Dual spatio-temporal regulation of axon growth and microtubule dynamics by RhoA signaling pathways.

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA-mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK signaling, which restrained growth cone microtubule assembly and protrusion.

Blockade of Rho-associated kinase prevents inhibition of axon regeneration of peripheral nerves induced by anti-ganglioside antibodies.

Anti-ganglioside antibodies are associated with delayed/poor clinical recovery in Guillain-Barrè syndrome, mostly related to halted axon regeneration. Cross-linking of cell surface gangliosides by anti-ganglioside antibodies triggers inhibition of nerve repair in in vitro and in vivo paradigms of axon regeneration. These effects involve the activation of the small GTPase RhoA/ROCK signaling pathways, which negatively modulate growth cone cytoskeleton, similarly to well stablished inhibitors of axon regeneration described so far. The aim of this work was to perform a proof of concept study to demonstrate the effectiveness of Y-27632, a selective pharmacological inhibitor of ROCK, in a mouse model of axon regeneration of peripheral nerves, where the passive immunization with a monoclonal antibody targeting gangliosides GD1a and GT1b was previously reported to exert a potent inhibitory effect on regeneration of both myelinated and unmyelinated fibers. Our results demonstrate a differential sensitivity of myelinated and unmyelinated axons to the pro-regenerative effect of Y-27632. Treatment with a total dosage of 9 mg/kg of Y-27632 resulted in a complete prevention of anti-GD1a/GT1b monoclonal antibody-mediated inhibition of axon regeneration of unmyelinated fibers to skin and the functional recovery of mechanical cutaneous sensitivity. In contrast, the same dose showed toxic effects on the regeneration of myelinated fibers. Interestingly, scale down of the dosage of Y-27632 to 5 mg/kg resulted in a significant although not complete recovery of regenerated myelinated axons exposed to anti-GD1a/GT1b monoclonal antibody in the absence of toxicity in animals exposed to only Y-27632. Overall, these findings confirm the in vivo participation of RhoA/ROCK signaling pathways in the molecular mechanisms associated with the inhibition of axon regeneration induced by anti-GD1a/GT1b monoclonal antibody. Our findings open the possibility of therapeutic pharmacological intervention targeting RhoA/Rock pathway in immune neuropathies associated with the presence of anti-ganglioside antibodies and delayed or incomplete clinical recovery after injury in the peripheral nervous system.

Gene Signature Associated with Nervous System in an Experimental Radiation- and Estrogen-Induced Breast Cancer Model.

Breast cancer is frequently the most diagnosed female cancer in the world. The experimental studies on cancer seldom focus on the relationship between the central nervous system and cancer. Despite extensive research into the treatment of breast cancer, chemotherapy resistance is an important issue limiting the efficacy of treatment. Novel biomarkers to predict prognosis or sensitivity to chemotherapy are urgently needed. This study examined nervous-system-related genes. The profiling of differentially expressed genes indicated that high-LET radiation, such as that emitted by radon progeny, in the presence of estrogen, induced a cascade of events indicative of tumorigenicity in human breast epithelial cells. Bioinformatic tools allowed us to analyze the genes involved in breast cancer and associated with the nervous system. The results indicated that the gene expression of the Ephrin A1 gene (EFNA1), the roundabout guidance receptor 1 (ROBO1), and the kallikrein-related peptidase 6 (KLK6) was greater in T2 and A5 than in the A3 cell line; the LIM domain kinase 2 gene (LIMK2) was greater in T2 than A3 and A5; the kallikrein-related peptidase 7 (KLK7), the neuroligin 4 X-linked gene (NLGN4X), and myelin basic protein (MBP) were greater than A3 only in T2; and the neural precursor cell expressed, developmentally down-regulated 9 gene (NEDD9) was greater in A5 than in the A3 and E cell lines. Concerning the correlation, it was found a positive correlation between ESR1 and EFNA1 in BRCA-LumA patients; with ROBO1 in BRCA-Basal patients, but this correlation was negative with the kallikrein-related peptidase 6 (KLK6) in BRCA-LumA and -LumB, as well as with LIMK2 and ROBO1 in all BRCA. It was also positive with neuroligin 4 X-linked (NLGN4X) in BRCA-Her2 and BRCA-LumB, and with MBP in BRCA-LumA and -LumB, but negative with KLK7 in all BRCA and BRCA-LumA and NEDD9 in BRCA-Her2. The differential gene expression levels between the tumor and adjacent tissue indicated that the ROBO1, KLK6, LIMK2, KLK7, NLGN4X, MBP, and NEDD9 gene expression levels were higher in normal tissues than in tumors; however, EFNA1 was higher in the tumor than the normal ones. EFNA1, LIMK2, ROBO1, KLK6, KLK7, and MBP gene expression had a negative ER status, whereas NEDD9 and NLGN4X were not significant concerning ER status. In conclusion, important markers have been analyzed concerning genes related to the nervous system, opening up a new avenue of studies in breast cancer therapy.

Congenital Zika Virus Infection Impairs Corpus Callosum Development.

Congenital Zika syndrome (CZS) is a set of birth defects caused by Zika virus (ZIKV) infection during pregnancy. Microcephaly is its main feature, but other brain abnormalities are found in CZS patients, such as ventriculomegaly, brain calcifications, and dysgenesis of the corpus callosum. Many studies have focused on microcephaly, but it remains unknown how ZIKV infection leads to callosal malformation. To tackle this issue, we infected mouse embryos in utero with a Brazilian ZIKV isolate and found that they were born with a reduction in callosal area and density of callosal neurons. ZIKV infection also causes a density reduction in PH3+ cells, intermediate progenitor cells, and SATB2+ neurons. Moreover, axonal tracing revealed that callosal axons are reduced and misrouted. Also, ZIKV-infected cultures show a reduction in callosal axon length. GFAP labeling showed that an in utero infection compromises glial cells responsible for midline axon guidance. In sum, we showed that ZIKV infection impairs critical steps of corpus callosum formation by disrupting not only neurogenesis, but also axon guidance and growth across the midline.

Histological Compatibility in Distal Neurotizations: A Systematic Review.

Considering the importance of defining the minimum number of axons between recipient and donor branches, that is, the definition of histological compatibility in distal neurotizations for the success of the procedure and the surgeon's freedom to choose individualized strategies for each patient, this systematic review was conducted to find out the most recent studies on the subject. The objective of this systematic review was to determine the importance of the number of axons and the relationship between axon counts in the donor and recipient nerves in the success of nerve transfer. A literature review was performed on five international databases: Web of Science, Scopus, Wiley (Cochrane Database), Embase, and PubMed. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed (2020 version), a guide designed to guide the elaboration of systematic literature reviews. One hundred and fifty-seven studies were found, and 23 were selected based on the eligibility criteria. The articles presented were conclusive in determining the importance of the number of axons in the success of nerve transfer. Still, the relationship between the number of axons in the donor and recipient nerves seems more relevant in the success of transfers and is not always explored by the authors. The review of the articles has provided compelling evidence that the number of axons is a critical determinant of the success of nerve transfer procedures. However, the relationship between the number of axons in the donor nerve and that in the recipient nerve appears to be even more crucial for successful transfers, a factor that is not always adequately explored by authors in the existing literature. Level of evidence : Level IV, therapeutic study.

Analogue signaling of somatodendritic synaptic activity to axon enhances GABA release in young cerebellar molecular layer interneurons.

Axons are equipped with the digital signaling capacity by which they generate and faithfully propagate action potentials (APs), and also with the analogue signaling capacity by which subthreshold activity in dendrites and soma is transmitted down the axon. Despite intense work, the extent and physiological role for subthreshold synaptic activity reaching the presynaptic boutons has remained elusive because of the technical limitation to record from them. To address this issue, we made simultaneous patch-clamp recordings from the presynaptic varicosities of cerebellar GABAergic interneurons together with their parent soma or postsynaptic target cells in young rat slices and/or primary cultures. Our tour-de-force direct functional dissection indicates that the somatodendritic spontaneous excitatory synaptic potentials are transmitted down the axon for significant distances, depolarizing presynaptic boutons. These analogously transmitted excitatory synaptic potentials augment presynaptic Ca++ influx upon arrival of an immediately following AP through a mechanism that involves a voltage-dependent priming of the Ca++ channels, leading to an increase in GABA release, without any modification in the presynaptic AP waveform or residual Ca++. Our work highlights the role of the axon in synaptic integration.

Quantitative Proteomics of Nervous System Regeneration: From Sample Preparation to Functional Data Analyses.

Mammals have a limited regenerative capacity, especially of the central nervous system. Consequently, any traumatic injury or neurodegenerative disease results in irreversible damage. An important approach to finding strategies to promote regeneration in mammals has been the study of regenerative organisms like Xenopus, the axolotl, and teleost fish. High-throughput technologies like RNA-Seq and quantitative proteomics are starting to provide valuable insight into the molecular mechanisms that drive nervous system regeneration in these organisms. In this chapter, we present a detailed protocol for performing iTRAQ proteomics that can be applied to the analysis of nervous system samples, using Xenopus laevis as an example. The quantitative proteomics protocol and directions for performing functional enrichment data analyses of gene lists (e.g., differentially abundant proteins from a proteomic study, or any type of high-throughput analysis) are aimed at the general bench biologist and do not require previous programming knowledge.

Necroptosis inhibition counteracts neurodegeneration, memory decline, and key hallmarks of aging, promoting brain rejuvenation.

Age is the main risk factor for the development of neurodegenerative diseases. In the aged brain, axonal degeneration is an early pathological event, preceding neuronal dysfunction, and cognitive disabilities in humans, primates, rodents, and invertebrates. Necroptosis mediates degeneration of injured axons, but whether necroptosis triggers neurodegeneration and cognitive impairment along aging is unknown. Here, we show that the loss of the necroptotic effector Mlkl was sufficient to delay age-associated axonal degeneration and neuroinflammation, protecting against decreased synaptic transmission and memory decline in aged mice. Moreover, short-term pharmacologic inhibition of necroptosis targeting RIPK3 in aged mice, reverted structural and functional hippocampal impairment, both at the electrophysiological and behavioral level. Finally, a quantitative proteomic analysis revealed that necroptosis inhibition leads to an overall improvement of the aged hippocampal proteome, including a subclass of molecular biofunctions associated with brain rejuvenation, such as long-term potentiation and synaptic plasticity. Our results demonstrate that necroptosis contributes to age-dependent brain degeneration, disturbing hippocampal neuronal connectivity, and cognitive function. Therefore, necroptosis inhibition constitutes a potential geroprotective strategy to treat age-related disabilities associated with memory impairment and cognitive decline.

Stem Cells and Tissue Engineering-Based Therapeutic Interventions: Promising Strategies to Improve Peripheral Nerve Regeneration.

Unlike the central nervous system, the peripheral one has the ability to regenerate itself after injury; however, this natural regeneration process is not always successful. In fact, even with some treatments, the prognosis is poor, and patients consequently suffer with the functional loss caused by injured nerves, generating several impacts on their quality of life. In the present review we aimed to address two strategies that may considerably potentiate peripheral nerve regeneration: stem cells and tissue engineering. In vitro studies have shown that pluripotent cells associated with neural scaffolds elaborated by tissue engineering can increase functional recovery, revascularization, remyelination, neurotrophin expression and reduce muscle atrophy. Although these results are very promising, it is important to note that there are some barriers to be circumvented: the host's immune response, the oncogenic properties attributed to stem cells and the duration of the pro-regenerative effects. After all, more studies are still needed to overcome the limitations of these treatments; those that address techniques for manipulating the lesion microenvironment combining different therapies seem to be the most promising and proactive ones.

Zebrafish Slit2 and Slit3 Act Together to Regulate Retinal Axon Crossing at the Midline.

Slit-Robo signaling regulates midline crossing of commissural axons in different systems. In zebrafish, all retinofugal axons cross at the optic chiasm to innervate the contralateral tectum. Here, the mutant for the Robo2 receptor presents severe axon guidance defects, which were not completely reproduced in a Slit2 ligand null mutant. Since slit3 is also expressed around this area at the stage of axon crossing, we decided to analyze the possibility that it collaborates with Slit2 in this process. We found that the disruption of slit3 expression by sgRNA-Cas9 injection caused similar, albeit slightly milder, defects than those of the slit2 mutant, while the same treatment in the slit2-/-mz background caused much more severe defects, comparable to those observed in robo2 mutants. Tracking analysis of in vivo time-lapse experiments indicated differential but complementary functions of these secreted factors in the correction of axon turn errors around the optic chiasm. Interestingly, RT-qPCR analysis showed a mild increase in slit2 expression in slit3-deficient embryos, but not the opposite. Our observations support the previously proposed "repulsive channel" model for Slit-Robo action at the optic chiasm, with both Slits acting in different manners, most probably relating to their different spatial expression patterns.

Autocrine/Paracrine Slit-Robo Signaling Controls Optic Lobe Development in Drosophila melanogaster.

Cell segregation mechanisms play essential roles during the development of the central nervous system (CNS) to support its organization into distinct compartments. The Slit protein is a secreted signal, classically considered a paracrine repellent for axonal growth through Robo receptors. However, its function in the compartmentalization of CNS is less explored. In this work, we show that Slit and Robo3 are expressed in the same neuronal population of the Drosophila optic lobe, where they are required for the correct compartmentalization of optic lobe neuropils by the action of an autocrine/paracrine mechanism. We characterize the endocytic route followed by the Slit/Robo3 complex and detected genetic interactions with genes involved in endocytosis and actin dynamics. Thus, we report that the Slit-Robo3 pathway regulates the morphogenesis of the optic lobe through an atypical autocrine/paracrine mechanism in addition to its role in axon guidance, and in association with proteins of the endocytic pathway and small GTPases.

Immunolocalization of kappa opioid receptors in the axon initial segment of a group of embryonic mesencephalic dopamine neurons.

The dopamine mesolimbic system is a major circuit involved in controlling goal-directed behaviors. Dopamine D2 receptors (D2R) and kappa opioid receptors (KOR) are abundant Gi protein-coupled receptors in the mesolimbic system. D2R and KOR share several functions in dopamine mesencephalic neurons, such as regulation of dopamine release and uptake, and firing of dopamine neurons. In addition, KOR and D2R modulate each other functioning. This evidence indicates that both receptors functionally interact, however, their colocalization in the mesostriatal system has not been addressed. Immunofluorescent assays were performed in cultured dopamine neurons and adult mice's brain tissue to answer this question. We observed that KOR and D2R are present in similar density in dendrites and soma of cultured dopamine neurons, but in a segregated manner. Interestingly, KOR immunolabelling was observed in the first part of the axon, colocalizing with Ankyrin in 20% of cultured dopamine neurons, indicative that KOR is present in the axon initial segment (AIS) of a group of dopaminergic neurons. In the adult brain, KOR and D2R are also segregated in striatal tissue. While the KOR label is in fiber tracts such as the striatal streaks, corpus callosum, and anterior commissure, D2R is located mainly within the striatum and nucleus accumbens, surrounding fiber tracts. D2R is also localized in some fibers that are mostly different from those positives for KOR. In conclusion, KOR and D2R are present in the soma and dendrites of mesencephalic dopaminergic neurons, but KOR is also found in the AIS of a subpopulation of these neurons.

Mitochondrial function in spinal cord injury and regeneration.

Many people around the world suffer from some form of paralysis caused by spinal cord injury (SCI), which has an impact on quality and life expectancy. The spinal cord is part of the central nervous system (CNS), which in mammals is unable to regenerate, and to date, there is a lack of full functional recovery therapies for SCI. These injuries start with a rapid and mechanical insult, followed by a secondary phase leading progressively to greater damage. This secondary phase can be potentially modifiable through targeted therapies. The growing literature, derived from mammalian and regenerative model studies, supports a leading role for mitochondria in every cellular response after SCI: mitochondrial dysfunction is the common event of different triggers leading to cell death, cellular metabolism regulates the immune response, mitochondrial number and localization correlate with axon regenerative capacity, while mitochondrial abundance and substrate utilization regulate neural stem progenitor cells self-renewal and differentiation. Herein, we present a comprehensive review of the cellular responses during the secondary phase of SCI, the mitochondrial contribution to each of them, as well as evidence of mitochondrial involvement in spinal cord regeneration, suggesting that a more in-depth study of mitochondrial function and regulation is needed to identify potential targets for SCI therapeutic intervention.

Transcriptomic characterization of the molecular mechanisms induced by RGMa during skeletal muscle nuclei accretion and hypertrophy.

BACKGROUND: The repulsive guidance molecule a (RGMa) is a GPI-anchor axon guidance molecule first found to play important roles during neuronal development. RGMa expression patterns and signaling pathways via Neogenin and/or as BMP coreceptors indicated that this axon guidance molecule could also be working in other processes and diseases, including during myogenesis. Previous works from our research group have consistently shown that RGMa is expressed in skeletal muscle cells and that its overexpression induces both nuclei accretion and hypertrophy in muscle cell lineages. However, the cellular components and molecular mechanisms induced by RGMa during the differentiation of skeletal muscle cells are poorly understood. In this work, the global transcription expression profile of RGMa-treated C2C12 myoblasts during the differentiation stage, obtained by RNA-seq, were reported. RESULTS: RGMa treatment could modulate the expression pattern of 2,195 transcripts in C2C12 skeletal muscle, with 943 upregulated and 1,252 downregulated. Among them, RGMa interfered with the expression of several RNA types, including categories related to the regulation of RNA splicing and degradation. The data also suggested that nuclei accretion induced by RGMa could be due to their capacity to induce the expression of transcripts related to 'adherens junsctions' and 'extracellular-cell adhesion', while RGMa effects on muscle hypertrophy might be due to (i) the activation of the mTOR-Akt independent axis and (ii) the regulation of the expression of transcripts related to atrophy. Finally, RGMa induced the expression of transcripts that encode skeletal muscle structural proteins, especially from sarcolemma and also those associated with striated muscle cell differentiation. CONCLUSIONS: These results provide comprehensive knowledge of skeletal muscle transcript changes and pathways in response to RGMa.

The sciatic and radial nerves seem to adapt similarly to different ladder-based resistance training protocols.

The present study aimed to compare the morphological response induced by different ladder-based resistance training (LRT) protocols on the peripheral nerve ultrastructure of young adult Wistar rats. Twenty-nine rodents were distributed into groups: control (CON), submaximal (SUBMAX [6 climbs/session, moderate intensity, 3x/week]) and maximum (MAX [> 4 climbs/session, maximum intensity, 3x/week]) LRT. After 8 weeks, the radial and sciatic nerves were removed and prepared for transmission electron microscopy. In the radial nerve, the myelinated fibers and axons, myelin sheath thickness, and unmyelinated axons were statistically greater in the SUBMAX and MAX. The MAX group had greater unmyelinated fibers than SUBMAX. The Schwann cell (SC) nuclei diameter was statistically larger in the SUBMAX than the CON. The number of microtubules and neurofilaments was statistically higher in the SUBMAX and MAX. In the sciatic nerve, the myelinated fibers, myelinated and unmyelinated axons, and myelin sheath thickness were statistically greater in the SUBMAX and MAX. The SUBMAX and MAX had more SC at the nuclei level than CON. The SC nuclei were statistically larger in the SUBMAX and MAX. The number of microtubules and neurofilaments was statistically higher in the SUBMAX and MAX. Total training load and total load per climb were not different between groups. The SUBMAX and MAX statistically increased maximum carried load (ML). In conclusion, the different LRT protocols induced similar morphological responses in radial and sciatic nerves, probably due to load progression and equal total load volume.

Extracellular matrix stiffness negatively affects axon elongation, growth cone area and F-actin levels in a collagen type I 3D culture.

Three dimensional (3D) in vitro neuronal cultures can better reproduce physiologically relevant phenotypes compared to 2D-cultures, because in vivo neurons reside in a 3D microenvironment. Interest in neuronal 3D cultures is emerging, with special attention to the mechanical forces that regulate axon elongation and sprouting in three dimensions. Type I collagen (Col-I) is a native substrate since it is present in the extracellular matrix and hence emulates an in vivo environment to study axon growth. The impact of its mechanical properties needs to be further investigated. Here, we generated Col-I 3D matrices of different mechanical stiffness and evaluated axon growth in three dimensions. Superior cervical ganglion (SCG) explants from neonatal rats were cultured in soft and stiff Col-I 3D matrices and neurite outgrowth was assessed by measuring: maximum neuritic extent; neuritic halo area and fasciculation. Axonal cytoskeletal proteins were examined. Axon elongation in stiff Col-I 3D matrices was reduced (31%) following 24 h in culture compared to soft matrices. In stiff matrices, neurites fasciculated and formed less dense halos. Consistently, almost no F-actin rich growth cones were recognized, and F-actin staining was strongly reduced in the axonal compartment. This study shows that stiffness negatively affects 3D neurite outgrowth and adds insights on the cytoskeletal responses upon mechanic interactions of axons with a 3D environment. Our data will serve to facilitate the development of model systems that are mechanically well-behaved but still mimic key physiologic properties observed in vivo.

Convergent molecular mechanisms underlying cognitive impairment in mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by pathogenic variants in the iduronate-2-sulfatase gene (IDS), responsible for the degradation of glycosaminoglycans (GAGs) heparan and dermatan sulfate. IDS enzyme deficiency results in the accumulation of GAGs within cells and tissues, including the central nervous system (CNS). The progressive neurological outcome in a representative number of MPSII patients (neuronopathic form) involves cognitive impairment, behavioral difficulties, and regression in developmental milestones. In an attempt to dissect part of the influence of axon guidance instability over the cognitive impairment presentation in MPS II, we used brain expression data, network propagation, and clustering algorithm to prioritize in the human interactome a disease module associated with the MPS II context. We identified new candidate genes and pathways that act in focal adhesion, integrin cell surface, laminin interactions, ECM proteoglycans, cytoskeleton, and phagosome that converge into functional mechanisms involved in early neural circuit formation defects and could indicate clues about cognitive impairment in patients with MPSII. Such molecular changes during neurodevelopment may precede the morphological and clinical evidence, emphasizing the importance of an early diagnosis and directing the development of potential drug leads. Furthermore, our data also support previous hypotheses pointing to shared pathogenic mechanisms in some neurodegenerative diseases.

Improved post-stroke spontaneous recovery by astrocytic extracellular vesicles.

Spontaneous recovery after a stroke accounts for a significant part of the neurological recovery in patients. However limited, the spontaneous recovery is mechanistically driven by axonal restorative processes for which several molecular cues have been previously described. We report the acceleration of spontaneous recovery in a preclinical model of ischemia/reperfusion in rats via a single intracerebroventricular administration of extracellular vesicles released from primary cortical astrocytes. We used magnetic resonance imaging and confocal and multiphoton microscopy to correlate the structural remodeling of the corpus callosum and striatocortical circuits with neurological performance during 21 days. We also evaluated the functionality of the corpus callosum by repetitive recordings of compound action potentials to show that the recovery facilitated by astrocytic extracellular vesicles was both anatomical and functional. Our data provide compelling evidence that astrocytes can hasten the basal recovery that naturally occurs post-stroke through the release of cellular mediators contained in extracellular vesicles.

Change in the neurochemical signature and morphological development of the parvocellular isthmic projection to the avian tectum.

Neurons can change their classical neurotransmitters during ontogeny, sometimes going through stages of dual release. Here, we explored the development of the neurotransmitter identity of neurons of the avian nucleus isthmi parvocellularis (Ipc), whose axon terminals are retinotopically arranged in the optic tectum (TeO) and exert a focal gating effect upon the ascending transmission of retinal inputs. Although cholinergic and glutamatergic markers are both found in Ipc neurons and terminals of adult pigeons and chicks, the mRNA expression of the vesicular acetylcholine transporter, VAChT, is weak or absent. To explore how the Ipc neurotransmitter identity is established during ontogeny, we analyzed the expression of mRNAs coding for cholinergic (ChAT, VAChT, and CHT) and glutamatergic (VGluT2 and VGluT3) markers in chick embryos at different developmental stages. We found that between E12 and E18, Ipc neurons expressed all cholinergic mRNAs and also VGluT2 mRNA; however, from E16 through posthatch stages, VAChT mRNA expression was specifically diminished. Our ex vivo deposits of tracer crystals and intracellular filling experiments revealed that Ipc axons exhibit a mature paintbrush morphology late in development, experiencing marked morphological transformations during the period of presumptive dual vesicular transmitter release. Additionally, although ChAT protein immunoassays increasingly label the growing Ipc axon, this labeling was consistently restricted to sparse portions of the terminal branches. Combined, these results suggest that the synthesis of glutamate and acetylcholine, and their vesicular release, is complexly linked to the developmental processes of branching, growing and remodeling of these unique axons.