Large Axons Research Articles

Immunocytochemical Evidence for Electrical Synapses in the Dorsal Descending and Dorsal Anterior Octaval Nuclei in the Goldfish, Carassius auratus

The dorsal portion of the descending octaval nucleus (dDO), the main first-order auditory nucleus in jawed fish, includes four lateral and three medial neuronal populations that project to the auditory midbrain. One medial population and one lateral population contain neurons that receive a remarkably large axon terminal from the utricular branch of the octaval nerve. Immunocytochemistry for connexin 35 (Cx35) was used to determine whether this connection includes electrical synapses. Although Cx35 was not localized to these large contacts, it was observed in the three other lateral dDO populations. Another first-order nucleus, the dorsal portion of the anterior octaval nucleus (dAO), primitively projects to the auditory midbrain in jawed fishes and contains neurons positive for Cx35. Utricular branch terminals were coincident with some Cx35 puncta in dDO and dAO. The results are discussed in light of what is known about the occurrence of electrical synapses in first-order auditory and vestibular nuclei in fish and tetrapods.

Cerebrospinal fluid neurofilament light chain tracks cognitive impairment in multiple sclerosis.

Cognitive impairment (CI) is a disabling symptom of multiple sclerosis (MS). Axonal damage disrupts neural circuits andmay play a role in determining CI, but its detection and monitoring are not routinely performed. Cerebrospinal fluid (CSF) neurofilament light chain (NfL) is a promising marker of axonal damage in MS. To retrospectively examine the relationship between CSF NfL and CI in MS patients. CSF NfL concentration was measured in 28 consecutive newly diagnosed MS patients who underwent a neuropsychological evaluation with the Brief Repeatable Battery of Neuropsychological tests (BRBN). CSF NfL was higher in patients with overall CI (947.8 ± 400.7 vs 518.4 ± 424.7pg/mL, p < 0.01), and with impairment in information processing speed (IPS) (820.8 ± 413.6 vs 513.6 ± 461.4pg/mL, p < 0.05) and verbal fluency (1292 ± 511 vs 582.8 ± 395.4pg/mL, p < 0.05), and it positively correlated with the number of impaired BRBN tests (r = 0.48, p = 0.01) and cognitive domains (r = 0.47, p = 0.01). Multivariate analyses taking into account potential confounders confirmed these findings. CSF NfL is higher in MS patients with CI and impaired IPS and verbal fluency. Large myelinated axons injury, causing neural disconnection, may be an important determinant of CI in MS and can be reliably measured through CSF NfL.

Propagation and Selectivity of Axonal Loss in Leber Hereditary Optic Neuropathy

Leber hereditary optic neuropathy (LHON) is a syndrome of subacute loss of central vision associated with mutations in mitochondrial DNA coding for components of complex I. LHON preferentially involves small axons in the temporal optic nerve, but the reason is unclear. We performed a Monte Carlo simulation of the spread of injury in LHON axons to better understand the predilection for small axons. Optic nerve slices were modeled as grids containing axons with sizes from reported regional distributions. The propagation of injury from a localized concentration of superoxide was simulated as the spread via passive diffusion from one axon to adjacent axons, with basal production and scavenging rate proportional to axonal area and volume, respectively. Axonal degeneration occurred when intra-axonal concentrations reached a toxic threshold. Simulations demonstrated that almost all small and medium axons degenerated by the time steady-state was reached, but about 50% of large axons were preserved. The location of initial injury affected time to steady state, with nasal injuries reaching steady state faster than temporal injuries. The pattern of axonal degeneration in the simulations mirrored both visual fields and optic nerve histology from patients with LHON. These results provide insight into the nature of axonal loss in LHON.

Pharmacological Attenuation of Electrical Effects in a Model of Compression Neuropathy.

Peripheral nerve compression and entrapment can be debilitating. Using a validated animal model of peripheral nerve compression, we examined the utility of 2 drugs approved for other uses in humans, 4-aminopyridine (4-AP) and erythropoietin (EPO), as treatments for surgically induced ischemia and as adjuvants to surgical decompression. Peripheral nerve compression was induced in wild-type mice by placing an inert silicone sleeve around the sciatic nerve. Decompression surgery was performed at 6 weeks with mice receiving 4-AP, EPO, or saline solution either during and after compression or only after decompression. A nerve conduction study and morphometric analyses were performed to compare the extent of the injury and the efficacy of the therapies, and the findings were subjected to statistical analysis. During peripheral nerve compression, there was a progressive decline in nerve conduction velocity compared with that in sham-treatment animals, in which nerve conduction velocity remained normal (∼55 m/s). Mice treated with 4-AP or EPO during the compression phase had significantly smaller declines in nerve conduction velocity and increased plateau nerve conduction velocities compared with untreated controls (animals that received saline solution). Histomorphometric analyses of newly decompressed nerves (i.e., nerves that underwent decompression on the day that the mouse was sacrificed) revealed that both treated groups had significantly greater proportions of large (>5-µm) axons than the untreated controls. Following surgical decompression, all animals recovered to a normal baseline nerve conduction velocity by day 15; however, treatment significantly accelerated improvement (in both the 4-AP and the EPO group), even when it was only started after decompression. Histomorphometric analyses at 7 and 15 days following surgical decompression revealed significantly increased myelin thickness and significantly greater proportions of large axons among the treated animals. Both the 4-AP and the EPO-treated group demonstrated improvements in tissue architectural and electrodiagnostic measurements, both during and after peripheral nerve compression, compared with untreated mice. Peripheral nerve decompression is one of the most commonly performed procedures in orthopaedic surgery. We believe that there is reason for some optimism about the translation of our findings to the clinical setting. Our findings in this murine model suggest that 4-AP and EPO may lessen the effects of nerve entrapment and that the use of these agents after decompression may speed and perhaps otherwise optimize recovery after surgery.

Histopathological Evaluation of Inferior Alveolar Neurovascular Bundle in Cases of Trigeminal Neuralgia.

Trigeminal neuralgia is a painful disease that has been afflicting mankind since time immemorial. The etiology and pathophysiology have been widely studied but poorly understood. There are well-documented researches analyzing ultrastructural changes in trigeminal root specimens obtained following microvascular decompression surgery. However, there are no studies evaluating microscopic changes following peripheral neurectomy. The present study examined microscopic changes in inferior alveolar neurovascular bundle in trigeminal neuralgia patients of mandibular division with no underlying cause. The biopsy specimens were obtained from peripheral neurectomy of 11 trigeminal neuralgia patients' refractory to anti-neuralgic medications. The autopsy specimens from 10 cadavers were used as control. The specimens were subjected to histopathological examination by hematoxylin and eosin, Masson trichrome and Luxol fast blue stains. All biopsy specimens reported luminal occlusion of small vessels, medial degeneration and intense mononuclear inflammatory infiltrate. Focal myelin digestion chambers were observed in large and small axons. No pathological alterations of either blood vessel or nerve fibers were reported in autopsy specimens. The demyelination of inferior alveolar nerve due to pathologic vascular changes in peripheral vasculature may have a role in initiation and precipitation of trigeminal neuralgia, and hence, peripheral neurectomy has a significant role in alleviating pain.

The Success and Failure of the Schwann Cell Response to Nerve Injury.

The remarkable plasticity of Schwann cells allows them to adopt the Remak (non-myelin) and myelin phenotypes, which are specialized to meet the needs of small and large diameter axons, and differ markedly from each other. It also enables Schwann cells initially to mount a strikingly adaptive response to nerve injury and to promote regeneration by converting to a repair-promoting phenotype. These repair cells activate a sequence of supportive functions that engineer myelin clearance, prevent neuronal death, and help axon growth and guidance. Eventually, this response runs out of steam, however, because in the long run the phenotype of repair cells is unstable and their survival is compromised. The re-programming of Remak and myelin cells to repair cells, together with the injury-induced switch of peripheral neurons to a growth mode, gives peripheral nerves their strong regenerative potential. But it remains a challenge to harness this potential and devise effective treatments that maintain the initial repair capacity of peripheral nerves for the extended periods typically required for nerve repair in humans.

A comparison between single and double cable neuron models applicable to deep brain stimulation

Computational models for activation assessment in deep brain stimulation (DBS) are commonly based on neuronal cable equations. The aim was to systematically compare the activation distance between a single cable model implemented in MATLAB, and a well-established double cable model implemented in NEURON. Both models have previously been used for DBS studies. The field distributions generated from a point source and a 3389 DBS lead were applied to the neuron models as input stimuli. Simulations (n = 670) were performed with intersecting axon diameters (D) between the models (2.0, 3.0, 5.7, 7.3, 8.7, 10.0 μm), variation in pulse shape and amplitude settings (0 to 5 in increments of 0.5 mA or V) with the single cable model as reference. Both models responded linearly to change of input (point source: 0.93 < R2 < 0.99, DBS source: R2 > 0.98), but with a systematic extended activation distance for the single cable model. The difference for a point source ranged from −0.2 mm (D = 2.0 μm) to −1.1 mm (D = 5.7 μm). For the DBS lead a D = 3.2 μm agreed with the commonly used double cable simulations D =5.7 μm in voltage mode. Possible reasons for the deviation at larger axons are the internodal length, the ion channel selection and physiological data behind the models. The single cable model covers a continuous range of small axon diameters and calculated the internodal length for each iteration, whereas the double cable models uses fixed defined axon diameters and tabulated data for the internodal length. Despite different implementations and model complexities, both models present similar sensitivity to pulse shape, amplitude and axon diameter. With awareness of the strength and weakness both models can be used to extract activation distance used to relate a specific electric field isolevel and thus estimate the volume of tissue activated in DBS simulation studies.

Early short-term PXT3003 combinational therapy delays disease onset in a transgenic rat model of Charcot-Marie-Tooth disease 1A (CMT1A).

The most common type of Charcot-Marie-Tooth disease is caused by a duplication of PMP22 leading to dysmyelination, axonal loss and progressive muscle weakness (CMT1A). Currently, no approved therapy is available for CMT1A patients. A novel polytherapeutic proof-of-principle approach using PXT3003, a low-dose combination of baclofen, naltrexone and sorbitol, slowed disease progression after long-term dosing in adult Pmp22 transgenic rats, a known animal model of CMT1A. Here, we report an early postnatal, short-term treatment with PXT3003 in CMT1A rats that delays disease onset into adulthood. CMT1A rats were treated from postnatal day 6 to 18 with PXT3003. Behavioural, electrophysiological, histological and molecular analyses were performed until 12 weeks of age. Daily oral treatment for approximately 2 weeks ameliorated motor deficits of CMT1A rats reaching wildtype levels. Histologically, PXT3003 corrected the disturbed axon calibre distribution with a shift towards large motor axons. Despite dramatic clinical amelioration, only distal motor latencies were improved and correlated with phenotype performance. On the molecular level, PXT3003 reduced Pmp22 mRNA overexpression and improved the misbalanced downstream PI3K-AKT / MEK-ERK signalling pathway. The improved differentiation status of Schwann cells may have enabled better long-term axonal support function. We conclude that short-term treatment with PXT3003 during early development may partially prevent the clinical and molecular manifestations of CMT1A. Since PXT3003 has a strong safety profile and is currently undergoing a phase III trial in CMT1A patients, our results suggest that PXT3003 therapy may be a bona fide translatable therapy option for children and young adolescent patients suffering from CMT1A.

Gallyas Silver Impregnation of Myelinated Nerve Fibers.

In the nervous system of vertebrates, nerve impulse propagation is accelerated by the ensheathment of neuronal axons with myelin. Myelin sheaths are molecularly specialized, lipid-rich plasma membrane extensions of Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system (CNS). To visualize myelinated nerve fibers and to allow for the morphological analyses of myelin in the brain and the spinal cord, an efficient method for silver impregnation of myelin has originally been developed by Ferenc Gallyas in 1979, referred to as Gallyas silver impregnation. Gallyas' method is based on the agyrophilic characteristic of myelin to form and bind silver particles, while this process is suppressed in tissues other than myelin. The silver particles are finally enhanced in a developing step ("physical developer"). The main advantage of this method is that it efficiently visualizes both large myelinated fiber tracts and individual myelinated axons. Here we provide our laboratory protocol that is suitable for paraffin embedded sections and the use of light microscopy based on Gallyas' original protocol and subsequent modifications by Pistorio and colleagues.

Local Acceleration of Neurofilament Transport at Nodes of Ranvier.

Myelinated axons are constricted at nodes of Ranvier. These constrictions are important physiologically because they increase the speed of saltatory nerve conduction, but they also represent potential bottlenecks for the movement of axonally transported cargoes. One type of cargo are neurofilaments, which are abundant space-filling cytoskeletal polymers that function to increase axon caliber. Neurofilaments move bidirectionally along axons, alternating between rapid movements and prolonged pauses. Strikingly, axon constriction at nodes is accompanied by a reduction in neurofilament number that can be as much as 10-fold in the largest axons. To investigate how neurofilaments navigate these constrictions, we developed a transgenic mouse strain that expresses a photoactivatable fluorescent neurofilament protein in neurons. We used the pulse-escape fluorescence photoactivation technique to analyze neurofilament transport in mature myelinated axons of tibial nerves from male and female mice of this strain ex vivo Fluorescent neurofilaments departed the activated region more rapidly in nodes than in flanking internodes, indicating that neurofilament transport is faster in nodes. By computational modeling, we showed that this nodal acceleration can be explained largely by a local increase in the duty cycle of neurofilament transport (i.e., the proportion of the time that the neurofilaments spend moving). We propose that this transient acceleration functions to maintain a constant neurofilament flux across nodal constrictions, much as the current increases where a river narrows its banks. In this way, neurofilaments are prevented from piling up in the flanking internodes, ensuring a stable neurofilament distribution and uniform axonal morphology across these physiologically important axonal domains.SIGNIFICANCE STATEMENT Myelinated axons are constricted at nodes of Ranvier, resulting in a marked local decrease in neurofilament number. These constrictions are important physiologically because they increase the efficiency of saltatory nerve conduction, but they also represent potential bottlenecks for the axonal transport of neurofilaments, which move along axons in a rapid intermittent manner. Imaging of neurofilament transport in mature myelinated axons ex vivo reveals that neurofilament polymers navigate these nodal axonal constrictions by accelerating transiently, much as the current increases where a river narrows its banks. This local acceleration is necessary to ensure a stable axonal morphology across nodal constrictions, which may explain the vulnerability of nodes of Ranvier to neurofilament accumulations in animal models of neurotoxic neuropathies and neurodegenerative diseases.

Progranulin reduces insoluble TDP-43 levels, slows down axonal degeneration and prolongs survival in mutant TDP-43 mice

BackgroundTAR DNA binding protein 43 (TDP-43) is the main disease protein in most patients with amyotrophic lateral sclerosis (ALS) and about 50% of patients with frontotemporal dementia (FTD). TDP-43 pathology is not restricted to patients with missense mutations in TARDBP, the gene encoding TDP-43, but also occurs in ALS/FTD patients without known genetic cause or in patients with various other ALS/FTD gene mutations. Mutations in progranulin (GRN), which result in a reduction of ~ 50% of progranulin protein (PGRN) levels, cause FTD with TDP-43 pathology. How loss of PGRN leads to TDP-43 pathology and whether or not PGRN expression protects against TDP-43-induced neurodegeneration is not yet clear.MethodsWe studied the effect of PGRN on the neurodegenerative phenotype in TDP-43(A315T) mice.ResultsPGRN reduced the levels of insoluble TDP-43 and histology of the spinal cord revealed a protective effect of PGRN on the loss of large axon fibers in the lateral horn, the most severely affected fiber pool in this mouse model. Overexpression of PGRN significantly slowed down disease progression, extending the median survival by approximately 130 days. A transcriptome analysis did not point towards a single pathway affected by PGRN, but rather towards a pleiotropic effect on different pathways.ConclusionOur findings reveal an important role of PGRN in attenuating mutant TDP-43-induced neurodegeneration.

Single-axon tracing of the corticosubthalamic hyperdirect pathway in primates.

Individual axons that form the hyperdirect pathway in Macaca fascicularis were visualized following microiontophoretic injections of biotinylated dextran amine in layer V of the primary motor cortex (M1). Twenty-eight singly labeled axons were reconstructed in 3D from serial sections. The M1 innervation of the subthalamic nucleus (STN) arises essentially from collaterals of long-ranged corticofugal axons en route to lower brainstem regions. Typically, after leaving M1, these large caliber axons (2-3µm) enter the internal capsule and travel between caudate nucleus and putamen without providing any collateral to the striatum. More ventrally, they emit a thin collateral (0.5-1.5µm) that runs lateromedially within the dorsal region of the STN, providing boutons en passant in the sensorimotor territory of the nucleus. In some cases, the medial tip of the collateral enters the lenticular fasciculus dorsally and yields a few beaded axonal branches in the zona incerta. In other cases, the collateral runs caudally and innervates the ventrolateral region of the red nucleus where large axon varicosities (up to 1.7µm in diameter) are observed, many displaying perisomatic arrangements. Our ultrastructural analysis reveals a high synaptic incidence (141%) of cortical VGluT1-immunoreactive axon varicosities on distal dendrites of STN neurons, and on various afferent axons. Our single-axon reconstructions demonstrate that the so-called hyperdirect pathway derives essentially from collaterals of long-ranged corticofugal axons that are rarely exclusively devoted to the STN, as they also innervate the red nucleus and/or the zona incerta.

Myelinated axons fail to develop properly in a genetically authentic mouse model of Charcot-Marie-Tooth disease type 2E

We have analyzed a mouse model of Charcot-Marie-Tooth disease 2E (CMT2E) harboring a heterozygous p.Asn98Ser (p.N98S) Nefl mutation, whose human counterpart results in a severe, early-onset neuropathy. Behavioral, electrophysiological, and pathological analyses were done on separate cohorts of NeflN98S/+ mutant mice and their wild type Nefl+/+ littermates between 8 and 48 weeks of age. The motor performance of NeflN98S/+ mice, as evidenced by altered balance and gait measures, was impaired at every age examined (from 6 to 25 weeks of age). At all times examined, myelinated axons were smaller and contained markedly fewer neurofilaments in NeflN98S/+ mice, in all examined aspects of the PNS, from the nerve roots to the distal ends of the sciatic and caudal nerves. Similarly, the myelinated axons in the various tracts of the spinal cord and in the optic nerves were smaller and contained fewer neurofilaments in mutant mice. The myelinated axons in both the PNS and the CNS of mutant mice had relatively thicker myelin sheaths. The amplitude and the nerve conduction velocity of the caudal nerves were reduced in proportion with the diminished sizes of myelinated axons. Conspicuous aggregations of neurofilaments were only seen in primary sensory and motor neurons, and were largely confined to the cell bodies and proximal axons. There was evidence of axonal degeneration and regeneration of myelinated axons, mostly in distal nerves. In summary, the p.N98S mutation causes a profound reduction of neurofilaments in the myelinated axons of the PNS and CNS, resulting in substantially reduced axonal diameters, particularly of large myelinated axons, and distal axon loss in the PNS.

F31. Longitudinal neurophysiological study of dominant intermediate Charcot-Marie-Tooth type C neuropathy

Dominant intermediate Charcot-Marie-Tooth neuropathy subtype C (DI-CMTC) is associated with mutations in the YARS gene, encoding tyrosyl-tRNA synthetase (TyrRS). In a prospectively designed study we were the first to report the phenotype and genotype of DI-CMTC from a U.S. and a Bulgarian family using electrodiagnostic (EDX) and morphological studies. Here we present longitudinal data for the U.S. family 16 years after the initial evaluation. After re-consenting, 13 of 21 original DI-CMTC participants underwent EDX and physical examinations. Data of 2000 and 2016 were compared for specific nerves using paired t-tests. General linear model univariate analysis was used to relate findings to patient age. Data are presented as mean ± SD. Four women and 9 men were examined at ages 8–77 years in 2000 and 24–93 years in 2016. During this time median nerve compound muscle action potential (CMAP) amplitudes decreased from 9.3 ± 2.9 mV to 6.0 ± 2.9 mV (p = 0.002), while distal motor latencies (DML) increased from 4.2 ± 0.6 ms to 4.5 ± 0.6 ms (p = 0.04); mean motor nerve conduction velocity (MNCV) remained largely unchanged at 37 m/s. Over the same period ulnar nerve CMAP amplitudes decreased from 9.2 ± 2.1 mV to 6.1 ± 1.8 mV (p = 0.004); mean MNCV remained the same at 39 m/s. In 2000 and 2016 all adult participants had absent peroneal motor responses, as well as absent tibial responses (except for 2 patients with CMAP amplitudes <1 mV and MNCV 32–35 m/s in 2000 and 28–32 m/s in 2016). In the interval sural sensory nerve action potential (SNAP) amplitudes changed from 2.0 ± 2.5 mV to 1.0 ± 2.4 mV (p = 0.10), and sensory nerve conduction velocities (SNCV) from 39.3 ± 1.0 to 35.5 ± 2.1 m/s (p = 0.13); this did not reach statistical significance. Median nerve sensory peak latencies increased from 3.4 ± 0.4 to 3.9 ± 0.4 ms (p = 0.01), SNAP amplitudes changed from 7.2 ± 5.3 to 6.5 ± 5.1 mV (p = 0.055), and SNCV from 46 ± 1.7 to 41.3 ± 7.6 m/s (p = 0.328). There was an age-dependent decrease of CMAP amplitudes of median (p = 0.044), peroneal and tibial (p < 0.001), and SNAP amplitudes of median(p = 0.008), radial (p = 0.013) and sural (p = 0.028). EMG revealed distal dominant acute and chronic denervation and reinnervation in lower limb muscles and some hand muscles. In this study MNCV remained stable over 16 years, but motor and sensory amplitudes decreased. DI-CMT C appears to produce a progressive age- and nerve length-dependent axonal degenerative process. Longitudinal EDX findings are consistent with our previous cross-sectional morphological study of sural nerves, which also revealed an age-dependent loss of large and small myelinated axons.

The Effect of Axon Resealing on Retrograde Neuronal Death after Spinal Cord Injury in Lamprey.

Failure of axon regeneration in the central nervous system (CNS) of mammals is due to both extrinsic inhibitory factors and to neuron-intrinsic factors. The importance of intrinsic factors is illustrated in the sea lamprey by the 18 pairs of large, individually identified reticulospinal (RS) neurons, whose axons are located in the same spinal cord tracts but vary greatly in their ability to regenerate after spinal cord transection (TX). The neurons that are bad regenerators also undergo very delayed apoptosis, signaled early by activation of caspases. We noticed that the neurons with a low probability of axon regeneration tend to be larger than the good regenerators. We postulate that the poorly regenerating larger neurons have larger caliber axons, which reseal more slowly, allowing more prolonged entry of toxic signals (e.g., Ca++) into the axon at the injury site. To test this hypothesis, we used a dye-exclusion assay, applying membrane-impermeable dyes to the cut ends of spinal cords at progressively longer post-TX intervals. Axons belonging to the very small neurons (not individually identified) of the medial inferior RS nucleus resealed within 15 min post-TX. Almost 75% of axons belonging to the medium-sized identified RS neurons resealed within 3 h. At this time, only 36% of the largest axons had resealed, often taking more than 24 h to exclude the dye. There was an inverse relationship between an RS neuron’s size and the probability that its axon would regenerate (r = −0.92) and that the neuron would undergo delayed apoptosis, as indicated by staining with a fluorescently labeled inhibitor of caspases (FLICA; r = 0.73). The artificial acceleration of resealing with polyethylene glycol (PEG) reduced retrograde neuronal apoptosis by 69.5% at 2 weeks after spinal cord injury (SCI), suggesting that axon resealing is a critical determinant of cell survival. Ca++-free Ringer’s solution with EGTA prolonged the sealing time and increased apoptotic signaling, suggesting that factors other than Ca++ diffusion into the injured tip contribute to retrograde death signaling. A longer distance of the lesion from the cell body reduced apoptotic signaling independent of the axon sealing time.

Immunohistochemestry for nerve morphometry?

Peripheral nerve biopsy is an invaluable aid to diagnosis carefully selected patients. The nerves histological evaluation gives clues for investigating diseases mechanisms and causes, as well as guides the therapeutic planning of inflammatory, infectious, demyelinating or degenerative lesions [1]. Nerves histological processing method that provides the best image quality is the epoxy resin embedding. Thus, nerve sections can be observed by light and/or transmission electron microscopy (TEM) and classes of nerve fibers can be morphometrically identified and studied. Epoxy resin embedding and TEM is considered the gold standard method for unmyelinated fibers identification and quantification. More recently, the immunohistochemistry technique (IHC) is being added to the inflammatory neuropathy investigation and the most important of the panel of antibodies employed are those needed to differentiate lymphocytes into T (CD3þve) and B cells (CD20þve). In addition, antibodies against macrophages, myelin basic protein, neurofilaments, and epithelial membrane antigen (EMA) (for perineurial cells) are included [1]. Neurotransmitters and their fiber “carrier” are much less explored. We suggest that it would be possible to use the IHC to identify nerve fiber classes as an alternative to TEM and morphometry, with the gain on functional information. To investigate this hypothesis we used IHQ and immunofluorescence (IF) to immunolabel small myelinated and unmyelinated fibers in sural nerves of rats. The nerves were surgically removed, immersed in paraformaldehyde 4% for 18 hours, and cryoprotected in increasing concentrations of sucrose solutions, before transversal cryosectioning (12μm). For IHC, sections were incubated in rabbit polyclonal antibody anti‐substance P (1:5.000, 18h) followed by anti‐rabbit IgG (1:500; 2h) and Extravidin‐HRP (1:1500, 2h). A reaction with diaminobenzidine intensified with nickel revealed immunoreactivity, observed by light microscopy. For IF, sections were incubated in rabbit polyclonal antibody anti‐substance P (1:1.000, 18h) followed by anti‐rabbit IgG (Alexa Fluor® 594) (1:200, 2h). The immunoreactivity was observed by fluorescence microscopy. Both protocols worked on transverse sections to identify substance P immunolabeled small myelinated and unmyelinated fibers. Results were compared with transverse sections prepared for TEM and the IHC results showed labeled small myelinated and unmyelinated axons intermingled with large unlabeled myelinated axons, with preservation of their myelin sheath. The IHC transverse sections were comparable to the epoxy resin embedded for small fiber localization in the endoneural space. Further studies on quantification of these fibers will be performed comparing both methods (IHC and IF) with the TEM images in order to show that IHC might also be a reliable method for quantifying axons.Support or Funding InformationFinancial support: FAPESP, CNPq, CAPES and FAEPAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phrenic afferent stimulation modulates cardiorespiratory output in the adult rat

Anatomical work from our laboratory indicates that small and large diameter phrenic nerve afferent fibers localize to the dorsal laminae, project throughout the ipsilateral spinal gray matter and occasionally reach the contralateral dorsal horn. In ongoing experiments, we are testing the hypothesis that activation of phrenic afferent neurons modulates contralateral phrenic motor output and arterial blood pressure in the adult rat. In anesthetized and ventilated adult Sprague‐Dawley rats, one phrenic nerve was electrically stimulated using a bipolar electrode, while respiratory bursting in the contralateral phrenic nerve and arterial blood pressure were recorded. Inspiratory triggered phrenic nerve stimulation was delivered for 20 seconds (approximately 18 breaths) at 40pps using a narrow (0.1 ms) pulse width to target activation of large diameter myelinated axons, or a wide (1.0 ms) pulse width to recruit small diameter myelinated and unmyelinated afferents. The experimental protocol consisted of eight currents (10, 25, 50, 70, 90, 120, 160, 220μA) delivered in a random order, each separated by a 5 minute recovery period (i.e. no stimulation). Each stimulus current caused a progressive increase in contralateral phrenic amplitude during the stimulation period. Phrenic amplitude remained above baseline for at least 1 minute after the cessation of stimulation. At lower currents (&lt;50μA), the wide pulse stimulation was more effective at increasing contralateral phrenic output compared to the narrow pulse stimulation, while higher currents (&gt;50μA) elicited a similar increase in contralateral amplitude regardless of pulse width. In addition, phrenic nerve stimulation was associated with a brief decrease, followed by an increase in mean arterial blood pressure during stimulation. In separate rats, a unilateral dorsal root rhizotomy (C3, C4, C5) was performed prior to phrenic nerve stimulation to eliminate afferent input to the spinal cord. This treatment prevented both the increase in contralateral phrenic amplitude and blood pressure at all currents and both pulse widths, indicating that the observed responses are due to activation of phrenic afferents. These preliminary results confirm that activation of phrenic afferents can modulate the activity of the contralateral phrenic motor pool, and suggest that phrenic afferents are capable of initiating phrenic motor plasticity.Support or Funding Information1F32NS095620‐01 (KS), T32‐ND043730 (MS), SPARC OT2 OD023854 (DDF), 1 R01 NS080180‐01A1 (DDF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Mauthner cell in a fish with top-performance and yet flexibly tuned C-starts. I. Identification and comparative morphology.

Archerfish use two powerful C-starts: one to escape threats, the other to secure prey that they have downed with a shot of water. The two C-starts are kinematically equivalent and variable in both phases, and the predictive C-starts - used in hunting - are adjusted in terms of the angle of turning and the final linear speed to where and when their prey will hit the water surface. Presently, nothing is known about the neural circuits that drive the archerfish C-starts. As the starting point for a neuroethological analysis, we first explored the presence and morphology of a pair of Mauthner cells, which are key cells in the teleost fast-start system. We show that archerfish have a typical Mauthner cell in each medullary hemisphere and that these send by far the largest axons down the spinal cord. Stimulation of the spinal cord caused short-latency all-or-none field potentials that could be detected even at the surface of the medulla and that had the Mauthner cell as its only source. The archerfish's Mauthner cell is remarkably similar morphologically to that of equally sized goldfish, except that the archerfish's ventral dendrite is slightly longer and its lateral dendrite thinner. Our data provide the necessary starting point for the dissection of the archerfish fast-start system and of any role potentially played by its Mauthner cell in the two C-start manoeuvres. Moreover, they do not support the recently expressed view that Mauthner cells should be reduced in animals with highly variable fast-start manoeuvres.

Transcriptional Reorganization of Drosophila Motor Neurons and Their Muscular Junctions toward a Neuroendocrine Phenotype by the bHLH Protein Dimmed.

Neuroendocrine cells store and secrete bulk amounts of neuropeptides, and display morphological and molecular characteristics distinct from neurons signaling with classical neurotransmitters. In Drosophila the transcription factor Dimmed (Dimm), is a prime organizer of neuroendocrine capacity in a majority of the peptidergic neurons. These neurons display large cell bodies and extensive axon terminations that commonly do not form regular synapses. We ask which molecular compartments of a neuron are affected by Dimm to generate these morphological features. Thus, we ectopically expressed Dimm in glutamatergic, Dimm-negative, motor neurons and analyzed their characteristics in the central nervous system and the neuromuscular junction. Ectopic Dimm results in motor neurons with enlarged cell bodies, diminished dendrites, larger axon terminations and boutons, as well as reduced expression of synaptic proteins both pre and post-synaptically. Furthermore, the neurons display diminished vesicular glutamate transporter, and signaling components known to sustain interactions between the developing axon termination and muscle, such as wingless and frizzled are down regulated. Ectopic co-expression of Dimm and the insulin receptor augments most of the above effects on the motor neurons. In summary, ectopic Dimm expression alters the glutamatergic motor neuron phenotype toward a neuroendocrine one, both pre- and post-synaptically. Thus, Dimm is a key organizer of both secretory capacity and morphological features characteristic of neuroendocrine cells, and this transcription factor affects also post-synaptic proteins.

Action Potential Dynamics in Fine Axons Probed with an Axonally Targeted Optical Voltage Sensor.

The complex and malleable conduction properties of axons determine how action potentials propagate through extensive axonal arbors to reach synaptic terminals. The excitability of axonal membranes plays a major role in neural circuit function, but because most axons are too thin for conventional electrical recording, their properties remain largely unexplored. To overcome this obstacle, we used a genetically encoded hybrid voltage sensor (hVOS) harboring an axonal targeting motif. Expressing this probe in transgenic mice enabled us to monitor voltage changes optically in two populations of axons in hippocampal slices, the large axons of dentate granule cells (mossy fibers) in the stratum lucidum of the CA3 region and the much finer axons of hilar mossy cells in the inner molecular layer of the dentate gyrus. Action potentials propagated with distinct velocities in each type of axon. Repetitive firing broadened action potentials in both populations, but at an intermediate frequency the degree of broadening differed. Repetitive firing also attenuated action potential amplitudes in both mossy cell and granule cell axons. These results indicate that the features of use-dependent action potential broadening, and possible failure, observed previously in large nerve terminals also appear in much finer unmyelinated axons. Subtle differences in the frequency dependences could influence the propagation of activity through different pathways to excite different populations of neurons. The axonally targeted hVOS probe used here opens up the diverse repertoire of neuronal processes to detailed biophysical study.