Lamprey Spinal Cord Research Articles

A new population of neurons with crossed axons in the lamprey spinal cord

Neurons with contralateral, rostrally and caudally projecting axons were studied in whole of lamprey spinal cord using retograde labelling techniques with fluorescent dextran-amines, cobalt-lysine or horseradish peroxidase. A previously unknown large population (180–300 cells per hemisegment) of small (<25 μm) cells with contralateral projecting axons is described. Their axons extend over less than 5 segments rostrally or caudally. The number of these cells per segment was relatively constant in the rostral half of the spinal cord, but increased significantly in the caudal half. In comparison, medium-sized cells with contralateral axons corresponding to previously identified premotor interneurons were far less numerous (14–21 per hemisegment) and their axons extended more than 5 segments. Contralaterally projecting edge cells (intraspinal stretch receptor neurons) with principal rostral or caudal axons plus short collaterals in the other direction were distributed throughout the length of the spinal cord, whereas large and giant cells with a varied morphology were found in the caudal half.

Axonal regeneration in the adult lamprey spinal cord

Larval sea lampreys recover from complete spinal transection by a process involving directionally specific axonal regeneration. In order to determine whether this is also true of adults, 14 adult lampreys were transected at the level of the 5th gill and allowed to recover for 10 weeks. Müller and Mauthner cells and their giant reticulospinal axons (GRAs) were impaled with microelectrodes and injected with horseradish peroxidase (HRP). The tissue was processed for HRP histochemistry and wholemounts of brain and spinal cord were prepared. All animals recovered coordinated swimming; 61 of 121 (50%) neurites emanating from 30 axons regenerated caudal to the scar into the distal stump. Of the neurites which had grown beyond the scar, 92% were correctly oriented, i.e., caudalward and ipsilateral to the parent axon. Retransection in two additional animals eliminated the recovered swimming. Thus, behavioral recovery in adult sea lampreys is accompanied by directionally specific axonal regeneration.

The need for cellular elements during axonal regeneration in the sea lamprey spinal cord

Spinal axons in the larval sea lamprey regenerate following a complete spinal transection. It is not known whether regenerating growth cones require contact with cellular elements or whether the basement membrane and collagenous meninx primitiva which surround the spinal cord are sufficient for neurite outgrowth. To determine this, a freeze lesion was made which severed axons, destroyed neuronal perikarya, and greatly reduced the number of glial cells. After at least 10 weeks of recovery, 50 neurites from 31 Müller and Mauthner axons were labeled by intracellular injection of HRP. Eighty-six percent of these neurites did not regenerate into the lesion site. No neurites grew through the lesion. No animals recovered coordinated swimming. These results suggest that glial and/or neuronal surfaces are required for axonal regeneration. Moreover, a monolayer of glial cells appears to be suboptimal and a three-dimensional matrix of cells may be necessary to promote regeneration in the lamprey spinal cord.

Presynaptic GABAA and GABAB Receptor-mediated Phasic Modulation in Axons of Spinal Motor Interneurons.

The lamprey spinal cord has been utilized to investigate the role of presynaptic inhibition in the control of the spinal motor system. Axons of the lamprey spinal cord are comparatively large because of their lack of myelination. Axons impaled with microelectrodes demonstrate depolarizing responses to the application of GABAA and GABAB receptor agonists, muscimol and baclofen. These depolarizing effects are counteracted by the specific GABAA and GABAB receptor antagonists, bicuculline and phaclofen. GABAA receptor activation leads to a gating of Cl- channels on the axons. However, the ionic mechanism leading to axonal depolarization following GABAB receptor activation is unknown. After initiation of fictive locomotion, these axons demonstrate oscillations in axonal membrane potential related to the locomotor cycle. During ficitive locomotion they depolarize in phase with the bursting of the ipsilateral ventral root of the same segment. These axonal membrane potential oscillations are due to a phasic GABAA and GABAB receptor-mediated gating of ion channels on the axonal membrane. Fictive locomotion in the lamprey spinal cord is largely unaffected by antagonism of one or other GABA receptor subtype alone, but is severely disrupted by simultaneous antagonism of both subtypes. In conclusions, we demonstrate, for the first time, an agonist-gated depolarization of a vertebrate presynaptic element measured by direct impalement of the axon under study. We also demonstrate that GABA-mediated presynaptic inhibition occurs in axons of spinal interneurons. It is not limited to the primary afferents as has previously been believed.

5-Hydroxytryptamine depresses reticulospinal excitatory postsynaptic potentials in motoneurons of the lamprey

Application of 5-hydroxytryptamine (5-HT) to the lamprey spinal cord in vitro reversibly depressed the chemical component of excitatory postsynaptic potentials recorded intracellularly in motoneurons and evoked by stimulation of single reticulospinal Müller cells. The depression could be produced either by local application of small volumes of 10 mM 5-HT to the surface of the spinal cord or by bath-application of 1 or 10 μM 5-HT. No effect on the input resistance of the postsynaptic cells or their sensitivity to glutamate, the suspected transmitter at this synapse, could be detected, suggesting the possibility of a presynaptic action of 5-HT at this synapse in the lamprey.

Intersegmental co-ordination of undulatory movements— a “trailing oscillator” hypothesis

Intersegmental co-ordination in undulatory locomotion was investigated in the lamprey spinal cord in vitro. It is characterized by a constant phase lag between the activation of consecutive segments. By increasing the excitability (NMDA level) locally, a "travelling wave" could be induced to start in any segment and to be conducted in both rostral and caudal directions. This leading segment, the one with the highest excitability, determined the frequency of all other segments, and the ascending and descending couplings were functionally symmetric. The intersegmental phase lag can be explained by a simple neural mechanism in which a leading oscillator will entrain the adjacent "trailing" segments, which in turn entrain their neighbours and so forth.

Synaptic effects of intraspinal stretch receptor neurons mediating movement-related feedback during locomotion

The flattened lamprey spinal cord contains stretch-sensitive edge cells located along the lateral margin, with dendritic processes sensing the lateral bending of the cord during each swim cycle. These intraspinal stretch receptor neurons provide movement-related sensory feedback input to the generator network for locomotion causing a powerful entertainment of the rhythm. In order to elucidate the synaptic effects of edge cells we have performed paired intracellular recordinngs and staining with Lucifer yellow. Monosynaptic connections that may explain entrainment were found to locomotor central pattern generator interneurons. Edge cells with an ipsilateral axon elicited excitatory postsynaptic potentials (EPSs) in ipsilateral interneurons. In addition, such edge cells evoked kainate/quisqualate receptor mediated EPSPs in ipsilateral motoneurons. This pathway mediates an intraspinal stretch reflex analogous to the muscle spindle stretch reflex of mammals. Edge cells with a contralateral axon produced monosynaptic glycinergic IPSPs in contralateral neurons, including contralateral edge cells.

Inhibition of a cAMP-dependent Ca-activated K conductance by forskolin prolongs Ca action potential duration in lamprey sensory neurons

Intracellular recordings from primary mechanosensory neurons (dorsal cells) of the lamprey spinal cord were made to test the membrane effects of forskolin, an activator of adenylate cyclase in these cells. At a concentration of 50 μM, forskolin was found to have a pronounced broadening effect on calcium action potentials (Ca APs) produced in the presence of voltage-activated K channel blockers (TEA, 3,4-DAP). Forskolin had no effect on passive membrane properties of the cells, such as resting potential or input resistance. Nor did it affect delayed rectification or Na APs and thus appeared not to block voltage-activated K channels. Forskolin's effect was evident only when a significant Ca component to the AP was present. It appeared not to increase the conductance of the Ca channel since its action was accompanied by a decrease in membrane conductance during the Ca AP, indicating instead an inhibition of a repolarizing Ca-activated conductance, other than a Ca-activated Cl conductance. The prolongation of Ca APs by forskolin, barium or the neurotransmitter GABA were all correlated in voltage-clamp with a decrease in outward current. Under the conductions used here, the major outward conductance in dorsal cells is a Ca-activated K conductance(gK(Ca))28, and it is concluded that the most probable mode of action for forskolin is via a cyclic AMP-mediated inhibition of this conductance. GABA has also been shown to prolong Ca APs in lamprey dorsal cells by inhibiting a repolarizinggK(Ca)28. Thus, the action of this transmitter may be mediated by an increase in intracellular cyclic AMP.

5-Hydroxytryptamine immunoreactive varicosities in the lamprey spinal cord have no synaptic specializations - an ultrastructural study

The distribution and fine structure of 5-hydroxytryptamine (5-HT) immunoreactive cell bodies and axonal varicosities have been studied in the lamprey spinal cord, using the peroxidase-antiperoxidase (PAP) immunohistochemical technique and subsequent analysis of ultrathin serial sections. Immunostained cell bodies were found in the ventral spinal cord close to the central canal. Immunostained varicosities were found throughout the spinal cord with the highest density in the ventromedial plexus and the dorsal horn. Only large granular vesicles could be clearly distinguished in immunostained cell bodies and varicosities, but it was concluded based on a comparison with unstained normal tissue that these boutons also contained small, pleomorphic agranular vesicles. Immunoreactive varicosities were studied in the ventromedial plexus, the dorsal horn, the dorsal column, the dorsolateral and ventrolateral funiculi and the grey matter. No morphological differences could be observed between varicosities in the different loci. The varicosities were in no case seen to make synaptic contact with surrounding neuronal elements, even when followed through serial sections. Consequently, 5-HT released from boutons in all parts of the spinal cord could be expected to act on 5-HT receptors located on nearby as well as distant receptors.

Control of respiratory motor pattern by sensory neurons in spinal cord of lamprey

Extracellular stimulation over the dorsal funiculus in the spinal cord of lampreys was found to selectively activate prolonged episodes of fictive arousal respiration. The induced episodes showed comparable increases in cycle frequency and motoneuron burst duration to the spontaneous arousal pattern observed in isolated brain preparations. Intracellular stimulation of primary sensory neurons with axons in the dorsal funiculus, called 'dorsal cells', also elicited the arousal pattern. Mechanoreceptive dorsal cells respond to cutaneous stimulation. When mechanical stimuli were applied to the skin of intact lampreys or to lampreys with ipsilateral vagotomy, arousal respiration was induced. Bilateral, but not unilateral, trigeminal lesion blocked dorsal cell induction of the arousal response. Spontaneous arousal respiration was recorded from intact, unrestrained lampreys. These results suggest that fictive arousal respiration is the in vitro correlate of natural arousal respiration in lampreys, and that one mechanism leading to arousal respiration may be the activity of sensory dorsal cells. A model for respiratory motor pattern switching in lamprey is proposed. The model suggests that the normal and arousal patterns are produced by separately engaging rostral or caudal pattern generators in the medulla, rather than by modifying one pattern generator.

Effects of bicuculline and strychnine on synaptic inputs to edge cells during fictive locomotion

Edge cells are mechanoreceptive neurones located in the lateral tracts of the lamprey spinal cord. Phasic activation of these cells by lateral bending can entrain the activity of the locomotor central pattern generator. During fictive locomotion induced by bath-applied NMDLA ( N-methyl- D,L-aspartate) or sensory stimulation, edge cells receive synaptic input. In this paper we provide evidence that the tonic inhibition received during sensory-induced fictive locomotion is glycinergic. We also show that during fictive locomotion induced by application of NMDLA to the spinal cord, bicuculline unveils phasic synaptic activity in edge cells. During sensory-evoked fictive locomotion, by contrast, only tonic synaptic activity is apparent in the presence of bicuculline.

CNQX and DNQX block non-NMDA synaptic transmission but not NMDA-evoked locomotion in lamprey spinal cord

The motor pattern underlying locomotion in the lamprey is activated and maintained by excitatory amino acid neurotransmission. The quinoxalinediones 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) are potent and selective antagonists ofnon-N-methyl- d-aspartate (NMDA) receptors in the mammalian central nervous system. In the lamprey, these compounds are now shown to block fast excitatory synaptic potentials elicited in neurones of the spinal ventral horn. They selectively antagonise responses to the application of selective kainate and quisqualate receptor agonists (kainate and α-amino-3-hydroxy-5-methyl-4-isoxalone (AMPA)) but do not influence NMDA receptor-mediated responses. Additionally, it is shown that the activation of NMDA receptors is sufficient to elicit and maintain fictive locomotion after blockade of non-NMDA receptors with either DNQX or CNQX. Conversely, activation of quisqualate receptors with AMPA, but not quisqualate leads to fictive locomotion with properties much like that activated by kainate.

Three types of GABA-immunoreactive cells in the lamprey spinal cord

Polyclonal antisera raised against conjugated GABA were used to study the distribution of GABAergic neurons in the spinal cords of lampreys ( Lampetra fluviatilis and Ichtyomyzon unicuspis) using immunofluorescence and peroxidase-antiperoxidase techniques. Three morphologically distinct types of GABA-immunoreactive (GABA-ir) cell bodies were observed, multipolar neurons in the lateral grey cell column, apparently bipolar cells in the ventral aspect of the dorsal horn, and small liquor-contacting cells surrounding the central canal. A high density of immunoreactive fibers of spinal origin were present in the lateral and ventral funiculi, whereas the dorsal column had a relatively low density. Dense GABA-ir plexuses were situated in the lateral spinal margin, and in the dorsal part of the dorsal horn. A chronic lesion of the rostral spinal cord did not result in any observable loss of GABA-ir fibers below or above the lesion, suggesting that the 3 types o f segmental GABA-ir neurons are the main sources of the GABAergic innervation of the lamprey spinal cord.

GABAergic control of rhythmic activity in the presence of strychnine in the lamprey spinal cord

Rhythmic ventral root activity has been induced in the spinal cord of the lamprey in vitro in the presence of strychnine. Bicuculline (a GABA A blocker) increases the initial frequency and decreases the episode duration of such activity, whereas diazepam (which potentiates GABA action) has the opposite effect. In addition, bicuculline slows the depolarisation of ventral horn neurones. However, voltage clamp at potentials positive to the interburst potential does not reveal net outward current, even in the presence of diazepam, indicating that the effects of GABA on the rhythmic activity must be presynaptic to the impaled cells.

Local sensitivity to excitatory and inhibitory amino acids in spinal interneurons of the lamprey

Sensitivity to glutamate, aspartate, glycine and GABA was examined in giant interneurons of the lamprey spinal cord. 1. The membrane potentials evoked by iontophoretic application decayed with varied time constants specific to amino acids: 2.5 sec for glutamate, 6.3 sec for glycine and 10.3 sec for GABA. li|2. Bath-applied amino acids reduced the input resistance by varying degrees; when glutamate effect was taken as 1, relative effects of aspartate, glycine and GABA were 0.28, 40.5 and 12.3, respectively. 3. Glutamate sensitivity was fairly uniform in both the soma and the dendrites. Glycine sensitivity, as well as GABA, was high in the soma and declined steeply along the dendrites by iontophoresis.

Evidence for functional regeneration in the adult lamprey spinal cord following transection

We report here that following partial spinal transections in adult lampreys, the fibers of the spinal cords can regenerate and restore some intersegmental coordination to the central pattern generator for locomotion, as tested in the isolated cord preparation. However, the regeneration by this test is not successful in all animals.

Endogenous activation of glycine and NMDA receptors in lamprey spinal cord during fictive locomotion

Strychnine is shown to abolish left-right alternation in fictive locomotion induced by sensory stimulation. Robust rhythmic activity, characterized by left-right coactivation at each segmental level, is seen in the presence of strychnine at all doses used (0.5-20 microM). The proportion of the cycle occupied by the ventral root bursts and the rostral-caudal coordination is similar to that seen in the absence of strychnine. Furthermore, the rhythm is abolished by cis-2,3-piperidine dicarboxylic acid (PDA), 2-amino-5-phosphonovalerate (APV), or the removal of Mg2+ from the perfusate, as in the absence of strychnine. Voltage clamp was applied to ventral horn neurons during stimulation in the presence of strychnine, with holding potentials negative to the plateau potential associated with a ventral root burst but positive to the potential in the interburst. Inward current was seen during the ventral root burst, but no outward current was seen at burst termination or during the interburst. The results indicate that in fictive locomotion induced by endogenous release of NMDA receptor agonists, left-right alternation is dependent on glycinergic transmission. Furthermore, evidence is provided that in the absence of glycinergic transmission, burst termination may depend on NMDA receptor-linked voltage-sensitive processes.

Identification of excitatory interneurons contributing to generation of locomotion in lamprey: structure, pharmacology, and function.

1. In the in vitro preparation of the lamprey spinal cord, paired intracellular recordings of membrane potential were used to identify interneurons producing excitatory postsynaptic potentials (EPSPs) on myotomal motoneurons. 2. Seventy-nine interneurons (8.4% of all neuron-motoneuron pairs tested) elicited unitary EPSPs that followed one-for-one at short, constant latencies and were therefore considered monosynaptic according to conventional criteria. Evidence was obtained for selectivity and divergence of excitatory interneuron (EIN) outputs and for convergence of EIN input to motoneurons. 3. The neurotransmitter released by EINs may be an excitatory amino acid such as glutamate, because the EPSPs were depressed by antagonists of excitatory amino acids. 4. Intracellular dye injection revealed that EINs have small cell bodies (average 11 x 27 microns), transversely oriented dendrites, and thin (less than 3 microns) slowly conducting axons (0.7 m/s) that project caudally and ipsilaterally. One EIN exhibited a system of thin multi-branching axon collaterals with periodic swellings. Ultrastructurally, these swellings contained clear spherical vesicles, and they apposed postsynaptic membrane specializations. 5. During fictive locomotion, the membrane-potential oscillations of EINs were greater in amplitude than, but similar in shape and timing to, those of their postsynaptic motoneurons. EINs fired action potentials during fictive locomotion and contributed to the depolarization of motoneurons. 6. These interneurons are proposed to be a source of excitation to motoneurons and interneurons in the lamprey spinal cord, participating in motor activity including locomotion.

Peculiarities of receptor-channel complexes for inhibitory mediators in the membranes of lamprey spinal cord neurones

Effects of inhibitory mediators on the membranes of isolated lamprey spinal cord neurones were investigated by means of whole-cell recording and concentration clamp techniques. Glycine and γ-aminobutyric acid (GABA) applications evoked desensitizing chloride currents. The concentrations at half-maximum effects were 16 μM for glycine- and 1.5 mM for GABA-activated currents. Ionic responses to applications of both amino acids demonstrated full cross-desensitization. Strychnine and bicuculline suppressed both glycine- and GABA-activated conductances to the same degree. We suggest that in the membranes of lamprey spinal cord neurones glycine and GABA act on the same receptor-channel complexes.

Increase in endogenous 5-hydroxytryptamine levels modulates the central network underlying locomotion in the lamprey spinal cord

To investigate the effects of an endogenous release of serotonin (5-HT) in the lamprey spinal cord, in vitro, the 5-HT uptake-blocker citalopram (1–10 μM) was added to the bathing solution. Samples taken from the physiological solution through high-performance liquid chromatography (HPLC) showed that 5-HT was released from the spinal cord. To study the effect of this endogenous release of 5-HT on the spinal network generating locomotion, ‘fictive locomotion’ was induced by bath application of N- methyl- d-aspartate (NMDA, 100 μM). It elicited a steady locomotor rhythm between 0.2 and 2.5 Hz. The effects of citalopram were the following: (1) the locomotor frequency slowed down, (2) the intensity of the ventral root bursts was increased and (3) the intersegmental phase lag was prolonged. The effects of citalopram and thus presumably of an endogenous release of 5-HT were similar to what has previously been observed during bath application of 5-HT. In the latter case the conditions are different, since all 5-HT receptors regardless of their location in relation to the 5-HT-containing boutons will be affected in a similar way.