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Neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) are characterized by the presence of protein inclusions in the affected neurons. Emerging data indicate that protein misfolding may be of mechanistic importance in these diseases.1 Mutations in the ubiquitously expressed superoxide dismutase (SOD1) gene account for 20% of cases of the familial form of ALS. More than 150 mutations in the SOD1 gene have been discovered, including the point mutations G93R and G85R.2 Recent studies also implicate SOD1 in the sporadic form of ALS and suggest a prionlike propagation of misfolded SOD1.3–5 Interestingly, some of the newly identified genes implicated in ALS, such as TARDBP and FUS, are also proteins that show a high propensity to misfold and prionlike activity.6 However, we still do not know the precise mechanism by which mutant proteins cause toxicity.5,7 The emerging consensus view is that multiple interacting pathophysiological factors, including protein misfolding, contribute to the neuronal toxicity in ALS.8,9 Despite progress in revealing multiple molecular processes involved in disease pathology, relatively little is known about when and how the disekease, which starts focally, spreads throughout the motor network.10–12 Interestingly, even in the subtypes of ALS caused by SOD1 mutations, there is considerable phenotypic heterogeneity. Ravits and La Spada12 hypothesized that despite disease heterogeneity, the disease poses common themes that may involve common mechanisms. They propose that ALS may in fact be an orderly, actively propagating process and that fundamental molecular mechanisms may be uniform. The zebrafish is emerging as a useful tool for studying neurological diseases relevant to humans. Previously, we had shown that mutant sod1 transgenic fish show the hallmarks of adult onset neurodegenerative ALS, including defective motor performance, motor neuron loss, a loss of neuromuscular connectivity, and muscle atrophy.13 The aforementioned observations demonstrate the usefulness of the zebrafish as a model for this disease. However, among the current limitations when working with in vivo models of ALS is the lack of a good readout for the presymptomatic course of the disease. The zebrafish offer great advantages in studying early disease processes, as they develop rapidly, reaching postembryonic life at around 3 days postfertilization (dpf), which is developmentally similar to the neonatal mouse (for a comparison of developmental stages in human, mouse, and zebrafish, see Table 1). Moreover, the embryonic and larval zebrafish spinal cord is functionally and anatomically similar to that of humans, yet it is also optically transparent and experimentally accessible, making it ideal for the study of spinal circuits in normal and pathophysiological conditions.14 TABLE 1 Comparison of Neural Developmental Stages in Humans, Mice, and Zebrafish In the current study, we monitored in vivo early neurological changes caused by mutant sod1 gene. The sod1 zebrafish ALS model harbors a fluorescent heat shock stress response (HSR) reporter gene (hsp70-DsRed). The HSR is an endogenous cellular pathway that attempts to refold the damaged proteins in stressed cells, although this response is not always sufficient or beneficial.15 Thus, the HSR-mediated DsRed fluorescence in the sod1 zebrafish model of ALS represents a useful tool for monitoring perturbations in cellular homeostasis caused by sod1 mutation. This facilitates the mapping of disease focality and spread through the central nervous system (CNS) by the spatiotemporal readout of the neuronal stress response in the spinal cord of mutant zebrafish and provides an understanding of the cells and networks involved in disease propagation in ALS. We present evidence that the HSR is an indicator of early pathogenic processes occurring in neurons. The HSR is first observed at embryonic stages, in discrete populations of inhibitory interneurons in the spinal cord, and is followed by dysregulation of glycine release from these inhibitory interneurons. Furthermore, we observe that following interneuron dysfunction, motor neurons start exhibiting neuronal stress. More interestingly, we show that motor neurons showing the HSR also show dysfunctional neuromuscular junctions (NMJs). Taken together, our observations suggest that the mutant sod1-induced HSR is a robust predictor of neuronal dysfunction and thus is a reliable marker of disease pathogenesis. Finally, we also show that the neuronal stress readout can be used to identify neuroprotective compounds such as riluzole and identify biological targets that may ameliorate early pathophysiological disease processes that are currently not well explored. Although the sod1 zebrafish model may by itself not be sufficient in developing new therapies for ALS, this model system would provide a rapid way to triage compounds for screening in higher vertebrate models, with the potential for more rapid identification of promising compounds for translation into human clinical trials.

Summary Activity-dependent changes in synaptic strength are well established as mediating long-term plasticity underlying learning and memory, but modulation of target neuron excitability could complement changes in synaptic strength and regulate network activity. It is thought that homeostatic mechanisms match intrinsic excitability to the incoming synaptic drive, but evidence for involvement of voltage-gated conductances is sparse. Here, we show that glutamatergic synaptic activity modulates target neuron excitability and switches the basis of action potential repolarization from Kv3 to Kv2 potassium channel dominance, thereby adjusting neuronal signaling between low and high activity states, respectively. This nitric oxide-mediated signaling dramatically increases Kv2 currents in both the auditory brain stem and hippocampus (>3-fold) transforming synaptic integration and information transmission but with only modest changes in action potential waveform. We conclude that nitric oxide is a homeostatic regulator, tuning neuronal excitability to the recent history of excitatory synaptic inputs over intervals of minutes to hours., Highlights ► Synaptic input drives NO-mediated modulation of voltage-gated potassium currents ► High synaptic activity switches the dominant delayed rectifier to Kv2 ► NO volume transmission tunes target neurons to excitatory synaptic drive ► This homeostatic regulation occurs broadly in the brain (brain stem and hippocampus)

SummaryNeuronal nitric oxide synthase (nNOS) is broadly expressed in the brain and associated with synaptic plasticity through NMDAR-mediated calcium influx. However, its physiological activation and the mechanisms by which nitric oxide (NO) influences synaptic transmission have proved elusive. Here, we exploit the unique input-specificity of the calyx of Held to characterize NO modulation at this glutamatergic synapse in the auditory pathway. NO is generated in an activity-dependent manner by MNTB principal neurons receiving a calyceal synaptic input. It acts in the target neuron and adjacent inactive neurons to modulate excitability and synaptic efficacy, inhibiting postsynaptic Kv3 potassium currents (via phosphorylation), reducing EPSCs and so increasing action potential duration and reducing transmission fidelity. We conclude that NO serves as a volume transmitter and slow dynamic modulator, integrating spontaneous and evoked neuronal firing, thereby providing an index of global activity and regulating information transmission across a population of active and inactive neurons.

The central auditory brainstem provides an efferent projection known as the medial olivocochlear (MOC) system, which regulates the cochlear amplifier and mediates protection on exposure to loud sound. It arises from neurons of the ventral nucleus of the trapezoid body (VNTB), so control of neuronal excitability in this pathway has profound effects on hearing. The VNTB and the medial nucleus of the trapezoid body are the only sites of expression for the Kv2.2 voltage-gated potassium channel in the auditory brainstem, consistent with a specialized function of these channels. In the absence of unambiguous antagonists, we used recombinant and transgenic methods to examine how Kv2.2 contributes to MOC efferent function. Viral gene transfer of dominant-negative Kv2.2 in wild-type mice suppressed outward K+currents, increasing action potential (AP) half-width and reducing repetitive firing. Similarly, VNTB neurons from Kv2.2 knock-out mice (Kv2.2KO) also showed increased AP duration. Control experiments established that Kv2.2 was not expressed in the cochlea, so any changes in auditory function in the Kv2.2KO mouse must be of central origin. Further,in vivorecordings of auditory brainstem responses revealed that these Kv2.2KO mice were more susceptible to noise-induced hearing loss. We conclude that Kv2.2 regulates neuronal excitability in these brainstem nuclei by maintaining short APs and enhancing high-frequency firing. This safeguards efferent MOC firing during high-intensity sounds and is crucial in the mediation of protection after auditory overexposure.

Summary Background During development, spinal networks undergo an intense period of maturation in which immature forms of motor behavior are observed. Such behaviors are transient, giving way to more mature activity as development proceeds. The processes governing age-specific transitions in motor behavior are not fully understood. Results Using in vivo patch clamp electrophysiology, we have characterized ionic conductances and firing patterns of developing zebrafish spinal neurons. We find that a kernel of spinal interneurons, the ipsilateral caudal (IC) cells, generate inherent bursting activity that depends upon a persistent sodium current (INaP). We further show that developmental transitions in motor behavior are accompanied by changes in IC cell bursting: during early life, these cells generate low frequency membrane oscillations that likely drive “coiling,” an immature form of motor output. As fish mature to swimming stages, IC cells switch to a sustained mode of bursting that permits generation of high-frequency oscillations during locomotion. Finally, we find that perturbation of IC cell bursting disrupts motor output at both coiling and swimming stages. Conclusions Our results suggest that neurons with unique bursting characteristics are a fundamental component of developing motor networks. During development, these may shape network output and promote stage-specific reconfigurations in motor behavior., Highlights ► IC cells of the developing zebrafish CPG exhibit INaP-dependent bursting activity ► IC cell bursting activity is stage specific ► At coiling stages, IC cells generate rhythmic membrane oscillations ► At swimming stages, IC cells generate high-frequency, sustained bursting

Principal neurons of the medial nucleus of the trapezoid body (MNTB) express a spectrum of voltage-dependent K(+) conductances mediated by Kv1-Kv4 channels, which shape action potential (AP) firing and regulate intrinsic excitability. Postsynaptic factors influencing expression of Kv channels were explored using organotypic cultures of brainstem prepared from P9-P12 rats and maintained in either low (5 mm, low-K) or high (25 mm, high-K) [K(+)](o) medium. Whole cell patch-clamp recordings were made after 7-28 days in vitro. MNTB neurons cultured in high-K medium maintained a single AP firing phenotype, while low-K cultures had smaller K(+) currents, enhanced excitability and fired multiple APs. The calyx of Held inputs degenerated within 3 days in culture, having lost their major afferent input; this preparation of calyx-free MNTB neurons allowed the effects of postsynaptic depolarisation to be studied with minimal synaptic activity. The depolarization caused by the high-K aCSF only transiently increased spontaneous AP firing (2 min) and did not measurably increase synaptic activity. Chronic depolarization in high-K cultures raised basal levels of [Ca(2+)](i), increased Kv3 currents and shortened AP half-widths. These events relied on raised [Ca(2+)](i), mediated by influx through voltage-gated calcium channels (VGCCs) and release from intracellular stores, causing an increase in cAMP-response element binding protein (CREB) phosphorylation. Block of VGCCs or of CREB function suppressed Kv3 currents, increased AP duration, and reduced Kv3.3 and c-fos expression. Real-time PCR revealed higher Kv3.3 and Kv1.1 mRNA in high-K compared to low-K cultures, although the increased Kv1.1 mRNA was mediated by a CREB-independent mechanism. We conclude that Kv channel expression and hence the intrinsic membrane properties of MNTB neurons are homeostatically regulated by [Ca(2+)](i)-dependent mechanisms and influenced by sustained depolarization of the resting membrane potential.