Presynaptic mechanisms in central synaptic transmission: 'biochemistry' of an intact glutamatergic synapse.
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Biomedical subjects
L Brodin
Publications and source records attributed to L Brodin.
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.
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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.
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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.
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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↗
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↗
Peptidergic neurons in the vertebrate spinal cord: evolutionary trends.
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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.
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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.
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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.
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Neuropeptide role of both peptide YY and neuropeptide Y in vertebrates suggested by abundant expression of their mRNAs in a cyclostome brain.
The evolution of the neuropeptide Y (NPY) family of peptides has been unclear despite sequence information from many vertebrates. We describe here two NPY-related peptides deduced from cDNA clones of the river lamprey (Lampetra fluviatilis), a cyclostome providing one of the best models of a primitive vertebrate brain. One peptide corresponds to NPY as it has 83% identity to human NPY and its mRNA is expressed in the lateral brainstem, dorsal spinal cord and retina. The second lamprey peptide corresponds anatomically to peptide YY (PYY) as its mRNA is found in gut cells and in medial brainstem neurons. Its sequence is 60-70% identical to both PYY and NPY of mammals. These data suggest that the gene duplication leading to NPY and PYY had already occurred in the ancestral vertebrate 450 million years ago. The expression of the presumed PYY homolog in both gut and central nervous system indicates that PYY has served the dual role as a hormone and a neuropeptide from an early stage in vertebrate evolution. The similarities in the location of NPY- and PYY-expressing cells between lamprey and mammals suggest that the functions of these peptides may have been conserved.
Amino Acid Sequence↗
Immunohistochemical analysis of the relation between 5-hydroxytryptamine- and neuropeptide-immunoreactive elements in the spinal cord of an amphibian (Xenopus laevis).
In mammals, a large proportion of the bulbospinal 5-hydroxytryptamine (5-HT) neurons also contain neuropeptides, such as substance P (SP) and galanin (GAL). To examine whether a similar coexistence occurs in an amphibian, an immunofluorescence double-labelling technique was employed on sections of the Xenopus laevis spinal cord. Antisera raised against SP, GAL, enkephalin (ENK), corticotropin-releasing factor (CRF), calcitonin gene-related peptide (CGRP), and cholecystokinin (CCK) produced a labelling of fibers at all rostrocaudal levels of the spinal cord, with the highest fiber densities for SP and ENK and intermediate densities for GAL, CCK, and CGRP, while CRF-immunoreactive fibers were barely detectable in intact animals. 5-HT-immunoreactive fibers were widely distributed in the spinal cord, and they often occurred in the vicinity of different types of peptide-immunoreactive fibers. However, no coexistence between 5-HT and the different peptide immunoreactivities could be detected, although SP and GAL immunoreactivities were sometimes found to be colocalized in the same fiber. Similar negative results were obtained when 5-HT+SP- and 5-HT+GAL-labelled sections were examined in single focal planes with a confocal microscope. After a spinal transection, (survival period 6 weeks to 4 months), almost all 5-HT-immunoreactive fibers below the lesion were lost, and a build-up of immunoreactive material occurred in fibers just rostral to the cut. In contrast, no significant loss of peptide-immunoreactive fibers occurred, although some swollen SP-, GAL-, ENK-, CRF-, and CCK-immunoreactive fibers were present rostral to the cut. The distribution of swollen peptide-immunoreactive fibers did not overlap with that of the swollen 5-HT-immunoreactive fibers. Although negative immunohistochemical data must be interpreted with caution, in conjunction with previous studies (Brodin et al. [1988] J. Comp. Neurol. 271:1-18; Sakamoto and Atsumi [1991] Cell Tissue Res. 264:221-230), the present results indicate that bulbospinal 5-HT neurons in nonmammalian vertebrates cocontain neuropeptides to a lesser extent than in mammals.
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Functional diversity of central glutamate synapses--pre- and post-synaptic mechanisms.
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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)
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Central glutamatergic transmission. A view from the presynaptic axon.
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Animals↗
Evolutionary conservation of synaptosome-associated protein 25 kDa (SNAP-25) shown by Drosophila and Torpedo cDNA clones.
The neuron-specific proteins SNAP-25 (synaptosome-associated protein 25 kDa), synaptobrevin and syntaxin, are localized to presynaptic terminals in mammals and have been found to associate with proteins involved in vesicle docking and membrane fusion. We describe here SNAP-25 cDNA clones from the fruit fly Drosophila melanogaster and the ray Torpedo marmorata. In situ hybridization showed that SNAP-25 mRNA is exclusively found in brain and ganglia in Drosophila with a pattern suggesting expression in most neurons. The Drosophila and Torpedo proteins show 61 and 81% amino acid identity to mouse SNAP-25, a degree of conservation similar to that previously reported for synaptobrevin. None of the SNAP-25 sequences has a membrane-spanning region, but all contain a cluster of cysteine residues that can be palmitoylated for membrane attachment. SNAP-25 displays sequence similarity to syntaxin A and B. These data show that SNAP-25 and synaptobrevin, which are both implicated in vesicle docking and/or membrane fusion, have both been highly conserved during evolution. This supports the existence of a basic molecular machinery for synaptic vesicle docking in vertebrate and invertebrate synapses.
Amino Acid Sequence↗
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↗
Computer simulations of NMDA and non-NMDA receptor-mediated synaptic drive: sensory and supraspinal modulation of neurons and small networks.
1. The segmental locomotor network in lamprey can generate the rhythmic burst pattern underlying locomotion when it is driven via synaptic glutamate receptors. Lower rates of activity can be evoked by activation of N-methyl-D-aspartate (NMDA) receptors, whereas a rapid activity can only be induced by non-NMDA receptors [kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)]. The reticulospinal and sensory inputs are known to act via both NMDA and non-NMDA receptors, but it is unclear how these inputs can provide an appropriate control of the locomotor rate. We have examined the effects of different types of excitatory synaptic input to neurons of the locomotor network with the use of a computer-simulated electrical neuron model, with Na+, K+, Ca(2+)-dependent K+ channels, and with inherent oscillatory properties linked to the NMDA conductance. Synapses were modeled as a modulated ionic conductance in the membrane of the postsynaptic cell comprising a voltage-dependent NMDA component (Na+, K+, Ca2+ conductances) of long duration, and/or a non-NMDA component (Na+, K+ conductance) of short duration. 2. By using two neurons to drive a postsynaptic cell with non-NMDA-type synapses, a continuous range of firing frequencies could be evoked in the postsynaptic cell, by altering the firing rate of the presynaptic cells. If a single presynaptic neuron was used, there was a tendency toward spike synchronization between the pre- and postsynaptic cells. 3. When a postsynaptic neuron was driven via NMDA synapses, an oscillatory burst activity could be evoked. The rate of the oscillations was, however, little affected by the presynaptic firing rate. When a drive neuron with mixed (NMDA and non-NMDA) synapses was used, the rate of the oscillations could be changed within a limited frequency range by altering the presynaptic firing rate. By adding another larger drive neuron, having a larger rheobase current and mixed synapses with smaller relative NMDA components, the frequency range of the postsynaptic oscillations could be markedly increased. The frequency range depended on the parameters selected for each of the two types of mixed synapses. 4. A small rhythm-generating neuronal network, comprising six cells connected as the principal interneurons of the lamprey spinal locomotor network, was used to test the role of a tonic NMDA and non-NMDA receptor activation to drive the network and produce bursting.(ABSTRACT TRUNCATED AT 400 WORDS)
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