Fast neurotransmitter release triggered by Ca influx through AMPA-type glutamate receptors

Feedback inhibition at reciprocal synapses between A17 amacrine cells and rod bipolar cells (RBCs) shapes light-evoked responses in the retina. Glutamate-mediated excitation of A17 cells elicits GABA (gamma-aminobutyric acid)-mediated inhibitory feedback onto RBCs, but the mechanisms that underlie GABA release from the dendrites of A17 cells are unknown. If, as observed at all other synapses studied, voltage-gated calcium channels (VGCCs) couple membrane depolarization to neurotransmitter release, feedforward excitatory postsynaptic potentials could spread through A17 dendrites to elicit 'surround' feedback inhibitory transmission at neighbouring synapses. Here we show, however, that GABA release from A17 cells in the rat retina does not depend on VGCCs or membrane depolarization. Instead, calcium-permeable AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs), activated by glutamate released from RBCs, provide the calcium influx necessary to trigger GABA release from A17 cells. The AMPAR-mediated calcium signal is amplified by calcium-induced calcium release (CICR) from intracellular calcium stores. These results describe a fast synapse that operates independently of VGCCs and membrane depolarization and reveal a previously unknown form of feedback inhibition within a neural circuit.     

Opioids block long-term potentiation of inhibitory synapses

Excitatory brain synapses are strengthened or weakened in response to specific patterns of synaptic activation, and these changes in synaptic strength are thought to underlie persistent pathologies such as drug addiction, as well as learning. In contrast, there are few examples of synaptic plasticity of inhibitory GABA (gamma-aminobutyric acid)-releasing synapses. Here we report long-term potentiation of GABA(A)-mediated synaptic transmission (LTP(GABA)) onto dopamine neurons of the rat brain ventral tegmental area, a region required for the development of drug addiction. This novel form of LTP is heterosynaptic, requiring postsynaptic NMDA (N-methyl-d-aspartate) receptor activation at glutamate synapses, but resulting from increased GABA release at neighbouring inhibitory nerve terminals. NMDA receptor activation produces nitric oxide, a retrograde signal released from the postsynaptic dopamine neuron. Nitric oxide initiates LTP(GABA) by activating guanylate cyclase in GABA-releasing nerve terminals. Exposure to morphine both in vitro and in vivo prevents LTP(GABA). Whereas brief treatment with morphine in vitro blocks LTP(GABA) by inhibiting presynaptic glutamate release, in vivo exposure to morphine persistently interrupts signalling from nitric oxide to guanylate cyclase. These neuroadaptations to opioid drugs might contribute to early stages of addiction, and may potentially be exploited therapeutically using drugs targeting GABA(A) receptors.     

Substance P-immunoreactive retinal ganglion cells and their central axon terminals in the rabbit

Retinal ganglion cells are the projection neurons that link the retina to the brain. Peptide immunoreactive cells in the ganglion cell layer (GCL) of the mammalian retina have been noted but their identity has not been determined. We now report that, in the rabbit, 25-35% of all retinal ganglion cells contain substance P-like (SP) immunoreactivity. They were identified by either retrograde transport of fluorescent tracers injected into the superior colliculus, or by retrograde degeneration after optic nerve section. SP immunoreactive cells are present in all parts of the retina and have medium to large cell bodies with dendrites that ramify extensively in the proximal inner plexiform layer. Their axons terminate in the dorsal lateral geniculate nucleus, superior colliculus and accessory optic nuclei, and these terminals disappear completely after contralateral optic nerve section and/or eye enucleation. In the dorsal lateral geniculate nucleus large, beaded, immunoreactive axons and varicosities make up a narrow plexus just below the optic tract, where they define a new geniculate lamina. The varicosities make multiple synaptic contacts with dendrites of dorsal lateral geniculate nucleus projection neurons and presumptive interneurons in complex glomerular neuropil. This is direct evidence that some mammalian retinal ganglion cells contain substance P-like peptides and strongly suggests that, in the rabbit, substance P (or related tachykinins) may be a transmitter or modulator in a specific population or populations of retinal ganglion cells.     

Glutamate spillover suppresses inhibition by activating presynaptic mGluRs

Metabotropic glutamate receptors (mGluRs) found on synaptic terminals throughout the brain are thought to be important in modulating neurotransmission. Activation of mGluRs by synaptically released glutamate depresses glutamate release from excitatory terminals but the physiological role of mGluRs on inhibitory terminals is unclear. We have investigated activation of mGluRs on inhibitory terminals within the cerebellar glomerulus, a structure in which GABA (gamma-aminobutyric acid)-releasing inhibitory terminals and glutamatergic excitatory terminals are in close apposition and make axo-dendritic synapses onto granule cells. Here we show that 'spillover' of glutamate, which is released from excitatory mossy fibres, inhibits GABA release from Golgi cell terminals by activating presynaptic mGluRs under physiological conditions. The magnitude of the depression of the inhibitory postsynaptic current is dependent on the frequency of mossy fibre stimulation, reaching 50% at 100 Hz. Furthermore, the duration of inhibitory postsynaptic current depression mirrors the time course of mossy fibre activity. Our results establish that mGluRs on inhibitory interneuron axons sense the activity of neighbouring excitatory synapses. This heterosynaptic mechanism is likely to boost the efficacy of active excitatory fibres by locally reducing the level of inhibition.     

3H-baclofen and 3H-GABA bind to bicuculline-insensitive GABA B sites in rat brain

The presence of a novel receptor for the neurotransmitter gamma-aminobutyric acid (GABA) on peripheral autonomic nerve terminals and in mammalian brain slices has been described recently. This receptor differs from the classical GABA site as it is unaffected by recognized GABA antagonists such as bicuculline and is not sensitive to the majority of accepted GABA-mimetics such as 3-aminopropanesulphonic acid (3-APS) or isoguvacine. We propose to designate the classical site as the GABA A and the novel site as the GABA B receptor. The beta-p-chlorophenyl derivative of GABA, baclofen, is stereospecifically active at the GABA B site whereas it is devoid of activity at the classical GABA A3 site. We now report that high-affinity saturable binding of 3H-baclofen and 3H-GABA to the GABA B site can be detected in fragments of crude synaptic membranes prepared from rat brain. The results support the concept of a novel GABA receptor within the mammalian brain and show that GABA and baclofen can compete for the same recognition site.     

Release of endogenous excitatory amino acids from turtle photoreceptors

Responses to light are transmitted from photoreceptors to second-order retinal neurons by chemical synapses that may use an excitatory amino acid (EAA) as the neurotransmitter. This hypothesis is based primarily on the pharmacological actions of EAA agonists and antagonists on the membrane potentials and light responses of second-order neurons. But the release of endogenous EAAs, which is a critical criterion for the identification of EAAs as transmitters, has not been demonstrated. Here we report the use of outside-out membrane patches excised from rat hippocampal neurons to detect the release of EAAs from synaptic terminals of isolated turtle photoreceptors. Electrical stimulation of or application of lanthanum chloride to photoreceptors induced an increase in the frequency of opening of 50-pS channels in the patches. These channels were identified as the class of glutamate-activated channels that are also gated by aspartate and NMDA (N-methyl-D-aspartate). In several photoreceptor-patch pairs, spontaneous channel activity was observed near the synaptic terminals. These results provide strong evidence to support the hypothesis that both rods and cones of the turtle use an EAA as their neurotransmitter.     

GABA may be a neurotransmitter in the vertebrate peripheral nervous system

gamma-Aminobutyric acid (GABA) is an inhibitory neurotransmitter in the peripheral nervous system of certain invertebrates and is thought to be a major transmitter in the vertebrate central nervous system. In this report we present evidence that GABA may also be a neurotransmitter in the vertebrate peripheral autonomic nervous system. We have used light and electron microscopic autoradiography to analyse high-affinity uptake of 3H-GABA into the myenteric plexus of the guinea pig taenia coli, both in situ and in a tissue culture preparation. In the isolated myenteric plexus, we have measured the specific activity of glutamic acid decarboxylase (GAD; EC 4.1.1.15), the enzyme responsible for conversion of glutamic acid to GABA in GABAergic neurones, and assessed the ability of this tissue to accumulate 3H-GABA newly synthesised from 3H-glutamic acid. Furthermore, we have measured the levels of endogenous GABA in strips of taenia coli containing the myenteric plexus.     

Mechanism of chloride interaction with neurotransmitter:sodium symporters

Neurotransmitter:sodium symporters (NSS) have a critical role in regulating neurotransmission and are targets for psychostimulants, anti-depressants and other drugs. Whereas the non-homologous glutamate transporters mediate chloride conductance, in the eukaryotic NSS chloride is transported together with the neurotransmitter. In contrast, transport by the bacterial NSS family members LeuT, Tyt1 and TnaT is chloride independent. The crystal structure of LeuT reveals an occluded binding pocket containing leucine and two sodium ions, and is highly relevant for the neurotransmitter transporters. However, the precise role of chloride in neurotransmitter transport and the location of its binding site remain elusive. Here we show that introduction of a negatively charged amino acid at or near one of the two putative sodium-binding sites of the GABA (gamma-aminobutyric acid) transporter GAT-1 from rat brain (also called SLC6A1) renders both net flux and exchange of GABA largely chloride independent. In contrast to wild-type GAT-1, a marked stimulation of the rate of net flux, but not of exchange, was observed when the internal pH was lowered. Equivalent mutations introduced in the mouse GABA transporter GAT4 (SLC6A11) and the human dopamine transporter DAT (SLC6A3) also result in chloride-independent transport, whereas the reciprocal mutations in LeuT and Tyt1 render substrate binding and/or uptake by these bacterial NSS chloride dependent. Our data indicate that the negative charge, provided either by chloride or by the transporter itself, is required during binding and translocation of the neurotransmitter, probably to counterbalance the charge of the co-transported sodium ions.     

Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Synaptic vesicles are loaded with neurotransmitter by means of specific vesicular transporters. Here we show that expression of BNPI, a vesicle-bound transporter associated with sodium-dependent phosphate transport, results in glutamate uptake by intracellular vesicles. Substrate specificity and energy dependence are very similar to glutamate uptake by synaptic vesicles. Stimulation of exocytosis--fusion of the vesicles with the cell membrane and release of their contents--resulted in quantal release of glutamate from BNPI-expressing cells. Furthermore, we expressed BNPI in neurons containing GABA (gamma-aminobutyric acid) and maintained them as cultures of single, isolated neurons that form synapses to themselves. After stimulation of these neurons, a component of the postsynaptic current is mediated by glutamate as it is blocked by a combination of the glutamate receptor antagonists, but is insensitive to a GABA(A) receptor antagonist. We conclude that BNPI functions as vesicular glutamate transporter and that expression of BNPI suffices to define a glutamatergic phenotype in neurons.     

Interactions between enkephalin and GABA in avian retina

In addition to conventional neurotransmitters such as acetylcholine, dopamine, glycine and gamma-aminobutyric acid (GABA), a number of peptide-immunoreactive substances have recently been localized in the vertebrate retina. The functional roles of these retinal peptides and their interactions with conventional neurotransmitters are largely unknown. We have previously shown that exogenous opiates affect both the release of GABA and the firing patterns of ganglion cells in the goldfish retina, and we have now begun a systematic characterization of the opioid pathways in the chicken retina, because, among vertebrate retinas, avian retinas contain the highest concentration of enkephalins. Monoclonal antibodies specific for enkephalin have been used to demonstrate that a subpopulation of enkephalin-containing amacrine cells exists in the chicken retina. This retina also synthesizes Met-enkephalin and releases it on cell depolarization. The enkephalin-induced inhibition of GABA release in goldfish retina led us to examine whether similar interactions occur in chicken, and if so, whether enkephalins and GABA coexist in the same amacrine cells. Our results, presented here, indicate that exogenous enkephalins do indeed inhibit GABA release in the chicken retina. Surprisingly, we found that although some amacrine cells contain both enkephalin and GABA, others contain only one or the other.     

Identification of a distinct synaptic glutamate receptor on horizontal cells in mudpuppy retina

The separation of ON and OFF channels and the development of an antagonistic surround occur at the first synapse in the vertebrate retina. This functional differentiation is mediated by the action of the photoreceptor neurotransmitter on the ON bipolar, OFF bipolar and horizontal cells, respectively. Glutamate mimics the action of the photoreceptor transmitter on all second-order neurones in fish, amphibian and mammalian retinas. The diversity of cellular responses produced by one neurotransmitter raises the possibility of multiple postsynaptic receptor-ionophore complexes. We reported previously that one glutamate analogue, 2-amino-4-phosphonobutyrate, reveals that the ON bipolar synaptic receptor is pharmacologically different from those of other second-order neurones. The results presented here demonstrate that another glutamate analogue, D-O-phosphoserine, selectively antagonizes the synaptic responses of horizontal cells. Taken together, these findings indicate that there are three glutamate-like receptor subtypes in the outer retina and suggest a correlation between receptor subtype and the physiological properties of second-order neurones.     

GAP-43在锦鲤荒漠沙蜥和雉鸡视网膜内分布的免疫组化研究

GAP-43具有多种功能,主要与神经元轴突的生长、再生、神经递质的释放及膜泡的吞噬有关.本研究用免疫组织化学的方法观察了正常成年锦鲤、荒漠沙蜥和雉鸡视网膜内GAP-43分布.结果显示GAP-43主要分布在内网层,另外,在内核层、外网层、光感受器细胞层因动物不同也呈现不同的分布特点,而在节细胞层中3种动物均未发现GAP-43阳性染色.在锦鲤视网膜中GAP-43主要分布在内网层和内核层的无长突细胞;在荒漠沙蜥视网膜中GAP-43主要分布在内网层和外网层,在雉鸡视网膜中GAP-43主要分布在内网层、外网层、外核层和光感受器外节,其中在雉鸡视网膜外核层和光感受器外节中发现阳性分布是在脊椎动物此层发现GAP-43的首次报道.     

Novel pathway connecting the outer and inner vertebrate retina

In many fish retinas, thin axons from the external horizontal cells extend through the inner nuclear layer and expand into large terminal processes that lie along the border of the inner nuclear and inner plexiform layers. Although the horizontal-cell axon terminals are structurally very prominent, their function is unknown. Here we report morphological and functional evidence that signals from catfish (Ictalurus punctatus) horizontal-cell axon terminals can be transmitted directly to amacrine cells. Current injected into horizontal-cell axon terminals produces responses from both transient and sustained amacrine cells very similar to those elicited by light stimuli. Electron microscope observations show chemical synapses from the axon terminals onto amacrine cell perikarya and processes. These data suggest that amacrine cells in the catfish retina receive two inputs, one from bipolar cells and the other from horizontal-cell axon terminals.     

Bipolar cells in the mudpuppy retina use an excitatory amino acid neurotransmitter

The bipolar cells of the vertebrate retina are the principal neuronal elements which transmit photoreceptor activity from the outer to the inner retina. An important function of the bipolars is to segregate photoreceptor input into independent ON and OFF channels which are subserved, respectively, by the depolarizing and hyperpolarizing bipolar subtypes. Ultrastructural and physiological observations suggest that chemical neurotransmission is the predominant means of bipolar input to the inner retina. Both ON and OFF bipolars apparently release excitatory transmitters. Histological studies with cytotoxic agents and physiological studies indicate that third-order neurones have excitatory amino acid receptors. In ON-OFF amacrine and ganglion cells, which receive input from both bipolars, ON and OFF excitation have a similar ionic basis, suggesting that the same transmitter may be released by both types of bipolars. We have now found that (+/-)cis-2,3-piperidine dicarboxylic acid (PDA), a new excitatory amino acid antagonist, blocks bipolar input to the inner retina and thus suggests that an excitatory amino acid is a bipolar cell transmitter.     

Expression cloning of a cocaine- and antidepressant-sensitive human noradrenaline transporter

At most synapses, chemical signalling is terminated by a rapid reaccumulation of neurotransmitter into presynaptic terminals. Uptake systems for the biogenic amines are the initial site of action for therapeutic antidepressants and drugs such as cocaine and the amphetamines. We have isolated a complementary DNA clone encoding a human noradrenaline transporter. The cDNA sequence predicts a protein of 617 amino acids, with 12-13 highly hydrophobic regions compatible with membrane-spanning domains. Expression of the cDNA clone in transfected HeLa cells indicates that noradrenaline transport activity is sodium-dependent and sensitive to selective noradrenaline transport inhibitors. Transporter RNA is localized to the brainstem and the adrenal gland. The predicted protein sequence demonstrates significant amino-acid identity with the Na+/gamma-aminobutyric acid transporter, thus identifying a new gene family for neurotransmitter transporter proteins. Analysis of its structure and function may lead to structure-based drug design for the treatment of human depression and could help determine whether transporter abnormalities underlie affective disorders.     

Drosophila Crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres

Drosophila Crumbs (Crb) is required for apical-basal polarity and is an apical determinant in embryonic epithelia. Here, we describe properties of Crb that control the position and integrity of the photoreceptor adherens junction and photosensitive organ, or rhabdomere. In contrast to normal photoreceptor adherens junctions and rhabdomeres, which span the depth of the retina, adherens junctions and rhabdomeres of Crb-deficient photoreceptors initially accumulate at the top of the retina and fail to maintain their integrity as they stretch to the retinal floor. We show that Crb controls localization of the adherens junction through its intracellular domain containing a putative binding site for a protein 4.1 superfamily protein (FERM). Although loss of Crb or overexpression of the FERM binding domain causes mislocalization of adherens junctions, they do not result in a significant loss of photoreceptor polarity. Mutations in CRB1, a human homologue of crb, are associated with photoreceptor degeneration in retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis (LCA). The intracellular domain of CRB1 behaves similarly to its Drosophila counterpart when overexpressed in the fly eye. Our studies may provide clues for mechanisms of photoreceptor degeneration in RP12 and LCA.     

Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles.

Neurotransmitter release at synapses between nerve cells is mediated by calcium-triggered exocytotic fusion of synaptic vesicles. Before fusion, vesicles dock at the presynaptic release site where they mature to a fusion-competent state. Here we identify Munc13-1, a brain-specific presynaptic phorbol ester receptor, as an essential protein for synaptic vesicle maturation. We show that glutamatergic hippocampal neurons from mice lacking Munc13-1 form ultrastructurally normal synapses whose synaptic-vesicle cycle is arrested at the maturation step. Transmitter release from mutant synapses cannot be triggered by action potentials, calcium-ionophores or hypertonic sucrose solution. In contrast, release evoked by alpha-latrotoxin is indistinguishable from wild-type controls, indicating that the toxin can bypass Munc13-1-mediated vesicle maturation. A small subpopulation of synapses of any given glutamatergic neuron as well as all synapses of GABA (gamma-aminobutyric acid)-containing neurons are unaffected by Munc13-1 loss, demonstrating the existence of multiple and transmitter-specific synaptic vesicle maturation processes in synapses.     

Identification of a photoreceptor-specific mRNA encoded by the gene responsible for retinal degeneration slow (rds)

Mutant mice homozygous for 'retinal degeneration slow' (rds/rds) are characterized phenotypically by abnormal development of photoreceptor outer segments in the retina, followed by gradual degeneration of photoreceptors. This process of degeneration is complete by one year, with preservation of all other retinal cells. The biochemical defect that leads to the mutant phenotype is not known. Our strategy for cloning the rds gene was based upon three previously reported observations. First, the rds locus maps to chromosome 17. Second, experimental rds/rds----+/+ and rds/+----+/+ tetra-parental mice manifest patchy photoreceptor changes in the retina, suggesting that the wild-type rds locus is expressed within cells of the photoreceptor lineage. Finally, the process of degeneration is specific to photoreceptors. On the basis of these observations, we predicted that the rds mRNA is encoded by a gene on chromosome 17 and is normally expressed exclusively within photoreceptors in the retina. We here present evidence that this is the case.     

Neuronal circuitry mechanism regulating adult quiescent neural stem-cell fate decision

Adult neurogenesis arises from neural stem cells within specialized niches. Neuronal activity and experience, presumably acting on this local niche, regulate multiple stages of adult neurogenesis, from neural progenitor proliferation to new neuron maturation, synaptic integration and survival. It is unknown whether local neuronal circuitry has a direct impact on adult neural stem cells. Here we show that, in the adult mouse hippocampus, nestin-expressing radial glia-like quiescent neural stem cells (RGLs) respond tonically to the neurotransmitter γ-aminobutyric acid (GABA) by means of γ2-subunit-containing GABAA receptors. Clonal analysis of individual RGLs revealed a rapid exit from quiescence and enhanced symmetrical self-renewal after conditional deletion of γ2. RGLs are in close proximity to terminals expressing 67-kDa glutamic acid decarboxylase (GAD67) of parvalbumin-expressing (PV+) interneurons and respond tonically to GABA released from these neurons. Functionally, optogenetic control of the activity of dentate PV+ interneurons, but not that of somatostatin-expressing or vasoactive intestinal polypeptide (VIP)-expressing interneurons, can dictate the RGL choice between quiescence and activation. Furthermore, PV+ interneuron activation restores RGL quiescence after social isolation, an experience that induces RGL activation and symmetrical division. Our study identifies a niche cell–signal–receptor trio and a local circuitry mechanism that control the activation and self-renewal mode of quiescent adult neural stem cells in response to neuronal activity and experience.     

Horizontal cell synapses onto glycine-accumulating interplexiform cells

Horizontal cells mediate lateral transmission of signals in the outer plexiform layer of the vertebrate retina, and are presumed to contribute to surround properties of photoreceptors and bipolar cells by chemical transmission. The cell bodies and dendrites of fish horizontal cells possess presynaptic specializations characteristic of conventional chemical synapses. Horizontal cell axon terminals have not so far been shown to contain presynaptic specializations nor have the targets of the somatic and dendritic synapses been fully characterized. Using electron microscope autoradiography of retinas labelled by high-affinity 3H-glycine uptake, we show here that goldfish horizontal cells make somatodendritic and axodendritic synapses on glycinergic interplexiform cells (Gly-IPCs) as apposed to dopaminergic interplexiform cells. Thus, horizontal cells have at least three postsynaptic targets: photoreceptors, bipolar cells and Gly-IPCs. Gly-IPCs may constitute a major alternative pathway for horizontal cell signals to reach the inner plexiform layer.