Assessing Rewiring of the Retinal Circuitry by Electroretinogram (ERG) After Inner Retinal Lesion in Adult Zebrafish.

Adult zebrafish respond to retinal injury with a regenerative response that replaces damaged neurons with Müller glia-derived regenerated neurons. The regenerated neurons are functional, appear to make appropriate synaptic connections, and support visually mediated reflexes and more complex behaviors. Curiously, the electrophysiology of damaged, regenerating, and regenerated zebrafish retina has only recently been examined. In our previous work, we demonstrated that electroretinogram (ERG) recordings of damaged zebrafish retina correlate with the extent of the inflicted damage and that the regenerated retina at 80 days post-injury exhibited ERG waveforms consistent with functional visual processing. In this paper we describe the procedure for obtaining and analyzing ERG recordings from adult zebrafish previously subjected to widespread lesions that destroy inner retinal neurons and engage a regenerative response that restores retinal function, in particular the synaptic connections between photoreceptor axon terminals and the dendritic trees of retinal bipolar neurons.

Dynamic functional and structural remodeling during retinal regeneration in zebrafish.

Introduction: Zebrafish regenerate their retinas following damage, resulting in restoration of visual function. Here we evaluate recovery of retinal function through qualitative and quantitative analysis of the electroretinogram (ERG) over time following retinal damage, in correlation to histological features of regenerated retinal tissue. Methods: Retinas of adult zebrafish were lesioned by intravitreal injection of 10 µM (extensive lesion; destroys all neurons) or 2 µM (selective lesion; spares photoreceptors) ouabain. Unlesioned contralateral retinas served as controls. Function of retinal circuitry was analyzed at selected timepoints using ERG recordings from live zebrafish, and whole eyes were processed for histological analyses immediately thereafter. Results: Qualitative and quantitative assessment of waveforms during retinal regeneration revealed dynamic changes that were heterogeneous on an individual level within each sampling time, but still followed common waveform recovery patterns on a per-fish and population-level basis. Early in the regeneration period (13-30 days post injury; DPI), for both lesion types, b-waves were essentially not detected, and unmasked increased apparent amplitudes, implicit times, and half-widths of a-waves (vs. controls). In control recordings, d-waves were not obviously detected, but apparent d-waves (OFF-bipolar responses) from regenerating retinas of several fish became prominent by 30DPI and dominated the post-photoreceptor response (PPR). Beyond 45DPI, b-waves became detectable, and the ratio of apparent d- to b-wave contributions progressively shifted with most, but not all, fish displaying a b-wave dominated PPR. At the latest timepoints (extensive, 90DPI; selective, 80DPI), recordings with measurable b-waves approached a normal waveform (implicit times and half-widths), but amplitudes were not restored to control levels. Histological analyses of the retinas from which ERGs were recorded showed that as regeneration progressed, PKCa + ON-bipolar terminals and parvalbumin + amacrine cell processes became more stereotypically positioned within the deep sublaminae of the INL over recovery time after each lesion type, consistent with the shift in PPR seen in the ERG recordings. Discussion: Taken together, these data suggest that photoreceptor-OFF-bipolar component/connectivity may functionally recover and mature earlier during regeneration compared to the photoreceptor-ON-bipolar component, though the timeframe in which such recovery happens is heterogeneous on a per-fish basis. Collectively our studies suggest gradual restoration of ON-bipolar functional circuitry during retinal regeneration.

Restoration of Dendritic Complexity, Functional Connectivity, and Diversity of Regenerated Retinal Bipolar Neurons in Adult Zebrafish.

Adult zebrafish (Danio rerio) are capable of regenerating retinal neurons that have been lost due to mechanical, chemical, or light damage. In the case of chemical damage, there is evidence that visually mediated behaviors are restored after regeneration, consistent with recovery of retinal function. However, the extent to which regenerated retinal neurons attain appropriate morphologies and circuitry after such tissue-disrupting lesions has not been investigated. Adult zebrafish of both sexes were subjected to intravitreal injections of ouabain, which destroys the inner retina. After retinal regeneration, cell-selective markers, confocal microscopy, morphometrics, and electrophysiology were used to examine dendritic and axonal morphologies, connectivities, and the diversities of each, as well as retinal function, for a subpopulation of regenerated bipolar neurons (BPs). Although regenerated BPs were reduced in numbers, BP dendritic spreads, dendritic tree morphologies, and cone-bipolar connectivity patterns were restored in regenerated retinas, suggesting that regenerated BPs recover accurate input pathways from surviving cone photoreceptors. Morphological measurements of bipolar axons found that numbers and types of stratifications were also restored; however, the thickness of the inner plexiform layer and one measure of axon branching were slightly reduced after regeneration, suggesting some minor differences in the recovery of output pathways to downstream partners. Furthermore, ERG traces from regenerated retinas displayed waveforms matching those of controls, but with reduced b-wave amplitudes. These results support the hypothesis that regenerated neurons of the adult zebrafish retina are capable of restoring complex morphologies and circuitry, suggesting that complex visual functions may also be restored.SIGNIFICANCE STATEMENT Adult zebrafish generate new retinal neurons after a tissue-disrupting lesion. Existing research does not address whether regenerated neurons of adults successfully reconnect with surrounding neurons and establish complex morphologies and functions. We report that, after a chemical lesion that ablates inner retinal neurons, regenerated retinal bipolar neurons (BPs), although reduced in numbers, reconnected to undamaged cone photoreceptors with correct wiring patterns. Regenerated BPs had complex morphologies similar to those within undamaged retina and a physiological measure of photoreceptor-BP connectivity, the ERG, was restored to a normal waveform. This new understanding of neural connectivity, morphology, and physiology suggests that complex functional processing is possible within regenerated adult retina and offers a system for the future study of synaptogenesis during adult retinal regeneration.

Inherited macular degeneration-associated mutations in CNGB3 increase the ligand sensitivity and spontaneous open probability of cone cyclic nucleotide-gated channels.

Cyclic nucleotide gated (CNG) channels are a critical component of the visual transduction cascade in the vertebrate retina. Mutations in the genes encoding these channels have been associated with a spectrum of inherited retinal disorders. To gain insight into their pathophysiological mechanisms, we have investigated the functional consequences of several CNGB3 mutations, previously associated with macular degeneration (Y469D and L595F) or complete achromatopsia (S156F, P309L, and G558C), by expressing these subunits in combination with wild-type CNGA3 in Xenopus oocytes and characterizing them using patch-clamp recordings in the inside-out configuration. These mutations did not prevent the formation of functional heteromeric channels, as indicated by sensitivity to block by L-cis-diltiazem. With the exception of S156F, each of the mutant channels displayed electrophysiological properties reflecting enhanced channel activity at physiological concentrations of cGMP (i.e., a gain-of-function phenotype). The increased channel activity produced by these mutations resulted from either increased functional expression levels, or increased sensitivity to cyclic nucleotides. Furthermore, L595F increased the spontaneous open probability in the absence of activating ligand, signifying a ligand independent gain-of-function change. In addition to the CNGB3 disease-associate mutations, we characterized the effects of several common CNGB3 and CNGA3 single-nucleotide polymorphisms (SNPs) on heteromeric CNGA3+CNGB3 channel function. Two of the SNPs examined (A3-T153M, and B3-W234C) produced decreased ligand sensitivity for heteromeric CNG channels. These changes may contribute to background disease susceptibility when combined with other genetic or non-genetic factors. Together, these studies help to define the underlying molecular phenotype for mutations relating to CNG channel disease pathogenesis.

Cyclic nucleotide-gated channel subunit glycosylation regulates matrix metalloproteinase-dependent changes in channel gating.

Cyclic-nucleotide gated (CNG) channels are essential for phototransduction within retinal photoreceptors. We have demonstrated previously that the enzymatic activity of matrix metalloproteinase-2 and -9, members of the matrix metalloproteinase (MMP) family of extracellular, Ca(2+)- and Zn(2+)-dependent proteases, enhances the ligand sensitivity of both rod (CNGA1 and CNGB1) and cone (CNGA3 and CNGB3) CNG channels. Additionally, we have observed a decrease in the maximal CNG channel current (Imax) that begins late during MMP-directed gating changes. Here we demonstrate that CNG channels become nonconductive after prolonged MMP exposure. Concurrent with the loss of conductive channels is the increased relative contribution of channels exhibiting nonmodified gating properties, suggesting the presence of a subpopulation of channels that are protected from MMP-induced gating effects. CNGA subunits are known to possess one extracellular core glycosylation site, located at one of two possible positions within the turret loop near the pore-forming region. Our results indicate that CNGA glycosylation can impede MMP-dependent modification of CNG channels. Furthermore, the relative position of the glycosylation site within the pore turret influences the extent of MMP-dependent proteolysis. Glycosylation at the site found in CNGA3 subunits was found to be protective, while glycosylation at the bovine CNGA1 site was not. Relocating the glycosylation site in CNGA1 to the position found in CNGA3 recapitulated CNGA3-like protection from MMP-dependent processing. Taken together, these data indicate that CNGA glycosylation may protect CNG channels from MMP-dependent proteolysis, consistent with MMP modification of channel function having a requirement for physical access to the extracellular face of the channel.

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels.

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.

State-dependent changes in cortical gain control as measured by auditory evoked responses to varying intensity stimuli.

STUDY OBJECTIVES: Auditory evoked potential (AEP) components correspond to sequential activation of brain structures within the auditory pathway and reveal neural activity during sensory processing. To investigate state-dependent modulation of stimulus intensity response profiles within different brain structures, we assessed AEP components across both stimulus intensity and state. DESIGN: We implanted adult female Sprague-Dawley rats (N = 6) with electrodes to measure EEG, EKG, and EMG. Intermittent auditory stimuli (6-12 s) varying from 50 to 75 dBa were delivered over a 24-h period. Data were parsed into 2-s epochs and scored for wake/sleep state. RESULTS: All AEP components increased in amplitude with increased stimulus intensity during wake. During quiet sleep, however, only the early latency response (ELR) showed this relationship, while the middle latency response (MLR) increased at the highest 75 dBa intensity, and the late latency response (LLR) showed no significant change across the stimulus intensities tested. During rapid eye movement sleep (REM), both ELR and LLR increased, similar to wake, but MLR was severely attenuated. CONCLUSIONS: Stimulation intensity and the corresponding AEP response profile were dependent on both brain structure and sleep state. Lower brain structures maintained stimulus intensity and neural response relationships during sleep. This relationship was not observed in the cortex, implying state-dependent modification of stimulus intensity coding. Since AEP amplitude is not modulated by stimulus intensity during sleep, differences between paired 75/50 dBa stimuli could be used to determine state better than individual intensities.

Cortical evoked responses associated with arousal from sleep.

STUDY OBJECTIVE: To determine if low-level intermittent auditory stimuli have the potential to disrupt sleep during 24-h recordings, we assessed arousal occurrence to varying stimulus intensities. Additionally, if stimulus-generated evoked response potential (ERP) components provide a metric of underlying cortical state, then a particular ERP structure may precede an arousal. DESIGN: Physiological electrodes measuring EEG, EKG, and EMG were implanted into 5 adult female Sprague-Dawley rats. We delivered auditory stimuli of varying intensities (50-75 dBa sound pressure level SPL) at random intervals of 6-12 s over a 24-hour period. Recordings were divided into 2-s epochs and scored for sleep/wake state. Following each stimulus, we identified whether the animal stayed asleep or woke. We then sorted the stimuli depending on prior and post-stimulus state, and measured ERP components. RESULTS: Auditory stimuli did not produce a significant increase in the number of arousals compared to silent control periods. Overall, arousal from REM sleep occurred more often compared to quiet sleep. ERPs preceding an arousal had decreased mean area and shorter N1 latency. CONCLUSION: Low level auditory stimuli did not fragment animal sleep since we observed no significant change in arousal occurrence. Arousals that occurred within 4 s of a stimulus exhibited an ERP mean area and latency had features similar to ERPs generated during wake, indicating that the underlying cortical tissue state may contribute to physiological conditions required for arousal.

Habituation-induced neural plasticity in the hippocampus and prefrontal cortex mediated by MMP-3.

Head-shake response (HSR) habituation was presently used to investigate the phenomena of spontaneous recovery and neural plasticity. Independent groups of rats were presented with five consecutive habituation sessions separated by inter-session intervals (ISIs) of 2, 24 or 72 h. At the conclusion of testing hippocampus and prefrontal cortex tissue samples were collected for determination of matrix metalloproteinase-3 (MMP-3:stromelysin-1) expression as a marker of neural plasticity. The results indicated that by the fifth session the 2 h ISI group showed no spontaneous recovery, the 72 h ISI group revealed nearly complete spontaneous recovery; while the 24 h ISI group showed intermediate recovery. MMP-3 expression in the hippocampus and prefrontal cortex was elevated in the 2 and 72 h ISI groups, but not in the 24 h group. A second experiment utilized 7-day osmotic pumps to intracerebroventricularly infuse an MMP-3 inhibitor for 6 days. The animals were then tested on the seventh day using the 2 h ISI protocol. Delivery of the MMP-3 inhibitor facilitated spontaneous recovery, thus compromising the animal's ability to appropriately habituate. This effect was accompanied by a significant inhibition of hippocampus and prefrontal cortex MMP-3 expression. These results suggest that elevations in hippocampus and prefrontal cortex MMP-3 expression contribute to this simplest form of learning and may be a mechanism underlying spontaneous recovery.

Hippocampal MMP-3 elevation is associated with passive avoidance conditioning.

Alterations in synaptic efficiency that underlie learning and memory consolidation appear to require an accompanying reconfiguration of the extracellular matrix (ECM). This restructuring of the ECM is carried out, in part, by a family of enzymes called, the matrix metalloproteinases, which includes matrix metalloproteinase-3 (MMP-3: stromelysin-1). The present study determined that a transient elevation in hippocampal MMP-3 expression occurred in rats following associative learning in the passive avoidance (PA) task. No change in MMP-3 was observed when rats were exposed either to the behavioral apparatus or the training stimulus alone. Furthermore, when an MMP-3 inhibitor was administered prior to PA training, dose-dependent learning deficits were observed, suggesting a causal relationship between learning-induced hippocampal MMP-3 elevation and associative memory formation. These findings suggest that increased hippocampal MMP-3 expression is an event that may play an important role in synaptic plasticity and memory consolidation.

Effects of matrix metalloproteinase inhibition on short- and long-term plasticity of schaffer collateral/CA1 synapses.

It is increasingly evident that matrix metalloproteinases (MMPs), a family of zinc containing extracellular endopeptidases, participate in processes supporting hippocampal synaptic plasticity. The purpose of this study was to further the understanding of MMPs involvement in hippocampal plasticity. Acute hippocampal slices, generated from 20- to 30-day-old male Sprague-Dawley rats, were subjected to various electrophysiologic stimulatory paradigms to produce either short-term or long-term modifications to synaptic efficacy. Slices exposed to broad-spectrum MMP inhibitor, FN-439, exhibited impairments in paired-pulse facilitation, theta-burst facilitation, and long-term depression. Additionally, we observed that MMP inhibition impaired both the induction and stability of long-term potentiation (LTP). Furthermore, evidence indicated that the effect of MMP inhibition on LTP maintenance is dependent upon integrin-directed adhesion, whereas the effects of MMP inhibition on LTP induction are independent of integrin-directed adhesion. Together, these data support a generalized role for MMPs in short-term and long-term hippocampal plasticity and indicate that MMPs are a necessary facet of integrin-mediated cell adhesion supporting LTP stabilization.

REM sleep deprivation attenuates actin-binding protein cortactin: a link between sleep and hippocampal plasticity.

Rapid eye-movement sleep (REMS) is thought to affect synaptic plasticity. Cortactin is a cytoskeletal protein critically involved in the regulation of actin branching and stabilization including the actin backbone of dendritic spines. Hippocampal cortactin levels, phosphorylation, and processing appear to be altered during learning and long-term potentiation (LTP); consistent with a role for cortactin in the dendritic restructuring that accompanies synaptic plasticity. In this study juvenile male Sprague-Dawley rats were selectively REMS-deprived (RD) for 48 h by the flowerpot method. Cage control (CC) and large pedestal control (PC) animals were used for comparison. Animals were euthanized immediately, or 12 h, after removal from the pedestal. The hippocampus was dissected, flash-frozen, and stored for subsequent Western blot or quantitative RT-PCR analysis of cortactin. Cortactin mRNA/cDNA levels initially rose in PC and RD rats but returned to CC levels by 12 h after removal from the pedestal. Predictably cortactin protein levels were initially unchanged but were up-regulated after 12 h. The PC group had more total and tyrosine-phosphorylated cortactin protein expression than the RD and CC groups. This increase in cortactin was likely due to the exposure of the rats to the novel environment of the deprivation chambers thus triggering plasticity events. The lack of REMS, however, severely hampered cortactin protein up-regulation and phosphorylation observed in the PC group suggesting an attenuation of plasticity-related events. Thus, these data support a functional link between REMS and cytoskeletal reorganization in the hippocampus, a process that is essential for synaptic plasticity.

Effects of extracellular matrix-degrading proteases matrix metalloproteinases 3 and 9 on spatial learning and synaptic plasticity.

Rats learning the Morris water maze exhibit hippocampal changes in synaptic morphology and physiology that manifest as altered synaptic efficacy. Learning requires structural changes in the synapse, and multiple cell adhesion molecules appear to participate. The activity of these cell adhesion molecules is, in large part, dependent on their interaction with the extracellular matrix (ECM). Given that matrix metalloproteinases (MMPs) are responsible for transient alterations in the ECM, we predicted that MMP function is critical for hippocampal-dependent learning. In support of this, it was observed that hippocampal MMP-3 and -9 increased transiently during water maze acquisition as assessed by western blotting and mRNA analysis. The ability of the NMDA receptor channel blocker MK801 to attenuate these changes indicated that the transient MMP changes were in large part dependent upon NMDA receptor activation. Furthermore, inhibition of MMP activity with MMP-3 and -9 antisense oligonucleotides and/or MMP inhibitor FN-439 altered long-term potentiation and prevented acquisition in the Morris water maze. The learning-dependent MMP alterations were shown to modify the stability of the actin-binding protein cortactin, which plays an essential role in regulating the dendritic cytoskeleton and synaptic efficiency. Together these results indicate that changes in MMP function are critical to synaptic plasticity and hippocampal-dependent learning.

Ethanol-induced impairment of spatial memory and brain matrix metalloproteinases.

The formation of spatial memory appears to be dependent upon an intact hippocampus capable of the specific biochemical changes associated with synaptic remodeling. Hippocampal damage results in the disruption of synaptic remodeling and the acquisition of spatial memory tasks. Ethanol also disrupts normal hippocampal functioning and spatial memory. The present investigation established a dose-response relationship between ethanol treatment and impairment of spatial memory as measured using the circular water maze task. Intraperitoneal ethanol doses of 1.5 and 2 g/kg significantly increased the latency and distance swam to find the submerged pedestal as compared with a 1 g/kg dose, and 0.15 M NaCl vehicle control treatments. On days 2, 4, and 6 of acquisition animals were sacrificed and brain tissues were retained from the hippocampus, prefrontal neocortex, and cerebellum for measurement of matrix metalloproteinases (MMPs). The results indicated that ethanol treatment interfered with MMP-9, but not MMP-2, activity in the hippocampus, and to a lesser degree in the prefrontal cortex. No changes in the cerebellum were measured. Elevations in MMP activity appear to be a prerequisite to reconfiguration of extracellular matrix cell adhesion molecules thought to be important in the process of synaptic plasticity, which in turn appears to be necessary for memory consolidation. Thus, ethanol-induced impairment in the acquisition of spatial memory tasks may, in part, be due to disruption of brain MMP activity.