Identification and characterization of a potentially novel dorsal cutaneous muscle in rodents.

In the course of performing a detailed dissection of adult rat to map the cutaneous nerves of cervical, thoracic, and lumbar levels a small and unexpected structure was isolated. It appeared to be a cutaneous striated muscle and was observed in both male and female rats and in mice but absent from cats and humans. With the skin reflected laterally from midline, the muscle lies closely apposed to the lateral border of the Thoracic Trapezius (Spinotrapezius) muscle and is easily missed in standard gross dissections. Focussed prosections were performed to identify the origin, insertion, and course of gross innervation. Identification of each of these elements showed them to be distinct from the nearby Trapezius and Cutaneous Trunci (Cutaneous Maximus in mouse) muscles. The striated muscle nature of the structure was validated with whole-mount microscopy. Consulting a range of published rodent anatomical atlases and gross anatomical experts revealed no prior descriptions. This preliminary report is an opportunity for the anatomical and research communities to provide input to either confirm the novelty of this muscle or refer to prior published descriptions in rodents or other species while the muscle, its innervation, and function are further characterized. Presuming this muscle is indeed novel, the name "Cutaneous Scapularis muscle" is proposed in accord with general principles of the anatomical field.

Construction of a Searchable Database for Gene Expression Changes in Spinal Cord Injury Experiments.

Spinal cord injury (SCI) is a debilitating condition with an estimated 18,000 new cases annually in the United States. The field has accepted and adopted standardized databases such as the Open Data Commons for Spinal Cord Injury (ODC-SCI) to aid in broader analyses, but these currently lack high-throughput data despite the availability of nearly 6000 samples from over 90 studies available in the Sequence Read Archive. This limits the potential for large datasets to enhance our understanding of SCI-related mechanisms at the molecular and cellular level. Therefore, we have developed a protocol for processing RNA-Seq samples from high-throughput sequencing experiments related to SCI resulting in both raw and normalized data that can be efficiently mined for comparisons across studies, as well as homologous discovery across species. We have processed 1196 publicly available RNA-Seq samples from 50 bulk RNA-Seq studies across nine different species, resulting in an SQLite database that can be used by the SCI research community for further discovery. We provide both the database as well as a web-based front-end that can be used to query the database for genes of interest, differential gene expression, genes with high variance, and gene set enrichments.

Construction of a searchable database for gene expression changes in spinal cord injury experiments.

Spinal cord injury (SCI) is a debilitating disease resulting in an estimated 18,000 new cases in the United States on an annual basis. Significant behavioral research on animal models has led to a large amount of data, some of which has been catalogued in the Open Data Commons for Spinal Cord Injury (ODC-SCI). More recently, high throughput sequencing experiments have been utilized to understand molecular mechanisms associated with SCI, with nearly 6,000 samples from over 90 studies available in the Sequence Read Archive. However, to date, no resource is available for efficiently mining high throughput sequencing data from SCI experiments. Therefore, we have developed a protocol for processing RNA-Seq samples from high-throughput sequencing experiments related to SCI resulting in both raw and normalized data that can be efficiently mined for comparisons across studies as well as homologous discovery across species. We have processed 1,196 publicly available RNA-seq samples from 50 bulk RNA-Seq studies across nine different species, resulting in an SQLite database that can be used by the SCI research community for further discovery. We provide both the database as well as a web-based front-end that can be used to query the database for genes of interest, differential gene expression, genes with high variance, and gene set enrichments.

Broad opioid antagonism amplifies disruption of locomotor function following therapy-like hindlimb stretching in spinal cord injured rats.

STUDY DESIGN: Preclinical pilot study. OBJECTIVES: To test the hypothesis that spinal opioidergic circuitry contributes to muscle stretch-induced locomotor deficits. SETTING: Kentucky Spinal Cord Injury Research Center, Louisville, KY, USA. METHODS: A pilot study with eight female Sprague-Dawley rats that received 25 g-cm T10 contusion injuries and recovered for 5 weeks. Rats were divided into two groups with one group receiving subcutaneous injections of naltrexone dissolved in saline (15 mg/kg) or an equal volume of saline. Each group received a daily 24-minute stretching protocol during weeks 6, 8, and 11 post-injury. Locomotor function was assessed throughout using the BBB Open Field Locomotor Scale. RESULTS: Consistent with previous findings, stretching reduced locomotor function in both naltrexone and saline groups. However, the loss of locomotor function appeared earlier in the naltrexone group. Animals in both groups had a similar rate of recovery following the termination of stretching. Interestingly, the administration of naltrexone did not influence acute thermal cutaneous nociceptive responses as measured by a tail-flick assay but caused a significant increase in spasticity following stretch. CONCLUSIONS: The results of this study suggest that the endogenous opioid system plays a role in modulating the negative impact of muscle stretch on spinal cord motor circuitry that is vulnerable due to loss of descending input. The observed actions of the broad-spectrum opioid antagonist naltrexone imply that pharmaceuticals targeting the endogenous opioid system post-SCI may have unintended consequences.

RNA-seq data of soleus muscle tissue after spinal cord injury under conditions of inactivity and applied exercise.

Reduced muscle mass and increased fatiguability are major complications after spinal cord injury (SCI), and often hinder the rehabilitation efforts of patients. Such detriments to the musculoskeletal system, and the concomitant reduction in level of activity, contribute to secondary complications such as cardiovascular disease, diabetes, bladder dysfunction and liver damage. As a result of decreased weight-bearing capacity after SCI, muscles undergo morphological, metabolic, and contractile changes. Recent studies have shown that exercise after SCI decreases muscle wasting and reduces the burden of secondary complications. Here, we describe RNA sequencing data for detecting chronic transcriptomic changes in the rat soleus after SCI at two levels of injury severity, under conditions of restricted in-cage activity and two methods of applied exercise, swimming or shallow water walking. We demonstrate that the sequenced data are of good quality and show a high alignment rate to the Rattus norvegicus reference assembly (Rn6). The raw data, along with UCSC Genome Browser tracks created to facilitate exploration of gene expression, are available in the NCBI Gene Expression Omnibus (GEO; GSE129694).

Transcriptome of dorsal root ganglia caudal to a spinal cord injury with modulated behavioral activity.

Spinal cord injury (SCI) is a devastating clinical condition resulting in significant disabilities. Apart from local injury within the spinal cord, SCI patients develop a myriad of complications including multi-organ dysfunction. Some of the dysfunctions may be directly or indirectly related to the sensory neurons of the dorsal root ganglia (DRG), which signal to both the spinal cord and the peripheral organs. After SCI, some classes of DRG neurons exhibit sensitization and undergo axonal sprouting both peripherally and centrally. Such physiological and anatomical re-organization after SCI contributes to both adaptive and maladaptive plasticity processes, which may be modulated by activity and exercise. In this study, we collected comprehensive gene expression data in whole DRG below the levels of the injury to compare the effects of SCI with and without two different forms of exercise in rats.

Activity/exercise-induced changes in the liver transcriptome after chronic spinal cord injury.

Multi-organ dysfunction is a major complication after spinal cord injury (SCI). In addition to local injury within the spinal cord, SCI causes major disruption to the peripheral organ innervation and regulation. The liver contains sympathetic, parasympathetic, and small sensory axons. The bi-directional signaling of sensory dorsal root ganglion (DRG) neurons that provide both efferent and afferent information is of key importance as it allows sensory neurons and peripheral organs to affect each other. SCI-induced liver inflammation precedes and may exacerbate intraspinal inflammation and pathology after SCI, which may be modulated by activity and exercise. In this study, we collected comprehensive gene expression data through RNA sequencing of liver tissue from rats with chronic SCI to determine the effects of activity and exercise on those expression patterns. The sequenced data are of high quality and show a high alignment rate to the Rn6 genome. Gene expression is demonstrated for genes associated with known liver pathologies. UCSC Genome Browser expression tracks are provided with the data to facilitate exploration of the samples.

Detection of Differentially Expressed Cleavage Site Intervals Within 3' Untranslated Regions Using CSI-UTR Reveals Regulated Interaction Motifs.

The length of untranslated regions at the 3' end of transcripts (3'UTRs) is regulated by alternate polyadenylation (APA). 3'UTRs contain regions that harbor binding motifs for regulatory molecules. However, the mechanisms that coordinate the 3'UTR length of specific groups of transcripts are not well-understood. We therefore developed a method, CSI-UTR, that models 3'UTR structure as tandem segments between functional alternative-polyadenylation sites (termed cleavage site intervals-CSIs). This approach facilitated (1) profiling of 3'UTR isoform expression changes and (2) statistical enrichment of putative regulatory motifs. CSI-UTR analysis is UTR-annotation independent and can interrogate legacy data generated from standard RNA-Seq libraries. CSI-UTR identified a set of CSIs in human and rodent transcriptomes. Analysis of RNA-Seq datasets from neural tissue identified differential expression events within 3'UTRs not detected by standard gene-based differential expression analyses. Further, in many instances 3'UTR and CDS from the same gene were regulated differently. This modulation of motifs for RNA-interacting molecules with potential condition-dependent and tissue-specific RNA binding partners near the polyA signal and CSI junction may play a mechanistic role in the specificity of alternative polyadenylation. Source code, CSI BED files and example datasets are available at: https://github.com/UofLBioinformatics/CSI-UTR.

Nociceptor-dependent locomotor dysfunction after clinically-modeled hindlimb muscle stretching in adult rats with spinal cord injury.

In the course of investigating how common clinical treatments and adaptive technologies affect recovery after spinal cord injury (SCI), we discovered that a clinically-modeled hindlimb stretching protocol dramatically, but transiently, reduces locomotor function. Nociceptive sensory input is capable of altering motor output at the spinal level, and nociceptive neurons are sensitized after SCI. Here we tested the hypotheses that stretch-induced locomotor deficits are dependent on nociceptive afferents by depleting TRPV1+ sensory afferents using capsaicin injections in neonatal rats. Following maturation, animals received 25g-cm contusive SCI at T10. After plateau of locomotor recovery at 6 weeks, daily stretching was performed for 3 weeks, followed by 2 weeks without stretch, and again for two additional weeks. Animals were sacrificed 2 h after the last stretching session for histological assessments. Consistent with previous findings, stretch-induced drops in locomotor function were observed in nociceptor-intact animals but were nearly absent in nociceptor-depleted animals. These functional changes were accompanied by corresponding increases in the number of c-Fos+ nuclei throughout the lumbar enlargement. As expected, nociceptor-depleted animals had very little CGRP+ axonal innervation of the dorsal horn. Nociceptor-intact stretched animals had significantly higher levels of CGRP+ as compared to non-stretched SCI rats, suggesting that stretching promoted intraspinal CGRP+ sprouting. These results indicate that stretch-induced locomotor dysfunction in animals with incomplete SCI involves C-fibers, adding a negative post-SCI role to their adaptive roles (e.g., bladder control), and suggesting that the clinical use of muscle stretching to combat contractures and spasticity may be unintentionally detrimental to locomotor function.

Framework for reanalysis of publicly available Affymetrix® GeneChip® data sets based on functional regions of interest.

BACKGROUND: Since the introduction of microarrays in 1995, researchers world-wide have used both commercial and custom-designed microarrays for understanding differential expression of transcribed genes. Public databases such as ArrayExpress and the Gene Expression Omnibus (GEO) have made millions of samples readily available. One main drawback to microarray data analysis involves the selection of probes to represent a specific transcript of interest, particularly in light of the fact that transcript-specific knowledge (notably alternative splicing) is dynamic in nature. RESULTS: We therefore developed a framework for reannotating and reassigning probe groups for Affymetrix® GeneChip® technology based on functional regions of interest. This framework addresses three issues of Affymetrix® GeneChip® data analyses: removing nonspecific probes, updating probe target mapping based on the latest genome knowledge and grouping probes into gene, transcript and region-based (UTR, individual exon, CDS) probe sets. Updated gene and transcript probe sets provide more specific analysis results based on current genomic and transcriptomic knowledge. The framework selects unique probes, aligns them to gene annotations and generates a custom Chip Description File (CDF). The analysis reveals only 87% of the Affymetrix® GeneChip® HG-U133 Plus 2 probes uniquely align to the current hg38 human assembly without mismatches. We also tested new mappings on the publicly available data series using rat and human data from GSE48611 and GSE72551 obtained from GEO, and illustrate that functional grouping allows for the subtle detection of regions of interest likely to have phenotypical consequences. CONCLUSION: Through reanalysis of the publicly available data series GSE48611 and GSE72551, we profiled the contribution of UTR and CDS regions to the gene expression levels globally. The comparison between region and gene based results indicated that the detected expressed genes by gene-based and region-based CDFs show high consistency and regions based results allows us to detection of changes in transcript formation.

Differential expression of ryanodine receptor isoforms after spinal cord injury.

Ryanodine receptors (RyRs) are highly conductive intracellular Ca2+ release channels and are widely expressed in many tissues, including the central nervous system. RyRs have been implicated in intracellular Ca2+ overload which can drive secondary damage following traumatic injury to the spinal cord (SCI), but the spatiotemporal expression of the three isoforms of RyRs (RyR1-3) after SCI remains unknown. Here, we analyzed the gene and protein expression of RyR isoforms in the murine lumbar dorsal root ganglion (DRG) and the spinal cord lesion site at 1, 2 and 7 d after a mild contusion SCI. Quantitative RT PCR analysis revealed that RyR3 was significantly increased in lumbar DRGs and at the lesion site at 1 and 2 d post contusion compared to sham (laminectomy only) controls. Additionally, RyR2 expression was increased at 1 d post injury within the lesion site. RyR2 and -3 protein expression was localized to lumbar DRG neurons and their spinal projections within the lesion site acutely after SCI. In contrast, RyR1 expression within the DRG and lesion site remained unaltered following trauma. Our study shows that SCI initiates acute differential expression of RyR isoforms in DRG and spinal cord.

Cutaneous tissue damage induces long-lasting nociceptive sensitization and regulation of cellular stress- and nerve injury-associated genes in sensory neurons.

Tissue damage is one of the major etiological factors in the emergence of chronic/persistent pain, although mechanisms remain enigmatic. Using incision of the back skin of adult rats as a model for tissue damage, we observed sensitization in a nociceptive reflex enduring to 28days post-incision (DPI). To determine if the enduring behavioral changes corresponded with a long-term impact of tissue damage on sensory neurons, we examined the temporal expression profile of injury-regulated genes and the electrophysiological properties of traced dorsal root ganglion (DRG) sensory neurons. The mRNA for the injury/stress-hub gene Activating Transcription Factor 3 (ATF3) was upregulated and peaked within 4 DPI, after which levels declined but remained significantly elevated out to 28 DPI, a time when the initial incision appears healed and tissue-inflammation largely resolved. Accordingly, stereological image analysis indicated that some neurons expressed ATF3 only transiently (mostly medium-large neurons), while in others it was sustained (mostly small neurons), suggesting cell-type-specific responses. In retrogradely-traced ATF3-expressing neurons, Calcium/calmodulin-dependent protein kinase type IV (CAMK4) protein levels and isolectin-B4 (IB4)-binding were suppressed whereas Growth Associated Protein-43 (GAP-43) and Neuropeptide Y (NPY) protein levels were enhanced. Electrophysiological recordings from DiI-traced sensory neurons 28 DPI showed a significant sensitization limited to ATF3-expressing neurons. Thus, ATF3 expression is revealed as a strong predictor of single cells displaying enduring pain-related electrophysiological properties. The cellular injury/stress response induced in sensory neurons by tissue damage and indicated by ATF3 expression is positioned to contribute to pain which can occur after tissue damage.

The Adaptor Protein CD2AP Is a Coordinator of Neurotrophin Signaling-Mediated Axon Arbor Plasticity.

Growth of intact axons of noninjured neurons, often termed collateral sprouting, contributes to both adaptive and pathological plasticity in the adult nervous system, but the intracellular factors controlling this growth are largely unknown. An automated functional assay of genes regulated in sensory neurons from the rat in vivo spared dermatome model of collateral sprouting identified the adaptor protein CD2-associated protein (CD2AP; human CMS) as a positive regulator of axon growth. In non-neuronal cells, CD2AP, like other adaptor proteins, functions to selectively control the spatial/temporal assembly of multiprotein complexes that transmit intracellular signals. Although CD2AP polymorphisms are associated with increased risk of late-onset Alzheimer's disease, its role in axon growth is unknown. Assessments of neurite arbor structure in vitro revealed CD2AP overexpression, and siRNA-mediated knockdown, modulated (1) neurite length, (2) neurite complexity, and (3) growth cone filopodia number, in accordance with CD2AP expression levels. We show, for the first time, that CD2AP forms a novel multiprotein complex with the NGF receptor TrkA and the PI3K regulatory subunit p85, with the degree of TrkA:p85 association positively regulated by CD2AP levels. CD2AP also regulates NGF signaling through AKT, but not ERK, and regulates long-range signaling though TrkA(+)/RAB5(+) signaling endosomes. CD2AP mRNA and protein levels were increased in neurons during collateral sprouting but decreased following injury, suggesting that, although typically considered together, these two adult axonal growth processes are fundamentally different. These data position CD2AP as a major intracellular signaling molecule coordinating NGF signaling to regulate collateral sprouting and structural plasticity of intact adult axons. SIGNIFICANCE STATEMENT: Growth of noninjured axons in the adult nervous system contributes to adaptive and maladaptive plasticity, and dysfunction of this process may contribute to neurologic pathologies. Functional screening of genes regulated during growth of noninjured axons revealed CD2AP as a positive regulator of axon outgrowth. A novel association of CD2AP with TrkA and p85 suggests a distinct intracellular signaling pathway regulating growth of noninjured axons. This may also represent a novel mechanism of generating specificity in multifunctional NGF signaling. Divergent regulation of CD2AP in different axon growth conditions suggests that separate mechanisms exist for different modes of axon growth. CD2AP is the first signaling molecule associated with adult sensory axonal collateral sprouting, and this association may offer new insights for NGF/TrkA-related Alzheimer's disease mechanisms.

Delayed intramuscular human neurotrophin-3 improves recovery in adult and elderly rats after stroke.

There is an urgent need for a therapy that reverses disability after stroke when initiated in a time frame suitable for the majority of new victims. We show here that intramuscular delivery of neurotrophin-3 (NT3, encoded by NTF3) can induce sensorimotor recovery when treatment is initiated 24 h after stroke. Specifically, in two randomized, blinded preclinical trials, we show improved sensory and locomotor function in adult (6 months) and elderly (18 months) rats treated 24 h following cortical ischaemic stroke with human NT3 delivered using a clinically approved serotype of adeno-associated viral vector (AAV1). Importantly, AAV1-hNT3 was given in a clinically-feasible timeframe using a straightforward, targeted route (injections into disabled forelimb muscles). Magnetic resonance imaging and histology showed that recovery was not due to neuroprotection, as expected given the delayed treatment. Rather, treatment caused corticospinal axons from the less affected hemisphere to sprout in the spinal cord. This treatment is the first gene therapy that reverses disability after stroke when administered intramuscularly in an elderly body. Importantly, phase I and II clinical trials by others show that repeated, peripherally administered high doses of recombinant NT3 are safe and well tolerated in humans with other conditions. This paves the way for NT3 as a therapy for stroke.

Transcriptional changes in sensory ganglia associated with primary afferent axon collateral sprouting in spared dermatome model.

Primary afferent collateral sprouting is a process whereby non-injured primary afferent neurons respond to some stimulus and extend new branches from existing axons. Neurons of both the central and peripheral nervous systems undergo this process, which contributes to both adaptive and maladaptive plasticity (e.g., [1], [2], [3], [4], [5], [6], [7], [8], [9]). In the model used here (the "spared dermatome" model), the intact sensory neurons respond to the denervation of adjacent areas of skin by sprouting new axon branches into that adjacent denervated territory. Investigations of gene expression changes associated with collateral sprouting can provide a better understanding of the molecular mechanisms controlling this process. Consequently, it can be used to develop treatments to promote functional recovery for spinal cord injury and other similar conditions. This report includes raw gene expression data files from microarray experiments in order to study the gene regulation in spared sensory ganglia in the initiation (7 days) and maintenance (14 days) phases of the spared dermatome model relative to intact ("naïve") sensory ganglia. Data has been deposited into GEO (GSE72551).

The effect of spinal cord injury on the neurochemical properties of vagal sensory neurons.

The vagus nerve is composed primarily of nonmyelinated sensory neurons whose cell bodies are located in the nodose ganglion (NG). The vagus has widespread projections that supply most visceral organs, including the bladder. Because of its nonspinal route, the vagus nerve itself is not directly damaged from spinal cord injury (SCI). Because most viscera, including bladder, are dually innervated by spinal and vagal sensory neurons, an impact of SCI on the sensory component of vagal circuitry may contribute to post-SCI visceral pathologies. To determine whether SCI, in male Wistar rats, might impact neurochemical characteristics of NG neurons, immunohistochemical assessments were performed for P2X3 receptor expression, isolectin B4 (IB4) binding, and substance P expression, three known injury-responsive markers in sensory neuronal subpopulations. In addition to examining the overall population of NG neurons, those innervating the urinary bladder also were assessed separately. All three of the molecular markers were represented in the NG from noninjured animals, with the majority of the neurons binding IB4. In the chronically injured rats, there was a significant increase in the number of NG neurons expressing P2X3 and a significant decrease in the number binding IB4 compared with noninjured animals, a finding that held true also for the bladder-innervating population. Overall, these results indicate that vagal afferents, including those innervating the bladder, display neurochemical plasticity post-SCI that may have implications for visceral homeostatic mechanisms and nociceptive signaling.

Distinct subclassification of DRG neurons innervating the distal colon and glans penis/distal urethra based on the electrophysiological current signature.

Spinal sensory neurons innervating visceral and mucocutaneous tissues have unique microanatomic distribution, peripheral modality, and physiological, pharmacological, and biophysical characteristics compared with those neurons that innervate muscle and cutaneous tissues. In previous patch-clamp electrophysiological studies, we have demonstrated that small- and medium-diameter dorsal root ganglion (DRG) neurons can be subclassified on the basis of their patterns of voltage-activated currents (VAC). These VAC-based subclasses were highly consistent in their action potential characteristics, responses to algesic compounds, immunocytochemical expression patterns, and responses to thermal stimuli. For this study, we examined the VAC of neurons retrogradely traced from the distal colon and the glans penis/distal urethra in the adult male rat. The afferent population from the distal colon contained at least two previously characterized cell types observed in somatic tissues (types 5 and 8), as well as four novel cell types (types 15, 16, 17, and 18). In the glans penis/distal urethra, two previously described cell types (types 6 and 8) and three novel cell types (types 7, 14, and 15) were identified. Other characteristics, including action potential profiles, responses to algesic compounds (acetylcholine, capsaicin, ATP, and pH 5.0 solution), and neurochemistry (expression of substance P, CGRP, neurofilament, TRPV1, TRPV2, and isolectin B4 binding) were consistent for each VAC-defined subgroup. With identification of distinct DRG cell types that innervate the distal colon and glans penis/distal urethra, future in vitro studies related to the gastrointestinal and urogenital sensory function in normal as well as abnormal/pathological conditions may be benefitted.

categoryCompare, an analytical tool based on feature annotations.

Assessment of high-throughput-omics data initially focuses on relative or raw levels of a particular feature, such as an expression value for a transcript, protein, or metabolite. At a second level, analyses of annotations including known or predicted functions and associations of each individual feature, attempt to distill biological context. Most currently available comparative- and meta-analyses methods are dependent on the availability of identical features across data sets, and concentrate on determining features that are differentially expressed across experiments, some of which may be considered "biomarkers." The heterogeneity of measurement platforms and inherent variability of biological systems confounds the search for robust biomarkers indicative of a particular condition. In many instances, however, multiple data sets show involvement of common biological processes or signaling pathways, even though individual features are not commonly measured or differentially expressed between them. We developed a methodology, categoryCompare, for cross-platform and cross-sample comparison of high-throughput data at the annotation level. We assessed the utility of the approach using hypothetical data, as well as determining similarities and differences in the set of processes in two instances: (1) denervated skin vs. denervated muscle, and (2) colon from Crohn's disease vs. colon from ulcerative colitis (UC). The hypothetical data showed that in many cases comparing annotations gave superior results to comparing only at the gene level. Improved analytical results depended as well on the number of genes included in the annotation term, the amount of noise in relation to the number of genes expressing in unenriched annotation categories, and the specific method in which samples are combined. In the skin vs. muscle denervation comparison, the tissues demonstrated markedly different responses. The Crohn's vs. UC comparison showed gross similarities in inflammatory response in the two diseases, with particular processes specific to each disease.

Identification of bladder and colon afferents in the nodose ganglia of male rats.

The sensory neurons innervating the urinary bladder and distal colon project to similar regions of the central nervous system and often are affected simultaneously by various diseases and disorders, including spinal cord injury. Anatomical and physiological commonalities between the two organs involve the participation of shared spinally derived pathways, allowing mechanisms of communication between the bladder and colon. Prior electrophysiological data from our laboratory suggest that the bladder also may receive sensory innervation from a nonspinal source through the vagus nerve, which innervates the distal colon as well. The present study therefore aimed to determine whether anatomical evidence exists for vagal innervation of the male rat urinary bladder and to assess whether those vagal afferents also innervate the colon. Additionally, the relative contribution to bladder and colon sensory innervation of spinal and vagal sources was determined. By using lipophilic tracers, neurons that innervated the bladder and colon in both the nodose ganglia (NG) and L6/S1 and L1/L2 dorsal root ganglia (DRG) were quantified. Some single vagal and spinal neurons provided dual innervation to both organs. The proportions of NG afferents labeled from the bladder did not differ from spinal afferents labeled from the bladder when considering the collective population of total neurons from either group. Our results demonstrate evidence for vagal innervation of the bladder and colon and suggest that dichotomizing vagal afferents may provide a neural mechanism for cross-talk between the organs.