Differential gene expression between callosal and ipsilateral projection neurons in the monkey dorsolateral prefrontal and posterior parietal cortices.

Reciprocal connections between primate dorsolateral prefrontal (DLPFC) and posterior parietal (PPC) cortices, furnished by subsets of layer 3 pyramidal neurons (PNs), contribute to cognitive processes including working memory (WM). A different subset of layer 3 PNs in each region projects to the homotopic region of the contralateral hemisphere. These ipsilateral (IP) and callosal (CP) projections, respectively, appear to be essential for the maintenance and transfer of information during WM. To determine if IP and CP layer 3 PNs in each region differ in their transcriptomes, fluorescent retrograde tracers were used to label IP and CP layer 3 PNs in the DLPFC and PPC from macaque monkeys. Retrogradely-labeled PNs were captured by laser microdissection and analyzed by RNAseq. Numerous differentially expressed genes (DEGs) were detected between IP and CP neurons in each region and the functional pathways containing many of these DEGs were shared across regions. However, DLPFC and PPC displayed opposite patterns of DEG enrichment between IP and CP neurons. Cross-region analyses indicated that the cortical area targeted by IP or CP layer 3 PNs was a strong correlate of their transcriptome profile. These findings suggest that the transcriptomes of layer 3 PNs reflect regional, projection type and target region specificity.

Mitral Cell Dendritic Morphology in the Adult Zebrafish Olfactory Bulb following Growth, Injury and Recovery.

The role of afferent target interactions in dendritic plasticity within the adult brain remains poorly understood. There is a paucity of data regarding the effects of deafferentation and subsequent dendritic recovery in adult brain structures. Moreover, although adult zebrafish demonstrate ongoing growth, investigations into the impact of growth on mitral cell (MC) dendritic arbor structure and complexity are lacking. Leveraging the regenerative capabilities of the zebrafish olfactory system, we conducted a comprehensive study to address these gaps. Employing an eight-week reversible deafferentation injury model followed by retrograde labeling, we observed substantial morphological alterations in MC dendrites. Our hypothesis posited that cessation of injury would facilitate recovery of MC dendritic arbor structure and complexity, potentially influenced by growth dynamics. Statistical analyses revealed significant changes in MC dendritic morphology following growth and recovery periods, indicating that MC total dendritic branch length retained significance after 8 weeks of deafferentation injury when normalized to individual fish physical characteristics. This suggests that regeneration of branch length could potentially function relatively independently of growth-related changes. These findings underscore the remarkable plasticity of adult dendritic arbor structures in a sophisticated model organism and highlight the efficacy of zebrafish as a vital implement for studying neuroregenerative processes.

Enhancing Motor and Sensory Axon Regeneration after Peripheral Nerve Injury Using Bioluminescent Optogenetics.

Introduction-Recovery from peripheral nerve injuries is poor even though injured peripheral axons can regenerate. Novel therapeutic approaches are needed. The most successful preclinical experimental treatments have relied on increasing the activity of the regenerating axons, but the approaches taken are not applicable to many nerve-injured patients. Bioluminescent optogenetics (BL-OG) is a novel method of increasing the excitation of neurons that might be similar to that found with activity-dependent experimental therapies. We investigated the use of BL-OG as an approach to promoting axon regeneration following peripheral nerve injury. Methods-BL-OG uses luminopsins, light-sensing ion channels (opsins) fused with a light-emitting luciferase. When exposed to a luciferase substrate, such as coelenterazine (CTZ), luminopsins expressed in neurons generate bioluminescence and produce excitation through their opsin component. Adeno-associated viral vectors encoding either an excitatory luminopsin (eLMO3) or a mutated form (R115A) that can generate bioluminescence but not excite neurons were injected into mouse sciatic nerves. After retrograde transport and viral transduction, nerves were cut and repaired by simple end-to-end anastomosis, and mice were treated with a single dose of CTZ. Results-Four weeks after nerve injury, compound muscle action potentials (M waves) recorded in response to sciatic nerve stimulation were more than fourfold larger in mice expressing the excitatory luminopsin than in controls expressing the mutant luminopsin. The number of motor and sensory neurons retrogradely labeled from reinnervated muscles in mice expressing eLMO3 was significantly greater than the number in mice expressing the R115A luminopsin and not significantly different from those in intact mice. When viral injection was delayed so that luminopsin expression was induced after nerve injury, a clinically relevant scenario, evoked M waves recorded from reinnervated muscles were significantly larger after injury in eLMO3-expressing mice. Conclusions-Treatment of peripheral nerve injuries using BL-OG has significant potential to enhance axon regeneration and promote functional recovery.

Individual arcuate nucleus proopiomelanocortin neurons project to select target sites.

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARH) are a diverse group of neurons that project widely to different brain regions. It is unknown how this small population of neurons organizes its efferent projections. In this study, we hypothesized that individual ARH POMC neurons exclusively innervate select target regions. To investigate this hypothesis, we first verified that only a fraction of ARH POMC neurons innervate the lateral hypothalamus (LH), the paraventricular nucleus of the hypothalamus (PVN), the periaqueductal gray (PAG), or the ventral tegmental area (VTA) using the retrograde tracer cholera toxin B (CTB). Next, two versions of CTB conjugated to distinct fluorophores were injected bilaterally into two of the regions such that PVN and VTA, PAG and VTA, or LH and PVN received tracers simultaneously. These pairs of target sites were chosen based on function and location. Few individual ARH POMC neurons projected to two brain regions at once, suggesting that there are ARH POMC neuron subpopulations organized by their efferent projections. We also investigated whether increasing the activity of POMC neurons could increase the number of ARH POMC neurons labeled with CTB, implying an increase in new synaptic connections to downstream regions. However, chemogenetic enhancement of POMC neuron activity did not increase retrograde tracing of CTB back to ARH POMC neurons from either the LH, PVN, or VTA. Overall, subpopulations of ARH POMC neurons with distinct efferent projections may serve as a way for the POMC population to organize its many functions.

Deafferentation-induced alterations in mitral cell dendritic morphology in the adult zebrafish olfactory bulb.

The removal of afferent input to the olfactory bulb by both cautery and chemical olfactory organ ablation in adult zebrafish results in a significant decrease in volume of the ipsilateral olfactory bulb. To examine the effects of deafferentation at a cellular level, primary output neurons of the olfactory bulb, the mitral cells, were investigated using retrograde tract tracing with fluorescent dextran using ex vivo brain cultures. Morphological characteristics including the number of major dendritic branches, total length of dendritic branches, area of the dendritic arbor, overall dendritic complexity, and optical density of the arbor were used to determine the effects of deafferentation on mitral cell dendrites. Following 8 weeks of permanent deafferentation there were significant reductions in the total length of dendritic branches, the area of the dendritic arbor, and the density of fine processes in the dendritic tuft. With 8 weeks of chronic, partial deafferentation there were significant reductions in all parameters examined, including a modified Sholl analysis that showed significant decreases in overall dendritic complexity. These results show the plasticity of mitral cell dendritic structures in the adult brain and provide information about the response of these output neurons following the loss of sensory input in this key model system.

Dual innervation may occur in a partially denervated muscle.

INTRODUCTION: With a view to simplifying surgical techniques for selective laryngeal reinnervation, we addressed the question of whether it is feasible to receive additional innervation by a partially denervated muscle using an infrahyoid muscle model. METHODS: In 90 rats (6 groups of 15), phrenic nerve transfer was used to reinnervate the sternothyroid muscle. In some cases, residual innervation by the original nerve was present. Three months later we performed electromyographic studies, contraction strength measurements, histologic assessment, and retrograde labeling. RESULTS: Muscles reinnervated by the phrenic nerve had a greater "dual-response" rate (in terms of nerve latency, contraction strength, and retrograde labeling) than muscles in the control groups. DISCUSSION: The phrenic nerve can impart its inspiratory properties to an initially denervated strap muscle-even when residual innervation is present. The preservation of contractile potential confirmed the feasibility of dual innervation in a previously injured muscle. Muscle Nerve 59:108-115, 2019.

Cauda equina repair in the rat: Part 3. Axonal regeneration across Schwann cell-Seeded collagen foam.

INTRODUCTION: Treatments for patients with cauda equina injury are limited. METHODS: In this study, we first used retrograde labeling to determine the relative contributions of cauda equina motor neurons to intrinsic and extrinsic rat tail muscles. Next, we transected cauda equina ventral roots and proceeded to bridge the proximal and distal stumps with either a type I collagen scaffold coated in laminin (CL) or a collagen-laminin scaffold that was also seeded with Schwann cells (CLSC). Regeneration was assessed by way of serial retrograde labeling. RESULTS: After accounting for the axonal contributions to intrinsic vs. extrinsic tail muscles, we noted a higher degree of double labeling in the CLSC group (58.0 ± 39.6%) as compared with the CL group (27.8 ± 16.0%; P = 0.02), but not the control group (33.5 ± 18.2%; P = 0.10). DISCUSSION: Our findings demonstrate the feasibility of using CLSCs in cauda equina injury repair. Muscle Nerve 57: E78-E84, 2018.

Facilitation of Contextual Fear Extinction by Orexin-1 Receptor Antagonism Is Associated with the Activation of Specific Amygdala Cell Subpopulations.

Background: Orexins are hypothalamic neuropeptides recently involved in the regulation of emotional memory. The basolateral amygdala, an area orchestrating fear memory processes, appears to be modulated by orexin transmission during fear extinction. However, the neuronal types within the basolateral amygdala involved in this modulation remain to be elucidated. Methods: We used retrograde tracing combined with immunofluorescence techniques in mice to identify basolateral amygdala projection neurons and cell subpopulations in this brain region influenced by orexin transmission during contextual fear extinction consolidation. Results: Treatment with the orexin-1 receptor antagonist SB334867 increased the activity of basolateral amygdala neurons projecting to infralimbic medial prefrontal cortex during fear extinction. GABAergic interneurons expressing calbindin, but not parvalbumin, were also activated by orexin-1 receptor antagonism in the basolateral amygdala. Conclusions: These data identify neuronal circuits and cell populations of the amygdala associated with the facilitation of fear extinction consolidation induced by the orexin-1 receptor antagonist SB334867.

Loss-of-function mutation of serine racemase attenuates excitotoxicity by intravitreal injection of N-methyl-D-aspartate.

Convincing data demonstrate that D-serine, a racemized product of serine racemase (SR), contributes to neurotoxicity. Furthermore, a line of evidence suggests that SR/D-serine contributes to retinal neurodegeneration in a diabetic retinopathy rat model and diabetic retinopathy patients. However, the connection between SR/D-serine and retinal neurodegeneration remains unclear. Herein, we report that intravitreal injection of N-methyl-D-aspartate (NMDA) induces excitotoxicity in rodent retina; this retinal neurodegeneration was attenuated in retina carrying a loss-of-function of mutation in Srr, the gene for SR, termed Srr(ochre269). Under the condition of NMDA injection, either posterior pole or middle - but not peripheral - retina from Srr(ochre269) mice was found to retain more retinal ganglion cells (RGC) than the counterpart from w/t (RGCs were identified with retrograde labeling). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining further demonstrated reduced RGC apoptosis from Srr(ochre269) compared to w/t mice under the condition of NMDA injection. Collectively, our studies demonstrate a pivotal role of SR/D-serine in retinal neurotoxicity. We demonstrated that loss-of-function mutation of the gene encoding serine racemase significantly attenuates excitotoxicity in retina; excitotoxicity accounts for retinal ganglion cell (RGC) demise in diabetic retinopathy (DR). We think that our findings deepen the current knowledge of the mechanisms of RGC degeneration.

A monoclonal natural human IgM protects axons in the absence of remyelination.

BACKGROUND: Whereas demyelination underlies early neurological symptoms in multiple sclerosis (MS), axonal damage is considered critical for permanent chronic deficits. Intracerebral infection of susceptible mouse strains with Theiler's murine encephalomyelitis virus (TMEV) results in chronic induced demyelinating disease (TMEV-IDD) with progressive axonal loss and neurologic dysfunction similar to progressive forms of MS. We previously reported that treatment of chronic TMEV-IDD mice with a neurite outgrowth-promoting natural human antibody, HIgM12, improved brainstem NAA concentrations and preserved functional motor activity. In order to translate this antibody toward clinical trial, we generated a fully human recombinant form of HIgM12, rHIgM12, determined the optimal in vivo dose for functional improvement in TMEV-IDD, and evaluated the functional preservation of descending spinal cord axons by retrograde labeling. FINDINGS: SJL/J mice at 45 to 90 days post infection (dpi) were studied. A single intraperitoneal dose of 0.25 mg/kg of rHIgM12 per mouse is sufficient to preserve motor function in TMEV-IDD. The optimal dose was 10 mg/kg. rHIgM12 treatment protected the functional transport in spinal cord axons and led to 40 % more Fluoro-Gold-labeled brainstem neurons in retrograde transport studies. This suggests that axons are not only present but also functionally competent. rHIgM12-treated mice also contained more mid-thoracic (T6) spinal cord axons than controls. CONCLUSIONS: This study confirms that a fully human recombinant neurite outgrowth-promoting monoclonal IgM is therapeutic in a model of progressive MS using multiple reparative readouts. The minimum effective dose is similar to that of a remyelination-promoting monoclonal human IgM discovered by our group that is presently in clinical trials for MS.

Correlated gene expression and target specificity demonstrate excitatory projection neuron diversity.

The neocortex contains diverse populations of excitatory neurons segregated by layer and further definable by their specific cortical and subcortical projection targets. The current study describes a systematic approach to identify molecular correlates of specific projection neuron classes in mouse primary somatosensory cortex (S1), using a combination of in situ hybridization (ISH) data mining, marker gene colocalization, and combined retrograde labeling with ISH for layer-specific marker genes. First, we identified a large set of genes with specificity for each cortical layer, and that display heterogeneous patterns within those layers. Using these genes as markers, we find extensive evidence for the covariation of gene expression and projection target specificity in layer 2/3, 5, and 6, with individual genes labeling neurons projecting to specific subsets of target structures. The combination of gene expression and target specificity imply a great diversity of projection neuron classes that is similar to or greater than that of GABAergic interneurons. The covariance of these 2 phenotypic modalities suggests that these classes are both discrete and genetically specified.

Topographic organization across foveal visual areas in macaques.

Introduction: While the fovea on the retina covers only a small region of the visual field, a significant portion of the visual cortex is dedicated to processing information from the fovea being a critical center for object recognition, motion control, and visually guided attention. Despite its importance, prior functional imaging studies in awake monkeys often focused on the parafoveal visual field, potentially leading to inaccuracies in understanding the brain structure underlying function. Methods: In this study, our aim is to unveil the neuronal connectivity and topography in the foveal visual cortex in comparison to the parafoveal visual cortex. Using four different types of retrograde tracers, we selectively injected them into the striate cortex (V1) or V4, encompassing the regions between the fovea and parafovea. Results: V1 and V4 exhibited intense mutual connectivity in the foveal visual field, in contrast to the parafoveal visual field, possibly due to the absence of V3 in the foveal visual field. While previous live brain imaging studies failed to reveal retinotopy in the foveal visual fields, our results indicate that the foveal visual fields have continuous topographic connectivity across V1 through V4, as well as the parafoveal visual fields. Although a simple extension of the retinotopic isoeccentricity maps from V1 to V4 has been suggested from previous fMRI studies, our study demonstrated that V3 and V4 possess gradually smaller topographic maps compared to V1 and V2. Feedback projections to foveal V1 primarily originate from the infragranular layers of foveal V2 and V4, while feedforward projections to foveal V4 arise from both supragranular and infragranular layers of foveal V1 and V2, consistent with previous findings in the parafoveal visual fields. Discussion: This study provides valuable insights into the connectivity of the foveal visual cortex, which was ambiguous in previous imaging studies.

Cell Type-Specific Profiles and Developmental Trajectories of Transcriptomes in Primate Prefrontal Layer 3 Pyramidal Neurons: Implications for Schizophrenia.

OBJECTIVE: In schizophrenia, impaired working memory is associated with transcriptome alterations in layer 3 pyramidal neurons (L3PNs) in the dorsolateral prefrontal cortex (DLPFC). Distinct subtypes of L3PNs that send axonal projections to the DLPFC in the opposite hemisphere (callosal projection [CP] neurons) or the parietal cortex in the same hemisphere (ipsilateral projection [IP] neurons) play critical roles in working memory. However, how the transcriptomes of these L3PN subtypes might shift during late postnatal development when working memory impairments emerge in individuals later diagnosed with schizophrenia is not known. The aim of this study was to characterize and compare the transcriptome profiles of CP and IP L3PNs across developmental transitions from prepuberty to adulthood in macaque monkeys. METHODS: The authors used retrograde labeling to identify CP and IP L3PNs in the DLPFC of prepubertal, postpubertal, and adult macaque monkeys, and used laser microdissection to capture these neurons for RNA sequencing. RESULTS: At all three ages, CP and IP L3PNs had distinct transcriptomes, with the number of genes differentially expressed between neuronal subtypes increasing with age. For IP L3PNs, age-related shifts in gene expression were most prominent between prepubertal and postpubertal animals, whereas for CP L3PNs such shifts were most prominent between postpubertal and adult animals. CONCLUSIONS: These findings demonstrate the presence of cell type-specific profiles and developmental trajectories of the transcriptomes of PPC-projecting IP and DLPFC-projecting CP L3PNs in monkey DLPFC. The evidence that IP L3PNs reach a mature transcriptome earlier than CP L3PNs suggests that these two subtypes differentially contribute to the maturation of working memory performance across late postnatal development and that they may be differentially vulnerable to the disease process of schizophrenia at specific stages of postnatal development.

Spontaneous regeneration after resection of various lengths of hypoglossal nerve in rats.

OBJECTIVES: The objective of this study was to investigate spontaneous neural regeneration and functional recovery after resection of various lengths of the hypoglossal (XII) nerve in adult rats. METHODS: Twelve weeks after XII nerve resection at lengths ranging from 0.0 to 15.8 mm, the tongue deviation angle of rats was measured to evaluate the severity of paralysis; thereafter, the XII neurons in the XII nucleus were labeled with Fluoro-Gold (FG), which was injected into the tongue to visualize regenerated XII neurons re-innervating the tongue muscles. RESULTS: In the XII nerve-resected rats, the regenerative rates, that is, the percentage of the total number of FG-positive neurons on the injured side relative to that on the uninjured side, were divided into two groups; the regenerative rates were more than 77% and less than 6%, respectively. Upon comparing the two groups, the boundary resection length was approximately 10.0 mm. Moreover, the former and latter groups demonstrated tongue deviation angles less than or greater than 15°, respectively. CONCLUSIONS: The critical nerve gap length for spontaneous neural regeneration was approximately 10.0 mm in XII nerve-resected adult rats, and nerve regeneration occurred in both morphological and functional aspects after resection at less than the critical length.

Evaluation of motor and sensory neuron populations in a mouse median nerve injury model.

BACKGROUND: Peripheral nerves can regenerate and restore function after injury but this process is hindered by many factors including chronic denervation, motor end-plate resorption and Schwann cell senescence. Forelimb injury models in rodents are becoming increasingly popular as they more accurately reflect the physiology and biomechanics of upper extremity nerve injuries. However several aspects of this surgical model remain poorly characterized. NEW METHOD: C57Bl/6 mice underwent enumeration of median nerve motor and sensory neuron pools using retrograde labeling with or without nerve transection. Distal histomorphometry of uninjured mouse median nerves was also examined. Baseline reference values of volitional forelimb grip strength measurements were determined and the rate of neural elongation was also estimated. RESULTS: We identified 1363 ± 165 sensory and 216 ± 16 motor neurons within the uninjured dorsal root ganglia (DRG) and ventral spinal cord, respectively. Eight days following injury, approximately 34% of motoneurons had elongated a distance of 5 mm beyond the repair site 8 days following injury. Volitional grip strength decreased 50% with unilateral median nerve transection and was negligible with contralateral flexor tendon tenotomy. COMPARISON WITH EXISTING METHOD: Our spinal cord and DRG harvesting technique presented here was technically straightforward and reliable. Estimates of motor and sensory neuron numbers for the mouse median nerve compared favourably with studies using intramuscular injection of retrograde neurotracer. Histomorphometry data was consistent with and reinforced reference values in the literature. CONCLUSIONS: This study provides data that further develops an increasingly popular surgical model for studying peripheral nerve injury and repair.

Successful retrograde regeneration using a sensory branch for motor nerve transfer.

OBJECTIVE: The objective of this study was to test whether regenerating motor axons from a donor nerve can travel in a retrograde fashion using sensory branches to successfully reinnervate a motor nerve end organ. METHODS: This study has two parts. In part I, rats (n = 30) were assigned to one of five groups for obturator nerve (ON)-to-femoral nerve transfer: group 1, ON-to-saphenous nerve (SN) distal stump; group 2, ON-to-SN proximal stump without femoral nerve proper (FNP) injury; group 3, ON-to-SN proximal stump with FNP crush injury; group 4, ON-to-SN proximal stump with FNP transection injury; and group 5, gold standard transfer, ON-to-motor femoral nerve (MFN) branch. At 8 weeks, retrograde labeling was done from the distal MFN, and the spinal cords were examined to assess the degree of obturator motor axon regeneration across the five groups. In part II, only group 4 was examined (n = 8). Through use of immunostaining and optical tissue clearing methods, the nerve transfer networks were cleared and imaged using light-sheet fluorescence microscopy to visualize the regeneration pathways in 2D and 3D models at 2- and 8-week time points. RESULTS: Proximal FNP transection (group 4) enabled a significantly higher number of retrogradely regenerated motor axons compared with control groups 1-3. Moreover, group 4 had modest, but nonsignificant, superiority of motor neuron counts compared with the positive control group, group 5. Optical tissue clearing demonstrated that the axons traveled in a retrograde fashion from the recipient sensory branch to the FNP mixed stump, then through complex turns, down to distal branches. Immunostaining confirmed the tissue clearing findings and suggested perineurium disruption as a means by which axons could traverse across fascicular boundaries. CONCLUSIONS: Sensory branches can transmit regenerating axons from donor nerves back to main mixed recipient nerves, then distally toward target organs. The extent of retrograde regeneration is markedly influenced by the type and severity of injury sustained by the recipient nerve. Using a sensory branch as a bridge for retrogradely regenerating axons can open new potential horizons in nerve repair surgery for severely injured mixed nerves.

A rabies virus-based toolkit for efficient retrograde labeling and monosynaptic tracing.

Analyzing the structure and function of the brain's neural network is critical for identifying the working principles of the brain and the mechanisms of brain diseases. Recombinant rabies viral vectors allow for the retrograde labeling of projection neurons and cell type-specific trans-monosynaptic tracing, making these vectors powerful candidates for the dissection of synaptic inputs. Although several attenuated rabies viral vectors have been developed, their application in studies of functional networks is hindered by the long preparation cycle and low yield of these vectors. To overcome these limitations, we developed an improved production system for the rapid rescue and preparation of a high-titer CVS-N2c-ΔG virus. Our results showed that the new CVS-N2c-ΔG-based toolkit performed remarkably: (1) N2cG-coated CVS-N2c-ΔG allowed for efficient retrograde access to projection neurons that were unaddressed by rAAV9-Retro, and the efficiency was six times higher than that of rAAV9-Retro; (2) the trans-monosynaptic efficiency of oG-mediated CVS-N2c-ΔG was 2-3 times higher than that of oG-mediated SAD-B19-ΔG; (3) CVS-N2c-ΔG could delivery modified genes for neural activity monitoring, and the time window during which this was maintained was 3 weeks; and (4) CVS-N2c-ΔG could express sufficient recombinases for efficient transgene recombination. These findings demonstrate that new CVS-N2c-ΔG-based toolkit may serve as a versatile tool for structural and functional studies of neural circuits.

Intraneural Topography of Rat Sciatic Axons: Implications for Polyethylene Glycol Fusion Peripheral Nerve Repair.

Peripheral nerve injuries are the most common type of nerve trauma. We have been working with a novel repair technique using a plasmalemmal fusogen, polyethylene glycol (PEG), to re-fuse the membranes of severed axons. PEG-fusion repair allows for immediate re-innervation of distal targets, prevents axonal degeneration, and improves behavioral recovery. PEG-fusion of severed axons is non-specific, and we have previously reported that following injury and PEG-fusion misconnections between spinal motoneurons and their distal targets were present. Surprisingly, appropriately paired proximal and distal motor axons were observed in all PEG-fused animals. We hypothesized that a topographic organization of axons contributing to the sciatic nerve could explain the incidence of appropriate connections. We traced the course of specific axon populations contributing to the sciatic nerve in young adult male and female rats. Following intraneural injection of Fast Blue into the tibial branch, labeled axons were confined to a discrete location throughout the course of the nerve. Following intramuscular injection of cholera toxin-conjugated horseradish peroxidase into the anterior tibialis, labeled axons were confined to a smaller but still discrete location throughout the nerve. In both cases, the relative locations of labeled axons were consistent bilaterally within animals, as well as across animals and sexes. Thus, the relatively consistent location of specific axon populations could allow for realignment of appropriate populations of axons, and enhanced behavioral recovery seen in PEG-fused animals. Knowing the organization of axons within the sciatic nerve promotes accurate territory realignment during repair, therefore aiding in recovery outcomes.

The effects of graft source and orientation on outcomes after ablation of a branched peripheral nerve.

Segmental peripheral nerve injuries (PNI) are the most common cause of enduring nervous system dysfunction. The peripheral nervous system (PNS) has an extensive and highly branching organization. While much is known about the factors that affect regeneration through sharp bisections and linear ablations of peripheral nerves, very little has been investigated or documented about PNIs that ablate branch points. Such injuries present additional complexity compared to linear segmental defects. This study compared outcomes following ablation of a branch point with branched grafts, specifically examining how graft source and orientation of the branched graft contributed to regeneration. The model system was Lewis rats that underwent a 2.5 cm ablation that started in the sciatic nerve trunk and included the peroneal/tibial branch point. Rats received grafts that were rat sciatic autograft, inbred sciatic allograft, and inbred femoral allograft, each of which was a branched graft of 2.5 cm. Allografts were obtained from Lewis rats, which is an inbred strain. Both branches of the sciatic grafts were mixed motor and sensory while the femoral grafts were smaller in diameter than sciatic grafts and one branch of the femoral graft is sensory and the other motor. All branched grafts were sutured into the defect in two orientations dictated by which branch in the graft was sutured to the tibial vs peroneal stumps in recipients. Outcome measures include compound muscle action potentials (CMAPs) and CatWalk gait analysis throughout the recovery period, with toluidine blue for intrinsic nerve morphometry and retrograde labeling conducted at the 36-week experimental end point. Results indicate that graft source and orientation does play a significant role earlier in the regenerative process but by 36 weeks all groups showed very similar indications of regeneration across multiple outcomes.

Chronic Alcohol Dysregulates Glutamatergic Function in the Basolateral Amygdala in a Projection-and Sex-Specific Manner.

Chronic intermittent ethanol and withdrawal (CIE/WD) produces alcohol dependence, facilitates anxiety-like behavior, and increases post-CIE alcohol intake. The basolateral amygdala (BLA) is one of several brain regions that regulates anxiety-like behavior and alcohol intake through downstream projections to the nucleus accumbens (NAC) and bed nucleus of the stria terminalis (BNST), respectively. Previous studies revealed that CIE/WD induces input- and sex-specific adaptations to glutamatergic function in the BLA. The BLA receives information from two distinct input pathways. Glutamatergic afferents from medial structures like the thalamus and prefrontal cortex enter the BLA through the stria terminalis whereas lateral cortical structures like the anterior insula cortex enter the BLA through the external capsule. CIE/WD increases presynaptic glutamatergic function at stria terminalis synapses and postsynaptic function at external capsule synapses. Previous studies sampled neurons throughout the BLA, but did not distinguish between projection-specific populations. The current study investigated BLA neurons that project to the NAC (BLA-NAC neurons) or the BNST (BLA-BNST neurons) as representative "reward" and "aversion" BLA neurons, and showed that CIE/WD alters glutamatergic function and excitability in a projection- and sex-specific manner. CIE/WD increases glutamate release from stria terminalis inputs only onto BLA-BNST neurons. At external capsule synapses, CIE/WD increases postsynaptic glutamatergic function in male BLA-NAC neurons and female BLA-BNST neurons. Subsequent experiments demonstrated that CIE/WD enhanced the excitability of male BLA-NAC neurons and BLA-BNST neurons in both sexes when glutamatergic but not GABAergic function was intact. Thus, CIE/WD-mediated increased glutamatergic function facilitates hyperexcitability in male BLA-NAC neurons and BLA-BNST neurons of both sexes.