Patrice D. Côté

Chimaeric mice deficient in dystroglycans develop muscular dystrophy and have disrupted myoneural synapses

Mutations in the dystrophin gene (DMD) and in genes encoding several dystrophin-associated proteins result in Duchenne and other forms of muscular dystrophy. α-Dystroglycan (Dg) binds to laminins in the basement membrane surrounding each myofibre and docks with β-Dg, a transmembrane protein, which in turn interacts with dystrophin or utrophin in the subplasmalemmal cytoskeleton. α- and β-Dgs are thought to form the functional core of a larger complex of proteins extending from the basement membrane to the intracellular cytoskeleton, which serves as a superstructure necessary for sarcolemmal integrity. Dgs have also been implicated in the formation of synaptic densities of acetylcholine receptors (AChRs) on skeletal muscle. Here we report that chimaeric mice generated with ES cells targeted for both Dg alleles have skeletal muscles essentially devoid of Dgs and develop a progressive muscle pathology with changes emblematic of muscular dystrophies in humans. In addition, many neuromuscular junctions are disrupted in these mice. The ultrastructure of basement membranes and the deposition of laminin within them, however, appears unaffected in Dg-deficient muscles. We conclude that Dgs are necessary for myofibre survival and synapse differentiation or stability, but not for the formation of the muscle basement membrane, and that Dgs may have more than a purely structural function in maintaining muscle integrity.

Voltage-gated sodium channels contribute to the b-wave of the rodent electroretinogram by mediating input to rod bipolar cell GABAc receptors

Voltage-gated sodium (Nav) channels are known to augment cone bipolar cell light responses, increasing the electroretinogram (ERG) b-wave in response to stimulus strengths above the cone threshold. However previous in vivo studies on a number of animal models have not found a role for Nav channels in augmenting the b-wave in scotopic conditions below the cone threshold. We recorded ERGs from mice and rats using a series of TTX concentrations and tested retinal output to ensure complete Nav channel block. We found that TTX concentrations sufficient to completely suppress retinal output caused large (~40%) decrease in the scotopic electroretinogram (ERG) response to high stimulus strengths (1.0 log cd s/m(2)). In addition the b-wave was reduced by ~20% even at stimulus strengths that should predominately excite the rod pathway (-2.2 log cd s/m(2)). Modulating stimulus strength and background luminance showed that Nav channel contribution to the b-wave is strongest in mesopic conditions with low strength stimuli. Blocking GABAc receptors indicted that Nav channels predominately contribute to the b-wave by supporting GABAc input to rod bipolar cells in addition to directly amplifying the light response of cone ON bipolar cells. We also determined that saturating levels of TTX reduced the rat b-wave below cone threshold. Nav channels increase the ERG b-wave in both rod and cone bipolar cell-dominated circuits. In circuits involving rod bipolar cells the effect is mediated indirectly via GABAergic inhibitory cells, while Nav channels directly located on cone bipolar cells amplify light responses in the cone pathways.

Physiological Maturation of Photoreceptors Depends on the Voltage-Gated Sodium Channel NaV1.6 (Scn8a)

Voltage-gated sodium channels (VGSCs) ensure the saltatory propagation of action potentials along axons by acting as signal amplifiers at the nodes of Ranvier. In the retina, activity mediated by VGSCs is important for the refinement of the retinotectal map. Here, we conducted a full-field electroretinogram (ERG) study on mice null for the sodium channel NaV1.6. Interestingly, the light-activated hyperpolarization of photoreceptor cells (the a-wave) and the major “downstream” components of the ERG, the b-wave and the oscillatory potentials, are markedly reduced and delayed in these mice. The functional deficit was not associated with any morphological abnormality. We demonstrate that Scn8a is expressed in the ganglion and inner nuclear layers and at low levels in the outer nuclear layer beginning shortly before the observed ERG deficit. Together, our data reveal a previously unappreciated role for VGSCs in the physiological maturation of photoreceptors.

Reduced Retinal Function in the Absence of Nav1.6

Background Mice with a function-blocking mutation in the Scn8a gene that encodes Nav1.6, a voltage-gated sodium channel (VGSC) isoform normally found in several types of retinal neurons, have previously been found to display a profoundly abnormal dark adapted flash electroretinogram. However the retinal function of these mice in light adapted conditions has not been studied. Methodology/Principal Findings In the present report we reveal that during light adaptation these animals are shown to have electroretinograms with significant decreases in the amplitude of the a- and b-waves. The percent decrease in the a- and b-waves substantially exceeds the acute effect of VGSC block by tetrodotoxin in control littermates. Intravitreal injection of CoCl2 or CNQX to isolate the a-wave contributions of the photoreceptors in littermates revealed that at high background luminance the cone-isolated component of the a-wave is of the same amplitude as the a-wave of mutants. Conclusions/Significance Our results indicate that Scn8a mutant mice have reduced function in both rod and the cone retinal pathways. The extent of the reduction in the cone pathway, as quantified using the ERG b-wave, exceeds the reduction seen in control littermates after application of TTX, suggesting that a defect in cone photoreceptors contributes to the reduction. Unless the postreceptoral component of the a-wave is increased in Scn8a mutant mice, the reduction in the b-wave is larger than can be accounted for by reduced photoreceptor function alone. Our data suggests that the reduction in the light adapted ERG of Scn8a mutant mice is caused by a combination of reduced cone photoreceptor function and reduced depolarization of cone ON bipolar cells. This raises the possibility that Nav1.6 augments signaling in cone bipolar cells.

D1 Dopamine receptors modulate cone ON bipolar cell Nav channels to control daily rhythms in photopic vision

In amphibians, voltage-gated sodium (Nav) channels in cone ON bipolar cells (ON-CBC) amplify cone signals in the dark and in mesopic background light. However, during light adaptation, dopamine, acting through D1 receptors (D1R), suppresses Nav channels and therefore act as a gain control mechanism. Curiously in rodents, Nav channel contributions to the ON-CBC-generated light-adapted electroretinogram (ERG) b-wave appear to exist even in fully light-adapted conditions. We sought to determine how rodent ON-CBC Nav channels are regulated by dopamine via D1R during light adaptation and during the circadian cycle. We first tested the sensitivity of Nav channels in mouse ON-CBCs to the modulation by dopamine via D1Rs. Although light-adaptation had little effect on Nav channel contributions to the b-wave, these channels were found to be modulated by D1Rs. We pharmacologically isolated the cone to ON-CBC circuit in fully light-adapted retinas to confirm these results. Retinal dopamine release following light adaptation has been previously shown to be increased in mice during circadian night. We first show that circadian fluctuations in ON-CBC function are suppressed in dark-adapted retinas, indicating that circadian fluctuations are a function of light adaptation. Secondly, we show that at night the mouse retina behaves similarly to those of frogs and salamanders with a gain control mechanism utilizing D1R modulation of Nav channels to suppress ON-CBC light responses in light-adapted conditions during circadian night. Taken together, these results suggest that circadian control of ON-CBC function contains an initial phase after approximately 18–30 h of dark adaptation, leading to substantial changes in b-wave amplitude after a relatively short time in free run which are dependent on D1R modulation of Nav channels.

Glial Reaction in the Spinal Cord of the Degenerating Muscle Mouse (Scn8a dmu )

Abstract The glial reaction was investigated in the spinal cord of the degenerating muscle (dmu) mouse, which harbours a null mutation in the voltage-gated sodium channel gene Scn8a and does not produce functional Nav1.6 channel. Glial fibrillary acidic protein (GFAP)- and Iba1-immunoreactivity were detected in numerous cells throughout the spinal cord of wild type mice. These cells had small cell bodies and ramified processes. The dmu mutation increased the number of GFAP-immunoreactive (-IR) cells and the length of their processes in the ventral horn but not in the dorsal horn of the lumbar spinal cord. The number of Iba1-IR cells was similar in cervical and lumbar spinal cords of wild type and dmu mice. However, Iba1-IR processes and their branches became thinner and showed a fine varinose appearance in dmu mice. The length of Iba1-IR processes was significantly reduced in dorsal and ventral horns of dmu mice. Double immunofluorescence also demonstrated the relationship between glial cells and motor neurons containing calcitonin gene-related peptide (CGRP), a marker for their degeneration. The dmu mutation caused increase in the length of GFAP-IR processes surrounding CGRP-IR motor neurons in the ventral horn. However, the thickness and length of Iba1-IR processes around CGRP-IR motor neurons were reduced by the mutation. The present study suggests that the dmu mutation causes astrocytic activation and microglial inactivation in the spinal cord. These changes may be associated with degeneration and activity of motor and sensory neuron in dmu mice.

Dopamine modulation of rod pathway signaling by suppression of GABAC feedback to rod‐driven depolarizing bipolar cells

Reducing signal gain in the highly sensitive rod pathway prevents saturation as background light levels increase, allowing the dark‐adapted retina to encode stimuli over a range of background luminances. Dopamine release is increased during light adaptation and is generally accepted to suppress rod signaling in light‐adapted retinas. However, recent research has suggested that dopamine, acting through D1 receptors, could additionally produce a sensitization of the rod pathway in dim light conditions via gamma‐aminobutyric acid (GABA) type C receptors. Here, we evaluated the overall activity of the depolarizing bipolar cell (DBC) population in vivo to ensure the integrity of long‐distance network interactions by quantifying the b‐wave of the electroretinogram in mice. We showed that dopamine, acting through D1 receptors, reduced the amplitude and sensitivity of rod‐driven DBCs during light adaptation by suppressing GABA type A receptor‐mediated serial inhibition onto rod DBC GABA type C receptors. Block of D1 receptors did not suppress rod‐driven DBC sensitivity when GABAA‐mediated serial inhibition was blocked by gabazine, suggesting that the reduction in rod‐driven DBC sensitivity in the absence of D1 receptors was due to disinhibition of serial inhibitory GABAergic circuitry rather than a direct facilitatory effect on GABA release onto rod‐driven DBC GABA type C receptors. Finally, the large population of GABAergic A17 wide‐field amacrine cells known to maintain reciprocal inhibition with rod DBCs could be excluded from the proposed disinhibitory circuit after treatment with 5,7‐dihydroxytryptamine.

Increase of CGRP Expression in Motor Endplates Within Fore and Hind Limb Muscles of the Degenerating Muscle Mouse (Scn8a (dmu) )

The distribution of calcitonin gene-related peptide (CGRP) was examined in skeletal muscles of fore and hind limb as well as in oral and cranio-facial regions of the degenerating muscle (dmu) mouse, which harbours a null mutation in the voltage-gated sodium channel gene Scn8a. In limb, oral and cranio-facial muscles of wild type mice, only a few motor endplates contained CGRP-immunoreactivity. However, many CGRP-immunoreactive motor endplates appeared in the triceps brachii muscle, the biceps brachii muscle, the brachialis muscle, and the gastrocnemius muscle of dmu mice. CGRP-immunoreactive density of motor endplates in the skeletal muscles was also elevated by the mutation. In these muscles, the atrophy of muscle fibers could be detected and the density of cell nuclei in the musculature increased. In the flexor digitorum profundus muscle, the flexor digitorum superficialis muscle, and the soleus muscle as well as in oral and cranio-facial muscles, however, the distribution of CGRP-immunoreactivity was barely affected by the mutation. The morphology of muscle fibers and the distribution of cell nuclei within them were also similar in wild type and dmu mice. In the lumbar spinal cord of dmu mice, CGRP-immunoreactive density of spinal motoneurons increased. These findings suggest that the atrophic degeneration in some fore and hind limb muscles of dmu mice may increase CGRP expression in their motoneurons.

Contribution of Nav1.8 sodium channels to retinal function

We examined the contribution of the sodium channel isoform Nav1.8 to retinal function using the specific blocker A803467. We found that A803467 has little influence on the electroretinogram (ERG) a- and b-waves, but significantly reduces the oscillatory potentials (OPs) to 40-60% of their original amplitude, with significant changes in implicit time in the rod-driven range. To date, only two cell types were found in mouse to express Nav1.8; the starburst amacrine cells (SBACs), and a subtype of retinal ganglion cells (RGCs). When we recorded light responses from ganglion cells using a multielectrode array we found significant and opposing changes in two physiological groups of RGCs. ON-sustained cells showed significant decreases while transient ON-OFF cells showed significant increases. The effects on ON-OFF transient cells but not ON-sustained cells disappeared in the presence of an inhibitory cocktail. We have previously shown that RGCs have only a minor contribution to the OPs (Smith et al., 2014), therefore suggesting that SBACs might be a significant contributor to this ERG component. Targeting SBACs with the cholinergic neurotoxin ethylcholine mustard aziridinium (AF64A) caused a reduction in the amplitude of the OPs similar to A803467. Our results, both using the ERG and MEA recordings from RGCs, suggest that Nav1.8 plays a role in modulating specific aspects of the retinal physiology and that SBACs are a fundamental cellular contributor to the OPs in mice, a clear demonstration of the dichotomy between ERG b-wave and OPs.