Clinical homeopathic use of RNA: evidence from two provings.

Two homeopathic drug provings (HDPs) of RNA have been published. The methods are reviewed. Our method is double-blind and placebo-controlled, conforms to GCP and includes run-in and follow-up phases. Symptoms are selected from diaries according to predefined criteria. The results of the provings are compared with each other and with the homeopathic clinical use of RNA. At this stage it is difficult to draw conclusions concerning the clinical use of RNA. Further HDPs are required, they should follow GCP and standard, transparent protocols.

Does the vagus nerve mediate the sixth sense?

Can sensations originating from the internal environment modulate attitude and behaviour? Can the feedback about the operation of the viscera provide a calming and relaxing influence? Information from the chest and abdomen is delivered continuously by the vagus nerve, the largest visceral sensory nerve in the body. Because various 'stress-related' diseases can be associated with impaired functions in sensory vagal fibres, a better understanding of how sensory vagal information is processed in the CNS might offer new strategies for the treatment and/or prevention of several disorders, including 'drug-resistant' forms of eating disorder, anxiety, chronic depression and epilepsy. A neuronal circuitry that has been suggested by experimental data to mediate sensory vagal inputs to those brain areas that are involved in the generation of 'stress-related' disorders is outlined.

Sciatic and vagal sensory inputs converge onto non-baroreceptive neurones of the rostral ventrolateral medulla.

Previous data suggested that somatic and vagal sensory afferent inputs may converge in the rostral ventrolateral medulla oblongata (RVLM). The aim of the present study was to establish the existence of convergence between inputs mediated via the cervical vagus and contralateral sciatic nerves using in vivo intracellular recordings. The majority of RVLM neurones that received input from the vagus or the sciatic nerves also responded to stimulation of the other nerve. In 72% of the neurones the response was excitation or inhibition to stimulation of both nerves, respectively. The most frequent response type was a short excitation in response to stimulation of both nerves. Only 8% of the neurones exhibited a visible response to stimulation of the aortic depressor nerve. The results provided experimental evidence that non-baroreceptive neurones of the RVLM are involved in coordination of somatic and visceral sensory inputs.

Synaptic interactions in neurones of the rat rostral ventrolateral medulla oblongata.

Increasing number of studies indicate that stimulation of peripheral nerves elicits complex postsynaptic responses in neurones of the ventral medulla oblongata. The present study describes a recommended protocol for intracellular recording of complex postsynaptic events in neurones of the ventral medulla oblongata. The aim was to provide surgical and experimental details that will enable successful recording of slow synaptic responses in vivo, in addition to the recording of fast evoked responses. The existence of slow inhibitory responses to stimulation of the cervical vagus and sciatic nerves have already been demonstrated together with the existence of convergent visceral and viscero-somatic inputs to neurones here. The data collected in over 200 neurones so far indicated that neurones of the rostral ventrolateral medulla oblongata play an important and versatile role in the integration of somato-visceral sensory inputs.

Gating of vagal inputs by sciatic afferents in nonspinally projecting neurons in the rat rostral ventrolateral medulla oblongata.

Integration and coordination of somato-visceral sensory information is crucial to achieve adaptive behavioural responses. We have recently shown that sensory vagal and somato-sensory (sciatic nerve) inputs converge in neurons of the rostral ventrolateral medulla oblongata, which was implicated in adjusting visceral activities to changing somatic performances. In the present study, the neuronal mechanism of interaction between sciatic and vagal sensory inputs was examined in the rostral ventrolateral medulla oblongata using in vivo intracellular recording and labelling. Conditioning stimulation of the contralateral sciatic nerve (2 V) led to a time-dependent inhibition of responses to vagal stimulation (100 microA) in each RVLM neuron that received convergent sciatic and vagal sensory inputs (n = 50). None of these neurons had direct spinal projections, and only 8% of them exhibited a visible response to stimulation of the aortic depressor nerve. A significant attenuation of the amplitude of vagal test responses was present for up to 800 ms of conditioning delay, although the duration of this sciatico-vagal inhibition was greatly dependent on the intensity of both stimuli. The electrophysiological data indicated that sciatico-vagal inhibition is mediated presynaptically, via activation of GABAB receptors. Morphological evidence of axo-axonic interactions that may underlie sciatico-vagal inhibition was subsequently found in the electron microscope. It is suggested that during movements of the hindleg, activation of sciatic sensory fibres leads to re-patterning of neuronal activity in RVLM neurons via inhibition of visceral sensory inputs. Sciatico-vagal inhibition is likely to affect the activity of those RVLM neurons that modulate higher neuronal activities via ascending projections.

Slow hyperpolarization in cortical neurons: a possible mechanism behind vagus nerve simulation therapy for refractory epilepsy?

PURPOSE: Recent studies have shown that chronic, intermittent stimulation of the left vagus nerve (VNS) decreases the frequency, duration, and/or intensity of seizures in some patients with medically refractory focal seizures. Although VNS is being used in an increasing number of patients, the neuronal mechanism behind VNS therapy of refractory epileptic seizures is yet unclear. METHODS: In vivo intracellular recordings were used to study responses elicited by the VNS in pyramidal neurons of the parietal association cortex in anesthetized rats. RESULTS: Low-intensity trains of VNS, which activated predominantly myelinated fibers (100 microA, 30 Hz, 0.5 millisecond, 20 seconds), elicited a slow hyperpolarization (onset latency 17.4 +/- 2.0 seconds, amplitude -4. 7 +/- 0.6 mV, duration 35 +/- 3.2 seconds; n = 19). Increasing the intensity of VNS to recruit nonmyelinated vagal fibers (200 microA) led to an increase in the magnitude of the response in some neurons while failed to evoke a response in others. On increasing the stimulus intensity to 500 microA, only one in nine neurons exhibited a visible response. All recorded and visualised neurons were pyramidal cells in cortical layer V. CONCLUSIONS: Stimulus intensities that activate predominantly myelinated fibers (less than 200 microA) were most effective to induce slow vagal hyperpolarization. It is suggested that slow hyperpolarization may be one of the mechanisms that underlie the seizure-reducing effect of VNS, by means of reducing the excitability in neurons that would be involved in propagation of seizure activity. As the balance of activity in myelinated and nonmyelinated primary vagal afferents influenced the effect of VNS stimulation, it is likely that the effect of VNS is modulated as changes occur in the underlying vagal tone.

Activation of cardiac vagal afferents facilitates late vagal inhibition in neurones of the rostral ventrolateral medulla oblongata bilaterally.

A reduced activity of cardiac vagal afferent fibres is considered as one of the pathophysiological causes of post-infarction complications [A. Head, Baroreflexes and cardiovascular regulation in hypertension. J. Cardiovasc. Pharmacol. 26 (1995) S7-S16]. The mechanism of how a reduction of cardiac vagal activity leads to enhanced sympathetic drive and systemic hypertension is however not yet clear. Experimental data have shown that the rostral ventrolateral medulla oblongata (RVLM) plays an important role in tonic blood pressure regulation, the control of sympathetic vasoconstriction and cardiac performance. The aim of the study was to determine whether activation of cardiac vagal afferents contributes to eliciting the long-lasting late inhibition that we have previously shown to occur in neurones of the RVLM [A. Zagon, K. Ishizuka, I. Rocha, K.M. Spyer, Late vagal inhibition in neurones of the ventrolateral medulla oblongata in the rat. Neurosci. 92 (1999) 877-888]. The experiments were carried out in terminally anaesthetised and artificially ventilated rats using in vivo intracellular recordings. The data confirmed that late vagal inhibition is elicited by cumulative activation of functionally different vagal afferents, including those that originate from cardiac receptors. It was also demonstrated that activation of cardiac afferents could lead to a significant increase in the duration of this long-lasting late response component. Facilitation of late vagal inhibition was observed in RVLM neurones both ipsi- and contralateral to the stimulated nerve. It is suggested that such facilitation of late vagal inhibition may be a mechanism of how pulse-synchronous activation of cardiac afferents leads to a tonic modulation of the activity of RVLM neurones. An attenuation of late vagal inhibition during reduced activity of cardiac vagal afferents could lead to enhanced excitability in these neurones which in turn can lead to an increase in medullary sympathetic outflows towards the heart and peripheral blood vessels.

Late vagal inhibition in neurons of the ventrolateral medulla oblongata in the rat.

Stimulation of cervical vagal afferents elicits long-lasting inhibitory effects in a variety of neuronal populations, although little is known concerning the cellular mechanisms that are involved in these effects. In the present study, the electrophysiological characteristics of responses elicited by cumulative activation of vagal afferents were examined in neurons of the rostral ventrolateral medulla oblongata, which play an important role in the coordination of cardiovascular and other visceral activities. The study has focused on the late-onset, slow inhibitory component of vagal responses, which is likely to affect the temporal modulation of postsynaptic effects. Vagal stimulation elicited four distinct response patterns in intracellularly penetrated neurons (n = 78): excitation, inhibition, excitation-inhibition and inhibition-inhibition. The late inhibitory component was encountered in 43 (55%) of the cells, including five putative medullospinal neurons. It was due to a postsynaptic hyperpolarization which reversed at potentials more negative than -83 mV. The voltage dependency, as well as the average onset latency (93+/-3.0 ms), duration (270+/-16.5 ms) and amplitude (1.3+/-0.2 mV as measured at resting membrane potentials), of late inhibition were clearly different from those of the short-latency inhibitory response. The differences in the voltage dependency and time-course of the short-latency responses and the late inhibition indicate that they are mediated by different central relays. In the majority of neurons, late inhibition could be elicited by stimulating only myelinated vagal afferents. The magnitude of the response was, however, significantly enhanced in 63% of the examined cells when the intensity of stimulation was raised to recruit further myelinated and non-myelinated fibres. This indicates that late vagal inhibition is often elicited by a cumulative activation of convergent afferent inputs. The intracellularly labelled vagally responsive neurons were present at all rostrocaudal levels of the rostral ventrolateral medulla, with an accumulation in the region of the lateral paragigantocellular nucleus. Neurons that exhibited late vagal inhibition were dominant in the juxtafacial region of this nucleus. Due to its slow time-course, late vagal inhibition may contribute to a tonic modulation of the activity of neurons in the rostral ventrolateral medulla oblongata. It is proposed that late vagal inhibition plays an important role in the temporal integration of sensory inputs in neurons of the rostral ventrolateral medulla oblongata. The time-course and strength of this modulatory effect are related to the level of activity in those visceral sensory inputs that converge onto the inhibitory interneurons that mediate late inhibition to rostral ventrolateral medulla oblongata neurons.

Vagal modulation of responses elicited by stimulation of the aortic depressor nerve in neurons of the rostral ventrolateral medulla oblongata in the rat.

Stimulation of cervical vagal afferents inhibits central sympathetic outflows in part by inhibiting the ongoing activity of putative baroreceptive neurons in the rostral ventrolateral medulla oblongata. The aim of the present study was to examine the electrophysiological characteristics of vagal responses and their interactions with responses elicited by stimulation of the aortic nerve in neurons there. The study focused on the role of the long-lasting, late-onset vagal inhibition, which is likely to play an important role in the tonic inhibitory effects of vagal afferent stimulation. In vivo intracellular recordings were obtained from 33 neurons that received convergent inputs from aortic and vagal afferents. Sixty-four percent of these neurons exhibited a late inhibition following electrical stimulation of myelinated vagal afferents (mean onset latency of 100+/-5 ms). The average duration of late inhibition (294+/-19 ms) exceeded the duration of the cardiac cycle. As a consequence of this, sustained vagal stimulation diminished the effect of rhythmic baroreceptor inputs in neurons that exhibited late vagal inhibition. Simultaneous activation of aortic and vagal afferents significantly increased the magnitude of late inhibition, even in those neurons where stimulation of the aortic nerve alone did not elicit a response (n = 15). This suggested that the convergence between vagal and aortic afferent inputs occurred in inhibitory inteneurons antecedent to the recorded rostral ventrolateral medulla oblongata neurons. Focal stimulation of the caudal part of the nucleus of the solitary tract also elicited a late-onset inhibition in 73% of the neurons that responded to stimulation of the aortic nerve. This inhibition appeared to be similar to the late vagal inhibition, except for its shorter average onset latency (64+/-7 ms). Based on this observation, it is proposed that inhibitory inteneurons that mediate late inhibition to rostral ventrolateral medulla oblongata neurons may lie within the caudal part of the nucleus of the solitary tract. The present study established that activation of myelinated vagal afferents exerts a complex modulation over the ongoing and evoked activity of neurons that respond to stimulation of the aortic nerve. The complex interaction that occurs between aortic and vagal inputs in neurons of the rostral ventrolateral medulla may be implicated in long-term modulation of sympathetic outflows in response to changes in the activation of visceral receptors supplied by vagus afferents. The modulation elicited by late vagal inhibition may help to adjust cardiovascular outflows according to requirements set by the thoraco-abdominal visceral environment.

Intrinsic membrane characteristics distinguish two subsets of nociceptive modulatory neurons in rat RVM.

Pain modulating neurons of the rostral ventromedial medulla (RVM) include three physiologically distinct classes of neurons in intact, anesthetized animals: and cells that change their activity before the onset of withdrawal reflexes and cells, which have activity unrelated to withdrawal reflexes. A previous in vitro intracellular study demonstrated that the RVM contains two types of neurons that are distinguished by their action-potential characteristics. The present in vivo intracellular study examined whether these intracellularly recorded action-potential characteristics are correlated with the physiological response properties of RVM neurons recorded. RVM neurons exhibited two distinct types of action potentials in vivo. Fast-spike (FS) neurons (n = 30) had short-duration action potentials (0.27 +/- 0.02 (SE) ms at half amplitude) and biphasic afterhyperpolarizations with a characteristic rapid overshooting spike repolarization. Slow-spike (SS) neurons (n = 25) had longer duration action potentials (0.44 +/- 0.02 ms at half-amplitude) due to a slower-spike repolarization rate and monophasic afterhyperpolarization. and cell classes included both FS and SS neurons. FS and neurons had an early onset response to noxious heat stimulation. SS and cells showed a delayed onset response to noxious heat. cells (n = 13) were all SS cells. Among the SS neurons, only cells had action potentials longer than 0. 45 ms (n = 9). FS and SS neurons were intermingled throughout the RVM. The majority of intracellularly labeled cells (n = 15) had fusiform somata with two to five fine caliber primary dendrites and a predominantly mediolateral orientation of the long axis of their dendritic tree. All labeled FS cells (n = 5) had large, multipolar somata with four to nine large caliber primary dendrites. The present study defines in vivo membrane and morphological characteristics of RVM neurons that correlate with physiological differences and may be used for identification of nociceptive modulatory RVM neurons in slice preparations.

Stimulation of aortic nerve evokes three different response patterns in neurons of rostral VLM of the rat.

Because the aortic nerve of the rat is believed to contain only barosensory fibers in a functionally significant number, stimulation of this nerve provides a convenient means to identify barosensitive neurons in the central nervous system. The aim of the present study was to describe the characteristics of responses to stimulation of the aortic nerve in the neurons of the ventrolateral medulla oblongata (VLM) from in vivo intracellular recordings. Data were obtained from 25 neurons that were either spontaneously active and exhibited pulse-synchronous discharge or were silent. Cells that showed a regular increase in their discharge rate during diastole responded with either an inhibitory postsynaptic potential (IPSP, group 1; n = 3) or a biphasic excitatory postsynaptic potential (EPSP)-IPSP (group 2; n = 10) to aortic nerve stimulation. Parameters of the inhibitory response were similar in both groups (27 +/- 2 and 31 +/- 2 ms for onset latency and 55 +/- 10 and 67 +/- 7 ms in duration, respectively). In group 2 cells, the inhibition was, however, preceded by a brief excitation (14 +/- 2 ms for onset latency and 13 +/- 1 ms in duration). Group 3 neurons, which showed a regular increase in their discharge rate during or before systole (n = 7), responded with an EPSP (20 +/- 2 ms for onset latency and 44 +/- 5 ms in average duration). Group 1 and 3 responses were also encountered in quiescent neurons (n = 5). Intracellular labeling revealed that neurons of all three types were localized within the rostral VLM. The functional significance of these findings is discussed.

Efferent connections from the anterior pretectal nucleus to the diencephalon and mesencephalon in the rat.

The anterior pretectal nucleus has been described as part of the visual pretectal complex. However, several electrophysiological and behavioural studies showed that this area is involved in somatosensory modulation, more specifically, antinociception. The efferents of the anterior pretectal nucleus have not been identified taking into account the different function of this nucleus in relation to the rest of the pretectal complex. In the study herein described, a sensitive anterograde tracer Phaseolus vulgaris leucoagglutinin was used to trace the mesencephalic and diencephalic efferents of the anterior pretectal nucleus in the rat. The majority of the connections were ipsilateral. Fibres with varicosities were observed in discrete areas of the thalamus (central lateral, posterior complex), hypothalamus (lateral, posterior and ventromedial), zona incerta, parvocellular red nucleus, intermediate and deep layers of the superior colliculus, central grey, deep mesencephalon, pontine parabrachial region, and pontine nuclei. Fibres en passant were detected in the medial lemniscus, from the level of the injection site to rostral medullary levels. Some labelled axons were seen coursing to the contralateral side through the posterior commissure and the decussation of the superior cerebellar peduncle. These results show that the anterior pretectal nucleus projects principally to areas involved in somatosensory and motor control in a manner that permits sensory modulation at higher and lower levels of the brain. These connections may explain the antinociceptive and antiaversive effects of stimulating the anterior pretectal nucleus in freely moving animals.

Internal connections in the rostral ventromedial medulla of the rat.

Physiological and pharmacological data suggest that the rostral ventromedial medulla (RVM) is an important site where integration between somatic and visceral functions might occur. The aim of the present study was to describe the interconnections between various nuclei of the rostral ventromedial medulla and thus reveal the possible anatomical basis for such functional interactions. The topography of anterogradely labelled internal projections was examined following iontophoretic microinjections of Phaseolus vulgaris leucoagglutinin (PHA-L). The results revealed that the nuclei of the rostral ventromedial medulla have strong interconnections and, to varying degrees, they also have bilateral projections into the rostral ventrolateral medulla. A particularly dense projection to widespread regions of the ventral medulla was traced from the raphe obscurus. Terminals, originating from the raphe pallidus were similarly dispersed but very low density in comparison. The focus of the projections of the gigantocellular nucleus pars ventralis and pars alpha shifted from the lateral paragigantocellular nucleus towards the RVM in rostral direction. Connections from the raphe magnus were altogether restricted to the RVM and the medial aspects of the lateral paragigantocellular nucleus. The diffuse and dense intramedullary connections of the raphe obscurus suggest that it might have an important role in coordinating the activity of rostral ventral medullary cells. The raphe pallidus and the ventral gigantocellular nuclei, areas that were innervated from widespread regions of the rostral ventral medulla but gave only limited projections there, are more likely to be involved in the direct descending control of spinal activities.

Direct projections from the anterior pretectal nucleus to the ventral medulla oblongata in rats.

The anterior pretectal nucleus has recently been implicated in the descending modulation of nociception. Electrical stimulation of the nucleus was found to reduce the nociceptive responses of deep dorsal horn neurons and to inhibit spinally integrated withdrawal reflexes. It is believed that at least part of the descending inhibitory effects of the anterior pretectal nucleus are mediated by reticulospinal cells of the ventrolateral medulla. The purpose of the present study was to trace the direct medullary projections of the anterior pretectal nucleus, to describe their topographical organization and to reveal the chemical nature of some of their putative target cells. The connections were studied using anterograde tract-tracing with Phaseolus vulgaris leucoagglutinin. Direct projections from the anterior pretectal nucleus to the ipsilateral rostral ventral medulla were found in all cases. A dense innervation of the dorsal inferior olive, the gigantocellular reticular nucleus pars ventralis and pars alpha and the ventral pontine reticular nucleus was found from all aspects of the anterior pretectal nucleus. Descending labelled terminals were also observed in the gigantocellular reticular nucleus proper and, laterally, in the lateral paragigantocellular nucleus and in the region of the A5 noradrenergic cell group. A relatively lower density of labelled terminals was noted in the medullary raphe nuclei and in the rostroventrolateral reticular nucleus. Following tract-tracer injections into five distinct subregions of the anterior pretectal nucleus, the topographical organization of the projection was examined and the relatively highest density and most widespread projection was found to originate from the caudoventral part of the anterior pretectal nucleus. A combined tract-tracing and immunolabelling study revealed that some of the descending, labelled terminals were in close proximity of tyrosine hydroxylase-immunoreactive dendrites in the C1 and A5 cell groups. Some labelled fibres were also noted among the serotonin-immunoreactive cells in the lateral extension of the B3 cell population. The existence of direct projections to the ventral medulla and pons correlates well with physiological data which showed that the descending, antinociceptive effects of the anterior pretectal nucleus are relayed via the rostral ventrolateral medulla. The data are also in keeping with pharmacological studies that suggested the role of catecholaminergic cells in the mediation of these descending effects. It is proposed that the rostral ventral medullary projections provide a path through which antinociceptive effects of the anterior pretectal nucleus are mediated to the spinal cord.

Direct projections from the ventrolateral medulla oblongata to the limbic forebrain: anterograde and retrograde tract-tracing studies in the rat.

Neurons in the ventrolateral medulla oblongata, a brain region implicated in central vasomotor regulation, have previously been reported to project to some forebrain limbic structures. The aim of the present study was (1) to describe the termination pattern of ventral medullary afferents in forebrain limbic areas using anterograde tract tracing, and (2) to determine the location and some morphological characteristics of the projection neurons using retrograde tract tracing from selected forebrain sites. Following ionophoretic microinjections of the anterograde tract tracer Phaseolus vulgaris leucoagglutinin into the rostral ventrolateral medulla, labelled afferents were observed in the hippocampus, entorhinal and retrosplenial cortices, dorsal septum, nucleus accumbens, and the medial prefrontal cortex. Anterogradely labelled axons, ascending from the caudal ventrolateral medulla, could be traced only to the rostral aspects of the investigated forebrain limbic structures. Here, the main target of the ascending projection was in the ventral septum. However, labelled terminals were also present in the nucleus accumbens, the dorsolateral septum, and in the infralimbic cortex. The density of the ventrolateral medullary projections into all examined forebrain areas was low. The location of the cells in the ventral medulla oblongata which give rise to direct forebrain projections was examined using retrograde tract tracing with wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP). Following WGA-HRP injections into the septo-accumbens region, retrogradely labelled cells were present in both the rostral and caudal ventrolateral medulla. When the tract tracer injection was restricted to the ventral region of the septal complex, the labelled cells were concentrated in the caudal aspects of the ventrolateral medulla (and the nucleus of the solitary tract). Following tracer injections into the anterior cingulate cortex or the hippocampus or the entorhinal cortex, retrogradely labelled cells in the medulla oblongata were predominantly in the rostral ventrolateral medulla. As a first attempt to reveal the chemical nature of the projection cells, the contribution of tyrosine hydroxylase-immunoreactive cells to the innervation of the septo-accumbens area was also investigated: tyrosine hydroxylase-immunoreactive cells of both the caudal ventrolateral medulla and the nucleus of the solitary tract were found to contribute to the innervation of the septo-accumbens area. The distribution of retrogradely labelled cells as well as the termination pattern of the anterogradely labelled terminals indicated that the innervation of the various forebrain limbic areas arises from cells, diffusely distributed in the rostral and/or the caudal ventrolateral medulla oblongata.(ABSTRACT TRUNCATED AT 400 WORDS)

Innervation of serotonergic medullary raphe neurons from cells of the rostral ventrolateral medulla in rats.

The rostral ventral medulla has been shown to consist of three distinct subregions: the midline or raphé region, the lateral paragigantocellular-gigantocellular region and the rostro-ventrolateral reticular nucleus. All three regions have been shown to contribute to central vaso-regulation and to project towards sympathetic preganglionic neurons of the thoracic spinal cord. Therefore it is of particular interest to describe the interconnections between the three regions and to see if local afferents reach cells which have been implicated in the regulation of descending inputs. Following injections of the anterograde tract tracer Phaseolus vulgaris leucoagglutinin into the lateral paragigantocellular nucleus or the rostroventrolateral reticular nucleus, labelled axons were traced into the medullary raphé nuclei and the contralateral rostral ventrolateral medulla. Efferents originating from both regions innervated the raphé pallidus, raphé obscurus and raphé magnus. However the distribution of terminals originating from the two regions was different in the contralateral ventrolateral medulla oblongata. The data indicate that the connection between the ipsi- and contralateral equivalents of both the lateral paragigantocellular-gigantocellular region and the rostroventrolateral reticular nucleus are stronger than the cross-connection between the ipsi- and contralateral parts of the two different regions. In the second part of the study, the existence of direct projections from the rostroventrolateral reticular nucleus and the lateral paragigantocellular-gigantocellular region onto serotonin-immunogold-labelled cells of the ventromedial medulla were investigated. The correlated light and electron microscopic analysis revealed direct synaptic contacts between axons originating from both the lateral paragigantocellular-gigantocellular region and the rostroventrolateral reticular nucleus, and serotonin-immunoreactive cells of the raphé obscurus and raphé pallidus. The results of the present light microscopic tract-tracing study revealed a different pattern of the intramedullary projection of the lateral paragigantocellular-gigantocellular region and the rostroventrolateral reticular nucleus. These data are in support of the proposed parcellation of the two cytoarchitectonically different areas of the rostral ventrolateral medulla into two functionally distinct subdivisions. Furthermore, the direct anatomical connection revealed in the present study between cells of the rostral ventrolateral and ventromedial medulla oblongata indicates the possibility that vasoregulatory effects of some cells of the rostral ventrolateral medulla oblongata might be executed via direct projections onto serotonin-immunoreactive cells of the medullary raphé nuclei.

Monosynaptic projections from the rostral ventrolateral medulla oblongata to identified sympathetic preganglionic neurons.

The rostral ventrolateral medulla oblongata plays an important role in the control of arterial blood pressure and it has strong descending projections into the intermediolateral nucleus of the thoracic spinal cord, where the majority of sympathetic preganglionic neurons are located. The purpose of this study was to see whether these projections form synaptic contacts with sympathetic preganglionic neurons in the rat. Projections from both the lateral part of the rostral ventrolateral medulla (rostroventrolateral reticular nucleus) and from the more medial region (lateral paragigantocellular nucleus) were investigated separately in view of their different functional roles in sympatho-regulation and their different chemical composition. Using anterograde tract-tracing of descending medullary pathways with Phaseolus vulgaris leucoagglutinin and retrograde labelling of sympatho-adrenal preganglionic neurons with cholera B chain conjugated to horseradish peroxidase, the existence of monosynaptic connections was sought by electron microscopy. Synaptic inputs from both the lateral and medial aspects of the rostral ventrolateral medulla oblongata were found on identified sympathetic preganglionic neurons. Synaptic specializations were of both the symmetrical and asymmetrical type. The targets of boutons forming asymmetrical synaptic contacts differed according to their origin: boutons originating from neurons in the rostroventrolateral reticular nucleus were mainly in contact with dendrites of sympathetic preganglionic neurons, while those originating from the lateral paragigantocellular nucleus mainly innervated the cell bodies. Our observations provide anatomical support for the view that there are two distinct classes of sympatho-regulatory cells in the rostral ventrolateral medulla, each of which can directly influence the activity of sympathetic preganglionic neurons; they also emphasize the importance of detailed investigation of the subregions of the ventrolateral medulla with respect to their sympatho-regulatory functions.

Evidence of a Monosynaptic Pathway Between Cells of the Ventromedial Medulla and the Motoneuron Pool of the Thoracic Spinal Cord in Rat: Electron Microscopic Analysis of Synaptic Contacts.

Previous electrophysiological and anatomical data have suggested the existence of a descending pathway from the ventromedial medulla into the thoracic motoneuron pool. However, systematic light and electron microscopic analysis have not yet been done to reveal such a projection. In the present study, the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L) was injected into several discrete regions of the medioventral medulla and descending PHA-L-labelled axons were investigated in the thoracic ventral horn using both light and electron microscopy. Light microscopic analysis of descending projections from 20 distinct areas of the medioventral medulla showed that neurons that project predominantly to the intermediate and ventral regions of the thoracic spinal grey matter are located caudal to the facial nucleus. Monosynaptic contacts were found between axons originating from five distinct regions of the medioventral medulla (containing raphé and/or gigantocellular reticular neurons) and cells in the thoracic motoneuron pool. PHA-L-labelled boutons formed synaptic contacts with large calibre dendrites and with somata. Seventy-two per cent of the investigated 32 boutons appeared to have symmetrical synaptic membrane specializations. The majority of the boutons contained only small, pleomorphic vesicles. Our findings show the existence of a direct monosynaptic pathway between the neurons of the ventromedial medulla and thoracic motor nuclei, providing anatomical support for previous physiological data.

Electron microscopic evidence of a monosynaptic pathway between cells in the caudal raphé nuclei and sympathetic preganglionic neurons in the rat spinal cord.

Electrophysiological and anatomical studies have suggested the existence of a pathway between the caudal raphé nuclei and regions of the spinal cord containing the sympathetic preganglionic neurons. However synaptic connections between cells in the raphé nuclei and identified sympathetic preganglionic neurons have not yet been shown. We have used a combination of anterograde tracing using Phaseolus vulgaris leucoagglutinin (PHA-L), retrograde tracing using a conjugate of cholera B chain and HRP and electron microscopy to look for such a pathway in rats. When PHA-L had been injected into the regions mainly restricted to the raphé pallidus and raphé magnus, synaptic contacts were found between PHA-L containing terminals and preganglionic neurons retrogradely labelled from the adrenal medulla. Out of the 43 synaptic contacts analysed, 26 were onto somata and 14 onto dendrites. 75% of the total appeared to have symmetric membrane specialisations, 20% asymmetric and the remainder could not be classified. Synaptic contacts were not seen in an animal in which the PHA-L injection site involved cells in the ventral raphé obscurus and surrounding gigantocellular reticular formation. These findings provide evidence of the existence of a direct monosynaptic pathway between cells in the raphé pallidus and/or caudal raphé magnus, and identified sympathetic preganglionic neurons and give further support for a role for the caudal raphé nuclei in sympathetic autonomic regulation.