How much does it cost? Teaching physiology of energy metabolism in mice using an indirect calorimetry system in a practical course for veterinary students.

In endothermic mammals total energy expenditure (EE) is composed of basal metabolic rate (BMR), energy spent for muscle activity, thermoregulation, any kind of production (such as milk, meat, or egg production), and the thermic effect of feeding. The BMR is predominantly determined by body mass and the surface-to-volume ratio of the body. The EE can be quantified by either direct or indirect calorimetry. Direct calorimetry measures the rate of heat loss from the body, whereas indirect calorimetry measures oxygen consumption and carbon dioxide production and calculates heat production from oxidative nutrient combustion. A deep and sustainable understanding of EE in animals is crucial for veterinarians to properly calculate and evaluate feed rations during special circumstances such as anesthesia or in situations with increased energy demands as commonly seen in high-yielding livestock. The practical class described in this article provides an experimental approach to understanding how EE can be measured and calculated by indirect calorimetry. Two important factors that affect the EE of animals (the thermic effect of feeding and the effect of ambient temperature) are measured. A profound knowledge about the energy requirements of animal life and its measurement is also relevant for education in general biology, animal and human physiology, and nutrition. Therefore, this teaching unit can equally well be implemented in other areas of life sciences.

Submucosal enteric neurons of the cavine distal colon are sensitive to hypoosmolar stimuli.

KEY POINTS: Neurons of the enteric submucous plexus are challenged by osmolar fluctuations during digestion and absorption of nutrients. Central neurons are very sensitive to changes in osmolality but knowledge on that issue related to enteric neurons is sparse. The present study focuses on investigation of osmosensitivity of submucosal neurons including potential molecular mediating mechanisms. Results show that submucosal neurons respond to hypoosmolar stimuli with increased activity which is partially mediated by the transient receptor potential vanilloid 4 channel. We provided important information on osmosensitive properties of enteric neurons. These data are fundamental to better explain the nerve-mediated control of the gastrointestinal functions during physiological and pathophysiological (diarrhoea) conditions. ABSTRACT: Enteric neurons are located inside the gut wall, where they are confronted with changes in osmolality during (inter-) digestive periods. In particular, neurons of the submucous plexus (SMP), located between epithelial cells and blood vessels may sense and respond to osmotic shifts. The present study was conducted to investigate osmosensitivity of enteric submucosal neurons and the potential role of the transient receptor potential vanilloid 4 channel (TRPV4) as a mediator of enteric neuronal osmosensitivity. Therefore, freshly dissected submucosal preparations from guinea pig colon were investigated for osmosensitivity using voltage-sensitive dye and Ca2+ imaging. Acute hypoosmolar stimuli (final osmolality reached at ganglia of 94, 144 and 194 mOsm kg-1 ) were applied to single ganglia using a local perfusion system. Expression of TRPV4 in the SMP was quantified using qRT-PCR, and GSK1016790A and HC-067047 were used to activate or block the receptor, respectively, revealing its relevance in enteric osmosensitivity. On average, 11.0 [7.0/17.0] % of submucosal neurons per ganglion responded to the hypoosmolar stimulus. The Ca2+ imaging experiments showed that glia responded to the hypoosmolar stimulus, but with a delay in comparison with neurons. mRNA expression of TRPV4 could be shown in the SMP and blockade of the receptor by HC-067047 significantly decreased the number of responding neurons (0.0 [0.0/6.3] %) while the TRPV4 agonist GSK1016790A caused action potential discharge in a subpopulation of osmosensitive enteric neurons. The results of the present study provide insight into the osmosensitivity of submucosal enteric neurons and strongly indicate the involvement of TRPV4 as an osmotransducer.

Modulation of intestinal calcium and phosphate transport in young goats fed a nitrogen- and/or calcium-reduced diet.

Feeding ruminants a reduced N diet is a common approach to reduce N output based on rumino-hepatic circulation. However, a reduction in N intake caused massive changes in Ca and inorganic phosphate (Pi) homoeostasis in goats. Although a single dietary Ca reduction stimulated intestinal Ca absorption in a calcitriol-dependent manner, a concomitant reduction of Ca and N supply led to a decrease in calcitriol, and therefore a modulation of intestinal Ca and Pi absorption. The aim of this study was to examine the potential effects of dietary N or Ca reduction separately on intestinal Ca and Pi transport in young goats. Animals were allocated to a control, N-reduced, Ca-reduced or combined N- and Ca-reduced diet for about 6-8 weeks, whereby N content was reduced by 25 % compared with recommendations. In Ussing chamber experiments, intestinal Ca flux rates significantly decreased in goats fed a reduced N diet, whereas Pi flux rates were unaffected. In contrast, a dietary Ca reduction stimulated Ca flux rates and decreased Pi flux rates. The combined dietary N and Ca reduction withdrew the stimulating effect of dietary Ca reduction on Ca flux rates. The expression of Ca-transporting proteins decreased with a reduced N diet too, whereas Pi-transporting proteins were unaffected. In conclusion, a dietary N reduction decreased intestinal Ca transport by diminishing Ca-transporting proteins, which became clear during simultaneous N and Ca reduction. Therefore, N supply in young ruminant nutrition is of special concern for intestinal Ca transport.

The recruitment of mechanosensitive enteric neurons in the guinea pig gastric fundus is dependent on ganglionic stretch level.

BACKGROUND: Serving as a reservoir, the gastric fundus can expand significantly, with an initial receptive and a following adaptive relaxation, controlled by extrinsic and intrinsic reflex circuits, respectively. We hypothesize that mechanosensitive enteric neurons (MEN) are involved in the adaptive relaxation, which is initiated when a particular gastric volume and a certain stretch of the stomach wall is reached. To investigate whether the responsiveness of MEN in the gastric fundus is dependent on tissue stretch, we performed mechanical stimulations in stretched versus ganglia "at rest". METHODS: Responses of myenteric neurons in the guinea pig gastric fundus were recorded with membrane potential imaging using Di-8-ANEPPS. MEN were identified by small-volume intraganglionic injection in ganglia stretched to different degrees using a self-constructed stretching tool. Immunohistochemical staining identified the neurochemical phenotype of MEN. Hexamethonium and capsaicin were added to test their effect on recruited MEN. KEY RESULTS: In stretched compared to "at rest" ganglia, significantly more MEN were activated. The change in the ganglionic area correlated significantly with the number of additional recruited MEN. The additional recruitment of MEN was independent from nicotinic transmission and the ratio of active MEN in stretched ganglia shifted towards a nitrergic phenotype. CONCLUSION AND INFERENCES: The higher number of active MEN with increasing stretch of the ganglia and their greater share of nitrergic phenotype might indicate their contribution to the adaptive relaxation. Further experiments are necessary to address the receptors involved in mechanotransduction.

Fecal supernatants from dogs with idiopathic epilepsy activate enteric neurons.

Introduction: Alterations in the composition and function of the gut microbiome have been reported in idiopathic epilepsy (IE), however, interactions of gut microbes with the enteric nervous system (ENS) in this context require further study. This pilot study examined how gastrointestinal microbiota (GIM), their metabolites, and nutrients contained in intestinal contents communicate with the ENS. Methods: Fecal supernatants (FS) from healthy dogs and dogs with IE, including drug-naïve, phenobarbital (PB) responsive, and PB non-responsive dogs, were applied to cultured myenteric neurons to test their activation using voltage-sensitive dye neuroimaging. Additionally, the concentrations of short-chain fatty acids (SCFAs) in the FS were quantified. Results: Our findings indicate that FS from all examined groups elicited neuronal activation. Notably, FS from PB non-responsive dogs with IE induced action potential discharge in a higher proportion of enteric neurons compared to healthy controls, which exhibited the lowest burst frequency overall. Furthermore, the highest burst frequency in enteric neurons was observed upon exposure to FS from drug-naïve dogs with IE. This frequency was significantly higher compared to that observed in PB non-responsive dogs with IE and showed a tendency to surpass that of healthy controls. Discussion: Although observed disparities in SCFA concentrations across the various FS samples might be associated with the induced neuronal activity, a direct correlation remains elusive at this point. The obtained results hint at an involvement of the ENS in canine IE and set the basis for future studies.

Mechanosensitive enteric neurons in the guinea pig gastric fundus and antrum.

BACKGROUND: Coping with the ingested food, the gastric regions of fundus, corpus, and antrum display different motility patterns. Intrinsic components of such patterns involving mechanosensitive enteric neurons (MEN) have been described in the guinea pig gastric corpus but are poorly understood in the fundus and antrum. METHODS: To elucidate mechanosensitive properties of myenteric neurons in the gastric fundus and antrum, membrane potential imaging using Di-8-ANEPPS was applied. A small-volume injection led to neuronal compression. We analyzed the number of MEN and their firing frequency in addition to the involvement of selected mechanoreceptors. To characterize the neurochemical phenotype of MEN, we performed immunohistochemistry. KEY RESULTS: In the gastric fundus, 16% of the neurons reproducibly responded to mechanical stimulation and thus were MEN. Of those, 83% were cholinergic and 19% nitrergic. In the antrum, 6% of the neurons responded to the compression stimulus, equally distributed among cholinergic and nitrergic MEN. Defunctionalizing the sensory extrinsic afferents led to a significant drop in the number of MEN in both regions. CONCLUSION: We provided evidence for MEN in the gastric fundus and antrum and further investigated mechanoreceptors. However, the proportions of the chemical phenotypes of the MEN differed significantly between both regions. Further investigations of synaptic connections of MEN are crucial to understand the hardwired neuronal circuits in the stomach.

Short- and Long-Term Effects of Cocaine on Enteric Neuronal Functions.

Cocaine is one of the most consumed illegal drugs among (young) adults in the European Union and it exerts various acute and chronic negative effects on psychical and physical health. The central mechanism through which cocaine initially leads to improved performance, followed by addictive behavior, has already been intensively studied and includes effects on the homeostasis of the neurotransmitters dopamine, partly mediated via nicotinic acetylcholine receptors, and serotonin. However, effects on the peripheral nervous system, including the enteric nervous system, are much less understood, though a correlation between cocaine consumption and gastrointestinal symptoms has been reported. The aim of the present study was to gain more information on the effects of cocaine on enteric neuronal functions and the underlying mechanisms. For this purpose, functional experiments using an organ bath, Ussing chamber and neuroimaging techniques were conducted on gastrointestinal tissues from guinea pigs. Key results obtained are that cocaine (1) exhibits a stimulating, non-neuronal effect on gastric antrum motility, (2) acutely (but not chronically) diminishes responses of primary cultured enteric neurons to nicotinic and serotonergic stimulation and (3) reversibly attenuates neuronal-mediated intestinal mucosal secretion. It can be concluded that cocaine, among its central effects, also alters enteric neuronal functions, providing potential explanations for the coexistence of cocaine abuse and gastrointestinal complaints.

Distension evoked mucosal secretion in human and porcine colon in vitro.

It was suggested that intestinal mucosal secretion is enhanced during muscle relaxation and contraction. Mechanisms of mechanically induced secretion have been studied in rodent species. We used voltage clamp Ussing technique to investigate, in human and porcine colonic tissue, secretion evoked by serosal (Pser) or mucosal (Pmuc) pressure application (2-60 mmHg) to induce distension into the mucosal or serosal compartment, respectively. In both species, Pser or Pmuc caused secretion due to Cl- and, in human colon, also HCO3- fluxes. In the human colon, responses were larger in proximal than distal regions. In porcine colon, Pmuc evoked larger responses than Pser whereas the opposite was the case in human colon. In both species, piroxicam revealed a strong prostaglandin (PG) dependent component. Pser and Pmuc induced secretion was tetrodotoxin (TTX) sensitive in porcine colon. In human colon, a TTX sensitive component was only revealed after piroxicam. However, synaptic blockade by ω-conotoxin GVIA reduced the response to mechanical stimuli. Secretion was induced by tensile rather than compressive forces as preventing distension by a filter inhibited the secretion. In conclusion, in both species, distension induced secretion was predominantly mediated by PGs and a rather small nerve dependent response involving mechanosensitive somata and synapses.

Good to Know: Baseline Data on Feed Intake, Fecal Pellet Output and Intestinal Transit Time in Guinea Pig as a Frequently Used Model in Gastrointestinal Research.

Guinea pigs are a traditional and frequently used species in gastrointestinal research. Comprehensive knowledge of basic parameters connected with their intestinal function, such as feed intake, fecal pellet output and gastrointestinal transit time, is important for evaluating results from basic gastrointestinal research that may be applied to practical problems in human and veterinary medicine, for example, when establishing diagnostic tools. Our study revealed that over a 24-h period, single-housed guinea pigs showed a continual but day-accentuated feeding activity, consuming 57% of the total feed during the light period, with pronounced peaks of feed intake during the beginning and end of the light period. This was mirrored by fecal pellet output during the light period and almost no defecation during the dark period, while potential coprophagy not measured in this study needs to be considered. A highly comparable feeding activity was recorded in pair-housed guinea pigs, with 60% of overall feed intake within the light period, indicating that such differences in housing conditions did not influence guinea pigs' feeding behavior. Intestinal transit time was successfully recorded by oral administration of carmine red and counted 5 h on average. Hence, this study provides important information on the basic functional parameters of guinea pigs' gastrointestinal tract physiology.

Compression and stretch sensitive submucosal neurons of the porcine and human colon.

The pig is commonly believed to be a relevant model for human gut functions-however, there are only a few comparative studies and none on neural control mechanisms. To address this lack we identified as one central aspect mechanosensitive enteric neurons (MEN) in porcine and human colon. We used neuroimaging techniques to record responses to tensile or compressive forces in submucous neurons. Compression and stretch caused Ca-transients and immediate spike discharge in 5-11% of porcine and 15-24% of human enteric neurons. The majority of these MEN exclusively responded to either stimulus quality but about 9% responded to both. Most of the MEN expressed choline acetyltransferase and substance P; nitric oxide synthase-positive MEN primarily occurred in distal colon. The findings reveal common features of MEN in human and pig colon which we interpret as a result of species-independent evolutionary conservation rather than a specific functional proximity between the two species.

Dietary nitrogen and calcium modulate bone metabolism in young goats.

Ruminants, possessing the rumino-hepatic circulation, are thought to cope easily with reduced dietary nitrogen (N) supply which is of economic and environmental interest to diminish N output. Nevertheless, feeding an N reduced diet to young goats resulted in a decrease in calcitriol and calcium (Ca) plasma concentrations. Although a dietary Ca reduction alone stimulated calcitriol synthesis and plasma Ca concentrations were restored, in combination with a reduced N supply this stimulating effect was abolished. Based on the important role bone tissue plays in maintaining Ca homeostasis, aim of the present study was to determine effects of an N reduced diet with or without a concomitant Ca reduction on bone metabolism in young goats. A dietary N reduction alone resulted in a significant rise in plasma concentrations of bone resorption marker C-terminal telopeptide of type I collagen (CTX) and bone formation marker osteocalcin (OC), while reduced intake of Ca as well as the combination of both dietary interventions increased bone markers only slightly. Bone mineral content and bone mineral density of metatarsi were decreased by reduced N intake, while Ca and phosphorus (P) content of dried bones remained unaffected. In contrast, a dietary Ca reduction alone led to decreased Ca and P content of dried bones. From these data it can be concluded that a dietary N reduction alone or in combination with a reduced dietary Ca supply modulated bone metabolism in young goats.

Expression of Tight Junction Proteins and Cadherin 17 in the Small Intestine of Young Goats Offered a Reduced N and/or Ca Diet.

Diets fed to ruminants should contain nitrogen (N) as low as possible to reduce feed costs and environmental pollution. Though possessing effective N-recycling mechanisms to maintain the N supply for rumen microbial protein synthesis and hence protein supply for the host, an N reduction caused substantial changes in calcium (Ca) and phosphate homeostasis in young goats including decreased intestinal transepithelial Ca absorption as reported for monogastric species. In contrast to the transcellular component of transepithelial Ca transport, the paracellular route has not been investigated in young goats. Therefore, the aim of the present study was to characterise the effects of dietary N and/or Ca reduction on paracellular transport mechanisms in young goats. Electrophysiological properties of intestinal epithelia were investigated by Ussing chamber experiments. The expression of tight junction (TJ) and adherens junction (AJ) proteins in intestinal epithelia were examined on mRNA level by qPCR and on protein level by western blot analysis. Dietary N reduction led to a segment specific increase in tissue conductances in the proximal jejunum which might be linked to concomitantly decreased expression of cadherin 17 mRNA. Expression of occludin (OCLN) and zonula occludens protein 1 was increased in mid jejunal epithelia of N reduced fed goats on mRNA and partly on protein level. Reduced dietary Ca supply resulted in a segment specific increase in claudin 2 and claudin 12 expression and decreased the expression of OCLN which might have been mediated at least in part by calcitriol. These data show that dietary N as well as Ca reduction affected expression of TJ and AJ proteins in a segment specific manner in young goats and may thus be involved in modulation of paracellular Ca permeability.

Modulation of aquaporin 2 expression in the kidney of young goats by changes in nitrogen intake.

In ruminants, a decrease of dietary nitrogen (N) is an appropriate feeding concept to reduce environmental pollution and costs. In our previous study, when goats were kept on an N-reduced diet, a decrease of plasma urea concentration and an increase of renal urea transporters were demonstrated. Renal urea absorption plays a crucial role for renal water absorption and urine concentration. Renal collecting duct water absorption is mainly mediated by the water channel aquaporin 1 and 2 (AQP1 and AQP2). Therefore, the aim of the present study was to investigate the effects of a dietary N reduction on expression of renal AQP1 and AQP2 in young goats. Twenty male White Saanen goats, 3 months old, were divided equally into two feeding groups, receiving either a diet with an adequate or a reduced-N supply. Goats fed a reduced-N diet showed significantly higher amounts of AQP1 mRNA in cortical tissue, and the expression of AQP2 mRNA and protein were highly elevated in renal outer medulla. An increase of vasopressin concentrations in plasma were detected for the N-reduced fed goats. Therefore, a stimulation of renal water absorption can be assumed. This might be an advantage for ruminants in times of N reduction due to higher urea concentrations in the tubular fluid and which might result in higher absorption of urea by renal urea transporters. Therefore, interplay of aquaporin water channels and urea transporters in the kidney may occur to maintain urea metabolism in times of N scarcity in young goats.