Molecular and Cellular Neuroscience

Mitochondrial dysfunction is an important cause for neurodegeneration, often associated with dyshomeostasis of reactive oxygen species, i.e. oxidative stress. However, apart from ATP production, mitochondria have many other functions the aberration of which may impact neurons in very different ways. Oxidative stress can cause the deterioration of axonal microtubule bundles, thus critically affecting the highways for life-sustaining transport and providing a potential path to neurodegeneration. We recently found that aberrant transport of mitochondria can have this effect by causing oxidative stress. We therefore asked which aberrations of mitochondrial physiology might impact microtubules, which of these might explain the observed consequences of aberrant mitochondrial transport, and whether mitochondria-induced microtubule phenotypes are always mediated by oxidative stress. Using one consistent Drosophila primary neuron system, we deleted 13 different mitochondrial factors known to be detrimental for neurons in vivo. Losses of five factors caused MT damage, all involving oxidative stress, hence supporting the path from mitochondria via oxidative stress to microtubule deterioration; we discuss Sod2 as potential candidate explaining effects of mitochondrial transport aberration. However, the loss of eight factors - seven of them important mitochondrial morphogenesis regulators - caused no microtubule damage, suggesting potential oxidative stress-independent pathways. Summary StatementAssessing mutant effects of 13 mitochondrial factors on axonal microtubule organisation to unravel potential mechanisms underpinning neurodegeneration

NMDA Receptors (NMDARs) have essential functions in the nervous system, including neuronal maturation, neurotransmission, synaptic plasticity, learning, and memory. Following membrane depolarization and glutamate activation, NMDARs mediate Ca2+ influx into neurons, activating Ca2+ signaling cascades with key roles in neuronal function. However, no studies have been reported on the roles of glutamate and NMDARs during early neuronal development. Although NMDARs classically act at the postsynaptic membrane, the present results indicate that neurons express functional NMDARs during polarity acquisition and localize them in the axonal compartment early in development; at this stage, cultured neurons spontaneously release glutamate. In addition, pharmacological and genetic experiments for NMDARs loss- and gain-of-function modulated neuronal polarization and axonal elongation antagonistically. An intracellular mechanism involving Ca2+ release from the endoplasmic reticulum, activation of the Rho GTPase Rac1, and actin cytoskeleton rearrangements at the axonal growth cone couples these morphological changes. Moreover, NMDAR activity regulates the physiological intracellular production of hydrogen peroxide (H2O2) via a Rac1/NADPH oxidase complex to support neuronal development. Optogenetic Rac1 activation simultaneously promoted lamellipodia formation and H2O2 production suggesting functional coupling between these seemingly unconnected events. The mechanism presented here involves a dual function for the Rac1 protein that depends on glutamate and NMDAR activity. Based on these findings, we suggest that early physiological and spontaneous glutamate release activates NMDARs to promote early neuronal development before synapse formation, indicating that glutamate is necessary for neurotransmission, early neuronal development, and axonal growth.

Caldendrin is a calmodulin-like Ca2+ binding protein that is expressed primarily in neurons and regulates multiple effectors including Cav1 L-type Ca2+ channels. Here, we tested the hypothesis that caldendrin regulates Cav1-dependent pathways that repress neurite growth in dorsal root ganglion neurons (DRGNs). By immunofluorescence, caldendrin was localized in medium- and large-diameter DRGNs. Consistent with an inhibitory effect of caldendrin on neurite growth, neurite initiation and growth was enhanced in dissociated DRGNs from caldendrin knockout (KO) mice compared to those from wild type (WT) mice. In an in vitro axotomy assay, caldendrin KO DRGNs grew longer neurites via a mechanism that was more sensitive to inhibitors of transcription as compared to WT DRGNs. Strong depolarization, which normally represses neurite growth through activation of Cav1 channels, had no effect on neurite growth in DRGN cultures from female caldendrin KO mice. Remarkably, DRGNs from caldendrin KO males were no different from those of WT males in terms of depolarization-dependent neurite growth repression. We conclude that caldendrin opposes neurite regeneration and growth, and this involves coupling of Cav1 channels to growth-inhibitory pathways in DRGNs of females but not males. Our findings suggest that caldendrin KO mice represent an ideal model in which to interrogate the transcriptional pathways controlling neurite regeneration and how these pathways may differ in males and females.

The endocannabinoid system (ECS) has a widespread role in the development and function of the central nervous system (CNS). Cannabinoid receptors like CB1 and CB2 can be activated with exogenous cannabinoids most popularly known as tetrahydrocannabinol (THC) or cannabis and cannabidiol (CBD). The components of the ECS are expressed early in fetal development, and prenatal exposure to cannabis can lead to structural changes in white matter. White matter is composed of neuronal axons ensheathed in myelin, a lipid-rich insulation that facilitates saltatory conduction and maintains axon integrity. In the CNS, myelin is made by specialized glial cells called oligodendrocytes (OLs), which in addition to neurons also express components of the ECS. However, while several studies have focused on how the ECS regulates neuronal development, there is a limited understanding of its impact on OL development or myelin formation. Therefore, our current study set out to understand how pharmacological activation of the ECS alters OL differentiation and myelin formation in vivo. We administered WIN 55,212-2 (WIN 55), a CB1 and CB2 agonist, to larval zebrafish and longitudinally analyzed OL development and myelination in vivo. Interestingly, we observed an increase in non-axonal ensheathments in the spinal cord, which appeared to be surrounding neuronal cell bodies. These non-axonal ensheathments were dependent on CB1, as the addition of WIN 55 in a global CB1 mutant prevented this phenotype. Furthermore, this ectopic cell body ensheathment occurred independently from normal myelination processes, as individual OLs did not exhibit changes in the number of myelin sheaths, sheath length, or total myelin output. This study shows that activation of CB receptors in vivo leads to increased non-axonal ensheathment without significantly changing OL differentiation or normal myelin formation. Future studies can further investigate the pathways that drive this phenotype to better understand how exogenous cannabinoid activation can regulate the precision of oligodendrocyte ensheathment.

Exposure to cyanotoxins has been linked to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimers, and Parkinsons disease. While the cyanotoxin {beta}-methylamino-L-alanine (BMAA) has received much attention, cyanobacteria produce many cyanotoxic compounds, several of which have been detected in nature alongside BMAA including 2,4-diaminobutyric acid (2,4-DAB), and N-(2-aminoethyl)glycine (AEG). Thus, the question of whether DAB and AEG also cause neurotoxic effects in vivo is of great interest, as is the question of whether they interact to enhance toxicity. Here, we evaluate the toxic and neurotoxic effects of these cyanotoxins alone or in combination by measuring zebrafish larval viability and behavior after exposure. 2,4-DAB was the most potent cyanotoxin as it decreased larval viability by approximately 50% at 6 days post fertilization, while BMAA and AEG decreased viability by just 16% and 8%, respectively. Although we only observed minor neurotoxic effects on spontaneous locomotion, BMAA and AEG enhanced acoustic startle sensitivity, and they interacted in an additive manner to exert their effects. 2,4-DAB, however, only modulated the startle kinematics, an indication of motor dysfunction. To investigate the mechanisms of 2,4-DABs effects, we analyzed the protein profile of larval zebrafish exposed to 500M 2,4-DAB at two time points and identified molecular signatures consistent with neurodegeneration, including disruption of metabolic pathways and downregulation of the ALS-associated genes SOD1 and UBQLN4. Together, our data demonstrate that BMAA and its isomers AEG and 2,4-DAB cause neurotoxic effects in vivo, with 2,4-DAB as the most potent of the three in the zebrafish model.

Calcium functions as an important second messenger in a wide variety of intracellular processes. In photoreceptor cells, calcium is involved in activation, deactivation, and adaptation in response to light stimuli. Calcium-binding protein 53E (Cbp53E, also known as calbindin-32 or cbn), a protein with 6 EF-Hand domains thought to act as a calcium buffer, was previously identified to have elevated expression levels in the eye of drosophila. While a recent study showed that transgenic flies lacking Cbp53E have aberrant axonal arborization at the neuromuscular junction, nothing is known about the role of Cbp53E in the visual system. We performed electroretinogram (ERG) recordings on Cbp53E mutant flies to test whether eye function was affected. Here, we report that Cbp53E null mutants exhibit a prolonged repolarization (or slow termination) phenotype which can be rescued by expressing Cbp53E in photoreceptor cells. The human homologs Calbindin 2, Calbindin 1, and S100G also rescue the Drosophila ERG phenotype. This supports a role for Cbp53E in regulating intracellular calcium levels of photoreceptor cells and contributing to normal sensory neuron response dynamics in vivo in Drosophila and suggests a similar function in human photoreceptor cells as well.

PKA signaling is essential for numerous processes but the subcellular localization of specific PKA isoforms has yet to be explored comprehensively in tissues. Expression of the C{beta} protein, in particular, has not been mapped previously at the tissue level. In this study we used retina as a window into PKA signaling in the brain and characterized localization of PKA C, C{beta}, RII, and RII{beta} subunits. Each subunit presented a distinct localization pattern. C and C{beta} were localized in all tissue layers, while RII and RII{beta} were enriched in the photoreceptor cells in contrast to the cell body and retinal portion of retinal ganglion cells. Only C was observed in photoreceptor outer segments and the cilia transition zone, while C{beta} was localized primarily to mitochondria and was especially prominent in the ellipsoid of the cone cells. In contrast to C, C{beta} also never colocalized with RII or RII{beta}. Using BaseScope technology to track expression of the C{beta} isoforms we find that C{beta}4 and C{beta}4ab are prominently expressed and, therefore, likely code for mitochondrial-C{beta} proteins. Our data indicates that PKA subunits are functionally nonredundant in the retina and suggesting that C{beta} might be important for mitochondrial-associated neurodegenerative diseases previously linked to PKA dysfunction.

Vesicular transport plays critical roles in photopigment delivery at photoreceptor outer segments and glutamate exocytosis at photoreceptor synapses. Previous studies into the role of photoreceptor SNAP proteins are limited in their characterizations into only gene/protein expression and do not delve further into their functional role. Here, we examine the expression and localization of SNAP-23 and SNAP-25 mRNA and protein. Using SNAP-23 and SNAP-25 conditional knockout mice, we further evaluated the morphological and functional consequences that the absence of these proteins has on vision. Although we found that the ubiquitously expressed SNAP-23 showed weak mRNA expression in photoreceptors, removal of SNAP-23 did not result in any observable phenotype. We found that neuronal SNAP-25 is developmentally regulated and SNAP-25 mRNA undergoes mRNA trafficking to the photoreceptor inner segments coinciding with the development of photoreceptor outer segments. Removal of SNAP-25 in photoreceptor cells led to changes in both outer segment protein trafficking and synaptic integrity, resulting in a complete loss of vision in SNAP-25 cKO mice. Our results conclude that SNAP-25, but not SNAP-23, is the essential isoform for photoreceptor survival and function.

Hypoxia, a condition of inadequate oxygen supply, is a common phenomenon affecting neurons and brain tissue, leading to significant implications for neuronal health and function. The prevalence of hypoxia in the brain is associated with various neurological conditions, making it a critical area of study. Neuritogenesis, the process of neurite outgrowth, is an essential aspect of neuronal development and connectivity and is particularly sensitive to hypoxic stress. Investigating how hypoxia affects neurite outgrowth is vital for understanding neuronal response and adaptation under low oxygen conditions. This study explores how hypoxic stress affects neurite regulation mediated by Contactin Associated Protein-1 (Caspr1) in primary mouse embryonic cortical neurons. Hypoxia, induced by culturing neurons in a 2% oxygen environment, significantly reduced neurite length and induced notable changes in growth cone morphology. Concurrently, we observed an upregulation in the expression of Caspr1 and its transcriptional regulator C/EBP, suggesting a compensatory role for Caspr1 in neurite extension under low oxygen conditions. Shorter hypoxia exposure periods revealed a dynamic biphasic response in Caspr1 levels, with an initial decrease followed by a substantial increase, correlating with corresponding changes in neurite length. This pattern emphasizes the critical involvement of Caspr1 in adapting neurite growth to fluctuating hypoxia duration. Furthermore, comparative analyses using wild-type and Caspr1 knockout Neuro2a cells demonstrated that the absence of Caspr1 mitigates hypoxia-induced neurite shortening, indicating a potential protective role against hypoxic stress. Additionally, hypoxia profoundly impacted mitochondrial morphology and function. Under hypoxic conditions, mitochondria transitioned to a more spherical shape. Mitochondrial respiration analysis revealed significant reductions in oxygen consumption rates (OCR), highlighting compromised mitochondrial function during hypoxia. These findings underscore the multifaceted role of Caspr1 in neurite regulation and mitochondrial adaptation to hypoxic stress. The study provides insights into the molecular mechanisms underpinning hypoxia-induced changes in neuronal morphology and function. Understanding these processes opens avenues for therapeutic strategies targeting Caspr1 in treating neurological disorders characterized by hypoxic stress. Future research will benefit from extending these investigations to more complex models, such as brain organoids, to further elucidate the metabolic and structural changes under hypoxia and their implications for neurodegenerative diseases.

Understanding of molecules and their role in neurite initiation and/or extension is not only helpful to prevent different neurodegenerative diseases but also can be important by which neuronal damages can be repaired. In this work we explored the role of TRPV2, a non-selective cation channel in the context of neurite functions. Using western blot, immunofluorescence, and live cell Ca2+-imaging; we confirm that functional TRPV2 is endogenously present in the F11 cell, a model system mimicking peripheral neuron. In F11 cells TRPV2 localizes in specific sub-cellular regions enriched with filamentous actin, such as in growth cone, filopodia, lamellipodia and in neurites. TRPV2 regulates actin cytoskeleton and also interacts with soluble actin. Ectopic expression of TRPV2-GFP but not GFP-only in F11 cell induces more primary and secondary neurites, confirming its role in neurite initiation, extension and branching events. TRPV2-mediated neuritogenesis is dependent on wild-type TRPV2 as cells expressing TRPV2 mutants reveal no neuritogenesis. However, TRPV2-mediated neuritogenesis is unperturbed by the chelation of intracellular Ca2+ by BAPTA-AM, and thus involves Ca2+-independent signaling events also. We demonstrate that pharmacological modulation of TRPV2 alters cellular cAMP levels. These findings are relevant to understand the sprouting of new neurites, neuroregeneration and neuronal plasticity at the cellular, subcellular and molecular level. Such understanding may have border implication in neurodegeneration and peripheral neuropathy.

Changes in mitochondrial distribution are a feature of numerous age-related neurodegenerative diseases. In Drosophila, reducing the activity of Cdk5 causes a neurodegenerative phenotype and is known to affect several mitochondrial properties. Therefore, we investigated whether alterations of mitochondrial distribution are involved in Cdk5-associated neurodegeneration. We find that reducing Cdk5 activity does not alter the balance of mitochondrial localization to the somatodendritic vs. axonal neuronal compartments of the mushroom body, the learning and memory center of the Drosophila brain. We do, however, observe changes in mitochondrial distribution at the axon initial segment (AIS), a neuronal compartment located in the proximal axon involved in neuronal polarization and action potential initiation. Specifically, we observe that mitochondria are partially excluded from the AIS in wild-type neurons, but that this exclusion is lost upon reduction of Cdk5 activity, concomitant with the shrinkage of the AIS domain that is known to occur in this condition. This mitochondrial redistribution into the AIS is not likely due to the shortening of the AIS domain itself but rather due to altered Cdk5 activity. Furthermore, mitochondrial redistribution into the AIS is unlikely to be an early driver of neurodegeneration in the context of reduced Cdk5 activity. Summary statementIn Drosophila, mitochondria are excluded from the axon iniCal segment, a neuronal compartment that regulates neuron polarity and axon potenCals, and this paYern is disrupted in a model of neurodegeneraCon.

The proper functioning of the nervous system is dependent on the establishment and maintenance of intricate networks of neurons that form functional neural circuits. Once neural circuits are assembled during development, a distinct set of molecular programs is likely required to maintain their connectivity throughout the lifetime of the organism. Here, we demonstrate that Fasciclin 3 (Fas3), an axon guidance cell adhesion protein, is necessary for the maintenance of the olfactory circuit in adult Drosophila. We utilized the TARGET system to spatiotemporally knockdown Fas3 in selected populations of adult neurons. Our findings show that Fas3 knockdown results in the death of olfactory circuit neurons and reduced survival of adults. We also demonstrated that Fas3 knockdown activates caspase-3 mediated cell death in olfactory local interneurons, which can be rescued by overexpressing p35, an anti-apoptotic protein. This work adds to the growing set of evidence indicating a critical role for axon guidance proteins in the maintenance of neuronal circuits in adults. SUMMARY STATEMENTLittle is known about the maintenance of adult neural circuits. We show that the continuous expression of Fasciclin 3, a cell adhesion protein involved in axon guidance, is required for neuronal survival in the adult olfactory circuit.

Variants of the CACNA1C voltage-gated calcium channel gene have been associated with autism and other neurodevelopmental disorders including bipolar disorder, schizophrenia, and ADHD. The Timothy syndrome mutation is a rare de novo gain-of-function variant in CACNA1C that causes autism with high penetrance, providing a powerful avenue into investigating the role of CACNA1C variants in neurodevelopmental disorders. In our previous work, we demonstrated that an egl-19(gof) mutation, that is equivalent to the Timothy syndrome mutation in the human homolog CACNA1C, can disrupt termination of the PLM axon in C. elegans. Here, we find that the egl-19(gof) mutation disrupts the polarity of process outgrowth in the ALM neuron of C. elegans. We also find that the egl-19(gof) mutation can disrupt termination of the ALM axon. These results suggest that the Timothy syndrome mutation can disrupt multiple steps of axon development. Further work exploring the molecular mechanisms that underlie these perturbations in neuronal polarity and axon termination will give us better understanding to how variants in CACNA1C contribute to the axonal defects that underlie autism.

PFTK1/Eip63E is a member of the Cyclin-dependent kinases (CDKs) family and plays an important role in normal cell cycle progression. Eip63E expresses primarily in postnatal and adult nervous system in Drosophila melanogaster but its role in CNS development remains unknown. We sought to understand the function of Eip63E in the CNS by studying the fly ventral nerve cord during development. Our results demonstrate that Eip63E regulates axogenesis in neurons and its deficiency leads to neuronal defects. Functional interaction studies performed using the same system identify an interaction between Eip63E and the small GTPase Rho1. Furthermore, deficiency of Eip63E homolog in mice, PFTK1, in a newly generated PFTK1 knockout mice results in increased axonal outgrowth confirming that the developmental defects observed in the fly model are due to defects in axogenesis. Importantly, RhoA phosphorylation and activity is affected by PFTK1 in primary neuronal cultures. We here report that GDP bound inactive RhoA is a substrate of PFTK1 and PFTK1 phosphorylation is required for RhoA activity. In conclusion, our work establishes an unreported neuronal role of PFTK1 in axon development mediated by phosphorylation and activation of GDP-bound RhoA. The results presented add to our understanding of the role of Cdks in the maintenance of RhoA mediated axon growth and its impact on CNS development and axonal regeneration.

The cerebellum regulates motor coordination and motor learning through highly organized circuits composed mainly of granule cells (GCs) and Purkinje cells (PCs). To investigate their distinct roles, we generated two lines of inducible transgenic mice in which either GCs or PCs could be selectively ablated in adulthood by administration of the progesterone receptor antagonist RU-486. This system combined a Cre recombinase-progesterone receptor fusion, in which Cre activity is induced in an RU-486-dependent manner, with a Cre-dependent diphtheria toxin A expression to achieve cell-type-specific ablation. High-dose RU-486 induced nearly complete loss of either GCs or PCs and resulted in severe ataxia. When partial ablation was induced by low-dose RU-486, different phenotypes emerged. Mice retaining about 20% of PCs were still able to improve motor coordination in the rotarod test and maintained performance in the balance beam test comparable to that of controls. In contrast, mice with about 30% of GCs remaining showed marked deficits, failing to improve across rotarod trials and exhibiting reduced latency to fall in the balance beam test. These results suggest that while both GCs and PCs are indispensable for motor coordination, a sufficient number of GCs is required for both motor coordination and motor learning. This inducible ablation model highlights the differential contributions of cerebellar neurons and provides a valuable tool to dissect circuit-specific functions in the adult brain.

The mammalian Ca2+/calmodulin-dependent protein kinase II (CAMK2) family consists of 4 different CAMK2 genes, encoding CAMK2A, CAMK2B, CAMK2D and CAMK2G, which have high structural homology. CAMK2A and CAMK2B are abundantly expressed in the brain; they play a unique role in proper neuronal functioning, since both CAMK2A and CAMK2B knockout mice show several behavioural and cellular phenotypes. However, our recent finding that deletion of both CAMK2A and CAMK2B is lethal indicates that they show redundancy and that the full spectrum of CAMK2 function in neurons remains to be uncovered. For example, it still remains unclear which overlapping functions are present at a single cell level in neuronal transmission and excitability. In order to get more insight into the full spectrum of CAMK2 functions in neurons, we performed whole-cell patch clamp experiments in inducible Camk2a/Camk2b double knockout mice, as well as the CAMK2A and CAMK2B knockout mice. We found that whereas deletion of only CAMK2A or CAMK2B did not change excitability, simultaneous deletion of CAMK2A and CAMK2B resulted in a decrease in excitability 10 days after deletion in CA1 pyramidal neurons, which reversed to increased excitability 21 days after deletion. Additionally, loss of both CAMK2A and CAMK2B resulted in a decreased frequency of both miniature excitatory and inhibitory postsynaptic currents (mEPSC and mIPSC) 21 days after deletion, but not 10 days after deletion, an effect not seen in the single mutants. Our results indicate that CAMK2 is critically important to maintain normal excitability of hippocampal CA1 pyramidal cells, as well as normal inhibitory and excitatory synaptic transmission. Together, these results lead to new insights in how CAMK2 regulates normal neuronal function and highlight the importance of having both CAMK2A and CAMK2B expressed in high levels in the brain.

Traumatic brain injury (TBI) can induce traumatic axonal injury in the optic nerve, which is referred to as traumatic optic neuropathy (TON). TON occurs in up to 5% of TBI cases and leads to irreversible visual deficits. TON-induced phosphorylation of eIF2, a downstream ER stress activator in the PERK pathway presents a potential point for therapeutic intervention. For eIF2 phosphorylation can lead to apoptosis or adaptation to stress. We hypothesized that dephosphorylation, rather than phosphorylation, of eIF2 would lead to reduced apoptosis and improved visual performance and retinal cell survival. Adult male mice were injected with Salubrinal (increases p-eIF2) or ISRIB (decreases p-eIF2) 60 minutes post-injury. Contrary to literature, both drugs hindered control animal visual function with minimal improvements in injured mice. Additionally, differences in eIF2 phosphorylation, antioxidant responses, and protein folding chaperones were different when examining protein expression between the retina and its axons in the optic nerve. These results reveal important compartmentalized ER stress responses to axon injury and suggest that interventions in the PERK pathway may alter necessary homeostatic regulation of the UPR in the retina.

Epileptogenesis, the process through which the brain becomes seizure-prone, is not well understood. Previous work identified a novel gene in Drosophila, julius seizure (jus), that when mutated or developmentally knocked down, leads to epilepsy in adult Drosophila, providing a useful model for dissecting epileptogenesis. Here we report that when a GFP-tagged version of Jus was used as bait in a co-immunoprecipitation (co-IP) assay, a complex of 23 associated proteins was identified that included ATPalpha and Nervana 3, two subunits of the Na+/K+ ATPase. RNAi-mediated knockdown of ATPalpha, Nervana 3, or any of 8 additional complex proteins enhanced seizure susceptibility. The critical period of Jus expression in epileptogenesis was further defined, occurring between pupal stages P4-7; remarkably, the jus seizure phenotype could be rescued by increasing neural activity of jus-expressing neurons during these mid-pupal stages, suggesting that altered neural activity in these neurons may contribute to the seizure phenotype. Our data support a model that, in wild type flies, a protein complex containing Na+/K+ ATPase and Jus prevents epileptogenesis, possibly by regulating neural activity.

Several lines of evidence demonstrate that increased neuronal excitability can enhance proteomic stress. For example, epilepsy can enhance the proteomic stress caused by the expression of certain aggregation-prone proteins implicated in neurodegeneration. However, unanswered questions remain concerning the mechanisms by which increased neuronal excitability accomplishes this enhancement. Here we test whether increasing neuronal excitability at a particular identified glutamatergic synapse, the Drosophila larval neuromuscular junction, can enhance the proteomic stress caused by mutations in the ER fusion gene atlastin (atl). It was previously shown that larval muscle from the atl2 null mutant is defective in autophagy and accumulates protein aggregates containing ubiquitin (poly-UB aggregates). To determine if increased neuronal excitability might enhance the increased proteomic stress caused by atl2, we activated the TrpA1-encoded excitability channel within neurons. We found that TrpA1 activation had no effect on poly-UB aggregate accumulation in wildtype muscle, but significantly increased poly-UB aggregate number in atl2muscle. Previous work has shown that atl loss from either neuron or muscle increases muscle poly-UB aggregate number. We found that neuronal TrpA1 activation enhanced poly-UB aggregate number when atl was removed from muscle, but not from neuron. Neuronal TrpA1 activation enhanced other phenotypes conferred by muscle atl loss, such as decreased pupal size and decreased viability. Taken together, these results indicate that the proteomic stress caused by muscle atl loss is enhanced by increasing neuronal excitability.

Visual deprivation by dark rearing in kittens and monkeys delays visual pathway development and prolongs the critical period. In contrast, receptive fields (RFs) in superior colliculus (SC) of Syrian hamsters (Mesocricetus auratus) refine normally with spontaneous activity alone, requiring only brief juvenile visual experience to maintain refined RFs in adulthood (Carrasco et al., 2005). Extending dark rearing past puberty leads to lower GAD and GABA levels due to reduced BDNF-TrkB signaling, resulting in RF re-enlargement (Carrasco et al., 2011; Mudd et al., 2019). Previous studies in kittens and monkeys have reported that dark rearing is associated with changes in both GABA ligand and GABAA receptor levels. Given the reduced GABA levels in SC of dark reared adult hamsters, we asked if dark rearing also causes changes in GABAA receptor levels. We examined expression of GABAA receptor subunits, their anchoring protein gephyrin, and the cation-chloride co-transporters KCC2 and NKCC1 in dark reared hamsters. Surprisingly, we found that dark rearing from birth until puberty had no effect on the levels of any of these postsynaptic elements, revealing a new form of maladaptive, presynaptic only inhibitory plasticity in which, rather than extending the critical period as seen in kittens and monkeys, hamster receptive fields refine normally and then lose refinement in adulthood. These results suggest that attempts to increase plasticity in adulthood for rehabilitation or recovery from injury should consider the possibility of unintended negative consequences. In addition, our results demonstrate the interesting finding that changes in neurotransmitter levels are not necessarily coordinated with changes in postsynaptic components.