Electrophysiology Properties Of The Cacnalf Mutated Rat’s Retina And Its Visual Signal Pathway Reorganization

X-linked congenital stationary night blindness (X-CSNB) is a congenitalnon-progressive retinal disease which characterized by the defective in rod signal system.The pathology mechanism of CSNB is not very lucid now and without any effectivetherapeutic methods. The aif subunit of L-type calcium channel encoded by Cacnalf gQtiQhas been considered as a key role in triggering off neurotransmitter vesicular release ofretinal neuron. Several mutations within the Cacnalf gene may cause disturbances ofvisual signal transmission and are associated with X-linked incomplete form CSNB(XL-iCSNB).The spontaneous mutated CSNB rat founded in our laboratory has beenidentified as Cacnalf gene mutated. It has electroretinogram (ERG) phenotype whichclosely resembles that of the iCSNB patients. Therefore CSNB rat would be an instrumental model for the explorations of iCSNB’s pathophysiologic mechanism and thecircuits of visual signal transmission.The multielectrode array (ME A) system has been used in the retinal studies such as therecording of electrophysiology activities of ganglion cells or photoreceptors. Due to itsensemble and non-invasive advantages, this technology bridges the gap between theconventional in vitro recording and the complicated in vivo recording, especially inelectrophysiological explorations that target ion channels.Using this instructive recordingsystem and the spontaneously mutated CSNB rats, the modified physiologic properties ofvisual signal pathway in the iCSNB retina and the underlying pathologic mechanisms ofretinal remolding were explored in the present study. As the anatomic architecture andphysiological properties of vertebrate retina have great in common，the research mightprovide proof for elucidation of iCSNB patients’ pathology mechanism and subsequentinvention of effective therapeutic measures.Methods and materialThe CSNB rats were obtained from the23rd inbred generation that derived from theoriginally identified mutant rat (8-10weeks, F23). Wild type (Wt) rats (Sprague-Dawley)were set as control and provided by Laboratory Animal Center of Fourth Military MedicalUniversity. Eyeball was enucleated from rats which were anesthetized and decapitatedatfer dark-adaptation and dissected immediately. The neural retina was gently removedfrom the pigment epithelium layer of the eye-cup and placed into the recording chamber，with the ganglion cell layer facing the ME A biochip electrode array. During recording, theretina samples were perfused with oxygenated Ringer’s solution. The chemicalcompounds were bath-applied through an active perfusion system if necessary.The LEDwere driven by a computed stimulator to provide the retinal specimen a uniform full ifeldillumination via a lens focus system. The light-induced responses were recorded by theMEA system. The analog extracellular neuronal signals from64channels were ACampliifed, sampled at20kHz and stored on a compatible computer. All recordings were subsequently subjected to off-line spike sorting and analysis using Olffine Sorter andNeuroexplorer sotfware. The clusters were firstly identified using a K-mean clusteralgorithm, and then manually edited for clustering errors. The interspike interval (ISI)histograms were analyzed as described previously for each unit to determine the regularityand distribution of the spikes, and thereby to elucidate the cell firing pattern. Theperi stimulus time histograms (PSTHs) and the raster plots of individual units were used tocategorize the ganglions. Inelectrical stimulated response recording, charge-balancedbiphasic current pulse trains were generated and applied to one electrode (No.28) at thecenter to stimulate the RCGs. The RGCs response intensities were measured by countingthe number of evoked spikes within10to50ms post stimulus. Each value was averagedfrom the10repeated stimulations.Results1.Typical electrical responses of wild type retinas were successfully elicited by light andharvested by each ME A electrode: a-wave, the main negative response after light onset(0.196士0.038mv，n=20); b-wave? the positive response immediately after a-wave.Generation of responsive a-waves was not blocked by100APB，while b-waves were.In Cacnai/mutated retinas, the light-induced a-waves can be still be detected though itsamplitude was significantly reduced compared to wild type retinas (0.078土0.026mv,n=20，Kruskal-Wallis test<, P0.01); b-wave could not be recorded in any Cacnalfmutated retinas.2.In both Wt and Cacnalf mutated ratinas，there were two types of RGC responsesevoked by electrical stimulus: the short-latency responses were evoked <5ms afterstimulation, while the long-latency responses were evoked approximately10?50ms afterstimulation. The reliably responding RGCs were categorized into different groups by thedistance between stimulating electrode and recording electrode and found that theresponse strength decreased as the distance between stimulating electrode and recordingelectrode increased;the number of evoked spikes within10?50ms post stimulus in each group was decreased in Cacna7/mutated rats compared to Wt rats.3.ME A recording monitored spontaneous extracellular firing spikes from30to90retinalRGCs simultaneously of each retina, and revealed that Cacwai/mutated retinal RGCs ifreat a much higher spontaneous frequency than wild type. A rightward shitf and decreasedinitial slope in cumulative frequency histograms of Cacna If mutated retinas indicatedincreased spontaneous firing rate of a larger RGCs proportion.4.Detected RGCs ranked among these four typical sorts: bursting cells, regularly firingcells, irregularly firing cells, and mixed type cells. The proportion of bursting and mixedtypes firing RGCs was found to be significantly increased in Cacnai/mutated retinas atthe expense of a decrease in the proportion of irregularly. The spike number of mixed typeRGCs is bigger in Cacna If mutated retinas (246553, n=20) than Wt retinas(112089, n二20,Kruskal-Wallis test, P <0.01).The spike numbers of other three RGCs type aresimilar(P>0.05)5.Mainly six categories of RGCs populations from both kinds of retinas weredistinguished by their responsive characters to light stimulus: responding predominantly tolight onset (ON), to light offset (OFF)，or to both (ON-OFF); sustained response to lightonset (sustained ON); sustained response to light on and offset (sustained ON-OFF);sluggish response to light ofFset(delayed OFF). The median amplitude of total light-drivenresponse was lower among Cacna Ifm utsAQd RGCs than Wt (Kruskal-Wallis test，P<0.01).Thus a lower signal-noise(S/N) ration was found in Cacnalfm utsited retinas.6.In Cacnalf mutated retinas, two types of cross-correlations were found: excitatorycorrelations with narrow-(<1ms) and medium-(<25ms) scale correlated activitiesrespectively. On the other hand, RGC pairs displaying correlated activity at broad-scaleintervals (40—100ms) could not be found in any of the recorded Cacnalf mutsAQd retinas.In the Wt retinas, all of the three types of cross-correlations were found in each specimen.In the Cacnalf mutsAQd retinas (n=5)，100|iM18p—GA caused a pronounced decrease inthe firing frequency of spontaneous firing spikes, whereas no appreciated effect was foundin any of the Wt retinas (n=5).7.According to the relative proportion of ON vs. OFF response amplitude that made up the total light response, the ganglion cells were divided into ON-dominant (ratio>0.5),OFF-dominant (ratio<0.5)，and equal (ration=0.5). More OFF-dominant cells and lessON-dominant cells were found in the Cacna Ifm utant retinas. The latency of both the OFFand ON responses significantly increased in the Cacna If mutant retinas (Kruskal-Wallistest<, P0.05). The majority of OFF response in both kinds of retinas were disrupted by theCNQX+PDA cocktail. The firing rate of total response decreased whereas the ifringfrequency of ON response increased in the Cacnalf mutated retinas. In addition，thelatency of ON responses was significantly increased in the Cacnalf mutant retinas atferthe application of CNQX+PDA cocktail. In both kinds of retinas, the OFF response of theOFF RGCs and the ON-OFF RGCs were disrupted by the CNQX+PDA cocktail, but theOFF response of the delayed OFF RGCs and the sustained ON-OFF RGCs were not.Conclusionl.In the Cacnalf mutated retinas, the light-induced a-waves can still be detected withsignificantly reduced amplitude, suggesting that the light-induced responses ofphotoreceptors are partly retained in the Cacnalf mutant retinas, and these photoreceptorsare partly functional atfer Cacna If mutation. The missed b-wave implies a disruption ofvisual signal transmission between the photoreceptors and the ON-bipolar cells2.Atfer electrical stimulation, long-latency responses are found to fire at lower frequencyin retinas from CSNB rats, which prove that indirect stimulation of synapses inputs fromouter retina has been affect by Cacnalf mutation, though the neurons in outer layer ofretina were stimulated by electrical current, these signals they generated could not betransmitted to ganglion cells successfully through synapses.3.The spontaneous hyperactivity found in Cacna If mutated retinas suggests a signiifcantalteration in ganglion cell electrophysiological properties. This hyperactivity should becaused by various alterations in the organization of inner retinal circuits presynaptic to theganglion cells.4.The proportions and the total spike number of and mixed type ifring RGCs are significantly higher in themutant retinas, suggests the mixed type RGCs mightbe responsible for the spontaneous hyperactivity of the Cacnalfm utant retinas.5.When a ganglion cell loses its primary drive through the photoreceptor to bipolar cell,one of the effects is a reduction in the S-N ratio，which indicates a loss of in the ability ofganglion cells to encode visual signals reliably and economically. Thus simultaneousphotoreceptor neurotransmitter release dysfunction and loss in signaling efficiency aremixed during iCSNB.6.Involvement of gap junction transmission in these synchronous RGCs discharges givesthe possibility that the firing patterns in Cacnalf mutated retina are strongly shaped byelectrical coupling in the inner retina. Under Cacnalf mutated conditions, the absentcontribution of broad scale cross-correlation to RGCs interactions points to vanishedpolysynaptic inputs from bipolar cells.7.ON pathway is somehow disconnected from ganglion cells while OFF pathways mightbe preferentially selected by Cacnalf mutated retina, providing evidences of functionalchanges consistent with the retinal network plasticity and remolding atfer congenital genemutation in outer retina. These results display that the ON pathway of Cacnalf mutate dretinas was suppressed by OFF pathway, and this suppression could be relieved to restorethe ON response when the robust OFF pathway are disrupted.In summary, malfunction of neurotransmitter release in OPL during early onset CSNBinteracts with visual signal pathway remolding in downstream inner retinal circuits.Improved understanding of such developmental mechanisms may facilitate improvementsin designing more effective therapeutic strategy for these disorders.