cells preferentially expressed beta 3 and gamma 2 subunit messenger RNAs; the mitral cells preferentially expressed beta 2 subunit messenger RNA; inner granule cells expressed moderate levels of beta 2, beta 3 and gamma 2 subunit messenger RNAs. Expression of beta 2, beta 3 and gamma 2 messenger RNAs was also anatomically co-existent at P5. In addition, significant expression of beta 1 and delta subunit messenger RNAs was apparent in hippocampus and entorhinal cortex. The identity of the gamma 2 expressed between E15 and E21 was shown to be mostly the short isoform of gamma 2 subunit messenger RNA. Expression of both forms was evident beginning around P3-5. These results indicate that during the late embryonic and early postnatal period of development, beta 2, beta 3 and gamma 2 subunit messenger RNAs are abundantly expressed and co-localized to most central nervous system regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Frequency modulation of transmitter release.

In 1952 Fatt and Katz recorded at a frog neuromuscular junction while stimulating the nerve and found "... that successive endplate potential responses varied in a step-like manner, corresponding to units of miniature endplate potentials" (J Physiol 117, 109-128). This led them to propose that fast neuromuscular transmission is 'quantal'. Quantal release is now commonly ascribed to a vesicular form of neurosecretion since vesicles have routinely been visualized in presynaptic terminals. The vesicular hypothesis (Del Castillo and Katz, 1955) assumes that quanta, or 'transmitter packets of standard size', are assembled and stored in the numerous vesicles routinely identified in micrographs of virtually all central and peripheral presynaptic nerve terminals. Simply stated, this model predicts that each one of the miniature synaptic signals (MSSs) follows from the exocytosis of one vesicle's contents. However, the time required for membrane fusion preceding exocytosis (Almers and Tse, 1990) and the variability in MSS amplitude and time course (Vautrin et al, 1992a,b) cannot readily be reconciled by a simple, exocytotic model of quantal release from preloaded vesicles. These difficulties with the original model have led us to re-evaluate MSSs generated at the classical peripheral synapse, the cholinergic neuromuscular junction of the mouse diaphragm, as well as at central synapses between embryonic hippocampal neurons mediated by gamma-aminobutyric acid (GABA). At these synapses, the release of GABA is also assumed to have classical quantal properties like peripheral acetylcholine release (Edwards et al, 1990). Our results show that at both synapses, progressive alterations in elementary signal properties can be induced in a remarkably rapid manner. The original report of preferred amplitudes and intervals in the spontaneous miniature signals (Fatt and Katz, 1952) has repeatedly been confirmed and is here incorporated into a dynamic model of fast synaptic transmission. Although MSSs exhibit variable rise-times and peak amplitudes, they can both be described in terms of synchronization of transmitter release. We have reviewed many experimental findings, which together strongly suggest that the original interpretation of Fatt and Katz (1952) regarding MSSs as reflecting the non-propagated 'neurogenic' activity of 'terminal spots' may be a useful concept to pursue since it may help to explain part of the underlying molecular basis of quantal release.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Calcium current block by (-)-pentobarbital, phenobarbital, and CHEB but not (+)-pentobarbital in acutely isolated hippocampal CA1 neurons: comparison with effects on GABA-activated Cl- current.

Block of a voltage-activated Ca2+ channel current by phenobarbital (PHB), 5-(2-cyclohexylideneethyl)-5-ethyl barbituric acid (CHEB), and the optical R(-)- and S(+)-enantiomers of pentobarbital (PB) was examined in freshly dissociated adult guinea pig hippocampal CA1 neurons; the effects of the barbiturates on GABA-activated Cl- current were also characterized in the same preparation. (-)-PB, PHB, and CHEB produced a reversible, concentration-dependent block of the peak Ca2+ channel current (3 mM Ba2+ as the charge carrier) evoked by depolarization from -80 to -10 mV (IC50 values, 3.5, 72, and 118 microM, respectively). In contrast, (+)-PB was nearly inactive at concentrations up to 1 mM. The inhibitory action of PHB was decreased at acid pH, indicating that the dissociated (anionic) form of the molecule is the active species. Block by (-)-PB was voltage dependent with the fractional block increasing at positive membrane potentials; calculations according to the method of Woodhull indicated that the (-)-PB blocking site senses approximately 40% of the transmembrane electric field. The time course and voltage dependence of activation of the Ca2+ channel current were unaffected by (-)-PB, PHB, and CHEB. The rate of inactivation was enhanced by (-)-PB and CHEB, with the major effect being acceleration of the slow phase of the biexponential decay of the current. GABA-activated Cl- current was potently enhanced by (-)-PB and PHB (EC50 values, 3.4 and 12 microM), whereas (+)-PB was only weakly active. At concentrations of (-)-PB > 100 microM and PHB > 200-300 microM, Cl- current responses were activated even in the absence of GABA. These results demonstrate that in CA1 hippocampal neurons, PB causes a stereoselective block of a voltage-activated Ca2+ current; PHB is also effective, but at higher concentrations. For (-)-PB, the effect on Ca2+ channel current occurred at similar concentrations as potentiation of GABA responses. In contrast, PHB was more potent as a GABA enhancer than as blocker of Ca2+ current, but the maximal potentiation of GABA responses was 40% of that obtained with (-)-PB. Consequently, the anticonvulsant action of PHB at clinically relevant concentrations may relate to modest enhancement of GABA responses and partial blockade of Ca2+ current, whereas the sedative effects that occur at higher concentrations could reflect stronger Ca2+ current blockade. The powerful sedative-hypnotic action of (-)-PB may reflect greater maximal enhancement of GABA responses in conjunction with strong inhibition of Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Sodium channels, GABAA receptors, and glutamate receptors develop sequentially on embryonic rat spinal cord cells.

It is not well understood when during embryonic development the elements of a cell's responsiveness first appear, nor the factors controlling their appearance. A strategy to approach this issue is to determine which aspects of neuronal development are highly stereotyped in presence, timing, or pattern across a variety of cell types, and which are more diversified by cell type, region, or other parameters. We have used a fluorescent potentiometric oxonol dye in conjunction with a digital video imaging system to record the emergence and distribution of specific forms of excitability in dissociated embryonic rat spinal cord cells. We studied the expression of responses to veratridine, a sodium channel activator; muscimol, a GABAA receptor agonist; and kainic acid, an agonist at a class of glutamate receptors. Responses were consistently detectable in a percentage of cells dissociated from the earliest age examined, embryonic day 13, and increased progressively in later ages. Cells were examined from four regions, with cervical-lumbosacral and ventrodorsal distinctions. In the population of cells from each region, functional sodium channels appeared prior to GABAA receptors, which in turn emerged prior to kainate-activated glutamate receptors. This pattern was common to all spinal cord regions and revealed ventrodorsal and rostrocaudal gradients reflecting the known pattern of spinal cord neurogenesis. Analysis of the individual cell responses indicated that the stereotypical pattern of sequential channel development occurs individually on most cells in each region.

Animals↗

PubMed1993

Characterization of the gamma-aminobutyric acidA receptor-channel complex composed of alpha 1 beta 2 and alpha 1 beta 3 subunits from rat brain.

The cloned alpha 1, beta 2 and beta 3 subunits of the gamma-aminobutyric acid (GABA)A receptor-channel complex from rat brain were coexpressed as alpha beta complexes in cultured Chinese hamster ovary cells. Electrophysiological characterization of alpha 1 beta 2 and alpha 1 beta 3 receptor subunit arrangements was performed utilizing patch electrodes in the whole-cell recording configuration. The reversal potential of the current activated by either GABA or muscimol corresponded to that expected for Cl- ions and was dependent on the Cl- gradient. The dose response to GABA for activation of Cl- currents by either subunit combination displayed similar potencies. Currents were partially blocked by the reversible antagonist bicuculline. (-)Pentobarbital was ineffective by itself, but potentiated responses to GABA. The steroid alphaxalone (3 alpha-hydroxy 5 alpha-pregnane 11,20-dione) produced just-detectable inward currents, but did not potentiate GABA-activated currents. Diazepam was completely ineffective. The kinetics and conductance of the Cl- ion channels were inferred from spectral analysis of agonist-induced current fluctuations. Both kinetics and conductance were dependent on agonist structure.

Animals↗

PubMed1992

Neuroepithelial cells in the rat spinal cord express glutamate decarboxylase immunoreactivity in vivo and in vitro.

It is unknown whether neuroepithelial cells in the mammalian central nervous system express neurotransmitter-synthesizing enzymes. In this study, expression of glutamate decarboxylase (GAD), the gamma-aminobutyric acid (GABA)-synthesizing enzyme, was examined in proliferative cells and postmitotic neuroblasts in embryonic rat spinal cord. Immunostaining coronal sections of the embryonic spinal cord with K2 antiserum, which recognizes GAD proteins encoded by the GAD67 gene, revealed intensely stained neuroepithelial cells in the basal plate at embryonic day (E) 13, in the intermediate plate between E 13-16, and last seen in the alar plate at E 16. Nissl counterstaining demonstrated that a small number of these GAD-immunoreactive cells adjacent to the neural tube lumen were mitotic. The ventral-to-dorsal gradient of GAD expression in precursor cells and postmitotic neuroblasts correlates anatomically and temporally with the sequential generation of motoneurons, commissural neurons, and interneurons in the dorsal horn. Some of these GAD-immunoreactive neuroepithelial cells may re-enter the mitotic cycle, while others are postmitotic neuroblasts presumably migrating to the intermediate zone to differentiate into young neurons. Double-immunostaining cells acutely dissociated from E 11-18 spinal cords with K2 and anti-bromodeoxyuridine antisera, following a bromodeoxyuridine pulse in vivo, revealed considerable numbers of DNA-synthesizing cells immunoreactive for GAD. The absolute number of double-stained cells peaked during E 12-15, coinciding with terminal cell division in most spinal neurons. These observations suggest that spinal neuronal precursors can synthesize GAD-related proteins prior to, or during, the terminal cell cycle. Although GAD immunoreactivity revealed by K2 antiserum was detected in proliferative cells and in migrating postmitotic neuroblasts, GABA immunoreactivity was never detectable in these cells. These early embryonic GAD-immunoreactive neuroepithelial cells may either synthesize levels of GABA that cannot be detected immunocytochemically, and/or express enzymatically inactive GAD-related proteins.

Animals↗

PubMed1992

Transient expression of GABA immunoreactivity in the developing rat spinal cord.

The development of GABAergic neurons in the spinal cord of the rat has been investigated by immunocytochemical staining of frozen sections with anti-gamma-aminobutyric acid (GABA) antiserum. In the cervical cord, GABA-immunoreactive fibers first appeared at embryonic day (E) 13 in the presumptive white matter within the ventral commissure, ventral funiculus, and dorsal root entrance zone, and in the ventral roots. There were no GABA-immunoreactive cell bodies detected at this age. By E14, motoneurons, the earliest generated spinal cells, were the first cell population to become GABA-immunoreactive at the cell body level. Thereafter, GABA-immunoreactive neurons increased progressively in number and extended from ventral to dorsal regions. GABA-immunoreactive relay neurons within lamina I of the dorsal horn were initially detected at E17. Interneurons in the substantia gelatinosa, the latest generated cells in the spinal cord, were also the last to express the GABA immunoreactivity at E18. Immunoreactive neurons peaked in intensity and extent at E18 and 19. GABA immunoreactivity was only detectable in neurons within the intermediate and marginal zones 1-3 days after they withdrew from the cell cycle. This contrasts to glutamate decarboxylase immunoreactivity, which is detected in precursor cells in the ventricular zone prior to, or during, withdrawal from the cell cycle. Toward the end of gestation, GABA immunoreactivity declined in intensity and extent. This regression began in the ventral horn of the cervical region and ended in the dorsal horn of the lumbosacral region. During the first week after birth, immunoreactivity in motoneurons and in many other neurons within the ventral horn, intermediate gray, and deeper layers of the dorsal horn disappeared, and only in those neurons predominantly within the superficial layers of the dorsal horn did it persist into adulthood. Thus, the expression and regression of GABA immunoreactivity in the spinal cord followed ventral-to-dorsal, rostral-to-caudal, and medial-to-lateral gradients. These observations indicate that the majority of embryonic spinal neurons pass through a stage of transient expression of GABA immunoreactivity. The functional significance of this transient expression is unknown, but it coincides with the period of intense neurite growth of motoneurons, sensory neurons, and interneurons, and of neuromuscular junction formation, suggesting that the transient presence of GABA may play an important role in the differentiation of sensorimotor neuronal circuits.

Animals↗

PubMed1992

Electrophysiological properties of early postnatal rat septal neurons in short-term culture.

Electrophysiological properties of septal neurons dissociated from PN1-PN7 rats were examined between 1 and 5 days in vitro (DIV) with whole-cell patch-clamp recording. The neurons had RMPs in the range -40 to -80 mV, resistances 0.5-1.5 G omega, and 50-90 mV action potentials. By 2-3 DIV, most neurons were spontaneously active, with some cells exhibiting rhythmic firing patterns. Depolarizations from -80 mV holding potential elicited TTX-sensitive inward Na+ currents, and transient and sustained outward K+ currents. Pharmacological dissection of the outward currents in PN6/7 neurons suggest the presence of multiple types of K+ currents (IA, IC, IK). L-type Ca2+ currents were observed in all PN6/7 neurons examined but were not always detectable in PN1/2 neurons. All PN1/2 and PN6/7 neurons were sensitive to glutamate and GABA but did not respond to ACh or NE applications. Responses to GABA were excitatory i.e., characteristic of immature neurons. Comparison of the results obtained in this study with the properties of adult neurons characterized in vivo or in brain slice preparations in vitro, suggest that septal neurons from PN1-PN7 rats are still in the process of differentiation of their mature electrical and chemosensitive membrane properties.

Adenosine Triphosphate↗

PubMed1992

Magainin 2, a natural antibiotic from frog skin, forms ion channels in lipid bilayer membranes.

We have examined the ion channel forming properties of magainin 2 by incorporating the peptide into artificial lipid bilayers held under voltage clamp. Magainin 2 increased lipid bilayer conductance in a concentration dependent manner with a Hill coefficient of 1.7. The magainin 2 conductance was selective for monovalent cations over anions with a ratio of 5:1 and had both voltage-sensitive and -insensitive components. Two structurally related but antibiotically less potent analogues, magainin 1 and Z-12, also increased lipid bilayer conductance with a similar ion selectivity but these peptides were less potent than magainin 2. We propose that the weak cation selectivity of the magainin channels can be accounted for by the inclusion of negatively charged lipids in the channel complex and suggest two possible structures for such a channel. The ionophoric properties of these peptides are likely to be proximal to their antibiotic activities.

Animals↗

PubMed1992

Differential and transient expression of GABAA receptor alpha-subunit mRNAs in the developing rat CNS.

The expression of mRNAs coding for alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of the GABAA neurotransmitter receptor was followed during the development of the rat CNS by in situ hybridization histochemistry. Expression of these subunit mRNAs in tissue sections of embryonic day 15 and 17 (E15, E17) whole rat and in brain at ages greater than E17 to adult were varied, transient, and region specific. Subunit mRNAs first detected at E15 were those coding for the alpha 2 and alpha 3 subunits. At E17, alpha 2, alpha 3, and alpha 5 mRNAs were present in abundance in numerous areas in the CNS, with lower but significant amounts of alpha 6 being present in the cortical neuroepithelial layers. However, alpha 6 subunit mRNA expression in the cortex declined until little or no alpha 6 mRNA was detected at E19. alpha 1 subunit mRNA first appeared at E19 in the cortex, followed by expression in the hippocampus by postnatal 5 (PN5). Particularly high expression of alpha 2 and alpha 5 subunit mRNAs was detected throughout the developing CNS, but they were most abundant in the olfactory bulb neurons. The high levels of alpha 2 and alpha 5 subunit mRNAs began to decline around PN5 to the amounts observed in adult. These results demonstrate that numerous GABAA receptor alpha-subunits are expressed before birth in a region- and age-specific manner. This complex and varied expression supports the hypothesis that GABA may play a role in cellular and synaptic differentiation.

Animals↗