AG

AA

bst

ract

sneurons; P<0.05). In contrast, UK14,304 had no effect on the amplitude of evoked inhibitory(GABAergic) postsynaptic currents (IPSCs) in any of 5 neurons tested (4.5±2.0% inhibition;P>0.05). Exposure to the adenylate cyclase stimulating agent, forskolin (10μM), for 5 minutes“uncovered” the ability of UK14,304 to inhibit GABAergic IPSCs in 3 of 3 neurons tested(8±1.4% inhibition in control vs 24±6.8% inhibition following forskolin; P<0.05). Thepresent data suggest that α2 adrenoceptors are present on the membrane of gastric-projectingDMV neurons, where their activation presumably inhibits vagal efferent outflow to thestomach. In addition, α2 adrenoceptors are present on the nerve terminals of excitatory andinhibitory nerve terminals impinging upon gastric-projecting DMV neurons. As with ourprevious studies investigating Gi/o coupled receptors within the brainstem, α2 adrenoceptoractivation modulates inhibitory synaptic transmission only after elevation of cAMP levelswithin the GABAergic terminal. This suggest that the response to α2 adrenoceptor activationwithin vagally-mediated gastric circuits will be dependent upon the “state of activation” ofvagal neurocircuits and may be modified drastically by neurotransmitters or neurohormonespositively coupled to adenylate cyclase. Supported by DK 55530

M1286

Vagally-Mediated Gastrointestinal Reflexes are Modulated by Central GlucoseLevelsKirsteen N. Browning

Ingested glucose induces gastric relaxation via the release of 5-hydroxytryptamine (5-HT;serotonin) from enteroendocrine cells. The released 5-HT activates 5-HT3 receptors presenton peripheral vagal afferent terminals resulting in vagally-mediated reflex relaxation of thestomach. Since 5-HT3 receptors are also abundantly present on the central terminals ofvagal afferents, the aim of the present study was to determine whether the glucose-inducedalterations in vagally-mediated gastric reflexes are also achieved, in part, via brainstem sitesof action. The receptive relaxation reflex is a classic vago-vagal reflex activated upon distentionof the esophagus that causes gastric relaxation and a delay in gastric emptying. A balloonwas used to distend the esophagus of adult rats and the resulting gastric relaxation measuredvia miniature strain gauges affixed to the stomach. Increasing blood glucose levels viaperipheral glucose clamp decreased the magnitude of the receptive relaxation from 0.6±0.1gat 3±0.2mM blood glucose to 0.4±0.1g and 0.3±0.1g at 6±0.5 and 10±1.8mM blood glucose,respectively (n=3). Similarly, altering brainstem glucose directly (via 4th ventricular applica-tion 3, 4.5 or 10mM glucose) decreased the receptive relaxation by 5±3, 15±0 and 20±3%,respectively (n=3). Brainstem application of the 5-HT3 antagonsist, ondansetron (30nmoles)increased the magnitude of the receptive relaxation by 16±1.4%, suggesting that 5-HT3receptors tonically decrease this reflex. The ability of elevated glucose levels to decreasereceptive relaxation was blocked by brainstem application of the 5-HT3 antagonist, ondanse-tron (30nmoles; 15±0.3% reduction to 10mM glucose vs 0±0.1% reduction in the presenceof ondansetron, n=3). These results suggest that alterations in brainstem glucose levels,either directly or indirectly, exerts profound effects upon vagally-mediated GI reflexes possiblyvia mechanism involving alterations in brainstem 5-HT3 levels. Supported by DK 55530

M1287

Ghrelin Increases Gastric Activity via Brainstem Sites of ActionKirsteen N. Browning, R. Alberto Travagli, Gregory M. Holmes

The neurohormone ghrelin, released from gastric mucosa, exert profound stimulatory effectsupon gastric motility and acid secretion as well as food intake and energy metabolism (Arigaet al, 2008; Wang et al, 2008) though an inhibitory effect has been reported for fundic tone(Kobashi et al, 2009). The present study aimed to elucidate (1) the effects of brainstemapplication of ghrelin on circular muscle contractions of the gastric corpus and antrum and(2) the actions of ghrelin on neurons of the dorsal motor nucleus of the vagus (DMV) thatprovide the preganglionic parasympathetic motor innervation to the stomach. After affixinga strain gauge to the gastric serosa overlying the circular muscle in an Inactin-anaesthetizedrat preparation, peripheral ghrelin (8 μg/kg, iv) elicted the predicted increase in gastricmotility. In a separate group of animals, ghrelin application to the floor of the fourth ventricle(3 - 100pmol) increased gastric circular muscle contractions in a concentration-dependentmanner (184 to 319%). Whole cell patch clamp recordings were made from DMV neuronsin thin brainstem slices. Ghrelin (100nM) was applied by superfusion for a period of timesufficient for the response to reach plateau (3-5min). Ghrelin had no effect upon the DMVneuronal membrane in any of the 9 neurons tested. In 3 neurons in which the effect ofghrelin upon the frequency and amplitude of spontaneous excitatory (glutamatergic) currentswas assessed, ghrelin increased the frequency (205±59%, P<0.05) but not the amplitude(106±3%; P>0.05) of spontaneous events in all 3 neurons. In contrast, in a further 3 neuronsghrelin had no effect upon the frequency (96±1%; P>0.05) or the amplitude (105±7%;P>0.05) of spontaneous inhibitory (GABAergic) synaptic currents. The present results verifythat centrally applied ghrelin elicits contractions of the gastric corpus and antrum. In addition,ghrelin exerts its gastric prokinetic effects via a facilitation of excitatory synaptic inputs toDMV neurons and not via a direct excitation of DMV motoneurons themselves. Furthermore,this indirect excitation appears selective since inhibitory synaptic transmission was notaffected in a similar fashion. Thus, we would suggest that the centrally-acting ghrelin mostlikely increases gastric activity via activation of excitatory synaptic inputs onto DMV neuronsresulting in increased efferent cholinergic vagal activity. Supported by NINDS 049177 (GMH)and NIDDK 055530 (AT)

S-372AGA Abstracts

M1288

Reduced Neurokinin-1 (Substance P) Receptor Binding in Patients WithIrritable Bowel Syndrome: A Positron Emission Tomography Study With[18f]SPA-RQJohanna M. Jarcho, Mark Mandelkern, Bahar Ebrat, Suzanne R. Smith, Bruce D. Naliboff,Jennifer S. Labus, Kirsten Tillisch, Emeran A. Mayer

BACKGROUND: Pre-clinical evidence suggests substance P and its receptor (neurokinin-1[NK1] receptor), are involved in visceral pain processing and modulation at the spinal andsupraspinal level. Stress induced visceral hyperalgesia in rats is associated with an upregul-ation of spinal NK1 receptors (Bradesi et al. Gastroenterology 2009), and NK1 receptorantagonists have been shown to reduce visceral hyperalgesia in several rodent models.However, these rodent findings have not translated into a significant effectiveness of NK1receptors to reduce IBS symptoms or perceptual responses to rectal balloon distension.AIMS: To test the hypothesis if alterations in central NK1 receptors in IBS may explain partof this translational failure. METHODS: Female patients (19-48 years of age) with IBS (N=9) and healthy controls (HCs;N=9) underwent positron emission tomography (PET) scanningwith the NK1 receptor antagonist radioligand, [18F]SPA-RQ, which has the capacity tolocalize and quantify NK1 receptors in the brain. Subjects also received a structural magneticresonance imaging scan, which was aligned with PET images to allow for more accuratespecification of brain regions of interest (ROIs). ROIs were specified using FMIRB's IntegratedRegistration Segmentation Tool, which defines subcortical brain regions. NK1 receptorbinding, a measure of receptor density, was estimated in each ROI using the simplifiedreference tissue model, with the cerebellum, a structure devoid of NK1 receptors, servingas a reference region. RESULTS: Compared with HCs, IBS patients had lower NK1 receptorbinding in all assessed ROIs, including brain stem, thalamus, amygdala, nucleus accumbens,putamen, caudate, and globus pallidus. These differences reached statistical significance inthe globus pallidus (p<.01 ). CONCLUSIONS: IBS patients have lower levels of bindingwith [18F]SPA-RQ in several brain regions, compared to HCs. Even though the etiologyand functional relevance of this reduction remains to be determined, these alterations maycontribute to the low levels of therapeutic success for agents that target this signaling system.This research was partially supported by Johnson & Johnson Pharmaceutical R&D, MerckPharmaceuticals R&D, and NIH R24 AT002681.

M1289

Functional Brain Mapping of Visceral Hypersensitivity Induced by ChronicWater Avoidance Stress in RatsZhuo Wang, Raina Pang, Yumei Guo, Sylvie Bradesi, Emeran A. Mayer, Daniel P.Holschneider

BACKGROUND: Chronic water avoidance stress (WAS) in rats has been shown to inducesustained visceral hypersensitivity measured as enhanced visceromotor response (VMR) tocolorectal distension (CRD). This animal model incorporates two characteristic features ofthe human irritable bowel syndrome (IBS), visceral hyperalgesia and a prominent role ofpsychosocial stress in the onset and exacerbation of IBS symptoms. However, in view ofthe multidimensional nature of the human pain experience, it is likely that VMR reflectsonly part of the nociceptive experience. AIM: To further validate WAS as a model for IBS,we used an autoradiographic perfusion method to map the brain correlates of WAS-inducedvisceral hyperalgesia and compare the results with human brain imaging findings thatcharacterize differences in functional brain responses to CRD between IBS patients andhealthy controls. METHODS: Four groups (n=6/group) of male Wistar rats were implantedwith telemetry transmitters to measure abdominal electromyography (EMG) and cannulatedfor IV injection. Baseline VMR to CRD at 10, 20, 40, and 60 mmHg was measured on day0. For the next 10 days rats were exposed to 1-hr WAS (2 groups) or sham WAS (2 groups).On day 11, VMR to CRD was measured again. Animals then received a 60- or 0-mmHgCRD during which they were administered the perfusion radiotracer, followed by rapideuthanasia. Regional cerebral blood flow (rCBF)-related tissue radioactivity was quantifiedby autoradiography and analyzed in the 3-D reconstructed brain by statistical parametricmapping. RESULTS: Rats exposed to WAS, but not sham, showed significant increase inVMR to CRD on day 11 compared to baseline (P<0.05), consistent with visceral hyperalgesia.In both sham and WAS rats, 60-mmHg CRD compared to 0-mmHg evoked significantincreases in rCBF in the insular, prelimbic, infralimbic cortices, as well as the amygdala andcaudate putamen. Compared to sham, WAS rats showed greater activation in the anteriorinsular, cingulate, and prelimbic cortices, lateral amygdala, locus coeruleus complex, andhypothalamus. CONCLUSIONS: Visceral hyperalgesia following chronic WAS was associatedwith greater activation in brain areas concerned with interoceptive processing and emotionalarousal. As homologous brain areas have previously been reported in human brain imagingstudies to show greater activation in IBS patients compared to healthy controls, these findingsprovide support for the face and construct validity of the chronic WAS model for IBS.Pharmacological targeting of these brain regions in the rat may have predictive validity forcandidate IBS drugs. Supported by NIH P50DK064539, R24AT002681.

M1290

Evidence for Less Effective Corticolimbic Inhibition During Pelvic VisceralDiscomfort in Female IBS Patients: A Functional Magnetic Resonance Imaging(FMRI) StudyLisa A. Kilpatrick, Jennifer S. Labus, Steven M. Berman, Brandall Y. Suyenobu, Bruce D.Naliboff, Emeran A. Mayer

Neuroimaging evidence suggests greater engagement of an emotional arousal network duringexpectation of visceral pain in female IBS compared to healthy controls (HC) (Labus etal 2008 NeuroImage). This study tested for a functional brain network associated withexperimentally induced pelvic visceral discomfort and characterized differences in connectiv-ity of this network between female patients and HC. Methods: Brain responses to controlledrectal distension were assessed by fMRI (1.5T) in 14 women with IBS-C and 12 age-matchedHC. Partial least squares (PLS) was employed to identify distributed patterns of brain regions