A novel unbiased counting method for the quantification of synapses in the mouse brain.

BACKGROUND: The numerical density of synapses and their ultrastructural features are best assessed with electron microscopy. Counting is done within counting frames placed on a pair of sections (disector technique). But this requires that the thin sections are taken from comparable brain regions and the disectors are placed in a uniform random fashion. Small brain areas like the polymorph layer of the mouse dentate gyrus are difficult to encounter, and manually moving the microscope stage for placing the micrographs seems arbitrary. NEW METHOD: Here the polymorph layer was approximated with 20µm thin, Nissl-stained vibratome sections. The subsequent vibratome section was processed for electron microscopy and serially thin sectioned. The microscope stage was moved using a random number generator, placing at least 20 disectors onto a pair of sections. The numerical synapse density, the numerical density of dense-core vesicles, and other ultrastructural features were compared between mice that had been kept in an enriched environment and mice kept under standard housing conditions. RESULTS: Environmental enrichment significantly decreased the numerical density of dense-core vesicles and synaptic cleft widths within the polymorph layer, associated with behavioral improvement in the Morris water maze, a hippocampus-dependent task of spatial learning and memory. COMPARISON WITH EXISTING METHODS: This procedure was easy to handle and enabled us to produce thin sections in small, defined brain areas. Furthermore, placing the disectors with random numbers excluded observer bias. CONCLUSIONS: Our procedure provides an uncomplicated way of assessing numerical densities in small brain areas in an unbiased manner.

Synergistic effects of NOD1 or NOD2 and TLR4 activation on mouse sickness behavior in relation to immune and brain activity markers.

Toll-like receptors (TLRs) and nuclear-binding domain (NOD)-like receptors (NLRs) are sensors of bacterial cell wall components to trigger an immune response. The TLR4 agonist lipopolysaccharide (LPS) is a strong immune activator leading to sickness and depressed mood. NOD agonists are less active but can prime immune cells to augment LPS-induced cytokine production. Since the impact of NOD and TLR co-activation in vivo has been little studied, the effects of the NOD1 agonist FK565 and the NOD2 agonist muramyl dipeptide (MDP), alone and in combination with LPS, on immune activation, brain function and sickness behavior were investigated in male C57BL/6N mice. Intraperitoneal injection of FK565 (0.001 or 0.003mg/kg) or MDP (1 or 3mg/kg) 4h before LPS (0.1 or 0.83mg/kg) significantly aggravated and prolonged the LPS-evoked sickness behavior as deduced from a decrease in locomotion, exploration, food intake and temperature. When given alone, FK565 and MDP had only minor effects. The exacerbation of sickness behavior induced by FK565 or MDP in combination with LPS was paralleled by enhanced plasma protein and cerebral mRNA levels of proinflammatory cytokines (IFN-γ, IL-1ß, IL-6, TNF-α) as well as enhanced plasma levels of kynurenine. Immunohistochemical visualization of c-Fos in the brain revealed that NOD2 synergism with TLR4 resulted in increased activation of cerebral nuclei relevant to sickness. These data show that NOD1 or NOD2 synergizes with TLR4 in exacerbating the immune, sickness and brain responses to peripheral immune stimulation. Our findings demonstrate that the known interactions of NLRs and TLRs at the immune cell level extend to interactions affecting brain function and behavior.

Neuropeptide Y and peptide YY protect from weight loss caused by Bacille Calmette-Guérin in mice.

BACKGROUND AND PURPOSE: Immune challenge of mice with Bacille Calmette-Guérin (BCG) has been reported to cause transient weight loss and a behavioural sickness response. Although BCG-induced depression involves the kynurenine pathway, weight loss occurs independently of this factor. Because neuropeptide Y (NPY) and peptide YY (PYY) are involved in the regulation of food intake, we hypothesized that they play a role in the BCG-induced weight loss. EXPERIMENTAL APPROACH: Male wild-type, PYY knockout (PYY-/-), NPY knockout (NPY-/-) and NPY-/-;PYY-/- double knockout mice were injected with vehicle or BCG (approximately 10(8) colony-forming units per mouse), and their weight, locomotion, exploration and ingestion were recorded for 2 weeks post-treatment. KEY RESULTS: Deletion of PYY and NPY aggravated the BCG-induced loss of body weight, which was most pronounced in NPY-/-;PYY-/- mice (maximum loss: 15%). The weight loss in NPY-/-;PYY-/- mice did not normalize during the 2 week observation period. BCG suppressed the circadian pattern of locomotion, exploration and food intake. However, these changes took a different time course than the prolonged weight loss caused by BCG in NPY-/-;PYY-/- mice. The effect of BCG to increase circulating IL-6 (measured 16 days post-treatment) remained unaltered by knockout of PYY, NPY or NPY plus PYY. CONCLUSIONS AND IMPLICATIONS: These data show that NPY and PYY are both required to protect from the action of BCG-evoked immune challenge to cause prolonged weight loss and disturb energy balance. The findings attest to an important role of NPY and PYY in orchestrating homeostatic reactions to infection and immune stimulation.

Environmental enrichment and gut inflammation modify stress-induced c-Fos expression in the mouse corticolimbic system.

Environmental enrichment (EE) has a beneficial effect on rodent behaviour, neuronal plasticity and brain function. Although it may also improve stress coping, it is not known whether EE influences the brain response to an external (psychological) stressor such as water avoidance stress (WAS) or an internal (systemic) stressor such as gastrointestinal inflammation. This study hence explored whether EE modifies WAS-induced activation of the mouse corticolimbic system and whether this stress response is altered by gastritis or colitis. Male C67BL/6N mice were housed under standard or enriched environment for 9 weeks, after which they were subjected to a 1-week treatment with oral iodoacetamide to induce gastritis or oral dextran sulfate sodium to induce colitis. Following exposure to WAS the expression of c-Fos, a marker of neuronal activation, was measured by immunocytochemistry. EE aggravated experimentally induced colitis, but not gastritis, as shown by an increase in the disease activity score and the colonic myeloperoxidase content. In the brain, EE enhanced the WAS-induced activation of the dentate gyrus and unmasked an inhibitory effect of gastritis and colitis on WAS-evoked c-Fos expression within this part of the hippocampus. Conversely, EE inhibited the WAS-evoked activation of the central amygdala and prevented the inhibitory effect of gastritis and colitis on WAS-evoked c-Fos expression in this region. EE, in addition, blunted the WAS-induced activation of the infralimbic cortex and attenuated the inhibitory effect of gastritis and colitis on WAS-evoked c-Fos expression in this area. These data reveal that EE has a region-specific effect on stress-induced c-Fos expression in the corticolimbic system, which is likely to improve stress resilience. The response of the prefrontal cortex - amygdala - hippocampus circuitry to psychological stress is also modified by the systemic stress of gut inflammation, and this interaction between external and internal stressors is modulated by the housing environment.

Prolonged depression-like behavior caused by immune challenge: influence of mouse strain and social environment.

Immune challenge by bacterial lipopolysaccharide (LPS) causes short-term behavioral changes indicative of depression. The present study sought to explore whether LPS is able to induce long-term changes in depression-related behavior and whether such an effect depends on mouse strain and social context. LPS (0.83 mg/kg) or vehicle was administered intraperitoneally to female CD1 and C57BL/6 mice that were housed singly or in groups of 4. Depression-like behavior was assessed with the forced swim test (FST) 1 and 28 days post-treatment. Group-housed CD1 mice exhibited depression-like behavior 1 day post-LPS, an effect that leveled off during the subsequent 28 days, while the behavior of singly housed CD1 mice was little affected. In contrast, singly housed C57BL/6 mice responded to LPS with an increase in depression-like behavior that was maintained for 4 weeks post-treatment and confirmed by the sucrose preference test. Group-housed C57BL/6 mice likewise displayed an increased depression-like behavior 4 weeks post-treatment. The behavioral changes induced by LPS in C57BL/6 mice were associated with a particularly pronounced rise of interleukin-6 in blood plasma within 1 day post-treatment and with changes in the dynamics of the corticosterone response to the FST. The current data demonstrate that immune challenge with LPS is able to induce prolonged depression-like behavior, an effect that depends on genetic background (strain). The discovery of an experimental model of long-term depression-like behavior after acute immune challenge is of relevance to the analysis of the epigenetic and pathophysiologic mechanisms of immune system-related affective disorders.

Sex-dependent control of murine emotional-affective behaviour in health and colitis by peptide YY and neuropeptide Y.

BACKGROUND AND PURPOSE: Peptide YY (PYY) and neuropeptide Y (NPY) are involved in regulating gut and brain function. Because gastrointestinal inflammation is known to enhance anxiety, we explored whether experimental colitis interacts with genetic deletion (knockout) of PYY and NPY to alter emotional-affective behaviour. EXPERIMENTAL APPROACH: Male and female wild-type, NPY (NPY(-/-) ), PYY (PYY(-/-) ) and NPY(-/-) ; PYY(-/-) double knockout mice were studied in the absence and presence of mild colitis induced by ingestion of dextran sulphate sodium (2%) in drinking water. Anxiety-like behaviour was tested on the elevated plus maze and open field, and depression-like behaviour assessed by the forced swim test. KEY RESULTS: In the absence of colitis, anxiety-like behaviour was increased by deletion of NPY but not PYY in a test- and sex-dependent manner, while depression-like behaviour was enhanced in NPY(-/-) and PYY(-/-) mice of either sex. The severity of DSS-induced colitis, assessed by colonic myeloperoxidase content, was attenuated in NPY(-/-) but not PYY(-/-) mice. Colitis modified anxiety- and depression-related behaviour in a sex-, genotype- and test-related manner, and knockout experiments indicated that NPY and PYY were involved in some of these behavioural effects of colitis. CONCLUSIONS AND IMPLICATIONS: These data demonstrate sex-dependent roles of NPY and PYY in regulation of anxiety- and depression-like behaviour in the absence and presence of colitis. Like NPY, the gut hormone PYY has the potential to attenuate depression-like behaviour but does not share the ability of NPY to reduce anxiety-like behaviour.

The COGITAT holeboard system as a valuable tool to assess learning, memory and activity in mice.

The comprehensive and stress-free assessment of various aspects of learning and memory is a prerequisite to evaluate mouse models for neuropsychiatric disorders such as Alzheimer's disease or attention deficit/hyperactivity disorder (ADHD). COGITAT is an automated holeboard system allowing simultaneous assessment of spatial working and reference-memory performance which we have adapted in this study to enable its usage with mice. The holeboard apparatus consists of an open-field chamber with a 25-hole floor insert, each hole being monitored by infrared light beams, located on three different levels, allowing the distinction between visits of holes, i.e. the animal reaches the bottom of the hole, or inspections, which means only superficial exploration of the hole. Across trials, animals learn a pattern of five baited holes. Here, we show that C57BL/6 mice readily acquire this task within 5 days when submitted to six trials per day. A number of individual parameters - overall exploratory activity, number of visits into or inspections of holes, number of baited, unbaited, or previously baited holes visited or inspected, reinspections of or revisits into any holes, number of pellets eaten, time to find pellets, and reference and working memory errors-are obtained simultaneously and results are immediately available after the end of each experiment. The muscarinic antagonist scopolamine impaired task performance, while the cognitive enhancer metrifonate (an acetylcholinesterase inhibitor) reduced error rates. Overall, our data indicate that this spatial learning task will be useful to characterize spatial memory in various genetic or pharmacological mouse models.

Gene-environment interaction influences anxiety-like behavior in ethologically based mouse models.

Ethologically based animal models are widely used; however, results from different laboratories vary significantly which may partly be due to the lack of standardization. Here, we examined the effects of circadian rhythm, lighting condition and mouse strain (BALB/c and C57BL/6, known to differ in measures of avoidance and risk assessment behavior) on two well established behavioral tests in mice: the Elevated Plus Maze (EPM) and the Open Field (OF). Parameters from both paradigms are commonly used as indices of anxiety-like behavior. BALB/c mice and C57BL/6 mice were independently tested in the morning and at night, in regular laboratory lighting and in the dark. We developed a novel method based on infrared lighting from below, coupled to respective video-tracking equipment, which facilitates standard testing of behavior interference-free in complete darkness. The two mouse strains differed in anxiety-related variables for the EPM in the dark, and for the OF in regular laboratory lighting. Moreover, BALB/c displayed greater anxiety-like behavior than C57BL/6 in the OF but less anxiety-like behavior than C57BL/6 in the EPM. Lighting condition has a major influence on both behavioral tests and this to a considerably larger extent than circadian rhythm. In addition, the lighting condition interacts strongly with the genetic background, producing discriminative differences in the anxiety-related variables depending on mouse strain and lighting condition. These results challenge the comparability of not sufficiently standardized tests of anxiety-like behavior and emphasize the need for controlling environmental variables in behavioral phenotyping.

Evidence from knockout mice that neuropeptide-Y Y2 and Y4 receptor signalling prevents long-term depression-like behaviour caused by immune challenge.

Neuropeptide Y participates in the acute behavioural responses to immune challenge, since Y2 receptor knockout (Y2⁻/⁻) mice are particularly sensitive to the short-term anxiogenic-like effect of bacterial lipopolysaccharide. The present exploratory study addressed the involvement of Y2 and Y4 receptors in the long-term behavioural responses to immune challenge. A single intraperitoneal injection of lipopolysaccharide (0.83 mg/kg) to control mice did not affect open field behaviour 3 h post-treatment but enhanced anxiety-like behaviour in Y2⁻/⁻ as well as Y4⁻/⁻ mice. Four weeks post-treatment this behavioural effect of lipopolysaccharide persisted in Y4⁻/⁻ mice but had gone in Y2⁻/⁻ mice. Depression-related behaviour in the forced swim test was enhanced 1 day post-lipopolysaccharide in control and Y2⁻/⁻ mice, but not in Y4⁻/⁻ mice. Four weeks post-treatment, the depressogenic-like effect of lipopolysaccharide had waned in control mice, persisted in Y2⁻/⁻ mice and was first observed in Y4⁻/⁻ mice. In summary, knockout of Y2 and/or Y4 receptors unmasks the ability of a single lipopolysaccharide injection to cause a delayed and prolonged increase in anxiety- and/or depression-like behaviour. These findings suggest that neuropeptide Y acting via Y2 and Y4 receptors prevents the development of long-term anxiety- and depression-like behaviour caused by acute immune challenge.

Alosetron, cilansetron and tegaserod modify mesenteric but not colonic blood flow in rats.

BACKGROUND AND PURPOSE: As the use of the 5-HT(3) receptor antagonist alosetron (GlaxoSmithKline) and the 5-HT(4) receptor agonist tegaserod (Novartis) in patients with irritable bowel syndrome has been associated with cases of ischaemic colitis, the effects of alosetron, cilansetron (Solvay) and tegaserod on the rat splanchnic circulation were evaluated. EXPERIMENTAL APPROACH: Phenobarbital-anaesthetised rats were instrumented to record blood flow in the superior mesenteric artery and transverse colon and to calculate mesenteric and colonic vascular conductance. KEY RESULTS: Intravenous alosetron (0.03-0.3 mg.kg(-1)) did not alter blood pressure or heart rate but reduced mesenteric blood flow and vascular conductance by 15-20%. This activity profile was also seen after intraduodenal alosetron and shared by the 5-HT(3) receptor antagonist cilansetron. In contrast, blood flow, vascular conductance and intraluminal pressure in the colon were not modified by alosetron and cilansetron. Intravenous or intraduodenal tegaserod (0.3-1.0 mg.kg(-1)) had no inhibitory effect on mesenteric and colonic blood flow. Peroral treatment of rats with alosetron or tegaserod for 7 days did not modify mesenteric haemodynamics at baseline and after blockade of nitric oxide synthesis. Mild inflammation induced by dextran sulphate sodium failed to provoke a vasoconstrictor effect of cilansetron in the colon. CONCLUSIONS AND IMPLICATIONS: Alosetron and cilansetron, not tegaserod, caused a small and transient constriction of the rat mesenteric vascular bed, whereas blood flow in the colon remained unaltered. The relevance of these findings to the treatment-associated occurrence of ischaemic colitis in patients with irritable bowel syndrome remains open.

Decreased anxiety in mice lacking the organic cation transporter 3.

The organic cation transporter 3 (OCT3; synonymous: extraneuronal monoamine transporter, EMT, Slc22a3) encodes an isoform of the organic cation transporters and is expressed widely across the whole brain. OCTs are a family of high-capacity, bidirectional, multispecific transporters of organic cations. These also include serotonin, dopamine and norepinephrine making OCTs attractive candidates for a variety of neuropsychiatric disorders including anxiety disorders. OCT3 has been implicated in termination of monoaminergic signalling in the central nervous system. Interestingly, OCT3 mRNA is however also significantly up-regulated in the hippocampus of serotonin transporter knockout mice where it might serve as an alternative reuptake mechanism for serotonin. The examination of the behavioural phenotype of OCT3 knockout mice thus is paramount to assess the role of OCT3. We have therefore subjected mice lacking the OCT3 gene to a comprehensive behavioural test battery. While cognitive functioning in the Morris water maze test and aggression levels measured with the resident-intruder paradigm were in the same range as the respective control animals, OCT3 knockout animals showed a tendency of increased activity and were significantly less anxious in the elevated plus-maze test and the open field test as compared to their respective wild-type controls arguing for a role of OCT3 in the regulation of fear and anxiety, probably by modulating the serotonergic tone in limbic circuitries.

Implication of neuropeptide-Y Y2 receptors in the effects of immune stress on emotional, locomotor and social behavior of mice.

Neuropeptide Y (NPY) is involved in the regulation of emotional behavior, and there is indirect evidence for a role of NPY in the cerebral responses to peripheral immune challenge. Since the NPY receptors involved in these reactions are not known, we investigated the effect of Escherichia coli lipopolysaccharide (LPS) on emotional, locomotor and social behavior, body temperature and circulating corticosterone in female Y2 (Y2-/-) and Y4 (Y4-/-) receptor knockout mice. LPS (0.1mg/kg injected IP 2.5h before testing) increased rectal temperature in control and Y4-/- mice to a larger degree than in Y2-/- animals. Both Y2-/- and Y4-/- mice exhibited reduced anxiety-related and depression-like behavior in the open field, elevated plus-maze and tail suspension test, respectively. While depression-like behavior was not changed by LPS, anxiety-related behavior was enhanced by LPS in Y2-/-, but not control and Y4-/- animals. Y2-/- mice were also particularly susceptible to the effect of LPS to attenuate locomotor behavior and social interaction with another mouse. The corticosterone response to LPS was blunted in Y2-/- mice which presented elevated levels of circulating corticosterone following vehicle treatment. These data show that Y2-/- mice are particularly sensitive to the effects of LPS-evoked immune stress to attenuate locomotion and social interaction and to increase anxiety-like behavior, while the LPS-induced rise of temperature and circulating corticosterone is suppressed by Y2 receptor knockout. Our observations attest to an important role of endogenous NPY acting via Y2 receptors in the cerebral response to peripheral immune challenge.

Deletion of the acid-sensing ion channel ASIC3 prevents gastritis-induced acid hyperresponsiveness of the stomach-brainstem axis.

Gastric acid challenge of the rat and mouse stomach is signalled to the brainstem as revealed by expression of c-Fos. The molecular sensors relevant to the detection of gastric mucosal acidosis are not known. Since the acid-sensing ion channels ASIC2 and ASIC3 are expressed by primary afferent neurons, we examined whether knockout of the ASIC2 or ASIC3 gene modifies afferent signalling of a gastric acid insult in the normal and inflamed stomach. The stomach of conscious mice (C57BL/6) was challenged with intragastric HCl; two hours later the activation of neurons in the nucleus tractus solitarii (NTS) of the brainstem was visualized by c-Fos immunocytochemistry. Mild gastritis was induced by addition of iodoacetamide (0.1%) to the drinking water for 7 days. Exposure of the gastric mucosa to HCl (0.25M) caused a 3-fold increase in the number of c-Fos-positive neurons in the NTS. This afferent input to the NTS remained unchanged by ASIC3 knockout, whereas ASIC2 knockout augmented the c-Fos response to gastric HCl challenge by 33% (P<0.01). Pretreatment of wild-type mice with iodoacetamide induced mild gastritis, as revealed by increased myeloperoxidase activity, and enhanced the number of NTS neurons responding to gastric HCl challenge by 41% (P<0.01). This gastric acid hyperresponsiveness was absent in ASIC3 knockout mice but fully preserved in ASIC2 knockout mice. The current data indicate that ASIC3 plays a major role in the acid hyperresponsiveness associated with experimental gastritis. In contrast, ASIC2 appears to dampen acid-evoked input from the stomach to the NTS.

Multidrug-resistance gene 1-type p-glycoprotein (MDR1 p-gp) inhibition by tariquidar impacts on neuroendocrine and behavioral processing of stress.

The multidrug-resistance gene 1-type p-glycoprotein (MDR1 p-gp) is a major gate-keeper at the blood-brain barrier (BBB), protecting the central nervous system from accumulation of toxic xenobiotics and drugs. In addition, MDR1 p-gp has been found to control the intracerebral access of glucocorticoid hormones and thus to modulate the activity of the hypothalamic-pituitary-adrenocortical (HPA) system. In view of the implication of glucocorticoids in the control of behavior, we examined how acute pharmacological inhibition of MDR1 p-gp at the BBB by tariquidar (XR9576; 12 mg/kg, PO) impacts the neuroendocrine and behavioral processing of stress in C57BL/6JIcoHim inbred mice. Inhibition of MDR1 p-gp at the BBB did not alter emotional behavior at baseline. However, mice that were sensitized by water-avoidance stress, a mild psychological stressor, displayed significantly reduced anxiety-related behavior in the elevated plus-maze test when treated with tariquidar. Tariquidar, however, had no effect on stress-coping performance assessed in the forced swim test. Investigating the impact of acute MDR1 p-gp inhibition on the glucocorticoid system, we observed a significant attenuation of the mild stress-induced increase of plasma corticosterone after tariquidar administration. In order to examine whether the anti-anxiety effect of tariquidar in sensitized animals is mediated by glucocorticoids, the animals were treated with corticosterone (1mg/kg, SC) immediately after exposure to water-avoidance stress. Corticosterone caused a significant anxiolytic-like effect in this stress-related anxiety protocol, whereas tariquidar could not further enhance corticosterone's anti-anxiety effects. The current data show for the first time that pharmacological inhibition of MDR1 p-gp at the murine BBB by tariquidar alters emotional behavior and HPA axis activity. By facilitating the entry of corticosterone into the brain, tariquidar enhances feedback inhibition of the HPA system and in this way improves anxiety-related stress processing. These findings highlight a novel approach to the treatment of stress-related affective disorders in humans.

Involvement of endothelial NO in the dilator effect of VIP on rat isolated pulmonary artery.

The endothelium and its interaction with smooth muscle play a central role in the local control of the pulmonary vasculature, and endothelial dysfunction is thought to contribute to pulmonary hypertension and chronic obstructive pulmonary disease. Vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, relaxes the rat pulmonary artery, but there is controversy as to whether or not this action of VIP depends on the endothelium. The aim of this study, therefore, was to investigate the role of the endothelium and nitric oxide (NO), the major endothelium-derived relaxing factor, in the dilator action of VIP on the rat isolated pulmonary artery. Pulmonary artery preparations pre-contracted by the alpha(1)-adrenoceptor agonist L-phenylephrine were relaxed by VIP (0.003-1 microM) and acetylcholine (0.003-10 microM) in a concentration-dependent manner. Mechanical removal of the endothelium reduced the maximal response to VIP by about 50% and practically abolished the response to acetylcholine. Inhibition of NO synthesis by N(omega)-nitro-L-arginine methyl ester (0.5 mM) had a similar effect, abolishing the vasorelaxation caused by acetylcholine and attenuating the vasorelaxation caused by VIP by about 50%. From these data it is concluded that the relaxant action of VIP on the rat isolated pulmonary artery depends in part on the presence of the endothelium and that this part is mediated by endothelial NO.

Selective increase of dark phase water intake in neuropeptide-Y Y2 and Y4 receptor knockout mice.

Neuropeptide-Y (NPY) is involved in the regulation of ingestive behaviour and energy homeostasis. Since deletion of the NPY Y2 and Y4 receptor gene increases and decreases food intake, respectively, we examined whether water intake during the light and dark phases is altered in Y2 and Y4 receptor knockout mice. The water consumption of mice staying in their home cages was measured by weighing the water bottles at the beginning and end of the light phase during 4 consecutive days. Control, Y2 and Y4 receptor knockout mice did not differ in their water intake during the light phase. However, during the dark phase Y2 and Y4 receptor knockout mice drank significantly more (46-63%, P<0.05) water than the control mice. The total daily water intake over 24 h was also enhanced. The enhanced water intake during the dark phase was not altered by the beta-adrenoceptor antagonist propranolol or the angiotensin AT1 receptor antagonist telmisartan (each injected intraperitoneally at 10 mg/kg). These data indicate that NPY acting via Y2 and Y4 receptors plays a distinctive role in the regulation of nocturnal water consumption. While beta-adrenoceptors and angiotensin AT1 receptors do not seem to be involved, water intake in Y2 and Y4 receptor knockout mice may be enhanced because presynaptic autoinhibition of NPY release and inhibition of orexin neurons in the central nervous system are prevented.

Differential effects of intragastric acid and capsaicin on gastric emptying and afferent input to the rat spinal cord and brainstem.

BACKGROUND: Hydrochloric acid (HCl) is a potential threat to the integrity of the gastric mucosa and is known to contribute to upper abdominal pain. We have previously found that gastric mucosal challenge with excess HCl is signalled to the rat brainstem, but not spinal cord, as visualized by expression of c-fos messenger ribonucleic acid (mRNA), a surrogate marker of neuronal excitation. This study examined whether gastric mucosal exposure to capsaicin, a stimulant of nociceptive afferents that does not damage the gastric mucosa, is signalled to both brainstem and spinal cord and whether differences in the afferent signalling of gastric HCl and capsaicin challenge are related to different effects on gastric emptying. RESULTS: Rats were treated intragastrically with vehicle, HCl or capsaicin, activation of neurons in the brainstem and spinal cord was visualized by in situ hybridization autoradiography for c-fos mRNA, and gastric emptying deduced from the retention of intragastrically administered fluid. Relative to vehicle, HCl (0.5 M) and capsaicin (3.2 mM) increased c-fos transcription in the nucleus tractus solitarii by factors of 7.0 and 2.1, respectively. Capsaicin also caused a 5.2-fold rise of c-fos mRNA expression in lamina I of the caudal thoracic spinal cord, although the number of c-fos mRNA-positive cells in this lamina was very small. Thus, on average only 0.13 and 0.68 c-fos mRNA-positive cells were counted in 0.01 mm sections of the unilateral lamina I following intragastric administration of vehicle and capsaicin, respectively. In contrast, intragastric HCl failed to induce c-fos mRNA in the spinal cord. Measurement of gastric fluid retention revealed that HCl suppressed gastric emptying while capsaicin did not. CONCLUSION: The findings of this study show that gastric mucosal exposure to HCl and capsaicin is differentially transmitted to the brainstem and spinal cord. Since only HCl blocks gastric emptying, it is hypothesized that the two stimuli are transduced by different afferent pathways. We infer that HCl is exclusively signalled by gastric vagal afferents whereas capsaicin is processed both by gastric vagal and intestinal spinal afferents.

Stomach-brain communication by vagal afferents in response to luminal acid backdiffusion, gastrin, and gastric acid secretion.

Vagal afferents play a role in gut-brain signaling of physiological and pathological stimuli. Here, we investigated how backdiffusion of luminal HCl or NH(4)OH and pentagastrin-stimulated acid secretion interact in the communication between rat stomach and brain stem. Rats were pretreated intraperitoneally with vehicle or appropriate doses of cimetidine, omeprazole, pentagastrin, dexloxiglumide (CCK(1) receptor antagonist), and itriglumide (CCK(2) receptor antagonist) before intragastric administration of saline or backdiffusing concentrations of HCl or NH(4)OH. Two hours later, neuronal activation in the nucleus of the solitary tract (NTS) and area postrema was visualized by c-Fos immunohistochemistry. Exposure of the rat gastric mucosa to HCl (0.15-0.5 M) or NH(4)OH (0.1-0.3 M) led to a concentration-dependent expression of c-Fos in the NTS, which was not related to gender, gastric mucosal injury, or gastropyloric motor alterations. The c-Fos response to HCl was diminished by cimetidine and omeprazole, enhanced by pentagastrin, and left unchanged by dexloxiglumide and itriglumide. Pentagastrin alone caused an omeprazole-resistant expression of c-fos, which in the NTS was attenuated by itriglumide and prevented by dexloxiglumide but in the area postrema was reduced by dexloxiglumide and abolished by itriglumide. We conclude that vagal afferents transmit physiological stimuli (gastrin) and pathological events (backdiffusion of luminal HCl or NH(4)OH) from the stomach to the brain stem. These communication modalities interact because, firstly, acid secretion enhances afferent signaling of gastric acid backdiffusion and, secondly, gastrin activates NTS neurons through stimulation of CCK(1) receptors on vagal afferents and of CCK(2) receptors on area postrema neurons projecting to the NTS.

Evaluation of peristalsis in multiple segments of the guinea-pig isolated small intestine: optimisation of tissue use by refined in vitro methodology.

Peristalsis is the aboral movement by which the intestine propels its contents. Since pharmacological research requires an experimental model with which drug-induced modifications of peristalsis can be reliably quantified, we set out to develop and validate an in vitro method for studying peristalsis in multiple gut segments. In our arrangement, up to four 10cm segments isolated from the guinea-pig jejunum and ileum can be set up in parallel and their lumens perfused. Peristalsis was elicited by pressure-evoked wall distension, and the peristalsis-induced changes in the intraluminal pressure were evaluated with software that determined the peristaltic pressure threshold, the frequency, maximal acceleration and amplitude of the peristaltic waves, and the residual baseline pressure. Validation experiments showed that the peristalsis parameters at baseline and after modification by morphine (0.01-10microM) did not differ between segments from the jejunum and ileum, or between segments examined in a consecutive manner. In conclusion, our work succeeded in optimising the use of the guinea-pig jejunum and ileum for multiple recordings of peristalsis in vitro, and in refining the recording and evaluation of peristaltic motility. This system promises to be particularly useful in the pharmacological screening and testing of drugs which modify peristalsis.