Reticulospinal Axons Research Articles

Las lampreas como modelo animal en estudios de regeneración tras lesión medular

Spinal cord injuries are an important sanitary and economical problem for the society. In mammals, including humans, a traumatic injury to the spinal cord leads to a loss of motor and sensorial function, which is irreversible due to the low regenerative ability of the central nervous system. In contrast to mammals, functional recovery occurs spontaneously after a complete spinal cord transection in lampreys. Functional recovery occurs because in these animals about 50% of the reticulospinal axons regenerate after injury and also because of the occurrence of processes of reorganization and plasticity of the spinal circuits. In this review, we first analyze the characteristics and regeneration ability of lampreys as compared to mammals. Then, we compile the knowledge about the process of recovery after a spinal cord injury acquired in studies using the lampreys as animal model and finally we provide some general perspectives about the molecular processes implicated in regeneration that can be investigated in a very advantageous way in this animal model and which knowledge could allow to develop new therapies for patients suffering spinal cord injury.

Regenerated synapses in lamprey spinal cord are sparse and small even after functional recovery from injury

Despite the potential importance that synapse regeneration plays in restoring neuronal function after spinal cord injury (SCI), even the most basic questions about the morphology of regenerated synapses remain unanswered. Therefore, we set out to gain a better understanding of central synapse regeneration by examining the number, distribution, molecular composition, and ultrastructure of regenerated synapses under conditions in which behavioral recovery from SCI was robust. To do so, we used the giant reticulospinal (RS) neurons of lamprey spinal cord because they readily regenerate, are easily identifiable, and contain large synapses that serve as a classic model for vertebrate excitatory neurotransmission. Using a combination of light and electron microscopy, we found that regenerated giant RS synapses regained the basic structures and presynaptic organization observed at control giant RS synapses at a time when behavioral recovery was nearly complete. However, several obvious differences remained. Most strikingly, regenerated giant RS axons produced very few synapses. In addition, presynaptic sites within regenerated axons were less complex, had fewer vesicles, and had smaller active zones than normal. In contrast, the densities of presynapses and docked vesicles were nearly restored to control values. Thus, robust functional recovery from SCI can occur even when the structures of regenerated synapses are sparse and small, suggesting that functional recovery is due to a more complex set of compensatory changes throughout the spinal network.

Axon regeneration in the absence of growth cones: Acceleration by cyclic AMP

Regenerative failure of spinal axons is commonly ascribed to signaling of F-actin depolymerization and growth cone collapse by molecules such as the myelin-associated growth inhibitors. cAMP is thought to promote regeneration at least in part by neutralizing this effect, either by direct action in the growth cone or indirectly by transcriptional mechanisms. In vivo evidence for this is based mainly on partial lesion studies in which it is sometimes difficult to distinguish regeneration of injured axons from collateral sprouting by uninjured axons. Moreover, previous observations on fixed lamprey central nervous system (CNS) suggested that regeneration may not involve growth cones. To distinguish actively growing axons from static or retracting ones, fluorescently labeled large reticulospinal axons were imaged in the living, transected lamprey cord with and without application of cAMP analogs and then studied by 2-photon microscopy. Axon tip movements over 2-48-hour intervals indicated: 1) regeneration was intermittent; 2) cAMP decreased initial axon retraction and increased subsequent regeneration up to 11-fold; 3) the increase in regeneration was due to an increase in velocity of axon growth, but not in the time spent in forward movement; 4) tips of actively regenerating axons were more sharply contoured than static tips but no filopodia or lamellipodia were observed, even in db-cAMP; and 5) during active growth, axon tips contained vesicle-like inclusions and were highly immunoreactive for neurofilaments. Staining for F-actin and microtubules was variable and F-actin was not concentrated at the leading edge. Thus, cAMP accelerates regeneration of lamprey spinal axons without inducing formation of growth cones.

Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey

The sea lamprey recovers normal-appearing locomotion after spinal cord transection and its spinal axons regenerate selectively in their correct paths. However, among identified reticulospinal neurons some are consistently bad regenerators and only about 50% of severed reticulospinal axons regenerate through the site of injury. We previously suggested (Shifman, M. I., and Selzer, M. E., 2000a. Expression of netrin receptor UNC-5 in lamprey brain; modulation by spinal cord transection. Neurorehabilitation and Neural Repair 14, 49–58; Shifman, M. I., and Selzer, M. E., 2000b. In situ hybridization in wholemounted lamprey spinal cord: localization of netrin mRNA expression. Journal of Neuroscience Methods 104, 19–25) that selective chemorepulsion might explain why some neurons are bad regenerators and others not. To explore the role of additional chemorepulsive axonal guidance molecules during regeneration, we examined the expression of the repulsive guidance molecule (RGM) and its receptor neogenin by in situ hybridization and quantitative PCR. RGM mRNA was expressed in the spinal cord, primarily in neurons of the lateral gray matter and in dorsal cells. Following spinal cord transection, RGM message was downregulated in neurons close (within 10 mm) to the transection at 2 and 4 weeks, although it was upregulated in reactive microglia at 2 weeks post-transection. Neogenin mRNA expression was unchanged in the brainstem after spinal cord transection, and among the identified reticulospinal neurons, was detected only in “bad regenerators”, neurons that are known to regenerate well never expressed neogenin. The downregulation of RGM expression in neurons near the transection may increase the probability that regenerating axons will regenerate through the site of injury and entered caudal spinal cord.

Schwann Cell Transplantation Improves Reticulospinal Axon Growth and Forelimb Strength after Severe Cervical Spinal Cord Contusion

Schwann cell (SC) implantation alone has been shown to promote the growth of propriospinal and sensory axons, but not long-tract descending axons, after thoracic spinal cord injury (SCI). In the current study, we examined if an axotomy close to the cell body of origin (so as to enhance the intrinsic growth response) could permit supraspinal axons to grow onto SC grafts. Adult female Fischer rats received a severe (C5) cervical contusion (1.1 mm displacement, 3 KDyn). At 1 week postinjury, 2 million SCs ex vivo transduced with lentiviral vector encoding enhanced green fluorescent protein (EGFP) were implanted within media into the injury epicenter; injury-only animals served as controls. Animals were tested weekly using the BBB score for 7 weeks postimplantation and received at end point tests for upper body strength: self-supported forelimb hanging, forearm grip force, and the incline plane. Following behavioral assessment, animals were anterogradely traced bilaterally from the reticular formation using BDA-Texas Red. Stereological quantification revealed a twofold increase in the numbers of preserved NeuN+ neurons rostral and caudal to the injury/graft site in SC implanted animals, corroborating previous reports of their neuroprotective efficacy. Examination of labeled reticulospinal axon growth revealed that while rarely an axon was present within the lesion site of injury-only controls, numerous reticulospinal axons had penetrated the SC implant/lesion milieu. This has not been observed following implantation of SCs alone into the injured thoracic spinal cord. Significant behavioral improvements over injury-only controls in upper limb strength, including an enhanced grip strength (a 296% increase) and an increased self-supported forelimb hanging, accompanied SC-mediated neuroprotection and reticulospinal axon growth. The current study further supports the neuroprotective efficacy of SC implants after SCI and demonstrates that SCs alone are capable of supporting modest supraspinal axon growth when the site of axon injury is closer to the cell body of the axotomized neuron.

Interaction between hindbrain and spinal networks during the development of locomotion in zebrafish

Little is known about the role of the hindbrain during development of spinal network activity. We set out to identify the activity patterns of reticulospinal (RS) neurons of the hindbrain in fictively swimming (paralyzed) zebrafish larvae. Simultaneous recordings of RS neurons and spinal motoneurons revealed that these were coactive during spontaneous fictive swim episodes. We characterized four types of RS activity patterns during fictive swimming: (i) a spontaneous pattern of discharges resembling evoked high-frequency spiking during startle responses to touch stimuli, (ii) a rhythmic pattern of excitatory postsynaptic potentials (EPSPs) whose frequency was similar to the motoneuron EPSP frequency during swim episodes, (iii) an arrhythmic pattern consisting of tonic firing throughout swim episodes, and (iv) RS cell activity uncorrelated with motoneuron activity. Despite lesions to the rostral spinal cord that prevented ascending spinal axons from entering the hindbrain (normally starting at approximately 20 h), RS neurons continued to display the aforementioned activity patterns at day 3. However, removal of the caudal portion of the hindbrain prior to the descent of RS axons left the spinal cord network unable to generate the rhythmic oscillations normally elicited by application of N-methyl-d-aspartate (NMDA), but in approximately 40% of cases chronic incubation in NMDA maintained rhythmic activity. We conclude that there is an autonomous embryonic hindbrain network that is necessary for proper development of the spinal central pattern generator, and that the hindbrain network can partially develop independently of ascending input.

G protein βγ-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties

Neurotransmitters are thought to be released as quanta, where synaptic vesicles deliver packets of neurotransmitter to the synaptic cleft by fusion with the plasma membrane. However, synaptic vesicles may undergo incomplete fusion. We provide evidence that G protein-coupled receptors inhibit release by causing such incomplete fusion. 5-hydroxytryptamine (5-HT) receptor signaling potently inhibits excitatory postsynaptic currents (EPSCs) between lamprey reticulospinal axons and their postsynaptic targets by a direct action on the vesicle fusion machinery. We show that 5-HT receptor-mediated presynaptic inhibition, at this synapse, involves a reduction in EPSC quantal size. Quantal size was measured directly by comparing unitary quantal amplitudes of paired EPSCs before and during 5-HT application and indirectly by determining the effect of 5-HT on the relationship between mean-evoked EPSC amplitude and variance. Results from FM dye-labeling experiments indicate that 5-HT prevents full fusion of vesicles. 5-HT reduces FM1-43 staining of vesicles with a similar efficacy to its effect on the EPSC. However, destaining of FM1-43-labeled vesicles is abolished by lower concentrations of 5-HT that leave a substantial EPSC. The use of a water-soluble membrane impermeant quenching agent in the extracellular space reduced FM1-43 fluorescence during stimulation in 5-HT. Thus vesicles contact the extracellular space during inhibition of synaptic transmission by 5-HT. We conclude that 5-HT, via free Gbetagamma, prevents the collapse of synaptic vesicles into the presynaptic membrane.

Zebrafish bashful/laminin‐α1 mutants exhibit multiple axon guidance defects

Laminin is known to provide a highly permissive substratum and in some cases directional information for axon outgrowth in vitro. However, there is still little known about laminin function in guiding axons in vivo. We investigated the axon guidance role of laminin-alpha1 in the developing zebrafish nervous system. Analysis of zebrafish bashful (bal)/laminin-a1 mutants revealed multiple functions for laminin-alpha1 in the outgrowth and guidance of central nervous system (CNS) axons. Most CNS axon pathways are defective in bal embryos. Some axon types, including retinal ganglion cell axons, early forebrain axons, and hindbrain reticulospinal axons, make specific pathfinding errors, suggesting laminin-alpha1 is required for directional decisions. Other axon tracts are defasciculated or not fully extended in bal embryos, suggesting a function for laminin-alpha1 in regulating adhesion or providing a permissive substratum for growth. In addition, some neurons have excessively branched axons in bal, indicating a potential role for laminin-alpha1 in branching. In contrast to CNS axons, most peripheral axons appear normal in bal mutants. Our results, thus, reveal important and diverse functions for laminin-alpha1 in guiding developing axons in vivo.

Homeodomain transcription factors in the development of subsets of hindbrain reticulospinal neurons

Hindbrain reticulospinal neurons are involved in complex neural functions that are mediated by spinal elements, including posture control and modulation of respiration and cardiovascular function. Recent descriptive studies with chick, mouse, and rat embryos have provided anatomical insight into the development of the different reticulospinal nuclei and the establishment of their axonal projection pathways into the spinal cord. In this study, we have addressed the molecular control of this process. Retrograde labeling of reticulospinal neurons in chick and mouse embryos combined with immunostaining for the homeodomain factors Lhx1/Lhx5, Lhx3/Lhx4, and Chx10 have defined transcriptional codes that label subsets of neurons with different axon projection patterns. Gain of function and loss of function experiments using in ovo electroporation implicate these transcription factors in the determination of reticulospinal neuron identity. Furthermore, our studies reveal novel gene interactions between the transcription factors analyzed that may determine the final patterns of reticulospinal axon projection.

Control of the temporal fidelity of synaptic transmission by a presynaptic high voltage-activated transient K current.

The type of K(+) channels controlling the waveform of the presynaptic spike and synaptic transmission were examined in the lamprey spinal cord. Reticulospinal neuron somata displayed a transient K(+) current with a high voltage-activation and inactivation. This current was selectively blocked by catechol at 100 microM. Reticulospinal axons also displayed a high voltage-activated fast K(+) current sensitive to catechol. The function of this presynaptic high voltage-activated fast K(+) current in controlling synaptic transmission was investigated by using paired intracellular recordings from reticulospinal axons and their targets. Blockade of this current by catechol (100 microM) prolonged the presynaptic spike elicited by a single stimulus leading to a potentiation of the postsynaptic EPSP. Calcium imaging of reticulospinal axons showed an increase in presynaptic calcium transients after blockade of the presynaptic K(+) current by catechol. During high frequency firing, catechol revealed an activity-dependent decrease in the spike duration, which resulted in a depression of synaptic transmission. These results suggest that the presynaptic high voltage-activated transient K(+) current acts to optimize the temporal fidelity of synaptic transmission by minimizing activity-dependent changes in the presynaptic spike waveform and calcium dynamics.

A pre-embedding immunogold approach for detection of synaptic endocytic proteins in situ

During the past decade, many molecular components of clathrin-mediated endocytosis have been identified and proposed to play various hypothetical roles in the process [Nat. Rev. Neurosci. 1 (2000) 161; Nature 422 (2003) 37]. One limitation to the evaluation of these hypotheses is the efficiency and resolution of immunolocalization protocols currently in use. In order to facilitate the evaluation of these hypotheses and to understand more fully the molecular mechanisms of clathrin-mediated endocytosis, we have developed a protocol allowing enhanced and reliable subcellular immunolocalization of proteins in synaptic endocytic zones in situ. Synapses established by giant reticulospinal axons in lamprey are used as a model system for these experiments. These axons are unbranched and reach up to 80–100 μm in diameter. Synaptic active zones and surrounding endocytic zones are established on the surface of the axonal cylinder. To provide access for antibodies to the sites of synaptic vesicle recycling, axons are lightly fixed and cut along their longitudinal axis. To preserve the ultrastructure of the synaptic endocytic zone, antibodies are applied without the addition of detergents. Opened axons are incubated with primary antibodies, which are detected with secondary antibodies conjugated to gold particles. Specimens are then post-fixed and processed for electron microscopy. This approach allows preservation of the ultrastructure of the endocytic sites during immunolabeling procedures, while simultaneously achieving reliable immunogold detection of proteins on endocytic intermediates. To explore the utility of this approach, we have investigated the localization of a GTPase, dynamin, on clathrin-coated intermediates in the endocytic zone of the lamprey giant synapse. Using the present immunogold protocol, we confirm the presence of dynamin on late stage coated pits [Nature 422 (2003) 37] and also demonstrate that dynamin is recruited to the coat of endocytic intermediates from the very early stages of the clathrin coat formation. Thus, our experiments show that the current pre-embedding immunogold method is a useful experimental tool to study the molecular mechanisms of synaptic vesicle recycling.

Functional synergies among neck muscles revealed by branching patterns of single long descending motor-tract axons

In this chapter, we describe our recent work on the divergent properties of single, long descending motor-tract neurons in the spinal cord, using the method of intra-axonal staining with horseradish peroxidase, and serial-section, three-dimensional reconstruction of their axonal trajectories. This work provides evidence that single motor-tract neurons are implicated in the neural implementation of functional synergies for head movements. Our results further show that single medial vestibulospinal tract (MVST) neurons innervate a functional set of multiple neck muscles, and thereby implement a canal-dependent, head-movement synergy. Additionally, both single MVST and reticulospinal axons may have similar innervation patterns for neck muscles, and thereby control the same functional sets of neck muscles. In order to stabilize redundant control systems in which many muscles generate force across several joints, the CNS routinely uses a combination of a control hierarchy and sensory feedback. In addition, in the head-movement system, the elaboration of functional synergies among neck muscles is another strategy, because it helps to decrease the degrees of freedom in this particularly complicated control system.

Diffusible signals and fasciculated growth in reticulospinal axon pathfinding in the hindbrain

We have addressed the control of longitudinal axon pathfinding in the developing hindbrain, including the caudal projections of reticular and raphe neurons. To test potential sources of guidance signals, we assessed axon outgrowth from embryonic rat hindbrain explants cultured in collagen gels at a distance from explants of midbrain–hindbrain boundary (isthmus), caudal hindbrain, or cervical spinal cord. Our results showed that the isthmus inhibited caudally directed axon outgrowth by 80% relative to controls, whereas rostrally directed axon outgrowth was unaffected. Moreover, caudal hindbrain or cervical spinal cord explants did not inhibit caudal axons. Immunohistochemistry for reticular and raphe neuronal markers indicated that the caudal, but not the rostral projections of these neuronal subpopulations were inhibited by isthmic explants. Companion studies in chick embryos showed that, when the hindbrain was surgically separated from the isthmus, caudal reticulospinal axon projections failed to form and that descending pioneer axons of the medial longitudinal fasciculus (MLF) play an important role in the caudal reticulospinal projection. Taken together, these results suggest that diffusible chemorepellent or nonpermissive signals from the isthmus and substrate-anchored signals on the pioneer MLF axons are involved in the caudal direction of reticulospinal projections and might influence other longitudinal axon projections in the brainstem.

Endogenous and exogenous dopamine presynaptically inhibits glutamatergic reticulospinal transmission via an action of D2-receptors on N-type Ca2+ channels.

In this study, the effects of exogenously applied and endogenously released dopamine (DA), a powerful modulator of the lamprey locomotor network, are examined on excitatory glutamatergic synaptic transmission between reticulospinal axons and spinal neurons. Bath application of DA (1-50 micro m) reduced the amplitude of monosynaptic reticulospinal-evoked glutamatergic excitatory postsynaptic potentials (EPSPs). The effect of DA was blocked by the D2-receptor antagonist eticlopride, and mimicked by the selective D2-receptor agonist 2,10,11 trihydroxy-N-propyl-noraporphine hydrobromide (TNPA). Bath application of the DA reuptake blocker bupropion, which increases the extracellular level of dopamine, also reduced the monosynaptic EPSP amplitude. This effect was also blocked by the D2-receptor antagonist eticlopride. To investigate if the action of DA was exerted at the presynaptic level, the reticulospinal axon action potentials were prolonged by administering K+ channel antagonists while blocking l-type Ca2+ channels. A remaining Ca2+ component, mainly dependent on N and P/Q channels, was depressed by DA. When DA (25-50 micro m) was applied in the presence of omega-conotoxin GVIA, a toxin specific for N-type Ca2+ channels, it failed to affect the monosynaptic EPSP amplitude. DA did not affect the response to extracellularly ejected d-glutamate, the postsynaptic membrane potential, or the electrical component of the EPSPs. DA thus acts at the presynaptic level to modulate reticulospinal transmission.

Magnetic resonance microimaging of intraaxonal water diffusion in live excised lamprey spinal cord.

Anisotropy of water diffusion in axon tracts, as determined by diffusion-weighted MRI, has been assumed to reflect the restriction of water diffusion across axon membranes. Reduction in this anisotropy has been interpreted as degeneration of axons. These interpretations are based primarily on a priori reasoning that has had little empirical validation. We used the experimental advantages of the sea lamprey spinal cord, which contains several very large axons, to determine whether intraaxonal diffusion is isotropic and whether anisotropy is attributable to restriction of water mobility by axon surface membranes. Through the application of magnetic resonance microimaging, we were able to measure the purely intraaxonal diffusion characteristics of the giant reticulospinal axons (20-40 microm in diameter). The intraaxonal apparent diffusion coefficients of water parallel (longitudinal ADC, l-ADC) and perpendicular (transverse ADC, t-ADC) to the long axis were 0.98 +/- 0.06 (10(-3) mm2 sec) and 0.97 +/- 0.11 (10(-3) mm2 sec), respectively. In white matter regions that included multiple axons, l-ADCs were almost identical regardless of axon density in the sampled axon tract. By comparison, t-ADCs were reduced and varied inversely with the number of axons (and thus axolemmas) in a fixed cross-sectional area. Thus, diffusion was found to be isotropic when measured entirely within a single axon and anisotropic when measured in regions that included multiple axons. These findings support the hypothesis that the cell membrane is the primary source of diffusion anisotropy in fiber tracts of the central nervous system.

Role of apamin-sensitive k(ca) channels for reticulospinal synaptic transmission to motoneuron and for the afterhyperpolarization.

Single motoneurons and pairs of a presynaptic reticulospinal axon and a postsynaptic motoneuron were recorded in the isolated lamprey spinal cord, to investigate the role of calcium-dependent K(+) channels (K(Ca)) during the afterhyperpolarization following the action potential (AHP), and glutamatergic synaptic transmission on the dendritic level. The AHP consists of a fast phase due to transient K(+) channels (fAHP) and a slower phase lasting 100-200 ms (sAHP), being the main determinant of spike frequency regulation. We now present evidence that the sAHP has two components. The larger part, around 80%, is abolished by superfusion of Cd(2+) (blocker of voltage-dependent Ca(2+) channels), by intracellular injection of 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA; fast Ca(2+) chelator), and by apamin (selective toxin for K(Ca) channels of the SK subtype). While 80% of the sAHP is thus due to K(Ca) channels, the remaining 20% is not mediated by Ca(2+), either entering through voltage-dependent Ca(2+) channels or released from intracellular Ca(2+) stores. This Ca(2+)-independent sAHP component has a similar time course as the K(Ca) portion and is not due to a Cl(-) conductance. It may be caused by Na(+)-activated K(+) channels. Glutamatergic excitatory postsynaptic potentials (EPSPs) evoked by single reticulospinal axons give rise to a local Ca(2+) increase in the postsynaptic dendrite, mediated in part by N-methyl-D-aspartate (NMDA) receptors. The Ca(2+) levels remain elevated for several hundred milliseconds and could be expected to activate K(Ca) channels. If so, this activation should cause a local conductance increase in the dendrite that would shunt EPSPs following the first EPSP in a spike train. We have tested this in reticulospinal/motoneuronal pairs, by stimulating the presynaptic axon with spike trains at different frequencies. We compared the first EPSP and the following EPSPs in the control and after blockade with apamin. No difference was observed in EPSP amplitude or shape before and after apamin, either in normal Ringer or in Mg(2+)-free Ringer removing the voltage-dependent block of NMDA receptors. In conclusion, the local Ca(2+) entry during reticulospinal EPSPs does not cause an activation of K(Ca) channels sufficient to affect the efficacy of synaptic transmission. Thus the integration of synaptic signals at the dendritic level in motoneurons appears simpler than would otherwise have been the case.

Functional Redundancy of Ventral Spinal Locomotor Pathways

Identification of long tracts responsible for the initiation of spontaneous locomotion is critical for spinal cord injury (SCI) repair strategies. Pathways derived from the mesencephalic locomotor region and pontomedullary medial reticular formation responsible for fictive locomotion in decerebrate preparations project to the thoracolumbar levels of the spinal cord via reticulospinal axons in the ventrolateral funiculus (VLF). However, white matter regions critical for spontaneous over-ground locomotion remain unclear because cats, monkeys, and humans display varying degrees of locomotor recovery after ventral SCIs. We studied the contributions of myelinated tracts in the VLF and ventral columns (VC) to spontaneous over-ground locomotion in the adult rat using demyelinating lesions. Animals received ethidium bromide plus photon irradiation producing discrete demyelinating lesions sufficient to stop axonal conduction in the VLF, VC, VLF-VC, or complete ventral white matter (CV). Behavior [open-field Basso, Beattie, and Bresnahan (BBB) scores and grid walking] and transcranial magnetic motor-evoked potentials (tcMMEP) were studied at 1, 2, and 4 weeks after lesion. VLF lesions resulted in complete loss or severe attenuation of tcMMEPs, with mean BBB scores of 18.0, and no grid walking deficits. VC lesions produced behavior similar to VLF-lesioned animals but did not significantly affect tcMMEPs. VC-VLF and CV lesions resulted in complete loss of tcMMEP signals with mean BBB scores of 12.7 and 6.5, respectively. Our data support a diffuse arrangement of axons within the ventral white matter that may comprise a system of multiple descending pathways subserving spontaneous over-ground locomotion in the intact animal.

Heterogeneity of the Population of Command Neurons in the Lamprey

The effects of signals transmitted from the brain to the spinal locomotor networks by a population of command neurons are determined by specific functional projections of each individual neuron. To reveal these projections, we used a simple vertebrate model, the lamprey, in which responses of the spinal networks to spikes in single reticulospinal axons were detected by using the spike-triggered averaging of the motoneuronal activity. We found that individual neurons exert a uniform effect on the segmental motor output along the whole extent of their axons. Twenty different patterns of effect, that is, combinations of influences on the segmental motoneuron pools, were found. The widespread projections and heterogeneity of the population of command neurons present a basis for formation of different gross motor synergies.

G proteins and Presynaptic Inhibition

Several neurotransmitters modulate synaptic release from presynaptic terminals by activating G-protein-coupled receptors. Blackmer et al. investigated the basic mechanisms underlying this modulation of the exocytotic machinery in neurons. Microinjection of G-protein ßγ-subunits (Gßγ) into lamprey reticulospinal axons inhibited synaptic transmission without decreasing the action potential-dependent influx of Ca 2+ . This result suggests a potentially new effector role for Gßγ through directly modulating the vesicle fusion machinery in the synaptic terminal. T. Blackmer, E. C. Larsen, M. Takahashi, T. F. J. Martin, S. Alford, H. E. Hamm, G protein ßγ subunit-mediated presynaptic inhibition: Regulation of exocytotic fusion downstream of Ca 2+ entry. Science 292 , 293-297 (2001). [Abstract] [Full Text]

Calcium influx‐independent depression of transmitter release by 5‐HT at lamprey spinal cord synapses

1. The mechanisms by which 5-hydroxytryptamine (5-HT) depresses transmitter release from lamprey reticulospinal axons were investigated. These axons make glutamatergic synapses onto spinal ventral horn neurons. 5-HT reduces release at these synapses, yet the mechanisms remain unclear. 2. Excitatory postsynaptic currents (EPSCs) evoked by stimulation of reticulospinal axons were recorded in ventral horn neurons. 5-HT depressed the EPSCs in a dose-dependent manner with an apparent Km of 2.3 microM. 3. To examine the presynaptic effect of 5-HT, electrophysiological and optical recordings were made from presynaptic axons. Action potentials evoked Ca(2+) transients in the axons loaded with a Ca(2+)-sensitive dye. 5-HT slightly reduced the Ca(2+) transient. 4. A third-power relationship between Ca(2+) entry and transmitter release was determined. However, presynaptic Ca(2+) currents were unaffected by 5-HT. 5. Further, in the presence of a K(+) channel blocker, 4-aminopyridine (4-AP), 5-HT left unaltered the presynaptic Ca(2+) transient, ruling out the possibility of its direct action on presynaptic Ca(2+) current. 5-HT activated a 4-AP-sensitive current with a reversal potential of -95 mV in these axons. 6. The basal Ca(2+) concentration did not affect 5-HT-mediated inhibition of release. Although 5-HT caused a subtle reduction in resting axonal [Ca(2+)]i, synaptic responses recorded during enhanced resting [Ca(2+)]i, by giving stimulus trains, were equally depressed by 5-HT. 7. 5-HT reduced the frequency of TTX-insensitive spontaneous EPSCs at these synapses, but had no effect on their amplitude. We propose a mechanism of inhibition for transmitter release by 5-HT that is independent of presynaptic Ca(2+) entry.