Biomedical subjects

J L Barker

Publications and source records attributed to J L Barker.

At least 19 recordsLinked to original sources

PubMed1995

Pharmacological properties of fetal rat hippocampal GABAA receptors.

Cells were cultured from embryonic (E) day 17 and 19 rat hippocampal tissues for one or more days in serum-supplemented growth medium. Intracellular recordings in the whole-cell configuration were made at room temperature. GABAA receptor agonists were applied by pressure pulses from closely positioned pipettes. Most of the cells recorded under these conditions responded to one or another ligand. When the equilibrium potential for Cl- was set near 0 mV, current responses to GABA and other GABAA receptor agonists reversed polarity near 0 mV, suggesting a dominant role for Cl- ions in the response. Many cells also responded to the general anesthetics (-)pentobarbital and alfaxalone (3 alpha-hydroxy-5 alpha-pregnane-11,20-dione). Micromolar concentrations of these drugs, like the transmitter GABA, elicited membrane current responses that reversed near 0 mV. Sequential exposure of individual neurons to both anesthetics revealed sensitivity to one but not the other agent as well as some responding to both. These results indicate: (1) the GABAA receptor function emerges pre-natally in rat hippocampal neurons; (2) barbiturates can have several sites for binding to in GABAA receptor complexes.

Anesthetics↗

PubMed1995

Acetylcholine esterase and peripherin mRNA level decrease in wobbler mouse.

Homozygote wobbler mice develop motoneurone degeneration. Throughout development the expression of choline acetyltransferase, of trkC receptor and F3 adhesion molecule genes is similar in wobbler and wild-type spinal cord. Acetylcholinesterase mRNA level instead is decreased to about 50% with respect to wild-type values in one forth of P5 and P10 wobbler progeny, putative wr/wr individuals; at P21 its expression is equally highly reduced in known homozygotes and it is reduced to 35% of normal values in about one half of the progeny, putative heterozygotes. Thus, similarly to medium neurofilament gene over-expression, reduced acetylcholinesterase gene expression is an early molecular marker for the wobbler mutation before onset of the illness.

Acetylcholinesterase↗

PubMed1995

Transient expression of glutamate decarboxylase and gamma-amino butyric acid in embryonic lens fibers of the rat.

We have determined the localization and developmental expression of glutamate decarboxylase (GAD67) in the rat lens. Immunofluorescence experiments showed that GAD67 was transiently expressed in the nuclear fiber cells of the lens between embryonic days (E) 15 and 20, with maximal immunostaining occurring on E17 and E18. gamma-amino butyric acid (GABA) co-localized with GAD67 in the embryonic nuclear fiber cells. Reverse transcription-polymerase chain reaction (RT-PCR) tests showed that at least three alternatively spliced forms of GAD67 mRNA, including mRNAs with and without the I80 and the I86 insert, were transiently co-expressed with GAD67 in the embryonic lens. The major GAD67 protein in the lens was 67 kDa. We conclude that enzymatically active GAD67 is transiently expressed in the lens nuclear fiber cells of the embryonic rat. The transient expression is regulated by transcriptional and/or posttranscriptional processes. We speculate on the basis of possible common gene regulatory elements for glutamate and ornithine decarboxylases and the involvement of these enzymes with polyamine synthesis, that the transient expression of GAD67 may be connected to nuclear and/or DNA breakdown during lens fiber cell differentiation.

Animals↗

PubMed1995

How can exocytosis account for the actual properties of miniature synaptic signals?

It is broadly accepted that a postsynaptic "miniature" is the most elementary chemically transmitted signal and results from the all-or-none release of transmitter packaged in a single presynaptic vesicle. Hitherto, it has not been possible to directly verify this renowned representation, although it is consistent with evidence of vesicle traffic and, following an intense period of release, vesicle depletion. However, vesicle traffic involving molecular components similar to those implicated in transmitter release has been attributed to other functions including membrane repair. Furthermore, as a number of investigators have recently proposed, miniature signals recorded at peripheral and central synapses may actually reflect several rather than a single discharge of transmitter. It is not clear whether such putative multiple-discharge miniatures represent near-synchronous exocytoses of several vesicles or a burst of openings in a pore that couples a vesicle with the outer membrane. In any case, despite the popularity of the vesicular hypothesis, the molecular mechanism involved in synchronizing fast elementary secretion has not yet been elucidated. Interdependencies among subminiature discharges composing a miniature have suggested that the underlying process is a regenerative signal restricted to a presynaptic terminal unit, confirming Fatt and Katz's first speculation on miniatures, which was not vesicular exocytosis [Fatt and Katz (1952), J. Physiol., 117:109-128]. Here we discuss the possibility that this regenerative signal might be a localized cytosolic Ca2+ transient and attempt to reconcile this hypothesis with the exocytotic models proposed to explain fast transmitter release.

Animals↗

PubMed1995

Exogenous GABA persistently opens Cl- channels in cultured embryonic rat thalamic neurons.

We recorded whole-cell Cl- currents in cultured embryonic rat thalamic neurons by brief applications of GABA or the structural analogue muscimol. In 17 of 141 neurons (12%) the Cl- current persisted for a minute or more after the pipette was removed from the bath. Cl- current never persisted after muscimol exposure even in those cells exhibiting persistent GABA-activated currents (PGC). The half decay times (T50) of PGCs were exponentially and asymptotically related to the duration of GABA exposure and could be interrupted or completely aborted by low-pressure application of saline. PGCs were insensitive to membrane potential, to Tiagabine, a nipecotic acid analogue known to block GABA uptake, and persisted in Cao(2+)-free medium. Fluctuation analysis revealed that PGCs exhibited inferred Cl- channel properties whose kinetic components and estimated average elementary conductance showed no significant difference from those estimated during GABA exposure. The relative contribution of low frequency components was consistently reduced and that of high frequency components modestly increased during PGC compared to those recorded during GABA exposure. Taken together, the results suggest the existence of a superficial compartment in these embryonic neurons that can momentarily accumulate and release exogenous GABA.

Animals↗

PubMed1995

The low efficacy gamma-aminobutyric acid type A agonist 5-(4-piperidyl)isoxazol-3-ol opens brief Cl- channels in embryonic rat olfactory bulb neurons.

4-PIOL is a structural analog of GABA that has low efficacy at GABAA receptor CI- channels and activates a nondesensitizing CI- conductance in central neurons. We investigated the biophysical mechanisms of its low efficacy in embryonic olfactory bulb neurons, which express a limited number of GABAA receptor subunit transcripts. Spectral analysis of GABA- and 4-PIOL-induced current fluctuations evoked in whole-cell recordings showed that three components with mean durations of approximately 0.7, 5, and 50 msec adequately describe the kinetics of the responses induced by both ligands. The contribution of the longest-lasting component was approximately 60% in the spectra of GABA-evoked responses but < 3% in the spectra of 4-PIOL-evoked responses. This is interpreted as a low incidence of long-lasting bursts in 4-PIOL-evoked responses. No difference was evident between the average inferred unitary conductances for 4-PIOL- and GABA-induced channels. These results at the level of the whole cell were confirmed and extended in outside-out single channel recordings. Taken together, the results indicate that the mechanism responsible for the low efficacy of 4-PIOL is the inability to produce frequent bursts of long duration.

Animals↗

PubMed1995

Developmental kinetics of GAD family mRNAs parallel neurogenesis in the rat spinal cord.

GABA (gamma-amino butyric acid), a fast-acting synaptic transmitter in the mature CNS, is synthesized from glutamate by GAD (glutamic acid decarboxylase). We have developed an ultrasensitive PCR technique to quantify the expression of GAD-related mRNAs during the development of the rat cervical spinal cord and have localized them using in situ hybridization. GAD65, GAD67, and an alternatively spliced variant of GAD67, EP10, were quantified each day from embryonic (E) day 11 through E21, and at postnatal days 0, 7, 14, and adult. GAD65 and GAD67 mRNAs were detected at E11 and increased exponentially over three orders of magnitude during embryonic development, then declined approximately threefold in the first-2 postnatal weeks. While the exponential growth phase coincided with the progressive appearance of GAD67 in situ signals in both the ventral and dorsal cord, the postnatal decline coincided with the virtual disappearance of expression in the ventral region. EP10 expression was prominent in the embryo, then declined markedly together with the mRNA encoding the neuroepithelial stem cell marker, nestin. The concerted appearance of GAD-related mRNAs paralleled transcripts encoding neuronal markers (light and heavy neurofilaments) and also closely correlated with the expression of GABA, mRNAs encoding GABAA receptor subunits, and depolarizing responses to GABA. We have used the results on GAD-related mRNA expressions to formulate a simple, minimal mathematical model that accounts for their kinetics in terms of positive and negative feedback loops.

Aging↗

PubMed1995

Complementary expressions of transcripts encoding GAD67 and GABAA receptor alpha 4, beta 1, and gamma 1 subunits in the proliferative zone of the embryonic rat central nervous system.

The developmental stage at which nerve cells initially express specific neurotransmitters and their corresponding receptors remains elusive. In the present study, the distribution patterns of transcripts for the GABA-synthesizing enzyme, glutamate decarboxylase (GAD67), and specific GABAA receptor subunits were examined in the proliferative zone of the rat central nervous system using in situ hybridization. In order to define the DNA synthetic zone of the germinal matrix, tissue sections were taken from embryos whose mothers had been injected with 5-bromo-2'-deoxyuridine (BrdU) and had survived for 1 hr. BrdU immunocytochemistry was used to locate the relative position of BrdU-immunoreactive nuclei within the ventricular zone (VZ). At embryonic day (E) 15 in the alar plate of the lumbar spinal cord, and at E17 and E20 in the dorsomedial sector of the neocortex, densely packed BrdU-immunoreactive nuclei were consistently detected in lateral portions of the inner half of the germinal matrix, indicating that the inner half of the germinal matrix corresponded to the VZ, while the outer half corresponded to the transitional (TZ) or subventricular zone (SV). In situ hybridization in tissue sections adjacent to BrdU-immunoreacted ones showed that the transcripts for GABAA receptor alpha 3, beta 3, and gamma 2 subunits were found exclusively in the mantle zone, while those for alpha 4, beta 1, and gamma 1 subunits were predominantly detected in the inner half of the germinal matrix (i.e., VZ). Furthermore, in the E15 germinal matrix of the lumbar spinal cord, cells exhibiting alpha 4 subunit mRNA were much more abundant in the receding intermediate plate, which contains mostly postmitotic cells, than in the alar plate comprised of many DNA-synthesizing cells, strongly suggesting that only those cells completing final cell division expressed the subunit mRNAs. In clear contrast, GAD67 mRNA was abundant in the outer half of the germinal matrix (i.e., TZ or SV), and in the intermediate zone as well. Immunocytochemical staining of E17 neocortex with anti-GABA antibody revealed a well defined band of GABA-immunoreactive cells and processes in the SV and occasional positive cells in the VZ. It appears that cells in the proliferative zone may express GABA at the migratory stage, whereas cells in the VZ may express mRNAs for GABAA receptor alpha 4, beta 1, and gamma 1 subunits at the premigratory stage, just after completing cell division.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Correlation of gp140trk expression and NGF-induced neuroblast chemotaxis in the embryonic rat spinal cord.

During rat embryogenesis, fibers containing nerve growth factor (NGF) are present near the target destinations of migratory spinal neuroblasts, suggesting that diffusible gradients of NGF provide signals to newly generated neurons in the developing cord. In vitro, pM concentrations of NGF induce neuroblast chemotaxis (directed migration along a chemical gradient), indicating evoked motility is mediated by high-affinity receptors. Binding of 125I-labelled NGF to fetal cord cells provides additional evidence that rat spinal neuroblasts express the high-affinity receptors; however, their presence has not been directly demonstrated. In the present study, we used immunocytochemistry to show that the high-affinity NGF receptor protein, gp140trk (trk) is detectable in embryonic spinal tissue sections and in cord dissociates. Correlation of trk expression with NGF-induced chemotaxis revealed that both the receptor protein expression and functional responses to NGF develop along a ventro-dorsal gradient that parallels the in vivo pattern of neurogenesis and migration. Analysis of the temporal changes in trk immunoreactivity demonstrated that expression of gp140trk is bimodal, possibly reflecting multiple effects of NGF during development. Chemotaxis to NGF was blocked by nM concentrations of the kinase inhibitor, K252a, suggesting that NGF stimulates motility via high-affinity receptors coupled to kinase activity. Elevated 3',5'-cyclic adenosine monophosphate (cAMP) also attenuated NGF-induced chemotaxis, presenting preliminary evidence that protein kinase A (PKA) may regulate motility responses to NGF.

Animals↗

PubMed1994

Fast presynaptic GABAA receptor-mediated Cl- conductance in cultured rat hippocampal neurones.

1. Hippocampal neurones cultured from the 18-day-old embryonic rat for 3 days to 3 weeks were recorded with Cl(-)-filled patch pipettes. Spontaneous synaptic currents, which reversed at the equilibrium potential for Cl- ions (ECl) and were blocked by the GABAA (gamma-aminobutyric acid) receptor antagonists bicuculline or picrotoxin, were recorded in every culture. At 25 degrees C and -80 mV they decayed with a time constant > or = 20 ms that invariably increased at positive potentials. After 2 weeks, 50-75% of all neurones were GABA immunoreactive. 2. In pairs-recordings, coincident synaptic currents in both cells were either spontaneous or evoked by stimulation of one cell. In the presence of tetrodotoxin and using pipettes containing lidocaine (lignocaine) N-ethyl bromide, coincident spontaneous Cl- transients still occurred in both neurones far more frequently than expected by chance. 3. Holding the potential of one neurone at a positive value reversed the synaptic transients in that cell and, in half of the cells, increased the frequency of coincident events in both cells. 4. In neurones where depolarization increased the frequency of coinciding events and all regenerative current apparent at the soma was abolished, short depolarizing pulses occasionally evoked all-or-none, pre- and postsynaptic currents with matching transmission failures and identical delays in transmission. 5. The results suggest that the same pulse of GABA simultaneously activates GABAA receptor-coupled Cl- channels on both sides of the same synaptic cleft, producing immediate auto-transmission in the absence of collaterals or interneurones.

Animals↗

PubMed1994

Transient increase in expression of GAD65 and GAD67 mRNAs during postnatal development of rat spinal cord.

Gamma-aminobutyric acid (GABA) is thought to be one of the classic neurotransmitters acting as a developmental signal. To understand the role for GABA in development, we investigated the expression of transcripts encoding two forms of the GABA-synthesizing enzyme glutamate decarboxylase (GAD65 and GAD67) in the cervical enlargement of the rat spinal cord at successive postnatal days--P0, P7, P14, P21, and P90 (adult)--by using in situ hybridization histochemistry. Cells hybridized with two oligonucleotide probes designed to detect GAD65 and GAD67 mRNAs were widely distributed in all laminae, except in motoneurons of the spinal cord. The integrated densities of hybridization signals were measured across all layers of the gray matter. The relative number of GAD mRNA-labeled cells was determined within each of four regions: laminae I-III, laminae IV-VI, laminae VII and VIII, and lamina X. There was a transient increase in both the integrated density and the relative number of hybridized cells between P7 and P14, after which there was a marked decline to adult levels (lowest). An overall decrease in the number of GAD mRNA-labeled cells was evident in all layers, but a dramatic drop occurred in a subpopulation of cells within ventral portions of the spinal cord. The distribution patterns and postnatal changes in expression of the mRNAs encoding GAD65 and GAD67 were similar and closely paralleled reported changes in the abundance of GAD65 and GAD67 proteins and their product, GABA. Transient increases in GAD mRNA expression during the early postnatal period coincide with, and may be linked to, synapse formation and synapse elimination of the developing spinal cord.

Animals↗

PubMed1994

Analysis of the anatomical distribution of GAD67 mRNA encoding truncated glutamic acid decarboxylase proteins in the embryonic rat brain.

During development of the central nervous system (CNS) the gene that encodes the 67 kDa form of glutamic acid decarboxylase (GAD) undergoes alternative splicing. The alternatively spliced variants include an exon (referred to as ES, for embryonic stop) that contains a premature stop codon. The detection of mRNA containing the ES exon in embryonic rat brain has been previously reported (Proc. Natl. Acad. Sci., 87 (1990) 8771-8775). We have used in situ hybridization to identify the anatomical distribution of ES mRNA in the embryonic rat brain during two stages of development, embryonic day 17 (E17) and E20. At E17, GAD67 mRNA was expressed in several CNS regions that were destined to contain GABAergic neurons when mature. ES transcripts were predominantly localized to ventricular zones and other regions associated with populations of proliferative cells at E17 and E20. At both ages, however, the alternatively spliced variants were also detected in regions of brain associated with migratory or post-mitotic neurons. GAD67 transcripts that did not include the ES exon were localized to anatomical areas that contained post-mitotic, and often post-migratory neurons. The temporal and spatial disappearance of mRNA containing the ES exon generally followed a caudal-to-rostral gradient which paralleled neuronal terminal mitosis and differentiation.

Animals↗

PubMed1994

Reciprocal expression of cell-cell coupling and voltage-dependent Na current during embryogenesis of rat telencephalon.

Using whole-cell patch-clamp techniques in situ (whole-tissue and tissue slices), we have studied two aspects of rat telencephalic cell development during the period of embryogenesis starting at E12. The first aspect was related to junctional coupling as revealed by low input resistance, intercellular dye spread and pharmacologic blockade. Coupling appeared to decrease with time, both in extent and occurrence. The second aspect dealt with cell excitability as revealed by voltage-dependent Na current (INa) expression. Immature action potentials and their underlying INaS were present in a small proportion of E12 cells. These currents were blocked 36% and 78% by 10(-7) M and 10(-6) M tetrodotoxin (TTX), respectively. From then onward, INaS got larger and more prevalent while no obvious changes in kinetics were observed. At E21, INaS were abolished by 10(-7) M TTX and channel density apparently was sufficient to support overshooting yet still immature action potentials.

Animals↗

PubMed1994

Development of the GABAergic phenotype in murine spinal cord-dorsal root ganglion cultures.

Murine spinal cord and dorsal root ganglion GABAergic neurons, derived from 12-day-old fetuses, were examined autoradiographically, biochemically and immunocytochemically in vitro to determine the timecourse of appearance and maturation of this phenotype and the extent and mode of its innervation of target neurons. Specific 3H-GABA uptake into spinal cord neurons was the first property to develop and was present at the earliest time studied, one day in vitro. Immunocytochemical localization of glutamic acid decarboxylase (GAD) revealed positively stained neurons beginning at four days. At five days in vitro, electron microscopic immunocytochemistry revealed GAD-immunoreactive (GAD-IMR) boutons investing neuronal perikarya as well as neuronal processes. By one week in vitro, GAD-IMR neurons constituted 27% of the total population and GAD-IMR boutons could be seen contacting every cell with a neuronal morphology. The mode of investment of target neurons by GAD-IMR boutons was not circumscribed to either soma or dendrites but usually involved the entire neuronal perimeter and did not change with time in culture. Three morphologically distinct types of GAD-IMR neurons were evident: a small, bipolar type; a medium-sized multipolar neuron which was the most common and a large, multipolar type, resembling a motoneuron. A small population (8%) of dorsal root ganglion neurons was found to contain GAD both biochemically and immunocytochemically but was never invested by GAD-IMR boutons. GAD activity in vitro paralleled in vivo levels with maximal activity being reached at four weeks in vitro and 10 days postnatally in the intact mouse spinal cord. Murine spinal cord GABAergic neurons are a morphologically diverse and abundant neuronal population with extensive, precocious innervation of all other neuronal phenotypes in vitro suggesting that GABA has a widespread influence over other developing neuronal systems in the murine spinal cord.

Animals↗

PubMed1994

Immature maxi-K channels exhibit heterogeneous properties in the embryonic rat telencephalon.

Using the cell-attached configuration of the patch-clamp technique, we recorded large-conductance K channels (LCKs) from intact telencephala and cortical slices of embryonic (E) 12-14 and E21 rats, respectively. Conductance (about 200 pS with K(+)-rich pipettes), kinetics and sensitivity to fatty acids (FAs) were reminiscent of some features of the Ca-activated 'maxi-K' channel (BK), yet less than half the LCKs were Ca-sensitive. At E12-14, an even smaller proportion of the channels were strictly voltage-sensitive, that proportion rising to about 50% at E21. Ca and voltage sensitivities were observed independent of one another. The open probability of voltage-sensitive LCKs increased exponentially with depolarization rates similar to those reported for classical BKs. It is postulated that embryonic LCKs may constitute immature forms of BKs whose gating is regulated by endogenous FAs.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

gamma-Aminobutyric acid type A receptor antagonists picrotoxin and bicuculline alter acetylcholine channel kinetics in cultured embryonic rat skeletal muscle.

The effects of the classical gamma-aminobutyric acid type A receptor antagonists picrotoxin and bicuculline on nicotinic acetylcholine receptors in cultured embryonic rat skeletal muscle were examined with whole-cell and cell-attached single-channel recording methods. Up to 600 microM picrotoxin had little or no effect on the amplitude of the whole-cell current, whereas bicuculline dose-dependently blocked it, with an IC50 value of 101.2 +/- 8.9 microM. Bicuculline reduced the maximum inducible acetylcholine current without changing the Kd value, suggesting that bicuculline uncompetitively blocked the binding of acetylcholine to its receptor. The elementary nicotinic acetylcholine receptor currents recorded in the cell-attached single-channel recording configuration exhibited properties typical of those recorded in embryonic muscle (approximately 36 pS and approximately 6 msec). Picrotoxin dramatically transformed individual channel openings into briefly interrupted bursts, so that the number of openings increased while the mean open time markedly decreased. Bicuculline decreased mean open time to a lesser but statistically significant degree. The dominant component of the closed time histogram in control recordings occurred at 17 msec, whereas that recorded with picrotoxin occurred at 0.5 msec. Bicuculline prolonged the closed time, with a dominant closed time component at 52 msec. Elementary conductance was not altered by either agent. In conclusion, we found that the gamma-aminobutyric acid type A channel antagonists picrotoxin and bicuculline were also blockers of embryonic nicotinic acetylcholine receptor channels in cultured rat muscle.

Animals↗

PubMed1994

GABA-induced chemokinesis and NGF-induced chemotaxis of embryonic spinal cord neurons.

During CNS development, neuroblasts proliferate within germinal zones of the neuroepithelium, and then migrate to their final positions. Although many neurons are thought to migrate along processes of radial glial fibers, increasing evidence suggests environmental factors also influence nerve cell movement. Extracellular matrix molecules are thought to be involved in guiding neuronal migration, and molecules such as NGF and GABA exert trophic effects on immature neurons. The nature of the signals that initiate and direct neuroblast migration, however, is unknown. In vitro, NGF and GABA promote neurite outgrowth from cultured cells, and NGF induces axonal chemotaxis (directed migration along a chemical gradient). At earlier developmental stages, these molecules could influence neuroblast movement. Therefore, we investigated whether these molecules induce embryonic neuronal migration. Using an in vitro microchemotaxis assay, we show that rat embryonic spinal cord neurons migrate toward picomolar NGF and femtomolar GABA beginning at embryonic day 13 (E13). Cells exhibit chemotactic responses to NGF while GABA stimulates chemokinesis (increased random movement). GABA effects are mimicked by muscimol and inhibited by bicuculline and picrotoxin, suggesting GABA motility signals are mediated by GABA receptor proteins. Expression of GABA receptors by embryonic cord cells has been previously reported (Mandler et al., 1990; Walton et al., 1993). We used polymerase chain reaction analysis to demonstrate the presence of NGF and trk mRNA in E13 and E14 cord cells, indicating the cells express message for both NGF and high-affinity NGF receptors. Immunohistochemistry of E13 spinal cord sections indicates that NGF and GABA colocalize in fibers close to the target destinations of migrating neurons, suggesting diffusible gradients of these molecules provide chemoattractant signals to migratory cells. Thus, in vitro, neuroblast migration is induced by specific signaling molecules that are present in the developing spinal cord, and may stimulate migration of embryonic neurons prior to synaptogenesis.

Animals↗

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↗