TRH stimulation and L-glutamic acid inhibition of proximal gastric motor activity in the rat dorsal vagal complex

The importance of the dorsal vagal complex (DVC) in the control of gastric motor activity has been previously established by electrical and chemical stimulation of this region. We have further evaluated excitatory and inhibitory influences on motor activity of the gastric corpus by microinjection of L-glutamic acid (GLU) and thyrotropin-releasing hormone (TRH) into the DVC. GLU and TRH were ejected by pressure (20-30 psi) in 1-10 nl vol. from multibarreled micropipettes and intraluminal pressure in the gastric corpus was measured using a manometric catheter placed into the stomach through the pylorus of urethane-chloralose anesthetized rats. Gastric motor activity was monitored while micropipettes were advanced from the surface of the dorsomedial medulla to a depth of 1 mm in 100 micron increments. Microinjections of GLU (1-10 pmol) at depths of 200-600 microns below the surface of the brainstem caused a decrease in tonic intraluminal pressure and amplitude of phasic contractions of the gastric corpus. Injection of TRH (1-10 pmol) at depths of 200-800 microns increased both tonic intraluminal pressure and amplitude of phasic contractions. The responses to GLU (10 pmol) and TRH (10 pmol) were abolished by hexamethonium and vagotomy; atropine abolished the effect of TRH and attenuated that of GLU. It is concluded that GLU evokes only vagally mediated inhibitory effects on tonic and phasic gastric motor activity when microinjected into the DVC. In contrast, injection of TRH at the same loci causes only vagal cholinergic increases in motor activity. Subpopulations of neurons in the DVC may, therefore, be activated by specific neurotransmitters having opposite effects on gastric motor activity.     

Raphe pallidus stimulation increases gastric contractility via TRH projections to the dorsal vagal complex in rats

The role of thyrotropin releasing hormone (TRH) in the dorsal vagal complex (DVC) in mediating the enhanced gastric contractility induced by glutamate (100 pmol) microinjected into the raphe pallidus (Rpa) was investigated in urethane-anesthetized rats acutely implanted with miniature strain gauge force transducers on the corpus of the stomach. Glutamate-induced stimulation of gastric contractility was dose-dependently inhibited by bilateral microinjection into the DVC of TRH antibody (0.17, 0.85 or 1.7 μg/100 nl/site) but not by vehicle. TRH antibody microinjected into the dorsal medullary reticular field had no effect. These data indicate that activation of Rpa neurons by glutamate increases gastric motor function through TRH release in the DVC.     

CRF microinjected into the dorsal vagal complex inhibits TRH analog- and kainic acid-stimulated gastric contractility in rats

The effect of CRF microinjected into the dorsal vagal complex (DVC) on centrally-stimulated gastric contractility was investigated in fasted, urethane-anesthetized rats. Miniature strain gauge force transducers were acutely implanted on the corpus of the stomach and contractility was analyzed by computer. Microinjection of the stable thyrotropin-releasing hormone (TRH) analog, RX 77368, (26 pmol) into the DVC induced a 12.2-fold stimulation of gastric contractility within 30 min. Corticotropin-releasing factor (CRF) (63-210 pmol) microinjected into the DVC concomitantly with RX 77368 (26 pmol) induced a dose-related inhibition of stimulated gastric contractility. Neither CRF alone (210 pmol) nor vehicle modified basal gastric contractility. Microinjection of kainic acid (141 pmol) into the raphe pallidus nucleus induced a 3.6-fold stimulation of gastric contractility after 45 min. This stimulation was suppressed by bilateral microinjection of CRF (105 pmol/site) into the DVC. These results demonstrate that CRF acts in the DVC to inhibit centrally-stimulated gastric contractility and suggest that TRH and CRF may interact in the DVC to regulate gastric motor function.     

Role of GABAA receptors in rat hindbrain nuclei controlling gastric motor function

It has been shown in cats that gastric motor control by the dorsal vagal complex and nucleus ambiguus is under a tonic GABAergic influence. Since much more work has been performed in rats to define vago-vagal reflexes controlling gastrointestinal function, an understanding of the potential inhibition by candidate neurotransmitters such as GABA (gamma aminobutyric acid) in the rat dorsal vagal complex (DVC) is essential to assess. Multiple-barrelled micropipettes were used to apply to the dorsal vagal complex the GABA_A antagonist, bicuculline methiodide (0.1–1 nmol), and a GABA_A agonist, muscimol (10 nmol) prior to micro-injection of the GABA_A antagonist. Micro-injections of bicuculline (353 pmol and 1 nmol), which were localized primarily in the dorsal motor nucleus of the vagus, produced significant increases in intragastric pressure and pyloric motility. These responses were abolished by vagotomy and by a prior micro-injection of muscimol. To determine whether GABAergic blockade in the dorsal vagal complex results in gastric motor excitation through excitatory amino acid receptors, kynurenic acid (5 nmol), a kainate/NMDA (N-methyl D -aspartic acid) receptor antagonist, was micro-injected prior to bicuculline. This abolished the increase in gastric motor function normally evoked by bicuculline. In the other two important hindbrain nuclei controlling gastric function, the nucleus raphe obscurus and nucleus ambiguus, bicuculline (353 pmol) significantly increased intragastric pressure via vagally mediated pathways. These data demonstrate that all three rat hindbrain nuclei known to influence gastric function via the vagus nerve are under tonic GABAergic control. In addition, in the dorsal vagal complex, relief from GABAergic inhibition results in increases in gastric motor function through kainate/NMDA receptor-mediated excitation.     

Bombesin microinjected into the dorsal vagal complex inhibits TRH-stimulated gastric contractility in rats

The effects of centrally injected bombesin on central and peripheral stimulated gastric contractility were investigated in fasted urethane-anesthetized rats. Miniature strain gauge force transducers were acutely implanted on the corpus of the stomach and gastric contractility was analyzed by computer. Intracisternal injection of the stable thyrotropin-releasing hormone (TRH)-analog RX 77368 (77 pmol) induced a stimulation of gastric contractility for 40 min. Intracisternal injection of bombesin (62-620 pmol) followed 30 min later by that of RX 77368 resulted in a dose-related inhibition of the TRH-analog-induced gastric contractility. Intracisternal injection of bombesin (620 pmol) did not modify gastric contractility stimulated by intravenous carbachol. Stimulation of gastric contractility induced by TRH-analog microinjected into the dorsal vagal complex (DVC) was dose-related suppressed by concomitant injections of bombesin (6.2-620 pmol). Neither bombesin alone (6.2 pmol) nor vehicle modified basal gastric contractility. These results demonstrate that bombesin acts within the brain to inhibit vagally stimulated gastric contractility and that the DVC is a sensitive site for bombesin inhibitory action. These findings suggest a possible interaction between TRH and bombesin in the central vagal regulation of gastric contractility.     

Microinjection of thyrotropin-releasing hormone in the paraventricular nucleus of the hypothalamus stimulates gastric contractility

Changes in gastric contractility following microinjection of thyrotropin-releasing hormone (TRH) into the paraventricular nucleus of the hypothalamus (PVN) were examined in fasted, urethane-anesthetized rats. Gastric contractility was measured with extraluminal force transducers and analysed by computer. Unilateral and bilateral PVN microinjections of TRH (0.5 and 1.0 μg) significantly increased the force index of gastric contractions from 0 to 60 min postinjection, when compared with animals microinjected with 0.1 μg TRH, 0.1% BSA or TRH (0.5 and 1.0 μg TRH) in sites adjacent to the PVN. The gastric force index was also significantly elevated from 61 to 120 min postinjection in rats receiving bilateral PVN microinjections of TRH (0.5 and 1.0 μg). Peak gastric responses occurred within 10–20 min postinjection and represented an approximately eight-fold increase over basal values. In the remaining groups, the force index was not significantly altered from preinjection values. The excitatory action of TRH (1.0 μg) on gastric contractility was completely abolished by subdiaphragmatic vagotomy. These results suggest that TRH acts within the PVN to stimulate gastric contractility via vagal-dependent pathways.     

Microinjection of thyrotropin-releasing hormone analogue into the central nucleus of the amygdala stimulates gastric contractility in rats

The effect on gastric contractility following bilateral microinjection of thyrotropin-releasing hormone (TRH) analog. RX 77368, into the central nucleus of the amygdala was examined in fasted. urethane-anesthetized rats. Extraluminal force transducers were used to measure gastric corpus contractility. Bilateral microinjection of RX 77368 (0.5 μg. 1.0 μg,n = 6 each) stimulated gastric contractility for up to 120 min post-injection,P < 0.05. Gastric contractility was not significantly stimulated by microinjection of 0.1 μg RX 77368. 0.1% bovine serum albumin (BSA) into the central nucleus or RX 77368 (0.5 μg. 1.0 μg) into sites adjacent to the central nucleus. Peak responses (1.0 μg) occurred 40 min post-injection and represented a 16-26-fold increase over basal values. The frequency of gastric contraction waves was attenuated for 0–90 min in rats receiving central amygdaloid microinjection of RX 77368 (0.1. 0.5 or 1.0 μg) versus rats microinjected with the vehicle or RX 77368 into sites adjacent to the central nuclei. The stimulatory effect of RX 77368 (1.0 μg) on gastric contractility was abolished by subdiaphragmatic vagotomy. These results indicate that the TRH analog. RX 77368, acts within the central amygdala to vagally stimulate gastric contractility.     

Inhibition of Gastric Motor Function by Circulating Corticotropin-Releasing Factor in Anesthetized Rats

We have investigated the influence of intravenous corticotropin-releasing factor (CRF) on intraluminal pressure of the body of the stomach and the neurohumoral pathways through which CRF inhibits gastric motor function in rats. CRF (0.21–210 pmol) injected intravenously produced a dose-dependent, long-lasting decrease in baseline tone and inhibited phasic gastric contractions measured manometrically in urethane-anesthetized rats. The inhibitory effect of CRF was abolished by administration of hexamethonium or by subdiaphragmatic vagotomy but was unaffected by perineural treatment of the vagus nerves with capsaicin, adrenalectomy, or celiac/superior mesenteric ganglionectomy. Intravenous administration of CRF (126 pmol) inhibited by 69% the emptying of a noncaloric liquid meal as measured by the phenol red technique in conscious rats. Hexamethonium pretreatment significantly attenuated the CRF -induced delay in gastric emptying; bretylium, naloxone, adrenalectomy, and celiac I superior mesenteric ganglionectomy had no such effect. In an isolated tissue preparation, CRF (10⁻¹¹ to 10⁻⁶ M) inhibited spontaneous, but not carbachol-induced, contractions of rat antral longitudinal muscle. The effect of CRF was dose-dependent and abolished in the presence of tetrodotoxin. These results suggest that peripheral CRF-induced inhibition of corpus and antral motor contractility may mediate delayed gastric emptying via an action involving a nicotinic synapse, presumably in the enteric nervous system, that requires a vagal input. The in vitro studies support the concept that the action of CRF is not direct on smooth muscle but involves neural transmission within the enteric nervous system.     

Distribution of nitric oxide synthase in rat dorsal vagal complex and effects of microinjection of nitric oxide compounds upon gastric motor function

Nitric oxide (NO) has received attention as a vagal nonadrenergic-noncholinergic (NANC) mediator of gastrointestinal relaxation. The dorsal vagal complex (DVC) is the primary hindbrain site of vagal control of the gastrointestinal tract, and yet the subnuclear distribution of NO and its physiological effects have not been analyzed in this nucleus. Therefore, this study estimates that relative number of of NO synthase (NOS)-containing neurons in subnuclear regions of the DVC, identifies NOS-containing vagal abdominal preganglionic neurons in the dorsal motor nucleus of the vagus, and defines a role of NO in the DVC in control of gastric motor function. The location of NADPH-diaphorase—positive staining (a marker of NOS activity) and NOS immunoreactivity overlap in the DVC. In the dorsal motor nucleus of the vagus there are positively stained cells caudal to the obex and at its most rostral extent, but not at the intermediate level. Intraperitoneal fluorogold combined with NADPH-diaphorase activity labels approximately 5% and 15% of fluorogold-immunoreactive cells in the caudal and rostral dorsal motor nucleus of the vagus, respectively. Thus, a portion of NOS-containing neurons are preganglionic vagal neurons projecting to the abdominal viscera. In the nucleus tractus solitarius, the majority of NADPH-diaphorase—positive cells are within the centralis, medial, and ventral/ventrolateral subnuclei. Fiber/terminal staining is present in the subnucleus centralis, subnucleus gelatinosus, subpostremal zone, and the medial nucleus tractus solitarius. The presence of NOS terminal staining implicates NO in afferent control of gastric function in the DVC (e.g., vago-vagal circuits in subnucleus gelatinosus). To determine a role of NO in the DVC, NO-related agents were microinjected into the DVC in α-chloralose—anesthetized rats while recording indices of gastric motor function. L -Arginine, microinjected into the DVC, significantly decreases intragastric pressure (−2.2 ± 0.4 cm², N = 12), and this effect is abolished by vagotomy. Microinjection of an NOS inhibitor, N^G-nitro-L -arginine methyl ester, increases intragastric pressure (1.9 ± 0.7 cm², N = 10), with the greatest effect in the DVC rostral to the obex. Overall, it was concluded that tonic release of NO in the DVC mediates gastric relaxation, at least in anesthetized animals, and NOS-containing preganglionic neurons in the dorsal motor nucleus of the vagus may be “command” NANC neurons which control a variety of gastrointestinal functions. J. Comp. Neurol. 377:49-69, 1997. © 1997 Wiley-Liss, Inc.     

α-Adrenergic activation of rat hypothalamic supraoptic neurons maintained in vitro

Extracellular recordings from 141 rat supraoptic nucleus neurons maintained in vitro in a perfused hypothalamic explant indicated that the excitability of 85% of cells was enhanced by the addition of 10–200 μM norepinephrine (NE) and the α-agonist methoxamine (MOXY), but not by isoproterenol, to the perfusion medium or by pressure application. Similar responses were observed from 7 cells maintained in synaptic isolation in medium containgng 15 mM Mg²⁺. At the lowest effective concentration (10 μM), NE and MOXY induced bursting activity without an overall increase in firing frequency; at higher concentrations an initial increase in firing frequency was followed by the appearance of phasic activity patterns. The actions of NE and MOXY were reversibly blocked by administration of the α-adrenergic antagonists phenoxybenzamine (1–10 μM), yohimbine (> 10 μM) and irreversibly blocked by prazosin (10 nM−1 μM). These observations suggest that NE has a predominatly facilitatory role to enhance the excitability and promote bursting activity in supraoptic neurosecretory neurons through an α-1 adrenoreceptor mechanism.     

Inhibition of carbachol microinjections of presumptive cholinergic PGO-on neurons in freely moving cats

The effects of microinjections of a cholinergic agonist, carbachol (0.2 μg/0.2 μl), were examined on a population of presumptive cholinergic mesopontine PGO-on neurons that presents a tonic pattern of discharge during waking and exhibits short bursts preceding the onset of dorsal lateral geniculate PGO waves during paradoxical sleep and slow wave sleep just prior to it. PGO-on neurons were activated antidromically by the stimulation of the dorsal lateral geniculate, pulvinar and/or medial and intralaminar nuclei. They were all characterized by a long spike duration and a slow conduction velocity. Microinjections of carbachol near unit recording sites in freely moving cats induced a complete suppression of the spontaneous tonic activity during waking, but did not suppress the spontaneous phasic burst activity during sleep. Carbachol microinjections also resulted in a marked reduction in responsiveness of PGO-on neurons to orthodromic stimulation. These spike depressant effects lasted for approximately 90–120 min and were reversed completely by a local or systemic administration of atropine sulfate. These findings point to a direct inhibition of central cholinergic PGO-on neurons via a muscarinic autoreceptor and a difference in the mechanisms underlying the generation of tonic and phasic burst activity of PGO-on neurons occurring during waking and sleep.     

Steroid Dependent Effects of Oxytocin on Spontaneous Motor Activity in Female Rats

In the present study, dose relationships for effects of oxytocin (OXY) on spontaneous motor activity in female rats were investigated. Ovariectomized (OVX) and cycling female Sprague-Dawley rats were given OXY 10–1000 μg/kg s.c. or saline, 10 min before registration of motor activity in an open-field arena. In the OVX rats, 100 μg/kg of OXY increased the activity in the center of the arena, whereas 1000 μg/kg decreased locomotor activity (LA). In the cycling rats, OXY 100–1000 μg/kg decreased LA during diestrus, while 1000 μg/kg also decreased LA during metestrus. The latter dose also reduced the exploratory behavior during estrus. In a second experiment, OVX rats were pretreated with estradiol benzoate (EB) and progesterone (P). When P levels were predominant, OXY 10–1000 μg/kg decreased LA. Oxytocin 10–100 μg/kg given after pretreatment with EB increased the activity in the center of the arena, whereas 1000 μg/kg given in the presence of both EB and P increased peripheral activity (PA). These results show that the effects of OXY on motor activity in female rats are modified by female sex steroid hormones.     

Effects of dorsal cord stimulation on stretch reflexes

The effects of dorsal cord stimulation on phasic and tonic stretch reflex activity in extensor muscles were studied in decerebrate cats. The tonic stretch response was depressed in both fore- and hindlimb (stimulation at levels C1 and T8, respectively) and this often persisted for 5–20 min after the end of 1–10 min of dorsal cord stimulation. Depression of the phasic stretch response was only consistently seen in the forelimb during stimulation (C1) and this rarely outlasted the period of stimulation. These results support the idea that dorsal cord stimulation can reduce muscle tone but provide no explanation for the long-lasting effects of chronic, continuous stimulation in spastic man.     

American Neurogastroenterology and Motility Society consensus statement on intraluminal measurement of gastrointestinal and colonic motility in clinical practice

Abstract Tests of gastric, small intestinal and colonic motor function provide relevant physiological information and are useful for diagnosing and guiding the management of dysmotilities. Intraluminal pressure measurements may include concurrent measurements of transit or intraluminal pH. A consensus statement was developed and based on reports in the literature, experience of the authors, and discussions conducted under the auspices of the American Neurogastroenterology and Motility Society in 2008. The article reviews the indications, methods, performance characteristics, and clinical utility of intraluminal measurements of pressure activity and tone in the stomach, small bowel and colon in humans. Gastric and small bowel motor function can be measured by intraluminal manometry, which may identify patterns suggestive of myopathy, neuropathy, or obstruction. Manometry may be most helpful when it is normal. Combined wireless pressure and pH capsules provide information on the amplitude of contractions as they traverse the stomach and small intestine. In the colon, manometry assesses colonic phasic pressure activity while a barostat assesses tone, compliance, and phasic pressure activity. The utility of colonic pressure measurements by a single sensor in wireless pressure/pH capsules is not established. In children with intractable constipation, colonic phasic pressure measurements can identify patterns suggestive of neuropathy and predict success of antegrade enemas via cecostomy. In adults, these assessments may be used to document severe motor dysfunction (colonic inertia) prior to colectomy. Thus, intraluminal pressure measurements may contribute to the management of patients with disorders of gastrointestinal and colonic motility.     

Thyrotropin-releasing hormone increases the number of muscarinic receptors in the lateral septal area of the rat brain

Stereotactic injection of acetylcholine (0.5–2 μg) into the lateral septal region of the rat brain produces a long-lasting sympathetic-mediated increase of the arterial blood pressure. This effect is mediated by muscarinic receptors since 1 μg atropine abolishes this response. In this same brain region, TRH (0.5–4 μg) did not elicit any significant change in the arterial blood pressure, but potentiated the effect of acetylcholine. This phenomenon is apparently due to an increase of the number of muscarinic receptors in the lateral septal area of the rat brain.     

Neuronal firing patterns of organotypic rat spinal cord cultures in normal and in ACTH/α-MSH(4–10) analog (BIM 22015)-supplemented medium

The spontaneous and evoked electrical patterns of spinal cord explants from 13- to 14-day old rat fetuses grown from 2 to 8 weeks in vitro were compared when fed either with a standard or with an adrenocorticotropic hormone/α-melanocyte stimulating hormone (4–10) analog (BIM 22015)-supplemented medium. Standard and BIM 22015-treated cultures developed similar patterns of extracellularly recorded activity which consisted of mostly phasic but also tonic discharges. The standard cultures when treated by BIM 22015 in acute experiments (100 μg/ml) showed a decrease in their frequency of discharges which fired in a regular tonic pattern. These effects were neither age- nor dose-dependent but were increased in Ca²⁺ free medium. The ventral cord neurons chronically fed with BIM 22015 showed a strongly bursting pattern resembling strychnine-induced synchronized bursts. Both these effects, inhibitory (acute) and excitatory (chronic), of the BIM upon spinal cord cultured ventral horn neurons are discussed as possible calcium-dependent phenomena.     

The effects of naloxone on the neural control of the urinary bladder of the cat

Naloxone in doses ranging from 0.5 to 512μg/kg i.v., enhanced reflex contractions of the urinary bladder of the cat. At the lowest doses (threshold, 0.5–5 μg/kg) the drug increased the frequency of spontaneous bladder contractions. In large doses (10–100 μg/kg) the drug produced an initial tonic contraction of bladder lasting 15–40 min followed by a period of high frequency rhythmic activity. Multiunit firing in parasympathetic postganglionic nervs on the surface of the urinary bladder was also enhanced. Bursts of firing which in untreated animals occured during large bladder contractions continously during the entire sustained contraction of the bladder following large doses of naloxone. Various evidence indicates that the site of action of naloxone is in the central nervous system. These findings suggest that the parasympathetic reflex pathway to the urinary bladder may be subject to tonic enkephalinergic inhibitory control.     

Stimulation of the nucleus raphe obscurus produces marked serotonin release into the dorsal medulla of fed but not fasted rats — glutamatergic dependence

Serotonin interacts with TRH at the dorsal vagal complex (DVC) to augment gastric functional parameters. To ascertain physiologic relevance, patterns of stimulated release at the terminal field were characterized. Stimulation of the nucleus raphe obscurus (nRO) by kainic acid (423 pmol/10 nl) produced marked release of serotonin into dorsal medullary dialysates containing the DVC in freely fed, but not 24-h fasted rats. Probe infusion of kynurenic acid (1 mM), but not acute bilateral cervical vagotomy attenuated nRO-stimulated serotonin release in fed animals. The results suggest that the fed state facilitates serotonin release into the dorsal medulla by a mechanism mediated by activation of excitatory amino acid receptors in the dorsal medulla. Enhanced serotonergic neurotransmission at the DVC may comprise a heretofore unrecognized component of the integrated vago-vagal response to a meal.     

GABA and bicuculline actions on mouse spinal cord and cortical neurons in cell culture

The neutral amino acid γ-aminobutyric acid (GABA) produced membrane hyperpolarization and increased membrane chloride ion conductance of spinal cord (SC) and cortical (CTX) neurons in cell culture. GABA dose-response curves were obtained for SC neurons by pressure applying known concentrations of GABA from micropipettes with large tips (miniperfusion pipettes). GABA response threshold was about 2 μM and large responses were elicited at GABA concentrations greater than 10 μM. Bicuculline (BICUC) (0.1–10 μM) reversibly antagonized GABA responses on both SC and CTX neurons with a half maximal inhibitory concentration of about 1 μM. BICUC antagonism of GABA responses was competitive (Lineweaver-Burke analysis). These results are compared with data on GABA and BICUC displacement of [³H]GABA binding to membranes of SC and CTX neurons in cell culture. It is suggested that high affinity GABA receptors are likely to be relevant for postsynaptic GABA responses while low affinity GABA receptors may be presynaptic.     

Manometric artefacts suggesting compression of the duodenum by the superior mesenteric artery in healthy humans

Multi-channel manometry offers the opportunity to study intestinal motor activity with high spatiotemporal resolution. We report tonic and phasic intraluminal pressure changes in the mid-portion of the horizontal part of the duodenum. In 10 healthy volunteers, we recorded 2 h of interdigestive duodenal motility using a water-perfused catheter. The assembly incorporated 12 duodenal sideholes at 1.5-cm intervals (D1-D12). Measurement of the antral and duodenal transmucosal potential difference (TMPD) was used to maintain a correct position of the catheter. The incidence of pressure waves (PWs) increased gradually from proximal (D1) to distal (D12) (P < 0.0001), while the mean amplitude of PWs decreased (P < 0.0001). In eight of 10 subjects, the signals recorded from D9 showed tonic pressure elevations with superimposed phasic pressure changes at heart-rate frequency, comprising 13.8% of total recording time. In the other two subjects, this phenomenon occurred in D8 (9.9% of time). D10 showed a lower incidence of PWs compared with neighbouring sideholes (D6-D9/D11-D12) (P < 0.035), with normal amplitudes. Fluoroscopy was performed in three subjects and showed that D9 was located at the midline. In healthy subjects manometric signals recorded from the horizontal part of the duodenum showed localized artefacts, presumably caused by compression by the superior mesenteric artery. In addition, a 'silent' region was present just distal to this site, the origin of which is uncertain.