Role of sensory-evoked NMDA plateau potentials in the initiation of locomotion.

Reticulospinal (RS) neurons constitute the main descending motor system of lampreys. This study reports on natural conditions whereby N-methyl-D-aspartate (NMDA)-mediated plateau potentials were elicited and associated with the onset of locomotion. Reticulospinal neurons responded in a linear fashion to mild skin stimulation. With stronger stimuli, large depolarizing plateaus with spiking activity were elicited and were accompanied by swimming movements. Calcium imaging revealed sustained intracellular calcium rise upon sensory stimulation. Blocking NMDA receptors on RS neurons prevented the plateau potentials as well as the associated rise in intracellular calcium. Thus, the activation of NMDA receptors mediates a switch from sensory-reception mode to a motor command mode in RS neurons.

Modulation of respiratory activity by locomotion in lampreys.

In vertebrates, locomotion is associated with changes in respiratory activity, but the neural mechanisms by which this occurs remain unknown. We began examining this in lampreys using a semi-intact preparation of young adult Petromyzon marinus, in which respiratory and locomotor behaviors can be recorded simultaneously with the activity of the underlying neural control systems. Spontaneous fictive respiration was recorded with suction electrodes positioned over the glossopharyngeal or the rostral vagal motor nucleus. In this preparation, locomotor activity, characterized by symmetrical tail movements (electromyogram recordings), was evoked by mechanical stimulation of the skin. During locomotion, the mean respiratory frequency and the mean area of the motor bursts were significantly increased (81.6+/-28.6% and 62.8+/-25.4%, respectively; P<0.05). The frequency returned to normal 92+/-51 s after the end of locomotion. There were fluctuations in the instantaneous respiratory and locomotor frequencies that were rhythmical but antiphasic for the two rhythmic activities. The changes in respiratory activity were also examined during bouts of locomotion occurring spontaneously, and it was found that a modification in respiratory activity preceded the onset of spontaneous locomotion by 3.5+/-2.6 s. This suggests that the early respiratory changes are anticipatory and are not caused by feedback generated by locomotion. The increase in respiratory frequency during locomotion induced by sensory stimulation persisted after removal of the mesencephalon. When both the mesencephalon and spinal cord were removed, resulting in the isolation of the rhombencephalon, changes in the respiratory activity were also present following skin stimulations that would have normally induced locomotion. Altogether, the results suggest that respiratory changes are programmed to adjust ventilation prior to motor activity, and that a central rhombencephalic mechanism is involved.

Opioid-induced depression in the lamprey respiratory network.

The role of opioid receptors in modulating respiratory activity was investigated in in vitro brainstem preparations of adult lampreys by bath application of agonists and antagonists. The vagal motor output was used to monitor respiratory activity. Neuronal recordings were also performed to characterize the rostrolateral trigeminal region that has been suggested to be critical for respiratory rhythmogenesis. Microinjections of the micro-opioid receptor agonist [d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) were also made into this region and at different locations within the brainstem. Bath application of DAMGO (0.5-2 microM) caused marked decreases in respiratory frequency up to complete apnea. Bath application of the delta-opioid receptor agonist [d-Pen(2,5)]-enkephalin (DPDPE) at 10-40 microM induced less pronounced depressant respiratory effects, while no changes in respiratory activity were induced by the kappa-opioid receptor agonist trans-(1S,2S)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide (U50488) at 10-40 microM. Bath application of the opioid receptor antagonists naloxone and naltrindole did not affect baseline respiratory activity, but prevented agonist-induced effects. DAMGO microinjections (1 mM; 0.5-1 nl) at sites rostrolateral to the trigeminal motor nucleus, where respiration-related neuronal activity was recorded, abolished the respiratory rhythm. The results show that opioids may have an important role in the lamprey respiratory network and that micro-opioid receptor activation is the most effective in causing respiratory depression. They also indicate that endogenous opioids are not required for the generation of baseline respiratory activity. Apneic responses induced by DAMGO microinjections support the hypothesis that a specific opioid-sensitive region rostrolateral to the trigeminal motor nucleus, that we have termed the paratrigeminal respiratory group (pTRG), likely has a pivotal role in respiratory rhythmogenesis. Since the lamprey diverged from the main vertebrate line around 450 million years ago, our results also imply that the inhibitory role of opioids on respiration is present at an early stage of vertebrate evolution.

Respiratory rhythms generated in the lamprey rhombencephalon.

Brainstem networks generating the respiratory rhythm in lampreys are still not fully characterized. In this study, we described the patterns of respiratory activities and we identified the general location of underlying neural networks. In a semi-intact preparation including the brain and gills, rhythmic discharges were recorded bilaterally with surface electrodes placed over the vagal motoneurons. The main respiratory output driving rhythmic gill movements consisted of short bursts (40.9+/-15.6 ms) of discharge occurring at a frequency of 1.0+/-0.3 Hz. This fast pattern was interrupted by long bursts (506.3+/-174.6 ms) recurring with an average period of 37.4+/-24.9 s. After isolating the brainstem by cutting all cranial nerves, the frequency of the short respiratory bursts did not change significantly, but the slow pattern was less frequent. Local injections of a glutamate agonist (AMPA) and antagonists (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or D,L-amino-5-phosphonopentanoic acid (AP5)) were made over different brainstem regions to influence respiratory output. The results were similar in the semi-intact and isolated-brainstem preparations. Unilateral injection of AP5 or CNQX over a rostral rhombencephalic region, lateral to the rostral pole of the trigeminal motor nucleus, decreased the frequency of the fast respiratory rhythm bilaterally or stopped it altogether. Injection of AMPA at the same site increased the rate of the fast respiratory rhythm and decreased the frequency of the slow pattern. The activity recorded in this area was synchronous with that recorded over the vagal motoneurons. After a complete transverse lesion of the brainstem caudal to the trigeminal motor nucleus, the fast rhythm was confined to the rostral area, while only the slow activity persisted in the vagal motoneurons. Our results support the hypothesis that normal breathing depends on the activity of neurons located in the rostral rhombencephalon in lampreys, whereas the caudal rhombencephalon generates the slow pattern.

Olfactory sensory neurons in the sea lamprey display polymorphisms.

The sea lamprey (Petromyzon marinus) is an ancient jawless fish phyletically removed from modern (teleost) fishes. It is an excellent organism in the study of olfaction due to its accessible olfactory pathway, which is susceptible to manipulation, and its important location in the evolution of vertebrates. There are many similarities in the olfactory systems of all fishes, and they also share characteristics with the olfactory system of mammals. Teleost fishes lack the distinctive vomeronasal organ of mammals; rather all odours are processed initially by olfactory sensory neurons (OSNs) of three morphotypes within the olfactory epithelium. We sought to identify olfactory sensory neuron polymorphisms in the sea lamprey. Using retrograde tracing with dyes injected into the olfactory bulb, we identified three morphotypes which are highly similar to those found in teleosts. This study provides the first evidence of morphotypes in the sea lamprey peripheral olfactory organ, and indicates that olfactory sensory neuron polymorphism may be a trait highly conserved throughout vertebrate evolution.

Spinal pattern generation.

Recent research in the field of spinal pattern generation has concentrated on three main areas: the effects of various transmitters on spinal rhythmic patterns in reduced preparations (neonatal rats, chick embryos, tadpole embryos, lampreys); the changes in membrane properties of different elements of the generating circuits; and the interactions between central generating mechanisms and afferent inputs. The important message is that new properties of neural membranes, as well as new reflex responses, have been identified that could not have been predicted in the absence of such rhythmic activity.

A cellular mechanism for the transformation of a sensory input into a motor command.

The initiation and control of locomotion largely depend on processing of sensory inputs. The cellular bases of locomotion have been extensively studied in lampreys where reticulospinal (RS) neurons constitute the main descending system activating and controlling the spinal locomotor networks. Ca(2+) imaging and intracellular recordings were used to study the pattern of activation of RS neurons in response to cutaneous stimulation. Pressure applied to the skin evoked a linear input/output relationship in RS neurons until a threshold level, at which a depolarizing plateau was induced, the occurrence of which was associated with the onset of swimming activity in a semi-intact preparation. The occurrence of a depolarizing plateau was abolished by blocking the NMDA receptors that are located on RS cells. Moreover, the depolarizing plateaus were accompanied by a rise in [Ca(2+)](i), and an intracellular injection of the Ca(2+) chelator BAPTA into single RS cells abolished the plateaus, suggesting that the latter are Ca(2+) dependent and rely on intrinsic properties of RS cells. The plateaus were shown to result from the activation of a Ca(2+)-activated nonselective cation current that maintains the cell in a depolarized state. It is concluded that this intrinsic property of the RS neuron is then responsible for the transformation of an incoming sensory signal into a motor command that is then forwarded to the spinal locomotor networks.

The trigeminal sensory relay to reticulospinal neurones in lampreys.

This study was carried out to identify lamprey neurones relaying trigeminal sensory inputs to reticulospinal cells. Double labeling with fluorescent tracers was used in vitro. Fluorescein-conjugated dextran amines were applied to the proximal stump of the cut trigeminal nerve on both sides, and Texas Red-conjugated dextran amines were injected unilaterally in the middle (MRRN) or the posterior (PRRN) rhombencephalic reticular nuclei. Texas Red retrogradely labeled cells were found ipsi- and contralateral to each injection. Any of these cells with the soma or at least a major dendrite among the fluorescein-labeled trigeminal afferent axons was considered a candidate relay cell. Of these two possibilities, only cells with their soma among the fluorescein-labeled trigeminal afferents were found. The candidate relay cells projecting to the MRRN were mostly clustered at the caudal vestibular nerve level within the trigeminal descending tract, whereas the majority of those projecting to the PRRN were located more caudally. The diameter of candidate relay cells ranged from 9.2 to 24.6 mum and 9.2 to 46.1 mum, after MRRN and PRRN injections, respectively. A possible relay function for these cells was tested with electrophysiological experiments. The intracellular responses to trigeminal nerve stimulation were recorded in reticulospinal cells under control conditions and after ejections of a combination of glutamate ionotropic receptor antagonists over the candidate relay cells in small areas along the sulcus limitans. The synaptic responses elicited in MRRN reticulospinal cells were maximally depressed when ejections were made at the level of the vestibular nerve, in accord with the anatomical data. The synaptic responses in PRRN reticulospinal cells showed maximal depression when ejections were made slightly more caudally. Altogether, these results suggest that cells located within the trigeminal descending tract and projecting to reticular nuclei are likely to be the sensory trigeminal relays to reticulospinal neurones in lampreys.

Relationship between vestibular primary afferents and vestibulospinal neurons in lampreys.

The distribution of vestibular primary afferents as well as their relationship with vestibulospinal and other brainstem neurons were studied in lampreys using anatomical tracers. Afferents from the anterior (aVIIIn) and the posterior (pVIIIn) branches of the vestibular nerve were located mainly in the ventral nucleus of the octavolateral area. The relationship between afferents and vestibulospinal neurons was studied by applying one fluorescent tracer to the whole vestibular nerve or one of its branches and applying another tracer to the spinal cord. Some afferents showed large, bulb-like enlargements (bulbs) and about 20 of these were found in the anterior and the intermediate octavomotor nucleus, whereas about 40 were found in the posterior octavomotor nucleus. Some of the bulbs made apparent contact with vestibulospinal neurons in the intermediate octavomotor nucleus and originated mostly from the aVIIIn, whereas bulbs in the posterior octavomotor nucleus originated from the pVIIIn. Applications of biocytin to hemisegments of rostral spinal cord labeled vestibulospinal neurons located in the ipsilateral intermediate octavomotor nucleus and the contralateral posterior octavomotor nucleus. In addition, vestibular primary afferents with bulbs in apparent contact with vestibulospinal neurons were transneuronally labeled by biocytin. They were observed in the ipsilateral aVIIIn and the contralateral pVIIIn and could be followed in the labyrinths, where they innervated the vertical and horizontal arms of the semicircular canal crests. Taken together, these results indicate that vestibular primary afferents from the aVIIIn innervate predominantly vestibulospinal neurons of the intermediate octavomotor nucleus, whereas afferents from the pVIIIn innervate vestibulospinal neurons in the posterior octavomotor nucleus. This anatomical organization suggests that afferents carrying bulbs convey dynamic information to vestibulospinal neurons, which, in turn, project to the spinal cord networks.

Dorsal root and dorsal column mediated synaptic inputs to reticulospinal neurons in lampreys: involvement of glutamatergic, glycinergic, and GABAergic transmission.

This study was aimed at characterizing the inputs from dorsal roots and dorsal columns to reticulospinal neurons within the posterior rhombencephalic reticular nucleus in the lamprey. The in vitro isolated brainstem and spinal cord preparation was used. Microstimulation of dorsal roots and columns on both sides induced, in identified reticulospinal neurons, synaptic responses which consisted of large IPSPs mixed with excitation, particularly from stimulation on the ipsilateral side. When the spinal cord was selectively exposed to kynurenic acid or to Ca2+ free Ringer's containing 2mM Mn2+, synaptic responses to stimulation of dorsal roots and columns were not modified, whereas the same responses were abolished when the brainstem was exposed selectively to kynurenic acid, thus suggesting that the responses were carried by long fibres ascending directly to the brainstem. The excitatory and inhibitory synaptic responses are relayed by interneurons located in the brainstem. The ascending excitatory inputs to inhibitory interneurons and, most likely, also to excitatory interneurons, use excitatory amino acid transmission. Inhibitory responses were abolished by adding the glycinergic antagonist strychnine (5 microM) to the physiological solution, thus suggesting that inhibitory interneurons use glycine transmission. The synaptic transmission was depressed by (-)-baclofen, a GABAB agonist, probably acting at a presynaptic site. Taken together, the present results suggest that dorsal root and dorsal column stimulations give rise to disynaptic inhibition and excitation of reticulospinal neurons mediated by excitatory and inhibitory amino acid transmission via brainstem interneurons.

Anatomical and physiological study of brainstem nuclei relaying dorsal column inputs in lampreys.

The course and sites of termination of dorsal column fibres in the lamprey brainstem are described along with their brainstem relays projecting to reticulospinal neurons. Dorsal column fibres ascend to the brainstem level where they intermingle with cells located in the alar plate close to the obex, a location that is analogous to that of the dorsal column nucleus in other vertebrates. Some dorsal column fibres continue further rostrally where they reach the octavolateralis and octavomotorii nuclei. Finally, a small contingent of fibres reach the cerebellum. Injections of cobalt-lysine into the posterior rhombencephalic reticular nucleus retrogradely label neurons within the dorsal column nucleus and within the octavolateralis and octavomotorii nuclei. Microstimulation of the dorsal column nucleus on either side elicits monosynaptic inhibitory responses in reticulospinal neurons while stimulation of octavolateralis and octavomotorius nuclei elicits excitation. By using intracellular recordings, it was shown that neurons within these alar plate nuclei receive monosynaptic inputs from the dorsal columns. It is thus proposed that disynaptic inputs from dorsal columns to reticulospinal neurons are relayed by these alar plate neurons: inhibition is relayed mainly by neurons in dorsal column nuclei and excitation by neurons in the octavolateralis and octavomotorii nuclei.

5-HT innervation of reticulospinal neurons and other brainstem structures in lamprey.

In order to determine if reticulospinal neurons involved in the control of locomotion and responsive to exogenously applied 5-hydroxytryptamine (5-HT) are innervated by fibers that contain serotonin, the serotoninergic innervation of reticulospinal neurons, identified by retrograde labeling with fluorescein-conjugated dextran-amine (FDA), was investigated by immunohistochemistry in the lamprey brainstem. A widespread distribution of 5-HT immunoreactive (5-HT-ir) fibers was seen within the basal plate of the brainstem, an area containing reticulospinal somata and dendritic aborizations. Numerous 5-HT varicose fibers were found in close relation to large reticulospinal cell bodies, particularly in the middle and anterior rhombencephalic reticular nuclei (MRRN and ARRN). Some of these reticulospinal somata were surrounded by a very dense pericellular 5-HT innervation. 5-HT-ir fibers were also seen in other brain structures that are known to influence reticulospinal neurons such as the rhombencephalic alar plate containing sensory relay interneurons, cranial nerves (III-X), cerebellum, and tectum. These findings suggest that, as in the spinal cord, motor behavior controlled by reticulospinal neurons may be subject to a serotoninergic modulation.

Anatomical study of vestibulospinal neurons in lampreys.

The present study was carried out to characterize anatomically the vestibulospinal (VS) system of lampreys. Cobalt-lysine or Texas Red dextran amines were applied in vitro to the rostral spinal cord. Two distinct populations of VS neurons were labeled in the ventral nucleus of the area octavolateralis. The rostral group, comprising the intermediate octavomotor nucleus (ION), contained between 100 and 150 neurons, having somata of variable size and morphology. Intracellular injections of Lucifer Yellow in single neurons revealed ION VS neurons with dendrites extending in the ventrolateral alar plate as well as medially in the basal plate. The caudal group, comprising the posterior octavomotor nucleus (PON), contained approximately 65 neurons, most of which were unipolar with round or oval somata. To study the projections of VS axons, cobalt-lysine was injected into the ION or PON regions in the brainstem. Axons from the ION projected to the ipsilateral spinal cord, whereas PON axons decussated within the basal plate giving out descending and ascending branches. The descending branch projected to the contralateral spinal cord. Injections of two fluorescent dextran-amines, each restricted to one side of the spinal cord, did not double-label VS cells in either octavomotor nuclei, indicating that the projections of each nucleus are restricted to one side. Injections of horseradish peroxidase further caudally in the spinal cord revealed that VS axons from the ION reached past the gill region. Our results indicate that the organization of the VS system of lampreys is similar to that observed in other vertebrates.

Organization of the six motor nuclei innervating the ocular muscles in lamprey.

The topography of motoneurons supplying each of the six ocular muscles of the lamprey, Lampetra fluviatilis, was studied by selective application of HRP to the cut nerves of identified muscles. In addition, the distributions of motoneuron populations to both eyes were studied simultaneously with fluorescein and rhodamine coupled dextran-amines (FDA and RDA) applied to cut ocular muscle nerves of either side. The motoneuron pool of the caudal oblique muscle is represented bilaterally in the trochlear (N IV) motor nucleus. The dorsal rectus muscle is innervated from a contralateral group of oculomotor (N III) motoneurons and the remaining four muscles exclusively from the ipsilateral side (N III and N VI). The inferior and posterior rectus muscles are both innervated by the abducens nerve. In contrast to all jawed vertebrates, only three eye muscles (the dorsal rectus, rostral rectus, and rostral oblique) are innervated by the oculomotor nerve in lampreys (N III). Lampreys have a motor nucleus similar to the accessory abducens nucleus previously described only in tetrapods. They lack the muscle homologous to the nasal rectus muscle of elasmobranchs and the medial rectus muscle of osteognathostomes. The distribution of the dendrites of different groups of motoneurons was studied and is considered in relation to inputs from tectum and the different cranial nerves.

Anatomical study of spinobulbar neurons in lampreys.

The present study was aimed at identifying spinal neurons ascending to the brainstem outside the dorsal columns in the lamprey. Two retrograde tracers (cobalt-lysine and horseradish peroxidase [HRP]) were injected in the brainstem or rostral spinal cord in vivo or in vitro. Labeled cells were distributed bilaterally with a contralateral dominance, along the whole rostrocaudal extent of the spinal cord. The density of cells markedly decreased rostrocaudally. Several classes of brainstem-projecting neurons were identified. Most cells with a short axon were small and formed columns, in the dorsolateral and ventrolateral gray matter, at the transition between the rhombencephalon and the spinal cord. Dorsal elongated cells were spindle shaped, located medially, in the first two spinal segments. Lateral elongated cells were medium to large size neurons, located in the intermediate and lateral gray matter, mainly contralateral to the injection site. Their axon emerging from the lateral part of the soma crossed the midline, ventral to the central canal. These cells were present throughout the rostral spinal cord. Cells were also labeled in the lateral white matter. Some of them had the typical dendritic arborizations of edge cells (intraspinal stretch receptor neurons) and were located in the most rostral segments, bilaterally. Other medium to large size neurons were identified dorsal and medial to most of the edge cells. We suggest that at least the group of lateral elongated cells exhibits rhythmic membrane potential oscillations during fictive locomotion. These cells may, together with the rostral edge cells, be responsible for the locomotor-related modulation of activity in reticulospinal and vestibulospinal neurons.

Anatomical organization of efferent neurons innervating various regions of the rabbit masseter muscle.

Previous studies have shown that the masseter muscle is supplied by motoneurons located in the anterodorsal region of the trigeminal motor nucleus and by an additional group of efferent neurons located in cell group k. The present experiments were performed on nine rabbits and were designed to establish the locations of neurons innervating the different regions of this muscle. Retrograde labeling with two fluorescent tracers (FluoroGold and Fast Blue) was applied to the central ends of cut branches of the masseter nerve. Serial coronal sections of the brainstem were viewed with fluorescence microscopy. The labeled cells were counted in all animals, and three-dimensional reconstructions of their distribution were made in five cases. In each successful experiment, labeled neurons were seen in the anterodorsal region of the trigeminal motor nucleus and in the two dorsal cell columns of cell group k (k1 and k3). Within-animal comparisons of the median position of populations innervating two distinct muscle regions in five rabbits showed that there were no significant differences in either the dorsoventral or rostrocaudal axes. However, in each case, there was a small but significant difference (83-173 microm) in the mediolateral axis within the motor nucleus but not within cell group k. Even in this axis, there was a 94-99% overlap of the two populations. Comparisons of the neuronal cross-sectional area showed that the deep regions were innervated by a larger proportion of small neurons from both nuclei than were the superficial and intermediate regions. Our results suggest that there is no simple topographical arrangement of motoneurons that corresponds to the peripheral pattern of nerve supply to the different regions of the masseter muscle.

Vestibulo-reticular projections in adult lamprey: their role in locomotion.

This study describes the anatomical projections from vestibular secondary neurons to reticulospinal neurons in the adult lamprey and the modulation of vestibular inputs during fictive locomotion. Anatomical tracers were applied in the posterior (PRRN) and middle rhombencephalic reticular nuclei as well as to the proximal stumps of cut vestibular nerve branches to identify the neurons projecting to the reticular nuclei that were in close proximity with vestibular primary afferents. Labeled neurons were found in the intermediate (ION) and posterior (PON) octavomotor nuclei, and were more numerous on the side of the injection (around 56-87 and 101-107 for the ION and the PON, respectively). Morphologies varied but cells were mostly round or oval. Axonal projections from the PON formed a dense bundle, whereas those from the ION were less densely packed. Based on their morphology and the distribution of their projections, most vestibulo-reticular neurons were presumed to be vestibulospinal cells. Reticulospinal cells from the PRRN were recorded intracellularly in the in vitro brainstem-spinal cord preparation and large excitatory post-synaptic potentials (EPSPs) were evoked following stimulation of the ipsilateral anterior and the contralateral posterior branches of the vestibular nerves, whereas inhibitory post-synaptic potentials (IPSPs) or smaller EPSPs were elicited by stimulation of the ipsilateral posterior or of the contralateral anterior branches. During fictive locomotion, both the excitatory and the inhibitory responses displayed phasic changes in amplitude such that the amplitude of the EPSPs was minimal when the spinal cord activity switched from the ipsilateral to the contralateral side of the recorded reticulospinal cell. The IPSPs were then of maximal amplitude. We propose that this modulation could serve to reduce the influence of vestibular inputs in response to head movements during locomotion.

An immunocytochemical and autoradiographic investigation of the serotoninergic innervation of trigeminal mesencephalic and motor nuclei in the rabbit.

The results of a previous experiment suggest that the cell bodies of many jaw closing muscle spindle afferents in the trigeminal mesencephalic nucleus of the rabbit are phasically inhibited during fictive mastication. The aim of this study was to investigate one possible neurotransmitter system that could be involved in this modulation, serotonin, by use of receptor autoradiography techniques and immunofluorescence combined with retrograde labelling of masseteric spindle afferents and motoneurons. A second objective was to compare the serotonin innervation of neurons in the trigeminal mesencephalic nucleus with that of masseteric motoneurons. Serotoninergic fibres were seen surrounding labelled masseteric spindle afferents, as well as unlabelled neurons, in the trigeminal mesencephalic nucleus. These fibres were close to the cell bodies and sometimes to the axon hillocks of the neurons. Although it has been reported that many neurons of the trigeminal nucleus are multipolar in some species, none of the labelled spindle afferent in this study had more than one process. Throughout the motor trigeminal nucleus, serotonin fibres were found in close proximity with cell bodies and with the proximal portions of axons and dendrites of labelled and unlabelled motoneurons. Serotonin fibres were also seen adjacent to cell bodies and processes of efferent neurons in cell group k. Autoradiography with several tritiated ligands was used to reveal the presence of receptors for serotonin as well as its uptake sites. Only serotonin2 receptors were found to be abundant in the trigeminal mesencephalic nucleus. The motor nucleus and cell group k contained serotonin2 and serotonin3 receptors, as well as serotonin uptake sites. Serotonin1A receptors appear to be absent from both nuclei. The findings suggest that release of serotonin from fibres in close proximity to trigeminal primary afferent somata could modify the transmission of action potentials from muscle spindle receptors during mastication through an action on serotonin2 receptors. In the motor nucleus and cell group k, serotonin may alter neuronal properties through actions on at least two receptor subtypes (serotonin2 and serotonin3).

A physiological study of brainstem and peripheral inputs to trigeminal motoneurons in lampreys.

The inputs to trigeminal motoneurons from sensory afferents and rhombencephalic premotor regions were studied in isolated brainstem preparations of adult lampreys (Petromyzon marinus). Stimulation of both trigeminal nerves, contralateral nucleus motorius nervi trigemini, nucleus sensibilis nervi trigemini and ipsilateral rostral reticular formation elicited large-amplitude excitatory postsynaptic potentials with short latencies. These were significantly attenuated by adding 6-cyano-7-nitroquinoxaline2,3-dione (10 microM) and 2-amino-5-phosphonopentanoate (200 microM) to the bath, suggesting participation of both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-D-aspartate receptors. The inputs from ipsilateral trigeminal afferents included a di- or oligosynaptic glycinergic inhibition. Sustained rhythmical membrane potential oscillations were observed in 52% of the recorded cells upon stimulation of trigeminal afferents or the contralateral nucleus sensibilis nervi trigemini. Two types of rhythm were obtained: (i) low-frequency oscillations (0.1-0.5 Hz), with peak-to-peak amplitudes between 8.5 and 17 mV; and (ii) higher frequency oscillations (1.0-2.8 Hz) with smaller amplitudes (1.8-5.1 mV). The two types of trigeminal rhythm could occur independently of fictive locomotion and fictive breathing. In a decerebrate semi-intact preparation, slow rhythmical trigeminal motoneuron potential oscillations were also evoked by stimulation of the oral disc. This study shows that trigeminal motoneurons receive excitatory synaptic inputs from several brainstem sites, and that membrane potential oscillations can be triggered upon stimulation of trigeminal afferents or the nucleus sensibilis nervi trigemini. We suggest that these oscillations recorded in vitro may represent the centrally generated components that underlie rhythmical feeding in lampreys.

Evidence that the masticatory muscles receive a direct innervation from cell group k in the rabbit.

These experiments have shown that a group of neurons lateral to the trigeminal motor nucleus innervates the muscles of mastication. The work began to describe the location of digastric last-order interneurons, using the technique of transneuronal labeling with wheatgerm agglutinin-conjugated horseradish peroxide injected into the left digastric muscle of rabbits under general anaesthesia. Four to eight days later, the animals were killed with an overdose of anaesthetic and perfused. Coronal sections of the frozen brainstem were cut at 20 microns thickness and processed for peroxidase activity. Motoneurons in the ventral and caudal divisions of the trigeminal motor nucleus were labeled in all animals as expected. An additional population of neurons located ventrolaterally to the motor nucleus in cell group k were also found to be labeled if the survival time was five days or more. In an attempt to determine whether cell group k neurons were labeled transynaptically, two series of control experiments were carried out. In the first, crystals of fluorescein- and rhodamine-conjugated dextran amines and horseradish peroxidase were applied directly to central ends of cut digastric nerves. In the second, central ends of cut digastric nerves were enclosed in cuffs containing 40-60% horseradish peroxidase solutions. Again, neurons in both the trigeminal motor nucleus and cell group k were labeled suggesting that neurons within cell group k project to the digastric muscle. Similar experiments using dextran amines and wheatgerm peroxidase were carried out on the masseter muscle. Motoneurons in the dorsomedial and rostral half of the trigeminal motor nucleus, as well as primary afferent cell bodies in the mesencephalic nucleus of the trigeminal nerve, were labeled in all experiments. In addition, a population of neurons in cell group k, dorsal to those associated with the digastric muscle, were found to contain each one of the reaction products. Since it is thought that only the wheatgerm agglutinin-conjugated horseradish peroxidase transferred from one neuron to another, we conclude that cell group k neurons provide an additional innervation to the digastric and masseter muscles.