Transient axonal side branches in the developing mammalian optic nerve

Optic axons were labelled with horseradish peroxidase to establish the presence of side branches and examine their distribution and morphology in the developing optic nerve of the quokka wallaby, Setonix brachyurus, the cat and rat at stages when axon numbers are at their peak. In each species, three quarters of the axons were essentially straight and lacked side branches. The remaining axons took significantly longer paths and bore side branches, mostly at points where axons undulated or changed direction. Side branches occurred at intervals of 28–43 µm, had lengths of 2–3 µm and were usually simple rather than branched. A minority (1%) of the axons crossed diagonally between fascicles and two thirds of these had more side branches (interval: 10–18 µm) on the interfascicular portion than were found on the forward-directed axons. A small number of axons (0.01%) doubled back to grow retrogradely towards the eye, these axons also bore relatively more side branches (interval: 8–22 µm), especially at points where the axons changed direction. Ultrastructural reconstruction showed that side branches resembled small axonal profiles and constituted 2% of the total axon number. It is suggested that side branches are involved in the fine-tuning of growth cone navigation. Most side branches are lost by adulthood, indicating their transient nature. The absence of retrogradely-directed axons from adults suggests that cells with such axons are removed by naturally occurring cell death.     

GABA and glutamate fluxes in developing nerve endings

Radiolabeled GABA and glutamate transport into 7 day, 14 day and adult cortical nerve ending preparations was examined. Transport was measured at several Na⁺ concentrations, 19, 27, 43 and 121 mM, and at two temperatures, 15 and 30°C. K_m and V_max values were calculated for all experimental conditions by means of Wilkinson (1961) analysis. A comparison of the day 14 and adult data shows higher K_m values at all Na⁺ concentrations on day 14 for both GABA and glutamate transport. In addition, the temperature dependence of transport was attenuated in the day 14 preparation. Finally, the specificity of GABA transport, as measured by the use of the transport inhibitors β-alanine and 2,4-diaminobutyric acid, was not different between the day 14 and adult preparations. Overall, it is concluded that both GABA and glutamate transport into day 14 nerve endings behave as if “adult” transporter molecules were existing in a more fluid lipid environment, which is the situation found in synaptic membranes prepared from day 14 nerve endings (Hitzemann and Johnson, 1983).Glutamate and GABA transport into 7 day nerve endings is complex and shows marked differences from the day 14 and adult data. Day 7 GABA transport was significantly more sensitive to β-alanine inhibition. Day 7 transport was more sensitive to Na⁺ manipulation and the temperature dependent kinetics show unique Na⁺ effects not seen in the day 14 or adult preparations. For example, at 19 mM Na⁺, 7 day glutamate transport was more temperature dependent than adult transport but as the Na⁺ concentration was increased the reverse was true. The opposite situation for temperature-Na⁺ effects was seen for GABA transport. Finally, no Ca⁺²-dependent component of GABA release could be found in 7 day nerve endings while a significant component was found at day 14. Overall, it is concluded that both glutamate and GABA fluxes in 7 day nerve endings differ both qualitatively from that seen in both day 14 and adult nerve endings.     

The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve

The ontogenetic appearance of the individual triplet polypeptides that comprise mammalian neurofilaments was studied in the developing rat optic nerve. Triton-insoluble cytoskeletal preparations from the optic nerves of rats of postnatal ages 1 Day (P1), 6 days (P6), 10 days (P10), 20 days (P20), and 3 months (adult) were analyzed for protein composition by one and two-dimensional gel electrophoresis. Results indicate that at P1, both the 150- and 68-kDa neurofilament subunit proteins are present. The 200-kDa subunit first becomes discernible at P20, but, at this age, it is still present in considerably less quantity than in the adult. Immunocytochemical verification of the presence of neurofilament protein was accomplished by staining tissue sections with specific antibodies against the 150- and the 68-kDa neurofilament subunits using the peroxidase-antiperoxidase technique. Results of the morphological analyses have shown that neurofilaments are not present in quantity until P10, which coincides with the time when the 68-kDa subunit increases in quantity by one dimensional gel analysis. Thus, the 150- and 68-kDa subunits can be detected prior to the appearance of neurofilaments, and the 200-kDa protein is not observed until sometime later. The potential physiological significance of the differential subunit transport is discussed with respect to neuronal differentiation in the developing mammalian CNS.     

Activation of epidermal growth factor receptors directs astrocytes to organize in a network surrounding axons in the developing rat optic nerve

In postnatal developing optic nerves, astrocytes organize their processes in a cribriform network to group axons into bundles. In neonatal rat optic nerves in vivo, the active form of EGFR tyrosine kinase is abundantly present when the organization of astrocytes and axons is most actively occurring. Blocking activity of EGFR tyrosine kinase during the development of rat optic nerves in vivo inhibits astrocytes from extending fine processes to surround axons. In vitro, postnatal optic nerve astrocytes, stimulated by EGF, organize into cribriform structures which look remarkably like the in vivo structure of astrocytes in the optic nerve. In addition, when astrocytes are co-cultured with neonatal rat retinal explants in the presence of EGF, astrocytes that are adjacent to the retinal explants, re-organize to an astrocyte-free zone into which neurites grow out from the retinal tissue. We hypothesize that in the developing optic nerve, EGFR activity directs the formation of a histo-architectural structure of astrocytes which surrounds axons and provides a permissive environment for axon development.     

Evidence that axon-derived neuregulin promotes oligodendrocyte survival in the developing rat optic nerve

It was previously shown that newly formed oligodendrocytes depend on axons for their survival, but the nature of the axon-derived survival signal(s) remained unknown. We show here that neuregulin (NRG) supports the survival of purified oligodendrocytes and aged oligodendrocyte precursor cells (OPCs) but not of young OPCs. We demonstrate that axons promote the survival of purified oligodendrocytes and that this effect is inhibited if NRG is neutralized. In the developing rat optic nerve, we provide evidence that delivery of NRG decreases both normal oligodendrocyte death and the extra oligodendrocyte death induced by nerve transection, whereas neutralization of endogenous NRG increases the normal death. These results suggest that NRG is an axon-associated survival signal for developing oligodendrocytes.     

A new synthesis of caged GABA compounds for studying GABAA receptors

A short and convergent synthetic approach to new photoactivatable precursors of γ-aminobutyric acid (GABA) is described. When photolyzed, the ‘caged’ GABA precursor efficiently releases GABA, as judged by depolarization measurements on the mammalian GABA_A receptor.     

Expression of GABA and glycine receptors by messenger RNAs from the developing rat cerebral cortex

The ontogenesis of mRNAs coding for GABA and glycine receptors in the cerebral cortex of the rat was examined by extracting poly(A)+ mRNA from the brains of embryonic, postnatal or adult rats and injecting it into Xenopus oocytes. The ability of a messenger to express functional receptors was then assayed by measuring the membrane currents elicited by the agonists. The size of the GABA-induced current increased progressively with age, being undetectable in oocytes injected with mRNA from embryonic day 15 and reaching a maximum in oocytes injected with mRNA from postnatal day 30. In contrast, the glycine-induced response was negligible in oocytes injected with mRNA from the cerebral hemispheres of embryos 15 days old; it increased sharply to a maximum with newborn animals and then decreased with age to become very small with mRNA from adult cortex. GABA and glycine receptors induced by mRNA from the cerebral cortex of all ages are associated with chloride channels.     

Molecular structure of the axolemma of developing axons following altered gliogenesis in rat optic nerve

Axonal and axolemmal development of fibers from rat optic nerves in which gliogenesis was severely delayed by systemic injection of 5-azacytidine (5-AZ) was examined by freeze-fracture electron microscopy. In neonatal (0-2 days) rat optic nerves, all fibers lack myelin, whereas in the adult, virtually all axons are myelinated. The axolemma of neonatal premyelinated fibers is relatively undifferentiated. The P-fracture face (P-face) displays a moderate (approximately 550/micron 2) density of intramembranous particles (IMPs), whereas the E-fracture face (E-face) has few IMPs (approximately 125/micron 2) present. By 14 days of age, approximately 25% of the axons within control optic nerves are ensheathed or myelinated, with the remaining axons premyelinated. The ensheathed and myelinated fibers display increased axonal diameter compared to premyelinated axons, and these larger caliber fibers exhibit marked axonal membrane differentiation. Notably, the P-face IMP density of ensheathed and myelinated fibers is substantially increased compared to premyelinated axolemma, and, at nodes of Ranvier, the density of E-face particles is moderately high (approximately 1300/micron 2), in comparison to internodal or premyelinated E-face axolemma. In optic nerves from 14-day-old 5-AZ-treated rats, few oligodendrocytes are present, and the percentage of myelinated fibers is markedly reduced. Despite delayed gliogenesis, some unensheathed axons within 5-AZ-treated optic nerves display an increased axonal diameter compared to premyelinated fibers. Most of these large caliber fibers also exhibit a substantial increase in P-face IMP density. Small (less than 0.4 micron) diameter unensheathed axons within treated optic nerves maintain a P-face IMP density similar to that of control premyelinated fibers. Regions of increased E-face particle density were not observed. The results demonstrate that some aspects of axolemma differentiation continue despite delayed gliogenesis and the absence of glial ensheathment, and suggest that axolemmal ultrastructure is, at least in part, independent of glial cell association.     

GABA in developing rat skeletal muscle and motor neurons

Protoplasma - In recent years, considerable evidence is accumulated pointing to participation of gamma-aminobutyric acid (GABA) in intercellular signaling in the peripheral nervous system,...     

γ—氨基丁酸受体的反应动力学及其功能意义

γ－氨基丁酸（ＧＡＢＡ）受体的反应动力学（激活,失敏和失活）是否在快速抑制性突触传递中起作用及怎样发挥作用是一个重要问题。随着分子生物学的发展,膜片钳技术及快速施药系统的应用,对ＧＡＢＡ受体的反应动力学的结构基础,单通道水平的发生机制及其人功能意义等开始形成较全面的认识,揭示了其反应动力学在调节抑制性突触后电流（ＩＰＳＣ）中的关键作用及在快速抑制性突触传递中的重要意义。     

A novel alpha subunit in rat brain GABAA receptors

Two cDNAs (alpha 1 and alpha 4) from rat brain cDNA libraries encode isoforms of the alpha subunit of the GABA/benzodiazepine receptor, which differ at 30% of their amino acid residues. Northern blot analysis and in situ hybridization histochemistry show that alpha 1 and alpha 4 mRNAs have distinct sizes and distinct regional and cellular distributions in rat brain: both mRNAs are found in the cortex and hippocampus; however, only the alpha 1 mRNA is detected in the cerebellum. We injected RNA transcribed from alpha 1 and alpha 4 cDNAs into Xenopus oocytes, together with an RNA for a rat beta subunit. We obtained GABA-dependent inward currents that were reversibly blocked by picrotoxin. Picrotoxin alone, applied to oocytes producing the alpha and beta polypeptides, elicited an outward current. We suggest that these polypeptides together produce GABA-gated ion channels that can also open spontaneously.     

Distribution of an endogenous lectin in the developing chick optic tectum

We determined the cellular localization of an endogenous lectin at various times during the development of a well-characterized region of chick brain, the optic tectum. This lectin is a carbohydrate-binding protein that interacts with lactose and other saccharides, undergoes striking changes in specific activity with development, and has previously been purified by affinity chromatography from extracts of embryonic chick brain and muscle. Cellular localization in the tectum was done by indirect immunofluoresecent staining, using immunoglobulin G derived from an antiserum raised against pure lectin. No lectin was detectable in the optic tectum examined at 5 days of embryonic development. From approximately 7 days of development, neuronal cell bodies and fibers were labeled by the antibody; and extracts of tectum contained hemagglutination activity that could be inhibited by lactose or by the antiserum. Lectin remained present in many tectal neuronal layers after hatching; but in 2-month-old chicks it was sparse or absent in most of the tectum except for prominent labeling of fibers in the stratum album centrale. The initial appearance of lectin in the optic tectum was not dependent on innervation by optic nerve fibers since bilateral enucleation during embryogenesis did not affect it. Lectin was detectable on the surface of embryonic optic tectal neurons dissociated with a buffer containing EDTA.     

Intracellular blockade of GABAA receptors in the rat hippocampal neurons

The intracellular blockade of GABA_A-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABA_A receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABA_A receptor channels substitute for Cl^? and in which Mg²⁺ and ATP are omitted to induce rundown of the GABA_A receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg²⁺- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABA_A-receptor-mediated synaptic currents and responses induced by the GABA_A receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AMPA receptors were not affected by the dialysis, whereas the NMDA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg²⁺, ATP-free solution either fully blocked giant depolarizing potentials (GDPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during GDPs and shifted the GDP reversal potential to more positive values (n = 5). The dialysis-resistant component of GDPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NMDA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of GDPs at negative voltages were very similar to those of AMPA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities.     

Structural and functional characterization of the gamma 1 subunit of GABAA/benzodiazepine receptors.

The GABAA receptor gamma 1 subunit of human, rat and bovine origin was molecularly cloned and compared with the gamma 2 subunit in structure and function. Both gamma subunit variants share 74% sequence similarity and are prominently synthesized in often distinct areas of the central nervous system as documented by in situ hybridization. When co-expressed with alpha and beta subunits in Xenopus oocytes and mammalian cells, the gamma variants mediate the potentiation of GABA evoked currents by benzodiazepines and help generate high-affinity binding sites for these drugs. However, these sites show disparate pharmacological properties which, for receptors assembled from alpha 1, beta 1 and gamma 1 subunits, are characterized by the conspicuous loss in affinity for neutral antagonists (e.g. flumazenil) and negative modulators (e.g. DMCM). These findings reveal a pronounced effect of gamma subunit variants on GABAA/benzodiazepine receptor pharmacology.     

Functional properties of recombinant rat GABAA receptors depend upon subunit composition.

GABA-gated chloride channels were expressed in human embryonic kidney cells following transfection of cDNAs encoding the alpha 1, beta 2, and gamma 2 subunits of the rat GABAA receptor (GABAR). Functional properties were determined using patch-clamp techniques in the whole-cell and outside-out configurations. Large whole-cell currents were observed in cells expressing the alpha 1 beta 2, alpha 1 gamma 2, and alpha 1 beta 2 gamma 2 subunit combinations. The unique characteristics of GABAR channels consisting of these subunit combinations depended upon the presence or absence of beta 2 and gamma 3 subunits. GABA-activated currents in cells expressing GABARs with the beta 2 subunit desensitized faster and showed greater outward rectification, and the channels had a shorter mean open time than GABARs composed of alpha 1 gamma 2 subunits. When the gamma 2 subunit was present the resulting GABAR channels had a larger conductance. The slope of the concentration-response curve was significantly steeper for GABARs composed of alpha 1 beta 2 gamma 2 subunits compared with GABARs consisting of alpha 1 beta 2 or alpha 1 gamma 2 subunit combinations.     

Histamine action on vertebrate GABAA receptors: direct channel gating and potentiation of GABA responses

Histamine is not only a crucial cytokine in the periphery but also an important neurotransmitter and neuromodulator in the brain. It is known to act on metabotropic H1-H4 receptors, but the existence of directly histamine-gated chloride channels in mammals has been suspected for many years. However, the molecular basis of such mammalian channels remained elusive, whereas in invertebrates, genes for histamine-gated channels have been already identified. In this report, we demonstrated that histamine can directly open vertebrate ion channels and identified beta subunits of GABA(A) receptors as potential candidates for histamine-gated channels. In Xenopus oocytes expressing homomultimeric beta channels, histamine evoked currents with an EC(50) of 212 microm (beta(2)) and 174 microm (beta(3)), whereas GABA is only a very weak partial agonist. We tested several known agonists and antagonists for the histamine-binding site of H1-H4 receptors and described for beta channels a unique pharmacological profile distinct from either of these receptors. In heteromultimeric channels composed of alpha(1)beta(2) or alpha(1)beta(2)gamma(2) subunits, we found that histamine is a modulator of the GABA response rather than an agonist as it potentiates GABA-evoked currents in a gamma(2) subunit-controlled manner. Despite the vast number of synthetic modulators of GABA(A) receptors widely used in medicine, which act on several distinct sites, only a few endogenous modulators have yet been identified. We show here for the first time that histamine modulates heteromultimeric GABA(A) receptors and may thus represent an endogenous ligand for an allosteric site.     

Cerebellar GABAB receptors modulate function of GABAA receptors.

Interactions between GABAA and GABAB receptors were studied using muscimol-stimulated uptake of 36Cl- by membrane vesicles from mouse cerebellum. Baclofen inhibited muscimol-stimulated 36Cl- uptake and this action was more pronounced with longer flux times (30 vs. 3 s) and after predesensitization of GABAA receptors. Baclofen also inhibited 36Cl- flux by cortical membranes but was more effective with cerebellar preparations. The action of baclofen was stereoselective, calcium-dependent, and blocked by the GABAB receptor antagonist 2-OH-saclofen. It was mimicked by GTP-gamma-S but not by GDP-beta-S, which suggests that baclofen may be acting via a G protein. The action of baclofen was inhibited by U73122, an inhibitor of phospholipase C. However, the potassium channel blockers tetraethylammonium or Ba2+ did not affect the action of baclofen. The results show that activation of GABAB receptors can inhibit the function of GABAA receptors and suggest that this action involves either a nondesensitizing subtype of GABAA receptor or the rate or recycling of desensitized to nondesensitized receptors. We speculate that this action of baclofen results from activation of phospholipase C and phosphorylation of a subtype of GABAA receptor by protein kinase C.