Involvement of gastrointestinal mechano- and intestinal chemoreceptors in vagal reflexes: an electrophysiological study

The role of gastrointestinal mechanoreceptors and intestinal chemoreceptors in the genesis of vago-vagal reflexes was assessed by recording single vagal efferent fibre discharge in the urethane-anaesthetized ferret during procedures known to activate discrete populations of gastrointestinal afferent fibres. Distension of the stomach, duodenum and jejunum was used to activate mechanoreceptors while perfusion of the intestinal loops with various chemical solutions was used to activate mucosal chemoreceptors. Mechanical stimulation of the stomach and/or intestine was effective in modulating vagal efferent discharge in 90% of units tested. The response (either excitation or inhibition of efferent firing) was characterized by its short-latency (less than 1 s), slow-adaptation, and rapid return on removal of the stimulus. In contrast, chemical stimulation was much less potent evoking clear-cut responses in only 26 of the 109 efferent units. Luminal HCl was the most effective stimulus accounting for 81% of the efferent responses although these were of long-latency (greater than 1 min), gradual in onset and poorly maintained. Other efferent responses to HCl and hypertonic saline were characterized by a long-latency, sudden increase in discharge associated with the prodrome of vomiting. We conclude that while the mechanosensitive afferent input is well represented in terms of the genesis of vagal reflexes, the chemosensitive afferent input may be more important in behavioural aspects of visceral stimuli like vomiting.     

Responses of the rat lower oesophageal sphincter (LOS) to vagal efferent activation

Nitric oxide (NO) is a major candidate in vagal-induced LOS relaxation. Vagal adrenergic fibres also innervate the gastrointestinal tract including the LOS. This study investigates the role of these two and other mechanisms in LOS responses to vagal activation in the rat, and provides functional and anatomical evidence for a smooth muscle LOS in this species. LOS, gastric and oesophageal pressures were measured in urethane anaesthetized rats during vagal stimulation. The LOS pressure (LOSP) response to vagal stimulation (5 mA, 10 Hz, 0.5 msec pulses, 5 sec) comprised three consecutive stages: (1) brief reduction of LOSP, (2) transient increase of LOSP and (3) prolonged reduction of LOSP. The influences of additive treatment with several antagonist drugs on the LOS response to vagal stimulation were investigated. L-NAME (100 mg kg⁻¹) reduced stage 1 and increased stage 2. Subsequent treatment with either phentolamine (1 mg kg⁻¹) or prazosin (200 μg kg⁻¹) abolished stage 1. After phentolamine, atropine treatment (400 μg kg⁻¹) abolished stage 2. Stage 3 was evident throughout experiments. In five additional studies, treatment with hexamethonium (30 mg kg⁻¹) abolished stages 2 and 3 leaving stage 1, which was later abolished by phentolamine or atropine. In the LOS response to vagal stimulation, the following major mechanisms are therefore evident: nicotinic transmission in both excitation and inhibition, alpha-adrenergic and NO-mediated inhibition, muscarinic excitation, and non-adrenergic, non-NO inhibition (not characterized further). Characteristics of these different neurotransmitter influences may be important in LOS relaxation associated with swallowing and gastro-oesophageal reflux.     

Differential release of vasopressin and oxytocin in response to abdominal vagal afferent stimulation or apomorphine in the ferret

The aim of this study was to investigate whether direct afferent stimulation of the abdominal vagus promote release of the neurohypophyseal hormones. The nucleus of the solitary tract is the major recipient of vagal afferent information, and this region of the brainstem may also be activated by stimulation of the area postrema. For this reason apomorphine, a D₂ dopaminergic agonist which acts on the area postrema, and can evoke vasopressin secretion in man, was also investigated for its effect on vasopressin and oxytocin release. Our results show that vasopressin, but not oxytocin is released in vast amounts in response to electrical afferent stimulation of the abdominal vagus. Administration of apomorphine also evoked a massive vasopressin release with less marked effects on oxytocin. The possible functional implications of these results are discussed especially in the context of nausea and vomiting.     

Responses of hepatic glucose output to electro-acupuncture stimulation of the hindlimb in anaesthetized rats

Responses of hepatic glucose output (HGO) to electro-acupuncture (EA) stimulation of the hindlimb were investigated in anaesthetized rats, focusing on involvement of the somatic afferent and autonomic efferent nerves. HGO was measured with a microdialysis probe implanted into the left lateral lobe of the liver. Stainless steel needles with a diameter of 0.25 mm were inserted into the right tibialis anterior muscle and connected to an electrical stimulator. The EA stimulation was delivered for 10 min at 10 mA, 20 Hz. Atropine was injected in order to block the action of the parasympathetic nerves, whereas phentolamine and propranolol were injected in order to block the action of the sympathetic nerves. Furthermore, adrenal sympathetic nerves were crushed bilaterally to block the reflex secretion of adrenal medullary hormones. The EA stimulation significantly increased HGO for 20 min after the onset of stimulation. The increases of HGO were abolished by severing the femoral and sciatic nerves, demonstrating that the responses are elicited via activation of somatic afferent nerves. Furthermore, the increases were diminished after severance of the adrenal sympathetic nerves, which regulate catecholamine secretion from the adrenal medulla. The increases were totally abolished after pretreatment with phentolamine, an alpha-adrenergic blocker, and propranolol, a beta-adrenergic blocker. On the other hand, the increases of HGO in response to the EA stimulation were augmented after pretreatment with atropine, a muscarinic cholinergic blocker. The present results demonstrate that EA stimulation to a hindlimb can reflexly increase HGO via activation of somatic afferents and, thereby, sympathetic efferents, including sympathetic efferents to the adrenal medulla. The present results further show that the increases of HGO in responses to EA stimulation are simultaneously reflexly inhibited via the parasympathetic nerves.     

Responses in human A and C fibres to repeated electrical intradermal stimulation

The responses of A and C fibres to electrical intradermal stimulation were recorded with microelectrodes inserted percutaneously into intact human skin nerves. Unitary discharges deriving from A fibres were often encountered and sometimes even single C unit deflections were identified. When several C fibres responded to the stimulation, a compact time presentation of the filtered and dot-converted spikes improved the discrimination of individual spikes time-locked to the stimulation, so that unitary C elements could often be identified in the treated records even if the signal-to-noise ratio of the original signals was low. Increases in latency or blockings were traced in both A and C unit responses to repeated excitation, but the influence of repeated activation was more pronounced in thin nerve fibres even at low stimulation frequencies. The decreased excitability of thin nerve endings on repetitive stimulation suggests that not only central factors but also excitation failure in peripheral thin nerve fibres might be responsible for the decrease in pain perception experienced during local intense electrical intradermal stimulation at high frequencies.     

Non-invasive stimulation of vagal afferents reduces gastric frequency

Metabolic feedback between the gut and the brain relayed via the vagus nerve contributes to energy homeostasis. We investigated in healthy adults whether non-invasive stimulation of vagal afferents impacts energy homeostasis via efferent effects on metabolism or digestion. In a randomized crossover design, we applied transcutaneous auricular vagus nerve stimulation (taVNS) while recording efferent metabolic effects using simultaneous electrogastrography (EGG) and indirect calorimetry. We found that taVNS reduced gastric myoelectric frequency (p = .008), but did not alter resting energy expenditure. We conclude that stimulating vagal afferents induces gastric slowing via vagal efferents without acutely affecting net energy expenditure at rest. Collectively, this highlights the potential of taVNS to modulate digestion by activating the dorsal vagal complex. Thus, taVNS-induced changes in gastric frequency are an important peripheral marker of brain stimulation effects.     

Sources of anterior gastric vagal efferent discharge in rats: an electrophysiological study.

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.     

Units in tegmental nuclei responding to stimulation of gastric vagal and greater splanchnic fibers in the cat

The response of neurons in the ventral and dorsal tegmental nuclei during electrical stimulation of the gastric vagal fibers which serve the proximal stomach and the left greater splanchnic fibers were evaluated in chloralose-anesthetized cats. The mean latency of 181 gastric vagally evoked unitary responses recorded in the tegmental nuclei was 352.2 ms, whereas the latency of the left greater splanchnic-evoked tegmental response was significantly less (63.2 ms). The unitary responses to the gastric vagal and greater splanchnic fibers stimulation were bilaterally distributed in the ventral and dorsal tegmental nuclei. Convergence of the gastric vagal input from the proximal stomach and the left greater splanchnic input was observed in 151 units (83 percent). Stimulation of the greater splanchnic nerve usually resulted in a short latency excitation followed by an inhibitory effect on gastric vagally evoked responses. The results suggested that some convergent splanchnic inhibition of gastric vagally evoked responses was mediated via an interneuron. Projections from the nucleus tractus solitarius and the parabrachial nucleus to the tegmental nuclei were also identified electrophysiologically by direct microstimulation of the two former areas. The significant number of gastric vagal and splanchnic evoked unitary responses recorded in the ventral and dorsal tegmental nuclei suggested that they may serve as an important pontine site for processing of visceral information between the nucleus tractus solitarius and forebrain sites.     

Responses of inspiratory neurons of the dorsal respiratory group to stimulation of expiratory muscle and vagal afferents

In decerebrate, paralyzed and ventilated cats, we monitored the intracellular responses of 30 inspiratory neurons of the dorsal respiratory group (DRG) to stimulation of vagal and expiratory muscle (internal intercostal and abdominal) afferents. We hypothesized that the inhibitory effects of stimulation of expiratory muscle afferents, previously reported, would block the excitatory responses of inspiratory neurons of the DRG to vagal stimulation. Although prolonged stimulus trains to expiratory muscle afferents caused respiratory phase-switching, single shocks or short trains elicited no responses in 17 bulbospinal neurons, excitatory responses in 6, and inhibitory responses in 2. Of the 4 propriobulbar neurons tested, 2 had inhibitory responses and 2 did not respond. In only 2 neurons, both bulbospinal, did conditioning stimuli to expiratory muscle afferents block or reduce the excitatory effects of vagal stimulation. These results suggest that interaction of vagal and expiratory muscle afferents, which might account for the absence of a change in inspiratory duration despite increased vagal afferent feedback at elevated end-expiratory lung volumes, does not occur within the DRG.     

Localization and acetylcholinesterase content of vagal efferent neurons

The acetylcholinesterase (AChE) content of rat vagal efferent neurons was studied. Retrograde transport of horseradish peroxidase (HRP) by cut vagal axons provided a means for localizing efferent cell bodies; tissue sections were then processed for the simultaneous visualization of HRP and AChE. A dorsal vagal efferent column contained the dorsal motor nucleus of the vagus, as a primary component, and extended caudally into the upper cervical spinal cord. A ventral column contained neurons in the nucleus ambiguus and the surrounding reticular formation. Although most of the vagal efferent neurons stained with moderate to heave intensity for AChE there were some HRP-labeled cells that contained little AChE and a small percentage in which AChE was absent. In spite of the fact that AChE has been demonstrated in certain non-cholinergic neurons, it has also been found in all cholinergic neurons. Therefore, the presence of AChE has been regarded as a necessary (but not sufficient) component for identifying cholinergic neurons. The absence of AChE in a small percentage of the vagal efferent neurons indicates that some preganglionic parasympathetic fibers in the vagus nerve are not cholinergic.     

Loss of efferent vagal activity in acute schizophrenia

An increased heart rate has been reported in schizophrenia. It is unclear whether this is due to disease-related autonomic nervous system changes or a side effect of antipsychotic medication. Altered cardiac autonomic function might to some extent account for the elevated cardiovascular mortality rate of schizophrenic patients. We assessed heart rate variability (HRV) in 30 acute unmedicated schizophrenic patients and matched controls. Patients were re-investigated 2--4 days after initiation of treatment to assess effects of medication. Our study demonstrates that non-medicated schizophrenic patients were significantly different in heart rate (increase) and parasympathetic parameters (loss of efferent vagal activity) at rest and during deep respiration. No significant effect was found after initiation of neuroleptic therapy. We found a correlation between duration of disease and parasympathetic parameters as well as very low frequency power (VLF) and delusion (SAPS subscale). These data suggest that schizophrenia is accompanied by a loss of vagal efferent activity, probably due to disturbed cortical-subcortical circuits modulating the autonomic nervous system in acute psychosis. The definite mechanisms by which vagal activity might be suppressed in schizophrenia are unknown. Parasympathetic hypoactivity might increase the risk for sudden cardiac death and arrhythmias in this disease. Future studies are warranted to investigate the interaction between cardiac autonomic function and schizophrenia and to identify patients on risk.     

The effect of selective electrical stimulation of non-myelinated vagal fibres on heart rate in the rabbit

The bradycardia evoked by electrical stimulation of the peripheral cut end of the rabbit vagus nerve is mediated by both myelinated and non-myelinated fibres. The purpose of this study was to assess the effects of non-myelinated fibres on heart rate in the rabbit using selective electrical stimulation techniques. In 8 rabbits selective activation of non-myelinated fibres using reversed polarity triangular shaped pulses (10 Hz, 20 s), resulted in a slowly developing fall in heart rate of 24.1 +/- 1.1 beats/min which outlasted the period of stimulation by 58.4 +/- 4.2 s. In 4 rabbits stimulation of myelinated fibres at 10 Hz for 20 s resulted in a fall in heart rate of 24.5 +/- 2.6 beats/min. On stimulation of both myelinated and non-myelinated fibres heart rate fell by 39.9 +/- 3.2 beats/min. Heart rate returned rapidly to control value following stimulation of myelinated fibres (5.6 +/- 0.5 s) but only slowly after stimulation of both myelinated and non-myelinated fibres (56.7 +/- 4.9 s). Atropine (5 mg/kg, i.v.) abolished all effects of vagal stimulation on heart rate. Hexamethonium (15 mg/kg, i.v.) abolished the effect of myelinated fibres on heart rate but did not affect the fall in heart rate produced by non-myelinated fibres. We suggest that the prolonged effects on stimulation of non-myelinated fibres may reflect the persistent action of a non-cholinergic excitatory transmitter at the cardiac parasympathetic ganglia.     

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.     

Role of endogenous bombesin-peptides during vagal stimulation of gastric acid secretion in the rat

The stimulatory effect of exogenous bombesin and its related mammalian peptides on gastric acid secretion and gastrin release has been examined in detail, while the regulatory role of endogenously released bombesin-like peptides is largely unknown. Accordingly we have determined the effect of a specific bombesin receptor antagonist during vagal stimulation of gastric acid secretion and gastrin release.In anesthetized rats electrical stimulation of the vagal nerves (10 V, 10 Hz, 1 ms) significantly increased plasma gastrin levels by 82 ± 11 pg/20 min (P < 0.01) and gastric acid output by 99.4 ± 9.9 μeq/20 min (P < 0.01). Intravenous infusion of the specific bombesin receptor antagonist d-Phe⁶-BN(6–13)OMe (400 nmol/kg/h) significantly reduced vagally induced increase of plasma gastrin levels by 70% to 29 ± 8 pg/20 min (P < 0.05 vs control) and vagally stimulated gastric acid output by 40% to 57.4 ± 10.6 μeq/20 min (P < 0.05 vs control). To demonstrate that the residual gastrin and acid response is due to non-bombesinergic mechanisms and not to an inadequate dose of the receptor antagonist, the latter was tested against gastrin-releasing peptide (GRP) at the maximally effective concentration of 300 pmol/kg/h, which resulted in an even 50% higher increase of plasma gastrin levels compared to vagal stimulation. The dose of the antagonist employed (400 nmol/kg/h) was sufficient to abolish GRP-induced stimulation of gastrin and gastric acid secretion.Previously it has been postulated that endogenous bombesin-peptides can stimulate acid secretion via gastrin-independent mechanisms. To investigate this possibility further the effect of the antagonist was examined on vagally induced acid secretion while gastrin levels were restored to the range of the respective control experiments. In presence of the antagonist the infusion of gastrin-17 (15 pmol/kg/h) in addition to vagal stimulation elevated plasma gastrin to levels not different from those during vagal stimulation alone. With identical plasma gastrin levels the bombesin receptor antagonist had no effect on vagally stimulated acid secretion (86.3 ± 10.7 μeq/20 min vs 99.4 ± 9.9 μeq/20 min in the controls; n.s.).In conclusion, the present data demonstrate for the first time that in rats in vivo endogenous bombesin peptides contribute to vagal stimulation of gastrin release and gastric acid secretion. Furthermore, endogenous bombesin-peptides exert their action on parietal cell function via an increase of gastrin release, while non-gastrinergic mechanisms are unimportant under the experimental conditions employed.     

Collaterals of recurrent laryngeal nerve fibres innervate the thymus: a fluorescent tracer and HRP investigation of efferent vagal neurons in the rat brainstem

The origin and course of efferent vagal fibers, which innervate the rat thymus, were investigated by a fluorescent retrograde double labeling method, using Fast blue (FB) and Diamidino yellow dihydrochloride (DY) as tracers. In the same animal, one tracer was injected into the cranial portion of the right lobe of the thymus and the other dye was deposited around the cut end of the right recurrent laryngeal nerve. The neuronal population giving origin to the recurrent nerve was mapped by using retrograde labeling with HRP applied to the central stump of the nerve. The HRP retrograde axonal transport showed that most efferent vagal fibers of the recurrent nerve have their perikarya in the nucleus retroambigualis (NRA), nucleus ambiguus (NA), and to a lesser extent in the nucleus retrofacialis (NRF). In fluorescent retrograde double labeling of thymus and recurrent laryngeal nerve both single and double labeled cells were found. The cells labeled by the injections into the thymus were colocalized with the neurons labeled by the tracer deposited in the recurrent laryngeal nerve to the NRA, NA, and NRF. Moreover along the rostrocaudal extent of the NRF and NA double labeled cells were present, showing that some of the thymic efferents are collaterals of the recurrent nerve fibers. Our experiments shown that some thymic vagal fibres originate from neurons of nucleus dorsalis nervi vagi (NDV) as demonstrated both by HRP and FB injected thymuses. The possible role of these efferents in thymic function is briefly discussed.     

Parabrachial nucleus: neuronal evoked responses to gastric vagal and greater splanchnic nerve stimulation.

Unitary responses were recorded extracellularly in the parabrachial nucleus (PBN) in anesthetized cats during electrical stimulation of the 1) gastric branches of the ventral and dorsal vagal trunks which serve the proximal stomach, and 2) left greater splanchnic nerve. The gastric vagally evoked parabrachial responses consisted of phasic single and multiple spike orthodromic discharges, which were bilaterally distributed, with a mean latency of 349 ms (S.D. +/- 38.5). The parabrachial-evoked splanchnic unitary responses had a much shorter latency with a bimodal distribution (mean latencies, 53 and 128 ms, respectively). Convergence of gastric vagal input from the proximal stomach and the left greater splanchnic nerve upon single neurons in the PBN was electrophysiologically demonstrated in 132 units. Eighty-seven percent of the gastric vagally evoked parabrachial unitary responses were inhibited by simultaneous electrical stimulation of the splanchnic nerve. The condition-test paradigm was used to evaluate the time course of the splanchnic inhibition of the gastric vagally evoked parabrachial response. Reciprocal connections between neuronal populations in the nucleus tractus solitarius (NTS) which received gastric vagal input and the PBN were also identified electrophysiologically by direct microstimulation of the former structure. The density and characteristics of the gastric vagal and greater splanchnic input to the PBN suggested that this nucleus receives and processes a substantial amount of visceral afferent input. The PBN may serve as an important site for integrating visceral information governing the proximal stomach and ingestive processes.     

Haemodynamic effects of withdrawal of efferent cervical vagal stimulation on anesthetized dogs--relative importance of chronotropic and non-chronotropic mechanisms

The aim of the present work was to study changes in cardiac output (CO) and arterial blood pressure (ABP) following either interruption of artificial efferent vagal stimulations (STOP), or suppression of negative chronotropic effects, during uninterrupted vagal stimulations (PACE). Experiments were performed on 7 anesthetized, open-chest dogs. A computerized data acquisition system was used to record CO (electromagnetic flowmeter), ABP, right atrial pressure and electrocardiogram; 9 parameters were automatically elaborated. The peripheral stumps of both vagus nerves, sectioned at the neck, were stimulated for long control periods (at least 3 min) with brief trains of stimuli triggered by atrial P waves. Records were started during steady-state vagal stimulations, and consisted of paired trials: in the first step the vagal stimulators were turned off (STOP); in the second step the heart was paced at the same rate reached at the end of the preceding step, but vagal stimulation was continued (PACE). Observations lasted two min after each step. Results indicate rapid rise in CO and ABP after STOP, up to 30% and 10%, respectively, in 10 s, followed by slow reduction in CO and further increase in ABP (22% and 15%, respectively, at 120 s). Thus STOP caused rapid and sustained improvements in the cardiac performance. After PACE changes in CO and ABP were smaller and followed a slower time-course. The greater effects of STOP with respect to PACE were attributed to non-chronotropic mechanisms, accounting for about 50% of the overall haemodynamic consequences of vagal withdrawal. Since peak aortic flow velocity and acceleration were increased after STOP, stroke volume was reduced much less than after PACE, despite equal rise in heart rate, and similar shortening in the ejection time. Evidence was presented of enhanced atrial and ventricular contractility after STOP. Experiments performed after beta-blockade in 5 dogs substantially confirmed the results. It is concluded that vagal withdrawal, which is an important aspect in many physiological situations, constitutes a rather powerful strategy for rapid enhancement of the cardiovascular performance, through different mechanisms, in addition to cardioacceleration.     

Expression of c-fos and NGFI-A messenger RNA in the medulla oblongata of the anaesthetized rat following stimulation of vagal and cardiovascular afferents.

Messenger RNA encoding the immediate early genes (IEGs) c-fos and NGFI-A was localized by in situ hybridization of specific 35S-labelled oligonucleotides to detect activated neurones in the medulla oblongata following unilateral electrical stimulation of the vagus (nX) and aortic depressor nerve (ADN), and following mechanical stimulation of the left carotid sinus (CS). In electrically stimulated rats, c-fos and NGFI-A mRNA was strongly expressed in the nucleus tractus solitarius (NTS) (predominantly ipsilaterally), area postrema (AP) and in a dorsal subregion of the paratrigeminal nucleus (PTN). Lower levels of c-fos and NGFI-A mRNA were seen in the ipsilateral NTS and PTN following mechanical stimulation of the left CS. In general these data correlate with the topography of innervation by the different nerve afferents, although the expression in the PTN (and in some cases the AP) would not be predicted on the basis of neuronal innervation patterns reported for the rat. Expression of these IEGs also occurred in the rostral and caudal ventrolateral medulla and inferior olive of both stimulated and sham-operated rats; presumably due to effects of the anaesthesia and surgical procedures. In conclusion the localization of the expression of c-fos and NGFI-A mRNAs represents a useful neuroanatomical technique for detecting the cell bodies of neurones that are activated by cardiovascular nerve afferents and should allow the further characterization of the neurochemical identity of these neurones.