Neurofibromatosis type I (NF1), caused by the mutation in the NF1 gene, is characterized by multiple pathological symptoms. Importantly, ~50% of NF1 patients also suffer learning difficulty. Although downstream pathways are well studied, regulation of the NF1-encoded neurofibromin protein is less clear. Here, we focused on the pathophysiology of Drosophila NF1 mutants in synaptic growth at neuromuscular junctions. Our analysis suggests that the Drosophila neurofibromin protein NF1 is required to constrain synaptic growth and transmission. NF1 functions downstream of the Drosophila focal adhesion kinase (FAK) Fak56 and physically interacts with Fak56. The N-terminal region of NF1 mediates the interaction with Fak56 and is required for the signaling activity and presynaptic localization of NF1. In presynapses, NF1 acts via the cAMP pathway, but independent of its GAP activity, to restrain synaptic growth. Thus, presynaptic FAK signaling may be disrupted, causing abnormal synaptic growth and transmission in the NF1 genetic disorder.

Gap junctions are ubiquitous throughout the nervous system, mediating critical signal transmission and integration, as well as emergent network properties. In mammalian retina, gap junctions within the Aii amacrine cell-ON cone bipolar cell (CBC) network are essential for night vision, modulation of day vision, and contribute to visual impairment in retinal degenerations, yet neither the extended network topology nor its conservation is well established. Here, we map the network contribution of gap junctions using a high-resolution connectomics dataset of an adult female rabbit retina. Gap junctions are prominent synaptic components of ON CBC classes, constituting 5%-25% of all axonal synaptic contacts. Many of these mediate canonical transfer of rod signals from Aii cells to ON CBCs for night vision, and we find that the uneven distribution of Aii signals to ON CBCs is conserved in rabbit, including one class entirely lacking direct Aii coupling. However, the majority of gap junctions formed by ON CBCs unexpectedly occur between ON CBCs, rather than with Aii cells. Such coupling is extensive, creating an interconnected network with numerous lateral paths both within, and particularly across, these parallel processing streams. Coupling patterns are precise with ON CBCs accepting and rejecting unique combinations of partnerships according to robust rulesets. Coupling specificity extends to both size and spatial topologies, thereby rivaling the synaptic specificity of chemical synapses. These ON CBC coupling motifs dramatically extend the coupled Aii-ON CBC network, with implications for signal flow in both scotopic and photopic retinal networks during visual processing and disease.SIGNIFICANCE STATEMENT Electrical synapses mediated by gap junctions are fundamental components of neural networks. In retina, coupling within the Aii-ON CBC network shapes visual processing in both the scotopic and photopic networks. In retinal degenerations, these same gap junctions mediate oscillatory activity that contributes to visual impairment. Here, we use high-resolution connectomics strategies to identify gap junctions and cellular partnerships. We describe novel, pervasive motifs both within and across classes of ON CBCs that dramatically extend the Aii-ON CBC network. These motifs are highly specific with implications for both signal processing within the retina and therapeutic interventions for blinding conditions. These findings highlight the underappreciated contribution of coupling motifs in retinal circuitry and the necessity of their detection in connectomics studies.

Adequate blood flow is essential to brain function, and its disruption is an early indicator in diseases, such as stroke and diabetes. However, the mechanisms contributing to this impairment remain unclear. To address this gap, in the diabetic and nondiabetic male mouse retina, we combined an unbiased longitudinal assessment of vasomotor activity along a genetically defined vascular network with pharmacological and immunohistochemical analyses of pericytes, the capillary vasomotor elements. In nondiabetic retina, focal stimulation of a pericyte produced a robust vasomotor response, which propagated along the blood vessel with increasing stimulus. In contrast, the magnitude, dynamic range, a measure of fine vascular diameter control, and propagation of vasomotor response were diminished in diabetic retinas from streptozotocin-treated mice. These functional changes were linked to several mechanisms. We found that density of pericytes and their sensitivity to stimulation were reduced in diabetes. The impaired response propagation from the stimulation site was associated with lower expression of connexin43, a major known gap junction unit in vascular cells. Indeed, selective block of gap junctions significantly reduced propagation but not initiation of vasomotor response in the nondiabetic retina. Our data establish the mechanisms for fine local regulation of capillary diameter by pericytes and a role for gap junctions in vascular network interactions. We show how disruption of this balance contributes to impaired vasomotor control in diabetes.SIGNIFICANCE STATEMENT Identification of mechanisms governing capillary blood flow in the CNS and how they are altered in disease provides novel insight into early states of neurological dysfunction. Here, we present physiological and anatomical evidence that both intact pericyte function as well as gap junction-mediated signaling across the vascular network are essential for proper capillary diameter control and vasomotor function. Changes to capillary blood flow precede other anatomical and functional hallmarks of diabetes establishing a significant window for prevention and treatment.

Glia are essential to protecting and enabling nervous system function and a key glial function is the formation of the glial sheath around peripheral axons. Each peripheral nerve in the Drosophila larva is ensheathed by three glial layers, which structurally support and insulate the peripheral axons. How peripheral glia communicate with each other and between layers is not well established and we investigated the role of Innexins in mediating glial function in the Drosophila periphery. Of the eight Drosophila Innexins, we found two (Inx1 and Inx2) are important for peripheral glia development. In particular loss of Inx1 and Inx2 resulted in defects in the wrapping glia leading to disruption of the glia wrap. Of interest loss of Inx2 in the subperineurial glia also resulted in defects in the neighboring wrapping glia. Inx plaques were observed between the subperineurial glia and the wrapping glia suggesting that gap junctions link these two glial cell types. We found Inx2 is key to Ca2+ pulses in the peripheral subperineurial glia but not in the wrapping glia, and we found no evidence of gap junction communication between subperineurial and wrapping glia. Rather we have clear evidence that Inx2 plays an adhesive and channel-independent role between the subperineurial and wrapping glia to ensure the integrity of the glial wrap.SIGNIFICANCE STATEMENT Gap junctions are critical for glia communication and formation of myelin in myelinating glia. However, the role of gap junctions in non-myelinating glia is not well studied, yet non-myelinating glia are critical for peripheral nerve function. We found the Innexin gap junction proteins are present between different classes of peripheral glia in Drosophila. Here Innexins form junctions to facilitate adhesion between the different glia but do so in a channel-independent manner. Loss of adhesion leads to disruption of the glial wrap around axons and leads to fragmentation of the wrapping glia membranes. Our work points to an important role for gap junction proteins in mediating insulation by non-myelinating glia.

Temporal contrast detected by rod photoreceptors is channeled into multiple retinal rod pathways that ultimately connect to cone photoreceptor pathways via Cx36 gap junctions or via chemical synapses. However, we do not yet understand how the different rod pathways contribute to the perception of temporal contrast (changes in luminance with time) at mesopic light levels, where both rods and cones actively respond to light. Here, we use a forced-choice, operant behavior assay to investigate rod-driven, temporal contrast sensitivity (TCS) in mice of either sex. Transgenic mice with desensitized cones (GNAT2 cpfl3 line) were used to identify rod contributions to TCS in mesopic lights. We found that at low mesopic lights (400 photons/s/μm2 at the retina), control and GNAT2 cpfl3 mice had similar TCS. Surprisingly, at upper mesopic lights (8000 photons/s/μm2), GNAT2 cpfl3 mice exhibited a relative reduction in TCS to low (<12 Hz) while maintaining normal TCS to high (12-36 Hz) temporal frequencies. The rod-driven responses to high temporal frequencies developed gradually over time (>30 min). Furthermore, the TCS of GNAT2 cpfl3 and GNAT2 cpfl3 ::Cx36-/- mice matched closely, indicating that transmission of high-frequency signals (1) does not require the rod-cone Cx36 gap junctions as has been proposed in the past; and (2) a Cx36-independent rod pathway(s) (e.g., direct rod to OFF cone bipolar cell synapses and/or glycinergic synapses from AII amacrine cells to OFF ganglion cells) is sufficient for fast, mesopic rod-driven vision. These findings extend our understanding of the link between visual circuits and perception in mouse.SIGNIFICANCE STATEMENT The contributions of specific retinal pathways to visual perception are not well understood. We found that the temporal processing properties of rod-driven vision in mice change significantly with light level. In dim lights, rods relay relatively slow temporal variations. However, in daylight conditions, rod pathways exhibit high sensitivity to fast but not to slow temporal variations, whereas cone-driven responses supplement the loss in rod-driven sensitivity to slow temporal variations. Our findings highlight the dynamic interplay of rod- and cone-driven vision as light levels rise from night to daytime levels. Furthermore, the fast, rod-driven signals do not require the rod-to-cone Cx36 gap junctions as proposed in the past, but rather, can be relayed by alternative Cx36-independent rod pathways.

Potassium channels comprise the most diverse family of ion channels. In nerve cells, their critical roles in synaptic integration and output generation have been demonstrated. Here, we provide evidence for a distribution that predicts a novel role of K+ channels in the CNS. Our experiments revealed a highly selective clustering of the Kv4.3 A-type K+ channel subunits at specialized junctions between climbing fibers and cerebellar GABAergic interneurons. High-resolution ultrastructural and immunohistochemical experiments demonstrated that these junctions are distinct from known chemical and electrical (gap junctions) synapses and also from puncta adherentia. Each cerebellar interneuron contains many such K+ channel-rich specializations, which seem to be distributed throughout the somatodendritic surface. We also show that such K+ channel-rich specializations are not only present in the cerebellum but are widespread in the rat CNS. For example, mitral cells of the main olfactory bulb establish Kv4.2 subunit-positive specializations with each other. At these specializations, both apposing membranes have a high density of K+ channels, indicating bidirectional signaling. Similar specializations with pronounced coclustering of the Kv4.2 and 4.3 subunits were observed between nerve cells in the medial nucleus of the habenula. Based on our results and on the known properties of A-type K+ channels, we propose that strategically clustered K+ channels at unique membrane specializations could mediate a novel type of communication between nerve cells.

By synchronizing neuronal activity, electrical transmission influences the coordination, pattern, and/or frequency of firing. In the hemaphroditic marine-snail, Aplysia calfornica, the neuroendocrine bag cell neurons use electrical synapses to synchronize a 30 min afterdischarge of action potentials for the release of reproductive hormone. During the afterdischarge, protein kinase C (PKC) is activated, although its impact on bag cell neuron electrical transmission is unknown. This was investigated here by monitoring electrical synapses between paired cultured bag cell neurons using dual whole-cell recording. Voltage clamp revealed a largely voltage-independent junctional current, which was enhanced by treating with a PKC activator, PMA, before recording. We also examined the transfer of presynaptic action potential-like waveforms (generated in voltage clamp) to the postsynaptic cell (measured in current clamp). For control pairs, the presynaptic spike-like waveforms mainly evoked electrotonic potentials; however, when PKC was triggered, these stimuli consistently produced postsynaptic action potentials. To assess whether this involved changes to postsynaptic responsiveness, single bag cell neurons were injected with junctional-like current mimicking that evoked by a presynaptic action potential. Unlike control neurons, which were less likely to spike, cells in PMA always fired action potentials to the junctional-like current. Furthermore, PKC activation increased a postsynaptic voltage-gated Ca2+ current, which was recruited even by modest depolarization associated with an electrotonic potential. Whereas PKC inhibits gap junctions in most systems, bag cell neurons are rather unique, as the kinase potentiates the electrical synapse; in turn, this synergizes with augmented postsynaptic Ca2+ current to promote synchronous firing.SIGNIFICANCE STATEMENT Electrical coupling is a fundamental form of communication. For the bag cell neurons of Aplysia, electrical synapses coordinate a prolonged burst of action potentials known as the afterdischarge. We looked at how protein kinase C, which is upregulated with the afterdischarge, influences information transfer across the synapse. The kinase activation increased junctional current, a remarkable finding given that this enzyme is largely considered inhibitory for gap junctions. There was also an augmentation in the ability of a presynaptic neuron to provoke postsynaptic action potentials. This increased excitability was, in part, due to enhanced postsynaptic voltage-dependent Ca2+ current. Thus, protein kinase C improves the fidelity of electrotonic transmission and promotes synchronous firing by modulating both junctional and membrane conductances.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) exhibit melanopsin-dependent light responses that persist in the absence of rod and cone photoreceptor-mediated input. In addition to signaling anterogradely to the brain, ipRGCs signal retrogradely to intraretinal circuitry via gap junction-mediated electrical synapses with amacrine cells (ACs). However, the targets and functions of these intraretinal signals remain largely unknown. Here, in mice of both sexes, we identify circuitry that enables M5 ipRGCs to locally inhibit retinal neurons via electrical synapses with a nonspiking GABAergic AC. During pharmacological blockade of rod- and cone-mediated input, whole-cell recordings of corticotropin-releasing hormone-expressing (CRH+) ACs reveal persistent visual responses that require both melanopsin expression and gap junctions. In the developing retina, ipRGC-mediated input to CRH+ ACs is weak or absent before eye opening, indicating a primary role for this input in the mature retina (i.e., in parallel with rod- and cone-mediated input). Among several ipRGC types, only M5 ipRGCs exhibit consistent anatomical and physiological coupling to CRH+ ACs. Optogenetic stimulation of local CRH+ ACs directly drives IPSCs in M4 and M5, but not M1-M3, ipRGCs. CRH+ ACs also inhibit M2 ipRGC-coupled spiking ACs, demonstrating direct interaction between discrete networks of ipRGC-coupled interneurons. Together, these results demonstrate a functional role for electrical synapses in translating ipRGC activity into feedforward and feedback inhibition of local retinal circuits.SIGNIFICANCE STATEMENT Melanopsin directly generates light responses in intrinsically photosensitive retinal ganglion cells (ipRGCs). Through gap junction-mediated electrical synapses with retinal interneurons, these uniquely photoreceptive RGCs may also influence the activity and output of neuronal circuits within the retina. Here, we identified and studied an electrical synaptic circuit that, in principle, could couple ipRGC activity to the chemical output of an identified retinal interneuron. Specifically, we found that M5 ipRGCs form electrical synapses with corticotropin-releasing hormone-expressing amacrine cells, which locally release GABA to inhibit specific RGC types. Thus, ipRGCs are poised to influence the output of diverse retinal circuits via electrical synapses with interneurons.

Neuromodulation of self-amplifying circuits directs context-dependent behavioral executions. Although recurrent networks are found throughout the Caenorhabditis elegans connectome, few reports describe the mechanisms that regulate reciprocal neural activity during complex behavior. We used C. elegans male copulation to dissect how a goal-oriented motor behavior is regulated by recurrently wired sensory-motor neurons. As the male tail presses against the hermaphrodite's vulva, cholinergic and glutamatergic reciprocal innervations of post cloaca sensilla (PCS) neurons (PCA, PCB, and PCC), hook neurons (HOA, HOB), and their postsynaptic sex muscles execute rhythmic copulatory spicule thrusts. These repetitive spicule movements continue until the male shifts off the vulva or genital penetration is accomplished. However, the signaling mechanism that temporally and spatially restricts repetitive intromission attempts to vulva cues was unclear. Here, we report that confinement of spicule insertion attempts to the vulva is facilitated by D2-like receptor modulation of gap-junctions between PCB and the hook sensillum. We isolated a missense mutation in the UNC-7(L) gap-junction isoform, which perturbs DOP-2 signaling in the PCB neuron and its electrical partner, HOA. The glutamate-gated chloride channel AVR-14 is expressed in HOA. Our analysis of the unc-7 mutant allele indicates that when DOP-2 promotes UNC-7 electrical communication, AVR-14-mediated inhibitory signals pass from HOA to PCB. As a consequence, PCB is less receptive to be stimulated by its recurrent synaptic partner, PCA. Behavioral observations suggest that dopamine neuromodulation of UNC-7 ensures attenuation of recursive intromission attempts when the male disengages or is dislodged from the hermaphrodite genitalia.

Clustering of Na(+) channels at the nodes of Ranvier is coordinated by myelinating glia. In the peripheral nervous system, axoglial contact at the nodes is mediated by the binding of gliomedin and glial NrCAM to axonal neurofascin 186 (NF186). This interaction is crucial for the initial clustering of Na(+) channels at heminodes. As a result, it is not clear whether continued axon-glial contact at nodes of Ranvier is required to maintain these channels at the nodal axolemma. Here, we report that, in contrast to mice that lack either gliomedin or NrCAM, absence of both molecules (and hence the glial clustering signal) resulted in a gradual loss of Na(+) channels and other axonal components from the nodes, the formation of binary nodes, and dysregulation of nodal gap length. Therefore, these mice exhibit neurological abnormalities and slower nerve conduction. Disintegration of the nodes occurred in an orderly manner, starting with the disappearance of neurofascin 186, followed by the loss of Na(+) channels and ankyrin G, and then βIV spectrin, a sequence that reflects the assembly of nodes during development. Finally, the absence of gliomedin and NrCAM led to the invasion of the outermost layer of the Schwann cell membrane beyond the nodal area and the formation of paranodal-like junctions at the nodal gap. Our results reveal that axon-glial contact mediated by gliomedin, NrCAM, and NF186 not only plays a role in Na(+) channel clustering during development, but also contributes to the long-term maintenance of Na(+) channels at nodes of Ranvier.

Converging lines of evidence suggest that the hypothalamic suprachiasmatic nucleus (SCN) is the site of the endogenous biological clock controlling mammalian circadian rhythms. To study the calcium responses of the cellular components that make up the clock, computer-controlled digital video and confocal scanning laser microscopy were used with the Ca2+ indicator dye fluo-3 to examine dispersed SCN cells and SCN explants with repeated sampling over time. Ca2+ plays an important second messenger role in a wide variety of cellular mechanisms from gene regulation to electrical activity and neurotransmitter release, and may play a role in clock function and entrainment. SCN neurons and astrocytes showed an intracellular Ca2+ increase in response to glutamate and 5-HT, two major neurotransmitters in afferents to the SCN. Astrocytes showed a marked heterogeneity in their response to the serial perfusion of different transmitters; some responded to both 5-HT and glutamate, some to neither, and others to only one or the other. Under constant conditions, most neurons showed irregular temporal patterns of Ca2+ transients. Expression of regular neuronal oscillations could be blocked by the inhibitory transmitter GABA. Astrocytes, on the other hand, showed very regular rhythms of cytoplasmic Ca2+ concentrations with periods ranging from 7 to 20 sec. This periodic oscillation could be initiated by in vitro application of glutamate, the putative neurotransmitter conveying visual input to the SCN critical for clock entrainment. Long-distance communication between glial cells, seen as waves of fluorescence moving from cell to cell, probably through gap junctions, was induced by glutamate, 5-HT, and ATP. These waves increased the period length of cellular Ca2+ rises to 45-70 sec. Spontaneously oscillating cells were common in culture medium, serum, or rat cerebrospinal fluid, but rare in HEPES buffer. One source for cytoplasmic Ca2+ increases was an influx of extracellular Ca2+, as seen under depolarizing conditions in about 75% of the astroglia studied. All neurotransmitter-induced Ca2+ fluxes were not dependent on voltage changes, as Ca2+ oscillations could be initiated under both normal and depolarizing conditions. Since neurotransmitters could induce a Ca2+ rise in the absence of extracellular Ca2+, the mechanisms of ultradian oscillations appear to depend on cycles of intracellular Ca2+ fluxes from Ca(2+)-sequestering organelles into the cytoplasm, followed by a subsequent Ca2+ reduction. In the adult SCN, fewer astrocytes are found than neurons, yet astrocytes frequently surround glutamate-immunoreactive axons in synaptic contact with SCN dendrites, isolating neurons from each other while maintaining contact with other astrocytes by gap junctions.(ABSTRACT TRUNCATED AT 400 WORDS)

Classic ON-OFF direction-selective ganglion cells (DSGCs) that encode the four cardinal directions were recently shown to also be orientation-selective. To clarify the mechanisms underlying orientation selectivity, we employed a variety of electrophysiological, optogenetic, and gene knock-out strategies to test the relative contributions of glutamate, GABA, and acetylcholine (ACh) input that are known to drive DSGCs, in male and female mouse retinas. Extracellular spike recordings revealed that DSGCs respond preferentially to either vertical or horizontal bars, those that are perpendicular to their preferred-null motion axes. By contrast, the glutamate input to all four DSGC types measured using whole-cell patch-clamp techniques was found to be tuned along the vertical axis. Tuned glutamatergic excitation was heavily reliant on type 5A bipolar cells, which appear to be electrically coupled via connexin 36 containing gap junctions to the vertically oriented processes of wide-field amacrine cells. Vertically tuned inputs are transformed by the GABAergic/cholinergic "starburst" amacrine cells (SACs), which are critical components of the direction-selective circuit, into distinct patterns of inhibition and excitation. Feed-forward SAC inhibition appears to "veto" preferred orientation glutamate excitation in dorsal/ventral (but not nasal/temporal) coding DSGCs "flipping" their orientation tuning by 90° and accounts for the apparent mismatch between glutamate input tuning and the DSGC's spiking response. Together, these results reveal how two distinct synaptic motifs interact to generate complex feature selectivity, shedding light on the intricate circuitry that underlies visual processing in the retina.

In higher sensory brain regions, slow oscillations (0.5-5 Hz) associated with quiet wakefulness and attention modulate multisensory integration, predictive coding, and perception. Although often assumed to originate via thalamocortical mechanisms, the extent to which subcortical sensory pathways are independently capable of slow oscillatory activity is unclear. We find that in the first station for auditory processing, the cochlear nucleus, fusiform cells from juvenile mice (of either sex) generate robust 1-2 Hz oscillations in membrane potential and exhibit electrical resonance. Such oscillations were absent prior to the onset of hearing, intrinsically generated by hyperpolarization-activated cyclic nucleotide-gated (HCN) and persistent Na+ conductances (NaP) interacting with passive membrane properties, and reflected the intrinsic resonance properties of fusiform cells. Cx36-containing gap junctions facilitated oscillation strength and promoted pairwise synchrony of oscillations between neighboring neurons. The strength of oscillations were strikingly sensitive to external Ca2+, disappearing at concentrations >1.7 mM, due in part to the shunting effect of small-conductance calcium-activated potassium (SK) channels. This effect explains their apparent absence in previous in vitro studies of cochlear nucleus which routinely employed high-Ca2+ extracellular solution. In contrast, oscillations were amplified in reduced Ca2+ solutions, due to relief of suppression by Ca2+ of Na+ channel gating. Our results thus reveal mechanisms for synchronous oscillatory activity in auditory brainstem, suggesting that slow oscillations, and by extension their perceptual effects, may originate at the earliest stages of sensory processing.

Backpropagating action potentials (bAPs) are indispensable in dendritic signaling. Conflicting Ca2+-imaging data and an absence of dendritic recording data means that the extent of backpropagation in thalamocortical (TC) and thalamic reticular nucleus (TRN) neurons remains unknown. Because TRN neurons signal electrically through dendrodendritic gap junctions and possibly via chemical dendritic GABAergic synapses, as well as classical axonal GABA release, this lack of knowledge is problematic. To address this issue, we made two-photon targeted patch-clamp recordings from rat TC and TRN neuron dendrites to measure bAPs directly. These recordings reveal that "tonic"' and low-threshold-spike (LTS) "burst" APs in both cell types are always recorded first at the soma before backpropagating into the dendrites while undergoing substantial distance-dependent dendritic amplitude attenuation. In TC neurons, bAP attenuation strength varies according to firing mode. During LTS bursts, somatic AP half-width increases progressively with increasing spike number, allowing late-burst spikes to propagate more efficiently into the dendritic tree compared with spikes occurring at burst onset. Tonic spikes have similar somatic half-widths to late burst spikes and undergo similar dendritic attenuation. In contrast, in TRN neurons, AP properties are unchanged between LTS bursts and tonic firing and, as a result, distance-dependent dendritic attenuation remains consistent across different firing modes. Therefore, unlike LTS-associated global electrical and calcium signals, the spatial influence of bAP signaling in TC and TRN neurons is more restricted, with potentially important behavioral-state-dependent consequences for synaptic integration and plasticity in thalamic neurons.SIGNIFICANCE STATEMENT In most neurons, action potentials (APs) initiate in the axosomatic region and propagate into the dendritic tree to provide a retrograde signal that conveys information about the level of cellular output to the locations that receive most input: the dendrites. In thalamocortical and thalamic reticular nucleus neurons, the site of AP generation and the true extent of backpropagation remain unknown. Using patch-clamp recordings, this study measures dendritic propagation of APs directly in these neurons. In either cell type, high-frequency low-threshold spike burst or lower-frequency tonic APs undergo substantial voltage attenuation as they spread into the dendritic tree. Therefore, backpropagating spikes in these cells can only influence signaling in the proximal part of the dendritic tree.

Fragile X mental retardation protein (FMRP) loss causes Fragile X syndrome (FXS), a major disorder characterized by autism, intellectual disability, hyperactivity, and seizures. FMRP is both an RNA- and channel-binding regulator, with critical roles in neural circuit formation and function. However, it remains unclear how these FMRP activities relate to each other and how dysfunction in their absence underlies FXS neurological symptoms. In testing circuit level defects in the Drosophila FXS model, we discovered a completely unexpected and highly robust neuronal dye iontophoresis phenotype in the well mapped giant fiber (GF) circuit. Controlled dye injection into the GF interneuron results in a dramatic increase in dye uptake in neurons lacking FMRP. Transgenic wild-type FMRP reintroduction rescues the mutant defect, demonstrating a specific FMRP requirement. This phenotype affects only small dyes, but is independent of dye charge polarity. Surprisingly, the elevated dye iontophoresis persists in shaking B mutants that eliminate gap junctions and dye coupling among GF circuit neurons. We therefore used a wide range of manipulations to investigate the dye uptake defect, including timed injection series, pharmacology and ion replacement, and optogenetic activity studies. The results show that FMRP strongly limits the rate of dye entry via a cytosolic mechanism. This study reveals an unexpected new phenotype in a physical property of central neurons lacking FMRP that could underlie aspects of FXS disruption of neural function.SIGNIFICANCE STATEMENT FXS is a leading heritable cause of intellectual disability and autism spectrum disorders. Although researchers established the causal link with FMRP loss >;25 years ago, studies continue to reveal diverse FMRP functions. The Drosophila FXS model is key to discovering new FMRP roles, because of its genetic malleability and individually identified neuron maps. Taking advantage of a well characterized Drosophila neural circuit, we discovered that neurons lacking FMRP take up dramatically more current-injected small dye. After examining many neuronal properties, we determined that this dye defect is cytoplasmic and occurs due to a highly elevated dye iontophoresis rate. We also report several new factors affecting neuron dye uptake. Understanding how FMRP regulates iontophoresis should reveal new molecular factors underpinning FXS dysfunction.