Distribution Of Axons Research Articles

Coarse-graining model reveals universal exponential scaling in axonal length distributions

Abstract The Exponential Distance Rule (EDR) is a well-documented phenomenon suggesting that the distribution of axonal lengths in the brain follows an exponential decay pattern. 
Nevertheless, individual-level axon data supporting this assertion is limited to Drosophila and mice, while inter-region connectome data is also accessible for macaques, marmosets, and humans. 
Although axon-level data in Drosophila and mice support the generality of the EDR, region-level data can significantly deviate from the exponential curve. 
In this study, we establish that the axon number-weighted length distribution of region-level connections converges onto a universal curve when rescaled to the mean axonal length, demonstrating similarities across different species. 
To explain these observations, we present a simple mathematical model that attributes the observed deviations from the EDR in the weighted length distribution of inter-regional connectomes to the inherent coarse-graining effect of translating from neuron-level to region-level connectomics.
We demonstrate that the qualitative predictions of the model are robust with respect to various aspects of brain region-geometry, including dimensionality, resolution, and curvature.
On the other hand, the performance of the model exhibits a monotonous dependence on the amount of region-geometry related detail incorporated into the model.
The findings validate the universality of the EDR rule across various species, paving the way for further in-depth exploration of this remarkably simple principle.

Topographical distribution and morphology of TH-IR and VAChT-IR axons in the antrum, pylorus, and duodenum of rats

The autonomic nervous system plays an essential role in controlling gastrointestinal functions including a variety of physiological functions such as the regulation of motility, gut secretion, and vascular flow. Tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT) are commonly used markers for sympathetic and parasympathetic innervation, respectively. However, the regional distribution and morphology of TH-IR and VAChT-IR axons in the submucosa and mucosa are not well documented. In this study, the rat antrum-pylorus-duodenum (APD) were transversely and longitudinally sectioned. A Zeiss M2 imager was used to scan the serial sections of each APD (each section montage consisted of 50–100 all-in-focus maximal projection images). To determine the detailed structures of TH-IR and VAChT-IR axons, we used the confocal microscope to scan the regions of interest. We found that (1) TH-IR axons innervated the muscular, submucosal, and mucosal layers. In the muscular layer, the submucosa and the mucosa, TH-IR varicose axons innervated the muscles and innervated blood vessels. In the villi, TH-IR axons were presented. (2) VAChT-IR axons heavily innervated the muscular, submucosal, and mucosal layers. In the muscular layer, VAChT-IR varicose axons densely innervated the muscles and myenteric neurons were VAChT positive. In the submucosa and mucosa, VAChT-IR axons innervated as well. In the villi, VAChT-IR axons were densely innervated. (3) TH-IR and VAChT-IR axons were found not co-localized in the muscular, submucosal, and mucosal layers. This work provided a comprehensive view of the distribution and morphology of TH-IR and VAChT-IR axons in the APD at single cell/axon/varicosity scale. This data will be used to create a 3D mapping of the TH-IR and VAChT-IR axons innervation of the APD in rats. This study was supported by NIH HEAL/SPARC U01 NS113867-01 and NIH R15HL137143-01A1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Remodeling of Ventricular Catecholaminergic Axons Following Chronic Intermittent Hypoxia in Mice

Chronic intermittent hypoxia (CIH) is a widely employed model for sleep apnea. The imbalance of autonomic control may contribute to CIH-induced cardiovascular diseases. Previously, we demonstrated that CIH increases sympathetic innervation of the atria. In this study, we determined whether CIH remodels peripheral cardiac tyrosine hydroxylase (TH, a marker for sympathetic efferent axons) innervation in the whole ventricles. The two challenges here were: 1) labeling the entire TH-IR axon innervation in the full thickness (~500 μm) of whole flat-mount ventricles. 2) Quantifying the topographical distribution of TH-IR axons through the thick ventricular walls at the single axon/varicosity scale. In this study, C57BL/6J mice (male, 2 months old, n=7/group) were exposed to room air (RA, 21% O2) or CIH (alternating between 21% and 5.7% O2 every 6 minutes for 10 hours/day) for 8-10 weeks. We prepared flat-mounts of the whole ventricles and septum and processed them with immunohistochemical labeling for TH. Using the Zeiss M2 Imager, confocal microscope, Zeiss Arivis Vision 4D, and Neurolucida system, we traced and digitized the TH-IR axons. We found that: 1) In RA and CIH mice, several large TH-immunoreactive (TH-IR) bundles from the base area entered the ventricles and septum. These large bundles branched into numerous smaller bundles as they traveled downward toward the apex and finally developed into fine varicose axons and terminals in the epicardial and myocardial layers. 2) Using Vision 4D, we were able to accurately detect, trace, and digitize all TH-IR axons in the ventricle walls with high accuracy. 3) CIH increased overall TH-IR axon density and network complexity of the whole ventricles and septum. 3) Abnormal CIH TH-IR axon terminal structures were frequently seen. 4) We geometrically and precisely registered the tracing data into a scaffold map for presentation and comparison in 3D. Altogether, CIH increased sympathetic innervation of the heart which may contribute to the augmented sympathetic drive. Our success in mapping data in the 3D heart scaffold will establish a heart-brain connectome/atlas. Supported by NIH 1 U01 NS113867-01 NIH R15 1R15HL137143-01A1, 2R01HL137832-05 and Carl Zeiss. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Electroacupuncture improves peripheral neuropathy by up-regulating Sirt1/PGC-1α/TFAM pathway in type 2 diabetes rats with peripheral neuropathy.

To observe the effect of electroacupuncture (EA) on activation of silent information regulator 1 (Sirt1)/peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α)/mitochondrial transcription factor A (TFAM) pathway in type 2 diabetes (T2DM) rats with peripheral neuropathy (DPN) , so as to explore its possible mechanisms underlying improvement of DPN. Thirty male SD rats were randomly divided into blank control group (n=8) and DPN model group (n=22) which were further divided into model group (n=8) and EA group (n=8) after successful modeling. The model of T2DM was established by high-fat diet and low-dose intraperitoneal injection of streptozocin (35 mg/kg). For rats of the EA group (anesthetized with isoflurane), EA stimulation (2 Hz/15 Hz, 2 mA) was applied to "Tianshu"(ST25) for 20 min, once daily, 6 times a week for 6 weeks. The blood glucose level, body weight, area under curve (AUC) of glucose tolerance test, and hind-paw mechanical pain threshold and thermal pain threshold were observed. The intra-epidermal nerve fiber density (IENFD) of the hind-foot pad was observed by immunofluorescence staining. The motor nerve conduction velocity (MNCV) of the sciatic nerve was measured by using electrophysiological method. H.E. staining was used to observe the histopathological changes of the sciatic nerve after modeling. Transmission electron microscopy (TEM) was used to observe the ultrastructural changes of the sciatic nerve. The protein expressions of energy-related Sirt1, PGC-1α and TFAM in the sciatic nerve was detected by Western blot. Compared with the blank control group, the model group had a higher blood glucose contents and AUC (P<0.001), a slower MNCV (P<0.01), and a decrease in the body weight and in the mechanical and thermal pain thresholds (P<0.001) and IENFD (P<0.001), and in the expression levels of Sirt1, PGC-1α and TFAM (P<0.05, P<0.01). In contrast to the model group, the EA group had a decrease in the blood glucose contents and AUC (P<0.05, P<0.01), and an increase in mechanical and thermal pain thresholds, MNCV, IENFD, and expression levels of Sirt1, PGC-1α and TFAM proteins (P<0.01, P<0.05). In addition, results of histopathological and ultrastructural changes of the sciatic nerve showed more fragmented and disordered distribution of axons on the transverse section, and extensive separation of myelin and axons, uneven myelin thickness, axonal degeneration and irregular shape in the model group, whereas in the EA group, the axons on the transverse section were relatively more dense and more complete, the myelin sheath of the sciatic nerve was relatively uniform, and the axonal shape was relatively regular with relatively milder lesions. EA up-regulates the expressions of Sirt1, PGC-1α, TFAM in T2DM rats with DPN, which may be associated with its functions in improving and repairing the injured peripheral nerves in rats with DPN.

F-box protein FBXB-65 regulates anterograde transport of the kinesin-3 motor UNC-104 through a PTM near its cargo-binding PH domain.

Axonal transport in neurons is essential for cargo movement between the cell body and synapses. Caenorhabditis elegans UNC-104 and its homolog KIF1A are kinesin-3 motors that anterogradely transport precursors of synaptic vesicles (pre-SVs) and are degraded at synapses. However, in C. elegans, touch neuron-specific knockdown of the E1 ubiquitin-activating enzyme, uba-1, leads to UNC-104 accumulation at neuronal ends and synapses. Here, we performed an RNAi screen and identified that depletion of fbxb-65, which encodes an F-box protein, leads to UNC-104 accumulation at neuronal distal ends, and alters UNC-104 net anterograde movement and levels of UNC-104 on cargo without changing synaptic UNC-104 levels. Split fluorescence reconstitution showed that UNC-104 and FBXB-65 interact throughout the neuron. Our theoretical model suggests that UNC-104 might exhibit cooperative cargo binding that is regulated by FBXB-65. FBXB-65 regulates an unidentified post-translational modification (PTM) of UNC-104 in a region beside the cargo-binding PH domain. Both fbxb-65 and UNC-104, independently of FBXB-65, regulate axonal pre-SV distribution, transport of pre-SVs at branch points and organismal lifespan. FBXB-65 regulates a PTM of UNC-104 and the number of motors on the cargo surface, which can fine-tune cargo transport to the synapse.

Polarized localization of kinesin-1 and RIC-7 drives axonal mitochondria anterograde transport.

Mitochondria transport is crucial for axonal mitochondria distribution and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 binding to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1, and metaxin2. We conclude that transport complexes containing kinesin-1 and RIC-7 polarize at the leading edge of mitochondria and are required for anterograde axonal transport in C. elegans.

The Reelin receptor ApoER2 is a cargo for the adaptor protein complex AP-4: Implications for Hereditary Spastic Paraplegia

Adaptor protein complex 4 (AP-4) is a heterotetrameric complex that promotes export of selected cargo proteins from the trans-Golgi network. Mutations in each of the AP-4 subunits cause a complicated form of Hereditary Spastic Paraplegia (HSP). Herein, we report that ApoER2, a receptor in the Reelin signaling pathway, is a cargo of the AP-4 complex. We identify the motif ISSF/Y within the ApoER2 cytosolic domain as necessary for interaction with the canonical signal-binding pocket of the µ4 (AP4M1) subunit of AP-4. AP4E1- knock-out (KO) HeLa cells and hippocampal neurons from Ap4e1-KO mice display increased co-localization of ApoER2 with Golgi markers. Furthermore, hippocampal neurons from Ap4e1-KO mice and AP4M1-KO human iPSC-derived cortical i3Neurons exhibit reduced ApoER2 protein expression. Analyses of biosynthetic transport of ApoER2 reveal differential post-Golgi trafficking of the receptor, with lower axonal distribution in KO compared to wild-type neurons, indicating a role of AP-4 and the ISSF/Y motif in the axonal localization of ApoER2. Finally, analyses of Reelin signaling in mouse hippocampal and human cortical KO neurons show that AP4 deficiency causes no changes in Reelin-dependent activation of the AKT pathway and only mild changes in Reelin-induced dendritic arborization, but reduces Reelin-induced ERK phosphorylation, CREB activation, and Golgi deployment. This work thus establishes ApoER2 as a novel cargo of the AP-4 complex, suggesting that defects in the trafficking of this receptor and in the Reelin signaling pathway could contribute to the pathogenesis of HSP caused by mutations in AP-4 subunits.

Using tissue clearing and light sheet fluorescence microscopy for the three-dimensional analysis of sensory and sympathetic nerve endings that innervate bone and dental tissue of mice.

Bone and dental tissues are richly innervated by sensory and sympathetic neurons. However, the characterization of the morphology, molecular phenotype, and distribution of nerves that innervate hard tissue has so far mostly been limited to thin histological sections. This approach does not adequately capture dispersed neuronal projections due to the loss of important structural information during three-dimensional (3D) reconstruction. In this study, we modified the immunolabeling-enabled imaging of solvent-cleared organs (iDISCO/iDISCO+) clearing protocol to image high-resolution neuronal structures in whole femurs and mandibles collected from perfused C57Bl/6 mice. Axons and their nerve terminal endings were immunolabeled with antibodies directed against protein gene product 9.5 (pan-neuronal marker), calcitonin gene-related peptide (peptidergic nociceptor marker), or tyrosine hydroxylase (sympathetic neuron marker). Volume imaging was performed using light sheet fluorescence microscopy. We report high-quality immunolabeling of the axons and nerve terminal endings for both sensory and sympathetic neurons that innervate the mouse femur and mandible. Importantly, we are able to follow their projections through full 3D volumes, highlight how extensive their distribution is, and show regional differences in innervation patterns for different parts of each bone (and surrounding tissues). Mapping the distribution of sensory and sympathetic axons, and their nerve terminal endings, in different bony compartments may be important in further elucidating their roles in health and disease.

Mitochondria Directed Oxidative Stress and Metal Abnormalities at the Center of Alzheimer’s Disease

For nearly four decades, our research has focused on dissecting the cytopathology of Alzheimer’s disease (AD) with the goal of developing a cure. We have used oxidative stress as a window to view and understand AD. Oxidative damage to sugars, proteins, lipids, and nucleic acids increases in neuronal cytoplasm. The same neuronal compartment has increased redoxactive iron and copper, which can catalyze oxidative damage, and likely derive from mitochondrial debris (in and outside lysosomes) including cytochromes, mitochondria-specific prosthetic groups, and mtDNA. Mitochondria show altered axonal transport, size distribution, energetics, fusion/fission, and degradation in AD that correlate with the extent of oxidative damage suggesting they are the origin. Synaptic mitochondria abnormalities correlate with synaptic vesicular changes. Surprisingly, amyloidβ and tau are quantitatively associated with reduced neuronal oxidative damage. Copper sequestration by amyloidβ blocks copper-mediated oxidation of lipids and vitamin C indicating amyloidβ can be a protective response rather than the initiator of AD. Instead of being bound to amyloidβ, iron is present as 10nm magnetite crystals with super-paramagnetic properties as well as abundant metallic iron, along with metallic copper. This is the first report of metallic iron and copper in humans. Not just amyloidβ, but also tau, may be protective responses induced in AD to maintain neurons with altered balance for decades. While these studies put oxidative stress at the center of AD, they also highlight a complexity of multifaceted alterations that is homeostatic and requires a deeper level of understanding before an effective cure.

Combined RNA interference and gene replacement therapy targeting MFN2 as proof of principle for the treatment of Charcot–Marie–Tooth type 2A

Mitofusin-2 (MFN2) is an outer mitochondrial membrane protein essential for mitochondrial networking in most cells. Autosomal dominant mutations in the MFN2 gene cause Charcot–Marie–Tooth type 2A disease (CMT2A), a severe and disabling sensory-motor neuropathy that impacts the entire nervous system. Here, we propose a novel therapeutic strategy tailored to correcting the root genetic defect of CMT2A. Though mutant and wild-type MFN2 mRNA are inhibited by RNA interference (RNAi), the wild-type protein is restored by overexpressing cDNA encoding functional MFN2 modified to be resistant to RNAi. We tested this strategy in CMT2A patient-specific human induced pluripotent stem cell (iPSC)-differentiated motor neurons (MNs), demonstrating the correct silencing of endogenous MFN2 and replacement with an exogenous copy of the functional wild-type gene. This approach significantly rescues the CMT2A MN phenotype in vitro, stabilizing the altered axonal mitochondrial distribution and correcting abnormal mitophagic processes. The MFN2 molecular correction was also properly confirmed in vivo in the MitoCharc1 CMT2A transgenic mouse model after cerebrospinal fluid (CSF) delivery of the constructs into newborn mice using adeno-associated virus 9 (AAV9). Altogether, our data support the feasibility of a combined RNAi and gene therapy strategy for treating the broad spectrum of human diseases associated with MFN2 mutations.

COMPARATIVE NEUROANATOMY OF DESCENDING NEURONS OF THE SUPRAESOPHAGEAL GANGLION OF COCKROACHES OF THE FAMILY BLABERIDAE (BLATTODEA)

A comparative study of the morphology of descending neurons connecting the supraesophageal ganglion and thoracic ganglia in cockroaches of the family Blaberidae, which differ in protective behavior and flight ability, was carried out. The neuronal structure of these families was compared with the descending neurons of the cockroach Periplaneta americana. The number, spatial distribution, and arrangement of axons and dendrites of descending neurons of cockroach Leucophaea maderae, Gromphadorhina portentosa, Blaberus craniifer, Nauphoeta cinerea (Blaberidae) were found to be similar. Neurons homologous to the ocellar, mechanosensory, and visual descending neurons described in the cockroach Periplaneta americana were found. It is suggested that during the evolution of the cockroach species, the adaptive behavior at danger was changed by transforming sensory inputs and motor responses, while the system of descending neurons remains stable.

Evolutionary scaling and cognitive correlates of primate frontal cortex microstructure.

Investigating evolutionary changes in frontal cortex microstructure is crucial to understanding how modifications of neuron and axon distributions contribute to phylogenetic variation in cognition. In the present study, we characterized microstructural components of dorsolateral prefrontal cortex, orbitofrontal cortex, and primary motor cortex from 14 primate species using measurements of neuropil fraction and immunohistochemical markers for fast-spiking inhibitory interneurons, large pyramidal projection neuron subtypes, serotonergic innervation, and dopaminergic innervation. Results revealed that the rate of evolutionary change was similar across these microstructural variables, except for neuropil fraction, which evolves more slowly and displays the strongest correlation with brain size. We also found that neuropil fraction in orbitofrontal cortex layers V-VI was associated with cross-species variation in performance on experimental tasks that measure self-control. These findings provide insight into the evolutionary reorganization of the primate frontal cortex in relation to brain size scaling and its association with cognitive processes.

Distribution and morphology of calcitonin gene-related peptide (CGRP) innervation in flat mounts of whole rat atria and ventricles

Calcitonin gene-related peptide (CGRP) is widely used as a marker for nociceptive afferent axons. However, the distribution of CGRP-IR axons has not been fully determined in the whole rat heart. Immunohistochemically labeled flat-mounts of the right and left atria and ventricles, and the interventricular septum (IVS) in rats for CGRP were assessed with a Zeiss imager to generate complete montages of the entire atria, ventricles, and septum, and a confocal microscope was used to acquire detailed images of selected regions. We found that 1) CGRP-IR axons extensively innervated all regions of the atrial walls including the sinoatrial node region, auricles, atrioventricular node region, superior/inferior vena cava, left pre-caval vein, and pulmonary veins. 2) CGRP-IR axons formed varicose terminals around individual neurons in some cardiac ganglia but passed through other ganglia without making appositions with cardiac neurons. 3) Varicose CGRP-IR axons innervated the walls of blood vessels. 4) CGRP-IR axons extensively innervated the right/left ventricular walls and IVS. Our data shows the rather ubiquitous distribution of CGRP-IR axons in the whole rat heart at single-cell/axon/varicosity resolution for the first time. This study lays the foundation for future studies to quantify the differences in CGRP-IR axon innervation between sexes, disease models, and species.

Single-subject electroencephalography measurement of interhemispheric transfer time for the in-vivo estimation of axonal morphology.

Assessing axonal morphology in vivo opens new avenues for the combined study of brain structure and function. A novel approach has recently been introduced to estimate the morphology of axonal fibers from the combination of magnetic resonance imaging (MRI) data and electroencephalography (EEG) measures of the interhemispheric transfer time (IHTT). In the original study, the IHTT measures were computed from EEG data averaged across a group, leading to bias of the axonal morphology estimates. Here, we seek to estimate axonal morphology from individual measures of IHTT, obtained from EEG data acquired in a visual evoked potential experiment. Subject-specific IHTTs are computed in a data-driven framework with minimal a priori constraints, based on the maximal peak of neural responses to visual stimuli within periods of statistically significant evoked activity in the inverse solution space. The subject-specific IHTT estimates ranged from 8 to 29 ms except for one participant and the between-session variability was comparable to between-subject variability. The mean radius of the axonal radius distribution, computed from the IHTT estimates and the MRI data, ranged from 0 to 1.09 μm across subjects. The change in axonal g-ratio with axonal radius ranged from 0.62 to 0.81 μm-α . The single-subject measurement of the IHTT yields estimates of axonal morphology that are consistent with histological values. However, improvement of the repeatability of the IHTT estimates is required to improve the specificity of the single-subject axonal morphology estimates.

Highly branched and complementary distributions of layer 5 and layer 6 auditory corticofugal axons in mouse.

The auditory cortex exerts a powerful, yet heterogeneous, effect on subcortical targets. Auditory corticofugal projections emanate from layers 5 and 6 and have complementary physiological properties. While several studies suggested that layer 5 corticofugal projections branch widely, others suggested that multiple independent projections exist. Less is known about layer 6; no studies have examined whether the various layer 6 corticofugal projections are independent. Therefore, we examined branching patterns of layers 5 and 6 auditory corticofugal neurons, using the corticocollicular system as an index, using traditional and novel approaches. We confirmed that dual retrograde injections into the mouse inferior colliculus and auditory thalamus co-labeled subpopulations of layers 5 and 6 auditory cortex neurons. We then used an intersectional approach to relabel layer 5 or 6 corticocollicular somata and found that both layers sent extensive branches to multiple subcortical structures. Using a novel approach to separately label layers 5 and 6 axons in individual mice, we found that layers 5 and 6 terminal distributions partially spatially overlapped and that giant terminals were only found in layer 5-derived axons. Overall, the high degree of branching and complementarity in layers 5 and 6 axonal distributions suggest that corticofugal projections should be considered as 2 widespread systems, rather than collections of individual projections.

Predicting the distribution of serotonergic axons: a supercomputing simulation of reflected fractional Brownian motion in a 3D-mouse brain model.

The self-organization of the brain matrix of serotonergic axons (fibers) remains an unsolved problem in neuroscience. The regional densities of this matrix have major implications for neuroplasticity, tissue regeneration, and the understanding of mental disorders, but the trajectories of its fibers are strongly stochastic and require novel conceptual and analytical approaches. In a major extension to our previous studies, we used a supercomputing simulation to model around one thousand serotonergic fibers as paths of superdiffusive fractional Brownian motion (FBM), a continuous-time stochastic process. The fibers produced long walks in a complex, three-dimensional shape based on the mouse brain and reflected at the outer (pial) and inner (ventricular) boundaries. The resultant regional densities were compared to the actual fiber densities in the corresponding neuroanatomically-defined regions. The relative densities showed strong qualitative similarities in the forebrain and midbrain, demonstrating the predictive potential of stochastic modeling in this system. The current simulation does not respect tissue heterogeneities but can be further improved with novel models of multifractional FBM. The study demonstrates that serotonergic fiber densities can be strongly influenced by the geometry of the brain, with implications for brain development, plasticity, and evolution.

Topographical distribution and morphology of SP-IR axons in the antrum, pylorus, and duodenum of mice.

Substance-P (SP) is a commonly used marker of nociceptive afferent axons, and it plays an important role in a variety of physiological functions including the regulation of motility, gut secretion, and vascular flow. Previously, we found that SP-immunoreactive (SP-IR) axons densely innervated the pyloric antrum of the flat-mount of the mouse whole stomach muscular layer. However, the regional distribution and morphology of SP-IR axons in the submucosa and mucosa were not well documented. In this study, the mouse antrum-pylorus-duodenum (APD) were transversely and longitudinally sectioned. A Zeiss M2 imager was used to scan the serial sections of each APD (each section montage consisted of 50-100 all-in-focus maximal projection images). To determine the detailed structures of SP-IR axons and terminals, we used the confocal microscope to scan the regions of interest. We found that 1) SP-IR axons innervated the muscular, submucosal, and mucosal layers. 2) In the muscular layer, SP-IR varicose axons densely innervated the muscles and formed varicose terminals which encircled myenteric neurons. 3) In the submucosa, SP-IR axons innervated blood vessels and submucosal ganglia and formed a network in Brunner's glands. 4) In the mucosa, SP-IR axons innervated the muscularis mucosae. Some SP-IR axons entered the lamina propria. 5) The muscular layer of the antrum and duodenum showed a higher SP-IR axon density than the pyloric sphincter. 6) SP-IR axons were from extrinsic and intrinsic origins. This work provided a comprehensive view of the distribution and morphology of SP-IR axons in the APD at single cell/axon/varicosity scale. This data will be used to create a 3D scaffold of the SP-IR axon innervation of the APD.

Stellate Ganglion Sympathetic postganglionic efferent innervation in the flat-mount of the whole heart of rats: Anterograde Tracing

The autonomic nervous system (sympathetic and parasympathetic) plays an essential role in controlling cardiac functions. Previously, we used anterograde tracing to examine the vagal afferent and efferent (preganglionic) innervations of the normal heart, their anatomical remodeling and their functional changes in disease models of rats and mice. In addition, we used vesicular acetylcholine transporter (VAChT, a parasympathetic marker;) and tyrosine hydroxylase (TH, a sympathetic marker,) to determine the parasympathetic and sympathetic postganglionic innervation of the whole heart (atria and ventricles). Immunohistochemical labeling with TH is useful for determining the broad distribution of sympathetic efferent axons in the heart. However, it is difficult to distinguish its extrinsic and intrinsic origins using TH labeling. Furthermore, the topographical organization and morphological terminal structures of cardiac sympathetic postganglionic efferent axons from the stellate ganglia (SG) remains unknown. To address this we anterogradely labeled individual sympathetic postganglionic axons projecting to the heart, using tracer Dextran-Biotin microinjections into the right SG of Sprague-Dawley rats (male, n=6, 3-6 months). Fourteen days after the injection, animals were euthanized, and their hearts were dissected into the left/right atria (LA and RA) and ventricles and prepared as flat-mounts. The tracer-labeled axons were digitized and analyzed using The Neurolucida 3D tracing system and imaged using a Zeiss M2 Imager. We found that stellate sympathetic neurons had projections to the LA and RA and left and right ventricles. Individual sympathetic axons formed complex arbors of varicose neurites in the areas of the sinoatrial node (SAN) and atrioventricular node (AVN) as well as the other areas of the atria and throughout the ventricles. Sympathetic axons also innervated blood vessels. Using the Neurolucida Digitization and Tracer system, we determined the regional density and characterized different types of axons. Some axons formed relatively simple terminal structures with only a few branches, whereas other axons produced very complex terminals with extensive branching. For the first time, we anterogradely labeled the sympathetic postganglionic axons and characterized their terminal architecture in the whole heart. These results will provide an anatomical foundation for future study This study was supported by NIH HEAL/SPARC U01 NS113867-01 and NIH R15HL137143-01A1. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Topographical mapping of catecholaminergic axon innervation in the flat-mounts of the mouse atria: a quantitative analysis

The sympathetic nervous system is crucial for controlling multiple cardiac functions. However, a comprehensive, detailed neuroanatomical map of the sympathetic innervation of the heart is unavailable. Here, we used a combination of state-of-the-art techniques, including flat-mount tissue processing, immunohistochemistry for tyrosine hydroxylase (TH, a sympathetic marker), confocal microscopy and Neurolucida 360 software to trace, digitize, and quantitatively map the topographical distribution of the sympathetic postganglionic innervation in whole atria of C57Bl/6 J mice. We found that (1) 4–5 major extrinsic TH-IR nerve bundles entered the atria at the superior vena cava, right atrium (RA), left precaval vein and the root of the pulmonary veins (PVs) in the left atrium (LA). Although these bundles projected to different areas of the atria, their projection fields partially overlapped. (2) TH-IR axon and terminal density varied considerably between different sites of the atria with the greatest density of innervation near the sinoatrial node region (P < 0.05, n = 6). (3) TH-IR axons also innervated blood vessels and adipocytes. (4) Many principal neurons in intrinsic cardiac ganglia and small intensely fluorescent cells were also strongly TH-IR. Our work provides a comprehensive topographical map of the catecholaminergic efferent axon morphology, innervation, and distribution in the whole atria at single cell/axon/varicosity scale that may be used in future studies to create a cardiac sympathetic-brain atlas.

MP60-13 VIRAL TRANSDUCTION EFFICACY OF PERIPHERAL NEURONS INNERVATING THE EXTERNAL URETHRAL SPHINCTER BY LOCAL INJECTION OF ADENO-ASSOCIATED VIRUS VECTORS

You have accessJournal of UrologyCME1 Apr 2023MP60-13 VIRAL TRANSDUCTION EFFICACY OF PERIPHERAL NEURONS INNERVATING THE EXTERNAL URETHRAL SPHINCTER BY LOCAL INJECTION OF ADENO-ASSOCIATED VIRUS VECTORS Firoj Alom, Richard Johnson, and Aaron Mickle Firoj AlomFiroj Alom More articles by this author , Richard JohnsonRichard Johnson More articles by this author , and Aaron MickleAaron Mickle More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003318.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Incomplete functional recovery of the lower urinary tract, including the external urethral sphincter (EUS), frequently occurs in spinal cord injury patients. Electrical neuromodulation of the lower urinary tract remains an attractive therapeutic target, but it has yet to be fully deployed clinically. The wide distribution of sensory and motor axons in the pudendal nerve innervating targets in the lower urinary tract but also other targets in other pelvic structures significantly limits the development of electrical neuromodulation strategies. We can address this limitation by using optogenetic techniques to modulate the lower urinary tract using light stimulation. First, we need to determine the viral vectors' efficiency and potential promoters to transduce the specific neuronal populations through local injection into regions of the lower urinary tract. In this study, we investigated the transduction efficiency of adeno-associated virus serotypes in the peripheral regions by local injection into the external urethral sphincter. METHODS: We injected different AAV vector serotypes along with a neural tracer into the external sphincter of rats. After five weeks, we evaluated the viral transduction efficiency of these AAV serotypes. RESULTS: We observed variable transduction of multiple peripheral neuron populations and showed different efficacies of neuronal transduction by these viruses compared with labeling with a neural tracer. We also found that this level of expression by local injection is also relevant for optogenetic-mediated modulation of the EUS function. CONCLUSIONS: Our preliminary results indicate that by using adeno-associated viruses, it is possible to transduce the neurons controlling the EUS by local injection. We think these studies will potentially help improve the viral targeting of neurons controlling lower urinary tract function. Source of Funding: This research was supported by the NIH NIBIB Trailblazer award (R21 EB031249) and in part by the Urology Care Foundation Research Scholar Award Program and Indian American Urological Association Sakti Das, MD Award © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e848 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Firoj Alom More articles by this author Richard Johnson More articles by this author Aaron Mickle More articles by this author Expand All Advertisement PDF downloadLoading ...