Calcium/calmodulin-dependent protein kinase II associates with the K+ channel isoform Kv4.3 in adult rat optic nerve.

Spikes are said to exhibit "memory" in that they can be altered by spikes that precede them. In retinal ganglion cell axons, for example, rapid spiking can slow the propagation of subsequent spikes. This increases inter-spike interval and, thus, low-pass filters instantaneous spike frequency. Similarly, a K+ ion channel blocker (4-aminopyridine, 4AP) increases the time-to-peak of compound action potentials recorded from optic nerve, and we recently found that reducing autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) does too. These results would be expected if CaMKII modulates spike propagation by regulating 4AP-sensitive K+ channels. As steps toward identifying a possible substrate, we test whether (i) 4AP alters optic nerve spike shape in ways consistent with reducing K+ current, (ii) 4AP alters spike propagation consistent with effects of reducing CaMKII activation, (iii) antibodies directed against 4AP-sensitive and CaMKII-regulated K+ channels bind to optic nerve axons, and (iv) optic nerve CaMKII co-immunoprecipitates with 4AP-sensitive K+ channels. We find that, in adult rat optic nerve, (i) 4AP selectively slows spike repolarization, (ii) 4AP slows spike propagation, (iii) immunogen-blockable staining is achieved with anti-Kv4.3 antibodies but not with antibodies directed against Kv1.4 or Kv4.2, and (iv) CaMKII associates with Kv4.3. Kv4.3 may thus be a substrate that underlies activity-dependent spike regulation in adult visual system pathways.

Extraretinal Spike Normalization in Retinal Ganglion Cell Axons.

Spike conduction velocity characteristically differs between myelinated and unmyelinated axons. Here we test whether spikes of myelinated and unmyelinated paths differ in other respects by measuring rat retinal ganglion cell (RGC) spike duration in the intraretinal, unmyelinated nerve fiber layer and the extraretinal, myelinated optic nerve and optic chiasm. We find that rapid spike firing and illumination broaden spikes in intraretinal axons but not in extraretinal axons. RGC axons thus initiate spikes intraretinally and normalize spike duration extraretinally. Additionally, we analyze spikes that were recorded in a previous study of rhesus macaque retinogeniculate transmission and find that rapid spike firing does not broaden spikes in optic tract. The spike normalization we find reduces the number of spike properties that can change during RGC light responses. However, this is not because identical spikes fire in all axons. Instead, our recordings show that different subtypes of RGC generate axonal spikes of different durations and that the differences resemble spike duration increases that alter neurotransmitter release from other neurons. Moreover, previous studies have shown that RGC spikes of shorter duration can fire at higher maximum frequencies. These properties should facilitate signal transfer by different mechanisms at RGC synapses onto subcortical target neurons.

Autophosphorylated CaMKII Facilitates Spike Propagation in Rat Optic Nerve.

Repeated spike firing can transmit information at synapses and modulate spike timing, shape, and conduction velocity. These latter effects have been found to result from voltage-induced changes in ion currents and could alter the signals carried by axons. Here, we test whether Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates spike propagation in adult rat optic nerve. We find that small-, medium-, and large-diameter axons bind anti-Thr286-phosphorylated CaMKII (pT286) antibodies and that, in isolated optic nerves, electrical stimulation reduces pT286 levels, spike propagation is hastened by CaMKII autophosphorylation and slowed by CaMKII dephosphorylation, single and multiple spikes slow propagation of subsequently activated spikes, and more frequent stimulation produces greater slowing. Likewise, exposing freely moving animals to flickering illumination reduces pT286 levels in optic nerves and electrically eliciting spikes in vivo in either the optic nerve or optic chiasm slows subsequent spike propagation in the optic nerve. By increasing the time that elapses between successive spikes as they propagate, pT286 dephosphorylation and activity-induced spike slowing reduce the frequency of propagated spikes below the frequency at which they were elicited and would thus limit the frequency at which axons synaptically drive target neurons. Consistent with this, the ability of retinal ganglion cells to drive at least some lateral geniculate neurons has been found to increase when presented with light flashes at low and moderate temporal frequencies but less so at high frequencies. Activity-induced decreases in spike frequency may also reduce the energy required to maintain normal intracellular Na+ and Ca2+ levels.SIGNIFICANCE STATEMENT By propagating along axons at constant velocities, spikes could drive synapses as frequently as they are initiated. However, the onset of spiking has been found to alter the conduction velocity of subsequent ("follower") spikes in various preparations. Here, we find that spikes reduce spike frequency in rat optic nerve by slowing follower spike propagation and that electrically stimulated spiking ex vivo and spike-generating flickering illumination in vivo produce net decreases in axonal Ca2+/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation. Consistent with these effects, propagation speed increases and decreases, respectively, with CaMKII autophosphorylation and dephosphorylation. Lowering spike frequency by CaMKII dephosphorylation is a novel consequence of axonal spiking and light adaptation that could decrease synaptic gain as stimulus frequency increases and may also reduce energy use.

Somatic and neuritic spines on tyrosine hydroxylase-immunopositive cells of rat retina.

Dopamine- and tyrosine hydroxylase-immunopositive cells (TH cells) modulate visually driven signals as they flow through retinal photoreceptor, bipolar, and ganglion cells. Previous studies suggested that TH cells release dopamine from varicose axons arborizing in the inner and outer plexiform layers after glutamatergic synapses depolarize TH cell dendrites in the inner plexiform layer and these depolarizations propagate to the varicosities. Although it has been proposed that these excitatory synapses are formed onto appendages resembling dendritic spines, spines have not been found on TH cells of most species examined to date or on TH cell somata that release dopamine when exposed to glutamate receptor agonists. By use of protocols that preserve proximal retinal neuron morphology, we have examined the shape, distribution, and synapse-related immunoreactivity of adult rat TH cells. We report here that TH cell somata, tapering and varicose inner plexiform layer neurites, and varicose outer plexiform layer neurites all bear spines, that some of these spines are immunopositive for glutamate receptor and postsynaptic density proteins (viz., GluR1, GluR4, NR1, PSD-95, and PSD-93), that TH cell somata and tapering neurites are also immunopositive for a γ-aminobutyric acid (GABA) receptor subunit (GABAA Rα1 ), and that a synaptic ribbon-specific protein (RIBEYE) is found adjacent to some colocalizations of GluR1 and TH in the inner plexiform layer. These results identify previously undescribed sites at which glutamatergic and GABAergic inputs may stimulate and inhibit dopamine release, especially at somata and along varicose neurites that emerge from these somata and arborize in various levels of the retina. J. Comp. Neurol. 525:1707-1730, 2017. © 2016 Wiley Periodicals, Inc.

Fixation strategies for retinal immunohistochemistry.

Immunohistochemical and ex vivo anatomical studies have provided many glimpses of the variety, distribution, and signaling components of vertebrate retinal neurons. The beauty of numerous images published to date, and the qualitative and quantitative information they provide, indicate that these approaches are fundamentally useful. However, obtaining these images entailed tissue handling and exposure to chemical solutions that differ from normal extracellular fluid in composition, temperature, and osmolarity. Because the differences are large enough to alter intercellular and intracellular signaling in neurons, and because retinae are susceptible to crush, shear, and fray, it is natural to wonder if immunohistochemical and anatomical methods disturb or damage the cells they are designed to examine. Tissue fixation is typically incorporated to guard against this damage and is therefore critically important to the quality and significance of the harvested data. Here, we describe mechanisms of fixation; advantages and disadvantages of using formaldehyde and glutaraldehyde as fixatives during immunohistochemistry; and modifications of widely used protocols that have recently been found to improve cell shape preservation and immunostaining patterns, especially in proximal retinal neurons.

Moniliform deformation of retinal ganglion cells by formaldehyde-based fixatives.

Protocols for characterizing cellular phenotypes commonly use chemical fixatives to preserve anatomical features, mechanically stabilize tissue, and stop physiological responses. Formaldehyde, diluted in either phosphate-buffered saline or phosphate buffer, has been widely used in studies of neurons, especially in conjunction with dyes and antibodies. However, previous studies have found that these fixatives induce the formation of bead-like varicosities in the dendrites and axons of brain and spinal cord neurons. We report here that these formaldehyde formulations can induce bead formation in the dendrites and axons of adult rat and rabbit retinal ganglion cells, and that retinal ganglion cells differ from hippocampal, cortical, cerebellar, and spinal cord neurons in that bead formation is not blocked by glutamate receptor antagonists, a voltage-gated Na(+) channel toxin, extracellular Ca(2+) ion exclusion, or temperature shifts. Moreover, we describe a modification of formaldehyde-based fixatives that prevents bead formation in retinal ganglion cells visualized by green fluorescent protein expression and by immunohistochemistry.

Dopamine and full-field illumination activate D1 and D2-D5-type receptors in adult rat retinal ganglion cells.

Dopamine can regulate signal generation and transmission by activating multiple receptors and signaling cascades, especially in striatum, hippocampus, and cerebral cortex. Dopamine modulates an even larger variety of cellular properties in retina, yet has been reported to do so by only D1 receptor-driven cyclic adenosine monophosphate (cAMP) increases or D2 receptor-driven cAMP decreases. Here, we test the possibility that dopamine operates differently on retinal ganglion cells, because the ganglion cell layer binds D1 and D2 receptor ligands, and displays changes in signaling components other than cAMP under illumination that should release dopamine. In adult rat retinal ganglion cells, based on patch-clamp recordings, Ca(2+) imaging, and immunohistochemistry, we find that 1) spike firing is inhibited by dopamine and SKF 83959 (an agonist that does not activate homomeric D1 receptors or alter cAMP levels in other systems); 2) D1 and D2 receptor antagonists (SCH 23390, eticlopride, raclopride) counteract these effects; 3) these antagonists also block light-induced rises in cAMP, light-induced activation of Ca(2+) /calmodulin-dependent protein kinase II, and dopamine-induced Ca(2+) influx; and 4) the Ca(2+) rise is markedly reduced by removing extracellular Ca(2+) and by an IP3 receptor antagonist (2-APB). These results provide the first evidence that dopamine activates a receptor in adult mammalian retinal neurons that is distinct from classical D1 and D2 receptors, and that dopamine can activate mechanisms in addition to cAMP and cAMP-dependent protein kinase to modulate retinal ganglion cell excitability.

Thy1 associates with the cation channel subunit HCN4 in adult rat retina.

PURPOSE: The membrane expression and gene promoter of the glycosylphosphatidylinositol (GPI)-anchored protein Thy1 have been widely used to examine the morphology and distribution of retinal ganglion cells in normal eyes and disease models. However, it is not known how adult mammalian retinal neurons use Thy1. Because Thy1 is not a membrane-spanning protein and, instead, complexes with structural and signaling proteins in other tissues, the aim of this study was to find protein partners of retinal Thy1. METHODS: Coimmunoprecipitation, immunohistochemistry, confocal imaging, and patch-clamp recording were used to test for association of Thy1 and HCN4, a cation channel subunit, in adult rat retina. RESULTS: Hyperpolarization of cells immunopanned by an anti-Thy1 antibody activated HCN channels. Confocal imaging showed that individual somata in the ganglion cell layer bound antibodies against Thy1 and HCN4, that the majority of these bindings colocalized, and that some of the immunopositive cells also bound antibody against a ganglion cell marker (Brn3a). Consistent with these results, Thy1 and HCN4 were coimmunoprecipitated by magnetic beads coated with either anti-Thy1 antibody or anti-HCN4 antibody. In control experiments, beads coated with these antibodies did not immunoprecipitate a photoreceptor rim protein (ABCR) and uncoated beads did not immunoprecipitate either Thy1 or HCN4. CONCLUSIONS: This is the first report that Thy1 colocalizes and coimmunoprecipitates with a membrane-spanning protein in retina, that Thy1 complexes with an ion channel protein in any tissue, and that a GPI-anchored protein associates with an HCN channel subunit protein.

Colocalization of hyperpolarization-activated, cyclic nucleotide-gated channel subunits in rat retinal ganglion cells.

The current-passing pore of mammalian hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels is formed by subunit isoforms denoted HCN1-4. In various brain areas, antibodies directed against multiple isoforms bind to single neurons, and the current (I(h)) passed during hyperpolarizations differs from that of heterologously expressed homomeric channels. By contrast, retinal rod, cone, and bipolar cells appear to use homomeric HCN channels. Here, we assess the generality of this pattern by examining HCN1 and HCN4 immunoreactivity in rat retinal ganglion cells, measuring I(h) in dissociated cells, and testing whether HCN1 and HCN4 proteins coimmunoprecipitate. Nearly half of the ganglion cells in whole-mounted retinae bound antibodies against both isoforms. Consistent with colocalization and physical association, 8-bromo-cAMP shifted the voltage sensitivity of I(h) less than that of HCN4 channels and more than that of HCN1 channels, and HCN1 coimmunoprecipitated with HCN4 from membrane fraction proteins. Finally, the immunopositive somata ranged in diameter from the smallest to the largest in rat retina, the dendrites of immunopositive cells arborized at various levels of the inner plexiform layer and over fields of different diameters, and I(h) activated with similar kinetics and proportions of fast and slow components in small, medium, and large somata. These results show that different HCN subunits colocalize in single retinal ganglion cells, identify a subunit that can reconcile native I(h) properties with the previously reported presence of HCN4 in these cells, and indicate that I(h) is biophysically similar in morphologically diverse retinal ganglion cells and differs from I(h) in rods, cones, and bipolar cells.

Inhibition of adult rat retinal ganglion cells by D1-type dopamine receptor activation.

The spike output of neural pathways can be regulated by modulating output neuron excitability and/or their synaptic inputs. Dopaminergic interneurons synapse onto cells that route signals to mammalian retinal ganglion cells, but it is unknown whether dopamine can activate receptors in these ganglion cells and, if it does, how this affects their excitability. Here, we show D(1a) receptor-like immunoreactivity in ganglion cells identified in adult rats by retrogradely transported dextran, and that dopamine, D(1)-type receptor agonists, and cAMP analogs inhibit spiking in ganglion cells dissociated from adult rats. These ligands curtailed repetitive spiking during constant current injections and reduced the number and rate of rise of spikes elicited by fluctuating current injections without significantly altering the timing of the remaining spikes. Consistent with mediation by D(1)-type receptors, SCH-23390 [R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine] reversed the effects of dopamine on spikes. Contrary to a recent report, spike inhibition by dopamine was not precluded by blocking I(h). Consistent with the reduced rate of spike rise, dopamine reduced voltage-gated Na(+) current (I(Na)) amplitude, and tetrodotoxin, at doses that reduced I(Na) as moderately as dopamine, also inhibited spiking. These results provide the first direct evidence that D(1)-type dopamine receptor activation can alter mammalian retinal ganglion cell excitability and demonstrate that dopamine can modulate spikes in these cells by a mechanism different from the presynaptic and postsynaptic means proposed by previous studies. To our knowledge, our results also provide the first evidence that dopamine receptor activation can reduce excitability without altering the temporal precision of spike firing.

HCN4-like immunoreactivity in rat retinal ganglion cells.

Antisera directed against hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels bind to somata in the ganglion cell layer of rat and rabbit retinas, and mRNA for different HCN channel isoforms has been detected in the ganglion cell layer of mouse retina. However, previous studies neither provided evidence that any of the somata are ganglion cells (as opposed to displaced amacrine cells) nor quantified these cells. We therefore tested whether isoform-specific anti-HCN channel antisera bind to ganglion cells labeled by retrograde transport of fluorophore-coupled dextran. In flat-mounted adult rat retinas, the number of dextran-backfilled ganglion cells agreed with cell densities reported in previous studies, and anti-HCN4 antisera bound to the somata of approximately 40% of these cells. The diameter of these somata ranged from 7 to 30 microm. Consistent with localization to cell membranes, the immunoreactivity formed a thin line that circumscribed individual somata. Optic fiber layer axon fascicles, and the proximal dendrites of some ganglion cells, also displayed binding of anti-HCN4 antisera. These results suggest that the response of some mammalian retinal ganglion cells to hyperpolarization may be modulated by changes in intracellular cAMP levels, and could thus be more complex than expected from previous voltage and current recordings.

Ih without Kir in adult rat retinal ganglion cells.

Antisera directed against hyperpolarization-activated mixed-cation ("I(h)") and K(+) ("K(ir)") channels bind to some somata in the ganglion cell layer of rat and rabbit retina. Additionally, the termination of hyperpolarizing current injections can trigger spikes in some cat retinal ganglion cells, suggesting a rebound depolarization arising from activation of I(h). However, patch-clamp studies showed that rat ganglion cells lack inward rectification or present an inwardly rectifying K(+) current. We therefore tested whether hyperpolarization activates I(h) in dissociated, adult rat retinal ganglion cell somata. We report here that, although we found no inward rectification in some cells, and a K(ir)-like current in a few cells, hyperpolarization activated I(h) in roughly 75% of the cells we recorded from in voltage clamp. We show that this current is blocked by Cs(+) or ZD7288 and only slightly reduced by Ba(2+), that the current amplitude and reversal potential are sensitive to extracellular Na(+) and K(+), and that we found no evidence of K(ir) in cells presenting I(h). In current clamp, injecting hyperpolarizing current induced a slowly relaxing membrane hyperpolarization that rebounded to a few action potentials when the hyperpolarizing current was stopped; both the membrane potential relaxation and rebound spikes were blocked by ZD7288. These results provide the first measurement of I(h) in mammalian retinal ganglion cells and indicate that the ion channels of rat retinal ganglion cells may vary in ways not expected from previous voltage and current recordings.

Circuits and properties of signal transmission in the retina.

This essay looks at the historical significance of three APS classic papers that are freely available online: Naka K-I and Nye PW. Role of horizontal cells in organization of the catfish retinal receptive field. J. Neurophysiol 34:785-801, 1971. Marmarelis PZ and Naka K-I. Nonlinear analysis and synthesis of receptive-field responses in the catfish retina. II. One-input white-noise analysis. J. Neurophysiol 36: 619-633, 1973. Naka K-I, Marmarelis PZ, and Chan RY. Morphological and functional identifications of catfish retinal neurons. III. Functional identification. J. Neurophysiol 38: 92-131, 1975.

DARPP-32-like immunoreactivity in AII amacrine cells of rat retina.

Previous studies demonstrated that the dopamine- and adenosine 3',5'-monophosphate-regulated phosphatase inhibitor known as "DARPP-32" is present in rat, cat, monkey, and human retinas. We have followed up these studies by asking what specific cell subtypes contain DARPP-32. Using a polyclonal antibody directed against a peptide sequence of human DARPP-32, we immunostained adult rat retinas that were either transretinally sectioned or flat mounted and found DARPP-32-like immunoreactivity in some cells of the amacrine cell layer across the entire retinal surface. We report here, based on the shape and spatial distribution of these cells, their staining by an anti-parvalbumin antibody, and their juxtaposition with processes containing tyrosine hydroxylase, that DARPP-32-like immunoreactivity is present in AII amacrine cells of rat retina. These results suggest that the response of AII amacrine cells to dopamine is not mediated as simply as previously supposed.

Dopamine receptor activation can reduce voltage-gated Na+ current by modulating both entry into and recovery from inactivation.

We tested whether dopamine receptor activation modulates the voltage-gated Na+ current of goldfish retinal ganglion cells, using a fast voltage-clamp amplifier, perforated-patch whole cell mode, and a physiological extracellular Na+ concentration. As found in other cells, activators of D1-type dopamine receptors and of protein kinase A reduced the amplitude of current activated by depolarizations from resting potential without altering the current kinetics or activation range. However, D1-type dopamine receptor activation also accelerated the rate of entry into inactivation during subthreshold depolarizations and slowed the rate of recovery from inactivation after single, brief depolarizations. Our results provide the first evidence in any preparation that D1-type receptor activation can produce both of these latter effects.

Dissociation of retinal ganglion cells without enzymes.

We describe here methods for dissociating retinal ganglion cells from adult goldfish and rat without proteolytic enzymes, and show responses of ganglion cells isolated this way to step-wise voltage changes and fluctuating current injections. Taking advantage of the laminar organization of vertebrate retinas, photoreceptors and other cells were lifted away from the distal side of freshly isolated goldfish retinas, after contact with pieces of membrane filter. Likewise, cells were sliced away from the distal side of freshly isolated rat retinas, after these adhered to a membrane filter. The remaining portions of retina were incubated in an enzyme-free, low Ca2+ solution, and triturated. After aliquots of the resulting cell suspension were plated, ganglion cells could be identified by dye retrogradely transported via the optic nerve. These cells showed no obvious morphological degeneration for several days of culture. Perforated-patch whole-cell recordings showed that the goldfish ganglion cells spike tonically in response to depolarizing constant current injections, that these spikes are temporally precise in response to fluctuating current injections, and that the largest voltage-gated Na+ currents of these cells were larger than those of ganglion cells isolated with a neutral protease.

Availability of low-threshold Ca2+ current in retinal ganglion cells.

Spiking in central neurons depends on the availability of inward and outward currents activated by depolarization and on the activation and priming of currents by hyperpolarization. Of these processes, priming by hyperpolarization is the least described. In the case of T-type Ca2+ current availability, the interplay of hyperpolarization and depolarization has been studied most completely in expression systems, in part because of the difficulty of pharmacologically separating the Ca2+ currents of native neurons. To facilitate understanding of this current under physiological conditions, we measured T-type current of isolated goldfish retinal ganglion cells with perforated-patch voltage-clamp methods in solutions containing a normal extracellular Ca2+ concentration. The voltage sensitivities and rates of current activation, inactivation, deactivation, and recovery from inactivation were similar to those of expressed alpha1G (CaV3.1) Ca2+ channel clones, except that the rate of deactivation was significantly faster. We reproduced the amplitude and kinetics of measured T currents with a numerical simulation based on a kinetic model developed for an alpha1G Ca2+ channel. Finally, we show that this model predicts the increase of T-type current made available between resting potential and spike threshold by repetitive hyperpolarizations presented at rates that are within the bandwidth of signals processed in situ by these neurons.

A single charged surface residue modifies the activity of ikitoxin, a beta-type Na+ channel toxin from Parabuthus transvaalicus.

We previously purified and characterized a peptide toxin, birtoxin, from the South African scorpion Parabuthus transvaalicus. Birtoxin is a 58-residue, long chain neurotoxin that has a unique three disulfide-bridged structure. Here we report the isolation and characterization of ikitoxin, a peptide toxin with a single residue difference, and a markedly reduced biological activity, from birtoxin. Bioassays on mice showed that high doses of ikitoxin induce unprovoked jumps, whereas birtoxin induces jumps at a 1000-fold lower concentration. Both toxins are active against mice when administered intracerebroventricularly. Mass determination indicated an apparent mass of 6615 Da for ikitoxin vs. 6543 Da for birtoxin. Amino acid sequence determination revealed that the amino-acid sequence of ikitoxin differs from birtoxin by a single residue change from glycine to glutamic acid at position 23, consistent with the apparent mass difference of 72 Da. This single-residue difference renders ikitoxin much less effective in producing the same behavioral effect as low concentrations of birtoxin. Electrophysiological measurements showed that birtoxin and ikitoxin can be classified as beta group toxins for voltage-gated Na+ channels of central neurons. It is our conclusion that the N-terminal loop preceding the alpha-helix in scorpion toxins is one of the determinative domains in the interaction of toxins with the target ion channel.