Oral administration of a centrally acting ghrelin receptor agonist to conscious rats triggers defecation.

Agonists of ghrelin receptors that cross the blood-brain barrier, but not ghrelin itself, administered peripherally (intravenous or subcutaneous), cause defecation by acting on centres in the lumbo-sacral spinal cord. It is not established whether orally administered ghrelin receptor agonists can have this action. We tested GSK894281 for its effectiveness at the ghrelin receptor and its ability to cross the blood-brain barrier. GSK894281 was effective at the human and rat ghrelin receptors at 1-10 nmol L(-1), but was >1000-fold less potent at the motilin receptor. It achieved a similar blood concentration by oral or intravenous administration. Oral bioavailability was 74% and brain : blood ratio at steady state was 0.7 : 1. GSK894281 administered orally (1-100 mg kg(-1)) caused a prompt, dose-related production of faecal pellets; at 10 mg kg(-1) faecal output was four times greater than after carrier. The output was the greatest in the first half hour and subsided over the next 90 min. At an oral dose of 10 mg kg(-1), the compound was effective on eight successive days. Faecal output was, on average, increased threefold over control in the 2 h after administration on each of the 8 days. This dose also significantly increased food consumption. Rats showed no adverse behavioural effects to the drug on a single application, but at the end of a week of administration they avoided the gavaging pipette. Oral administration of ghrelin receptor agonists that enter the central nervous system could possibly be used to relieve acute cases of constipation or to clear the bowel for colonoscopy.

Effects of NMDA receptor antagonists on visceromotor reflexes and on intestinal motility, in vivo.

Antagonists of NMDA receptors can inhibit both the transmission of pain signals from the intestine and enteric reflexes. However, it is unknown whether doses of the NMDA antagonist, ketamine, that are used in anaesthetic mixtures suppress motility reflexes and visceromotor responses (VMRs). In fact, whether intestinal motility is affected by NMDA receptor blockers in vivo has been little investigated. We studied the effects of ketamine and memantine, administered intravenously or intrathecally. Rats were maintained under alpha-chloralose plus xylazine or pentobarbitone anaesthesia; VMR and jejunal motility were measured. Under alpha-chloralose/xylazine anaesthesia, i.v. ketamine inhibited VMRs at 6 mg kg h(-1), but not at 3 mg kg h(-1). It did not inhibit propulsive reflexes in the jejunum at 10 mg kg h(-1), but reduced them by 30% at 20 mg kg h(-1). Under alpha-chloralose/pentobarbitone anaesthesia, i.v. ketamine reduced propulsive reflexes at 40 mg kg h(-1) and VMR at 10 mg kg h(-1). Memantine inhibited VMRs at 20 mg kg h(-1) and propulsion at 2 mg kg h(-1). Ketamine and memantine, intrathecally, prevented VMRs, but not jejunal propulsion. We conclude that peripherally administered ketamine reduces both VMR and motility reflexes, but not at doses used in anaesthetic mixes (1.8-2.4 mg kg h(-1)). Effects on motility reflexes are likely to be due to non-NMDA receptor actions, possibly on nicotinic receptors.

Motor patterns and propulsion in the rat intestine in vivo recorded by spatio-temporal maps.

We have used spatio-temporal maps derived from video images to investigate propagated contractions of the rat small intestine in vivo. The abdomen, including an exteriorized segment of jejunum, was housed in a humid chamber with a viewing window. Video records were converted to spatio-temporal maps of jejunal diameter changes. Intraluminal pressure and fluid outflow were measured. Contractions occupied 3.8 +/- 0.2 cm of intestine and propagated anally at 3.1 +/- 0.2 mm s(-1) when baseline pressure was 4 mmHg. Contractions at any one point lasted 8.7 +/- 0.6 s. Contractions often occurred in clusters; within cluster frequencies were 2.28 +/- 0.04 min(-1). Pressure waves, with amplitudes greater than about 9 mmHg, expelled fluid when the baseline pressure was 4 mmHg. In the presence of L-NAME, circular muscle contractions occurred at a high frequency, but they were not propagated. We conclude that video recording methods give good spatio-temporal resolution of intestinal movement when applied in vivo. They reveal neurally-mediated propulsive contractions, similar to those previously recorded from intestinal segments in vitro. The propagated contractions had speeds of propagation that were slower and frequencies of occurrence that were less than speeds and frequencies of slow waves in the rat small intestine.

A quantitative approach to recording peristaltic activity from segments of rat small intestine in vivo.

We have developed methods that allow correlation of propulsive reflexes of the intestine with measurements of intraluminal pressure, fluid movement and spatio-temporal maps of intestinal wall movements for the first time in vivo. A segment of jejunum was cannulated and set up in a Trendelenburg recording system while remaining connected to the vascular and nerve supply of the anaesthetized rat. The resting intraluminal pressure in intact intestine was 2-4 mmHg. Hydrostatic pressures of 2, 4, 8 and 16 mmHg were imposed. At a baseline pressure of 4 mmHg, propulsive waves generated pressures of 9 +/- 1 mmHg, that progressed oral to anal at 2-5 mm s(-1). Individual propulsive waves propelled 0.8 +/- 0.4 mL of fluid. The frequency of propulsive waves increased with pressure, but peristaltic efficiency (mL per contraction) decreased with pressure increase between 4 and 16 mmHg. Atropine, as a bolus, transiently blocked peristalsis, but caused maintained block when infused. Hexamethonium blocked propulsive contractions. Inhibition of nitrergic transmission converted regular peristalsis to non-propulsive contractions. These studies demonstrate the utility of an adapted Trendelenburg method for quantitative investigation of motility and pharmacology of enteric reflexes in vivo.

Effects of the peripherally acting NK receptor antagonist, SB-235375, on intestinal and somatic nociceptive responses and on intestinal motility in anaesthetized rats.

We investigated the effects of the selective NK(3) tachykinin receptor antagonist, SB-235375, on noxious signalling from gut and skin and on intestinal motility in anaesthetized rats. We also measured penetrance into brain and spinal cord. Nociceptive responses in reaction to colorectal distension and skin pinch were assessed by recording the electromyogram (EMG) from the external oblique muscle (a visceromotor response). Motility was measured by recording intraluminal pressure waves during changes in baseline pressure in the jejunum. Colorectal compliance was assessed by measuring luminal pressure change during isovolumic distension. SB-235375 (20 mg kg(-1), by i.v. bolus) reduced the EMG response to colorectal distension by over 90%. The reduction was slow at onset, peaked at about 60 min, and lasted for over 2 h. Responses to noxious skin pinch were unchanged. Amplitudes of propulsive waves in the jejunum were slightly reduced, but their frequency of occurrence was unchanged. SB-235375 decreased colorectal compliance by 5-10%. There was undetectable penetration of i.v. SB-235375 into brain or spinal cord. We conclude that SB-235375 acts peripherally to substantially reduce nociceptive signalling from colorectum without affecting noxious signalling from skin and with little effect on intestinal motility.

Analysis of factors that determine the compliance of rat jejunum to distension in vivo.

Distension of the intestine is commonly used to elicit reflex responses at other sites in the gastrointestinal tract, and also to evaluate pain of intestinal origin. The sensory neurones, that initiate the reflexes or pain responses, react to the forces generated in the wall of the intestine. Thus, the responses of the intestine at the site of distension, particularly changes in contractile activity, influence the signals from the gut. In the present work we have analysed the relationship between distension and pressure changes in the jejunum of the rat, in vivo. Isovolumic distension for 5 min caused an initial pressure increase which declined quickly in the first 30 s, and then declined more slowly. Phasic pressure increases were superimposed on the baseline pressure change. Hexamethonium blocked the phasic pressure increases, whereas the initial rapid and subsequent slower pressure decline during distension persisted. Inhibition of nitric oxide synthase (NOS) increased intraluminal pressure and caused increased frequency and irregularity of phasic pressure increases. However, the decline in jejunal pressure during distension was not changed by inhibition of NOS. The pressure decline during isovolumic distension was similar whether saline or paraffin oil were used to distend the intestine, indicating that the decline was not due to increased hydrostatic pressure causing water and electrolyte to cross the mucosal epithelium from the lumen to the intestinal interstitium. Hyoscine had no significant effect on the pressure profile when the intestine was distended. However, when the systemic or the local circulation of the jejunum was infused with nicardipine, the pressure that was achieved during isovolumic distension was less, although the rate of change in pressure during the slow decline was similar. It is concluded that distension evokes phasic pressure increases in the jejunum, that are nerve-mediated, and increases the tension in the wall through a stretch-activated increase in contractile force generated by the circular muscle. The decline in pressure during maintained distension is primarily a consequence of visco-elastic properties of the wall of the intestine.

Volume expansion does not activate neuronal projections from the NTS or depressor VLM to the RVLM.

We investigated whether a monosynaptic connection from the nucleus tractus solitarius (NTS) or the depressor ventrolateral medulla (VLM) to the pressor region of the rostral VLM (RVLM) constituted part of the reflex pathway activated by cardiopulmonary baroreceptors. Volume expansion in the conscious rabbit, which elicits renal nerve inhibition predominantly via cardiac mechanoreceptors, was used as the stimulus. The protein Fos was used as a marker of neuronal activation. The retrogradely transported tracer rhodamine-tagged microspheres, previously injected into the pressor region of the RVLM, identified medullary neurons that projected to that region. Volume expansion significantly increased the number of Fos-positive cell nuclei in the NTS and in the depressor VLM. Neurons that projected to the RVLM were found throughout the depressor region of the VLM and in the NTS but were not activated by volume expansion. Thus, although the central reflex pathways activated by volume expansion include the NTS and the depressor region of the VLM, we could not find evidence for a monosynaptic connection between those regions and the RVLM.

Neurons in the hypothalamic paraventricular nucleus send collaterals to the spinal cord and to the rostral ventrolateral medulla in the rat.

The hypothalamic paraventricular nucleus (PVN) projects to the rostral ventrolateral medulla (RVLM) and to the intermediolateral cell column (IML) of the spinal cord. The present study determined whether the same neurons can innervate both regions. In each rat, two retrogradely-transported tracers, microspheres tagged with fluorescein or rhodamine, were injected into the left lower thoracic/upper lumbar IML (fluorescein) and into the pressor region of the left RVLM (rhodamine). In the PVN over 90% of the neurons labelled with either tracer were found ipsilateral to the injection site. Double labelled cells averaged almost one-third of the spinally-projecting cells in four of the five animals. In the remaining animal, there were few double-labelled cells. The results suggest that a population of PVN neurons innervates both the lower-thoracic/upper lumbar IML and the RVLM.

Functional anatomy of sympathetic premotor cell groups in the medulla.

Pre- and postganglionic sympathetic neurons are organized into discrete functional channels, according to the target they supply. The activity patterns which they show differ between channels, implying that the CNS pathways driving them are not the same. Premotor neurons probably play a key role. This article focuses on what recent evidence tells us about the organization of premotor neurons which control specific sympathetic outflows. Cells that drive muscle vasoconstrictor (MVC), cutaneous vasoconstrictor (CVC), visceral vasoconstrictor (VVC) and renal sympathetic (RSN) outflows have been identified among the premotor neurons of the rostral ventrolateral medulla (RVLM). Other vasomotor, cardiac or adrenal drives are also represented in that cell group. Neurons driving sudomotor responses have been localized in the rostral ventromedial medulla. Evidence on the specific functions of other premotor cell groups is briefly discussed.

Is CRF a ganglionic transmitter or modulator in the cat sudomotor pathway?

The presence of corticotrophin releasing factor (CRF)-like immunoreactivity distinguishes a subset of cat sympathetic preganglionic neurons which supplies sweat glands. It is abundant in their terminals in the stellate ganglion. We sought first to determine whether this immunoreactivity is due to true CRF, then to test whether CRF plays any role in ganglionic transmission in the cat sudomotor pathway. CRF-immunoreactive material extracted from cat stellate ganglia and hypothalamus were eluted on HPLC at equivalent retention times, slightly less than that of standard sheep CRF. In chloralose-anaesthetised cats, sheep CRF (0.13, 1.3 and 13 micrograms/kg, i.v.) raised plasma immunoreactive ACTH levels by between 3- and 300-fold. Submaximal stimulus trains delivered to pre- or postganglionic nerves of the right stellate ganglion evoked electrodermal responses (EDR, a measure of sweat gland activity) in the right forepaw pad as well as increases in heart rate and blood pressure. Exogenous sheep CRF (dose range 130 ng/kg to 13 mg/kg) given close-arterially to the stellate ganglion in 5 chloralose-anaesthetised cats had no consistent effect on either baseline or preganglionically-evoked EDR. Given i.v. at 13 micrograms/kg to four further cats, sheep CRF caused no significant change in mean baseline or mean preganglionically-evoked EDP (P > 0.05; CUSUM test). Hexamethonium (10 or 30 mg/kg i.v.) abolished the EDR to preganglionic nerve stimulation (7/7 cats). Increasing preganglionic stimulus voltage, frequency and train duration failed to show any hexamethonium-resistant component of the EDR, although such effects were evident in the cardioaccelerator pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

CRF-like immunoreactivity selectively labels preganglionic sudomotor neurons in cat.

Immunohistochemical and neuronal tracing methods were used in cats to determine which type of postganglionic sympathetic neuron is innervated by preganglionic neurons which contain corticotrophin releasing factor-like immunoreactivity (CRF-LI). Preganglionic neurons with CRF-LI have their cell bodies at two restricted levels of the spinal cord and terminate in the stellate and lower lumbar ganglia. CRF-LI terminal baskets in stellate and lumbar ganglia surrounded cell bodies, 96-99% of which showed no tyrosine hydroxylase (TH)-LI (presumptive cholinergic neurons). Calcitonin gene-related peptide (CGRP)-LI was used to label the cholinergic ganglion cells which innervate sweat glands: 96-99% of those were confirmed as lacking TH-LI, while the remainder showed weak staining. Every one of over 6000 CRF-LI terminal baskets counted in 4 stellate and 6 lumbar ganglia was found to surround a cell body with CGRP-LI; conversely, 81-86% of the cell bodies showing CGRP-LI were surrounded by CRF-LI terminal baskets. In 3 cats, the retrograde tracer fluorogold was used to label postganglionic neurons projecting to the paw pads (a population which includes both cholinergic sudomotor neurons and noradrenergic vasoconstrictor neurons). Between 26 and 38% of the retrogradely labelled ganglion cells were surrounded by CRF-LI terminal baskets. We conclude that in cats, preganglionic sympathetic neurons with CRF-LI are sudomotor in function.

Olfactory bulb output neurons excited from a basal forebrain magnocellular nucleus.

We present intracellular data which demonstrates a unique facilitatory centrifugal influence on the output cells of the olfactory bulb; the source being the lateral component of the nucleus of the horizontal limb of the diagonal band (HDB), part of the basal forebrain magnocellular complex. Damage to this facilitatory HDB influence may explain the loss of olfactory sensitivity seen early in Alzheimer's disease in which pathological changes occur in the basal forebrain.

Hemorrhage induces c-fos immunoreactivity in spinally projecting neurons of cat subretrofacial nucleus.

Neuronal expression of c-fos product in the cat rostral ventrolateral medulla was studied by immunohistochemistry. Spinally projecting neurons of the subretrofacial (SRF) nucleus were pre-labeled by retrograde transport of fluorescent latex microspheres from the lumbar cord. In 3 animals which were bled by approximately 25% blood volume from a carotid cannula, 48-62% of SRF-spinal neurons expressed c-fos immunoreactivity. In 2 control animals, the corresponding values were 2% and 4%. The data show that bulbospinal neurons of presumed vasomotor function express c-fos in response to hemorrhage.

Intracellular responses of olfactory bulb granule cells to stimulating the horizontal diagonal band nucleus.

The effects of centrifugal afferents on membrane potentials of identified granule cell layer using evoked field potential profiles, and trans-synaptic activation via antidromic stimulation of output cell axon collaterals. Intracellular recordings maintained for 4-30 min showed complex spontaneous spike discharges and allowed characterization of the cell's input resistance, and on some occasions its morphology following intracellular injection of Lucifer Yellow. Stimulation in the nucleus of the horizontal limb of the diagonal band, but not surrounding regions, produced hyperpolarizing responses in 13 of 27 cells in the granule cell layer; four of these were morphologically identified as granule cells of two types, in five the responses had reversal potentials more negative than the resting potential, and six were identified as granule cells by monosynaptic activation from output axon collaterals. A different set of three cells in the granule cell layer responded with depolarization. The results are consistent with the inhibition of tonic activity of granule cells by the nucleus of the horizontal limb of the diagonal band, leading to disinhibition of mitral and tufted cells via dendrodendritic synapses of granule cells on mitral/tufted cell secondary dendrites.

Intrastriatal dopaminergic agents, muscarinic stimulation, and GABA antagonism compared for rotation responses in rats.

We tested the common hypothesis that rotation on systemic injection of dopaminergic agents in rats with unilateral 6-hydroxydopamine lesions of the substantia nigra is attributable to unequal stimulation of dopamine receptors between the two striata. No rotation occurred when dopamine, apomorphine or amphetamine were injected into dorsal striatum or nucleus accumbens of intact, unanesthetized rats. Intrastriatal haloperidol elicited ipsiversive postural deviation only in conjunction with hypermotility induced by systemic amphetamine. In unilateral 6-hydroxydopamine-lesioned rats, intrastriatal apomorphine elicited rotation directed away from the side of its injection, whether intact or lesioned. Carbachol elicited short-latency rotation, contraversive to injection in dorsal striatum or nucleus accumbens, in both intact and 6-hydroxydopamine-lesioned rats. The rotation response to carbachol was suppressed by atropine administered systemically or into the site of intrastriatal carbachol. Picrotoxin or bicuculline produced contraversive rotation or contralateral myoclonic jerks on injection into the striatum in intact rats. The results show that asymmetric stimulation of striatal dopamine receptors is not sufficient to cause rotation, unless the receptors have been denervated. On the other hand, asymmetric stimulation muscarinic receptors is in itself enough to produce the imbalance of gamma-aminobutyric acid (GABA)ergic striatal outputs responsible for rotation.

Effect of stimulating the nucleus of the horizontal limb of the diagonal band on single unit activity in the olfactory bulb.

The effects of centrifugal afferents on single unit discharge in the main olfactory bulb were studied in anaesthetized rats. Recording with extracellular micropipettes revealed spontaneous firing in all bulb layers. Units were located to different laminae using evoked field-potential profiles and histological verification. Output neurons were identified by antidromic response to stimulation of the lateral olfactory tract. Single- or brief multiple-pulse stimulation in the nucleus of the horizontal limb of the diagonal band, but not in adjacent regions, facilitated 17 out of 27 mitral cells with no effect on 10, but inhibited 21 out of 33 granule cell layer units with no effect on 12. Of 13 presumed tufted cells, six were facilitated and the rest unaffected. In contrast, stimulation of olfactory cortex inhibited mitral cells and facilitated most granule layer cells. The results are consistent with an inhibition of tonic granule cell discharge by the horizontal diagonal band nucleus, with resultant disinhibition of mitral cells via the dendrodendritic synapses of granule cells on mitral cell secondary dendrites.