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O Shupliakov

Publications and source records attributed to O Shupliakov.

At least 37 records · Page 2Linked to original sources
PubMed1996

Monosynaptic input from cutaneous sensory afferents to fin motoneurons in lamprey.

The sensory control of lamprey dorsal fin motoneurons was studied by using paired intracellular recordings combined with a morphological analysis. Dorsal cells innervating the skin of the dorsal fin and fin motoneurons were retrogradely labeled by injecting fluoresceincoupled dextran amines into the dorsal fin. Labeled motoneurons and dorsal cells showed close appositions, suggesting that the dorsal cells innervating the fin region make monosynaptic connections with fin motoneurons. By using conventional electrophysiological criteria, monosynaptic excitatory connections were found between fin dorsal cells and fin motoneurons. In addition, Lucifer yellow injection followed by confocal three-dimensional (3-D) reconstructions of monosynaptically connected pairs, revealed close apposition between dorsal cell axons and the distal dendrites of fin motoneurons. Each fin motoneuron received monosynaptic excitatory input from at least four different afferents. The amplitude of the monosynaptic excitatory postsynaptic potential (EPSP)s was reduced by administration of the N-methyl-D-aspartate (NMDA) receptor antagonist DL,2 amino-5-phosphovaleric acid (APV). Sensory stimulation could also elicit di- or oligosynaptic inhibitory postsynaptic potential (IPSP)s, which were blocked by the glycine antagonist strychnine, resulting in the appearance of large monosynaptic EPSPs, which could induce action potentials.

2-Amino-5-phosphonovalerate↗
PubMed1996

Qualitative and quantitative analysis of glycine- and GABA-immunoreactive nerve terminals on motoneuron cell bodies in the cat spinal cord: a postembedding electron microscopic study.

The distribution of glycine- and gamma-aminobutyric acid (GABA)-like immunoreactivity (LI) in nerve terminals on the cell soma of motoneurons in the aldehyde-fixed cat L7 spinal cord was examined using postembedding immunogold histochemistry in serial ultrathin sections. Quantitative examination of 405 terminals on eight neurons of alpha-motoneuron size in the L7 motor nuclei from one animal was performed. A majority of the terminals (69%) were immunoreactive to glycine and/or GABA. These terminals contained flat or oval synaptic vesicles, thus classifying them as F type or as C type in one case. In no case was a type-F terminal unlabeled for both glycine and GABA. Most of the immunolabeled terminals were immunoreactive to glycine only (62.5%), whereas 35.4% contained both glycine- and GABA-LI. A very small number of immunolabeled terminals (2%) were immunoreactive to GABA only. In those terminals, where glycine- and GABA-LI coexisted, the gold particle density for each amino acid was only half of that seen in boutons containing only one of the two amino acids. The involvement of glycine and GABA in postsynaptic inhibition of spinal alpha-motoneurons is discussed, with particular reference to the possibility that these two inhibitory amino acids may be coreleased from a significant proportion of the nerve terminals impinging on the cell bodies.

Animals↗
PubMed1996

Immunocytochemical localization of glycine in the lamprey spinal cord with reference to GABAergic and glutamatergic synapses: a light and electron microscopic study.

The distribution of glycine immunoreactivity in the lamprey (Lampetra fluviatilis and Ichthyomyzon unicuspis) spinal cord was studied at the light and electron microscopic levels by use of postembedding techniques and antibodies against glutaraldehyde-conjugated glycine. To determine if glycine may be co-stored with other amino acid transmitters, the levels of glycine immunolabeling in identified GABAergic and glutamatergic synapses were examined. The most intense glycine labeling occurred in axon profiles of different diameter distributed throughout the ventral and lateral columns, with the highest density in the areas bordering the lateral cell column. Intermediate levels of glycine labeling were present in certain interneurons in the lateral cell column and in stretch receptors (edge cells) at the lateral margin of the spinal cord. Most other cell bodies, including glutamatergic dorsal cells, were virtually unlabeled. Examination of adjacent sections incubated with GABA antiserum revealed that many of the glycine-containing cells and fibers also contained high levels of GABA. At the ultrastructural level, the glycine immunolabeling was accumulated in two morphologically distinct types of terminal, one of which co-contained GABA. The terminals which exhibited glycine, but not GABA immunoreactivity, contained flattened synaptic vesicles and formed symmetrical synaptic specializations. The terminals that exhibited both GABA and glycine labeling contained pleomorphic synaptic vesicles and had either symmetrical or asymmetrical synaptic specializations. In both cases the glycine labeling was accumulated over the synaptic vesicles. Examination of identified glutamatergic axons in glycine-labeled sections did not provide any evidence for an accumulation of glycine in the synaptic vesicles or other structures of these exons. The present study provide the first morphological description of the localization of glycine in the lamprey spinal cord. The results confirm previous physiological and pharmacological studies, which have implicated glycine as a major fast inhibitory transmitter in the interneuronal network for locomotion, and in a proportion of stretch receptor neurons. The data also show that a significant proportion of the GABAergic synapses, but not the glutamatergic synapses, may utilize glycine as co-transmitter.

Animals↗
PubMed1995

Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice.

Synapsin I has been proposed to be involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis. To further understand the role of synapsin I in the function of adult nerve terminals, we studied synapsin I-deficient mice generated by homologous recombination. The organization of synaptic vesicles at presynaptic terminals of synapsin I-deficient mice was markedly altered: densely packed vesicles were only present in a narrow rim at active zones, whereas the majority of vesicles were dispersed throughout the terminal area. This was in contrast to the organized vesicle clusters present in terminals of wild-type animals. Release of glutamate from nerve endings, induced by K+,4-aminopyridine, or a Ca2+ ionophore, was markedly decreased in synapsin I mutant mice. The recovery of synaptic transmission after depletion of neurotransmitter by high-frequency stimulation was greatly delayed. Finally, synapsin I-deficient mice exhibited a strikingly increased response to electrical stimulation, as measured by electrographic and behavioral seizures. These results provide strong support for the hypothesis that synapsin I plays a key role in the regulation of nerve terminal function in mature synapses.

Animals↗
PubMed1995

Distinct pools of synaptic vesicles in neurotransmitter release.

Nerve terminals are unique among cellular secretory systems in that they can sustain vesicular release at a high rate. Although little is known about the mechanisms that account for the distinctive features of neurotransmitter release, it can be assumed that neuron-specific proteins are involved. One such protein family, the synapsins, are believed to regulate neurotransmitter release through phosphorylation-dependent interactions with synaptic vesicles and cytoskeletal elements. Here we show that clusters of vesicles at synaptic release sites are composed of two pools, a distal pool containing synapsin and a proximal pool devoid of synapsin and located adjacent to the presynaptic membrane. Presynaptic injection of synapsin antibodies resulted in the loss of the distal pool, without any apparent effect on the proximal pool. Depletion of this distal pool was associated with a marked depression of neurotransmitter release evoked by high-frequency (18-20 Hz) but not by low-frequency (0.2 Hz) stimulation. Thus the availability of the synapsin-associated pool of vesicles seems to be required to sustain release of neurotransmitter in response to high-frequency bursts of impulses.

Animals↗
PubMed1995

Synaptic vesicle depletion in reticulospinal axons is reduced by 5-hydroxytryptamine: direct evidence for presynaptic modulation of glutamatergic transmission.

5-hydroxytryptamine (5-HT; serotonin) is known to depress glutamatergic synaptic transmission in the spinal cord of vertebrates. To test directly whether 5-HT inhibits synaptic glutamate release, we examined its effect on the ultrastructure of synaptic vesicle clusters in giant reticulospinal axons in a lower vertebrate (lamprey; Lampetra fluviatilis). The size of these axons makes it possible to selectively expose only a part of the presynaptic element to 5-HT, while another part of the same axon is maintained in control solution. Action potential stimulation at 20 Hz for 20 min caused a marked reduction in the number of synaptic vesicles in active zones maintained in control solution, while in the part exposed to 5-HT (20 microM) the number of synaptic vesicles per active zone was approximately 3-fold higher. In contrast, 5-HT had no effect on the number of vesicles in resting axons. To examine whether 5-HT acts by reducing presynaptic Ca2+ influx, intra-axonal recordings of Ba2+ potentials were performed. No reduction of the axonal Ba2+ potential could be detected after application of 20 or 200 microM 5-HT. The present results show that 5-HT reduces the rate of synaptic exocytosis in reticulospinal axons. The effect appears to be mediated by a mechanism distinct from the presynaptic Ca2+ channels.

Action Potentials↗
PubMed1995

Control of lamprey locomotor neurons by colocalized monoamine transmitters.

Neurons in the central nervous system (CNS) often store more than one neurotransmitter, but as yet the functional significance of this type of coexistence is poorly understood. 5-Hydroxytryptamine (5-HT) modulates calcium-dependent K+ channels (KCa) responsible for the postspike afterhyperpolarization in different regions of the CNS. In lamprey, 5-HT neurons control apamine-sensitive KCa channels in spinal locomotor network interneurons, thereby in addition regulating the duration of locomotor bursts. We report here that these spinal 5-HT neurons also contain dopamine. Like 5-HT, dopamine causes a reduction of the afterhyperpolarization, but in this case it is due to a reduction of calcium entry during the action potential, which results in a reduced activation of KCa. 5-HT and dopamine are both released from these midline neurons, and both reduce the afterhyperpolarization through two distinctly different, but complementary cellular mechanisms. The net effect of dopamine (10-100 microM) on the locomotor network is similar to that of 5-HT, and the effects of dopamine and 5-HT are additive at the network level.

Action Potentials↗
PubMed1995

Presynaptic glutamate levels in tonic and phasic motor axons correlate with properties of synaptic release.

Synaptic glutamate release involves the accumulation of cytoplasmic glutamate in synaptic vesicles, whereafter it is released by triggered exocytosis. As glutamatergic terminals are known to be functionally diverse it was of interest to examine whether the presynaptic glutamate supply differs between individual axon terminals with distinct release properties. The glutamatergic terminals in the crustacean neuromuscular system system comprise a "phasic" type which shows fatigue of release during repetitive stimulation, and a "tonic" type which can maintain transmission for long periods. Quantitative immunogold analysis showed that the axons in a tonic nerve innervating slow muscles in the abdomen contained two times higher levels of glutamate labeling over axoplasmic matrix and over mitochondria, as compared to the corresponding elements in a phasic nerve. Similar results were obtained when adjacent phasic and tonic axons in a mixed nerve innervating leg muscles were compared. In the terminal regions of tonic and phasic axons the glutamate labeling differed correspondingly over axoplasmic matrix and mitochondria, while the synaptic vesicles showed a similar strong accumulation of labeling in both types of terminal. The level of labeling for glutamine, a glutamate precursor, was closely similar in phasic and tonic axons. The axoplasmic glutamate concentration was estimated to be in the low millimolar range, through comparison with coprocessed conjugates with known glutamate concentration. These results show that fatigue-resistant tonic axons and terminals contain higher levels of glutamate than fatiguable phasic axons, presumably representing an adaptation to the markedly different impulse activities in the two types of neuron. The axonal glutamate concentrations are in the range of the Km value for vesicular glutamate transport. Thus in tonic axons the high glutamate level appears to promote an efficient refilling of synaptic vesicles during sustained release, while in phasic axons the refilling should be slower which is compatible with an infrequent release.

Animals↗
PubMed1995

Quantification of excitatory amino acid uptake at intact glutamatergic synapses by immunocytochemistry of exogenous D-aspartate.

To study the localization and efficiency of glutamate/aspartate membrane transport in the vicinity of intact glutamatergic synapses, the avascular lamprey spinal cord was incubated with D-aspartate, a metabolically inert transporter substrate. The exogenous D-aspartate was localized by immunocytochemistry after aldehyde fixation. Incubation at 50 or 500 microM D-aspartate for 1 hr caused a prominent D-aspartate labeling of glial processes at glutamatergic synapses, while presynaptic axons and postsynaptic dendrites remained unlabeled. The glial processes surrounding glutamatergic sensory axons with a predominantly tonical firing pattern contained significantly higher levels of D-aspartate than did processes surrounding glutamatergic reticulospinal axons, which fire rarely and in brief bursts. Preparations incubated for 10 hr with 500 microM D-aspartate showed D-aspartate immunolabeling in glia as well as in the two types of glutamatergic axon, but no evidence was obtained for uptake into synaptic vesicles. Nor was such evidence obtained after high-frequency electrical stimulation. The observations suggest that excitatory amino acids delivered diffusely to the extracellular space in the intact CNS are transported almost exclusively into glia. The avid uptake in glial processes, combined with their spatial arrangement around glutamatergic synapses, appears to limit the access of exogenous D-aspartate to the nerve terminal glutamate/aspartate transporter. In physiological conditions, the glial processes are likely to impede the exchange of glutamate between the synaptic cleft and the rest of the extracellular space. The transport was more efficient in glial processes located near tonically active synapses than in ones located near synapses releasing transmitter sporadically. D-Aspartate is not a substrate of vesicular glutamate transport sites at these intact synapses.

Animals↗
PubMed1994

Extrasynaptic localization of taurine-like immunoreactivity in the lamprey spinal cord.

Taurine is an endogenous amino acid that can occur in nerve terminals in the central nervous system and that can produce inhibitory neuronal responses. It is unclear, however, whether this amino acid can function as a synaptic transmitter. To examine the distribution of taurine at high anatomical resolution in a vertebrate, light and electron microscopic immunocytochemical postembedding techniques were applied to the lamprey spinal cord (Ichtyomyzon unicuspis and Lampetra fluviatilis), which contains many large, unmyelinated axons. The most intense immunolabeling occurred in a population of liquor-contacting cells (tanycytes), located around the central canal, which extended processes to the dorsal, lateral, and ventral margins of the spinal cord. In addition, a proportion of the taurine-immunoreactive cells contained gamma-aminobutyric acid (GABA)-like immunoreactivity. A moderate level of taurine immunoreactivity was also present in ependymal cells, located around the central canal, as well as in astrocytes throughout all regions of the spinal cord. At the ultrastructural level, the taurine immunoreactivity showed an even distribution in the cytoplasm of the labeled cells. In contrast to the glial labeling, neuronal cell bodies and axons exhibited very low levels of taurine labeling, which were similar to the level of background labeling. The synaptic vesicle clusters within the axons did not show any clear accumulation of taurine immunoreactivity. These results suggest that taurine may have metabolic roles in the lamprey spinal cord, and, as in other systems, it may take part in osmoregulation. However, the lack of immunolabeling in presynaptic elements is not consistent with a role of taurine as a synaptic transmitter.

Animals↗
PubMed1994

Immunohistochemical evidence for coexistence of glycine and GABA in nerve terminals on cat spinal motoneurones: an ultrastructural study.

Previous electrophysiological and pharmacological studies have suggested the involvement of both glycine and gamma-aminobutyric acid (GABA) as transmitters in the reflex pathway mediating recurrent inhibition of cat spinal motoneurones. By use of the postembedding immunogold technique and antibodies against glutaraldehyde-conjugated amino acids it is shown that glycine- and GABA-immunoreactive nerve terminals occur in contact with cell bodies of alpha motoneurone size, as well as in the surrounding neuropil in the motor nuclei of the cat spinal cord. A substantial proportion of these terminals harbours both glycine- and GABA-LI. The enrichment of immunolabelling over synaptic vesicles in such terminals strongly suggests that both of these amino acids are used as transmitter substances.

Animals↗
PubMed1994

The reticulospinal glutamate synapse in lamprey: plasticity and presynaptic variability.

1. The glutamatergic synapses formed between the unbranched giant reticulospinal axons onto spinal neurons in lamprey offer a central vertebrate synapse in which the presynaptic element can be impaled with one or several microelectrodes, which may be used for recording as well as microinjection of different substances. To provide a basis for the use of this synapse in studies of release mechanisms, we have examined the use-dependent modulation of the synaptic response under conditions of conventional cell body stimulation, and during direct stimulation of the presynaptic axon. 2. To examine the stability of the mixed electrotonic and chemical reticulospinal excitatory postsynaptic potential (EPSP) over time, action potentials were evoked at a rate of 1 Hz for 800-1000 trials. In three out of seven synapses the chemical component remained at a similar amplitude, while in four cases a progressive decrease (up to 35%) occurred. The electrotonic component remained at a similar amplitude in all cases. 3. During paired pulse stimulation of the reticulospinal cell body (pulse interval 65 ms) the chemical EPSP component showed a net facilitation in all cases tested [from 0.64 +/- 0.35 to 0.89 +/- 0.48 (SD) mV, n = 13], while the peak amplitude of the electrotonic component was unchanged (1.37 +/- 0.68 and 1.36 +/- 0.66 mV, respectively). Recording of the axonal action potential during paired pulse stimulation showed that the width of the first and second action potential did not differ [1/2 width (2.48 +/- 0.39 ms and 2.48 +/- 0.42 ms, respectively; n = 8)]. 4. The degree of facilitation varied markedly between different synapses, ranging from an increase of a few percent to a two-fold increase (24 +/- 16% mean change of total EPSP amplitude, corresponding to 44 +/- 26% mean change of chemical EPSP amplitude). This type of variability was also observed in synapses made from the same unbranched reticulospinal axon onto different postsynaptic cells. 5. When paired pulse stimulation was applied to the reticulospinal axon in the very vicinity of the synaptic area (0.1-1 mm) a net depression of the chemical component occurred in 11 out of 19 cases, and in the remaining cases the level of net facilitation was lower as compared with cell body stimulation (range between +17 and -23% change of total EPSP amplitude; mean -5%; n = 19). 6. To test if the change of the EPSP plasticity during local stimulation correlated with an increased transmitter release, two microelectrodes were placed in the same reticulospinal axon at different distances from the synaptic area.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Possible morphological substrates for GABA-mediated presynaptic inhibition in the lamprey spinal cord.

Gamma-aminobutyric acid (GABA) neurons intrinsic to the lamprey spinal cord are known to modulate synaptic transmission from interneurons active during locomotion and from mechanosensory dorsal cells. Many of these physiological effects are presynaptic. To establish the morphological substrates for these axo-axonic interactions, an ultrastructural analysis was performed with an antiserum to fixed GABA. The GABA immunoreactivity (ir) was detected by postembedding peroxidase-antiperoxidase and immunogold techniques. GABA-ir terminals were found to make appositions with unlabelled axons located in the dorsal columns and in the ventrolateral aspect of the spinal cord. In the ventrolateral part of the cord, similar appositions between different GABA-ir terminals were also observed. The immunolabelled terminals contained spherical to pleomorphic synaptic vesicles, and also glycogen granules and dense core vesicles. In some cases, the fine structure of the contacts between immunogold-labelled terminals and unlabelled axons suggested a synaptic relationship. Such a relation was found in a relatively small proportion (2-3%) of the appositions studied. These specializations were always observed in close relation to an output synapse of the postsynaptic axon. It is suggested that the axo-axonal contacts described may provide an effective modulation of the synaptic transmission from axons in the lamprey spinal cord.

Animals↗
PubMed1993

Origin of phasic synaptic inhibition in myotomal motoneurons during fictive locomotion in the lamprey.

The periodic membrane potential fluctuations in motoneurons during fictive locomotion in the lamprey, a primitive vertebrate, involve phasic synaptic excitation and inhibition. This paper investigates the origin of the phasic synaptic input to lamprey myotomal motoneurons in the in vitro spinal cord preparation with regard to the relative contribution of descending propriospinal input from interneurons in the local segment. The synaptic drive to myotomal motoneurons in the most rostral and the most caudal part of the spinal cord preparation are compared before and after selective spinal cord lesions. Current clamp recordings of the same cell before and after lesion showed that neither the excitatory phase nor the inhibitory phase was abolished after interruption of the descending or the ascending ipsilateral input, or after interrupting crossing segmental input by a local longitudinal midline incision. None of these sources thus appears to be alone responsible for the phasic synaptic drive. To quantitatively evaluate these effects, and in particular the contribution from the descending propriospinal fibres to the inhibitory phase, voltage clamp recordings were made in combination with a spinal cord hemisection just rostral to the motoneuron. The input from propriospinal interneurons in approximately 15 rostral segments may be responsible for as much as 70% of the phase of inhibitory current during the locomotor cycle. In accordance with these findings, a similar voltage clamp analysis of rostrally and caudally located motoneurons showed that the average peak-to-peak amplitude of the current fluctuations in rostral cells was approximately 50% of that in caudal cells.

Animals↗
PubMed1993

Immunocytochemical localization of amino acid neurotransmitter candidates in the ventral horn of the cat spinal cord: a light microscopic study.

The distribution of immunoreactivities to six amino acids, possibly related to synaptic function, was investigated in the motor nucleus of the cat L7 spinal cord (laminae VII and IX) using a postembedding peroxidase-antiperoxidase technique. Consecutive 0.5 micron transverse sections of plastic-embedded tissue were incubated with antisera raised against protein-glutaraldehyde conjugates of gamma-aminobutyric acid (GABA), glycine, aspartate, glutamate, homocysteate, and taurine. This method allowed localization of the different immunoreactivities in individual cell profiles. The results showed that all these amino acids, except homocysteate, could be clearly detected in either neuronal or glial elements in the ventral horn. In cell bodies of neurons in lamina VII, immunoreactivity was observed for aspartate, glutamate, GABA, and glycine. Adjacent section analysis revealed that combinations of immunoreactivity for glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate and glutamate/aspartate, respectively, may occur in one and the same cell. In the motor nuclei (lamina IX), immunoreactivity to amino acids was observed in two types of neuron. Large cells, probably representing alpha-motoneurons, were harboring immunoreactivity to both glutamate and aspartate, while a few small neurons in this area displayed a colocalization of glycine, glutamate, and aspartate. Dendrites and axons in the motor nuclei contained glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate, and glutamate/aspartate immunoreactivities. In both laminae VII and IX, taurine-like immunoreactivity was absent in neuronal cell bodies, but highly concentrated in perivascular cells and small cells with a morphology resembling that of glial cells. A punctate immunolabeling, in all probability representing labeling of nerve terminals, could be demonstrated in the ventral horn for GABA, glycine, and glutamate, but not with certainty for aspartate or taurine. A quantitative estimate of the covering of cell bodies of alpha-motoneuron size by immunoreactive puncta revealed that glycine immunoreactive terminal-like structures were most abundant (covering 26-42% of the somatic membrane), while glutamate immunoreactive terminals were seen least frequently (5-9% covering). GABA-immunoreactive terminals covered from 10 to 24% of the soma surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗