Effect Of Neuromuscular Function In Rat With Cacna1f Mutation

The congenital stationary blindness (CSNB) is a non-progressive disorder ofthe retina characterized by the loss of night vision. Its clinical features mainlyinclude impaired night vision. By ERGs testing, X-linked form of CSNB（XLCSNB）exhibited a typical negative b-wave of standard combined ERG response.There are no progressive changes in retinal fundus and impairments of motorfunction. One of phenotype in a majority of patients affected muscular dystrophyDuchenne (DMD) is the abnormal scotopic electroretinogram[1，2]. The phenotypeof ERG in DMD patients is similar to CSNB disease, but the patients had normaldark adaptation. In current, there was no report about whether CSNB patient havedeficient muscle strength or not. The inbred strain of the CSNB rats with retinaldysfunction had built up[3，4], which is caused by a mutation in the Cacna1f gene,was X-link recessive inheritance[5]. In behavior tests for our CSNB rats, theresults indicated that there are changes not only in visual related tests, but aslo inmotor function tests. The Cacna1f gene mutation affected the number of ribbon body (previous study) and the expression of synapses associated proteins inactive zone (CAZ) of retina; and caused the abnormal synaptic conduction lead totpyical phenotype of CSNB. The CAZ is also an important structure inneuromuscular junction (NMJ). And there is special structure dense body whosefunction is similar to ribbon body and synapses associated proteins. The reportshowed the Cacna1f encoded the Cav1.4protein not only expressed in retina butalso express in skeletal muscle[6,7]. The function of Cav1.4protein in skeletalmuscle is not known clearly. In consideration of the low level of muscleendurance were performed in CSNB rats, we hypothesize that the mutation ofCacna1f probably affect the active zone of NMJ, lead to the change oftransmission function of synapses in slow muscle. The CSNB rats not only amodel animal in retinal degeneration, but also give us chance to further study thefunction of Cacna1f gene.The research carried out the four respects to verify the hypothesis step by step.The four steps are as follows:1.the character changes of structure and function indevelping retina by Cacna1f mutation.2. Build up F1hybrid to verify muscleendurance.3. Measurements fatigue resistance of soleus muscle in CSNB rat.4.Immunoreactivity of synapses associated protein in retina and skeletal muscle ofCacna1f mutation rat.Methods and materialThe retinal degenaration CSNB rat models and SD rats as the control wereused in present reseach. Using technology of vision electrophysiology, observesthe function changes of retina in development of CSNB rat. Building up F1hybrid to learn the phenotype of ERG and behavior performance and know thehereditary feature of abnormal muscle endurance in CSNB rat. Isolated soleus muscle strips were perfused ex vivo and different modes of contraction wereinduced by electrical stimulation to measure fatigue resistance of soleus musclein CSNB rat. Taking immunohistochemical staining to learn the expression ofCav1.4and synapses associated protein in skeletal muscle and other tissues;using Western Blot technique to get the semiquantitative expression of synapsesassociated protein in muscle.Results1. The character changes of structure and function in develping retina byCacna1f mutation.The phenotypes of ERGs are similar in control rats and CSNB at the openingeyes day (about14day) and the day before. After two days from openingeyes, thechanges of control rat were notable: rod ERG, b-wave of Max ERG, and PhotopicERG were appeared; hereafter, the waveform of ERG changed slightly in controlrats. The waveform of ERGs remained similarly from the one day before openingeyes, and the Photopic ERG was appeared in Postnatal26days (P26) in CSNBrats. Flicker ERG existed in one day before opening eyes.The vision function of developing retina showed: The amplitudes of a-waveof Max ERG were similar in control and CSNB rats from before one day ofopening eyes to P26, but the latencies of CSNB rats were prolonged (P <0.05).The amplitudes and latencies of Photopic ERG in CSNB rats were significantdifference compared with control rats (P <0.01). In control rats, the amplitudesof a-wave of Max ERG and b-wave of cone response peaked at3mon, and thewaveforms still existed at17mon. In CSNB rats, the amplitudes of a-wave ofMax ERG and b-wave of cone response peaked at1mon and disappeared at10mon. The results of HE staining showed that the structure of early developing retinawere similar in control and CSNB rats, but the OPL was thiner in CSNB rat thanin control rat. At the opening eyes day, there were no expression of the synapsesassociated proteins SYN and CtBP2in CAZ of retina. After opening eyes, thepunctate expressions of the two proteins were reduced in CSNB rat comparedwith control rat. SYN always accompaning CtBP2, and the expressions of thetwo proteins were increased in early developming retina. The number of rodbipolar cells was similar in two kinds of rats. And from the two days afteropening eyes the dendrites in control rats were more than that in CSNB rats.2. Build up F1hybrid to verify muscle endurance.all male F1offspring rats had the phenotype of CSNB-like ERG responses,whereas all the female F1rats displayed normal ERG responses. Theperformances of hanging wire test showed that the gripping time of male ratswere significant reduced compared with female rats in every development period.3. Measurements fatigue resistance of soleus muscle in CSNB rat.The optimal frequency of CSNB rats was similar to control rat, it was100Hz.In the0.8%duty cycle and30%duty cycle intermittent tetanic contraction, theresults demonstrated normal fatigue resistance of CSNB rats SOL in intermittenttetanic contraction recordings. In the45sec100%continuous tetanic contraction,tension rose to peak and declined quickly in the CSNB soleus; but the peaktetanic force (P30/P0) was normal.4. Immunoreactivity of synapses associated protein in retina and skeletalmuscle of Cacna1f mutation rat.In retina, the C-terminal of Cav1.4protein was eliminated from the OPL, butthe N-terminal of Cav1.4was expressed weakly in CSNB rat retina; Thepunctate labering with SYN1presented intense expression in OPL of control rat retina, whereas the quantity of labering in CSNB rat retina was reduced. Inskeletal muscle, SYN and Cav1.4presented at muscle membrance; theimmunoreactivity expression of N-terminal of Cav1.4was significantly reducedin affected rats, and the expression of C-terminal of Cav1.4was roughlydisappeared in the soleus cross-and longitudinal-sections. There were two band ofSYN in Western Blot results; the SYN with high molecular weight weresignificantly reduced in CSNB rats（P <0.05）, but the SYN with low molecularweight were normal in control and CSNB rats. Within the gray matter, intenseC-terminal of Cav1.4was located into spinal dorsal horn and anterior horn ofcontrol rat, whereas the antibody only expressed in spinal dorsal horn of CSNBrat with weaker immunoreactivity. The distributions of C-terminal of Cav1.4protein were undetectable in the brain of both control and CSNB rats.Conclusions1. during opening eyes and before periods, the phenotype of ERGs are similar incontrol and CSNB rats, and the biggest differences happenies in after2days fromopening eyes comparison the two kinds of rats. The vision function of CSNB ratdisappears quickly. The number of the synapses of rod bipolar cells is reduced.Cacna1f mutation affects the expression of synapses associated proteins in CAZof CSNB rat.2. The muscle endurance is indeed declined in CSNB rat. And the muscleendurance of F1generation is X-linked mode of inheritance accompanyed withCSNB.3. There is change in the function of muscle strips indeed, but the affected extentof contraction function is slight.4. The Cacna1F mutation affects the Cav1.4and synapses associated proteins (SYN) in CAZ of NMJ in CSNB rat. Therefore, we primarily validate thehypothesis that the mutation of Cacna1f probably affect the active zone of NMJ,lead to the change of transmission function of synapses in slow muscle.