Inhibitory masking controls the threshold sensitivity of retinal ganglion cells.

KEY POINTS: Retinal ganglion cells (RGCs) in dark-adapted retinas show a range of threshold sensitivities spanning ∼3 log units of illuminance. Here, we show that the different threshold sensitivities of RGCs reflect an inhibitory mechanism that masks inputs from certain rod pathways. The masking inhibition is subserved by GABAC receptors, probably on bipolar cell axon terminals. The GABAergic masking inhibition appears independent of dopaminergic circuitry that has been shown also to affect RGC sensitivity. The results indicate a novel mechanism whereby inhibition controls the sensitivity of different cohorts of RGCs. This can limit and thereby ensure that appropriate signals are carried centrally in scotopic conditions when sensitivity rather than acuity is crucial. ABSTRACT: The responses of rod photoreceptors, which subserve dim light vision, are carried through the retina by three independent pathways. These pathways carry signals with largely different sensitivities. Retinal ganglion cells (RGCs), the output neurons of the retina, show a wide range of sensitivities in the same dark-adapted conditions, suggesting a divergence of the rod pathways. However, this organization is not supported by the known synaptic morphology of the retina. Here, we tested an alternative idea that the rod pathways converge onto single RGCs, but inhibitory circuits selectively mask signals so that one pathway predominates. Indeed, we found that application of GABA receptor blockers increased the sensitivity of most RGCs by unmasking rod signals, which were suppressed. Our results indicate that inhibition controls the threshold responses of RGCs under dim ambient light. This mechanism can ensure that appropriate signals cross the bottleneck of the optic nerve in changing stimulus conditions.

Gap junction-mediated death of retinal neurons is connexin and insult specific: a potential target for neuroprotection.

Secondary cell death via gap junctions (GJs) plays a role in the propagation of neuronal loss under a number of degenerative disorders. Here, we examined the role of GJs in neuronal death in the retina, which has arguably the most diverse expression of GJs in the CNS. Initially, we induced apoptotic death by injecting single retinal ganglion cells and glia with cytochrome C and found that this resulted in the loss of neighboring cells to which they were coupled via GJs. We next found that pharmacological blockade of GJs eradicated nearly all amacrine cell loss and reduced retinal ganglion cell loss by ∼70% after induction of either excitotoxic or ischemic insult conditions. These data indicate that the GJ-mediated secondary cell death was responsible for the death of most cells. Whereas genetic deletion of the GJ subunit Cx36 increased cell survivability by ∼50% under excitotoxic condition, cell loss in Cx45 knock-out mouse retinas was similar to that seen in wild-type mice. In contrast, ablation of Cx45 reduced neuronal loss by ∼50% under ischemic insult, but ablation of Cx36 offered no protection. Immunolabeling of the connexins showed differential changes in protein expression consistent with their differing roles in propagating death signals under the two insults. These data indicate that secondary cell death is mediated by different cohorts of GJs dependent on the connexins they express and the type of initial insult. Our results suggest that targeting specific connexins offers a novel therapeutic strategy to reduce progressive cell loss under different neurodegenerative conditions.

Neuroprotection of Retinal Ganglion Cells Suppresses Microglia Activation in a Mouse Model of Glaucoma.

Purpose: Microglial activation has been implicated in many neurodegenerative eye diseases, but the interrelationship between cell loss and microglia activation remains unclear. In glaucoma, there is no consensus yet whether microglial activation precedes or is a consequence of retinal ganglion cell (RGC) degeneration. We therefore investigated the temporal and spatial appearance of activated microglia in retina and their correspondence to RGC degeneration in glaucoma. Methods: We used an established microbead occlusion model of glaucoma in mouse whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to immunolabel microglia in resting and activated states. To block retinal gap junction (GJ) communication, which has been shown previously to provide significant neuroprotection of RGCs, the GJ blocker meclofenamic acid was administered or connexin36 (Cx36) GJ subunits were ablated genetically. We then studied microglial activation at different time points after microbead injection in control and neuroprotected retinas. Results: Histochemical analysis of flatmount retinas revealed major changes in microglia morphology, density, and immunoreactivity in microbead-injected eyes. An early stage of microglial activation followed IOP elevation, as indicated by changes in morphology and cell density, but preceded RGC death. In contrast, the later stage of microglia activation, associated with upregulation of major histocompatibility complex class II expression, corresponded temporally to the initial loss of RGCs. However, we found that protection of RGCs afforded by GJ blockade or genetic ablation largely suppressed microglial changes at all stages of activation in glaucomatous retinas. Conclusions: Together, our data strongly suggest that microglia activation in glaucoma is a consequence, rather than a cause, of initial RGC degeneration and death.

A Robust Microbead Occlusion Model of Glaucoma for the Common Marmoset.

Purpose: To establish a robust experimental model of glaucoma in the common marmoset (Callithrix jacchus), a New World primate, using an intracameral microbead injection technique. Methods: Elevated intraocular pressure (IOP) was induced by an injection of polystyrene microbeads. Morphologic changes in the retina and optic nerve of glaucomatous eyes were assessed and electroretinogram (ERG) recordings were performed to evaluate functional changes. Results: Microbead injections induced a sustained IOP elevation for at least 10 weeks in a reproducible manner. At the end of the 10-week experimental period, there was significant loss of retinal ganglion cells (RGCs) in all quadrants and eccentricities, although it was more prominent in the mid-peripheral and peripheral regions. This was consistent with a thinning of the Retinal nerve fiber layer (RNFL) seen in spectral domain optical coherence tomography scans. Surviving RGCs showed marked changes in morphology, including somatic shrinkage and dendritic atrophy. Retinas also showed significant gliosis. The amplitude of the ERG photopic negative response, with subsequent a- and b-wave changes, was reduced in glaucomatous eyes. The optic nerve of glaucomatous eyes showed expanded cupping, disorganization of the astrocytic matrix, axonal loss, and gliosis. Conclusions: We developed a robust and reproducible model of glaucoma in the marmoset. The model exhibits both structural and functional alterations of retina and optic nerve characteristic of glaucoma in humans and animal models. Translational Relevance: The glaucoma model in the marmoset described here forms a robust method to study the disease etiology, progression, and potential therapies in a nonhuman primate, allowing for more effective translation of animal data to humans.

Masked excitatory crosstalk between the ON and OFF visual pathways in the mammalian retina.

A fundamental organizing feature of the visual system is the segregation of ON and OFF responses into parallel streams to signal light increment and decrement. However, we found that blockade of GABAergic inhibition unmasks robust ON responses in OFF α-ganglion cells (α-GCs). These ON responses had the same centre-mediated structure as the classic OFF responses of OFF α-GCs, but were abolished following disruption of the ON pathway with L-AP4. Experiments showed that both GABA(A) and GABA(C) receptors are involved in the masking inhibition of this ON response, located at presynaptic inhibitory synapses on bipolar cell axon terminals and possibly amacrine cell dendrites. Since the dendrites of OFF α-GCs are not positioned to receive excitatory inputs from ON bipolar cell axon terminals in sublamina-b of the inner plexiform layer (IPL), we investigated the possibility that gap junction-mediated electrical synapses made with neighbouring amacrine cells form the avenue for reception of ON signals. We found that the application of gap junction blockers eliminated the unmasked ON responses in OFF α-GCs, while the classic OFF responses remained. Furthermore, we found that amacrine cells coupled to OFF α-GCs display processes in both sublaminae of the IPL, thus forming a plausible substrate for the reception and delivery of ON signals to OFF α-GCs. Finally, using a multielectrode array, we found that masked ON and OFF signals are displayed by over one-third of ganglion cells in the rabbit and mouse retinas, suggesting that masked crossover excitation is a widespread phenomenon in the inner mammalian retina.

Neuroprotection of the Inner Retina Also Prevents Secondary Outer Retinal Pathology in a Mouse Model of Glaucoma.

Purpose: We examined structural and functional changes in the outer retina of a mouse model of glaucoma. We examined whether these changes are a secondary consequence of damage in the inner retina and whether neuroprotection of the inner retina also prevents outer retinal changes. Methods: We used an established microbead occlusion model of glaucoma whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to label rod and cone bipolar cells (BCs), horizontal cells (HCs), and retinal ganglion cells (RGCs), as well as synaptic components in control and glaucomatous eyes, to assess structural damage and cell loss. ERG recordings were made to assess outer retina function. Results: We found structural and functional damage of BCs, including significant cell loss and dendritic/axonal remodeling of HCs, following IOP elevation. The first significant loss of both BCs occurred at 4 to 5 weeks after microbead injection. However, early changes in the dendritic structure of RGCs were observed at 3 weeks, but significant changes in the rod BC axon terminal structure were not seen until 4 weeks. We found that protection of inner retinal neurons in glaucomatous eyes by pharmacological blockade of gap junctions or genetic ablation of connexin 36 largely prevented outer retinal damage. Conclusions: Together, our results indicate that outer retinal impairments in glaucoma are a secondary sequalae of primary damage in the inner retina. The finding that neuroprotection of the inner retina can also prevent outer retinal damage has important implications with regard to the targets for effective neuroprotective therapy.

Glutamate-induced internalization of Ca(v)1.3 L-type Ca(2+) channels protects retinal neurons against excitotoxicity.

Glutamate-induced rise in the intracellular Ca(2+) level is thought to be a major cause of excitotoxic cell death, but the mechanisms that control the Ca(2+) overload are poorly understood. Using immunocytochemistry, electrophysiology and Ca(2+) imaging, we show that activation of ionotropic glutamate receptors induces a selective internalization of Ca(v)1.3 L-type Ca(2+) channels in salamander retinal neurons. The effect of glutamate on Ca(v)1.3 internalization was blocked in Ca(2+)-free external solution, or by strong buffering of internal Ca(2+) with BAPTA. Downregulation of L-type Ca(2+) channel activity in retinal ganglion cells by glutamate was suppressed by inhibitors of dynamin-dependent endocytosis. Stabilization of F-actin by jasplakinolide significantly reduced the ability of glutamate to induce internalization suggesting it is mediated by Ca(2+)-dependent reorganization of actin cytoskeleton. We showed that the Ca(v)1.3 is the primary L-type Ca(2+) channel contributing to kainate-induced excitotoxic death of amacrine and ganglion cells. Block of Ca(v)1.3 internalization by either dynamin inhibition or F-actin stabilization increased vulnerability of retinal amacrine and ganglion cells to kainate-induced excitotoxicity. Our data show for the first time that Ca(v)1.3 L-type Ca(2+) channels are subject to rapid glutamate-induced internalization, which may serve as a negative feedback mechanism protecting retinal neurons against glutamate-induced excitotoxicity.

CaV1.3 L-type Ca2+ channels modulate depression-like behaviour in mice independent of deaf phenotype.

Mounting evidence suggests that voltage-gated L-type Ca2+ channels can modulate affective behaviour. We therefore explored the role of CaV1.3 L-type Ca2+ channels in depression- and anxiety-like behaviours using CaV1.3-deficient mice (CaV1.3-/-). We showed that CaV1.3-/- mice displayed less immobility in the forced swim test as well as in the tail suspension test, indicating an antidepressant-like phenotype. Locomotor activity in the home cage or a novel open-field test was not influenced. In the elevated plus maze (EPM), CaV1.3-/- mice entered the open arms more frequently and spent more time there indicating an anxiolytic-like phenotype which was, however, not supported in the stress-induced hyperthermia test. By performing parallel experiments in Claudin 14 knockout mice (Cldn14-/-), which like CaV1.3-/- mice are congenitally deaf, an influence of deafness on the antidepressant-like phenotype could be ruled out. On the other hand, a similar EPM behaviour indicative of an anxiolytic phenotype was also found in the Cldn14-/- animals. Using electroretinography and visual behavioural tasks we demonstrated that at least in mice, CaV1.3 channels do not significantly contribute to visual function. However, marked morphological changes were revealed in synaptic ribbons in the outer plexiform layer of CaV1.3-/- retinas by immunohistochemistry suggesting a possible role of this channel type in structural plasticity at the ribbon synapse. Taken together, our findings indicate that CaV1.3 L-type Ca2+ channels modulate depression-like behaviour but are not essential for visual function. The findings raise the possibility that selective modulation of CaV1.3 channels could be a promising new therapeutic concept for the treatment of mood disorders.

Amacrine cells coupled to ganglion cells via gap junctions are highly vulnerable in glaucomatous mouse retinas.

We determined whether the structural and functional integrity of amacrine cells (ACs), the largest cohort of neurons in the mammalian retina, are affected in glaucoma. Intraocular injection of microbeads was made in mouse eyes to elevate intraocular pressure as a model of experimental glaucoma. Specific immunocytochemical markers were used to identify AC and displaced (d)ACs subpopulations in both the inner nuclear and ganglion cell layers, respectively, and to distinguish them from retinal ganglion cells (RGCs). Calretinin- and γ-aminobutyric acid (GABA)-immunoreactive (IR) cells were highly vulnerable to glaucomatous damage, whereas choline acetyltransferase (ChAT)-positive and glycinergic AC subtypes were unaffected. The AC loss began 4 weeks after initial microbead injection, corresponding to the time course of RGC loss. Recordings of electroretinogram (ERG) oscillatory potentials and scotopic threshold responses, which reflect AC and RGC activity, were significantly attenuated in glaucomatous eyes following a time course that matched that of the AC and RGC loss. Moreover, we found that it was the ACs coupled to RGCs via gap junctions that were lost in glaucoma, whereas uncoupled ACs were largely unaffected. Our results suggest that AC loss in glaucoma occurs secondary to RGC death through the gap junction-mediated bystander effect. J. Comp. Neurol. 527:159-173, 2019. © 2016 Wiley Periodicals, Inc.

Disruption of actin cytoskeleton causes internalization of Ca(v)1.3 (alpha 1D) L-type calcium channels in salamander retinal neurons.

PURPOSE: To study the influence of actin cytoskeleton reorganization on the subcellular distribution of Ca(v)1.3 L-type Ca2+ channels in salamander retinal third-order neurons. METHODS: Immunocytochemistry with confocal microscopy was used to demonstrate internalization of the Ca(v)1.3 isoform of L-type voltage-gated Ca2+ channels in third-order retinal neurons. A specificity of antibody was confirmed with Western blotting and in control experiments preabsorbing antibody wit its respective peptide. Whole-cell patch clamp technique was applied to record L-type currents from ganglion cells in slice preparations in the presence of N- and P/Q type Ca2+ channel blockers. RESULTS: A high level of Ca(v)1.3 labeling was present in cone photoreceptor terminals in the outer plexiform layer (OPL), as aggregates of puncta. Punctate Ca(v)1.3 labeling was evident throughout the IPL and around the cell bodies in the outer nuclear (ONL), inner nuclear (INL) and on somas and axons of ganglion cells labeled with rhodamine-conjugated dextran. Doubly labeled sections for synaptophysin and Ca(v)1.3 revealed colocalization in the OPL and IPL. Depolymerization of the actin cytoskeleton caused a dynamin-dependent internalization of Ca(v)1.3 but not Ca(v)1.2 subtype of voltage-gated Ca2+ channels in dissociated neurons. In ganglion cells, the inhibition of L-type Ca2+ currents by F-actin disrupters was mediated by Ca2+ channel internalization. Treatment with cytochalasin D protected retinal neurons against kainate-induced excitotoxicity. CONCLUSIONS: Actin cytoskeleton dynamics plays an important role in the regulation of subcellular distribution and function of Ca(v)1.3 L-type Ca2+ channels in salamander retinal neurons. Ca2+-dependent actin depolymerization may serve as a negative feedback mechanism to reduce excessive Ca2+ influx and thereby protect neurons against glutamate-induced excitotoxicity.

Targeting neuronal gap junctions in mouse retina offers neuroprotection in glaucoma.

The progressive death of retinal ganglion cells and resulting visual deficits are hallmarks of glaucoma, but the underlying mechanisms remain unclear. In many neurodegenerative diseases, cell death induced by primary insult is followed by a wave of secondary loss. Gap junctions (GJs), intercellular channels composed of subunit connexins, can play a major role in secondary cell death by forming conduits through which toxic molecules from dying cells pass to and injure coupled neighbors. Here we have shown that pharmacological blockade of GJs or genetic ablation of connexin 36 (Cx36) subunits, which are highly expressed by retinal neurons, markedly reduced loss of neurons and optic nerve axons in a mouse model of glaucoma. Further, functional parameters that are negatively affected in glaucoma, including the electroretinogram, visual evoked potential, visual spatial acuity, and contrast sensitivity, were maintained at control levels when Cx36 was ablated. Neuronal GJs may thus represent potential therapeutic targets to prevent the progressive neurodegeneration and visual impairment associated with glaucoma.

Activity-dependent phosphorylation of tyrosine hydroxylase in dopaminergic neurons of the rat retina.

We studied in vivo activity-dependent phosphorylation of tyrosine hydroxylase (TH) in dopaminergic (DA) neurons of the rat retina. TH phosphorylation (TH-P) was evaluated by immunocytochemistry, using antibodies specific for each of three regulated phosphorylation sites. TH synthesis rate was measured by dihydroxyphenylalanine (DOPA) accumulation in the presence of NSD-1015, an inhibitor of aromatic amino acid decarboxylase. TH-P was increased markedly by light or after intraocular injection of GABA(A) and glycine inhibitors. All three phosphospecific antibodies responded similarly to test drugs or light. A 30 min exposure to light increased DOPA accumulation by threefold over that seen after 30 min in darkness. Immunostaining to an anti-panNa channel antibody was found in all parts of the DA neuron. TTX blocked TH-P induced by light or GABA/glycine inhibitors but only in varicosities of the DA axon plexus, not in perikarya or dendrites. Veratridine increased TH-P in all parts of the DA neuron. The distribution of the monoamine vesicular transporter 2 was shown by immunocytochemistry to reside in varicosities of the DA plexus but not in dendrites, indicating that the varicosities are sites of dopamine release. Collectively, these data indicate that, in the retina, dopamine synthesis in varicosities is affected by the spiking activity of retinal neurons, possibly including that of the DA neurons themselves.

Calcium and retinal function.

We survey the primary roles of calcium in retinal function, including photoreceptor transduction, transmitter release by different classes of retinal neuron, calcium-mediated regulation of gap-junctional conductance, activation of certain voltage-gated channels for K+ and Cl-, and modulation of postsynaptic potentials in retinal ganglion cells. We discuss three mechanisms for changing [Ca2+]i, which include flux through voltage-gated calcium channels, through ligand-gated channels, and by release from stores. The neuromodulatory pathways affecting each of these routes of entry are considered. The many neuromodulatory mechanisms in which calcium is a player are described and their effects upon retinal function discussed.

Activation of Protein Kinase C Modulates Light Responses in Horizontal Cells of the Turtle Retina.

The effect of phorbol esters on the light-evoked responses of horizontal cells were studied in the turtle eyecup preparation. Phorbol esters caused a reduction in receptive field size and a significant decrease in the amplitude of responses to annular and full-field illumination; however, they caused only minor changes in responses to small spots in the receptive field centre. The dark membrane potential was not affected. The results suggest that phorbol esters may affect both coupling resistance and membrane resistance in horizontal cells. The effects of phorbol esters were blocked by the protein kinase C inhibitor staurosporine, and inactive phorbol ester had no effect, making it very likely that the phorbol ester effects were mediated through activation of protein kinase C. The above effects of the phorbol esters were considerably reduced by the dopamine antagonists haloperidol and fluphenazine, suggesting that they were in part mediated by release of dopamine.

Differential modulation of light-evoked on- and off-EPSCs by paired-pulse stimulation in salamander retinal ganglion cells.

Short-term plasticity of On- and Off-EPSPs, and its potential role in regulation of signal processing was studied in salamander retinal On-Off ganglion cells by whole-cell recording. Paired-pulse light stimulation resulted in a depression of On-, and an enhancement of Off-EPSCs. Recovery from depression and enhancement was exponential and complete by 20 s. Paired-pulse enhancement, but not depression, was abolished with increasing stimulus duration. Blockade of On-EPSC by L-2-amino-4-phosphonobutyrate (AP-4), an agonist at group III mGluRs, significantly increased Off-EPSCs evoked by short (<2 s) duration conditioning light stimuli, resulting in a reversal of the paired-pulse enhancement to depression. The acetylcholinesterase inhibitor eserine reduced Off-EPSC1 and increased the ratio of enhancement. An opposite effect was observed in the presence of the nACh receptor antagonist d-tubocurarine. AP-7, an antagonist of NMDA receptors attenuated the enhancement of Off-EPSCs. In current clamp mode paired-pulse stimulation resulted in a modulation of light evoked, as well as the depolarization-induced spike firing pattern of ganglion cells. The present study suggests that paired light stimulation differently modulates On and Off EPSPs, and the light-evoked spike firing pattern of On-Off ganglion cells.

Calcium homeostasis and cone signaling are regulated by interactions between calcium stores and plasma membrane ion channels.

Calcium is a messenger ion that controls all aspects of cone photoreceptor function, including synaptic release. The dynamic range of the cone output extends beyond the activation threshold for voltage-operated calcium entry, suggesting another calcium influx mechanism operates in cones hyperpolarized by light. We have used optical imaging and whole-cell voltage clamp to measure the contribution of store-operated Ca(2+) entry (SOCE) to Ca(2+) homeostasis and its role in regulation of neurotransmission at cone synapses. Mn(2+) quenching of Fura-2 revealed sustained divalent cation entry in hyperpolarized cones. Ca(2+) influx into cone inner segments was potentiated by hyperpolarization, facilitated by depletion of intracellular Ca(2+) stores, unaffected by pharmacological manipulation of voltage-operated or cyclic nucleotide-gated Ca(2+) channels and suppressed by lanthanides, 2-APB, MRS 1845 and SKF 96365. However, cation influx through store-operated channels crossed the threshold for activation of voltage-operated Ca(2+) entry in a subset of cones, indicating that the operating range of inner segment signals is set by interactions between store- and voltage-operated Ca(2+) channels. Exposure to MRS 1845 resulted in approximately 40% reduction of light-evoked postsynaptic currents in photopic horizontal cells without affecting the light responses or voltage-operated Ca(2+) currents in simultaneously recorded cones. The spatial pattern of store-operated calcium entry in cones matched immunolocalization of the store-operated sensor STIM1. These findings show that store-operated channels regulate spatial and temporal properties of Ca(2+) homeostasis in vertebrate cones and demonstrate their role in generation of sustained excitatory signals across the first retinal synapse.

Calcium channel and glutamate receptor activities regulate actin organization in salamander retinal neurons.

Intracellular Ca2+ regulates a variety of neuronal functions, including neurotransmitter release, protein phosphorylation, gene expression and synaptic plasticity. In a variety of cell types, including neurons, Ca2+ is involved in actin reorganization, resulting in either actin polymerization or depolymerization. Very little, however, is known about the relationship between Ca2+ and the actin cytoskeleton organization in retinal neurons. We studied the effect of high-K+-induced depolarization on F-actin organization in salamander retina and found that Ca2+ influx through voltage-gated L-type channels causes F-actin disruption, as assessed by 53 +/- 5% (n = 23, P < 0.001) reduction in the intensity of staining with Alexa-Fluor488-phalloidin, a compound that permits visualization and quantification of polymerized actin. Calcium-induced F-actin depolymerization was attenuated in the presence of protein kinase C antagonists, chelerythrine or bis-indolylmaleimide hydrochloride (GF 109203X). In addition, phorbol 12-myristate 13-acetate (PMA), but not 4alpha-PMA, mimicked the effect of Ca2+ influx on F-actin. Activation of ionotropic AMPA and NMDA glutamate receptors also caused a reduction in F-actin. No effect on F-actin was exerted by caffeine or thapsigargin, agents that stimulate Ca2+ release from internal stores. In whole-cell recording from a slice preparation, light-evoked 'off' but not 'on' EPSCs in 'on-off' ganglion cells were reduced by 60 +/- 8% (n = 8, P < 0.01) by cytochalasin D. These data suggest that elevation of intracellular Ca2+ during excitatory synaptic activity initiates a cascade for activity-dependent actin remodelling, which in turn may serve as a feedback mechanism to attenuate excitotoxic Ca2+ accumulation induced by synaptic depolarization.

Effect of hypothalamic proline-rich-polypeptide on voltage-gated Ca2+ currents in retinal ganglion cells.

Neurotrophins are molecules that regulate neuronal survival, nervous system plasticity, and many other physiological functions of neuronal and glial cells. Here we studied the physiological action of a novel neurosecretory polypeptide proline-rich polypeptide (PRP), isolated from bovine neurohypophysis neurosecretory granules, on voltage-gated Ca currents and spike firing activity of retinal ganglion cells. PRP reversibly increased high voltage-activated L-type Ca current, but was without effect on low voltage-activated T-type current. PRP also increased the spike after hyperpolarization and reduced the frequency of spike firing, most likely by affecting a Ca-dependent potassium current.