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.

Muscarinic receptor activation elicits sustained, recurring depolarizations in reticulospinal neurons.

In lampreys, brain stem reticulospinal (RS) neurons constitute the main descending input to the spinal cord and activate the spinal locomotor central pattern generators. Cholinergic nicotinic inputs activate RS neurons, and consequently, induce locomotion. Cholinergic muscarinic agonists also induce locomotion when applied to the brain stem of birds. This study examined whether bath applications of muscarinic agonists could activate RS neurons and initiate motor output in lampreys. Bath applications of 25 microM muscarine elicited sustained, recurring depolarizations (mean duration of 5.0 +/- 0.5 s recurring with a mean period of 55.5 +/- 10.3 s) in intracellularly recorded rhombencephalic RS neurons. Calcium imaging experiments revealed that muscarine induced oscillations in calcium levels that occurred synchronously within the RS neuron population. Bath application of TTX abolished the muscarine effect, suggesting the sustained depolarizations in RS neurons are driven by other neurons. A series of lesion experiments suggested the caudal half of the rhombencephalon was necessary. Microinjections of muscarine (75 microM) or the muscarinic receptor (mAchR) antagonist atropine (1 mM) lateral to the rostral pole of the posterior rhombencephalic reticular nucleus induced or prevented, respectively, the muscarinic RS neuron response. Cells immunoreactive for muscarinic receptors were found in this region and could mediate this response. Bath application of glutamatergic antagonists (6-cyano-7-nitroquinoxaline-2,3-dione/D-2-amino-5-phosphonovaleric acid) abolished the muscarine effect, suggesting that glutamatergic transmission is needed for the effect. Ventral root recordings showed spinal motor output coincides with RS neuron sustained depolarizations. We propose that unilateral mAchR activation on specific cells in the caudal rhombencephalon activates a circuit that generates synchronous sustained, recurring depolarizations in bilateral populations of RS neurons.

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.

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.

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.

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.

Anatomical and physiological study of respiratory motor innervation in lampreys.

The innervation of gill muscles of lampreys was investigated in a semi-intact preparation in which the respiratory rhythm was maintained for more than 2 days. Lesion experiments showed that the muscles of gill 1 are innervated by nerves VII (facial) and IX (glossopharyngeal), and those of gill 2 by nerve IX and the first branchial branch of nerve X (vagal). The other gills are supplied by the other branchial branches of nerve X. Retrograde tracers, injected in peripheral respiratory nerves, showed that branchial muscles are innervated by VII, IX and X motoneurons. Within the X nucleus, the motoneuron pools were branchiotopically organized, but with considerable rostro-caudal overlap. Electrophysiological recordings were used to show that the onset of activation of the branchial muscles was increasingly delayed with the distance from the brainstem, but that motoneuronal activity recorded with surface electrodes began at approximately the same time in all pools. The conduction velocity of VII and caudal X motor axons was found to be the same. Differences in the length of motoneuron axons appear to account for the rostro-caudal delay in gill contraction. The data presented here provide a much needed anatomical and physiological basis for further studies on the neural network controlling respiration in lampreys.

Genome organization of the densovirus from Bombyx mori (BmDNV-1) and enzyme activity of its capsid.

Bombyx mori densovirus (BmDNV-1), on the basis of the previously reported genome sequence, constitutes by itself a separate genus (Iteravirus) within the Densovirinae subfamily of parvoviruses. Inconsistencies in the genome organization, however, necessitated its reassessment. The genome sequence of new clones was determined and resulted in a completely different genome organization. The corrected sequence also contained conserved sequence motifs found in other parvoviruses. Some amino acids in the highly conserved domain in the unique region of VP1 were shared by critical amino acids in the catalytic site and Ca(2+)-binding loop of secreted phospholipase A2, such as from snake and bee venoms. Expression of this domain and determination of enzyme activity demonstrated that capsids have a phospholipase A2 activity thus far unknown to occur in viruses. This viral phospholipase A2, which is required shortly after entry into the cell, showed a substrate preference for phosphatidylethanolamine and phosphatidylcholine over phosphatidylinositol.

Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi-intact lampreys.

The role of the mesencephalic locomotor region (MLR) in initiating and controlling the power of swimming was studied in semi-intact preparations of larval and adult sea lampreys. The brain and the rostral portion of the spinal cord were exposed in vitro, while the intact caudal two-thirds of the body swam freely in the Ringer's-containing chamber. Electrical microstimulation (2-10 Hz; 0. 1-5.0 microA) within a small periventricular region in the caudal mesencephalon elicited well-coordinated and controlled swimming that began within a few seconds after the onset of stimulation and lasted throughout the stimulation period. Swimming stopped several seconds after the end of stimulation. The power of swimming, expressed by the strength of the muscle contractions and the frequency and the amplitude of the lateral displacement of the body or tail, increased as the intensity or frequency of the stimulating current were increased. Micro-injection of AMPA, an excitatory amino acid agonist, into the MLR also elicited active swimming. Electrical stimulation of the MLR elicited large EPSPs in reticulospinal neurons (RS) of the middle rhombencephalic reticular nucleus (MRRN), which also displayed rhythmic activity during swimming. The retrograde tracer cobalt-lysine was injected into the MRRN and neurons (dia. 10-20 microm) were labelled in the MLR, indicating that this region projects to the rhombencephalic reticular formation. Taken together, the present results indicate that, as higher vertebrates, lampreys possess a specific mesencephalic region that controls locomotion, and the effects onto the spinal cord are relayed by brainstem RS neurons.

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.

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.

Fictive rhythmic motor patterns induced by NMDA in an in vitro brain stem-spinal cord preparation from an adult urodele.

An in vitro brain stem-spinal cord preparation from an adult urodele (Pleurodeles waltl) was developed in which two fictive rhythmic motor patterns were evoked by bath application of N-methyl-D-aspartate (NMDA; 2.5-10 microM) with D-serine (10 microM). Both motor patterns displayed left-right alternation. The first pattern was characterized by cycle periods ranging between 2.4 and 9. 0 s (4.9 +/- 1.2 s, mean +/- SD) and a rostrocaudal propagation of the activity in consecutive ventral roots. The second pattern displayed longer cycle periods (8.1-28.3 s; 14.2 +/- 3.6 s) with a caudorostral propagation. The two patterns were inducible after a spinal transection at the first segment. Preliminary experiments on small pieces of spinal cord further suggested that the ability for rhythm generation is distributed along the spinal cord of this preparation. This study shows that the in vitro brain stem-spinal cord preparation from Pleurodeles waltl may be a useful model to study the mechanisms underlying the different axial motor patterns and the flexibility of the neural networks involved.

Distribution of cholinergic neurons in cell group K of the rabbit brainstem.

The cell bodies of efferent neurons supplying the masseter and digastric muscles of the rabbit are located in two brainstem nuclei: the trigeminal motor nucleus and cell group k. The latter also contains neurons innervating muscles of the middle ear and Eustachian tube, as well as neurons that project to the cerebellum and the oculomotor complex. As part of an attempt to identify the functional subpopulations within the three cell divisions (kl-k3) that make up cell group k, we have investigated the distribution of neurons containing choline acetyltransferase, because these are likely to be motoneurons. Five rabbits anaesthetized with sodium pentobarbital (90 mg/kg, i.v.) were used in this study. They were perfused with 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4). Two animals were used for preliminary studies. In the other three cases, serial Vibratome coronal sections of the brainstem were cut at 50 microm and two series of alternating sections were collected. The first was stained with a monoclonal antibody (code AB8, Incstar) directed against choline acetyltransferase, using the avidin-biotin-peroxidase method. The other was stained with Cresyl Violet. Cell counts and three-dimensional reconstructions were made for both series to determine positions and ratios of cholinergic and non-cholinergic neurons within the trigeminal motor nucleus and the subdivisions of cell group k. The results showed that the numbers of choline acetyltransferase- and Nissl-stained neurons within the trigeminal motor nucleus were almost identical. In cell group k, significantly fewer choline acetyltransferase-stained cells were counted in all three animals (ratios of choline acetyltransferase/Nissl=0.53-0.71). In addition, the distribution of cholinergic neurons was not uniform throughout cell group k. Subdivisions kl and k3 contained proportionately fewer choline acetyltransferase-positive cells (ratios of choline acetyltransferase/Nissl=0.23-0.64) than did k2 (ratios choline acetyltransferase/ Nissl=0.75-0.88). Within each subdivision, there were significant differences in the spatial coordinates of Nissl- and choline acetyltransferase-positive neurons. We conclude that cell group k contains at least two populations of neurons which are unevenly distributed between and within the three subdivisions. While the majority of neurons in subgroup k2 contain choline acetyltransferase and thus are likely to be motoneurons, more than half of the neurons in subgroups k1 and k3 are not cholinergic. It remains to be determined whether these are the neurons that project to the cerebellum and to other CNS regions.

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.

An anatomical study of brainstem projections to the trigeminal motor nucleus of lampreys.

This study was undertaken to identify and describe populations of brainstem neurons that project to the area of the nucleus motorius nervi trigemini in lampreys as a first step in the study of neurons that control feeding behavior in this species. To identify these neurons, the retrograde tracer cobalt-lysine was injected into the nucleus motorius nervi trigemini on one side of the in vitro isolated brainstem preparation of seven spawning adult lampreys (Petromyzon marinus). Transport times ranged from 42 to 48 h. Retrogradely labeled neurons were found within the rostral spinal cord, the rhombencephalon, the mesencephalon and the caudal diencephalon. This study concentrates on the labeled neurons in the rhombencephalon, since the essential circuits for mastication and swallowing are confined to this region in higher vertebrates. Within the rhombencephalon, labeled cells were in the nucleus sensibilis nervi trigemini on both sides. A densely packed column of labeled neurons was found medial to the nucleus motorius nervi trigemini on the ipsilateral side, extending further rostrally in the isthmic region. Continuous columns of labeled cells were observed in the lateral reticular formation on each side in the basal plate ventral to rhombencephalic cranial motor nuclei. They extended from the rostral trigeminal region down into the rostral spinal cord. A comparison with data from cats and rats shows that the distribution of neurons that project to the nucleus motorius nervi trigemini is very similar in mammals and in agnathes. We conclude that the organization of the motor command network of the trigeminal system is well preserved throughout phylogeny and that the in vitro isolated brainstem of lampreys should be a useful model for the study of vertebrate feeding behavior.

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.

Spinal inputs from lateral columns to reticulospinal neurons in lampreys.

This study characterizes the inputs from the lateral columns of the spinal cord to reticulospinal neurons in the lampreys, using the in vitro isolated brainstem and spinal cord preparation. Synaptic responses to the electrical stimulation of the lateral columns were recorded in reticulospinal neurons of the posterior and middle rhombencephalic reticular nuclei. The responses consisted of a mixture of excitation and inhibition. They were markedly potentiated when using trains of two to five pulses, suggesting that the larger part of these synaptic responses was mediated via an oligosynaptic pathway. An early component, however, persisted when using twin pulses at 10-20 Hz on the ipsilateral side, suggesting the presence of an early mono- or disynaptic component. When increasing the stimulation strength, an early fast rising excitatory component appeared. It most likely resulted from an antidromic activation of vestibulospinal axons in the lateral tracts, which make en passant synaptic contacts with reticulospinal neurons. Responses were practically abolished by adding CNQX and AP5 to the Ringer's solution. The late component of excitatory responses was decreased by AP5, suggesting that NMDA receptors were activated. The NMDA receptor-mediated component was larger when using trains of stimuli or in Mg2+-free Ringer's. The application of NMDA depolarized reticulospinal neurons. The glycinergic inhibitory component was markedly increased in Mg2+-free Ringer's. Moreover, GABAB-receptor activation with (-)-baclofen abolished both excitatory and inhibitory responses. Taken together, the present results indicate that ascending lateral column axons generate large excitatory and inhibitory synaptic potentials in reticulospinal neurons. The possible role of these inputs in modulating the activity of reticulospinal neurons during locomotion is discussed.

Electrophysiological and neuropharmacological study of tectoreticular pathways in lampreys.

Tectoreticular (TR) cells along the diencephalic-mesencephalic border are the origin of prominent crossed and uncrossed pathways that project to the middle (MRRN) and posterior (PRRN) rhombencephalic reticular nuclei in juvenile and adult lampreys [I.C. Zompa, R. Dubuc, Diencephalic and mesencephalic projections to rhombencephalic reticular nuclei in lampreys, Brain Res. (1998) in press.]. This study investigated the synaptic contacts between TR axons and the reticular cells. Intracellular recordings were carried out in reticular neurones (n=124) while microstimulating the TR regions. Tectoreticular inputs were recorded in all reticular cells studied (248 PSPs); although stronger responses were evoked in the MRRN neurones. The majority of responses were excitatory, but increasingly mixed and inhibitory when recorded in the middle and caudal part of the reticular nuclei. The excitation had the shortest onset latencies and sharpest slopes measured in both reticular nuclei, while the inhibition was longer and smoother. The characteristics of TR inputs to different reticular cell types is also presented. The transmission of evoked responses was isolated to the crossed and uncrossed TR pathways by studying the effects of 1% Xylocaine ejections and surgical lesions. The TR inputs were transmitted to reticular cells through monosynaptic and polysynaptic contacts. The synaptic transmission involved excitatory amino acids, acting through AMPA and NMDA receptors, while the inhibition was glycinergic. Comparisons with other sensory systems in lampreys are discussed.

Diencephalic and mesencephalic projections to rhombencephalic reticular nuclei in lampreys.

Behavioral studies in lampreys of the northern genera, Ichthyomyzon, reveal that sensory inputs initiate and modulate locomotion by activation of reticulospinal (RS) neurones, which constitute the primary descending system involved in motor activity. The interneurones relaying afferent vestibular, trigeminal, lateral line, cutaneous and proprioceptive inputs are localized in the rhombencephalic region of the lamprey brainstem, unlike the visual inputs that are relayed in the mesencephalic region. The knowledge of diencephalic-mesencephalic cell distributions that project to the RS neurones is limited. They were isolated by iontophoretically injecting cobalt-lysine in vitro into the middle (MRRN) and posterior (PRRN) rhombencephalic reticular nuclei of Petromyzon marinus and Ichthyomyzon unicuspis, Fourteen of 31 injections were successful (MRRN, 7; PRRN, 7). Cell groups were labeled ipsilateral to the injection site in the thalamus (corpi geniculati; pars dorsalis thalami lateralis and medialis; nucleus (n.) subhabenularis lateralis), in the epithalamus (n. commissura posteriori) and in the pretectum. Cell groups were labeled bilaterally within the dorsal region along the diencephalic-mesencephalic border (caudal pretectum and rostral tectum opticum), in tectum opticum, torus semicircularis, and tegmentum mesencephali. There were more backfilled cells from MRRN injections (538-6466 cells) than from PRRN injections (53-553 cells) (MW Rank Sum, p < 0.001). The cell bodies were less than 40 microns long ipsilateral to the injection site, and longer contralaterally. Those greater than 50 microns were backfilled from PRRN injections. The location and organization of the cell groups identified is comparable to that of other vertebrates.