Disappearance of Rohon-Beard neurons from the spinal cord of larval Xenopus laevis

Rohon-Beard neurons are primary sensory cells located in the spinal cord of embryonic lower vertebrates. The kinetics of their normal, gradual, but complete disappearance in Xenopus tadpoles has been followed. Levels of acid phosphatase activity, a common histochemical correlate of cell death, were assayed and found to increase at the time of onset of disappearance of Rohon-Beard cells. Ultrastructural examination revealed the presence of numerous secondary lysosomes, swelling of endoplasmic reticulum and mitochondria, and a decrease in nuclear density. The disappearance of Rohon-Beard neurons may be attributed to autophagic cell death involving lysosomal acid hydrolases. This process begins only a few days after the maturation of voltage- and neurotransmitter-dependent membrane conductances and the electrical uncoupling of these neurons. The loss of Rohon-Beard neurons in embryos whose development was arrested by crowding was appropriate for the developmental stage of the animals rather than their chronological age.     

A quantitative description of excitatory amino acid neurotransmitter responses on cultured embryonic Xenopus spinal neurons

We have performed a quantitative analysis of excitatory amino acid neurotransmitter receptors on cultured embryonic Xenopus spinal neurons using the whole-cell patch-clamp technique. Neuroblasts and underlying mesodermal cells isolated from spinal regions of neural plate-stage embryos were placed into dissociated cell culture, and responses were studied soon after the appearance of neurites on embryonic neurons. Glutamate (Glu) receptors were separated into two general classes based on responses to the characteristic agonists quisqualate (Quis), kainate (Ka) and N-methyl-D-aspartate (NMDA); these were NMDA receptors (those activated by NMDA) and non-NMDA receptors (those activated by Ka and Quis). Half-maximal responses to Glu and other agonists on NMDA and non-NMDA receptors were determined from Hill analysis of dose response relations. The order of sensitivities observed was: GluNMDA (ED50 = 5.1 microM) greater than Glunon-NMDA (ED50 = 28 microM), and for Glu receptor agonists, Quis (ED50 = 1.5 microM) greater than NMDA (ED50 = 41 microM) greater than Ka (ED50 = 58 microM). The order of response amplitudes recorded at concentrations near the appropriate ED50s was GluNMDA greater than Glunon-NMDA, and Ka greater than NMDA greater than Quis. A 10-fold decrease in external [Na+] shifted the reversal potentials for Glunon-NMDA, Ka, and Quis to more negative voltages. Increasing external [Ca2+] shifted the reversal potential for NMDA responses to more positive potentials, an observation consistent with Ca2+ permeation of the embryonic NMDA-activated channel. NMDA-evoked currents could not be recorded in nominally glycine (Gly)-free media. Addition of Gly to external solutions potentiated NMDA responses (ED50 = 644 nM). NMDA responses were blocked by DL-2-amino-5-phosphonovaleric acid (APV; ED50 = 1.9 microM) and by Mg2+ at negative potentials. In their sensitivities to agonists and antagonists, and ionic dependences, amino acid neurotransmitter responses on embryonic Xenopus neurons closely resembled those previously observed for mature Xenopus and mammalian central neurons. The GluNMDA receptors present on these immature neurons were sufficiently sensitive to be activated by endogenous concentrations of extracellular Glu, suggesting a possible role for receptor activation in modulating early neural development.     

DL-Quisqualic and L-aspartic acids activate separate excitatory conductances in cultured spinal cord neurons

Intracellular recordings were made from mouse spinal cord neurons in dissociated tissue cultures. Input conductance (G_M) of these neurons was assessed using injected constant current pulses before and during applications of excitatory amino acids. Using this technique, it was demonstrated that dl-quisqualic acid depolized and excited these neurons, as did l-aspartic acid, but the evoked change in G_M differed significantly between responses to these amino acids. In neurons where l-aspartic acid activated a voltage-dependent decrease (apparent) in G_M^6–8dl-quisqualic increased G_M by a relatively voltage-independent mechanism and resembled most closely the effects of another amino acid analogue dl-kainic acid.Quisqualic acid, as a consequence, was unable to induce regenerative spikes or burst which are characteristic of responses to a variety of excitatory amino acids^6–8 such as l-aspartic.     

Unmyelinated primary afferent fibers in dorsal funiculi of cat sacral spinal cord

The present study tests the hypothesis that there are numerous unmyelinated primary afferent fibers in cat posterior funiculi. The animals have unilateral dorsal rhizotomies from L6 to Ca3. One week later the axons of both S2 dorsal funiculi are counted. The data indicate that there are approximately 22,500 myelinated and 8,500 unmyelinated axons on the unoperated side and 11,000 myelinated and 3,900 unmyelinated axons on the operated side. On this basis, we suggest that 51% of the myelinated and 54% of the unmyelinated axons in cat dorsal funiculi arise from dorsal root ganglion cells and thus are primary afferent axons. If this is correct, then 71% of the primary afferent axons in the cat dorsal funiculus are myelinated and 29% are unmyelinated. The function of this large group of previously unsuspected fine sensory axons remains to be determined.     

Marginal neurons of the spinal cord: types, afferent synaptology and functional considerations

Electron microscopic studies of the marginal zone of the dorsal horn in the cat spinal cord revealed 4 different types of synaptic endings contacting marginal neurons. These included boutons with clear spherical vesicles, boutons with clear flattened vesicles, boutons with a mixture of clear spherical and flattened vesicles and boutons with a mixture of clear spherical and dense-cored vesicles.The origin of the synaptic input to marginal neurons was determined by means of degeneration studies at the ultrastructural level following various surgical lesions. It was found that primary afferents contribute approximately 40% of the boutons on the distal dendrites, 20% on the proximal dendrites and 10% on the soma of marginal neurons, while the axons of the tract of Lissauer, which stem from gelatinosa neurons and are inhibitory in function, contribute approximately 20% of the boutons on the distal dendrites, 60% of those on the proximal dendrites and 50% of the somatic endings on marginal neurons. Cranial nerve afferents contribute approximately 10% of the boutons on each area of the marginal neurons within the first cervical segment; this converging afferent input suggests that these cells function as non-specific central nociceptive relay neurons.Fluorescence histochemical studies for the demonstration of biogenic amines revealed a fairly dense array of fluorescent fibers and terminals in the marginal zone. There was no change in this fluorescence following the surgical lesions and their origin remains unknown.Although the observed synaptology of the marginal zone is more complex than that upon which the central inhibitory balance theory of pain was originally proposed, our data provide good anatomic support for that theory.     

Inhibitory amino acid transmitters associated with axons in presynaptic apposition to cutaneous primary afferent axons in the cat spinal cord

The purpose of the present study was to characterize the transmitter content of structures in presynaptic apposition to the central terminals of cutaneous afferent fibers in the dorsal horn of the spinal cord. Axons in the Aalphabeta conduction velocity range were identified in adult cats, stained intra-axonally with horseradish peroxidase, and prepared for combined light and electron microscopy. In total, we labeled two slowly adapting (Type 1) axons, two hair-follicle afferents, and one rapidly adapting (Krause) afferent. Ninety-nine labeled boutons were examined through complete series of serial sections. Approximately 80% of boutons originating from rapidly adapting and hair-follicle afferents were postsynaptic to other axons, but only 50% of boutons from slowly adapting axons were associated with this type of arrangement. Postembedding immunogold reactions revealed that between 80% (for slowly adapting axons) and 100% (for rapidly adapting axons) of boutons presynaptic to primary afferents were immunoreactive for gamma-aminobutyric acid (GABA). The vast majority of these terminals (in excess of 80%) were also enriched with glycine. Therefore, presynaptic inhibition of these three functional classes of Aalphabeta cutaneous primary afferents is mediated principally by the subgroup of GABAergic interneuron that also contains glycine.     

Developing descending neurons of the early Xenopus tail spinal cord in the caudal spinal cord of early Xenopus

The enzyme horseradish peroxidase (HRP) was used to describe and identify neurons, axons of which initiate the earliest descending pathways of the tail spinal cord of Xenopus embryos and larvae. Spinal cords were pierced at different rostrocaudal levels with fine insect pins coated with HRP. The resulting pattern of cellular labeling indicated that primitive sensory (Rohon-Beard) axons were at the lead of developing descending tracts followed by axons of primary motor neurons. Axons of these two neuron types travel in widely separated fascicles located dorso- and ventrolaterally, respectively. Subsequently, axons of several morphologically distinct intersegmental interneurons establish several additional fascicles positioned dorsal to the descending motor axons. Descending supraspinal axons appear only later. The distinctive morphological characteristics of each of the early descending cell types are illustrated along with some stages in their early differentiation. These observations establish the temporal pattern by which new axons are added to descending pathways beginning with the simplest level of the amphibian spinal cord and determine the identity of neurons to which axons at early stages in this sequence belong.     

The excitatory amino acid receptor antagonist MK-801 prevents the hypersensitivity induced by spinal cord ischemia in the rat.

Protection by the NMDA receptor antagonist MK-801 against transient spinal cord ischemia-induced hypersensitivity was studied in rats. The spinal ischemia was initiated by vascular occlusion resulting from the interaction between the photosensitizing dye Erythrosin B and an argon laser beam. The hypersensitivity, termed allodynia, where the animals reacted by vocalization to nonnoxious mechanical stimuli in the flank area, was consistently observed during several days after induction of the ischemia. Pretreatment with MK-801 (0.1-0.5 mg/kg, iv) 10 min before laser irradiation dose dependently prevented the occurrence of allodynia. The neuroprotective effect of MK-801 was not reduced by maintaining normal body temperature during and after irradiation. There was a significant negative correlation between the delay in the administration of MK-801 after irradiation and the protective effect of the drug. Histological examination revealed slight morphological damage in the spinal cord in 38% of control rats after 1 min of laser irradiation without pretreatment with MK-801. No morphological abnormalities were observed in rats after pretreatment with MK-801 (0.5 mg/kg). The present results provide further evidence for the involvement of excitatory amino acids, through activation of the NMDA receptor, in the development of dysfunction following ischemic trauma to the spinal cord.     

Metamorphosis and the regenerative capacity of spinal cord axons in Xenopus laevis

Throughout the vertebrate subphylum, the regenerative potential of central nervous system axons is greatest in embryonic stages and declines as development progresses. For example, Xenopus laevis can functionally recover from complete transection of the spinal cord as a tadpole but is unable to do so after metamorphosing into a frog. Neurons of the reticular formation and raphe nucleus are among those that regenerate axons most reliably in tadpole and that lose this ability after metamorphosis. To identify molecular factors associated with the success and failure of spinal cord axon regeneration, we pharmacologically manipulated thyroid hormone (TH) levels using methimazole or triiodothyronine, to either keep tadpoles in a permanently larval state or induce precocious metamorphosis, respectively. Following complete spinal cord transection, serotonergic axons crossed the lesion site and tadpole swimming ability was restored when metamorphosis was inhibited, but these events failed to occur when metamorphosis was prematurely induced. Thus, the metamorphic events controlled by TH led directly to the loss of regenerative potential. Microarray analysis identified changes in hindbrain gene expression that accompanied regeneration-permissive and -inhibitory conditions, including many genes in the permissive condition that have been previously associated with axon outgrowth and neuroprotection. These data demonstrate that changes in gene expression occur within regenerating neurons in response to axotomy under regeneration-permissive conditions in which normal development has been suspended, and they identify candidate genes for future studies of how central nervous system axons can successfully regenerate in some vertebrates.     

Alterations in spinal cord excitatory amino acid receptors in amyotrophic lateral sclerosis patients.

Excitatory amino acids (EAA) have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). We have analyzed the distribution of the N-methyl-D-aspartate (NMDA) 1-(1-(2-thienyl)-cyclohexyl) piperidine (TCP), kainate and alpha-amino-3-hydroxy-5-methyl-4 isoxazole propionic acid (AMPA) quisqualate subtypes of EAA receptors using quantitative receptor autoradiography in the cervical and thoracic spinal cords of patients who have died with ALS, and of controls. We observed that in control spinal cords [3H]TCP/NMDA binding sites were located both in the ventral and dorsal horns with the highest densities being situated in lamina II. [3H]AMPA and [3H]kainate binding sites were present almost exclusively in the substantia gelatinosa of the dorsal horn. In ALS, the distribution of these 3 types of receptors was unchanged, but [3H]TCP/NMDA binding was decreased both in the dorsal and ventral horns. [3H]kainate binding was possibly decreased in substantia gelatinosa, of ALS cords. However, the limited sample size available for [3H]kainate binding did not permit statistical analysis. [3H]AMPA binding sites were unaltered in ALS. These results indicate that there is a preferential reduction in NMDA receptors in ALS. We suggest that should an excitotoxic mechanism be involved in the pathogenesis of ALS, then NMDA receptors may be the target of this effect.     

Ultrastructure and synaptic connections of cutaneous afferent fibres in the spinal cord

The combination of intracellular recording and staining techniques with electron microscopy offers a unique opportunity for correlating physiological and ultrastructural properties of single neurones. The spinal terminations of cutaneous primary afferent fibres have been studied by this method, and examination of the ultrastructural synaptology of their boutons sheds light on how sensory information may be processed at the first synapse in the spinal cord. The combination of intracellular staining techniques with other neuroanatomical tract-tracing methods can reveal some of the neuronal networks of the spinal cord and, particularly, the contributions made by primary afferent fibres to these networks.     

The role of putative excitatory amino acid neurotransmitters in the initiation of locomotion in the lamprey spinal cord. I. The effects of excitatory amino acid antagonists

The activation of N-methyl-D-aspartate (NMDA) and kainate receptors will evoke fictive locomotion in the appropriate motor pattern for locomotion in the isolated lamprey spinal cord, but not a selective activation of quisqualate receptors. The present experiments test whether the initiation of locomotion in response to sensory stimulation depends on these types of receptors. An in vitro preparation of the lamprey spinal cord with part of its tailfin left innervated has been used. In this preparation a sequence of fictive locomotion (i.e. alternating bursts in the segmental ventral roots with a rostrocaudal phase lag) can be elicited by continual sensory stimulation of the tailfin. The effects of excitatory amino acid antagonists were studied by recordings from ventral roots (extracellularly) and motoneurones (intracellularly). It was found that the strong initial bursts of each swimming sequence induced by sensory stimulation were depressed by combined NMDA/kainate antagonists (cis-2,3-piperidine dicarboxylate (PDA) and gamma-D-glutamylglycine (gamma-DGG] whereas the less intense burst activity, occurring particularly towards the end of each swimming sequence, was depressed by a selective NMDA antagonist, 2-amino-5-phosphonovalerate (2-APV). This condition could be mimicked in an isolated spinal cord preparation by an application of L-glutamate; the low-level fictive locomotion induced by low doses of L-Glu (less than 100 microM) was depressed by a NMDA antagonist (2-APV), and, if higher doses were applied, the activity was only depressed by PDA/gamma-DGG. The mode and time course of the depression (by excitatory amino acid antagonists) of fictive locomotion, induced by sensory stimulation, shows that the putative excitatory amino acid neurotransmitter directly or indirectly acts at the pattern generating circuitry within the spinal cord.     

Differential induction of immediate early genes by excitatory amino acid receptor types in primary cultures of cortical and striatal neurons.

In primary cultures of neurons from cerebral cortex and striatum, 30 s stimulation with the excitatory amino acid glutamate elicited a 5 to 9-fold increase in immediate early gene (IEG) mRNAs. Glutamate increased c-fos, c-jun, jun-B, and NGFI-A (zif/268) mRNAs by binding to both alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor types, and increased c-fos, jun-B, and NGFI-A mRNAs by binding to the metabotropic receptor. NMDA receptor activation elicited IEG expression by a transmembrane calcium influx; AMPA receptor-induced depolarization played a permissive role for the opening of the NMDA receptor channel. The protein kinase C (PKC) inhibitor H-7 (but not inhibitors of cyclic nucleotide-dependent and calcium/calmodulin-dependent protein kinases) partially blocked IEG expression induced by glutamate.     

The early development of neurons with GABA immunoreactivity in the CNS of Xenopus laevis embryos

We have used an antibody to glutaraldehyde fixation complexes of gamma-amino butyric acid (GABA) to stain the developing central nervous system of Xenopus laevis embryos. Neuronal somata, growth cones, axons, and dendrites were found with GABA-like immunoreactivity. Transmission electron microscope (TEM) observations were made of axons and synapses. By observation of the earliest stages of differentiation of neurons, seven classes of putative GABAergic interneurons were discerned. 1) Ascending neurons are first stained in the hindbrain at stage 26 and later extend caudally in the spinal cord. They have ascending ipsilateral axons. 2) Midhindbrain reticulospinal neurons are first stained at stage 25 and develop as a compact group with descending ipsilateral and contralateral axons. 3) Vestibular complex commissural neurons are first stained at stage 29/30 in a dorsal position near the entry of the seventh and eighth cranial nerves. They have ventral commissural axons that descend contralaterally and their somata form a compact mass. 4) Rostral hindbrain commissural neurons are first stained at stage 33/34 just rostral to the entry of the trigeminal nerve. They each have a decussating projection. 5) Rostral midbrain neurons are first stained in the midbrain at stage 29/30 and are later associated with prominent dorsal and ventral commissures. 6) Optic tract and 7) rostral forebrain neurons are found in the forebrain associated with strongly stained axon tracts. The direction of axonal growth from its earliest stages was distinct for each class of hindbrain and spinal cord neuron.     

Domoic acid induced spinal cord lesions in adult mice: evidence for the possible molecular pathways of excitatory amino acids in spinal cord lesions

Domoic acid (DA) is an excitatory amino acids (EAAs) analog which induced excitotoxicity lesion to central nervous system, but whether induced adult animal spinal cord is not known, furthermore, previous studies have shown that EAAs play an important role in spinal cord lesion, however, the molecular pathways in spinal cord lesion are not fully known. Therefore, a motor neuron-like cell culture system and a DA-induced spinal cord lesioned mice model were used to study the effect of DA on spinal cord in adult mice and the possible molecular pathways of EAAs in spinal cord lesions. Exposure of motor neuron-like cells NSC34 to DA dramatically increased reactive oxygen species (ROS) production by the DCF fluorescent oxidation assay, reduced mitochondrial function by MTT assay, cell viability by trypan blue exclusion assay, and was accompanied by an increase of cell apoptosis by histone protein release assay. In DA-induced spinal cord lesioned mice model, we showed that the decrease of proteasome activity, increase of UCP4 expression by immunohistochemistry and neural cell apoptosis by TUNEL staining, and was accompanied by an decrease of motor disturbance grade during the different stages of DA treatment. Taken together, the in vitro and in vivo data presented in the current report demonstrated that DA induces spinal cord lesions in adult mice, and the multiple molecular pathways promoted by EAAs in spinal cord lesions, at least partially was associated with ROS generation increase, mitochondrial dysfunction, proteasome activity decrease and UCP4 expression increase.     

Sympathetic preganglionic neurones in neonatal rat spinal cord in vitro: electrophysiological characteristics and the effects of selective excitatory amino acid receptor agonists

Intracellular recordings were made from 52 lateral horn neurones in thin slices of neonatal rat thoracolumbar spinal cord. Of these neurones 12 were spontaneously active and the remainder silent. A number of these cells could be activated antidromically by stimulation of ventral roots. The conduction velocity of the antidromic potential was estimated to be 0.9–2 m/s which is within the range reported for axons of sympathetic preganglionic neurones (SPNs). The membrane properties of antidromically identified SPNs were similar to other lateral horn neurones included in this study and comparable to those reported for SPNs by others. Spontaneous burst firing was recorded in 3 neurones and activity in a further 5 neurones was characterized by the discharge of an action potential followed by an afterhyperpolarization potential (AHP) of peak amplitude 3–13 mV and duration 0.5–4 s. The AHP had an initial fast component (fAHP) which was sensitive to the potassium channel blocker tetraethylammonium (TEA), and a second slower component (sAHP) which was both sensitive to extracellular calcium and TEA. The effects of the selective excitatory amino acid receptor agonists N-methyl-d-aspartate (NMDA), kainate and quisqualate were investigated by superfusion of the agonists, at known concentrations (100 nM to 100 μM). These agonists induced concentration-dependent depolarizations which were primarily associated with a reduction in neuronal input resistance. NMDA-induced depolarizations were potentiated in the absence of magnesium. In a number of neurones NMDA, kainate and quisqualate (1–50 μM) induced, in addition to a depolarizing response, the discharge of small (1.5–6.5 mV), brief (7–20 ms) IPSPs which were reversed just below resting membrane potential at −64 mV. These results suggest that both NMDA and non-NMDA receptors may have an important role in the excitation of SPNs and of inhibitory interneurones presynaptic to SPNs.     

Development of excitatory amino acid induced cytotoxicity in cultured neurons

The neurotoxicity of the excitatory amino acids (EAAs) L-glutamate (L-glu), L-aspartate (L-asp), N-methyl-D-aspartate (NMDA), kainate (KA), quisqualate (QA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA) was followed as a function of development in primary cultures of cerebral cortex neurons and cerebellar granule cells. These two types of neurons express, respectively, glutamate receptor subtypes with sensitivity to all of these excitatory amino acids or only to glutamate and aspartate. None of the EAAs were toxic in cerebral cortex neurons at 2 days in culture, whereas at culture day 4 the neurons became sensitive to glutamate, at day 5 to KA followed by sensitivity to QA at day 6, and finally to NMDA, L-asp and AMPA at day 7. The rank order of potency of the EAAs was in cerebral cortex neurons cultured for 12 days: L-asp (ED50 = 0.5 microM) = L-glu (ED50 = 1 microM) greater than AMPA (ED50 = 10 microM) greater than NMDA (ED50 = 65 microM) greater than QA = KA (ED50 = 100 microM). Cerebellar granule cells were insensitive to all of the EAAs at 3 and 5 days in culture but at day 8 the cells became sensitive to toxicity induced by L-glu (ED50 = 70 microM) and L-asp (ED50 = 30 microM). In order to determine ED50 values for L-asp and L-glu accurately, media in these experiments also contained 500 microM of the glutamate uptake inhibitor L-aspartate-beta-hydroxamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cascades of response vectors of olfactory receptor neurons in Xenopus laevis tadpoles

Olfactory receptor neurons (ORNs) of Xenopus laevis tadpoles respond to water-born stimuli such as amino acids. Their sensitivity spectra with respect to amino acids have recently been shown to become more selective over ontogenetic stages [Manzini & Schild (2004) J. Gen. Physiol., 123, 99-107]. In this paper, we undertake a theoretical analysis of this data set and determine the correlational relationships among odorant responses represented as binary response vectors. We first show that, on the one hand, the number of 204 ORN classes (out of 283 recorded ORNs) cannot be explained by a random expression pattern of olfactory receptors (ORs). On the other hand, this number does not appear to be reconcilable with the idea that individual ORNs express one type of OR each. The covariance matrix of stimulus responses shows that the responses to some stimuli are correlated to those of others. Furthermore, the response vectors show positive as well as negative correlations among each other. While the positive correlations can partly be explained by the differing response frequencies to the odorants used, the negative ones cannot. Finally, we analyse the similarity among responses using the Hamming distance as a distance measure, the result being that most response vectors differ from others by small Hamming distances. Such vectors are shown to form pattern cascades, possibly reflecting a decreasing number of ORs being expressed over ontogenetic stages.