Motor Axon Synapses on Renshaw Cells Contain Higher Levels of Aspartate than Glutamate

Motoneuron synapses on spinal cord interneurons known as Renshaw cells activate nicotinic, AMPA and NMDA receptors consistent with co-release of acetylcholine and excitatory amino acids (EAA). However, whether these synapses express vesicular glutamate transporters (VGLUTs) capable of accumulating glutamate into synaptic vesicles is controversial. An alternative possibility is that these synapses release other EAAs, like aspartate, not dependent on VGLUTs. To clarify the exact EAA concentrated at motor axon synapses we performed a quantitative postembedding colloidal gold immunoelectron analysis for aspartate and glutamate on motor axon synapses (identified by immunoreactivity to the vesicular acetylcholine transporter; VAChT) contacting calbindin-immunoreactive (-IR) Renshaw cell dendrites. The results show that 71% to 80% of motor axon synaptic boutons on Renshaw cells contained aspartate immunolabeling two standard deviations above average neuropil labeling. Moreover, VAChT-IR synapses on Renshaw cells contained, on average, aspartate immunolabeling at 2.5 to 2.8 times above the average neuropil level. In contrast, glutamate enrichment was lower; 21% to 44% of VAChT-IR synapses showed glutamate-IR two standard deviations above average neuropil labeling and average glutamate immunogold density was 1.7 to 2.0 times the neuropil level. The results were not influenced by antibody affinities because glutamate antibodies detected glutamate-enriched brain homogenates more efficiently than aspartate antibodies detecting aspartate-enriched brain homogenates. Furthermore, synaptic boutons with ultrastructural features of Type I excitatory synapses were always labeled by glutamate antibodies at higher density than motor axon synapses. We conclude that motor axon synapses co-express aspartate and glutamate, but aspartate is concentrated at higher levels than glutamate.     

Glutamatergic transmission between antagonistic motor neurones in the locust

The pharmacology of the direct central connections between the fast extensor and flexor motor neurones of a locust (Schistocerca gregaria) hind leg was studied. A spike in the fast extensor produces an EPSP in the flexor motor neurones. Glutamate depolarized the flexor motor neurones when injected into the neuropil. Quisqualate, but not by kainate or NMDA, also depolarized the flexor motor neurones. The fast extensor was also depolarized by glutamate, and also by kainate, but not by quisqualate, AMPA or NMDA. The glutamate response in the flexor motor neurones and the EPSP evoked by a spike in FETi both had similar reversal potentials. The FETi-evoked EPSP was blocked by bath application of the glutamate antagonist glutamic acid diethyl ester. The responses of extrasynaptic somata receptors to glutamate were compared to the neuropil responses. Glutamate usually hyperpolarized the somata of FETi and the flexor motor neurones. The response of a flexor motor neurone to glutamate was abolished at potentials less negative than -90 mV. The results provide evidence for glutamate transmission at central synapses in the locust, and show that presumed synaptic receptors in the neuropil differ to the extrasynaptic soma response     

GABA and glutamate-like immunoreactivity at synapses received by dorsal unpaired median neurones in the abdominal nerve cord of the locust

Dorsal unpaired median (DUM) neurones in the abdominal ganglia of the locust were impaled with microelectrodes and some were injected intracellularly with horseradish peroxidase so that their synapses could be identified in the electron microscope. Simultaneous recordings from DUM neurones in different abdominal ganglia revealed that they received common postsynaptic potentials from descending interneurones. Post-embedding immunocytochemistry using antibodies against GABA and glutamate was carried out on ganglia containing HRP-stained neurones. GABA-like immunoreactivity was found in 39% (n=82) of processes presynaptic to abdominal DUM neurones and glutamate-like immunoreactivity in 21% (n=42) of presynaptic processes. Output synapses from the DUM neurites were rarely observed within the neuropile. Structures resembling presynaptic dense bars but not associated with synaptic vesicles, were seen in some large diameter neurites.     

Cholinergic interneurons mediate fast VGluT3-dependent glutamatergic transmission in the striatum

The neurotransmitter glutamate is released by excitatory projection neurons throughout the brain. However, non-glutamatergic cells, including cholinergic and monoaminergic neurons, express markers that suggest that they are also capable of vesicular glutamate release. Striatal cholinergic interneurons (CINs) express the Type-3 vesicular glutamate transporter (VGluT3), although whether they form functional glutamatergic synapses is unclear. To examine this possibility, we utilized mice expressing Cre-recombinase under control of the endogenous choline acetyltransferase locus and conditionally expressed light-activated Channelrhodopsin2 in CINs. Optical stimulation evoked action potentials in CINs and produced postsynaptic responses in medium spiny neurons that were blocked by glutamate receptor antagonists. CIN-mediated glutamatergic responses exhibited a large contribution of NMDA-type glutamate receptors, distinguishing them from corticostriatal inputs. CIN-mediated glutamatergic responses were insensitive to antagonists of acetylcholine receptors and were not seen in mice lacking VGluT3. Our results indicate that CINs are capable of mediating fast glutamatergic transmission, suggesting a new role for these cells in regulating striatal activity.     

Effect of the microiontophoretic application of acetylcholine on the formation of conditioned reactions in motor cortex neurons

In alert rabbits the activity of the motor cortex neurones was recorded with simultaneous application of acetylcholine to them in the process of defensive conditioning. Conditioned reorganization, mainly of activation type, were found in 60% of neurones. In most cases conditionally reacting cells were sensitive to acetylcholine. Ionophoretic application of the transmitter promoted the formation of conditioned neuronal responses and increased them in comparison with conditioned reactions evoked in absence of acetylcholine. It is supposed that the influence of acetylcholine on conditioned cellular process is realized due to its action on the state of excitability of the cortical neurones.     

Relation between fast and slow elemental synaptic potentials in command neurons in Helix lucorum taurica L

In experiments on a semi-intact snail preparation and a preparation of the snail isolated CNS, after spikes (Sp) evoked in presynaptic neurones by depolarizing current, not only rapid (R) EPSPs emerged in the command neurones of the defensive reaction of closing the pneumostome, but they were also followed by slow (S) EPSPs lasting over 2 min. For each single synaptic contact, the R and S EPSP amplitudes were in a good linear correspondence. In different synapses no direct connection was observed between R EPSP and S EPSP. It is suggested that R and S EPSPs may set in as a result of the action of different substances on the command neurones. Functional significance of S EPSPs with different amplitudes in different command neurones may consist in a prolonged specific preparation of the neurones for the action of stimuli.     

Down-regulation of vesicular glutamate transporters precedes cell loss and pathology in Alzheimer's disease

Alzheimer's disease (AD) is characterized pathologically by plaques, tangles, and cell and synapse loss. As glutamate is the principle excitatory neurotransmitter of the CNS, the glutamatergic system may play an important role in AD. An essential step in glutamate neurotransmission is the concentration of glutamate into synaptic vesicles before release from the presynaptic terminal. Recently a group of proteins responsible for uptake has been identified - the vesicular glutamate transporters (VGLUTs). The generation of antibodies has facilitated the study of glutamatergic neurones. Here, we used antibodies to the VGLUTs together with immunohistochemistry and western blotting to investigate the status of glutamatergic neurones in temporal, parietal and occipital cortices of patients with AD; these regions were chosen to represent severely, moderately and mildly affected regions at the end stage of the disease. There was no change in expression of the synaptic markers in relation to total protein in the temporal cortex, but a significant reduction in synaptophysin and VGLUT1 was found in both the parietal and occipital cortices. These changes were found to relate to the number of tangles in the temporal cortex. There were no correlations with either mental test score or behaviour syndromes, with the exception of depression.     

Cholinergic mechanisms of interneuronal transmission in the retina

Acetylcholine, applied to the isolated perfused frog and human retina, induces a corneopositive potential. This electrogenic action of acetylcholine, in conjunction with existing data, confirms the view that transmission in the retina is cholinergic. The magnitude and the temporal course of the potential evoked by acetylcholine depends both on its concentration and on the state of adaptation of the retina. Photic stimulation reduces the response to acetylcholine; under these circumstances flashes are more effective than a steady illumination. On the other hand the response of the retina to light decreases during perfusion with acetylcholine. The positive component of the ERG is particularly strongly inhibited, leaving only the negative P_III. The results indicate that acetylcholine acts on synapses between the first and second retinal neurons. They can be explained in terms of the hypthesis of the desensitization of cholinergic receptors in the retina.     

Reflection of the plastic properties of the simplest neuronal associations in statistical characteristics of the spike activity of their elements. Polysynaptically linked neurons

Changes of crosscorrelation histograms of trains of action potentials and mean interspike intervals of polysynaptically connected neurones were studied by means of mathematical modelling of synaptic neuronal interaction at changes of efficiency of interneuronal monosynaptic connections, at changes of neuronal excitability, and at changes of total action on them of independent disorderly afferent synaptic inflows. Increase of amplitude of the main maximum (minimum) of the normalized crosscorrelation histogram of trains of action potentials accompanied by reduction of mean interspike intervals of both neurones, was shown to be a unsignificant indication of an increase of efficiency of polysynaptic excitatory (inhibitory) connections between the neurones (due to modification of synapses or to a change of the functional state of interneurones).     

Does the destruction of the catecholaminergic neurons in newborn rat pups influence the modulatory function of the cholinergic system?]

On outbred ratlings aged 21-31 days the influence was studied of the destruction of catecholaminergic (CA) system on the reactions of the neurones of the cortical somatosensory zone, elicited by the stimulation of the ischiatic nerve and modulation of these reactions after stimulation of the basal nuclei area (the source of the neocortex cholinergic innervation) and acetylcholine (ACh) microiontophoretic application. It is shown that destruction of CA system in newborn ratlings increases the reactivity of the somatosensory cortical neurones in 21-31 days old animals to sensory stimulation; it does not influence the efficiency of modulating action of the cholinergic system of the forebrain and leads to the increase of modulating influence of the applicated ACh. It is postulated that as the result of perinatal destruction of CA brain system, in the neocortex a specific morpho-functional organization is formed of structures and processes at which the modulating function of the forebrain cholinergic system turns out, by quantitative criterion, at least, to be compensated.     

Axonal branching pattern and coupling mechanisms of the cerebral giant neurones in the snail,lymnaea stagnalis

The axonal branching pattern of the two cerebral giant neurones (CGCs) of Lymnaea stagnalis was studied with intrasomatically applied horseradish peroxidase. The cells are symmetrical. Each CGC projects to the ipsilateral n. labialis medius and n. arteriae labialis, the subcerebral commissure, and to all ipsi- and contralateral buccal nerves. The contralateral buccal nerves are reached via the ipsilateral cerebro-buccal connective and the buccal commissure. The CGC fire action potentials 1:1 in a driver-follower relationship. Each cell is capable of both driving and following. The relationship depends on the membrane potentials of the somata. In driving CGC spikes are initiated in a cerebral spike trigger zone located near the soma. In following cells spikes are initiated in a distal zone located in the buccal ganglia. The buccal zone is only affected by the partner CGC. CGC are synchronized by three coupling mechanisms: mutual excitatory chemical synapses, electrotonic coupling, and common input. The chemical and electrotonic connections are located in the buccal ganglia. All spikes are relayed to the partner cell via the chemical synapses. The electrotonic coupling improves the efficiency of the chemical synapses. The dual connection selectively synchronizes the CGC-axonal spikes from each side of the buccal mass. Common excitatory input affects the cerebral spike trigger zones and can initiate simultaneous spikes in both cells. This results in bilateral synchrony of spikes in the CGC-axons in both the buccal and the lip nerves.     

Morphine excitation in the cerebral cortex.

Microiontophoretic administrations of morphine to cholino-excitable neurones in the cerebral cortex of decerebrate cats evoked a weak excitation which became more prominent upon repeated administrations of the alkaloid. This effect was not antagonized by naloxone. Iontophoresis of methylatropine prevented the excitation induced with acetylcholine and morphine, leaving that caused by glutamate relatively unaltered. Similar applications of morphine to neurones which were not excited by test applications of acetylcholine did not result in excitation but elicited mainly a depression of glutamate-evoked firing. It is suggested that the muscarinic effect of morphine in the cortex may be related to the excitation and convulsions, but not the analgesia, which occurs upon systemic administrations of the narcotic.     

Pharmacological and electrophysiological characteristics of neurones in the paramedian reticular nucleus.

1. Neurones in the paramedian reticular nucleus of decerebrate, unanaesthetised cats have been identified by microelectrode recording combined with antidromic activation of their axons in the ipsilateral inferior cerebellar peduncle. Most paramedian reticular neurones were not influenced by somatic stimulation. 2. When applied by iontophoresis from multibarrelled micropipettes acetylcholine and 5-hydroxytryptamine excited all but a few paramedian reticular neurones while l-noradrenaline inhibited almost all the neurones investigated. The excitatory response to acetylcholine could be antagonised by gallamine. 3. The paramedian reticular nucleus appears to be a relatively homogeneous group of neurones, pharmacologically as well as anatomically.     

Neurotransmitter release at ribbon synapses in the retina

The synapses of photoreceptors and bipolar cells in the retina are easily identified ultrastructurally by the presence of synaptic ribbons, electron-dense bars perpendicular to the plasma membrane at the active zones, extending about 0.5 microm into the cytoplasm. The neurotransmitter, glutamate, is released continuously (tonically) from these 'ribbon synapses' and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven bursts of release at conventional synapses. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but a few differences have been identified that may be important determinants of tonic transmitter release. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.     

Control of Cleft Glutamate Concentration and Glutamate Spill-Out by Perisynaptic Glia: Uptake and Diffusion Barriers

Most glutamatergic synapses in the mammalian central nervous system are covered by thin astroglial processes that exert a dual action on synaptically released glutamate: they form physical barriers that oppose diffusion and they carry specific transporters that remove glutamate from the extracellular space. The present study was undertaken to investigate the dual action of glia by means of computer simulation. A realistic synapse model based on electron microscope data and Monte Carlo algorithms were used for this purpose. Results show (1) that physical obstacles formed by glial processes delay glutamate exit from the cleft and (2) that this effect is efficiently counteracted by glutamate uptake. Thus, depending on transporter densities, the presence of perisynaptic glia may result in increased or decreased glutamate transient in the synaptic cleft. Changes in temporal profiles of cleft glutamate concentration induced by glia differentially impact the response of the various synaptic and perisynaptic receptor subtypes. In particular, GluN2B- and GluN2C-NMDA receptor responses are strongly modified while GluN2A-NMDA receptor responses are almost unaffected. Thus, variations in glial transporter expression may allow differential tuning of NMDA receptors according to their subunit composition. In addition, simulation data suggest that the sink effect generated by transporters accumulation in the vicinity of the release site is the main mechanism limiting glutamate spill-out. Physical obstacles formed by glial processes play a comparatively minor role.     

Plasticity and its physiological markers in the microsystem of cortical neurons during repeated local stimulation

Separate neuronal microsystems in the sensomotor cerebral cortex are able to exhibit the functional plasticity under conditions of repeated action of acetylcholine applied microiontophoretically. The characteristic properties of dynamics in activity of single cortex neurones are determined mainly by the initial reactivity to transmitter and by the state of nervous cells of the surrounding microsystem. The composition and succession of response components as well the duration of excitatory stage of reaction to acetylcholine serve as physiological markers of plastic properties of neurones in cortical microsystem.     

Reactions of neurons in the sensory-motor cortex on different amounts of administered acetylcholine

As a result of acetylcholine iontophoresis with different currents 3-fold increase of transmitter compared with the threshold one for reaction has been shown not to result in change of a type of reaction pattern more than in 80.3% of neurones. Such increase of action force is quite enough for the significant lengthening of the reaction excitatory components in the most of investigated neurones. After the following repeated application of smaller quantity of transmitter the number of neurones with growth of frequency of excited impulsive activity recovers as well as the level of firing frequency decrease in the course of repetitive administration of transmitter. The effect of large doses of transmitter results in aftereffect expressed by increasing probability of excitatory component reduction during the repetitive applications of acetylcholine.     

Neurotoxicity of L-Glutamate and DL-Threo-3-Hydroxyaspartate in the Rat Striatum

Destruction of the glutamatergic corticostriatal pathway potentiates the neurotoxic action of 1 mumol L-glutamate injected into the rat striatum, whereas the toxic effects of 10 nmol kainate are markedly attenuated. Injection of 170 nmol of the glutamate uptake inhibitor, DL-threo-3-hydroxyaspartate, into the intact striatum also causes neuronal degeneration, which is accompanied by a reduction in markers for cholinergic and GABAergic neurones. Prior removal of the corticostriatal pathway destroys the ability of DL-threo-3-hydroxyaspartate to cause lesions in the striatum. These results indicate that removal, or blockade, of uptake sites for glutamate increase the vulnerability of striatal neurones to the toxic effects of synaptically released glutamate.     

Synaptic and vesicular co-localization of the glutamate transporters VGLUT1 and VGLUT2 in the mouse hippocampus

Vesicular glutamate transporters (VGLUTs) are essential to glutamatergic synapses and determine the glutamatergic phenotype of neurones. The three known VGLUT isoforms display nearly identical uptake characteristics, but the associated expression domains in the adult rodent brain are largely segregated. Indeed, indirect evidence obtained in young VGLUT1-deficient mice indicated that in cells that co-express VGLUT1 and VGLUT2, the transporters may be targeted to different synaptic vesicles, which may populate different types of synapses formed by the same neurone. Direct evidence for a systematic segregation of VGLUT1 and VGLUT2 to distinct synapses and vesicles is lacking, and the mechanisms that may convey this segregation are not known. We show here that VGLUT1 and VGLUT2 are co-localized in many layers of the young hippocampus. Strikingly, VGLUT2 co-localizes with VGLUT1 in the mossy fibers at early stages. Furthermore, we show that a fraction of VGLUT1 and VGLUT2 is carried by the same vesicles at these stages. Hence, hippocampal neurones co-expressing VGLUT1 and VGLUT2 do not appear to sort them to separate vesicle pools. As the number of transporter molecules per vesicle affects quantal size, the developmental window where VGLUT1 and VGLUT2 are co-expressed may allow for greater plasticity in the control of quantal release.