PubMed Health⌕ Search

Biomedical subjects

Gary Matthews

Publications and source records attributed to Gary Matthews.

At least 19 recordsLinked to original sources
PubMed2007

Polarized distribution of ion channels within microdomains of the axon initial segment.

Voltage-gated sodium (Na(v)) channels accumulate at the axon initial segment (IS), where their high density supports spike initiation. Maintenance of this high density of Na(v) channels involves a macromolecular complex that includes the cytoskeletal linker protein ankyrin-G, the only protein known to bind Na(v) channels and localize them at the IS. We found previously that Na(v)1.6 is the predominant Na(v) channel isoform at IS of adult rodent retinal ganglion cells. However, here we report that Na(v)1.6 immunostaining is consistently reduced or absent in short regions of the IS proximal to the soma, although both ankyrin-G and pan-Na(v) antibodies stain this region. We show that this proximal IS subregion is a unique axonal microdomain, containing an accumulation of Na(v)1.1 channels that are spatially segregated from the Na(v)1.6 channels of the distal IS. Additionally, we find that axonal K(v)1.2 potassium channels are present within the distal IS, but are also excluded from the Na(v)1.1-enriched proximal IS microdomain. Because ankyrin-G was prominent in both proximal and distal subcompartments of the IS, where it colocalized with either Na(v)1.1 or Na(v)1.6, respectively, mechanisms other than association with ankyrin-G must mediate differential targeting of Na(v) channel subtypes to achieve the spatial precision observed within the IS. This precise arrangement of ion channels within the axon initial segment is likely an important determinant of the firing properties of ganglion cells and other mammalian neurons.

Action Potentials↗
PubMed2006

The synaptic vesicle cycle: is kissing overrated?

In this issue of Neuron, Granseth et al. re-examine the mechanism of endocytosis at hippocampal synapses using a new optical reporter, sypHy. They conclude that only a single slow mode of endocytosis operates at this synapse and that retrieval after physiological stimuli is largely, if not solely, dominated by the clathrin-mediated pathway. These conclusions dispute previous assertions that "kiss-and-run" is a major mechanism of vesicle recycling at hippocampal synapses.

Animals↗
PubMed2006

Characterization of a novel cyclic nucleotide-gated channel from zebrafish brain.

Cyclic nucleotide-gated (CNG) channels have been well characterized in the sensory receptors of vision and olfaction, but their characteristics in other tissues remain largely unknown. Here, we report characterization of a novel brain-specific CNG channel from zebrafish. Unique among CNG channels, the transcript is expressed mainly in the brain. When expressed in Xenopus oocytes, the channel's electrophysiological properties are distinct compared to CNG channels from either rods (CNGA1), olfactory receptors (CNGA2), or cones (CNGA3). The channel is less sensitive to cAMP than cGMP (K(1/2) of 280 and 7 microM, respectively), with a maximum cAMP efficacy at least 80% of that with saturating levels of cGMP. The single-channel conductance of 58pS is larger than most other CNG channels. Like other CNG channels the channel is relatively nonselective among monovalent cations. However, unlike other CNG channels, there was rundown of the macroscopic current within 30-100 min after patch excision.

Amino Acid Sequence↗
PubMed2006

Impaired firing and cell-specific compensation in neurons lacking nav1.6 sodium channels.

The ability of neurons to fire precise patterns of action potentials is critical for encoding inputs and efficiently driving target neurons. At the axon initial segment and nodes of Ranvier, where nerve impulses are generated and propagated, a high density of Na(v)1.2 sodium channels is developmentally replaced by Na(v)1.6 channels. In retinal ganglion cells (GCs), this isoform switch coincides with the developmental transition from single spikes to repetitive firing. Also, Na(v)1.6 channels are required for repetitive spiking in cerebellar Purkinje neurons. These previous observations suggest that the developmental appearance of Na(v)1.6 underlies the transition to repetitive spiking in GCs. To test this possibility, we recorded from GCs of med (Na(v)1.6-null) and wild-type mice during postnatal development. By postnatal day 18, when the switch to Na(v)1.6 at GC initial segments is normally complete, the maximal sustained and instantaneous firing rates were lower in med than in wild-type GCs, demonstrating that Na(v)1.6 channels are necessary to attain physiologically relevant firing frequencies in GCs. However, the firing impairment was milder than that reported previously in med Purkinje neurons, which prompted us to look for differences in compensatory sodium channel expression. Both Na(v)1.2 and Na(v)1.1 channels accumulated at initial segments and nodes of med GCs, sites normally occupied by Na(v)1.6. In med Purkinje cells, only Na(v)1.1 channels were found at initial segments, whereas in other brain regions, only Na(v)1.2 was detected at med initial segments and nodes. Thus, compensatory mechanisms in channel isoform distribution are cell specific, which likely results in different firing properties.

Action Potentials↗
PubMed2006

Expression of sodium channels Nav1.2 and Nav1.6 during postnatal development of the retina.

During the second and third postnatal weeks, there is a developmental switch from sodium channel isoform Na(v)1.2 to isoform Na(v)1.6 at initial segments and nodes of Ranvier in rat retinal ganglion cells. We used quantitative, real-time PCR to determine if the developmental appearance of Na(v)1.6 channels is accompanied by an increase in steady-state level of Na(v)1.6 mRNA in the retina. Between postnatal day 2 (P2) and P10, Na(v)1.6 levels did not change, but between P10 and P19, there was an approximately three-fold increase in Na(v)1.6 transcript levels. This coincides with the appearance of Na(v)1.6 channels in the retina and optic nerve. The steady-state level of Na(v)1.2 mRNA also increased during this same period, which suggests that the rise in Na(v)1.6 may be part of a general increase in sodium channel transcripts at about the time of eye opening at P14. The results are consistent with a developmental increase in steady-state transcripts giving rise to a corresponding increase in sodium channel protein expression.

Animals↗
PubMed2006

Identification of calcium channel alpha1 subunit mRNA expressed in retinal bipolar neurons.

PURPOSE: Glutamate release from goldfish bipolar cell terminals is driven by Ca2+ influx through L-type calcium channels that exhibit several uncommon features, including rapid kinetics of activation and deactivation, slow inactivation, and activation at an unusually negative voltage range for L-type channels. The purpose of this study was to establish the molecular identities of the alpha1 subunits responsible for these distinctive properties. METHODS: Transcripts for calcium channel alpha1 subunits expressed in individual goldfish ON-type bipolar cells were identified using single-cell reverse transcriptase polymerase chain reaction (RT-PCR). After cloning the goldfish homologs of the zebrafish and mammalian subunits, we designed sets of nested primers that are specific for Cav1.3a, and Cav1.3b L-type calcium channels. RESULTS: Large-terminal, ON-type bipolar cells express transcripts of Cav1.3a and/or Cav1.3b. CONCLUSIONS: The endogenous expression of only one or both subunits in a single cell raises the possibility of functionally distinct classes of bipolar cells that differ in calcium current properties.

Amino Acid Sequence↗
PubMed2005

Novel clustering of sodium channel Na(v)1.1 with ankyrin-G and neurofascin at discrete sites in the inner plexiform layer of the retina.

Voltage-gated sodium channels cluster at sites of action potential generation and propagation by interacting with partner proteins such as neurofascin, an adhesion molecule in the L1 family, and ankyrin-G, a spectrin-binding protein required for sodium channel accumulation at axon initial segments. Here, we describe in the inner plexiform layer of the retina a novel site of high-density sodium channel clustering, marked by ankyrin-G and neurofascin. The sodium channel isoform at this site is Na(v)1.1, instead of the Na(v)1.6 channels more commonly found in association with the clustering machinery. During development, Na(v)1.2 channels first associate with ankyrin-G in the inner plexiform layer but are later replaced by Na(v)1.1, similar to the switch from Na(v)1.2 to Na(v)1.6 at nodes of Ranvier and initial segments. This represents the first instance of high-density clustering of Na(v)1.1 channels, which may contribute to synaptic interactions among retinal neurons in the inner plexiform layer.

Animals↗
PubMed2005

Structure and function of ribbon synapses.

Sensory neurons with short conduction distances can use nonregenerative, graded potentials to modulate transmitter release continuously. This mechanism can transmit information at much higher rates than spiking. Graded signaling requires a synapse to sustain high rates of exocytosis for relatively long periods, and this capacity is the special virtue of ribbon synapses. Vesicles tethered to the ribbon provide a pool for sustained release that is typically fivefold greater than the docked pool available for fast release. The current article, which is part of the TINS Synaptic Connectivity series, reviews recent evidence for this fundamental computational strategy and its underlying cell biology.

Action Potentials↗
PubMed2004

Visualizing synaptic ribbons in the living cell.

Visual and auditory information is encoded by sensory neurons that tonically release neurotransmitter at high rates. The synaptic ribbon is an essential organelle in nerve terminals of these neurons. Its precise function is unknown, but if the ribbon could be visualized in a living terminal, both its own dynamics and its relation to calcium and vesicle dynamics could be studied. We designed a short fluorescent peptide with affinity for a known binding domain of RIBEYE, a protein unique to the ribbon. When introduced via a whole-cell patch pipette, the peptide labeled structures at the presynaptic plasma membrane of ribbon-type terminals. The fluorescent spots match in size, location, number, and distribution the known features of synaptic ribbons. Furthermore, fluorescent spots mapped by confocal microscopy directly match the ribbons identified by electron microscopy in the same cell. Clearly the peptide binds to the synaptic ribbon, but even at saturating concentrations it affects neither the morphology of the ribbon nor its tethering of synaptic vesicles. It also does not inhibit exocytosis. Using the peptide label, we observed that the ribbon is immobile over minutes and that calcium influx is concentrated at the ribbon. Finally, we find that each ribbon in a retinal bipolar cell contains approximately 4000 molecules of RIBEYE, indicating that it is the major component of the synaptic ribbon.

Amino Acid Motifs↗
PubMed2004

Cycling the synapse: scenic versus direct routes for vesicles.

What happens to synaptic vesicles after they release their neurotransmitter content? Recent work on a variety of synaptic systems shows that there is no single answer to this question. Rather, it seems that neurons use a variety of methods to retrieve and reuse synaptic vesicles after they have undergone exocytosis. The challenge now is to establish the molecular mechanisms and to decipher the rules that govern which cycling pathway is used in a given functional context.

Animals↗
PubMed2004

Vesicle priming and depriming: a SNAP decision.

Synapses have a limited pool of vesicles that are docked and primed for rapid release. In neuroendocrine cells, splice variants of the SNARE protein SNAP-25 and phosphorylation of SNAP-25 independently influence the size of the releasable vesicle pool, possibly by altering the rate of vesicle depriming. Pre- and posttranslational modifications of SNAP-25 may therefore affect synaptic strength.

Animals↗
PubMed2003

Endocytosis and vesicle recycling at a ribbon synapse.

At ribbon synapses, where exocytosis is regulated by graded depolarization, vesicles can fuse very rapidly with the plasma membrane (complete discharge of the releasable pool in approximately 200 msec). Vesicles are also retrieved very rapidly (time constant of approximately 1 sec), leading us to wonder whether their retrieval uses an unusual mechanism. To study this, we exposed isolated bipolar neurons from goldfish retina to cationized ferritin. This electron-dense marker uniformly decorated the cell membrane and was carried into the cell during membrane retrieval. Endocytosis was activity-dependent and restricted to the synaptic terminal. The labeling pattern was consistent with direct retrieval from the plasma membrane of large, uncoated endosomes 60-200 nm in diameter. Even after extensive synaptic activity lasting several minutes, most of the ferritin remained in large endosomes and was present in only approximately 10% of the small vesicles that constitute the reserve pool. By contrast, after brief stimulation at a conventional terminal, ferritin did not reside in endosomes but was present in approximately 63% of the small vesicles. We suggest that the bipolar ribbon synapse sustains its rapid exocytosis by retrieving membrane in larger "bites" than the clathrin-dependent mechanism thought to dominate at conventional synapses. The resulting large endosomes bud off small vesicles, which reenter the reserve pool and finally the releasable pool.

Action Potentials↗
PubMed2003

Functional specialization of the axon initial segment by isoform-specific sodium channel targeting.

Voltage-dependent sodium channels cluster at high density at axon initial segments, where propagating action potentials are thought to arise, and at nodes of Ranvier. Here, we show that the sodium channel Na(v)1.6 is precisely localized at initial segments of retinal ganglion cells (RGCs), whereas a different isoform, Na(v)1.2, is found in the neighboring unmyelinated axon. During development, initial segments first expressed Na(v)1.2, and Na(v)1.6 appeared later, approximately in parallel with the onset of repetitive RGC firing. In Shiverer mice, Na(v)1.6 localization at the initial segment was unaffected, although Na(v)1.6 expression was severely disrupted in the aberrantly myelinated optic nerve. Targeting or retention of Na(v)1.6 requires molecular interactions that normally occur only at initial segments and nodes of Ranvier. Expression at nodes but not initial segments exhibits an additional requirement for intact myelination. Because of their high density at the initial segment, Na(v)1.6 channels may be crucial in determining neuronal firing properties.

Animals↗
PubMed2003

Retinal bipolar neurons express the cyclic nucleotide-gated channel of cone photoreceptors.

Cyclic nucleotide-gated (CNG) channels link intracellular cyclic nucleotides to changes in membrane ionic conductance in a variety of physiological contexts. In the retina, in addition to their central role in phototransduction, CNG channels may be involved in nitric oxide signaling in bipolar neurons or in the hyperpolarizing synaptic response to glutamate in ON-type (depolarizing) bipolar cells. Despite their potential physiological significance, however, expression of CNG channels has not yet been demonstrated in bipolar cells. To identify CNG channel subtypes in retinal bipolar neurons, we used single-cell molecular biological techniques in morphologically distinctive ON bipolar cells from goldfish retina. Both single-cell in situ hybridization and single-cell RT-PCR demonstrated in ON bipolar cells the presence of mRNA for the CNG channel subtype that is also found in cone photoreceptors. Other bipolar cells, which likely represent OFF cells, did not express the cone CNG channel. Thus the CNG channel of cone photoreceptors is expressed in ON bipolar cells, where it may be involved in physiological responses to nitric oxide, or in the sign-inverting glutamatergic synapse that gives rise to the ON visual pathway.

Amino Acid Sequence↗
PubMed2002

Synaptic vesicle exocytosis. Does a lingering kiss lead to fusion?

Direct optical measurements of single synaptic vesicles undergoing exocytosis at a synapse reveal rapid and complete transfer of membrane marker from the vesicle to the plasma membrane (; this issue of Neuron). Contact between the two membranes is consistent with free lipid exchange, such as might result from full fusion of the vesicle and plasma membranes.

Animals↗
PubMed2002

Differences in stimulus induced calcium increases in lacrimal gland acinar cells from normal and NZB/NZW F1 female mice.

PURPOSE: To compare the changes in intracellular free calcium in response to both cholinergic and adrenergic agonists in cells isolated from exorbital lacrimal glands of NZB/NZW F1 female mice (NZB/W) and normal Swiss Webster mice (SW). METHODS: We have loaded cells with Fura-2 and measured total intracellular calcium using ratiomicrofluorometric methods. Isolated cells were also patch-clamped using perforated patch. RESULTS: In all cells, both carbachol (CCh) and phenylephrine (PE) increased intracellular calcium. The calcium increase to both CCh and PE was less in cells from NZB/W animals than in cells from SW animals. In cells from young animals, the baseline calcium levels were the same, but in cells from older (6 months) NZB/W animals, the baseline levels of calcium were 50% higher than those seen in SW animals. CONCLUSION: These data suggest that cells from the NZB/W mice have different calcium dynamics, which could contribute to their compromised fluid secretion.

Adrenergic alpha-Agonists↗
PubMed2002

Calcium dependence of exocytosis in lacrimal gland acinar cells.

Simultaneous measurements of membrane capacitance and intracellular calcium concentration were used to examine the calcium dependence of exocytosis in single acinar cells from mouse lacrimal gland and to establish the quantitative relation between calcium concentration and rate of exocytosis. Application of adrenergic or muscarinic agonists elevated intracellular calcium and evoked exocytosis, as indicated by an increase in membrane capacitance of single cells. The capacitance response to agonist stimulation was eliminated by internal dialysis with the calcium buffer EGTA, which demonstrated that the increase in intracellular calcium was necessary for agonist-evoked exocytosis. When internal calcium was elevated by application of the calcium ionophore ionomycin, exocytosis was evoked in the absence of agonist stimulation. Thus an increase in intracellular calcium was necessary and sufficient for exocytosis in single acinar cells. The rate of change of membrane capacitance increased as approximately the third power of the calcium concentration, which is similar to the dependence of exocytosis rate on calcium concentration in other secretory cells.

Adrenergic Agonists↗