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Aims Lysophosphatidic acid (LPA) is released from injured tissue and cancer cells and is involved in the induction of neuropathic pain. The present study explores whether LPA plays a role in the development of osteocarcinoma-induced pain. Main methods The bone cancer model was established using the Walker 256 mammary gland carcinoma cell line, and cancer-related behavioral and physiological changes were observed using von Frey, X-ray and immunohistochemical methods. The role of LPA in the bone cancer model and related mechanisms were examined by using in vitro single fiber recording and western blot. Key findings Rats exhibited severe hyperalgesia 2 weeks after the cancer cell implantation. Several changes were observed at this time point including: ipsilateral dorsal root ganglion (DRG) neurons were labeled by injured neurons marker ATF3; LPA 1 receptor expression in DRG neurons was increased; sural C-fibers were more sensitive to LPA stimuli, and this response could be blocked by LPA receptor and substance P receptor antagonists. Significance These data indicate that LPA is involved in the induction of bone cancer pain through mechanisms of peripheral C-fibers sensitization. LPA and its downstream molecules possibly are promising therapeutic targets for treatment of cancer pain.

Nutrients in the intestine initiate changes in secretory and motor function of the gastrointestinal (GI) tract. The nature of the 'sensors' in the intestinal wall is not well characterized. Intestinal lipid stimulates the release of cholecystokinin (CCK) from mucosal entero-endocrine cells, and it is proposed that CCK activates CCK A receptors on vagal afferent nerve terminals. There is evidence that chylomicron components are involved in this lipid transduction pathway. The aim of the present study was to determine (1) the pathway mediating reflex inhibition of gastric motility and (2) activation of duodenal vagal afferents in response to chylomicrons. Mesenteric lymph was obtained from awake rats fitted with lymph fistulas during intestinal perfusion of lipid (Intralipid, 170 micromol h(-1), chylous lymph) or a dextrose and/or electrolyte solution (control lymph). Inhibition of gastric motility was measured manometrically in urethane-anaesthetized recipient rats in response to intra-arterial injection of lymph close to the upper GI tract. Chylous lymph was significantly more potent than control lymph in inhibiting gastric motility. Functional vagal deafferentation by perineural capsaicin or CCK A receptor antagonist (devazepide, 1 mg kg(-1), i.v.) significantly reduced chylous lymph-induced inhibition of gastric motility. The discharge of duodenal vagal afferent fibres was recorded from the dorsal abdominal vagus nerve in an in vitro preparation of the duodenum. Duodenal vagal afferent nerve fibre discharge was significantly increased by close-arterial injection of CCK (1-100 pmol) in 43 of 83 units tested. The discharge of 88% of CCK-responsive fibres was increased by close-arterial injection of chylous lymph; devazepide (100 microg, i.a.) abolished the afferent response to chylous lymph in 83% of these units. These data suggest that in the intestinal mucosa, chylomicrons or their products release endogenous CCK which activates CCK A receptors on vagal afferent nerve fibre terminals, which in turn initiate a vago-vagal reflex inhibition of gastric motor function.

To prevent the blood-borne interference and reflex actions via neighboring organs and the central nervous system, the study was conducted in an in vitro isolated stomach-gastric vagus nerve preparation obtained from overnight-fasted, urethan-anesthetized rats. Afferent unit action potentials were recorded from the gastric branch of the vagus nerve. The left gastric artery was catheterized for intra-arterial injection. In vitro we found that 1) 55/70 gastric vagal afferents (GVAs) were polymodal, responding to CCK-8 and mechanical stimuli, 13 were mechanoreceptive, and 2 were CCK-responsive; 2) sequential or randomized intra-arterial injections of CCK-8 (0.1–200 pmol) dose-dependently increased firing rate and reached the peak rate at 100 pmol; 3) the action was suppressed by CCK-A (Devazepide) but not by CCK-B (L-365,260) receptor antagonist; 4) neither antagonist blocked the mechanosensitivity of GVA fibers. These results are consistent with corresponding in vivo well-documented findings. Histological data indicate that the layered structure of the stomach wall was preserved in vitro for 6–8 h. Based on these results, it seems reasonable to use the in vitro preparation for conducting a study that is usually difficult to be performed in vivo. For instance, because there was no blood supply in vitro, the composition of the interstitial fluid, i.e., the ambient nerve terminals, can be better controlled and influenced by intra-arterial injection of a defined solution. Here we report that acutely changing the ambient CCK level by a conditioning stimulus (a preceding intra-arterial injection of increasing doses of CCK-8) reduced the CCK sensitivity of GVA terminals to a subsequent test stimulus (a constant dose of CCK-8 intra-arterial injection).

The effect of short-term lower esophageal distension on intragastric pressure (IGP) and the related neural pathways involved were investigated in urethane-anesthetized rats in which enteric nervous system connections were interrupted by ligations of the pylorus and the gastroesophageal junction while keeping the gastric vagus nerve trunks intact. Under these conditions, lower esophageal distension with a bolus of 0.2 to 0.5 ml saline in 0.1 ml step increments, raised the inside esophagus balloon pressure from 1.89 +/- 0.17 to 4.21 +/- 0.13 cm H2O and reduced IGP from -0.42 +/- 0.08 to -0.77 +/- 0.12 cm H2O, respectively. Bilateral cervical vagotomy partly blocked the gastric relaxation induced by 0.5 ml esophageal distension from -0.77 +/- 0.12 to -0.34 +/- 0.02 cm H2O; in contrast, a further bilateral splanchnectomy partly rebounded the effect of 0.5 ml esophageal distension from -0.34 +/- 0.02 to -0.46 +/- 0.05 cm H2O. These results suggest that the enteric nervous system may not play a prominent role in acute esophageal distension induced-gastric relaxation. However, more than 50% of this effect is central nervous system mediated (via the long vago-vagal reflex). The other 40% can be maintained without central and enteric nervous systems involvement, probably via a proposed gastric vagal afferent-esophageal collateral reflex.

1. Single-unit impulse activity evoked by transient, focal application of hydrogen peroxide (H2O2) to identified visceral receptive fields has been characterized in an in vitro rat splanchnic nerve-mesentery preparation. In addition to H2O2 responsiveness, units were characterized in terms of sensitivity to mechanical stimuli, warming, and bradykinin. 2. Mesenteric receptive fields of single splanchnic afferent C fibers in vitro were located with the use of warm (approximately 45 degrees C saline) or mechanical search stimuli. After delimitation of the warm-sensitive and/or mechanosensitive receptive field, units were tested for responsiveness to transient, focal application of H2O2. Microliter volumes (usually 1 microliter) of H2O2 (88-880 mM) evoked responses in 25 of 42 (60%) units with identified warm-sensitive and/or mechanosensitive receptive fields, and in an additional 10 units for which H2O2 was the only effective stimulus. 3. Tachyphylaxis to repeated H2O2 stimulation was observed with interstimulus intervals 2 min) response latencies to focal application of H2O2 to defined receptive fields. 7. Excitation of splanchnic neurons by H2O2 may be relevant to the modulation of reactive oxygen species production by immunocompetent cells, because sensory neuropeptides contained in these afferent fibers are known to influence the respiratory burst of macrophages and neutrophils.

Our previous studies indicated that in rats about 10% of ventral gastric vagal efferent discharges do not originate from supracervical neural elements. To determine the origin of these efferent activities, an in vitro subdiaphragmatic vagus nerve-esophagus preparation was used. Action potentials with the same amplitude and waveform, and behaving ‘all or none’ characteristic are considered to be recorded from a nerve fiber and defined as an unit activity. Because these centrifugal unit activities were recorded from the proximal cut end of the ventral gastric vagal strands, they are ostensibly considered to be efferent activities. However, about 50% of unit action potential samples (21 out of 40) behave like unit activities recorded from mechanoreceptive afferent fibers. They have spot-like or diffuse mechanoreceptive fields on the subdiaphragmatic esophagus. When these receptive fields were stimulated the sensory nerve terminals in the fields generate afferent unit action potentials. These afferent potentials not only propagate orthodromically to the central nerve system, but also can be transmitted centrifugally to the gastric branches of the same vagal afferent neuron. Together with the efferent discharges of gastric vagal motor neurons, these centrifugal sensory potentials can be intercepted from the proximal cut end of gastric vagal nerve strands at gastroesophageal junction. Three types of mechanoresponsive centrifugal afferent unit activities were observed: rapidly adapting ( n = 8), with or without after-discharge; slowly adapting ( n = 8), with or without after-discharge, and initial high frequency followed by a plateau, with long-lasting after-discharge ( n = 5). Of the tested units ( n = 24), 25% were either activated or inhibited by esophageal inflation and 23% ( n = 22) by esophageal deflation. It is evident that not all centrifugal unit action potentials recorded from the proximal cut end of gastric vagal nerve strands are generated from the vagal motor neurons, the recorded centrifugal unit activities may contain antidromic unit action potentials generated from the esophageal collateral branches of the gastric vagal afferent nerve fibers. These results suggest that gastric vagal afferent neurons possess collateral branches innervating the esophagus, activation of esophageal terminals may exert an effect on the gastric terminals via collateral reflex, analogous to the ‘axon reflex’ mechanism.

The influence of intravenous (iv) bombesin on multiunit activities recorded from the ventral gastric branch of the vagus was investigated in urethan-anesthetized rats. Consecutive injections of bombesin (0.062, 0.62, 6.2, 62, and 620 pmol iv) decreased dose dependently gastric vagal efferent discharges to 79.8 +/- 4.9, 68.3 +/- 10.2, 47.0 +/- 6.7, 41.6 +/- 4.7, and 36.5 +/- 8.9%, respectively, from preinjection levels. Saline injection had no effect. Bombesin (62 pmol iv) reduced cervical vagal efferent discharges to 25 +/- 6% before and 67 +/- 5% after bilateral cervical vagotomy distal to the recording site. Bombesin (62 and 620 pmol iv) increased gastric vagal afferent discharges by 45 and 93%, respectively. These data show that systemic injection of bombesin potently decreases gastric and cervical vagal efferent activity in part through vagal-dependent mechanisms that may involve the increase in gastric vagal afferent activity.

The source of vagal efferent discharge (VED) in the anterior branch of the gastric vagus was investigated in urethane-chloralose anesthetized rats using successive and selective vagal cuts. After cutting the right cervical vagus, the basal VEDs were increased in 15 out of 21 cases by 4–53% (median 18%). After both cervical vagi were cut, VEDs were reduced by 10–95% (median 90%) in 14 of 17 experiments and a subcervical basal VED was observed in all rats. Additional cut of the distal end of the anterior gastric branch did not induce a consistent effect. A small segment of subdiaphragmatic anterior gastric vagus (4–5 mm) was further isolated by a fourth cut at the proximal end of the anterior gastric vagus; abolition of the subcervical VED occurred in only 4 of 14 successful cuts whereas in the other 10 experiments, the VED was reduced by 38–94% (median 87%). Histological examination revealed the presence of neurons in a paraganglion lying within the isolated nerve segment. These findings indicate that the stomach not only receives VED descending directly from medullary vagal motor neurons (about 90%), but also (approximately 10%) from neural elements located between subcervical to upper abdomen levels (the ‘subcervical VED’) and/or between the bifurcation of the accessory celiac branch to the gastro-esophageal junction (the ‘residual VED’). In rats there is little crossed gastric vagal innervation, in agreement with anatomical observations, although there is a robust inhibitory influence from contralateral vagal afferents on medullary vagal motor neurons.

Clusters of glomus cells, isolated from rat carotid bodies, were cultured for up to 2 weeks. Afterwards, we simultaneously measured the intracellular pH (pHi) and membrane potentials (Em) of single cells with pH-sensitive and KCl-filled microelectrodes. In 89 control cells (bathed in saline equilibrated with 50% O2 in N2) pHi was 6.87 +/- 0.014 (SE) and Em -36.3 +/- 0.45 mV. In 42 cells, switching to air (about 20% O2) lowered pHi in 60% of them by as much as 0.14 unit (mean decrease, 0.05). In the remaining cells, pHi increased by as much as 0.18 unit (mean increase, 0.06). Application of 2.5% CO2 in 50% O2 (balance N2) reduced the pHi in 90% of 47 cells by as much as 0.44 unit (mean decrease, 0.14). pHi increased to a maximum of 0.05 unit (mean increase, 0.04) in the others. Either stimulus depolarized or hyperpolarized glomus cells (-5 to 8 mV) in approximately equal proportions. There was a significant and positive correlation between delta Em and delta pHi. This observation confirms the idea that the Em of glomus cell is H(+)-dependent. Results do not agree with the acidic hypothesis for chemoreception.

Activation of corticotropin releasing factor 1 (CRF1) receptors is involved in stress related responses and visceral pain, while activation of CRF2 receptors dampens the endocrine and some behavioural stress responses. We hypothesised that CRF2 receptor activation may influence visceral pain induced by colorectal distension (CRD) in conscious rats, and assessed the possible sites and mechanisms of action.Male Sprague-Dawley rats were exposed to CRDs (60 mm Hg, 10 minutes twice, with a 10 minute rest interval). Visceromotor responses (VMR) were measured by electromyography or visual observation. Spinal (L6-S1) extracellular signal regulated kinase 1/2 (ERK 1/2) activation following in vivo CRD and CRF2 receptor gene expression in the T13-S1 dorsal root ganglia (DRG) and spinal cord were determined. Inferior splanchnic afferent (ISA) activity to CRD (0.4 ml, 20 seconds) was assessed by electrophysiological recording in an in vitro ISA nerve-inferior mesenteric artery (intra-arterial)-colorectal preparation.In controls, VMR to the second CRD was mean 31 (SEM 4)% higher than that of the first (p0.05). The selective CRF2 agonist, human urocortin 2 (hUcn 2, at 10 and 20 microg/kg), injected intravenous after the first distension, prevented sensitisation and reduced the second response by 8 (1)% and 30 (5)% (p0.05) compared with the first response, respectively. RT-PCR detected CRF2 receptor gene expression in the DRG and spinal cord. CRD (60 mm Hg for 10 minutes) induced phosphorylation of ERK 1/2 in neurones of lumbosacral laminae I and IIo and the response was dampened by intravenous hUcn 2. CRD, in vitro, induced robust ISA spike activity that was dose dependently blunted by hUcn 2 (1-3 microg, intra-arterially). The CRF2 receptor antagonist, astressin2-B (200 microg/kg subcutaneously or 20 microg intra-arterially) blocked the hUcn 2 inhibitory effects in vivo and in vitro.Peripheral injection of hUcn 2 blunts CRD induced visceral pain, colonic afferent, and spinal L6-S1 ERK 1/2 activity through CRF2 receptor activation in rats.

Background & Aims: Corticotropin-releasing factor (CRF) exerts its action through CRF receptors 1 and 2 (CRF-R1 and CRF-R2). CRF has preferential affinity for CRF-R1, whereas urocortin displays high affinity for both. We investigated changes in colonic motor function after intraperitoneal (IP) injection of CRF-related peptides. Methods: Colonic motility was recorded in vivo in conscious rats equipped with electrodes chronically implanted in the cecum and proximal colon or in vitro in distal colon; fecal output was monitored in naive rats. Results: Rat CRF, rat urocortin, and amphibian sauvagine (10 μg/kg, IP) induced a new pattern of cecocolonic myoelectric activity characterized by clustered spike bursts of long duration; the percentage of occurrence was highest after CRF. The rank order of potency to increase fecal pellet output after IP peptide injection (0.3–10 μg/kg, IP) was CRF > urocortin=sauvagine. The CRF-R1/R2 antagonist astressin (33 μg/kg, IP) and the CRF-R1 antagonist CP-154,526 (20 mg/kg, subcutaneously) inhibited IP CRF-induced changes in cecocolonic myoelectric activity and IP CRF- and water avoidance stress–induced fecal output. In vitro, CRF injected into the inferior mesenteric artery increased distal colonic myoelectric activity compared with saline injection. Conclusions: These results demonstrate that CRF acts peripherally to stimulate colonic motility and that CRF-R1 is primarily involved in mediating IP CRF/urocortin– and water avoidance stress–induced colonic motor response. GASTROENTEROLOGY 2000;119:1569-1579

Leptin administered (either intracerebroventricularly, icv, or intraperitoneally, ip) acts in synergy with CCK to suppress food intake and body weight in lean mice or rats. The potentiating effect induced by the co-injection of ip CCK and leptin to inhibit food consumption in mice is mediated by the CCK-A receptor and capsaicin sensitive afferents. In vitro, studies in rats showed that a subset of gastric vagal afferent fibers responded to leptin injected directly into the gastric artery only after a prior intra-arterial CCK injection. Moreover, the tonic activity of gastric-related neurons in the nucleus tractus solitarius (NTS) increased when leptin was delivered into the gastric chamber of an in vitro stomach–brainstem preparation. CCK co-injected with leptin potentiated Fos expression selectively in the area postrema, NTS and paraventricular nucleus of the hypothalamus (PVN), which points to the PVN as part of the afferent and efferent limbs of the circuitry involved in the synergistic interaction between leptin and CCK. The dampening of CCK or leptin inhibitory action on ingestive behavior when either factor is not present or their receptors are non functional supports the notion that such leptin-CCK interaction may have a physiological relevance. These observations provide a mean through which leptin and CCK integrate short- and mid-term meal-related input signals into long-term control of energy balance.

Adelson, David W., Jen Yu Wei, Mahrokh Yashar, T. J. O-Lee, and Yvette Taché. Central autonomic activation by intracisternal TRH analogue excites gastric splanchnic afferent neurons. J. Neurophysiol. 81: 682–691, 1999. Intracisternal (ic) injection of thyrotropin-releasing hormone (TRH) or its stable analogue RX 77368 influences gastric function via stimulation of vagal muscarinic pathways. In rats, the increase in gastric mucosal blood flow evoked by a low ic dose of RX 77368 occurs via release of calcitonin gene-related peptide from capsaicin-sensitive afferent neurons, most probably of spinal origin. In this study, the effect of low ic doses of RX 77368 on afferent impulse activity in splanchnic single fibers was investigated. The cisterna magna of overnight-fasted, urethan-anesthetized Sprague-Dawley rats was acutely cannulated, and fine splanchnic nerve twigs containing at least one fiber responsive to mechanical probing of the stomach were isolated at a site immediately distal to the left suprarenal ganglion. Unit mechanoreceptive fields were encountered in all portions of the stomach, both superficially and in deeper layers. Splanchnic afferent unit impulse activity was recorded continuously during basal conditions and in response to consecutive ic injections of saline and RX 77368 (15–30 min later; 1.5 or 3 ng). Basal discharge rates ranged from 0 to 154 impulses/min (median = 10.2 impulses/min). A majority of splanchnic single units with ongoing activity increased their mean discharge rate by ≥20% after ic injection of RX 77368 at either 1.5 ng (6/10 units; median increase 63%) or 3 ng (19/24 units; median increase 175%). Five units lacking impulse activity in the 5-min before ic RX 77368 (3 ng) were also excited, with the onset of discharge occurring within 1.0–5.0 min postinjection. In units excited by ic RX 77368, peak discharge occurred 15.6 ± 1.3 min after injection and was followed by a decline to stable activity levels ≤20–40 min thereafter. In a few cases (4/24), ic RX 77368 (3 ng) inhibited the impulse activity of initially active units, with a time course comparable to that seen in units excited by the same treatment. The pattern of discharge in most units was not suggestive of mechanical modulation of activity by rhythmic gastric contractions. The data demonstrate that low ic doses of TRH analogue induce sustained increases in afferent discharge in a substantial proportion of splanchnic neurons innervating the rat stomach. These findings support the notion that splanchnic afferent excitation occurs concomitantly with vasodilatory peptide release from gastric splanchnic afferent nerve terminals after ic TRH-induced autonomic activation.

Leptin is a circulating protein involved in the long-term regulation of food intake and body weight. Cholecystokinin (CCK) is released postprandially and elicits satiety signals. We investigated the interaction between leptin and CCK-8 in the short-term regulation of food intake induced by 24-hr fasting in lean mice. Leptin, injected intraperitoneally (i.p.) at low doses (4–120 μg/kg), which did not influence feeding behavior for the first 3 hr postinjection, decreased food intake dose dependently by 47–83% during the first hour when coinjected with a subthreshold dose of CCK. Such an interaction was not observed between leptin and bombesin. The food-reducing effect of leptin injected with CCK was not associated with alterations in gastric emptying or locomotor behavior. Leptin–CCK action was blocked by systemic capsaicin at a dose inducing functional ablation of sensory afferent fibers and by devazepide, a CCK-A receptor antagonist but not by the CCK-B receptor antagonist, L-365,260. The decrease in food intake which occurs 5 hr after i.p. injection of leptin alone was also blunted by devazepide. Coinjection of leptin and CCK enhanced the number of Fos-positive cells in the hypothalamic paraventricular nucleus by 60%, whereas leptin or CCK alone did not modify Fos expression. These results indicate the existence of a functional synergistic interaction between leptin and CCK leading to early suppression of food intake which involves CCK-A receptors and capsaicin-sensitive afferent fibers.

In vitro gastric vagal afferents' (GVAs) unit activities were recorded from the ventral GVA nerve strands in rats. The responsiveness of 16 GVA terminals to close intra-arterial injection of vehicle (0.1 ml), leptin (350 pmol), and cholecystokinin (CCK)-8 (10 pmol) was analyzed to generate a spike count-versus-time histogram. Data of 5-min spike counts before and after each treatment were normalized by dividing the latter by the former. A quotient (Q) > 1 indicates an excitatory effect, Q < 1 indicates an inhibitory effect, and Q close to 1 indicates no effect. Two types of GVA terminals were identified. Type 1 (n = 8) responded to leptin with Q > 1; CCK-8 pretreatment did not consistently alter leptin sensitivity. In contrast, Type 2 (n = 8) responded to leptin with Q < 1 or close to 1, and CCK-8 pretreatment increased the leptin sensitivity so that the terminals responded to subsequent leptin with Q > 1. These data suggest that Type 1 and Type 2 GVA terminals may provide afferent neural signals, which, in turn, will be involved in body weight and food intake control systems, respectively.

Adelson, David W., Jen Yu Wei, and Lawrence Kruger. Warm-sensitive afferent splanchnic C-fiber units in vitro. J. Neurophysiol. 77: 2989–3002, 1997. Receptive fields of 41 slowly conducting sensory fibers were located using a thermal (warm) search stimulus in an in vitro splanchnic nerve-mesentery preparation. Warm-sensitive receptive fields were punctate and were densest in the region surrounding the prevertebral ganglia, an area with prominent deposits of brown adipose tissue, where the abdominal aorta branches into the major trunks supplying the abdominal viscera. Impulse activity was recorded while applying a warm stimulus to identified receptive fields (RFs). The warm stimulus consisted of a warming ramp (10–15°C in 1–2 s to a 42–49°C peak temperature) followed by a 10- to 30-s period during which the RF was maintained at this peak temperature (plateau phase). Eighty percent (33/41) of warm-sensitive units responded to warming with discharge comprising both a phasic and a tonic component (slowly adapting warm-sensitive, or SA-W, units). The remainder (8/41) responded with only phasic discharge (rapidly adapting warm-sensitive, or RA-W, units). Units' adaptation characteristics were consistent from trial to trial and when applying stimuli from different positions. Fifty percent of SA-W units (8/16) and 17% of RA-W units (1/6) were activated by transient exposure to 9–90 nM bradykinin (BK). Twenty-seven percent (9/33) of SA-W units and 12%(1/8) of RA-W units were activated by probing their RF with von Frey hairs with bending forces 2 SD greater than the mean pre-BK response, indicating sensitization. This sensitization was transient, the response to warming returning to within one standard deviation of the pretrial mean or less over the course of the next 5–10 min. Changes in background activity, mechanical sensitivity, BK sensitivity, and BK-induced sensitization were noted in various splanchnic units over the course of prolonged observations, suggesting that these indices may not reliably distinguish unit type, but instead may indicate the functional state of the sense organ. Splanchnic neurons responsive to the intense warming used in the present in vitro experiments may participate in the cardiovascular responses observed in vivo in heat-stressed rats. The dense distribution of warm-receptive fields in the vicinity of the celiac-superior mesenteric ganglionic complex is consistent with the localization of splanchnic thermosensitive units previously noted in vivo in the rabbit.

The membrane potential (E(m)) was used as an indicator to evaluate the effect of stem cell factor (SCF) on the membrane integrity of peritoneal mast cells (PMCs). PMCs were harvested from the peritoneal lavage of Sprague-Dawley rats, purified more than 95% and cultured with or without the presence of SCF (2 x 10(-8) M). E(m) values were measured with conventional intracellular recording techniques. Results from day 1 to day 4 in culture were compared. Significant differences in average E(m) (aE(m)) (P < 0.01, analysis of variance) were seen on days 3 and 4 (means +/- SE in millivolts): -67.4 +/- 8.0 and -59.4 +/- 4.8 with SCF vs. -24.8 +/- 7.9 and -7.6 +/- 3.9 without SCF, respectively. Moreover, after culture with SCF for > 1 wk, the aE(m) values of purified PMCs had a tendency to reach plateau values similar to that of unpurified PMCs on day 1 (at -20 mV). The morphological appearances of PMCs can be correlated with the results of aE(m) measurements. PMCs with a smooth spherical shape and highly refractive appearance, and better tolerance to electrode impalement, showed E(m) with greater negative values and lesser fluctuations. These results indicate that SCF can maintain the membrane properties and viability of purified PMCs in a long-term culture.

O-Lee, T. J., J. Y. Wei and Y. TachE. Intracisternal Trh and Rx 77368 potently activate gastric vagal efferent discharge in rats. Peptides 18(2) 213–219, 1997.—The influence of intracisternal (ic) TRH and the stable TRH analog, RX 77368, on gastric vagal efferent discharge (GVED) was investigated in urethane-anesthetized rats. Consecutive IC injections of TRH (3, 30, and 300 ng) at 60 min intervals stimulated dose dependently multi-unit GVED with a peak increase of 90 ± 21%, 127 ± 18% and 145 ± 16% respectively. In two separate studies, IC injection of RX 77368 at 1.5 or 15 ng stimulated multi-unit GVED by 142 ± 24% and 244 ± 95% respectively. Saline injection IC had no effect on GVED. RX 77368 (1.5 ng, ic) action was long lasting (84 ± 13 min) compared with TRH (3 ng: 44 ± 7 min). Single-unit analysis also showed that 13 of 13 units responded to ic RX 77368 (1.5 ng) by an increase in activity. These data indicate that low doses of TRH injected ic stimulate vagal efferent outflow to the rat stomach and that RX 77368 action is more potent than TRH.

Uptake of material from surrounding medium by activated rat peritoneal mast cells (PMCs) was studied using in vitro peritoneal eluate cells, the vital fluorescent dye sulforhodamine B (SFRM-B), secretagogue compound 48/80, and an imaging technique. PMCs, which undergo different states of degranulation, are shown to possess the ability to take up (by endocytosis) SFRM-B in an activity-dependent manner. The endocytosed dye is incorporated in the granules and can be discharged into the medium when the cells are reactivated. Both the uptake and the discharge processes are calcium-dependent. The reactivity of mast cells to secretagogue is not altered by the application of the dye. SFRM-B, a negatively charged, nonspecific protein stain, displays greater photostability and less leakage than the positively charged acridine orange, and its fluorescence persists for hours, whereas acridine orange fluorescence fades within 1 min when exposed to ultraviolet illumination. The fluorescent image of the dye-loaded mast cells can be preserved overnight in a container at room temperature. SFRM-B elicits no detectable damaging influence on the activated afferent discharge of splanchnic afferent nerve fibers with mesenteric terminals. This enables the use of SFRM-B for studying the interactions between mesenteric afferent terminals and their surrounding mast cells.

The central nervous system action of bombesin to influence basal gastric vagal efferent discharge (GVED) was investigated in urethane-anesthetized rats. Bombesin (62, 620, and 6200 pmol) injected intracisternally (IC) decreased GVED to 78 ± 10% , 50 ± 4% , and 43 ± 3% of preinjection levels, respectively. Bombesin (620 pmol) injected IV also reduced GVED to 36 ± 6% . Pretreatment with bombesin monoclonal antibody 2A11 completely prevented the decrease in GVED induced by bombesin (620 pmol) given IV but not IC. These data indicate that both IC and IV injections of bombesin decrease basal GVED, and that the inhibitory effect of IC injection represents a central nervous system-mediated action.

The influence of acidity on the resting potential (Em) of cultured rat glomus cells was studied within a narrow range (7.32-7.54) of extracellular pH (pH0). Under these conditions, the intracellular pH (pHi) ranged from 6.3 to 7.21. Resting potentials were most negative when pHi was lowest, and least negative when pHi was highest. These effects were determined by the equilibrium potential of H+ ions (EH) since Em linearly followed EH. However, EH was little affected by pH0. Therefore, intracellular, and not extracellular, acidity was most important in contributing to the Em of glomus cells.

Clusters of carotid body (glomus) cells cultured from a few days to 3 weeks, maintained their morphological characteristics during this period. The resting potential ( E m ) and input resistance ( R o ) did not change for 2 weeks but both declined afterwards. The intracellular pH (pH i ) of glomus cells, measured with glass microelectrodes filled with an H + ion exchanger, was 6.34–6.96 at extracellular pH (pH o ) of 7.32–7.53. Changes in pH o from normal to about 5.5 depolarized most cells but the fall in pH i was less marked than predicted by the Nernst equation. Conversely, shifting pH o to 8.5 hyperpolarized the cells with an increase in pH i which was more acid than predicted. E H (the calculated equilibrium potential for H + (Nernst equation) was more positive than E m during acidity and more negative during normal or alkaline pH o . The kinetics of H + ion distribution was assessed by brief exposures to NH 4 Cl. It is concluded that hydrogen ions are not passively distributed across the glomus cell membranes and that E m is dependent on H + ions.

Calcitonin gene related peptide (CGRP) was one of the first examples of a biologically active peptide to be identified by recombinant DNA and molecular biological approach (Rosenfeld et al., 1983; Amara et al., 1985). It was also the first demonstration of tissue-specific alternative processing of a gene. In 1983, Rosenfeld et al. initially reported that the RNA transcript from the calcitonin gene is processed in rat central and peripheral neural tissues to a mRNA encoding the precursor to a previously unknown 37-residue peptide called CGRP or α-CGRP (Rosenfeld et al., 1983). In thyroidal cells, the calcitonin gene generates a mRNA which encodes a calcitonin precursor protein expressing the calcium-regulating hormone, calcitonin (Rosenfeld et al., 1983). Subsequently, both intra- and interspecies variants have been discovered. A second calcitonin gene generating a mRNA expressing β-CGRP or CGRP II was identified in rats (Amara et al., 1985). Rat β and α forms differ in only one amino acid residue at position 35. The same mechanism of specific alternate RNA processing was described in human tissue.

A comparison was made between different categories of mechanically sensitive, ventrolateral spinal axons to assess their sensitivity to the itch-producing substance cowhage. Of 52 wide-dynamic-range (WDR) units, 17 had contralateral, 22 had ipsilateral, and 13 had bilateral receptive fields. Of the 5 low-threshold units, 1 had an ipsilateral receptive field and the remainder were bilateral. Among the high-threshold units, 10 were contralateral, 6 ipsilateral, and 5 bilateral. Although there was no evidence of cowhage sensitivity in either low- or high-threshold spinal axons, neurons with WDR properties were reactive to cowhage. WDR neurons were subclassified on the basis of their resting discharge pattern as having intermittent, continuous, or no resting discharge. WDR units with an intermittent pattern of resting discharge demonstrated a significant sensitivity to active cowhage and hence might be regarded as pruritogen-responsive spinal axons. Inactive cowhage was used as a control stimulus. In some WDR units with large receptive fields, there were observations suggesting convergence of chemoreceptive and mechanoreceptive inputs, which produced inhibitory as well as excitatory effects.

infected animals showed enhanced gas accumulation in the proximal colon. Immunohistochemical detection of BDV-antioen revealed staining exclusively in the ENS and not in the muscle layers. The selective staining of neural structures was confirmed by neuron specific enolase (NSE) antibodies. In both the submucous and the myentedc plexus BDV-positive nerve fibers and cell bodies were found only in the infected animals. In the submucous plexus, BDV-positive nerve fibres and occasional BDV-positive cell bodies were found 4 weeks pJ.. The number of BDV-posifive nerve fibres and cell bodies increased dramatically between week 4 and 6 pJ. and resulted in positive staining of up to 30% of cell bodies in the submucous plexus. This proportion further increased to 50% at 14 weeks p.i. Infection of the myenteric plexus with BDV became also apparent 4 weeks p.i., but no dramatic increase with extended infection periods were observed. On average, 10% of myenteric neumnes ware BDV-positive after 4 weeks pJ.. This ratio increased up to 18% at week 14 Immunohistocheroical demonstration of choline acetyifransferase (CHAT) revealed that all BDV infected submucous neurones and 84% of the infected myenteric neurones were ChAT-positive. These data indicate that BDV colonizes the gut and dramatically affects the ENS. Like in the CNS,only subpopuiations of enteric neurones contain the BDV antigen. These neurones seemed to be primarily excitatory cholinergic neurones. Therefore it is concluded that infection of the ENS with BOV might lead to gastrointestinal dysfunctions. Furthermore, intestinal biposies may be used as

BACKGROUND: NMDA receptors (NMDA-R) are expressed on the peripheral terminals of extrinsic afferent nerves innervating the rat colon, and NMDA-R antagonists, acting peripherally, inhihit hehavioral pain responses to colorectal distension (CRD). AIM: To determine whether peripheral NMDA-R are directly involved in CRD-mediated afferent nerve activity using an isolated colon-inferior splanchnic nerve preparation. METHODS: About 4 cm of distal colon and 1 cm proximal rectum with attached inferior mesenteric artery, ganglion and inferior splanchnic nerve branches were isolated and transferred into the main chamber of an organ bath. Unit action potentials were recorded from thin nerve filaments teased from inferior splanchnic branches or the mesentery nerve. CRD was provided hy a mini-latex balloon made from a piece of fatigued latex membrane that had a diameter of ~gmm when inflated with O.4ml air. The response of units to quick inflation and withdrawal of 0.3-0.5ml of air with raising and failing phases 0.5 sec and a 2g sec plateau was tested. Distensions were repeated at intervals not less than 4 min apart. Drugs were directly administered via the inferior mesenteric artery in a volume of 0.1 ml. RESULTS: Neither vehicle, nor the non-compstitive NMDA-R open channel blocker memantine (2g/~g) administered via intra-arterial injection had any effect on ongoing basal activity. However, memantine suppressed the responses of all units to subsequent CRD more than 65%. After intra-arterial washing, the responsiveness to CRO partially recovered (-50%) indicating that the effect of memantine was reveraihle. Intra-arterial injection of 10 p.g NMDA together with 5 p.g D-serine (a required co-agonist) in low Mg 2+ Ringers (to remove voltage-dependent inhibition) transiently increased spontaneous activity more than 3-fold over basal levels. Furthermore, prior treatment with a subthreshold dose of NMDA (5 ~g) in combination with D-serine enhanced CRD mediated responses >30%. Injection of D-serine alone in low Mg 2. Ringers had no effect of basal or evoked activity. CONCLUSIONS: These results strongly suggest that NMDA-R expressed on peripheral terminals of extrinsic primary afferents of the rat colon modulate, and may be directly involved, in mechanically induced nociception in this tissue. [Supported by NIH grants DK58173 and DK48476, and funds from AstraZeneca R&D, MOIndal]

Plots were made of multiunit activity versus ankle joint position for receptors in each of the 12 muscles crossing the cat ankle joint, except peroneus tertius, by recording from populations of afferent fibers in muscle nerves. The discharge was measured 15 or 30 sec after terminating the movements that altered the position of the joint. These recordings were dominated by large-spike activity that would be expected to originate mainly from primary spindle endings. Seven of the 12 muscles also cross other joints. Their responses at a given ankle joint position were so altered by changes in the position of the knee or toe joints that they could not reliably signal the position of the ankle joint. As judged from multiunit recording, receptors in each of the five muscles specific to the ankle joint were influenced by more than one axis of ankle joint displacement. Single-unit recording from dorsal root filaments was used to determine whether primary or secondary spindle receptors in soleus and tibialis anterior could selectively signal one axis of ankle joint rotation. Individual soleus receptors were tested both on the flexion-extension axis and with a combined adduction-eversion movement. For 38 of the 70 soleus receptors examined (54%), firm adduction-eversion produced a level of activity greater than that caused by 10 degrees of flexion, and for 77% the level of activity was greater than that caused by 5 degrees of flexion. For 168 of the 184 tibialis anterior receptors studied (91%), firm abduction-inversion produced a level of activity greater than that caused by 10 degrees of extension. Thus few receptors were found that responded exclusively to one axis of rotation. One way in which the position of the ankle joint could be specified in the face of multiaxial receptor activity is by examining the receptor discharge from more than one muscle. A suggestion for how the nervous system might do this is given in the discussion.

Psychophysical experiments have shown that signals from slowly adapting subcutaneous receptors are used to sense limb position under static conditions (i.e., when the joints are stationary). The ascending collaterals of the slowly adapting primary sensory neurons supplying the deep tissues of the hindlimb do not project to the brain via the fasciculus gracilis. In experiments on cats, we have found a population of axons in the lateral fasciculus that signal the position of the ipsilateral hindlimb with a slowly adapting discharge. In the lower thoracic cord these fibers lie between the spinocervical tract and the ventral roots. Although plentiful in the lower thoracic cord, they are sparse or absent below L3. In addition, a few position signaling axons with crossed input were found in the ventral part of the lateral white matter and in the ventral columns. Since the clinical evidence suggests that the spinal pathway for position sense is uncrossed, we propose that information used for conscious judgments of limb position when the joints are stationary initially ascends via the dorsal columns and then relays to the lateral fasciculus on the same side. These slowly adapting signals also may be used to judge limb position when the joints are moving. To determine whether this slowly adapting discharge originates from muscle or joint receptors, the tendons crossing the ankle joint were exposed but left in continuity and then pulled on while the joint was stationary. In this way individual lateral fascicular axons that signaled ankle flexion, extension, abduction or adduction could be shown to receive a strong excitatory input from muscle receptors. After the muscle tendons crossing the ankle joint were cut, tract fibers signaling ankle flexion, extension, abduction or adduction could no longer be found in this portion of the spinal white matter. Axons signaling clockwise or counterclock-wise twist of the ankle were reduced in number but a few were still present. These results suggest that muscle receptors provide the predominant signal used to sense ankle flexion, extension, abduction and adduction and that receptors in articular tissues may signal ankle twist.

The effect of indenting the skin at different rates on the perceived intensity of the stimulus was studied by indenting the skin of the fingertip with two triangular waveforms, given as a pair. The subjects were asked to judge which member of the pair was more intense. Perceived intensity was found to increase both with the depth and the speed of the indentation. In contrast, changes in the rate of skin indentation had little influence on perceived skin indentation depth. This suggests that intensity and depth are different attributes of tactile sensibility. Since the skin is viscous, a rapid indentation is more forceful than a slow indentation of the same depth, raising the possibility that perceived intensity is related to stimulus force. Even though intensity judgments were more closely correlated with the force of a stimulus than with the indentation it produced, a rapidly increasing force was felt as more intense than one that increased more slowly but attained the same final magnitude. When mechanoreceptors in the palmar aspect of the monkey's hand were excited with triangular stimuli like those used in the psychophysical studies, their discharge frequency increased with the rate of skin indentation. However, the receptors were distinctly more rate sensitive than the human judgments of stimulus intensity, suggesting that impulse summation in the central nervous system summates (integrates in the mathematical sense) the receptor input so as to enhance, relatively, the perceived intensity of the slower stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

The conduction velocities of muscle spindle afferent fibers have a bimodal distribution, and classifications of spindle receptors based on afferent fiber diameter have therefore divided these receptors into two groups, the well known primary and secondary endings. However, measures of spindle function that are likely to be important for kinesthetic sensibility such as dynamic response, adaptation and linear directionality (hysteresis) are distributed rather uniformly. Therefore, from this functional perspective it might be argued that muscle spindle receptors should not be subdivided at all. On the other hand, different receptors demonstrate these properties to varying degrees, and there are simple, linear correlations among log (dynamic response), log (adaptation), linear directionality and conduction velocity. Thus, the receptors can be divided into as many as 5-10 different subpopulations that differ significantly in one or more of these properties.

Within the sampled population of cutaneous unmyelinated afferent neurons (n = 94), only the C-polymodal nociceptor population was reactive to the pruritogen cowhage. Of 62 C-polymodal neurons tested, 11 were unresponsive to cowhage. No C-polymodal neurons were more responsive to inactive, than to active, cowhage (n = 17) and all were responsive to mechanical (n = 62) stimuli and noxious heat (n = 24). The range of conduction velocities obtained by single-unit recording techniques was similar to that found by signal averaging the activity from larger strands of nerve. Hence, it is concluded that our recording technique was capable of recording from the smallest afferent fibers in a cutaneous nerve and it was unlikely that we would have missed finding a slowly conducting, pruritus-signaling neuron due to sampling bias. A search of slowly conducting afferents (n = 314) using electrocutaneous stimulation gave no evidence to suggest the existence of an unknown population of unmyelinated fibers that might signal pruritus. A number of alternative mechanisms by which the sensation of itch might be encoded were discussed, the most favored being the activation of a subset of the C-polymodal nociceptive population.

Expiratory discharges in cervical afferent vagal fibres during spontaneous respiration were observed in anesthetized animals (17 rabbits, 4 cats and 2 monkeys). The percentages of such units among the total observed fibres was 11% in rabbits, 5% in monkeys, 2% in cats. All the experiments were done after section of the recurrent laryngeal nerve and the abdominal branches of the vagus nerve. Changing the intraesophageal pressure from +15 mm Hg to -25 mm Hg by injection or suction of air into or out of the esophagus, of which the abdominal end had been ligated, did not affect the expiratory discharges significantly suggesting that the receptors were not in the esophagus. Injection of air into the lungs to elevate the intratracheal pressure to 5 mm, 10 mm or 15 mm Hg could not excite such receptors. Collapse of the lungs caused by artificial pneumothorax produced continuous discharges in such fibres. Inflation of collapsed lungs by an artificial respiration pump stopped the sustained discharges immediately. The average conduction velocity of the afferent fibres was 25.5 m/s. It seems that this is a type of slowly adapting, low threshold pulmonary receptor with medium sized afferent fibres. The adequate stimulus of such receptors is deflation of the lungs. The possible advantage of participation of such receptors, in addition to the pulmonary stretch (inflation) receptors, in regulation of normal respiration is discussed in the light of the concept of 'paired receptors'.

Nerve impulses were recorded from sensory fibers supplying the tibialis anterior and soleus muscles of anesthetized cats as the ankle joint was moved from one end of the flexion-extension axis to the other and back again in steps of 6-7 degrees. The rate of movement from one position to the next was 40 deg/s and each position was held for 16-18 s. Plots were made of receptor discharge frequency as a function of ankle joint angle during joint movement (dynamic input-output (I-O) functions) as well as 2 and 15 s after movement terminated (2 and 15 s static I-O functions). Only receptors with a sustained (5s) static response within the physiological range were studied. A total of 229 tibialis anterior receptors met this criterion, of which 11 were identified as tendon organs. One hundred and five soleus receptors were studied, of which 6 were tendon organs. Thus tendon organ activity accounted for only a small part of the muscle afferent signal under passive conditions. The spindle receptors in soleus and tibialis anterior divided the ankle flexion-extension range about equally between them, those in soleus signaling over the flexion half of the range and those in tibialis anterior over the extension half. At angles where the receptors in a particular muscle did not signal joint angle, the tendon of the muscle was observed to be slack. Thus the total muscle afferent discharge in a relaxed animal is high at one end of the range, declines progressively as the ankle is displaced to an intermediate position, and then increases again as the joint moves toward the opposite end of the range. The spindle receptors within an individual muscle were recruited rather early as the muscle came under tension so that over most of a muscle's signaling range joint angle could have been coded by changes in receptor discharge frequency but not by which spindle receptors were active. To evaluate the information signaled by individual muscle spindle receptors, the following measurements were made from plots of impulse frequency vs joint angle: dynamic response, defined as the frequency difference between the dynamic and 2 s static I-O functions during muscle lengthening; adaptation, defined as the frequency difference between the 2 and 15 s static I-O functions during muscle lengthening.(ABSTRACT TRUNCATED AT 400 WORDS)

Within the sampled population of cutaneous unmyelinated afferent neurons (n = 94), only the C-polymodal nociceptor population was reactive to the pruritogen cowhage. Of 62 C-polymodal neurons tested, 11 were unresponsive to cowhage. No C-polymodal neurons were more responsive to inactive, than to active, cowhage (n = 17) and all were responsive to mechanical (n = 62) stimuli and noxious heat (n = 24). The range of conduction velocities obtained by single-unit recording techniques was similar to that found by signal averaging the activity from larger strands of nerve. Hence, it is concluded that our recording technique was capable of recording from the smallest afferent fibers in a cutaneous nerve and it was unlikely that we would have missed finding a slowly conducting, pruritus-signaling neuron due to sampling bias. A search of slowly conducting afferents (n = 314) using electrocutaneous stimulation gave no evidence to suggest the existence of an unknown population of unmyelinated fibers that might signal pruritus. A number of alternative mechanisms by which the sensation of itch might be encoded were discussed, the most favored being the activation of a subset of the C-polymodal nociceptive population.