Non-technical summary Obesity is known to result from energy intake in excess of expenditure. What is not known is how individuals are able to eat in excess of their energy needs. We show that after chronic consumption of a high fat diet (which causes obesity), intestinal sensory nerves are less responsive to chemicals released from the gut during a meal (cholecystokinin and 5-hydroxytryptamine) as well as to distension of the gut as might occur during a meal. This appears to be due to the fact that the ability of the nerve cells to be excited is impaired. This suggests that consumption of an unhealthy diet that leads to obesity causes decreased signalling from the intestine, which may lead to increased food intake and contribute to further weight gain, or allow the maintenance of excess weight and obesity.Abstract Gastrointestinal vagal afferents transmit satiety signals to the brain via both chemical and mechanical mechanisms. There is indirect evidence that these signals may be attenuated in obesity. We hypothesized that responses to satiety mediators and distension of the gut would be attenuated after induction of diet induced obesity. Obesity was induced by feeding a high fat diet (60% kcal from fat). Low fat fed mice (10% kcal from fat) served as a control. High fat fed mice were obese, with increased visceral fat, but were not hyperglycaemic. Recordings from jejunal afferents demonstrated attenuated responses to the satiety mediators cholecystokinin (CCK, 100 nM) and 5-hydroxytryptamine (5-HT, 10 μM), as was the response to low intensity jejunal distension, while responses to higher distension pressures were preserved. We performed whole cell patch clamp recordings on nodose ganglion neurons, both unlabelled, and those labelled by fast blue injection into the wall of the jejunum. The cell membrane of both labelled and unlabelled nodose ganglion neurons was less excitable in HFF mice, with an elevated rheobase and decreased number of action potentials at twice rheobase. Input resistance of HFF neurons was also significantly decreased. Calcium imaging experiments revealed reduced proportion of nodose ganglion neurons responding to CCK and 5-HT in obese mice. These results demonstrate a marked reduction in afferent sensitivity to satiety related stimuli after a chronic high fat diet. A major mechanism underlying this change is reduced excitability of the neuronal cell membrane. This may explain the development of hyperphagia when a high fat diet is consumed. Improving sensitivity of gastrointestinal afferent nerves may prove useful to limit food intake in obesity.

Understanding bladder afferent pathways may reveal novel targets for therapy of lower urinary tract disorders such as overactive bladder syndrome and cystitis. Several potential candidate molecules have been postulated as playing a significant role in bladder function. One such candidate is the transient receptor potential vanilloid 1 (TRPV1) ion channel. Mice lacking the TRPV1 channel have altered micturition thresholds suggesting that TRPV1 channels may play a role in the detection of bladder filling. The aim of this study was therefore to investigate the role of TRPV1 receptors in controlling bladder afferent sensitivity in the mouse using pharmacological receptor blockade and genetic deletion of the channel. Multiunit afferent activity was recorded in vitro from bladder afferents taken from wild-type (TRPV+/+) mice and knockout (TRPV1−/−) mice. In wild-type preparations, ramp distension of the bladder to a maximal pressure of 40 mmHg produced a graded increase in afferent activity. Bath application of the TRPV1 antagonist capsazepine (10 μM) caused a significant attenuation of afferent discharge in TRPV1+/+ mice. Afferent responses to distension were significantly attenuated in TRPV1−/− mice in which sensitivity to intravesical hydrochloric acid (50 mM) and capsaicin (10 μM) were also blunted. Altered mechanosensitivity occurred in the absence of any changes in the pressure-volume relationship during filling indicating that this was not secondary to a change in bladder compliance. Single-unit analysis was used to classify individual afferents into low-threshold and high-threshold fibres. Low threshold afferent responses were attenuated in TRPV1−/− mice compared to the TRPV1+/+ littermates while surprisingly high threshold afferent sensitivity was unchanged. While TRPV1 channels are not considered to be mechanically gated, the present study demonstrates a clear role for TRPV1 in the excitability of particularly low threshold bladder afferents. This suggests that TRPV1 may play an important role in normal bladder function.

Folate intake is strongly influenced by various methods of cooking that can degrade the natural forms of the vitamin in foods. The aim of the present study was to determine the effect of different cooking methods on folate retention in various foods that contribute to folate intake in the UK diet. Typical purchasing and cooking practices of representative food folate sources were determined from a questionnaire survey of local shoppers (n 100). Total folate was determined by microbiological assay (Lactobacillus casei NCIMB 10463) following thermal extraction and tri-enzyme (a-amylase, protease and conjugase) treatment in raw foods and after typical methods of cooking. Boiling for typical time periods resulted in only 49 % retention of folate in spinach (191·8 and 94·4 mg/100 g for raw and boiled spinach respectively; P, 0·005), and only 44 % in broccoli (177·1 and 77·0 mg/100 g for raw and boiled broccoli respectively, P, 0·0001). Steaming of spinach or broccoli, in contrast, resulted in no significant decrease in folate content, even for the maximum steaming periods of 4·5 min (spinach) and 15·0 min (broccoli). Prolonged grilling of beef for the maximum period of 16·0 min did not result in a significant decrease in folate content (54·3 and 51·5 mg/100 g for raw and grilled beef respectively). Compared with raw values, boiling of whole potatoes (skin and flesh) for 60·0 min did not result in a significant change in folate content (125·1 and 102·8 mg/100 g for raw and boiled potato respectively), nor was there any effect on folate retention whether or not skin was retained during boiling. These current results show that the retention of folate in various foods is highly dependent both on the food in question and the method of cooking. Thus, public health efforts to increase folate intake in order to improve folate status should incorporate practical advice on cooking.

The prevalence of lower urinary tract storage disorders such as overactive bladder syndrome and urinary incontinence significantly increase with age. Previous studies have demonstrated age-related changes in detrusor function and urothelial transmitter release but few studies have investigated how the urothelium and sensory pathways are affected. The aim of this study was to investigate the effect of ageing on urothelial-afferent signalling in the mouse bladder. Three-month-old control and 24-month-old aged male mice were used. In vivo natural voiding behaviour, sensory nerve activity, urothelial cell function, muscle contractility, transmitter release and gene and protein expression were measured to identify how all three components of the bladder (neural, contractile and urothelial) are affected by ageing. In aged mice, increased voiding frequency and enhanced low threshold afferent nerve activity was observed, suggesting that ageing induces overactivity and hypersensitivity of the bladder. These changes were concurrent with altered ATP and acetylcholine bioavailability, measured as transmitter overflow into the lumen, increased purinergic receptor sensitivity and raised P2X3 receptor expression in the urothelium. Taken together, these data suggest that ageing results in aberrant urothelial function, increased afferent mechanosensitivity, increased smooth muscle contractility, and changes in gene and protein expression (including of P2X3). These data are consistent with the hypothesis that ageing evokes changes in purinergic signalling from the bladder, and further studies are now required to fully validate this idea.

Objective• To investigate the direct effect of onabotulinumtoxinA (OnaBotA) on bladder afferent nerve activity and release of ATP and acetylcholine (ACh) from the urothelium. Materials and Methods• Bladder afferent nerve activity was recorded using an in vitro mouse preparation enabling simultaneous recordings of afferent nerve firing and intravesical pressure during bladder distension.• Intraluminal and extraluminal ATP, ACh, and nitric oxide (NO) release were measured using the luciferin-luciferase and Amplex ® Red assays (Molecular Probes, Carlsbad, CA, USA), and fluorometric assay kit, respectively.• OnaBotA (2U), was applied intraluminally, during bladder distension, and its effect was monitored for 2 h after application.• Whole-nerve activity was analysed to classify the single afferent units responding to physiological (low-threshold [LT] afferent <15 mmHg) and supra-physiological (high-threshold [HT] afferent >15 mmHg) distension pressures. Results• Bladder distension evoked reproducible pressure-dependent increases in afferent nerve firing.• After exposure to OnaBotA, both LT and HT afferent units were significantly attenuated.• OnaBotA also significantly inhibited ATP release from the urothelium and increased NO release. Conclusion• These data indicate that OnaBotA attenuates the bladder afferent nerves involved in micturition and bladder sensation, suggesting that OnaBotA may exert its clinical effects on urinary urgency and the other symptoms of overactive bladder syndrome through its marked effect on afferent nerves.

Both TRPV1 and P2X receptors present on bladder sensory nerve fibres have been implicated in mechanosensation during bladder filling. The aim of this study was to determine possible interactions between these receptors in modulating afferent nerve activity. In wildtype (TRPV1 +/+ ) and TRPV1 knockout (TRPV1 −/− ) mice, bladder afferent nerve activity, intravesical pressure, and luminal ATP and acetylcholine levels were determined and also intracellular calcium responses of dissociated pelvic DRG neurones and primary mouse urothelial cells (PMUCs). Bladder afferent nerve responses to the purinergic agonist αβMethylene-ATP were depressed in TRPV1 As the bladder fills, the degree of distension is detected by mechanosensitive ion channels located either on sensory nerve terminals or on the specialised epithelial lining of the bladder, the urothelium. This evokes an afferent signal that is conveyed to the CNS via the dorsal root ganglia. Initially this signal lies below the level of consciousness, supplying the autonomic reflexes responsible for controlled bladder filling. As bladder volume increases, however, a sensation of fullness and a desire to void is perceived. In continent individuals this desire can be deferred until it is convenient to empty the bladder, however in patients with lower urinary tract conditions such as overactive bladder syndrome (OAB), bladder pain syndrome (BPS) or interstitial cystitis (IC), this sensation is difficult to defer, occurs when the bladder is not yet full and can even be accompanied by pain (BPS/IC) suggesting that a defect in the ability of the bladder to detect filling may underlie these conditions. Sensory innervation from the bladder is conveyed by myelinated low threshold Aδ fibres and unmyelinated high threshold C fibres carried in the pelvic, hypogastric and pudendal afferent nerves 1 . However, the complex mechanisms which determine how the afferent nerves detect bladder filling remain poorly understood, limiting the development of effective treatments for these bladder sensory defects.There is compelling evidence that purinergic receptors play an active role in mechanosensation of the bladder in rodents. Firstly bladder projecting afferents express a number of P2X puringeric receptors, of which P2X2 and P2X3 are the main subtype. Secondly, ATP is able to activate pelvic nerve afferents arising from the rat urinary bladder and initiate bladder overactivity 2,3 . Thirdly, the P2X1 and P2X3 agonist, αβMethylene-ATP increases firing in both nociceptive and non-nociceptive populations of bladder afferents 4 . Finally, it has been shown that stretch or distension of the urothelium evokes the graded release of non-neuronal ATP 5 which activates purinergic receptors located on the afferent terminal to control afferent firing. Previous studies using P2X3 or P2X2/3 null mice found profound attenuation of the afferent response to bladder filling, increased bladder capacity and decreased voiding frequency, supporting a role for ATP in bladder sensation 5,6 . Purinergic receptors ...

The incidence of bladder conditions such as overactive bladder syndrome and its associated urinary incontinence is highly prevalent in the elderly. However, the mechanisms underlying these disorders are unclear. Studies suggest that the urothelium forms a ‘sensory network’ with the underlying innervation, alterations in which, could compromise bladder function. As the accumulation of reactive oxygen species can cause functional alterations with age, the aim of this study was to investigate whether oxidative stress alters urothelial sensory signalling and whether the mechanism underlying the effect of oxidative stress on the urothelium plays a role in aging. Five-month-old(young) and 24-month-old (aged) mice were used. H2O2, used to induce oxidative stress, resulted in an increase in bladder afferent nerve activity and urothelial intracellular calcium in preparations from young mice. These functional changes were concurrent with upregulation of TRPM8 in the urothelium. Moreover, application of a TRPM8 antagonist significantly attenuated the H2O2-induced calcium responses. Interestingly, an upregulation of TRPM8 was also found in the urothelium from aged mice, where high oxidative stress levels were observed, together with a greater calcium response to the TRPM8 agonist WS12. Furthermore, these calcium responses were attenuated by pretreatment with the antioxidant N-acetyl-cysteine. This study shows that oxidative stress affects urothelial function involving a TRPM8-mediated mechanism and these effects may have important implications for aging. These data provide an insight into the possible mechanisms by which oxidative stress causes physiological alterations in the bladder, which may also occur in other organs susceptible to aging.

Recent studies provide further evidence that afferent control of the bladder may be dependent on integration of excitatory and inhibitory mediators from the urothelium such as ATP and nitric oxide. A number of studies have examined the role cholinergic and adrenergic mechanisms play in bladder afferent function, and several new potential mechanisms involving the cannabinoid receptors and transient receptor potential channels have emerged as areas which warrant further investigation. A better understanding of afferent mechanisms in the bladder will hopefully lead to more effective treatments of lower urinary tract disorders.