An adeno-associated viral labeling approach to visualize the meso- and microanatomy of mechanosensory afferents and autonomic innervation of the rat urinary bladder.

The urinary bladder is supplied by a rich network of sensory and autonomic axons, commonly visualized by immunolabeling for neural markers. This approach demonstrates overall network patterning but is less suited to understanding the structure of individual motor and sensory terminals within these complex plexuses. There is a further limitation visualizing the lightly myelinated (A-delta) class of sensory axons that provides the primary mechanosensory drive for initiation of voiding. Whereas most unmyelinated sensory axons can be revealed by immunolabeling for specific neuropeptides, to date no unique neural marker has been identified to immunohistochemically label myelinated visceral afferents. We aimed to establish a non-surgical method to visualize and map myelinated afferents in the bladder in rats. We found that in rats, the adeno-associated virus (AAV), AAV-PHP.S, which shows a high tropism for the peripheral nervous system, primarily transduced myelinated dorsal root ganglion neurons, enabling us to identify the structure and regional distribution of myelinated (mechanosensory) axon endings within the muscle and lamina propria of the bladder. We further identified the projection of myelinated afferents within the pelvic nerve and lumbosacral spinal cord. A minority of noradrenergic and cholinergic neurons in pelvic ganglia were transduced, enabling visualization and regional mapping of both autonomic and sensory axon endings within the bladder. Our study identified a sparse labeling approach for investigating myelinated sensory and autonomic axon endings within the bladder and provides new insights into the nerve-bladder interface.

Sex differences in c-Fos and EGR-1/Zif268 activity maps of rat sacral spinal cord following cystometry-induced micturition.

Storage and voiding of urine from the lower urinary tract (LUT) must be timed precisely to occur in appropriate behavioral contexts. A major part of the CNS circuit that coordinates this activity is found in the lumbosacral spinal cord. Immediate early gene (IEG) activity mapping has been widely used to investigate the lumbosacral LUT-related circuit, but most reports focus on the effects of noxious stimulation in anesthetized female rats. Here we use c-Fos and EGR-1 (Zif268) activity mapping of lumbosacral spinal cord to investigate cystometry-induced micturition in awake female and male rats. In females, after cystometry c-Fos neurons in spinal cord segments L5-S2 were concentrated in the sacral parasympathetic nucleus (SPN), dorsal horn laminae II-IV, and dorsal commissural nucleus (SDCom). Comparisons of cystometry and control groups in male and female revealed sex differences. Activity mapping suggested dorsal horn laminae II-IV was activated in females but showed net inhibition in males. However, inhibition in male rats was not detected by EGR-1 activity mapping, which showed low coexpression with c-Fos. A class of catecholamine neurons in SPN and SDCom neurons were also more strongly activated by micturition in females. In both sexes, most c-Fos neurons were identified as excitatory by their absence of Pax2 expression. In conclusion, IEG mapping in awake male and female rats has extended our understanding of the functional molecular anatomy of the LUT-related circuit in spinal cord. Using this approach, we have identified sex differences that were not detected by previous studies in anesthetized rats.

Recording of Electrically Evoked Neural Activity and Bladder Pressure Responses in Awake Rats Chronically Implanted With a Pelvic Nerve Array.

Bioelectronic medical devices are well established and widely used in the treatment of urological dysfunction. Approved targets include the sacral S3 spinal root and posterior tibial nerve, but an alternate target is the group of pelvic splanchnic nerves, as these contain sacral visceral sensory and autonomic motor pathways that coordinate storage and voiding functions of the bladder. Here, we developed a device suitable for long-term use in an awake rat model to study electrical neuromodulation of the pelvic nerve (homolog of the human pelvic splanchnic nerves). In male Sprague-Dawley rats, custom planar four-electrode arrays were implanted over the distal end of the pelvic nerve, close to the major pelvic ganglion. Electrically evoked compound action potentials (ECAPs) were reliably detected under anesthesia and in chronically implanted, awake rats up to 8 weeks post-surgery. ECAP waveforms showed three peaks, with latencies that suggested electrical stimulation activated several subpopulations of myelinated A-fiber and unmyelinated C-fiber axons. Chronic implantation of the array did not impact on voiding evoked in awake rats by continuous cystometry, where void parameters were comparable to those published in naïve rats. Electrical stimulation with chronically implanted arrays also induced two classes of bladder pressure responses detected by continuous flow cystometry in awake rats: voiding contractions and non-voiding contractions. No evidence of tissue pathology produced by chronically implanted arrays was detected by immunohistochemical visualization of markers for neuronal injury or noxious spinal cord activation. These results demonstrate a rat pelvic nerve electrode array that can be used for preclinical development of closed loop neuromodulation devices targeting the pelvic nerve as a therapy for neuro-urological dysfunction.

Insights into the neurochemical signature of the Innervation of Beige Fat.

OBJECTIVE: The potential for brown adipose tissue (BAT) to be targeted as a therapeutic option to combat obesity has been heightened by the discovery of a brown-like form of inducible "beige" adipose tissue in white fat which has overlapping structural and functional properties to "classical" BAT. The likelihood that both beige and brown fat are recruited functionally by neural mechanisms, taken together with the lack of a detailed understanding of the nature of changes in the nervous system when white adipose tissue (WAT) is transformed to brown, provides the impetus for this study. Here, we aim to identify whether there is a shift in the gene expression profile in neurons directly innervating inguinal white adipose tissue (iWAT) that has undergone "beiging" to a signature that is more similar to neurons projecting to BAT. METHODS: Two groups of rats, one housed at thermoneutrality (27 °C) and the other exposed to cold (8 °C) for 7 days, were killed, and their T13/L1 ganglia, stellate ganglion (T1/T2), or superior cervical ganglion (SCG, C2/3) removed. This approach yielded ganglia containing neurons that innervate either beiged white fat (8 °C for 7 days), inguinal WAT (27 °C for 7 days), BAT (both 27 °C and 8 °C for 7 days) or non-WAT (8 °C for 7 days), the latter included to isolate changes in gene expression that were more aligned with a response to cold exposure than the transformation of white to beige adipocytes. Bioinformatics analyses of RNA sequencing data was performed followed by Ingenuity Pathway Analysis (IPA) to determine differential gene expression and recruitment of biosynthetic pathways. RESULTS: When iWAT is "beiged" there is a significant shift in the gene expression profile of neurons in sympathetic ganglia (T13/L1) innervating this depot toward a gene neurochemical signature that is similar to the stellate ganglion projecting to BAT. Bioinformatics analyses of "beiging" related genes revealed upregulation of genes encoding neuropeptides proopiomelanocortin (POMC) and calcitonin-gene related peptide (CGRP) within ganglionic neurons. Treatment of differentiated 3T3L1 adipocytes with αMSH, one of the products cleaved from POMC, results in an elevation in lipolysis and the beiging of these cells as indicated by changes in gene expression markers of browning (Ucp1 and Ppargc1a). CONCLUSION: These data indicate that, coincident with beiging, there is a shift toward a "brown-like" neurochemical signature of postganglionic neurons projecting to inguinal white fat, an increased expression of POMC, and, consistent with a causative role for this prohormone in beiging, an αMSH-mediated increase in beige gene markers in isolated adipocytes.

Characterization of the central neural projections to brown, white, and beige adipose tissue.

The functional recruitment of classic brown adipose tissue (BAT) and inducible brown-like or beige fat is, to a large extent, dependent on intact sympathetic neural input. Whereas the central neural circuits directed specifically to BAT or white adipose tissue (WAT) are well established, there is only a developing insight into the nature of neural inputs common to both fat types. Moreover, there is no clear view of the specific central and peripheral innervation of the browned component of WAT: beige fat. The objective of the present study is to examine the neural input to both BAT and WAT in the same animal and, by exposing different cohorts of rats to either thermoneutral or cold conditions, define changes in central neural organization that will ensure that beige fat is appropriately recruited and modulated after browning of inguinal WAT (iWAT). At thermoneutrality, injection of the neurotropic (pseudorabies) viruses into BAT and WAT demonstrates that there are dedicated axonal projections, as well as collateral axonal branches of command neurons projecting to both types of fat. After cold exposure, central neural circuits directed to iWAT showed evidence of reorganization with a greater representation of command neurons projecting to both brown and beiged WAT in hypothalamic (paraventricular nucleus and lateral hypothalamus) and brainstem (raphe pallidus and locus coeruleus) sites. This shift was driven by a greater number of supraspinal neurons projecting to iWAT under cold conditions. These data provide evidence for a reorganization of the nervous system at the level of neural connectivity following browning of WAT.-Wiedmann, N. M., Stefanidis, A., Oldfield, B. J. Characterization of the central neural projections to brown, white, and beige adipose tissue.