Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys

In lampreys as in other vertebrates, brainstem centres play a key role in the initiation and control of locomotion. One such centre, the mesencephalic locomotor region (MLR), was identified physiologically at the mesopontine border. Descending inputs from the MLR are relayed by reticulospinal neurons in the pons and medulla, but the mechanisms by which this is carried out remain unknown. Because previous studies in higher vertebrates and lampreys described cholinergic cells within the MLR region, we investigated the putative role of cholinergic agonists in the MLR-controlled locomotion. The local application of either acetylcholine or nicotine exerted a direct dose-dependent excitation on reticulospinal neurons as well as induced active or fictive locomotion. It also accelerated ongoing fictive locomotion. Choline acetyltransferase-immunoreactive cells were found in the region identified as the MLR of lampreys and nicotinic antagonists depressed, whereas physostigmine enhanced the compound EPSP evoked in reticulospinal neurons by electrical stimulation of this region. In addition, cholinergic inputs from the MLR to reticulospinal neurons were found to be monosynaptic. When the brainstem was perfused with d-tubocurarine, the induction of swimming by MLR stimulation was depressed, but not prevented, in a semi-intact preparation. Altogether, the results support the hypothesis that cholinergic inputs from the MLR to reticulospinal cells play a substantial role in the initiation and the control of locomotion.     

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 neuronal substrate for a state‐dependent modulation of sensory inputs in the brainstem

Central networks modulate sensory transmission during motor behavior. Sensory inputs may thus have distinct impacts according to the state of activity of the central networks. Using an in‐vitro isolated lamprey brainstem preparation, we investigated whether a brainstem locomotor center, the mesencephalic locomotor region (MLR), modulates sensory transmission. The synaptic responses of brainstem reticulospinal (RS) cells to electrical stimulation of the sensory trigeminal nerve were recorded before and after electrical stimulation of the MLR. The RS cell synaptic responses were significantly reduced by MLR stimulation and the reduction of the response increased with the stimulation intensity of the MLR. Bath perfusion of atropine prevented the depression of sensory transmission, indicating that muscarinic receptor activation is involved. Previous studies have shown that, upon stimulation of the MLR, behavioral activity switches from a resting state to an active‐locomotor state. Therefore, our results suggest that a state‐dependent modulation of sensory transmission to RS cells occurs in the behavioral context of locomotion and that muscarinic inputs from the MLR are involved.     

Phasic modulation of transmission from vestibular inputs to reticulospinal neurons during fictive locomotion in lampreys

The aim of this study was to determine whether the transmission from sensory inputs to reticulospinal neurons is modulated during fictive locomotion in lampreys. Reticulospinal neurons play a key role in the control of locomotion; modulation of sensory transmission to these neurons might be of importance for the adaptation of the control they exert during locomotion. In this series of experiments, intracellular synaptic responses of reticulospinal neurons of the posterior rhombencephalic reticular nucleus elicited by electrical stimulation of vestibular nerves on each side were studied during fictive locomotion induced by 50 microM N-methyl-D-aspartate (NMDA). Interestingly, shortly after NMDA had reached the bath and much before locomotor discharges were apparent in the recorded ventral roots, there was a significant depression of the synaptic transmission from vestibular nerves. The effect was reversed by washing out the NMDA and persisted in the isolated brainstem after spinal transection at the first segmental level. As locomotor discharges appeared in the ventral roots, synaptic responses elicited by vestibular nerve stimulation showed a clear phasic modulation of their amplitude during the locomotor cycle. Responses to stimulation of the ipsilateral vestibular nerve were smaller during the ipsilateral burst discharge than during the contralateral activity, whilst responses to stimulation of the contralateral vestibular nerve were minimal during contralateral activity and maximal during ipsilateral activity. This opposite pattern of modulation observed in the same reticulospinal neuron suggests that the phasic modulation of vestibular transmission is not due to changes in the membrane properties of the reticulospinal cell but is produced at a pre-reticular level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Descending brain neurons in larval lamprey: Spinal projection patterns and initiation of locomotion

In larval lamprey, partial lesions were made in the rostral spinal cord to determine which spinal tracts are important for descending activation of locomotion and to identify descending brain neurons that project in these tracts. In whole animals and in vitro brain/spinal cord preparations, brain-initiated spinal locomotor activity was present when the lateral or intermediate spinal tracts were spared but usually was abolished when the medial tracts were spared. We previously showed that descending brain neurons are located in eleven cell groups, including reticulospinal (RS) neurons in the mesenecephalic reticular nucleus (MRN) as well as the anterior (ARRN), middle (MRRN), and posterior (PRRN) rhombencephalic reticular nuclei. Other descending brain neurons are located in the diencephalic (Di) as well as the anterolateral (ALV), dorsolateral (DLV), and posterolateral (PLV) vagal groups. In the present study, the Mauthner and auxillary Mauthner cells, most neurons in the Di, ALV, DLV, and PLV cell groups, and some neurons in the ARRN and PRRN had crossed descending axons. The majority of neurons projecting in medial spinal tracts included large identified Müller cells and neurons in the Di, MRN, ALV, and DLV. Axons of individual descending brain neurons usually did not switch spinal tracts, have branches in multiple tracts, or cross the midline within the rostral cord. Most neurons that projected in the lateral/intermediate spinal tracts were in the ARRN, MRRN, and PRRN. Thus, output neurons of the locomotor command system are distributed in several reticular nuclei, whose neurons project in relatively wide areas of the cord.     

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

In order to determine if reticulospinal neurons involved in the control of locomotion and responsive to exogenously applied 5-hydroxtryptamine (5-HT) are innervated by fibers that contain serotonin, the serotoninergic innervation of reticulospinal neurons, identified by retrograde labeling with fluorescein-conjugated dextran-amine (FDA), was investigated by immunohistochemistry in the lamprey brainstem. A widespread distribution of 5-HT immunoreactive (5-HT-ir) fibers was seen within the basal plate of the brainstem, an area containing reticulospinal somata and dendritic arborizations. Numerous 5-HT varicose fibers were found in close relation to large reticulospinal cell bodies, particularly in the middle and anterior rhombencephalic reticular nuclei (MRRN and ARRN). Some of these reticulospinal somata were surrounded by a very dense pericellular 5-HT innervation. 5-HT-ir fibers were also seen in other brain structures that are known to influence reticulospinal neurons such as the rhombencephalic alar plate containing sensory relay interneurons, cranial nerves (III–X), cerebellum, and tectum. These findings suggest that, as in the spinal cord, motor behavior controlled by reticulospinal neurons may be subject to a serotoninergic modulation. © 1994 Wiley-Liss, Inc.     

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.     

The mesencephalic locomotor region sends a bilateral glutamatergic drive to hindbrain reticulospinal neurons in a tetrapod

In vertebrates, stimulation of the mesencephalic locomotor region (MLR) on one side evokes symmetrical locomotor movements on both sides. How this occurs was previously examined in detail in a swimmer using body undulations (lamprey), but in tetrapods the downstream projections from the MLR to brainstem neurons are not fully understood. Here we examined the brainstem circuits from the MLR to identified reticulospinal neurons in the salamander Notophthalmus viridescens. Using neural tracing, we show that the MLR sends bilateral projections to the middle reticular nucleus (mRN, rostral hindbrain) and the inferior reticular nucleus (iRN, caudal hindbrain). Ca²⁺ imaging coupled to electrophysiology in in vitro isolated brains revealed very similar responses in reticulospinal neurons on both sides to a unilateral MLR stimulation. As the strength of MLR stimulation was increased, the responses increased in size in reticulospinal neurons of the mRN and iRN, but the responses in the iRN were smaller. Bath‐application or local microinjections of glutamatergic antagonists markedly reduced reticulospinal neuron responses, indicating that the MLR sends glutamatergic inputs to reticulospinal neurons. In addition, reticulospinal cells responded to glutamate microinjections and the size of the responses paralleled the amount of glutamate microinjected. Immunofluorescence coupled with anatomical tracing confirmed the presence of glutamatergic projections from the MLR to reticulospinal neurons. Overall, we show that the brainstem circuits activated by the MLR in the salamander are organized similarly to those previously described in lampreys, indicating that the anatomo‐physiological features of the locomotor drive are well conserved in vertebrates. J. Comp. Neurol. 524:1361–1383, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.     

The role of spinal cord inputs in modulating the activity of reticulospinal neurons during fictive locomotion in the lamprey

Lamprey reticulospinal neurons are rhythmically modulated during fictive swimming. The present study examines the possibility that this modulation may originate from the spinal cord locomotor networks rather than from the brainstem. To test this, the in vitro preparation of the lamprey brainstem-spinal cord was separated into two compartments which could be exposed to different chemical environments. Locomotor activity was induced pharmacologically in the caudal spinal cord compartment and reticulospinal (RS) neurons from the posterior rhombencephalic reticular nucleus (PRRN) were recorded intracellularly in the rostral compartment containing normal lamprey Ringer. Under these conditions, the membrane potential of RS neurons showed clear rhythmic oscillations which are correlated with the ongoing locomotor activity in the caudal spinal cord bath, although no locomotor discharges were present in the ventral roots of the rostral bath. Such oscillations were not present in the absence of locomotion. These results indicate that the spinal cord locomotor networks can contribute to the rhythmic oscillations which occur in RS neurons during fictive locomotion. Moreover, the latter oscillations of membrane potential are due to both phasic excitation and Cl- -dependent inhibition in the opposite phase.     

Serotoninergic modulation of sensory transmission to brainstem reticulospinal cells

Sensory inputs are subjected to modulation by central neural networks involved in controlling movements. It has been shown that serotonin (5-HT) modulates sensory transmission. This study examines in lampreys the effects of 5-HT on sensory transmission to brainstem reticulospinal (RS) neurons and the distribution of 5-HT cells that innervate RS cells. Cells were recorded intracellularly in the in vitro isolated brainstem of larval lampreys. Trigeminal nerve stimulation elicited disynaptic excitatory responses in RS neurons, and bath application of 5-HT reduced the response amplitude with maximum effect at 10 μ m . Local ejection of 5-HT either onto the RS cells or onto the relay cells decreased sensory-evoked excitatory postsynaptic potentials (EPSPs) in RS cells. The monosynaptic EPSPs elicited from stimulation of the relay cells were also reduced by 5-HT. The reduction was maintained after blocking either N -methyl- d -aspartate (NMDA) or α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. The local ejection of glutamate over RS cells elicited excitatory responses that were only slightly depressed by 5-HT. In addition, 5-HT increased the threshold for eliciting sustained depolarizations in response to trigeminal nerve stimulation but did not prevent them. Combined 5-HT immunofluorescence with axonal tracing revealed that the 5-HT innervation of RS neurons of the middle rhombencephalic reticular nucleus comes mainly from neurons in the isthmic region, but also from neurons located in the pretectum and caudal rhombencephalon. Our results indicate that 5-HT modulates sensory transmission to lamprey brainstem RS cells.     

Effects of stimulating the reticular formation during fictive locomotion in lampreys

The effects of stimulating the reticular formation were studied during fictive locomotion in lampreys (Ichthyomyzon unicuspis). The in vitro isolated preparation of the brainstem and spinal cord was used and fictive locomotion was induced by bath application of N-methyl-

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-aspartate (NMDA; 50–100 μM). During different phases of the locomotor cycle, short trains of stimuli (10 pulses at 80–100 Hz; 10 μA) were delivered through glass-coated tungsten microelectrodes positioned within the middle rhombencephalic reticular nucleus (MRRN) and their effects were studied on ipsi- and contralateral ventral root locomotor discharges. Irrespective of the locomotor phase during which the stimulation train was delivered, a resetting effect occurred. It was characterized by a re-synchronization of the locomotor discharges with a constant latency for each ventral root on the ipsilateral side. The latency increased as the recorded root was located further caudally. This increase in latency was in the range of the phase lag observed between roots during control bouts of locomotion. These results suggest that reticulospinal neurones exert strong resetting effects on spinal locomotor networks. These effects may play a significant role with respect to changes of direction during swimming.     

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, GABA_B-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.     

The spinobulbar system in lamprey

Locomotor networks in the spinal cord are controlled by descending systems which in turn receive feedback signals from ascending systems about the state of the locomotor networks. In lamprey, the ascending system consists of spinobulbar neurons which convey spinal network signals to the two descending systems, the reticulospinal and vestibulospinal neurons. Previous studies showed that spinobulbar neurons consist of both ipsilaterally and contralaterally projecting cells distributed at all rostrocaudal levels of the spinal cord, though most numerous near the obex. The axons of spinobulbar neurons ascend in the ventrolateral spinal cord and brainstem to the caudal mesencephalon and within the dendritic arbors of reticulospinal and vestibulospinal neurons. Compared to mammals, the ascending system in lampreys is more direct, consisting of excitatory and inhibitory monosynaptic inputs from spinobulbar neurons to reticulospinal neurons. The spinobulbar neurons are rhythmically active during fictive locomotion, representing a wide range of timing relationships with nearby ventral root bursts including those in phase, out of phase, and active during burst transitions between opposite ventral roots. The spinobulbar neurons are not simply relay cells because they can have mutual synaptic interactions with their reticulospinal neuron targets and they can have synaptic outputs to other spinal neurons. Spinobulbar neurons not only receive locomotor inputs but also receive direct inputs from primary mechanosensory neurons. Due to the relative simplicity of the lamprey nervous system and motor control system, the spinobulbar neurons and their interactions with reticulospinal neurons may be advantageous for investigating the general organization of ascending systems in the vertebrate.     

Activity in the mesencephalic locomotor region during locomotion

The activity of single neurons in the mesencephalic locomotor region (MLR) was recorded extracellularly in cats during spontaneous locomotion on a treadmill. Although stimulation of the MLR is required to induce locomotion on a treadmill after a precollicular-postmamillary brain stem transection in the cat, spontaneous locomotion may occur after a precollicular-premamillary transection. The activity of flexor and extensor muscles of each limb also was recorded by EMG. Nearly 50% of the MLR neurons exhibited rhythmic firing patterns during locomotion. In about one-half of those cells, unit firing patterns could be correlated with the EMG activity in one or more muscles by using spike-triggered averaging. Single MLR neurons were found to be correlated to EMG activity in a single limb, and others were related to the EMG from muscles in two limbs or in all four limbs. Passive movement or stoppage of the limb(s) did not abolish rhythmicity in these neurons. In addition, somatosensory stimulation did not appear to affect the firing patterns of MLR neurons. Averaged EMGs of correlated forelimb muscles revealed a postspike mean latency of 7.1 ms. These measurements agreed well with reports of a 1- to 1.5-ms delay in MLR projections to reticulospinal neurons and a 5- to 6-ms delay (postspike) in reticulospinal activity correlated to EMGs during locomotion. These findings suggest that (a) MLR neurons are rhythmically active during locomotion, (b) the activity of MLR neurons can be correlated with that of EMGs in one or more limbs, (c) rhythmicity in MLR neurons may be independent of phasic sensory input, and (d) the downstream influence of the MLR may be relayed, at least in part, via reticulospinal neurons.     

The mesencephalic locomotor region. II. Projections to reticulospinal neurons

Single neurons were recorded extracellularly in an area of the medioventral medulla known to receive descending mesencephalic locomotor region (MLR) input. A large number (47%) of these cells were found to receive short-latency orthodromic input following stimulation of the physiologically identified MLR. Of the medioventral medulla neurons studied, 34% were found to project to the spinal cord (determined by antidromic activation following stimulation of the ventrolateral funiculus). Approximately one-half (17% of the total population) of these reticulospinal cells were found to receive short-latency orthodromic input from the MLR. The regional distribution of this group of reticulospinal cells corresponded with the area found to receive descending projections from the MLR in previous anatomical studies. In addition, electrical and chemical activation of the same region was found to elicit locomotion on a treadmill in a companion study. The present findings demonstrate that descending MLR projections influence a large number of medioventral medulla cells, some of which have direct spinal projections, and suggest that this area is a primary relay in the manifestation of MLR function.     

Biophysical properties of descending brain neurons in larval lamprey

In the brains of larval lamprey, biophysical properties of reticulospinal (RS) neurons were determined by applying depolarizing and hyperpolarizing current pulses under current clamp conditions. In response to above threshold depolarizing current pulses, almost all RS neurons produced an initial relatively high spiking frequency (F_i) followed by a variable decay to a steady-state firing frequency (F_ss). Spike-frequency adaptation (SFA), defined as [(F_i−F_ss)/F_i]×100%, was minimal at the lowest currents and more pronounced with larger applied current pulses. Some RS neurons, particularly those in the posterior rhombencephalic reticular nucleus (PRRN), either adapted very quickly, and stopped firing, or fired in short bursts during a constant depolarizing current pulse. Several types of RS neurons, including some Muller cells and unidentified neurons in the middle rhombencephalic reticular nucleus (MRRN), displayed delayed excitation (DE) in which spiking in response to a depolarizing current pulse was delayed if preceded by a hyperpolarizing prepulse. Very few neurons fired action potentials following a hyperpolarizing pulse, such as in the case of post-inhibitory rebound (PIR), and no neurons were found that displayed plateau potentials. The possible contributions of these properties to the descending activation of spinal locomotor networks is discussed.     

Dopaminergic modulation of olfactory-evoked motor output in sea lampreys (Petromyzon marinus L.)

Detection of chemical cues is important to guide locomotion in association with feeding and sexual behavior. Two neural pathways responsible for odor-evoked locomotion have been characterized in the sea lamprey (Petromyzon marinus L.), a basal vertebrate. There is a medial pathway originating in the medial olfactory bulb (OB) and a lateral pathway originating from the rest of the OB. These olfactomotor pathways are present throughout the life cycle of lampreys, but olfactory-driven behaviors differ according to the developmental stage. Among possible mechanisms, dopaminergic (DA) modulation in the OB might explain the behavioral changes. Here, we examined DA modulation of olfactory transmission in lampreys. Immunofluorescence against DA revealed immunoreactivity in the OB that was denser in the medial part (medOB), where processes were observed close to primary olfactory afferents and projection neurons. Dopaminergic neurons labeled by tracer injections in the medOB were located in the OB, the posterior tuberculum, and the dorsal hypothalamic nucleus, suggesting the presence of both intrinsic and extrinsic DA innervation. Electrical stimulation of the olfactory nerve in an in vitro whole-brain preparation elicited synaptic responses in reticulospinal cells that were modulated by DA. Local injection of DA agonists in the medOB decreased the reticulospinal cell responses whereas the D2 receptor antagonist raclopride increased the response amplitude. These observations suggest that DA in the medOB could modulate odor-evoked locomotion. Altogether, these results show the presence of a DA innervation within the medOB that may play a role in modulating olfactory inputs to the motor command system of lampreys.     

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

The course and sites of termination of dorsal column fibres in the lamprey brainstem are described along with their brainstem relays projecting to reticulospinal neurons. Dorsal column fibres ascend to the brainstem level where they intermingle with cells located in the alar plate close to the obex, a location that is analogous to that of the dorsal column nucleus in other vertebrates. Some dorsal column fibres continue further rostrally where they reach the octavolateralis and octavomotorii nuclei. Finally, a small contingent of fibres reach the cerebellum. Injections of cobalt-lysine into the posterior rhombencephalic reticular nucleus retrogradely label neurons within the dorsal column nucleus and within the octavolateralis and octavomotorii nuclei. Microstimulation of the dorsal column nucleus on either side elicits monosynaptic inhibitory responses in reticulospinal neurons while stimulation of octavolateralis and octavomotorius nuclei elicits excitation. By using intracellular recordings, it was shown that neurons within these alar plate nuclei receive monosynaptic inputs from the dorsal columns. It is thus proposed that disynaptic inputs from dorsal columns to reticulospinal neurons are relayed by these alar plate neurons: inhibition is relayed mainly by neurons in dorsal column nuclei and excitation by neurons in the octavolateralis and octavomotorii nuclei. © 1993 Wiley-Liss, Inc.     

Visual Potentiation of Vestibular Responses in Lamprey Reticulospinal Neurons

The lamprey normally swims with the dorsal side up. Illumination of one eye shifts the set-point of the vestibular roll control system, however, so that the animal swims with a roll tilt towards the source of light (the dorsal light response). A tilted orientation is often maintained for up to 1 min after the stimulation. In the present study, the basis for this behaviour was investigated at the neuronal level. The middle rhombencephalic reticular nucleus (MRRN) is considered a main nucleus for the control of roll orientation in lampreys. Practically all MRRN neurons receive vestibular and visual input and project to the spinal cord. Earlier extracellular experiments had shown that optic nerve stimulation potentiates the response to vestibular stimulation in the ipsilateral MRRN. This most likely represents a neural correlate of the dorsal light response. Experiments were carried out in vitro on the isolated brainstem of the silver lamprey (Ichthyomyzon unicuspis). MRRN cells were recorded intracellularly, and the overall activity of descending systems was monitored with bilateral extracellular electrodes. The responses to 10 Hz optic nerve stimulation and 1 Hz vestibular nerve stimulation, and the influence of optic nerve stimulation on the vestibular responses, were investigated. In most preparations, optic nerve stimulation excited practically all ipsilateral MRRN cells. After stimulation, the cell was typically depolarized and showed an increased level of synaptic noise for up to 80 s. In contralateral MRRN neurons, optic nerve stimulation usually evoked hyperpolarization or no response. Vestibular nerve stimulation evoked compound excitatory postsynaptic potentials (EPSPs) or spikes in -90% of the cells, both ipsilaterally and contralaterally. A smaller subpopulation of MRRN cells (-10%) received vestibular inhibition. In 26 of 48 recorded MRRN cells, the response to vestibular stimulation was potentiated after ipsilateral optic nerve stimulation. The potentiation was seen in cells receiving either excitatory or inhibitory vestibular input as an increase in EPSP amplitudelspiking (85%) and a decrease in inhibitory postsynaptic potential amplitude (15%) respectively. In most cases the vestibular responses did not return to control levels during the testing period (10–30 min), and thus the visual stimulation most likely induced long-lasting changes in the functional connectivity of the roll control network, in addition to the short-lasting afteractivity. In four of the 11 cells recorded contralateral to the stimulated optic nerve, a depression of the vestibular response could be seen. In potentiated cells, single vestibular pulses often evoked longer episodes of large synaptic noise and sometimes spiking. In the latter case, the action potentials appeared with highly variable latency after each stimulation pulse. This indicates that an important mechanism underlying the potentiation may be a long-lasting increase in excitability in a pool of unidentified interneurons located either upstream of the MRRN cells, relaying vestibular and visual inputs, or downstream, providing positive feedback.     

Glutamate metabotropic receptor mediated depression of synaptic inputs to lamprey reticulospinal neurones

The transmission of vestibular inputs to reticulospinal (RS) neurones of the posterior rhombencephalic nucleus (PRRN) has been shown to be depressed by the bath application of N-methyl-D-aspartate (NMDA). The aim of this study was to investigate the pharmacological mechanism involved using patch clamp recordings of reticulospinal neurones. It is demonstrated that the chemical component of vestibular inputs to the PRRN is mediated by glutamatergic synapses utilizing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors on the PRRN neurones. Monosynaptic excitatory postsynaptic currents (EPSCs) from octavomotorius relay cells to RS neurones are markedly depressed by the application of NMDA, a depression which was insensitive to competitive and non-competitive NMDA receptor antagonists. The effect of NMDA was eliminated by inactivation of G proteins. A similar depressive effect was observed following application of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) to the superfusate. It is concluded that NMDA acts at a metabotropic receptor located most likely presynaptically to reticulospinal neurones on terminals of octavomotorius relay cells.