Dense Innervation Research Articles

What the hippocampus tells the HPA axis: Hippocampal output attenuates acute stress responses via disynaptic inhibition of CRF+ PVN neurons

The hippocampus exerts inhibitory feedback on the release of glucocorticoids. Because the major hippocampal efferent projections are excitatory, it has been hypothesized that this inhibition is mediated by populations of inhibitory neurons in the hypothalamus or elsewhere. These regions would be excited by hippocampal efferents and project to corticotropin-releasing factor (CRF) cells in the paraventricular nucleus of the hypothalamus (PVN). A direct demonstration of the synaptic responses elicited by hippocampal outputs in PVN cells or upstream GABAergic interneurons has not been provided previously. Here, we used viral vectors to express channelrhodopsin (ChR) and enhanced yellow fluorescent protein (EYFP) in pyramidal cells in the ventral hippocampus (vHip) in mice expressing tdTomato in GABA- or CRF-expressing neurons. We observed dense innervation of the bed nucleus of the stria terminalis (BNST) by labeled vHip axons and sparse labeling within the PVN. Using whole-cell voltage-clamp recording in parasagittal brain slices containing the BNST and PVN, photostimulation of vHip terminals elicited rapid excitatory postsynaptic currents (EPSCs) and longer-latency inhibitory postsynaptic currents (IPSCs) in both CRF+ and GAD + cells. The ratio of synaptic excitation and inhibition was maintained in CRF + cells during 20 Hz stimulus trains. Photostimulation of hippocampal afferents to the BNST and PVN in vivo inhibited the rise in blood glucocorticoid levels produced by acute restraint stress. We thus provide functional evidence suggesting that hippocampal output to the BNST contributes to a net inhibition of the hypothalamic-pituitary axis, providing further mechanistic insights into this process using methods with enhanced spatial and temporal resolution.

Healthy Human Laryngopharyngeal Sensory Innervation Density Correlates with Age.

Somatosensory feedback from upper airway structures is essential for swallowing and airway defense but little is known about the identities and distributions of human upper airway neurons. Furthermore, whether sensory innervation modifies with aging is unknown. In this study, we quantify neuronal and chemosensory cell density in upper airway structures and correlate with age. Participants underwent biopsies from base of tongue, lateral and midline pharyngeal wall, epiglottis, and arytenoids (N=25 13 female/12 male; 20-80 years, mean 51.4 years without clinical diagnosis of dysphagia or clinical indication for biopsy). Tissue sections were labeled with antibodies for all neurons, myelinated neurons, and chemosensory cells. Densities of lamina propria innervation, epithelial innervation, solitary chemosensory cells, and taste buds were calculated and correlated with age. Arytenoid had the highest density of innervation and chemosensory cells across all measures compared to other sites. Taste buds were frequently observed in arytenoid and epiglottis. Base of tongue, lateral pharynx, and midline posterior pharynx had minimal innervation and few chemosensory cells. Epithelial innervation was present primarily in close proximity to chemosensory cells and taste buds. Overall innervation and myelinated fibers in the arytenoid lamina propria decline with aging. Findings establish the architecture of healthy adult sensory innervation and demonstrate the varied distribution of laryngopharyngeal innervation, necessary steps toward understanding the sensory basis for swallowing and airway defense. We also document age-related decline in arytenoid innervation density. These findings suggest that sensory afferent denervation of the upper airway may be a contributing factor to presbyphagia. NA Laryngoscope, 133:773-784, 2023.

Sex-specific differences in metabolic outcomes after sleeve gastrectomy and intermittent fasting in obese middle-aged mice.

Despite the high prevalence of obesity among middle-aged subjects, it is unclear if sex differences in middle age affect the metabolic outcomes of obesity therapies. Accordingly, in this study, middle-aged obese female and male mice were randomized to one of three groups: sleeve gastrectomy (SG), sham surgery ad libitum (SH-AL), or sham surgery with weight matching to SG through intermittent fasting with calorie restriction (SH-IF). Comprehensive measures of energy and glucose homeostasis, including energy intake, body weight, energy expenditure, glucose and insulin tolerance, and interscapular brown adipose tissue (iBAT) sympathetic innervation density were obtained. At the end of 8 wk, SG and SH-IF females had better metabolic outcomes than their male counterparts. SG females had improved weight loss maintenance, preservation of fat-free mass (FFM), higher total energy expenditure (TEE), normal locomotor activity, and reduced plasma insulin and white adipose tissue (WAT) inflammatory markers. SH-IF females also had lower plasma insulin and WAT inflammatory markers, and higher TEE than SH-IF males, despite their lower FFM. In addition, SH-IF females had higher iBAT sympathetic nerve density than SG and SH-AL females, whereas there were no differences among males. Notably, SH-IF mice of both sexes had the most improved glucose tolerance, highlighting the benefits of fasting, irrespective of weight loss. Results from this study demonstrate that in middle-aged obese mice, female sex is associated with better metabolic outcomes after SG or IF with calorie restriction. Clinical studies are needed to determine if sex differences should guide the choice of obesity therapies.NEW & NOTEWORTHY SG or IF with calorie restriction produces better metabolic outcomes in females than in males. IF with calorie restriction prevents metabolic adaptation, even in the face of fat-free mass loss. IF with calorie restriction in females only, is associated with increased iBAT sympathetic innervation, which possibly mitigates reductions in energy expenditure secondary to fat-free mass loss. Lastly, IF leads to better glucose homeostasis than SG, irrespective of sex.

Characterisation of deep dorsal horn projection neurons in the spinal cord of the Phox2a::Cre mouse line.

Projection neurons belonging to the anterolateral system (ALS) underlie the perception of pain, skin temperature and itch. Many ALS cells are located in laminae III-V of the dorsal horn and the adjacent lateral white matter. However, relatively little is known about the excitatory synaptic input to these deep ALS cells, and therefore about their engagement with the neuronal circuitry of the region. We have used a recently developed mouse line, Phox2a::Cre, to investigate a population of deep dorsal horn ALS neurons known as “antenna cells”, which are characterised by dense innervation from peptidergic nociceptors, and to compare these with other ALS cells in the deep dorsal horn and lateral white matter. We show that these two classes differ, both in the density of excitatory synapses, and in the source of input at these synapses. Peptidergic nociceptors account for around two-thirds of the excitatory synapses on the antenna cells, but for only a small proportion of the input to the non-antenna cells. Conversely, boutons with high levels of VGLUT2, which are likely to originate mainly from glutamatergic spinal neurons, account for only ∼5% of the excitatory synapses on antenna cells, but for a much larger proportion of the input to the non-antenna cells. VGLUT1 is expressed by myelinated low-threshold mechanoreceptors and corticospinal axons, and these innervate both antenna and non-antenna cells. However, the density of VGLUT1 input to the non-antenna cells is highly variable, consistent with the view that these neurons are functionally heterogeneous.

Dopaminergic and cholinergic innervation in the mouse cochlea after noise-induced or age-related synaptopathy

Cochlear synaptopathy, the loss of or damage to connections between auditory-nerve fibers (ANFs) and inner hair cells (IHCs), is a prominent pathology in noise-induced and age-related hearing loss. Here, we investigated if degeneration of the olivocochlear (OC) efferent innervation is also a major aspect of the synaptopathic ear, by quantifying the volume and spatial organization of its cholinergic and dopaminergic components, using antibodies to vesicular acetylcholine transporter (VAT) and tyrosine hydroxylase (TH), respectively. CBA/CaJ male mice were examined 1 day to 8 months after a synaptopathic noise exposure, and compared to unexposed age-matched controls and unexposed aged mice at 24-28 months. In normal ears, cholinergic lateral (L)OC terminals were denser in the apical half of the cochlea and on the modiolar side of the inner hair cells (IHCs), where ANFs of low-spontaneous rate are typically found, while dopaminergic terminals were more common in the basal third of the cochlea and, re the IHC axes, were offset towards the habenula with respect to cholinergic terminals. The noise had only small and transient effects on the density of LOC innervation, its spatial organization around the IHC axes, or the extent to which TH and VAT signal were colocalized. The synaptopathic noise also had relatively small and transient effects on cholinergic innervation density in the outer hair cell (OHC) area, which normally peaks in the 16 kHz region and falls monotonically towards higher and lower frequencies. In contrast, in the aged ears, there was massive degeneration of OHC efferents, especially in the apical half of the cochlea, where there was also significant loss of OHCs. In the IHC area, there was significant loss of cholinergic terminals in both apical and basal regions and of dopaminergic innervation in the basal half. Furthermore, the cholinergic terminals in the aged ears spread from their normal clustering near the IHC basolateral pole, where the ANF synapses are found, to positions up and down the IHC somata and regions of the neuropil closer to the habenula. This apparent migration was most striking in the apex, where the hair cell pathology was greatest, and may be a harbinger of impending hair cell death.

Fascial Innervation: A Systematic Review of the Literature.

Currently, myofascial pain has become one of the main problems in healthcare systems. Research into its causes and the structures related to it may help to improve its management. Until some years ago, all the studies were focused on muscle alterations, as trigger points, but recently, fasciae are starting to be considered a new, possible source of pain. This systematic review has been conducted for the purpose of analyze the current evidence of the muscular/deep fasciae innervation from a histological and/or immunohistochemical point of view. A literature search published between 2000 and 2021 was made in PubMed and Google Scholar. Search terms included a combination of fascia, innervation, immunohistochemical, and different immunohistochemical markers. Of the 23 total studies included in the review, five studies were performed in rats, four in mice, two in horses, ten in humans, and two in both humans and rats. There were a great variety of immunohistochemical markers used to detect the innervation of the fasciae; the most used were Protein Gene Marker 9.5 (used in twelve studies), Calcitonin Gene-Related Peptide (ten studies), S100 (ten studies), substance P (seven studies), and tyrosine hydroxylase (six studies). Various areas have been studied, with the thoracolumbar fascia being the most observed. Besides, the papers highlighted diversity in the density and type of innervation in the various fasciae, going from free nerve endings to Pacini and Ruffini corpuscles. Finally, it has been observed that the innervation is increased in the pathological fasciae. From this review, it is evident that fasciae are well innerved, their innervation have a particular distribution and precise localization and is composed especially by proprioceptors and nociceptors, the latter being more numerous in pathological situations. This could contribute to a better comprehension and management of pain.

Neuroimmune Interaction: A Widespread Mutual Regulation and the Weapons for Barrier Organs.

Since the embryo, the nervous system and immune system have been interacting to regulate each other’s development and working together to resist harmful stimuli. However, oversensitive neural response and uncontrolled immune attack are major causes of various diseases, especially in barrier organs, while neural-immune interaction makes it worse. As the first defense line, the barrier organs give a guarantee to maintain homeostasis in external environment. And the dense nerve innervation and abundant immune cell population in barrier organs facilitate the neuroimmune interaction, which is the physiological basis of multiple neuroimmune-related diseases. Neuroimmune-related diseases often have complex mechanisms and require a combination of drugs, posing challenges in finding etiology and treatment. Therefore, it is of great significance to illustrate the specific mechanism and exact way of neuro-immune interaction. In this review, we first described the mutual regulation of the two principal systems and then focused on neuro-immune interaction in the barrier organs, including intestinal tract, lungs and skin, to clarify the mechanisms and provide ideas for clinical etiology exploration and treatment.

Dissecting the Neural Substrate of Decreased Reward Sensitivity in a Model of Chronic Pain

Chronic pain is a debilitating condition that shades the lives of approximately 20% of the global population. Patients with chronic pain often display impairments in cognitive function, such as cognitive inflexibility, that negatively affect treatment efficacy. Understanding the mechanism underlying chronic pain and its behavioral impacts is therefore crucial for the development of successful treatments on symptoms beyond the physical aspect. However, current study on the circuitous underpinning of pain-associated behavioral inflexibility is still insufficient. Chronic pain is associated with a hypodopaminergic state in patients and animal models; the medial prefrontal cortex (mPFC) and nucleus accumbens shell (NAcSh) are two brain regions that receive dense dopaminergic innervation, and dopamine exquisitely modulates synaptic plasticity and excitability in this pathway. Projections from the medial prefrontal cortex (mPFC) to nucleus accumbens shell (NAcSh) specifically are essential for encoding reward associations and flexibly updating behavior in response to reward. Therefore, we hypothesize that a pain-associated hypodopaminergic state would modulate function of the mPFC–NAcSh pathway and lead to impairment in reward-related behavioral flexibility. Whether maladaptive plasticity of the mPFC–NAc pathway contributes to the behavioral inflexibility associated with chronic pain has not been well understood. Here, we will test the hypothesis that decreased synaptic strength of the mPFC–NAcSh pathway leads to decreased reward-based effort allocation following induction of a chronic pain and impaired behavioral flexibility under conditions of reward uncertainty. These behavioral deficits were associated with decreased excitatory synaptic transmission into the nucleus accumbens in a cell-type-specific manner, providing a potential neural substrate of these effects. Together, these results provide evidence linking a pain-associated hypodopaminergic state to downstream dopamine-dependent adaptations in cortico-striatal circuits that could contribute to impairments in reward-based behavior. In the future, it is possible that this pathway could be harnessed for neuromodulation strategies to treat cognitive or affective symptoms of chronic pain. Grant support from Rita Allen Scholar Award in Pain (to M.C.C.). Chronic pain is a debilitating condition that shades the lives of approximately 20% of the global population. Patients with chronic pain often display impairments in cognitive function, such as cognitive inflexibility, that negatively affect treatment efficacy. Understanding the mechanism underlying chronic pain and its behavioral impacts is therefore crucial for the development of successful treatments on symptoms beyond the physical aspect. However, current study on the circuitous underpinning of pain-associated behavioral inflexibility is still insufficient. Chronic pain is associated with a hypodopaminergic state in patients and animal models; the medial prefrontal cortex (mPFC) and nucleus accumbens shell (NAcSh) are two brain regions that receive dense dopaminergic innervation, and dopamine exquisitely modulates synaptic plasticity and excitability in this pathway. Projections from the medial prefrontal cortex (mPFC) to nucleus accumbens shell (NAcSh) specifically are essential for encoding reward associations and flexibly updating behavior in response to reward. Therefore, we hypothesize that a pain-associated hypodopaminergic state would modulate function of the mPFC–NAcSh pathway and lead to impairment in reward-related behavioral flexibility. Whether maladaptive plasticity of the mPFC–NAc pathway contributes to the behavioral inflexibility associated with chronic pain has not been well understood. Here, we will test the hypothesis that decreased synaptic strength of the mPFC–NAcSh pathway leads to decreased reward-based effort allocation following induction of a chronic pain and impaired behavioral flexibility under conditions of reward uncertainty. These behavioral deficits were associated with decreased excitatory synaptic transmission into the nucleus accumbens in a cell-type-specific manner, providing a potential neural substrate of these effects. Together, these results provide evidence linking a pain-associated hypodopaminergic state to downstream dopamine-dependent adaptations in cortico-striatal circuits that could contribute to impairments in reward-based behavior. In the future, it is possible that this pathway could be harnessed for neuromodulation strategies to treat cognitive or affective symptoms of chronic pain. Grant support from Rita Allen Scholar Award in Pain (to M.C.C.).

SPARC: The structure of vagal nociceptive nerve fibers in the mouse esophagus

The esophagus is innervated by nociceptive sensory afferent nerve fibers originating from the vagus nerve. The nature of the vagal nociceptive fiber innervation, specifically which esophageal layers, however, remains unknown. We hypothesized that the vagal nociceptive TRPV1+ afferent fibers innervate the esophageal mucosa. We evaluated our hypothesis by using genetic neuronal tracing in several mutant mice, including two new strains we generated. The wholemount preparations of separated esophageal mucosa and muscle were immunostained for appropriate fluorescent proteins and imaged by spinning‐disk confocal microscopy. In the TRPV1‐Flp/RC: FLTG mice(N=8) in which TRPV1‐expressing cells express tdTomato the TRPV1+ fibers with varicose appearance densely innervated both the mucosa and the muscle. In the mucosa the TRPV1+ fibers are located just beneath the epithelium, run in parallel registers of two or more fibers, have craniocaudal orientation and branch repeatedly. In the muscle the TRPV1+ fibers form a dense network in the myenteric plexus innervating virtually all myenteric ganglia forming intraganglionic varicose endings (IGVEs). Many TRPV1+ fibers connecting IGVEs run in parallel to the fibers of the outer and inner striated muscle. In the TRPV1‐Flp/Tac1‐Cre/RC:FLTG mice(N=4) in which GFP is selectively expressed in cells positive for both TRPV1 and Tac1 (the marker of esophageal nociceptors derived from neural crest) the esophageal innervation by TRPV1+/Tac1+ fibers mimicked the dense innervation by TRPV1+ fibers. In contrast, in the TRPV1‐Flp/P2X2‐Cre/RC:FLTG mice(N=4) in which GFP is selectively expressed in cells positive for both TRPV1 and P2X2 (the marker of esophageal nociceptors derived from placodes) the TRPV1+/P2X2+ fibers were almost absent in the esophagus. Consistent with this finding, in the P2X2‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP virus into vagal ganglia(N=2) in which GFP is selectively expressed in vagal P2X2+ fibers, the nodose TRPV1‐negative tension mechanosensor nerve terminals (intraganglionic laminar endings) were visualized in the esophageal muscle. Finally, in the TRPV1‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP virus into vagal ganglia(N=3), in which only the vagal TRPV1+ fibers express GFP, the vagal TRPV1+ fibers had the same pattern as TRPV1+/Tac1+ fibers, but with lower density and patchy distribution. Our data indicate that vagal nociceptive TRPV1+ fibers innervate both the esophageal mucosa and myenteric ganglia and originate from neural crest‐derived jugular portion of the vagal jugular‐nodose ganglia complex.

Peripheral Neuro-immune Interactions in Atopic Dermatitis

Atopic dermatitis (AD) is a chronic inflammatory skin disease, characterized by clinical manifestations of dry skin, chronic eczema and severe itching, often combined with asthma and allergic rhinitis. AD can affect as many as 20% of children and 1–3% of adults. Skin barrier dysfunction, inflammatory and immunological abnormalities have long been noted in patients with AD. However, conflicting evidence suggests that enhanced innervation of sensory neurons may contribute to disease pathology in AD. To better understand this phenomenon, we investigated the role of sensory neurons and their relationship with mast cells in itch sensations at different ages of AD initiation. AD was induced by epicutaneous exposure of extracts from Aspergillus fumigatus (ASP) in a sensory neuron reporter animal (Pirt;tdTomato) initiated either at postnatal day14 (P14) or P35. Mice were exposed to ASP extract (or saline) for 3 weeks via patches on the back skin. We performed immunohistochemistry on back skin for mast cell protease 6 (MCPT-6), assessed trans-epidermal water loss (TEWL), and determined scratching behaviors using an artificial intelligence system. We demonstrated an increased total innervation in the skin of ASP treated mice started at P35 but not at P14. There was significant increase in TEWL and thickness of epidermal layers in both AD groups. However, increased thickening at P35 was 2x greater than that observed at P14. This resulted in decreased sensory innervation density specifically in the epidermal layer at P35. We also found increased MCs at the epidermal/dermal border in both ASP treated groups. Surprisingly, ASP treated mice at P14 did not develop robust scratching behaviors as observed at P35. Results suggest that distinctions in innervation density in the epidermis vs dermis in association with MCs may underlie scratching behaviors that contribute to AD progression and could reveal novel treatment strategies for AD. Grant support from 5U19 AI70235-15 R01NS105715 R01NS113965.

Sympathetic Modulation of Oral Cancer Pain via Adrenergic Signaling in Trigeminal Primary Afferent Neurons

Head and neck squamous cell carcinoma (HNSCC) often codevelops with severe functional pain that significantly impacts patients' quality of life. Previous research demonstrated dense innervation of sympathetic and sensory nerves within the oral tumor microenvironment. We hypothesize that nociceptive afferents innervating oral cancer develop adrenergic sensitivity resulting in sympathetically maintained pain. Patient-reported pain in tandem with patient blood and tumor samples were used to explore the relationship between tumor-innervating sympathetic nerves and cancer pain. A syngeneic tongue transplant mouse oral cancer (MOC2) model and an orofacial behavioral assay using gnaw-time as an index of pain were used to quantify cancer-induced nociceptive behavior. qPCR was performed on trigeminal ganglia (TGs) from MOC2-tumor-bearing mice to investigate the effect of oral cancer on adrenergic receptor gene expression. Sympathetic nerve fibers, identified using tyrosine hydroxylase (TH) immunoreactivity, comprised 4.73± 1.3% of total nerve area across 13 HNSCC tumor samples. We found a positive correlation (r2=0.309) between TH nerve presence and patient-reported pain using the FACT-H&N questionnaire. Furthermore, we quantified a 2-fold increase in platelet NE concentration isolated from HSNCC patient plasma compared to healthy controls and there was a positive correlation (r2=0.687) between NE concentration and patient reported pain. MOC2 tumor progression evoked orofacial nociceptive behavior in female mice; there was a 198.8±20.5% and 478.5±146.8% increase in gnaw-time at post inoculation day (PID) 7 and 14 respectively compared to sham mice. PID14 was also associated with significant changes in adrenergic gene expression in TG from tumor-bearing female compared to control mice; there was a 2-fold increase in genes for the following adrenergic receptor subtypes: Adra1b, Adra2b, and Adrb2. Future investigation using adrenergic blockade will further support the role of sympathetic modulation of oral cancer pain and allow for better and more focused treatment options for these patients. Grant support from NIH NIDCR R01DE030892.

Prefrontal‐medullary circuit activation attenuates stress reactivity

Organismal survival and adaptation to stress rely on brainstem catecholaminergic neurons. In particular, catecholaminergic neurons in the rostral ventrolateral medulla (RVLM) drive sympathetic activity and enable physiological adaptations, including vasoconstriction, corticosterone release, and glycemic mobilization. However, it is unclear how brain regions involved in the cognitive appraisal of stress regulate the activity of RVLM neurons. Our previous studies found that the rodent infralimbic prefrontal cortex (IL) integrates behavioral and physiological responses to stress. Thus, a potential IL‐to‐RVLM connection would represent a crucial link between stress appraisal and sympathetic reactivity. In the current study, we investigated a direct IL‐to‐RVLM circuit by targeting a genetically‐encoded anterograde tracer under the control of the CaMKIIα promoter to the IL of adult male and female rats. Analysis revealed that IL terminals apposed RVLM neurons expressing the catecholamine‐synthesizing enzyme dopamine beta hydroxylase. Further, IL input to catecholamine cells was widespread throughout the rostral to caudal extent of the VLM in both male and female rodents. Quantification of innervation density revealed that males had a larger proportion of VLM catecholamine neurons receiving IL inputs relative to female rats. Additionally, IL appositions were identified on GABAergic and glycinergic neurons in both sexes. Accordingly, we hypothesized that IL projections may activate local RVLM inhibitory cells to limit sympathetic output. To test this hypothesis, we injected a viral vector coding for channelrhodopsin‐2 (ChR2) in the IL of males and females. Next, a fiber optic cannula was implanted dorsal to the RVLM to evoke IL synaptic glutamate release. Animals then received photostimulation during restraint stress with blood sampled to determine stress reactivity. Compared to controls, male rats expressing ChR2 on IL terminals had suppressed glycemic stress responses (p < 0.05). In contrast, stimulation of the IL‐RVLM circuit in females did not affect glucose mobilization (p < 0.05). However, ChR2 decreased corticosterone responses to stress relative to control rats in both sexes (males, p < 0.01; females, p < 0.05). Thus, both male and female rats have a direct circuit from the IL portion of the mPFC to catecholamine‐synthesizing cells of the RVLM that limits glucocorticoid stress responses, likely through the activation of local inhibitory neurons. However, the density of this circuit is greater in males, potentially accounting for reduced glycemic responses. Ultimately, excitatory/inhibitory balance at IL synapses in the RVLM may be critical for the health consequences of stress.

Diabetic visceral neuropathy of gastroparesis: Gastric mucosal innervation and clinical significance.

The pathogenesis of diabetic gastroparesis due to visceral neuropathy involves multidimensional mechanisms with limited exploration of gastric mucosal innervation. This study aimed to examine quantitatively this topic and its relationship with gastroparesis symptoms and gastric emptying in diabetes. We prospectively enrolled 22 patients with type 2 diabetes and gastroparesis symptoms and 25 age- and gender-matched healthy controls for comparison. The assessments included: (i) neuropathology with quantification of gastric mucosal innervation density (MID) on endoscopic biopsy; (ii) clinical manifestations based on the Gastroparesis Cardinal Symptom Index (GCSI) questionnaire; and (iii) functional tests of gastric emptying scintigraphy (GES). In patients with diabetes, stomach fullness, bloating and feeling excessively full after meals constituted the most common GCSI symptoms. Seven patients with diabetes (32%) had prolonged gastric emptying patterns. In diabetes, gastric MID was significantly lower in all the regions examined compared with the controls: antrum (294.8±237.0 vs. 644.0±222.0mm/mm3 ; p<0.001), body (292.2±239.0 vs. 652.6±260.9mm/mm3 ; p<0.001), and fundus (238.0±109.1 vs. 657.2±332.8mm/mm3 ; p<0.001). Gastric MID was negatively correlated with gastroparesis symptoms and total scores on the GCSI (p<0.001). Furthermore, gastric MID in the fundus was negatively correlated with fasting glucose and glycated hemoglobin levels. Gastric emptying variables, including half emptying time and gastric retention, were prolonged in patients with diabetes, and gastric retention at 3h was correlated with fasting glucose level. In diabetes, gastric MID was reduced and GES parameters were prolonged. Both were correlated with gastroparesis symptoms and glycemic control. These findings provide pathology and functional biomarkers for diabetic visceral neuropathy of gastroparesis and underlying pathophysiology.

Immunohistochemical and ultrastructural study of the immune cell system and epithelial surfaces of the respiratory organs in the bimodally breathing African sharptooth catfish (Clarias gariepinus Burchell, 1822).

Acetylcholine (Ach) represents the old neurotransmitter in central and peripheral nervous system. Its muscarinic and nicotinic receptors (mAChRs and nAChRs) constitute an independent cholinergic system that is found in immune cells and play a key role in the regulation of the immune function and cytokine production. Gas exchanging surfaces of the gills and air-breathing organs (ABOs) of the sharptooth catfish Clarias gariepinus were investigated using ultrastructural and confocal immunofluorescence techniques. This study was predominantly focused on the structure of the immune cell types, the expression of their neurotransmitters, including the antimicrobial peptide piscidin 1, and the functional significance of respiratory gas exchange epithelia. A network of immune cells (monocytes, eosinophils, and mast cells) was observed in the gill and the ABO epithelia. Eosinophils containing 5-hydroxytryptamine (5-HT) immunoreactivity were seen in close association with mast cells expressing acetylcholine (Ach), 5-HT, neuronal nitric oxide synthase, and piscidin 1. A rich and dense cholinergic innervation dispersing across the islet capillaries of the gas exchange barrier and the localization of Ach in the squamous pavement cells covering the capillaries were evidenced byVAChT antibodies. We report for the first time that piscidin 1 (Pis 1)-positive mast cells interact with Pis 1-positive nerves found in the epithelia of the respiratory organs. Pis 1 immunoreactivity was also observed in the covering respiratory epithelium of the ABOs and associated with a role in local mucosal immune defense. The above results anticipate future studies on the neuro-immune interactions at mucosal barrier surfaces, like the gill and the skin of fish, areas densely populated by different immune cells and sensory nerves that constantly sense and adapt to tissue-specific environmental challenges.

SARM1 Suppresses Axon Branching Through Attenuation of Axonal Cytoskeletal Dynamics.

Axon branching is a fundamental aspect of neuronal morphogenesis, neuronal circuit formation, and response of the nervous system to injury. Sterile alpha and TIR motif containing 1 (SARM1) was initially identified as promoting Wallerian degeneration of axons. We now report a novel function of SARM1 in postnatal sensory neurons; the suppression of axon branching. Axon collateral branches develop from axonal filopodia precursors through the coordination of the actin and microtubule cytoskeleton. In vitro analysis revealed that cultured P0-2 dorsal root ganglion sensory neurons from a SARM1 knockout (KO) mouse exhibit increased numbers of collateral branches and axonal filopodia relative to wild-type neurons. In SARM1 KO mice, cutaneous sensory endings exhibit increased branching in the skin in vivo with normal density of innervation. Transient axonal actin patches serve as cytoskeletal platforms from which axonal filopodia emerge. Live imaging analysis of axonal actin dynamics showed that SARM1 KO neurons exhibit increased rates of axonal actin patch formation and increased probability that individual patches will give rise to a filopodium before dissipating. SARM1 KO axons contain elevated levels of drebrin and cortactin, two actin regulatory proteins that are positive regulators of actin patches, filopodia formation, and branching. Live imaging of microtubule plus tip dynamics revealed an increase in the rate of formation and velocity of polymerizing tips along the axons of SARM1 KO neurons. Stationary mitochondria define sites along the axon where branches may arise, and the axons of SARM1 KO sensory neurons exhibit an increase in stationary mitochondria. These data reveal SARM1 to be a negative regulator of axonal cytoskeletal dynamics and collateral branching.

Morphologies, dimensions and targets of gastric nitric oxide synthase neurons

We investigated the distributions and targets of nitrergic neurons in the rat stomach, using neuronal nitric oxide synthase (NOS) immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Nitrergic neurons comprised similar proportions of myenteric neurons, about 30%, in all gastric regions. Small numbers of nitrergic neurons occurred in submucosal ganglia. In total, there were ~ 125,000 neuronal nitric oxide synthase (nNOS) neurons in the stomach. The myenteric cell bodies had single axons, type I morphology and a wide range of sizes. Five targets were identified, the longitudinal, circular and oblique layers of the external muscle, the muscularis mucosae and arteries within the gastric wall. The circular and oblique muscle layers had nitrergic fibres throughout their thickness, while the longitudinal muscle was innervated at its inner surface by fibres of the tertiary plexus, a component of the myenteric plexus. There was a very dense innervation of the pyloric sphincter, adjacent to the duodenum. The muscle strands that run between mucosal glands rarely had closely associated nNOS nerve fibres. Both nNOS immunohistochemistry and NADPH histochemistry showed that nitrergic terminals did not provide baskets of terminals around myenteric neurons. Thus, the nitrergic neuron populations in the stomach supply the muscle layers and intramural arteries, but, unlike in the intestine, gastric interneurons do not express nNOS. The large numbers of nNOS neurons and the density of innervation of the circular muscle and pyloric sphincter suggest that there is a finely graded control of motor function in the stomach by the recruitment of different numbers of inhibitory motor neurons.

Evolution of inner ear neuroanatomy of bats and implications for echolocation.

Phylogenomics of bats suggests that their echolocation either evolved separately in the bat suborders Yinpterochiroptera and Yangochiroptera, or had a single origin in bat ancestors and was later lost in some yinpterochiropterans1-6. Hearing for echolocation behaviour depends on the inner ear, of which the spiral ganglion is an essential structure. Here we report the observation of highly derived structures of the spiral ganglion in yangochiropteran bats: a trans-otic ganglion with a wall-less Rosenthal's canal. This neuroanatomical arrangement permits a larger ganglion with more neurons, higher innervation density of neurons and denser clustering of cochlear nerve fascicles7-13. This differs from the plesiomorphic neuroanatomy of Yinpterochiroptera and non-chiropteran mammals. The osteological correlates of these derived ganglion features can now be traced into bat phylogeny, providing direct evidence of how Yangochiroptera differentiated from Yinpterochiroptera in spiral ganglion neuroanatomy. These features are highly variable across major clades and between species of Yangochiroptera, and in morphospace, exhibit much greater disparity in Yangochiroptera than Yinpterochiroptera. These highly variable ganglion features may be a neuroanatomical evolutionary driver for their diverse echolocating strategies4,14-17 and are associated with the explosive diversification of yangochiropterans, which include most bat families, genera and species.

Clodronate Treatment Prevents Vaginal Hypersensitivity in a Mouse Model of Vestibulodynia.

IntroductionImproved understanding of vestibulodynia pathophysiology is required to develop appropriately targeted treatments. Established features include vulvovaginal hyperinnervation, increased nociceptive signalling and hypersensitivity. Emerging evidence indicates macrophage-neuron signalling contributes to chronic pain pathophysiology. Macrophages are broadly classified as M1 or M2, demonstrating pro-nociceptive or anti-nociceptive effects respectively. This study investigates the impact of clodronate liposomes, a macrophage depleting agent, on nociceptive signalling in a mouse model of vestibulodynia.MethodsMicroinjection of complete Freund’s adjuvant (CFA) at the vaginal introitus induced mild chronic inflammation in C57Bl/6J mice. A subgroup was treated with the macrophage depleting agent clodronate. Control mice received saline. After 7 days, immunolabelling for PGP9.5, F4/80+CD11c+ and F4/80+CD206+ was used to compare innervation density and presence of M1 and M2 macrophages respectively in experimental groups. Nociceptive signalling evoked by vaginal distension was assessed using immunolabelling for phosphorylated MAP extracellular signal-related kinase (pERK) in spinal cord sections. Hyperalgesia was assessed by visceromotor response to graded vaginal distension.ResultsCFA led to increased vaginal innervation (p < 0.05), increased pERK-immunoreactive spinal cord dorsal horn neurons evoked by vaginal-distension (p < 0.01) and enhanced visceromotor responses compared control mice (p < 0.01). Clodronate did not reduce vaginal hyperinnervation but significantly reduced the abundance of M1 and M2 vaginal macrophages and restored vaginal nociceptive signalling and vaginal sensitivity to that of healthy control animals.ConclusionsWe have developed a robust mouse model of vestibulodynia that demonstrates vaginal hyperinnervation, enhanced nociceptive signalling, hyperalgesia and allodynia. Macrophages contribute to hypersensitivity in this model. Macrophage-sensory neuron signalling pathways may present useful pathophysiological targets.

Specializations of somatosensory innervation in the skin of humpback whales (Megaptera novaeangliae).

Cetacean behavior and life history imply a role for somatosensory detection of critical signals unique to their marine environment. As the sensory anatomy of cetacean glabrous skin has not been fully explored, skin biopsy samples of the flank skin of humpback whales were prepared for general histological and immunohistochemical (IHC) analyses of innervation in this study. Histology revealed an exceptionally thick epidermis interdigitated by numerous, closely spaced long, thin diameter penicillate dermal papillae (PDP). The dermis had a stratified organization including a deep neural plexus (DNP) stratum intermingled with small arteries that was the source of intermingled nerves and arterioles forming a more superficial subepidermal neural plexus (SNP) stratum. The patterns of nerves branching through the DNP and SNP that distribute extensive innervation to arteries and arterioles and to the upper dermis and PDP provide a dense innervation associated through the whole epidermis. Some NF-H+ fibers terminated at the base of the epidermis and as encapsulated endings in dermal papillae similar to Merkel innervation and encapsulated endings seen in terrestrial mammals. However, unlike in all mammalian species assessed to date, an unusual acellular gap was present between the perineural sheaths and the central core of axons in all the cutaneous nerves perhaps as mechanism to prevent high hydrostatic pressure from compressing and interfering with axonal conductance. Altogether the whale skin has an exceptionally dense low-threshold mechanosensory system innervation most likely adapted for sensing hydrodynamic stimuli, as well as nerves that can likely withstand high pressure experienced during deep dives.

Management of Psychiatric Disorders with HIV and Dermatological Disorders.

Management of Psychiatric Disorders with HIV and Dermatological Disorders.