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A physiological study of brainstem and peripheral inputs to trigeminal motoneurons in lampreys
Pergamon PII:
Neuroscience Vol. 91, No. 1, pp. 379–389, 1999 Copyright 䉷 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 S0306-4522(98)00634-4
A PHYSIOLOGICAL STUDY OF BRAINSTEM AND PERIPHERAL INPUTS TO TRIGEMINAL MOTONEURONS IN LAMPREYS D. PETROPOULOS,*† J. P. LUND*† and R. DUBUC*‡§ *Centre de recherche en sciences neurologiques, Universite´ de Montre´al, Montre´al, Que´bec, Canada, H3C 3J7 †Faculty of Dentistry, McGill University, Montre´al, Que´bec, Canada, H3A 2B2 ‡De´partement de Kinanthropologie, Universite´ du Que´bec a` Montre´al, C.P. 8888, Succ. Centre-ville, Montre´al, Que´bec, Canada, H3C 3P8
Abstract—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 mM) and 2-amino-5-phosphonopentanoate (200 mM) to the bath, suggesting participation of both a-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. 䉷 1999 IBRO. Published by Elsevier Science Ltd. Key words: trigeminal system, excitatory amino acids, electrotonic synapse, glycine, feeding behavior, lampreys.
Parasitic adult lampreys feed by rhythmical co-ordinated movements of a sucker equipped with numerous regularly arranged teeth, and a tonguelike structure called the apicalis. 12,15 Contraction of the annularis muscle around the toothed sucker, together with an intrastomal vacuum, allows blood-feeding species (such as Petromyzon marinus) to remain attached to larger fish for days. Forward and backward movements of the toothed apicalis are largely responsible for the destruction of the host tissue. 29 These specialized co-ordinated feeding mechanisms utilize a complex and unique group of muscles that control the sucker, apicalis, pharynx §To whom correspondence should be addressed at the De´partement de Kinanthropologie, Universite´ du Que´bec a` Montre´al. Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; 2-AP5, 2-amino-5-phosphonopentanoate; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; EMG, electromyogram; EPSP, excitatory postsynaptic potential; IPSP, inhibitory postsynaptic potential; NMDA, N-methyl-d-aspartate; nVm, nucleus motorius nervi trigemini; nVs, nucleus sensibilis nervi trigemini; RF, reticular formation; RFc, caudal reticular formation; RFi, intermediate reticular formation; RFr, rostral reticular formation.
and velum. 12 All of these muscles are innervated by trigeminal motoneurons. The anatomy of the trigeminal system in lampreys has been well described 20 and trigeminal motoneurons have been shown to supply only muscles on the ipsilateral side. 17 In a previous study, 14a the populations of brainstem neurons that project to the nucleus motorius nervi trigemini (nVm) of postmetamorphic lampreys were identified as a first step in characterizing the neuronal circuitry that controls trigeminal motoneurons. The retrograde tracer cobalt–lysine was injected in vitro in the nVm. Neurons were labeled in the contralateral nVm, as well as in the nucleus sensibilis nervi trigemini (nVs) on both sides. Further caudally, a continuous column of labeled cells was observed in the reticular formation (RF) on both sides, ventral and lateral to the rhombencephalic motor nuclei. The present study was undertaken to determine whether these groups of cells make synaptic contacts with trigeminal motoneurons, using microstimulation of these brainstem regions combined with intracellular recordings of the synaptic responses from trigeminal motoneurons. Some of the results
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of this study have been published in abstract form. 24,25 EXPERIMENTAL PROCEDURES
Experiments were performed on a total of 18 young adult lampreys (Petromyzon marinus; from the Great Lakes and Lake Champlain). All surgical and experimental procedures conform to the Canadian Medical Research Council guidelines and were approved by the University Animal Care and Use Committee. Each animal was anesthetized with tricaine methanesulfonate (MS-222; 100 mg/l) and eviscerated. All muscle tissue was resected and the brain and spinal cord (1–2 cm from the obex) was isolated in vitro, with the underlying cranium and notochord fixed to Sylgard gel at the bottom of a recording chamber filled with freshly oxygenated Ringer’s kept at 4–9⬚C. Trigeminal motoneurons were recorded intracellularly with sharp microelectrodes filled with 4 M potassium acetate (R 70–100 MV). The signals were amplified with a bridge circuit (Axoclamp 2A, Axon Instruments). Recording sites within the nVm were related to the somatotopic organization of motoneuronal pools according to Homma. 13 Glass-coated tungsten electrodes (5–15 mm exposed tip; 0.5–2.0 MV) connected to a stimulus isolation unit (PSIU6, Grass Instruments) were used to microstimulate the trigeminal nerves and the brainstem sites (for schematic representation, see Fig. 1A) that contained trigeminal premotor interneurons (Huard H. et al., unpublished observations). The nVs on the ipsilateral side was rarely stimulated because the insertion of the tungsten electrode disturbed the intracellular recording. Stimulated sites included both the nVm and nVs on the contralateral side and the RF on both sides, ventral to the rhombencephalic cranial motor nuclei. The RF was subdivided into three zones: rostral (RFr), intermediate (RFi) and caudal (RFc), according to Huard et al. (unpublished observations). The stimuli consisted of rectangular pulses of 1 ms duration applied every 3–6 s. Trains of two to three shocks (15–100 Hz) were used at intensities of 2 × threshold for synaptic responses (never exceeding 15 mA). In order to determine whether the RF synaptic responses resulted from the activation of cell bodies or fibers, we tested the effects of microejections of 10 mM dl-glutamate (Sigma). Micropipettes were fitted into a sealed holder attached to a micromanipulator and the tip was broken to a diameter of 20–30 mm. Fast Green (5%) was added to the glutamate solution for visualization. The solution was pressure ejected (Picospritzer, General Valve Corporation) over the RF sites and synaptic responses were recorded in trigeminal motoneurons (n 5). The size and location of the injections were redrawn during the experiment from the dissecting microscope placed over the preparation. In six experiments, suction electrodes were placed on the surface of the nVm, and in contact with the vagal nerve and selected ventral roots, to determine whether nVm membrane potential oscillations in one motoneuron were correlated with activity in other trigeminal motoneuron pools, respiratory or locomotor rhythms. Eight experiments were carried out to identify the transmitters and the receptor subtypes involved in generating the synaptic responses in trigeminal motoneurons from both trigeminal afferents and from the different brainstem sites. The effects on these responses of antagonists to amino acid transmission and manipulations of ion concentrations in Ringer’s solution were investigated. The following compounds were used: 6-cyano-7-nitroquinoxaline-2,3dione (CNQX; 10 mM; Research Biochemicals International), an a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist; 2-amino-5phosphonopentanoate (2-AP5; 200 mM; Research Biochemicals International), an N-methyl-d-aspartate (NMDA)
receptor antagonist, and strychnine (5 mM; Sigma), a glycine receptor antagonist. Mg 2⫹-free Ringer’s was used in some experiments to enhance NMDA responses. To identify electrotonic components, Ca 2⫹ ions were removed from the bath. In four experiments, the muscles controlling the sucker were left intact (see Fig. 6A) and electromyograms (EMGs) were recorded with bipolar 100-mm Teflon-coated stainless steel wires inserted into the left and right sides of the annularis muscle. Brief pressure (less than 1 s) was applied to the teeth and the oral fimbriae with a fine smooth-tipped probe. The intracellular and extracellular nerve recordings and EMGs were amplified and stored on a VHS tape via a digitizing board (Vetter Digital 4000A) for later analysis. Area measurements of the synaptic responses were made using Microcal Origin software (version 4.0). The response latencies were not normally distributed, and the median was used to describe central tendencies rather than the mean. Differences between groups were tested using a Mann– Whitney rank order test. Measurements of burst and cycle durations were made with Axoscope version 1.1 software (Axon Instruments). RESULTS
Effects of stimulating trigeminal afferents Synaptic responses evoked by electrical stimulation of the trigeminal nerves and the different rhombencephalic sites were recorded in 64 trigeminal motoneurons. The responses were predominantly excitatory (Table 1). Pure inhibitory potentials were only recorded in two motoneurons, and four mixed responses were found. Stimulation of either trigeminal nerve (n 28) typically caused sharply rising short-latency excitatory postsynaptic potentials (EPSPs; arrows in Fig. 1B1, B4) followed by a subsequent low-amplitude excitatory component, occasionally mixed with inhibition upon stimulation of the ipsilateral side. Overall, the ipsilateral latency was significantly shorter (median 8.0 ms vs 11.3 ms; P ⬍ 0.01). The early ipsilateral trigeminal nerve-evoked EPSP followed twin pulse stimulation at 20 and 30 Hz (arrows in Fig. 1B2, B3), suggesting a monosynaptic connection, but the response from the contralateral side did not follow high frequency (Fig. 1B4, B5, B6). The role of AMPA receptors was tested by adding 10 mM CNQX to the perfusate, which greatly depressed the amplitude of the early EPSPs caused by stimulation of both trigeminal nerves (n 5; Fig. 1C). Upon stimulating the ipsilateral trigeminal nerve, a slow-rising, long-lasting depolarizing potential remained after CNQX (arrow in Fig. 1C), but this was abolished by the specific NMDA receptor antagonist 2-AP5 (200 mM). This suggests that, under control conditions, the late excitation is reduced by inhibition. This was confirmed by adding strychnine, which increased the area of the response dramatically (380%; Fig. 1D, left panel). Adding CNQX and 2-AP5 to a bath already containing strychnine abolished all synaptic responses to the ipsilateral trigeminal nerve. CNQX did not enhance responses to contralateral trigeminal nerve
Inputs to trigeminal motoneurons in lampreys
Fig. 1. (A) Illustration showing all the rhombencephalic sites stimulated while recording intracellularly from trigeminal motoneurons. Abbreviations: cont, contralateral; ipsi, ipsilateral; V, trigeminal; RF r., RF i., RF c., rostral, intermediate and caudal reticular formation, respectively; n. V m., contralateral nucleus motorius nervi trigemini; n. V s., contralateral nucleus sensibilis nervi trigemini; MRRN and PRRN, middle and posterior rhombencephalic reticular nucleus, respectively. (B) Synaptic responses of trigeminal motoneurons to stimulation of ipsilateral and contralateral trigeminal nerve at different frequencies. (C, D) Effects of CNQX, 2-AP5 and strychnine on trigeminal nerve responses. Each trace is the average of eight responses. The control response was redrawn (dotted line) over the response under CNQX in C.
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Site of stimulation
Motoneurons
EPSP
IPSP
Oscillatory behavior
Ipsilateral Trigeminal nerve RFr RFi RFc
27 22 9 10
27 22 9 10
2 1 0 0
12 0 0 0
Contralateral Trigeminal nerve nVm nVs RFr RFi RFc
28 31 30 10 6 6
28 30 29 10 6 6
0 1 2 0 0 0
13 0 14 0 0 0
stimulation and strychnine was also without effect (Fig. 1D, right panel). Effects of stimulating brainstem sites Contralateral nVs, nVm and ipsilateral RFr inputs were studied in most detail, because they had stronger postsynaptic effects than those from more caudal sites. EPSPs caused by the stimulation of the contralateral nVs (n 29) and contralateral nVm (n 30) followed twin pulse stimulation at 15 Hz, and showed evidence of temporal summation when trains of three shocks at 100 Hz were used (not shown). The rising phase of the synaptic response to the ipsilateral RFr was particularly sharp (Fig. 2A, B, Fig. 3A) and was followed, at times, by a second excitatory response (Fig. 2B; arrow in Fig. 3A). The earliest component followed stimulation up to 100 Hz, but only the late component showed temporal summation with trains of stimuli (not shown). All EPSPs (Fig. 2A) were depressed significantly by CNQX (10 mM; n 4). The contralateral nVs response was almost abolished and the area of the nVm response was reduced significantly (70%). In the case of the ipsilateral RFr, a sharp early component remained (Fig. 2A), although there was also a clear depression (64%). EPSPs were prolonged in Mg 2⫹-free Ringer’s, an effect that was reversed by 2-AP5 (200 mM), suggesting the presence of NMDA receptors in the synaptic pathway (Fig. 2B; n 3). In the cases of the nVm and ipsilateral RFr, a presumed inhibitory component after the early excitation was seen under Mg 2⫹-free Ringer’s (Fig. 2B, arrows). EPSPs could be evoked by stimulation of the RF on both sides from just caudal to the nVm to the obex (Fig. 3A). The latency was shortest on the ipsilateral side and it progressively increased as the stimulating electrode was moved caudally. When the ipsilateral RF was stimulated (n 5) in the absence of Ca 2⫹ ions, the earliest fast-rising excitatory component remained (Fig. 3B). This component was also CNQX resistant (Fig. 2A, right panel). The
larger, long-lasting excitatory component was absent in the Ca 2⫹-free solution. When glutamate was microejected into the RF (Fig. 4), large EPSPs (3–10 mV) with a latency ranging from 0.2 to 1.8 s were evoked in all five cells tested. The time to peak ranged from 0.9 to 3 s and the responses persisted for 7–12 s. Responses to stimulation of the contralateral RFr, contralateral RFc and ipsilateral RFr were biphasic. Spread of the green stain in the glutamate solution never exceeded 200 mm around the tip of the electrode, which was never closer than 1.0 mm to the recorded cell. Rhythmical activity Membrane potential oscillations were evoked by low-intensity shocks (0.6–4.0 mA) to the trigeminal nerves or contralateral nVs (Fig. 5A) in 27 motoneurons. In these cases, the initial synaptic responses were not followed by full repolarization. Instead, depolarization increased gradually until a series of oscillations began. These lasted from 20 s to 4 min (Fig. 5A). The oscillations were usually triggered by a single stimulus, but sometimes repetitive stimulation was needed. Stimulation of the other brainstem sites never elicited oscillatory activity (Fig. 5A, Table 1). The rhythms fell into two frequency ranges: high (n 17), ranging from 1.0 to 2.8 Hz (Fig. 5B1), and low (n 15), between 0.1 and 0.5 Hz (Fig. 5B2). Five motoneurons showed both types of oscillations (Fig. 5Ca–e) and switches from one type to another could occur within the same sequence (Fig. 5B3). The division into high- and low-frequency cycles was not arbitrary. As can be seen in the plot of the frequency distribution of all the oscillations that were recorded, a gap separates the low- and highfrequency ranges (Fig. 5C, arrow in top panel). The mean peak-to-peak amplitudes are also separated by a gap (Fig. 5C, arrow in bottom panel). The highfrequency oscillatory potentials ranged in amplitude from 1.8 to 5.1 mV, whereas those of the lowfrequency oscillations ranged from 8.5 to 17 mV.
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Fig. 2. Synaptic responses elicited by stimulation of the contralateral nVs, contralateral nVm and ipsilateral RFr. (A) Effect of CNQX. Traces are averages of five responses on each condition. (B) Effect of Mg 2⫹-free Ringer’s and 2-AP5. Averages of five responses per trace. For abbreviations, see legend to Fig. 1.
Intracellular motoneuronal membrane potentials were compared with activity in (i) populations of trigeminal motoneurons recorded with a surface electrode, (ii) the vagal nerve and (iii) one or two rostral ventral roots. The top panel of Fig. 5D shows that membrane potential oscillations caused by the stimulation of the contralateral trigeminal nerve were synchronous with bursts of activity in the ipsilateral nVm and from the ipsiventral roots immediately after the stimulus. However, there were no bursts in the ventral root corresponding to the three subsequent bursts seen in the ipsilateral nVm and in the trigeminal motoneuron. When rhythmical activity was present in all three motoneuronal groups (trigeminal, vagal and spinal), the membrane potential oscillations in the trigeminal motoneurons seemed to be independent of either of the other two rhythms in 75% of cases (Fig. 5D, middle panel).
Oscillations were seen in motoneurons in the regions of the nVm that innervate the basilaris/ velar (44%), apicalis (66%) and annularis (52%) muscles. Moreover, both types (low and high frequencies) could be triggered from either of the trigeminal nerves or contralateral nVs, suggesting that the rhythms were not specific to a particular input. Simultaneous recordings of intracellular motoneuronal membrane potentials from the annularis region of the nVm and burst activity from the other two motoneuronal populations of the ipsilateral nVm were made in a brain transected at the level of the obex to cut inputs from the spinal cord (Fig. 5D, bottom panel). These showed that rhythmic spiking of the annularis motoneuron was synchronous with bursts of activity in the basilaris/ velum and apicalis populations of motoneurons. Brief mechanical stimuli applied to the teeth or
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Fig. 3. (A) Synaptic responses elicited in trigeminal motoneurons by stimulation of six sites in the RF. Each record includes six to 10 superimposed traces for each case. Histograms of latency of the first excitatory component are presented on the right. (B) Responses to the stimulation of three RF sites in normal Ringer’s and after a 55-min exposure in Ca 2⫹-free Ringer’s. For abbreviations, see legend to Fig. 1.
the fimbriae of the sucker (Fig. 6A) sometimes caused synchronous bursts of EMG activity on both sides of the annularis muscle in the semi-intact preparation (Fig. 6B). In 57% of the cases, the first burst was followed by one to four others (Fig. 6C1), and the frequency varied between 0.1 and 1.5 Hz (median 0.32 Hz; Fig. 6C2). The bursts could last for up to 7 s (median 2.1 s; Fig. 6C3), but their duration was very variable. DISCUSSION
The present study shows that electrical stimulation of trigeminal afferents and several brainstem sites that contain premotor interneurons 14a elicits short-latency synaptic responses in trigeminal motoneurons. It was particularly interesting to find that stimulation of either trigeminal nerve or the contralateral nVs often triggered membrane potential oscillations.
Synaptic responses to stimulation of primary afferents and cell groups Stimulation of trigeminal afferents on the ipsilateral side elicited large excitatory synaptic responses in trigeminal motoneurons. The responses were characterized by an early component with a short time to peak that could be monosynaptic. This component was not an antidromic initial segment spike, since its amplitude varied with stimulus intensity and it was abolished by CNQX. It followed twin pulse stimulation at a frequency of 30 Hz, which is far beyond the 10 Hz frequency which has been suggested as the cut-off point for monosynaptic conduction in this species. 27 This suggests that trigeminal motoneurons receive monosynaptic inputs from primary afferents. In this respect, they resemble fin motoneurons, which have been shown 10 to receive monosynaptic inputs from dorsal cells, a particular group of primary afferent neurons with
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Fig. 4. Synaptic responses evoked in a trigeminal motoneuron by local pressure ejection of 10 mM dl-glutamate at five RF sites. Arrows show onsets of pressure ejection. Shaded areas represent the approximate spread of glutamate at each site. For abbreviations, see legend to Fig. 1.
cell bodies in the CNS. 6 However, the vast majority of lamprey motoneurons are not excited monosynaptically by sensory afferents. The contralateral trigeminal nerve excitatory inputs appear to be at least disynaptic, which is not surprising since primary afferent fibers project only on the ipsilateral side of the lamprey brainstem. 11,17,22 The large amplitude and relatively short latency of the responses suggest that the pathway includes commissural interneurons in the nVs (Huard H. et al., unpublished observations), which appear to be the shortest link between the afferent terminals in the nucleus descendens nervi trigemini and motoneurons in the contralateral nVm. The responses in trigeminal motoneurons to trigeminal afferent inputs appear to be mediated by excitatory amino acids. The early component
depends on AMPA receptors. The late, long-lasting depolarization that is enhanced by CNQX is NMDA mediated, since it disappears after 2-AP5. Afferent inputs to trigeminal motoneurons in mammals also depend on AMPA and NMDA receptors. 5 The longlasting depolarization that was enhanced with CNQX when stimulating the ipsilateral nerve suggests that CNQX eliminated a concurrent inhibitory postsynaptic potential (IPSP). The simplest hypothesis is that the IPSP is due to an AMPAmediated excitation of inhibitory premotor neurons by primary afferents, and these are likely to be glycinergic, since the depolarization was greatly enhanced by strychnine. In mammals, glycinergic interneurons that project to the nVm have been found in regions that receive trigeminal afferent inputs, particularly the rostral division of the
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Fig. 5. Membrane potential oscillations elicited in trigeminal motoneurons. (A) Slow frequency oscillations were elicited by a single pulse stimulation (1.0 mA) to the contralateral nVs (top trace). Single pulses (1.0 mA) delivered to the contralateral nVm or ipsilateral RFr did not trigger membrane potential oscillations (middle and lower traces). (B1) High-frequency oscillations after one pulse (3.0 mA) to the contralateral trigeminal nerve. (B2) Low-frequency oscillations elicited by stimulation (1.0 mA) of the contralateral nVs. (B3) Low- and highfrequency oscillations after stimulation (3.6 mA) of the ipsilateral trigeminal nerve. (C) Distribution of the oscillatory events (mean ^ S.D.) according to frequency (top panel) and amplitude (bottom panel). Events are arranged in order of increasing oscillatory frequency. Events marked with the same letter were recorded in the same motoneuron. (D1) Simultaneous recordings of the membrane potential in a trigeminal motoneuron (top trace) and of surface potentials in the same (middle trace) and an ipsilateral ventral root (bottom trace). (D2) Simultaneous recordings of the membrane potential in a trigeminal motoneuron (top trace), of an ipsilateral ventral root (middle trace) and of the vagal (x) nerve (bottom trace). (D3) Simultaneous recordings of the membrane potential in an annularis motoneuron (top trace) and of surface potentials from basilaris/velum (middle trace) and apicalis (bottom trace) motoneuronal pools.
trigeminal spinal nucleus, the rostral division of the parvocellular RF (which corresponds to our RFr group) and the shell of neurons that surround the nVm. 30 Stimulation of cells in the latter region in brainstem slices causes IPSPs in trigeminal motoneurons that are sensitive to strychnine. 16 We have already commented on the fact that all levels of the lateral RF contain trigeminal premotor interneurons in mammals 18 and lampreys. 14a The present data show that stimulation of all levels of the RF on both sides elicits EPSPs in trigeminal motoneurons, and that responses from rostral sites
are not caused solely by stimulation of fibers of passage from the RFc, since local ejections of glutamate are also effective. The early EPSPs evoked by ipsilateral RF stimulation were of very short latency when elicited from rostral sites. The earliest response had a fast rise time, even when the RFc was stimulated. Entrainment of the early EPSP was observed without attenuation or amplification at 100 Hz, and it remained in Ca 2⫹-free Ringer’s. This is strong evidence that this component is electrotonic. The second chemical component was of slightly longer latency and was potentiated by
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Fig. 6. Rhythmical EMG activity recorded in the annularis muscle after mechanical stimulation of the oral disc. (A) Drawing showing the semi-intact decerebrate preparation, with stimulation and recording sites. (B) EMG bursts elicited by stimulation (arrow) of fimbriae on the right side. (C) Histograms prepared with data from four experiments. (C1) Number of bursts. (C2) Frequency. (C3) Burst duration.
repetitive stimulation. As with nVs and trigeminal nerve stimulation, RF responses involve excitatory amino acid transmission of both AMPA and NMDA receptor subtypes. As in mammals, 4 inputs from the RF were not exclusively excitatory. Inhibition was revealed by using Mg 2⫹-free Ringer’s, an observation noted previously for inputs to reticulospinal
neurons in lampreys. 9 Inhibitory inputs from the RF impinging upon trigeminal motoneurons have also been observed in higher vertebrates. Electrotonic synapses are not rare in lamprey and mixed electrotonic/chemical postsynaptic potentials have been found in the brainstem and the spinal cord. Reticulospinal cells receive mixed electrical/
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chemical inputs from the cells within the alar plate relaying vestibular inputs, 1,2,28 and in turn make similar synaptic contacts with myotomal motoneurons and interneurons of the spinal cord. 3,23,27 Stimulation of the nVm also elicited large EPSPs in contralateral trigeminal motoneurons. Although glutamate ejections also evoked a similar response (Petropoulos D., Lund J. P. and Dubuc R., unpublished observations), it is unlikely that stimulation of motoneurons caused this response, because they do not project to the opposite nucleus. 14a However, there are many small neurons within and surrounding the nVm in lampreys and mammals that do project to the contralateral side. 7,8,14,14a,19,21,31 Some of these have recently been shown to act on ipsilateral trigeminal motoneurons through AMPA receptors in mammals. 16 Oscillations The two patterns of membrane potential oscillations recorded in trigeminal motoneurons in the in vitro isolated brainstem preparation may be the neural correlates of feeding patterns observed in intact lampreys. Feeding behavior in this species is characterized by rhythmical movements of the apicalis, accompanied by co-contraction of the major head muscles that control the sucker, and of the basilaris muscle, which regulates the volume of the pharynx. 12,15,29 To accomplish this, the annularis, basilaris and apicalis motoneurons of the nVm must be active during feeding movements. We found that oscillatory potentials could be triggered in motoneurons in the three regions of the nVm that supply these muscles. We also observed synchronous rhythmical activity in the three groups of motoneurons. The basic feeding rhythm of intact animals varies between 0.05 and 0.3 Hz, which is compatible with the low-frequency oscillatory membrane potentials recorded in trigeminal motoneurons in the isolated brainstem, but bouts of higher frequency movements have also been observed. 15 Suction measurements in the intact animals revealed that the low-frequency cycles generated much higher pressure than the higher frequency cycles. 15 It is therefore of interest that the low-frequency oscillations in trigeminal motoneurons recorded in this study had twice the amplitude of the high-frequency potentials. Although we have observed that trigeminal motoneurons can be entrained by the locomotor rhythm in
vitro (Bussie`res N., Lund J. P. and Dubuc R., unpublished observations), membrane potential oscillations of nVm neurons were usually seen in the absence of activity in the ventral roots in the present experiments. Locomotor bouts were not frequent during trigeminal oscillatory activity elicited by stimulation of the trigeminal nerves or contralateral nVs, and in most cases when bursts of fictive locomotion were evoked, there was no co-ordination between the two rhythms. It was also clear that, most of the time, trigeminal motoneuron activity was independent of the respiratory rhythm. The motor activity elicited by mechanical stimulation of the teeth and oral fimbriae was recorded from the annularis muscle in semi-intact preparations. The frequency of the rhythmical muscle activity in the head was within the range of the low-frequency oscillations elicited in trigeminal motoneurons in isolated brainstem preparations. This suggests that sensory inputs from the head region play an important role in the control of feeding, as they do in higher vertebrates. 26 CONCLUSIONS
This paper presents evidence that neurons in regions of the lamprey brainstem that are retrogradely labeled after injections of cobalt–lysine into the trigeminal motor nucleus 14a have postsynaptic effects on trigeminal motoneurons. Most neurons are excitatory and exert their action through the release of excitatory amino acids that act on AMPA and NMDA postsynaptic receptor subtypes. Inputs from neurons of the ipsilateral RF include an electrotonic component. Glycinergic inhibition from a smaller number of sites contributes to the trigeminal motoneuronal response. Rhythmical membrane potential fluctuations that can be triggered in trigeminal motoneurons by sensory inputs appear to be the corollary of feeding behavior in this species. Acknowledgements—This work was supported by a Group Grant (Neurological Sciences) from the Canadian Medical Research Council, as well as from FCAR (Que´bec). We wish to express our gratitude to D. Veilleux and S. Lepage for help with the experiments, and to J. Jodoin and G. Delforge for technical assistance. We are also grateful to S. Dupuis and E´. Cle´ment for computer programming. We would like to express our gratitude to J. E. Gersmehl from the U.S. Fish and Wildlife Service, as well as to Dr J. G. Seelye, Mr W. D. Swink and Mrs M. K. Jones from the Lake Huron Biological Station for the kind supply of lampreys.
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Chandler S. H. and Trueblood P. R. (1995) Pharmacological mechanisms controlling trigeminal motoneuronal excitability. In Brain and Oral Functions (eds Morimoto T., Matsuya T. and Takada K.), pp. 99–105. Elsevier, Amsterdam. ˚ . and Grillner S. (1988) The dorsal cell, one class of primary sensory neuron in the lamprey 6. Christenson J., Lagerba¨ck P.-A spinal cord. II. A light and electron microscopical study. Brain Res. 440, 9–17. 7. Donga R. and Lund J. P. (1991) Discharge patterns of trigeminal commissural last-order interneurons during locomotion. J. Neurophysiol. 66, 1564–1578. 8. Donga R., Lund J. P. and Veilleux D. (1990) An electrophysiological study of trigeminal commissural interneurons in the anaesthetized rabbit. Brain Res. 515, 351–354. 9. Dubuc R., Ohta Y. and Grillner S. (1988) Supraspinal and spinal inputs to the posterior rhombencephalic reticular nucleus (PRRN): anatomy, physiology and pharmacology. Acta physiol. scand. 134 (Suppl. 575), 39. 10. El Manira A., Shupliakov O., Fagerstedt P. and Grillner S. (1996) Monosynaptic input from cutaneous sensory afferents to fin motoneurons in lamprey. J. comp. Neurol. 369, 533–542. 11. Finger T. E. and Rovainen C. M. (1982) Spinal and medullary dorsal cell axons in the trigeminal nerve in lampreys. Brain Res. 240, 331–333. 12. Hardisty M. W. and Rovainen C. M. (1982) Morphological and functional aspects of the muscular system. In The Biology of Lampreys (eds Hardisty M. W. and Potter I. C.), Vol. 4A, pp. 137–231. Academic, London. 13. Homma S. (1978) Organization of the trigeminal motor nucleus before and after metamorphosis in lampreys. Brain Res. 140, 33–42. 14. Huard H., Lund J. P. and Dubuc R. (1995) A study of trigeminal premotor neurones in lampreys. Soc. Neurosci. Abstr. 21, 142. 14a. Huard H., Lund J. P., Veilleux D. and Dubuc R. (1999) An anatomical study of brainstem projections to the trigeminal motor nucleus of lampreys.. Neuroscience 91, 363–378. 15. Kawasaki R. and Rovainen C. M. (1988) Feeding behavior by parasitic phase lampreys, Ichthyomyzon unicuspis. Brain Behav. Evol. 32, 317–329. 16. Kolta A. (1997) In vitro investigation of synaptic relations between interneurons surrounding the trigeminal motor nucleus and masseteric motoneurons. J. Neurophysiol. 78, 1720–1725. 17. Koyama H., Kishida R., Goris R. C. and Kusunoki T. (1987) Organization of sensory and motor nuclei of the trigeminal nerve in lampreys. J. comp. Neurol. 264, 437–448. ˚ . and Westberg K.-G. (1986) Bulbar neurones with axonal projections to the trigeminal motor 18. Landgren S., Olsson K. A nucleus in the cat. Expl Brain Res. 65, 98–111. 19. Li Y.-Q., Takada M., Kaneko T. and Mizuno N. (1995) Premotor neurons for trigeminal motor nucleus neurons innervating the jaw-closing and jaw-opening muscles: differential distribution in the lower brainstem of the rabbit. J. comp. Neurol. 356, 563–579. 20. Lindstro¨m T. (1949) On the cranial nerves of the cyclostomes with special reference to n. trigeminus. Acta zool. 30, 315–458. 21. Mizuno N., Nomura S., Itoh K., Nakamura Y. and Konishi A. (1978) Commissural interneurons for masticatory motoneurons: a light and electron microscope study using the horseradish peroxidase tracer technique. Expl Neurol. 59, 254–262. 22. Northcutt R. G. (1979) Experimental determination of the primary trigeminal projections in lampreys. Brain Res. 163, 323– 327. 23. Ohta Y. and Grillner S. (1989) Monosynaptic excitatory amino acid transmission from the posterior rhombencephalic reticular nucleus to spinal neurons involved in the control of locomotion in lamprey. J. Neurophysiol. 62, 1079–1089. 24. Petropoulos D., Lund J. P. and Dubuc R. (1996) An electrophysiological study of brainstem projections to trigeminal motoneurons in lampreys. Soc. Neurosci. Abstr. 22, 1847. 25. Petropoulos D., Lund J. P. and Dubuc R. (1997) The neural correlate of fictive feeding in lampreys. Soc. Neurosci. Abstr. 23, 206. 26. Rossignol S., Lund J. P. and Drew T. (1988) The role of sensory inputs in regulating patterns of rhythmical movements in higher vertebrates. A comparison between locomotion, respiration, and mastication. In Neural Control of Rhhythmic Movements in Vertebrates (eds Cohen A. H., Rossignol S. and Grillner S.), pp. 201–283. John Wiley, New York. 27. Rovainen C. M. (1974) Synaptic interactions of reticulospinal neurons and nerve cells in the spinal cord of the sea lamprey. J. comp. Neurol. 154, 207–224. 28. Rovainen C. M. (1979) Electrophysiology of vestibulospinal and vestibuloreticulospinal systems in lampreys. J. Neurophysiol. 42, 745–765. 29. Rovainen C. M. (1996) Feeding and breathing in lampreys. Brain Behav. Evol. 48, 297–305. 30. Turman J. E. and Chandler S. H. (1994) Immunohistochemical evidence for GABA and glycine-containing trigeminal premotoneurons in the guinea pig. Synapse 18, 7–20. ˚ . (1995) Integration in trigeminal premotor interneurones in the cat. 3. Input 31. Westberg K.-G., Sandstro¨m G. and Olsson K. A characteristics and synaptic actions of neurones in subnucleus-g of the oral nucleus of the spinal trigeminal tract with a projection to the masseteric motoneurone subnucleus. Expl Brain Res. 104, 449–461. (Accepted 12 October 1998)
Neuroscience Vol. 91, No. 1, pp. 379–389, 1999 Copyright 䉷 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 S0306-4522(98)00634-4
A PHYSIOLOGICAL STUDY OF BRAINSTEM AND PERIPHERAL INPUTS TO TRIGEMINAL MOTONEURONS IN LAMPREYS D. PETROPOULOS,*† J. P. LUND*† and R. DUBUC*‡§ *Centre de recherche en sciences neurologiques, Universite´ de Montre´al, Montre´al, Que´bec, Canada, H3C 3J7 †Faculty of Dentistry, McGill University, Montre´al, Que´bec, Canada, H3A 2B2 ‡De´partement de Kinanthropologie, Universite´ du Que´bec a` Montre´al, C.P. 8888, Succ. Centre-ville, Montre´al, Que´bec, Canada, H3C 3P8
Abstract—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 mM) and 2-amino-5-phosphonopentanoate (200 mM) to the bath, suggesting participation of both a-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. 䉷 1999 IBRO. Published by Elsevier Science Ltd. Key words: trigeminal system, excitatory amino acids, electrotonic synapse, glycine, feeding behavior, lampreys.
Parasitic adult lampreys feed by rhythmical co-ordinated movements of a sucker equipped with numerous regularly arranged teeth, and a tonguelike structure called the apicalis. 12,15 Contraction of the annularis muscle around the toothed sucker, together with an intrastomal vacuum, allows blood-feeding species (such as Petromyzon marinus) to remain attached to larger fish for days. Forward and backward movements of the toothed apicalis are largely responsible for the destruction of the host tissue. 29 These specialized co-ordinated feeding mechanisms utilize a complex and unique group of muscles that control the sucker, apicalis, pharynx §To whom correspondence should be addressed at the De´partement de Kinanthropologie, Universite´ du Que´bec a` Montre´al. Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; 2-AP5, 2-amino-5-phosphonopentanoate; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; EMG, electromyogram; EPSP, excitatory postsynaptic potential; IPSP, inhibitory postsynaptic potential; NMDA, N-methyl-d-aspartate; nVm, nucleus motorius nervi trigemini; nVs, nucleus sensibilis nervi trigemini; RF, reticular formation; RFc, caudal reticular formation; RFi, intermediate reticular formation; RFr, rostral reticular formation.
and velum. 12 All of these muscles are innervated by trigeminal motoneurons. The anatomy of the trigeminal system in lampreys has been well described 20 and trigeminal motoneurons have been shown to supply only muscles on the ipsilateral side. 17 In a previous study, 14a the populations of brainstem neurons that project to the nucleus motorius nervi trigemini (nVm) of postmetamorphic lampreys were identified as a first step in characterizing the neuronal circuitry that controls trigeminal motoneurons. The retrograde tracer cobalt–lysine was injected in vitro in the nVm. Neurons were labeled in the contralateral nVm, as well as in the nucleus sensibilis nervi trigemini (nVs) on both sides. Further caudally, a continuous column of labeled cells was observed in the reticular formation (RF) on both sides, ventral and lateral to the rhombencephalic motor nuclei. The present study was undertaken to determine whether these groups of cells make synaptic contacts with trigeminal motoneurons, using microstimulation of these brainstem regions combined with intracellular recordings of the synaptic responses from trigeminal motoneurons. Some of the results
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of this study have been published in abstract form. 24,25 EXPERIMENTAL PROCEDURES
Experiments were performed on a total of 18 young adult lampreys (Petromyzon marinus; from the Great Lakes and Lake Champlain). All surgical and experimental procedures conform to the Canadian Medical Research Council guidelines and were approved by the University Animal Care and Use Committee. Each animal was anesthetized with tricaine methanesulfonate (MS-222; 100 mg/l) and eviscerated. All muscle tissue was resected and the brain and spinal cord (1–2 cm from the obex) was isolated in vitro, with the underlying cranium and notochord fixed to Sylgard gel at the bottom of a recording chamber filled with freshly oxygenated Ringer’s kept at 4–9⬚C. Trigeminal motoneurons were recorded intracellularly with sharp microelectrodes filled with 4 M potassium acetate (R 70–100 MV). The signals were amplified with a bridge circuit (Axoclamp 2A, Axon Instruments). Recording sites within the nVm were related to the somatotopic organization of motoneuronal pools according to Homma. 13 Glass-coated tungsten electrodes (5–15 mm exposed tip; 0.5–2.0 MV) connected to a stimulus isolation unit (PSIU6, Grass Instruments) were used to microstimulate the trigeminal nerves and the brainstem sites (for schematic representation, see Fig. 1A) that contained trigeminal premotor interneurons (Huard H. et al., unpublished observations). The nVs on the ipsilateral side was rarely stimulated because the insertion of the tungsten electrode disturbed the intracellular recording. Stimulated sites included both the nVm and nVs on the contralateral side and the RF on both sides, ventral to the rhombencephalic cranial motor nuclei. The RF was subdivided into three zones: rostral (RFr), intermediate (RFi) and caudal (RFc), according to Huard et al. (unpublished observations). The stimuli consisted of rectangular pulses of 1 ms duration applied every 3–6 s. Trains of two to three shocks (15–100 Hz) were used at intensities of 2 × threshold for synaptic responses (never exceeding 15 mA). In order to determine whether the RF synaptic responses resulted from the activation of cell bodies or fibers, we tested the effects of microejections of 10 mM dl-glutamate (Sigma). Micropipettes were fitted into a sealed holder attached to a micromanipulator and the tip was broken to a diameter of 20–30 mm. Fast Green (5%) was added to the glutamate solution for visualization. The solution was pressure ejected (Picospritzer, General Valve Corporation) over the RF sites and synaptic responses were recorded in trigeminal motoneurons (n 5). The size and location of the injections were redrawn during the experiment from the dissecting microscope placed over the preparation. In six experiments, suction electrodes were placed on the surface of the nVm, and in contact with the vagal nerve and selected ventral roots, to determine whether nVm membrane potential oscillations in one motoneuron were correlated with activity in other trigeminal motoneuron pools, respiratory or locomotor rhythms. Eight experiments were carried out to identify the transmitters and the receptor subtypes involved in generating the synaptic responses in trigeminal motoneurons from both trigeminal afferents and from the different brainstem sites. The effects on these responses of antagonists to amino acid transmission and manipulations of ion concentrations in Ringer’s solution were investigated. The following compounds were used: 6-cyano-7-nitroquinoxaline-2,3dione (CNQX; 10 mM; Research Biochemicals International), an a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist; 2-amino-5phosphonopentanoate (2-AP5; 200 mM; Research Biochemicals International), an N-methyl-d-aspartate (NMDA)
receptor antagonist, and strychnine (5 mM; Sigma), a glycine receptor antagonist. Mg 2⫹-free Ringer’s was used in some experiments to enhance NMDA responses. To identify electrotonic components, Ca 2⫹ ions were removed from the bath. In four experiments, the muscles controlling the sucker were left intact (see Fig. 6A) and electromyograms (EMGs) were recorded with bipolar 100-mm Teflon-coated stainless steel wires inserted into the left and right sides of the annularis muscle. Brief pressure (less than 1 s) was applied to the teeth and the oral fimbriae with a fine smooth-tipped probe. The intracellular and extracellular nerve recordings and EMGs were amplified and stored on a VHS tape via a digitizing board (Vetter Digital 4000A) for later analysis. Area measurements of the synaptic responses were made using Microcal Origin software (version 4.0). The response latencies were not normally distributed, and the median was used to describe central tendencies rather than the mean. Differences between groups were tested using a Mann– Whitney rank order test. Measurements of burst and cycle durations were made with Axoscope version 1.1 software (Axon Instruments). RESULTS
Effects of stimulating trigeminal afferents Synaptic responses evoked by electrical stimulation of the trigeminal nerves and the different rhombencephalic sites were recorded in 64 trigeminal motoneurons. The responses were predominantly excitatory (Table 1). Pure inhibitory potentials were only recorded in two motoneurons, and four mixed responses were found. Stimulation of either trigeminal nerve (n 28) typically caused sharply rising short-latency excitatory postsynaptic potentials (EPSPs; arrows in Fig. 1B1, B4) followed by a subsequent low-amplitude excitatory component, occasionally mixed with inhibition upon stimulation of the ipsilateral side. Overall, the ipsilateral latency was significantly shorter (median 8.0 ms vs 11.3 ms; P ⬍ 0.01). The early ipsilateral trigeminal nerve-evoked EPSP followed twin pulse stimulation at 20 and 30 Hz (arrows in Fig. 1B2, B3), suggesting a monosynaptic connection, but the response from the contralateral side did not follow high frequency (Fig. 1B4, B5, B6). The role of AMPA receptors was tested by adding 10 mM CNQX to the perfusate, which greatly depressed the amplitude of the early EPSPs caused by stimulation of both trigeminal nerves (n 5; Fig. 1C). Upon stimulating the ipsilateral trigeminal nerve, a slow-rising, long-lasting depolarizing potential remained after CNQX (arrow in Fig. 1C), but this was abolished by the specific NMDA receptor antagonist 2-AP5 (200 mM). This suggests that, under control conditions, the late excitation is reduced by inhibition. This was confirmed by adding strychnine, which increased the area of the response dramatically (380%; Fig. 1D, left panel). Adding CNQX and 2-AP5 to a bath already containing strychnine abolished all synaptic responses to the ipsilateral trigeminal nerve. CNQX did not enhance responses to contralateral trigeminal nerve
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Fig. 1. (A) Illustration showing all the rhombencephalic sites stimulated while recording intracellularly from trigeminal motoneurons. Abbreviations: cont, contralateral; ipsi, ipsilateral; V, trigeminal; RF r., RF i., RF c., rostral, intermediate and caudal reticular formation, respectively; n. V m., contralateral nucleus motorius nervi trigemini; n. V s., contralateral nucleus sensibilis nervi trigemini; MRRN and PRRN, middle and posterior rhombencephalic reticular nucleus, respectively. (B) Synaptic responses of trigeminal motoneurons to stimulation of ipsilateral and contralateral trigeminal nerve at different frequencies. (C, D) Effects of CNQX, 2-AP5 and strychnine on trigeminal nerve responses. Each trace is the average of eight responses. The control response was redrawn (dotted line) over the response under CNQX in C.
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Site of stimulation
Motoneurons
EPSP
IPSP
Oscillatory behavior
Ipsilateral Trigeminal nerve RFr RFi RFc
27 22 9 10
27 22 9 10
2 1 0 0
12 0 0 0
Contralateral Trigeminal nerve nVm nVs RFr RFi RFc
28 31 30 10 6 6
28 30 29 10 6 6
0 1 2 0 0 0
13 0 14 0 0 0
stimulation and strychnine was also without effect (Fig. 1D, right panel). Effects of stimulating brainstem sites Contralateral nVs, nVm and ipsilateral RFr inputs were studied in most detail, because they had stronger postsynaptic effects than those from more caudal sites. EPSPs caused by the stimulation of the contralateral nVs (n 29) and contralateral nVm (n 30) followed twin pulse stimulation at 15 Hz, and showed evidence of temporal summation when trains of three shocks at 100 Hz were used (not shown). The rising phase of the synaptic response to the ipsilateral RFr was particularly sharp (Fig. 2A, B, Fig. 3A) and was followed, at times, by a second excitatory response (Fig. 2B; arrow in Fig. 3A). The earliest component followed stimulation up to 100 Hz, but only the late component showed temporal summation with trains of stimuli (not shown). All EPSPs (Fig. 2A) were depressed significantly by CNQX (10 mM; n 4). The contralateral nVs response was almost abolished and the area of the nVm response was reduced significantly (70%). In the case of the ipsilateral RFr, a sharp early component remained (Fig. 2A), although there was also a clear depression (64%). EPSPs were prolonged in Mg 2⫹-free Ringer’s, an effect that was reversed by 2-AP5 (200 mM), suggesting the presence of NMDA receptors in the synaptic pathway (Fig. 2B; n 3). In the cases of the nVm and ipsilateral RFr, a presumed inhibitory component after the early excitation was seen under Mg 2⫹-free Ringer’s (Fig. 2B, arrows). EPSPs could be evoked by stimulation of the RF on both sides from just caudal to the nVm to the obex (Fig. 3A). The latency was shortest on the ipsilateral side and it progressively increased as the stimulating electrode was moved caudally. When the ipsilateral RF was stimulated (n 5) in the absence of Ca 2⫹ ions, the earliest fast-rising excitatory component remained (Fig. 3B). This component was also CNQX resistant (Fig. 2A, right panel). The
larger, long-lasting excitatory component was absent in the Ca 2⫹-free solution. When glutamate was microejected into the RF (Fig. 4), large EPSPs (3–10 mV) with a latency ranging from 0.2 to 1.8 s were evoked in all five cells tested. The time to peak ranged from 0.9 to 3 s and the responses persisted for 7–12 s. Responses to stimulation of the contralateral RFr, contralateral RFc and ipsilateral RFr were biphasic. Spread of the green stain in the glutamate solution never exceeded 200 mm around the tip of the electrode, which was never closer than 1.0 mm to the recorded cell. Rhythmical activity Membrane potential oscillations were evoked by low-intensity shocks (0.6–4.0 mA) to the trigeminal nerves or contralateral nVs (Fig. 5A) in 27 motoneurons. In these cases, the initial synaptic responses were not followed by full repolarization. Instead, depolarization increased gradually until a series of oscillations began. These lasted from 20 s to 4 min (Fig. 5A). The oscillations were usually triggered by a single stimulus, but sometimes repetitive stimulation was needed. Stimulation of the other brainstem sites never elicited oscillatory activity (Fig. 5A, Table 1). The rhythms fell into two frequency ranges: high (n 17), ranging from 1.0 to 2.8 Hz (Fig. 5B1), and low (n 15), between 0.1 and 0.5 Hz (Fig. 5B2). Five motoneurons showed both types of oscillations (Fig. 5Ca–e) and switches from one type to another could occur within the same sequence (Fig. 5B3). The division into high- and low-frequency cycles was not arbitrary. As can be seen in the plot of the frequency distribution of all the oscillations that were recorded, a gap separates the low- and highfrequency ranges (Fig. 5C, arrow in top panel). The mean peak-to-peak amplitudes are also separated by a gap (Fig. 5C, arrow in bottom panel). The highfrequency oscillatory potentials ranged in amplitude from 1.8 to 5.1 mV, whereas those of the lowfrequency oscillations ranged from 8.5 to 17 mV.
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Fig. 2. Synaptic responses elicited by stimulation of the contralateral nVs, contralateral nVm and ipsilateral RFr. (A) Effect of CNQX. Traces are averages of five responses on each condition. (B) Effect of Mg 2⫹-free Ringer’s and 2-AP5. Averages of five responses per trace. For abbreviations, see legend to Fig. 1.
Intracellular motoneuronal membrane potentials were compared with activity in (i) populations of trigeminal motoneurons recorded with a surface electrode, (ii) the vagal nerve and (iii) one or two rostral ventral roots. The top panel of Fig. 5D shows that membrane potential oscillations caused by the stimulation of the contralateral trigeminal nerve were synchronous with bursts of activity in the ipsilateral nVm and from the ipsiventral roots immediately after the stimulus. However, there were no bursts in the ventral root corresponding to the three subsequent bursts seen in the ipsilateral nVm and in the trigeminal motoneuron. When rhythmical activity was present in all three motoneuronal groups (trigeminal, vagal and spinal), the membrane potential oscillations in the trigeminal motoneurons seemed to be independent of either of the other two rhythms in 75% of cases (Fig. 5D, middle panel).
Oscillations were seen in motoneurons in the regions of the nVm that innervate the basilaris/ velar (44%), apicalis (66%) and annularis (52%) muscles. Moreover, both types (low and high frequencies) could be triggered from either of the trigeminal nerves or contralateral nVs, suggesting that the rhythms were not specific to a particular input. Simultaneous recordings of intracellular motoneuronal membrane potentials from the annularis region of the nVm and burst activity from the other two motoneuronal populations of the ipsilateral nVm were made in a brain transected at the level of the obex to cut inputs from the spinal cord (Fig. 5D, bottom panel). These showed that rhythmic spiking of the annularis motoneuron was synchronous with bursts of activity in the basilaris/ velum and apicalis populations of motoneurons. Brief mechanical stimuli applied to the teeth or
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Fig. 3. (A) Synaptic responses elicited in trigeminal motoneurons by stimulation of six sites in the RF. Each record includes six to 10 superimposed traces for each case. Histograms of latency of the first excitatory component are presented on the right. (B) Responses to the stimulation of three RF sites in normal Ringer’s and after a 55-min exposure in Ca 2⫹-free Ringer’s. For abbreviations, see legend to Fig. 1.
the fimbriae of the sucker (Fig. 6A) sometimes caused synchronous bursts of EMG activity on both sides of the annularis muscle in the semi-intact preparation (Fig. 6B). In 57% of the cases, the first burst was followed by one to four others (Fig. 6C1), and the frequency varied between 0.1 and 1.5 Hz (median 0.32 Hz; Fig. 6C2). The bursts could last for up to 7 s (median 2.1 s; Fig. 6C3), but their duration was very variable. DISCUSSION
The present study shows that electrical stimulation of trigeminal afferents and several brainstem sites that contain premotor interneurons 14a elicits short-latency synaptic responses in trigeminal motoneurons. It was particularly interesting to find that stimulation of either trigeminal nerve or the contralateral nVs often triggered membrane potential oscillations.
Synaptic responses to stimulation of primary afferents and cell groups Stimulation of trigeminal afferents on the ipsilateral side elicited large excitatory synaptic responses in trigeminal motoneurons. The responses were characterized by an early component with a short time to peak that could be monosynaptic. This component was not an antidromic initial segment spike, since its amplitude varied with stimulus intensity and it was abolished by CNQX. It followed twin pulse stimulation at a frequency of 30 Hz, which is far beyond the 10 Hz frequency which has been suggested as the cut-off point for monosynaptic conduction in this species. 27 This suggests that trigeminal motoneurons receive monosynaptic inputs from primary afferents. In this respect, they resemble fin motoneurons, which have been shown 10 to receive monosynaptic inputs from dorsal cells, a particular group of primary afferent neurons with
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Fig. 4. Synaptic responses evoked in a trigeminal motoneuron by local pressure ejection of 10 mM dl-glutamate at five RF sites. Arrows show onsets of pressure ejection. Shaded areas represent the approximate spread of glutamate at each site. For abbreviations, see legend to Fig. 1.
cell bodies in the CNS. 6 However, the vast majority of lamprey motoneurons are not excited monosynaptically by sensory afferents. The contralateral trigeminal nerve excitatory inputs appear to be at least disynaptic, which is not surprising since primary afferent fibers project only on the ipsilateral side of the lamprey brainstem. 11,17,22 The large amplitude and relatively short latency of the responses suggest that the pathway includes commissural interneurons in the nVs (Huard H. et al., unpublished observations), which appear to be the shortest link between the afferent terminals in the nucleus descendens nervi trigemini and motoneurons in the contralateral nVm. The responses in trigeminal motoneurons to trigeminal afferent inputs appear to be mediated by excitatory amino acids. The early component
depends on AMPA receptors. The late, long-lasting depolarization that is enhanced by CNQX is NMDA mediated, since it disappears after 2-AP5. Afferent inputs to trigeminal motoneurons in mammals also depend on AMPA and NMDA receptors. 5 The longlasting depolarization that was enhanced with CNQX when stimulating the ipsilateral nerve suggests that CNQX eliminated a concurrent inhibitory postsynaptic potential (IPSP). The simplest hypothesis is that the IPSP is due to an AMPAmediated excitation of inhibitory premotor neurons by primary afferents, and these are likely to be glycinergic, since the depolarization was greatly enhanced by strychnine. In mammals, glycinergic interneurons that project to the nVm have been found in regions that receive trigeminal afferent inputs, particularly the rostral division of the
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Fig. 5. Membrane potential oscillations elicited in trigeminal motoneurons. (A) Slow frequency oscillations were elicited by a single pulse stimulation (1.0 mA) to the contralateral nVs (top trace). Single pulses (1.0 mA) delivered to the contralateral nVm or ipsilateral RFr did not trigger membrane potential oscillations (middle and lower traces). (B1) High-frequency oscillations after one pulse (3.0 mA) to the contralateral trigeminal nerve. (B2) Low-frequency oscillations elicited by stimulation (1.0 mA) of the contralateral nVs. (B3) Low- and highfrequency oscillations after stimulation (3.6 mA) of the ipsilateral trigeminal nerve. (C) Distribution of the oscillatory events (mean ^ S.D.) according to frequency (top panel) and amplitude (bottom panel). Events are arranged in order of increasing oscillatory frequency. Events marked with the same letter were recorded in the same motoneuron. (D1) Simultaneous recordings of the membrane potential in a trigeminal motoneuron (top trace) and of surface potentials in the same (middle trace) and an ipsilateral ventral root (bottom trace). (D2) Simultaneous recordings of the membrane potential in a trigeminal motoneuron (top trace), of an ipsilateral ventral root (middle trace) and of the vagal (x) nerve (bottom trace). (D3) Simultaneous recordings of the membrane potential in an annularis motoneuron (top trace) and of surface potentials from basilaris/velum (middle trace) and apicalis (bottom trace) motoneuronal pools.
trigeminal spinal nucleus, the rostral division of the parvocellular RF (which corresponds to our RFr group) and the shell of neurons that surround the nVm. 30 Stimulation of cells in the latter region in brainstem slices causes IPSPs in trigeminal motoneurons that are sensitive to strychnine. 16 We have already commented on the fact that all levels of the lateral RF contain trigeminal premotor interneurons in mammals 18 and lampreys. 14a The present data show that stimulation of all levels of the RF on both sides elicits EPSPs in trigeminal motoneurons, and that responses from rostral sites
are not caused solely by stimulation of fibers of passage from the RFc, since local ejections of glutamate are also effective. The early EPSPs evoked by ipsilateral RF stimulation were of very short latency when elicited from rostral sites. The earliest response had a fast rise time, even when the RFc was stimulated. Entrainment of the early EPSP was observed without attenuation or amplification at 100 Hz, and it remained in Ca 2⫹-free Ringer’s. This is strong evidence that this component is electrotonic. The second chemical component was of slightly longer latency and was potentiated by
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Fig. 6. Rhythmical EMG activity recorded in the annularis muscle after mechanical stimulation of the oral disc. (A) Drawing showing the semi-intact decerebrate preparation, with stimulation and recording sites. (B) EMG bursts elicited by stimulation (arrow) of fimbriae on the right side. (C) Histograms prepared with data from four experiments. (C1) Number of bursts. (C2) Frequency. (C3) Burst duration.
repetitive stimulation. As with nVs and trigeminal nerve stimulation, RF responses involve excitatory amino acid transmission of both AMPA and NMDA receptor subtypes. As in mammals, 4 inputs from the RF were not exclusively excitatory. Inhibition was revealed by using Mg 2⫹-free Ringer’s, an observation noted previously for inputs to reticulospinal
neurons in lampreys. 9 Inhibitory inputs from the RF impinging upon trigeminal motoneurons have also been observed in higher vertebrates. Electrotonic synapses are not rare in lamprey and mixed electrotonic/chemical postsynaptic potentials have been found in the brainstem and the spinal cord. Reticulospinal cells receive mixed electrical/
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chemical inputs from the cells within the alar plate relaying vestibular inputs, 1,2,28 and in turn make similar synaptic contacts with myotomal motoneurons and interneurons of the spinal cord. 3,23,27 Stimulation of the nVm also elicited large EPSPs in contralateral trigeminal motoneurons. Although glutamate ejections also evoked a similar response (Petropoulos D., Lund J. P. and Dubuc R., unpublished observations), it is unlikely that stimulation of motoneurons caused this response, because they do not project to the opposite nucleus. 14a However, there are many small neurons within and surrounding the nVm in lampreys and mammals that do project to the contralateral side. 7,8,14,14a,19,21,31 Some of these have recently been shown to act on ipsilateral trigeminal motoneurons through AMPA receptors in mammals. 16 Oscillations The two patterns of membrane potential oscillations recorded in trigeminal motoneurons in the in vitro isolated brainstem preparation may be the neural correlates of feeding patterns observed in intact lampreys. Feeding behavior in this species is characterized by rhythmical movements of the apicalis, accompanied by co-contraction of the major head muscles that control the sucker, and of the basilaris muscle, which regulates the volume of the pharynx. 12,15,29 To accomplish this, the annularis, basilaris and apicalis motoneurons of the nVm must be active during feeding movements. We found that oscillatory potentials could be triggered in motoneurons in the three regions of the nVm that supply these muscles. We also observed synchronous rhythmical activity in the three groups of motoneurons. The basic feeding rhythm of intact animals varies between 0.05 and 0.3 Hz, which is compatible with the low-frequency oscillatory membrane potentials recorded in trigeminal motoneurons in the isolated brainstem, but bouts of higher frequency movements have also been observed. 15 Suction measurements in the intact animals revealed that the low-frequency cycles generated much higher pressure than the higher frequency cycles. 15 It is therefore of interest that the low-frequency oscillations in trigeminal motoneurons recorded in this study had twice the amplitude of the high-frequency potentials. Although we have observed that trigeminal motoneurons can be entrained by the locomotor rhythm in
vitro (Bussie`res N., Lund J. P. and Dubuc R., unpublished observations), membrane potential oscillations of nVm neurons were usually seen in the absence of activity in the ventral roots in the present experiments. Locomotor bouts were not frequent during trigeminal oscillatory activity elicited by stimulation of the trigeminal nerves or contralateral nVs, and in most cases when bursts of fictive locomotion were evoked, there was no co-ordination between the two rhythms. It was also clear that, most of the time, trigeminal motoneuron activity was independent of the respiratory rhythm. The motor activity elicited by mechanical stimulation of the teeth and oral fimbriae was recorded from the annularis muscle in semi-intact preparations. The frequency of the rhythmical muscle activity in the head was within the range of the low-frequency oscillations elicited in trigeminal motoneurons in isolated brainstem preparations. This suggests that sensory inputs from the head region play an important role in the control of feeding, as they do in higher vertebrates. 26 CONCLUSIONS
This paper presents evidence that neurons in regions of the lamprey brainstem that are retrogradely labeled after injections of cobalt–lysine into the trigeminal motor nucleus 14a have postsynaptic effects on trigeminal motoneurons. Most neurons are excitatory and exert their action through the release of excitatory amino acids that act on AMPA and NMDA postsynaptic receptor subtypes. Inputs from neurons of the ipsilateral RF include an electrotonic component. Glycinergic inhibition from a smaller number of sites contributes to the trigeminal motoneuronal response. Rhythmical membrane potential fluctuations that can be triggered in trigeminal motoneurons by sensory inputs appear to be the corollary of feeding behavior in this species. Acknowledgements—This work was supported by a Group Grant (Neurological Sciences) from the Canadian Medical Research Council, as well as from FCAR (Que´bec). We wish to express our gratitude to D. Veilleux and S. Lepage for help with the experiments, and to J. Jodoin and G. Delforge for technical assistance. We are also grateful to S. Dupuis and E´. Cle´ment for computer programming. We would like to express our gratitude to J. E. Gersmehl from the U.S. Fish and Wildlife Service, as well as to Dr J. G. Seelye, Mr W. D. Swink and Mrs M. K. Jones from the Lake Huron Biological Station for the kind supply of lampreys.
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