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.

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.

Effect of sex and sex steroids on brown adipose tissue heat production in humans.

OBJECTIVE: Retrospective studies suggest that women have more active brown adipose tissue (BAT) than men, but little is known of the effect of fluctuating sex steroids across the menstrual cycle on thermogenesis in women. DESIGN: To characterise the effects of sex and sex steroids on BAT activity we recruited healthy weight men (n = 14) and women at two stages of the menstrual cycle (luteal, n = 9; follicular, n = 11). METHODS: Infrared thermography measured supraclavicular temperature to index BAT thermogenesis in response to both cold (immersion of one hand in water at 15°C) and meal (Ensure, 10 kcal/kg body weight) stimuli. RESULTS: Adaptive BAT temperature responses were greater (P < 0.05) in women than men, irrespective of stage of menstrual cycle. Whereas during cold exposure, the increase in BAT temperature was abrogated (P < 0.05) in women during follicular phase compared to men and women during luteal phase. Plasma concentrations of progesterone, 17ß-estradiol, testosterone and cortisol were measured. Regression analyses demonstrated that baseline BAT temperature was positively correlated (P < 0.05) with progesterone levels, but was inversely associated (P < 0.05) with cortisol concentration. Both cold- and meal-induced changes in BAT temperature mildly correlated (P = 0.07; P < 0.05) with 17ß-estradiol levels, but not with testosterone concentrations. CONCLUSIONS: Baseline supraclavicular temperature is elevated in women during the luteal phase of the menstrual cycle, which correlated with elevated progesterone concentrations. Women exhibited greater thermogenic responses than men, irrespective of the state of the menstrual cycle, which was associated with plasma levels of 17ß-estradiol. We conclude that sex steroids may regulate BAT thermogenesis in healthy adults.

Adipose and skeletal muscle thermogenesis: studies from large animals.

The balance between energy intake and energy expenditure establishes and preserves a 'set-point' body weight. The latter is comprised of three major components including metabolic rate, physical activity and thermogenesis. Thermogenesis is defined as the cellular dissipation of energy via heat production. This process has been extensively characterised in brown adipose tissue (BAT), wherein uncoupling protein 1 (UCP1) creates a proton leak across the inner mitochondrial membrane, diverting protons away from ATP synthesis and resulting in heat dissipation. In beige adipocytes and skeletal muscle, thermogenesis can occur independent of UCP1. Beige adipocytes have been shown to produce heat via UCP1 as well as via both futile creatine and calcium cycling pathways. On the other hand, the UCP1 homologue UCP3 is abundant in skeletal muscle and post-prandial thermogenesis has been associated with UCP3 and the futile calcium cycling. This review will focus on the differential contributions of adipose tissue and skeletal muscle in determining total thermogenic output and energy expenditure in large mammals. Sheep and pigs do not have a circumscribed brown fat depot but rather possess white fat depots that contain brown and beige adipocytes interspersed amongst white adipose tissue. This is representative of humans, where brown, beige and white adipocytes have been identified in the neck and supraclavicular regions. This review will describe the mechanisms of thermogenesis in pigs and sheep and the relative roles of skeletal muscle and adipose tissue thermogenesis in controlling body weight in larger mammals.

Exercise counteracts the homeostatic decrease in thermogenesis caused by caloric restriction in sheep.

Caloric restriction causes a homeostatic reduction in thermogenesis. We aimed to determine whether exercise could counteract this. We studied four groups of normal-weight ewes ( n = 5), including control sedentary fed ad libitum, exercise fed ad libitum (30 min/d, 5 d/wk), diet-restricted (70% of ad libitum food intake), and combined diet and exercise. Temperature probes implanted in sternal and retroperitoneal adipose tissue and skeletal muscle measured thermogenesis. After the 4-wk intervention, hypothalami were collected for in situ hybridization, and fat and muscle biopsies were collected for real-time PCR and Western blotting. Combined diet and exercise reduced adiposity ( P < 0.05). Caloric restriction alone reduced overnight temperatures in sternal and retroperitoneal fat ( P < 0.05), which was counteracted by exercise ( P < 0.05). Exercise did not induce expression of cellular markers of browning in adipose tissue. There was no effect of diet or exercise on skeletal muscle thermogenesis. Combined diet and exercise increased the expression of neuropeptide Y and agouti-related protein in the hypothalamic arcuate nucleus ( P < 0.05), consistent with reduced adiposity. Gene expressions of key hypothalamic appetite-regulating peptides were not associated with altered thermogenesis. We demonstrate that exercise counteracts the inhibitory effect of caloric restriction to restore thermogenesis in adipose tissue of sheep.-Fuller-Jackson, J.-P., Clarke, I. J., Rao, A., Henry, B. A. Exercise counteracts the homeostatic decrease in thermogenesis caused by caloric restriction in sheep.

Ontogeny and Thermogenic Role for Sternal Fat in Female Sheep.

Brown adipose tissue acting through a unique uncoupling protein (UCP1) has a critical role in preventing hypothermia in newborn sheep but is then thought to rapidly disappear during postnatal life. The extent to which the anatomical location of fat influences postnatal development and thermogenic function in adulthood, particularly following feeding, is unknown, and we examined both in our study. Changes in gene expression of functionally important pathways (i.e., thermogenesis, development, adipogenesis, and metabolism) were compared between sternal and retroperitoneal fat depots together with a representative skeletal muscle over the first month of postnatal life, coincident with the loss of brown fat and the accumulation of white fat. In adult sheep, implanted temperature probes were used to characterize the thermogenic response of fat and muscle to feeding and the effects of reduced or increased adiposity. UCP1 was more abundant in sternal fat than in retroperitoneal fat and was retained only in the sternal depot of adults. Distinct differences in the abundance of gene pathway markers were apparent between tissues, with sternal fat exhibiting some similarities with muscle that were not apparent in the retroperitoneal depot. In adults, the postprandial rise in temperature was greater and more prolonged in sternal fat than in retroperitoneal fat and muscle, a difference that was maintained with altered adiposity. In conclusion, sternal adipose tissue retains UCP1 into adulthood, when it shows a greater thermogenic response to feeding than do muscle and retroperitoneal fat. Sternal fat may be more amenable to targeted interventions that promote thermogenesis in large mammals.