Regulation of the Chloride Store in the Retinal Amacrine Cells

Nitric Oxide (NO) is a key gaseous messenger that has been shown to be produced by multiple cell types in the vertebrate retina. Research in our lab is aimed at unlocking critical synaptic functions of NO. A major finding from our lab is that NO affects synaptic responses in amacrine cells by altering the plasma membrane gradient for Cl-. This is due to release of Cl- from an internal store and this in turn dependent on a decrease in cytosolic pH. Determining the factors regulating cytosolic Cl- in neurons is fundamental to our understanding of the function of GABAergic and glycinergic synapses. This is because the Cl- distribution across the postsynaptic plasma membrane determines the sign and strength of postsynaptic voltage responses. Here, my goals were to confirm the compartmental nature of the internal Cl- store and to test the hypothesis that Cl- is being released from acidic organelles such as the Golgi, synaptic vesicles, endosomes or lysosomes. To accomplish this, I made whole cell voltage clamp recordings from cultured chick retinal amacrine cells and used GABA-gated currents to track changes in cytosolic Cl-. The compartmental pH was monitored using LysoSensor imaging. My results demonstrate that increasing compartmental pH with internal weak bases leads to release of Cl- into the cytosol and subsequent addition of NO causes a reduction rather than the usual increase in cytosolic Cl-. In contrast, collapsing proton gradients and thus proton-dependent membrane potentials completely blocked the ability of NO to release compartmental Cl-. These results indicate that maintenance of internal proton gradients is critical to the mechanisms of Cl- release and that Cl- is likely to come from acidic organelles. Additionally, I tested the hypothesis that the store can be emptied and refilled. I determined the conditions under which the store can be depleted and the dependence of refilling on extracellular Cl-. These results demonstrate that the regulation of cytosolic Cl- is closely linked to multiple regulatory processes and further our understanding of repertoire of NO signaling mechanisms

A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl \u3csup\u3e–\u3c/sup\u3e from acidic organelles in amacrine cells

© 2017 the American Physiological Society. γ-Amino butyric acid (GABA) and glycine typically mediate synaptic inhibition because their ligandgated ion channels support the influx of Cl – . However, the electrochemical gradient for Cl – across the postsynaptic plasma membrane determines the voltage response of the postsynaptic cell. Typically, low cytosolic Cl – levels support inhibition, whereas higher levels of cytosolic Cl – can suppress inhibition or promote depolarization. We previously reported that nitric oxide (NO) releases Cl – from acidic organelles and transiently elevates cytosolic Cl – , making the response to GABA and glycine excitatory. In this study, we test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the NO-dependent efflux of organellar Cl – . We first establish the mRNA and protein expression of CFTR in our model system, cultured chick retinal amacrine cells. Using whole cell voltage- clamp recordings of currents through GABA-gated Cl – channels, we examine the effects of pharmacological inhibition of CFTR on the NO-dependent release of internal Cl – . To interfere with the expression of CFTR, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. We find that both pharmacological inhibition and CRISPR/Cas9-mediated knockdown of CFTR block the ability of NO to release Cl – from internal stores. These results demonstrate that CFTR is required for the NO-dependent efflux of Cl – from acidic organelles. NEW & NOTEWORTHY Although CFTR function has been studied extensively in the context of epithelia, relatively little is known about its function in neurons. We show that CFTR is involved in an NO-dependent release of Cl – from acidic organelles. This internal function of CFTR is particularly relevant to neuronal physiology because postsynaptic cytosolic Cl – levels determine the outcome of GABA- and glycinergic synaptic signaling. Thus the CFTR may play a role in regulating synaptic transmission

Multimodal Evaluation of TMS - Induced Somatosensory Plasticity and Behavioral Recovery in Rats With Contusion Spinal Cord Injury

Introduction: Spinal cord injury (SCI) causes partial or complete damage to sensory and motor pathways and induces immediate changes in cortical function. Current rehabilitative strategies do not address this early alteration, therefore impacting the degree of neuroplasticity and subsequent recovery. The following study aims to test if a non-invasive brain stimulation technique such as repetitive transcranial magnetic stimulation (rTMS) is effective in promoting plasticity and rehabilitation, and can be used as an early intervention strategy in a rat model of SCI.Methods: A contusion SCI was induced at segment T9 in adult rats. An rTMS coil was positioned over the brain to deliver high frequency stimulation. Behavior, motor and sensory functions were tested in three groups: SCI rats that received high-frequency (20 Hz) rTMS within 10 min post-injury (acute-TMS; n = 7); SCI rats that received TMS starting 2 weeks post-injury (chronic-TMS; n = 5), and SCI rats that received sham TMS (no-TMS, n = 5). Locomotion was evaluated by the Basso, Beattie, and Bresnahan (BBB) and gridwalk tests. Motor evoked potentials (MEP) were recorded from the forepaw across all groups to measure integrity of motor pathways. Functional MRI (fMRI) responses to contralateral tactile hindlimb stimulation were measured in an 11.7T horizontal bore small-animal scanner.Results: The acute-TMS group demonstrated the fastest improvements in locomotor performance in both the BBB and gridwalk tests compared to chronic and no-TMS groups. MEP responses from forepaw showed significantly greater difference in the inter-peak latency between acute-TMS and no-TMS groups, suggesting increases in motor function. Finally, the acute-TMS group showed increased fMRI-evoked responses to hindlimb stimulation over the right and left hindlimb (LHL) primary somatosensory representations (S1), respectively; the chronic-TMS group showed moderate sensory responses in comparison, and the no-TMS group exhibited the lowest sensory responses to both hindlimbs.Conclusion: The results suggest that rTMS therapy beginning in the acute phase after SCI promotes neuroplasticity and is an effective rehabilitative approach in a rat model of SCI

Expression and Localization of CLC Chloride Transport Proteins in the Avian Retina

Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl−/H+ antiporters, ClCs 3–7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl− can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue

A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl −

© 2017 the American Physiological Society. γ-Amino butyric acid (GABA) and glycine typically mediate synaptic inhibition because their ligandgated ion channels support the influx of Cl – . However, the electrochemical gradient for Cl – across the postsynaptic plasma membrane determines the voltage response of the postsynaptic cell. Typically, low cytosolic Cl – levels support inhibition, whereas higher levels of cytosolic Cl – can suppress inhibition or promote depolarization. We previously reported that nitric oxide (NO) releases Cl – from acidic organelles and transiently elevates cytosolic Cl – , making the response to GABA and glycine excitatory. In this study, we test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the NO-dependent efflux of organellar Cl – . We first establish the mRNA and protein expression of CFTR in our model system, cultured chick retinal amacrine cells. Using whole cell voltage- clamp recordings of currents through GABA-gated Cl – channels, we examine the effects of pharmacological inhibition of CFTR on the NO-dependent release of internal Cl – . To interfere with the expression of CFTR, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. We find that both pharmacological inhibition and CRISPR/Cas9-mediated knockdown of CFTR block the ability of NO to release Cl – from internal stores. These results demonstrate that CFTR is required for the NO-dependent efflux of Cl – from acidic organelles. NEW & NOTEWORTHY Although CFTR function has been studied extensively in the context of epithelia, relatively little is known about its function in neurons. We show that CFTR is involved in an NO-dependent release of Cl – from acidic organelles. This internal function of CFTR is particularly relevant to neuronal physiology because postsynaptic cytosolic Cl – levels determine the outcome of GABA- and glycinergic synaptic signaling. Thus the CFTR may play a role in regulating synaptic transmission

Peripheral Nerve Injury Induces Changes in the Activity of Inhibitory Interneurons as Visualized in Transgenic GAD1-GCaMP6s Rats

Peripheral nerve injury induces cortical remapping that can lead to sensory complications. There is evidence that inhibitory interneurons play a role in this process, but the exact mechanism remains unclear. Glutamate decarboxylase-1 (GAD1) is a protein expressed exclusively in inhibitory interneurons. Transgenic rats encoding GAD1–GCaMP were generated to visualize the activity in GAD1 neurons through genetically encoded calcium indicators (GCaMP6s) in the somatosensory cortex. Forepaw denervation was performed in adult rats, and fluorescent Ca2+ imaging on cortical slices was obtained. Local, intrahemispheric stimulation (cortical layers 2/3 and 5) induced a significantly higher fluorescence change of GAD1-expressing neurons, and a significantly higher number of neurons were responsive to stimulation in the denervated rats compared to control rats. However, remote, interhemispheric stimulation of the corpus callosum induced a significantly lower fluorescence change of GAD1-expressing neurons, and significantly fewer neurons were deemed responsive to stimulation within layer 5 in denervated rats compared to control rats. These results suggest that injury impacts interhemispheric communication, leading to an overall decrease in the activity of inhibitory interneurons in layer 5. Overall, our results provide direct evidence that inhibitory interneuron activity in the deprived S1 is altered after injury, a phenomenon likely to affect sensory processing

Regulation of Electromagnetic Perceptive Gene Using Ferromagnetic Particles for the External Control of Calcium Ion Transport

Developing synthetic biological devices to allow the noninvasive control of cell fate and function, in vivo can potentially revolutionize the field of regenerative medicine. To address this unmet need, we designed an artificial biological “switch” that consists of two parts: (1) the electromagnetic perceptive gene (EPG) and (2) magnetic particles. Our group has recently cloned the EPG from the Kryptopterus bicirrhis (glass catfish). The EPG gene encodes a putative membrane-associated protein that responds to electromagnetic fields (EMFs). This gene’s primary mechanism of action is to raise the intracellular calcium levels or change in flux through EMF stimulation. Here, we developed a system for the remote regulation of [Ca2+]i (i.e., intracellular calcium ion concentration) using streptavidin-coated ferromagnetic particles (FMPs) under a magnetic field. The results demonstrated that the EPG-FMPs can be used as a molecular calcium switch to express target proteins. This technology has the potential for controlled gene expression, drug delivery, and drug developments