[Dorsal root afferent fiber termination in the spinal cord of the turtle Testudo horsfieldi and 3-dimensional reconstruction of the sensory-motoneuron connection].

HRP tracing methods and computer reconstruction were used to study the structural organization of sensory-motoneuron connections in the turtle. HRP was applied through suction electrodes to thin dorsal and ventral root filaments of superfused isolated lumbar spinal cord of the turtle Testudo horsefieldi. Single motoneurons were labeled iontophoretically with the use of intracellular glass microelectrodes. Labeled elements were examined by light microscopy. The Eutectic Neuron Tracing System and its associated program were used for 3-D reconstructions and morphometry. The distribution of dorsal root afferent fibers and their terminations were presented in a new scheme in which, beside the well known zones, new ones were shown in the Lissauer zone, motor nuclei, ventrolateral funiculus and in the contralateral medial gray matter (IV--V laminae). Unlike in the frog, the motoneuron dendritic field in the turtle was restricted to an ellipsoid space having a short axis in the rostro-caudal direction (300-500 microm). The dorsal root afferent fibers connected to motoneurons produced very short branches (50-70 microm) in a restricted rostro-caudal direction. One dorsal root fiber collateral had about 80 synapselike enlargements (approximately 10-fold fewer than in the frog). The putative sensory-motoneuron contacts were found on the I--VII order dendritic segments of the dorsal and ventromedial dendritic trees. It was shown that in the turtle only one first order collateral of the dorsal root fiber participated in the sensory-motoneuron connection with a small number (about 4) of putative contacts, which was also less than in the frog by a factor of 10. The simplicity of the synapse structure in the turtle is likely to be compensated through the higher efficiency of the signal transmission which is comparable to that in mammals.

[The role of group II and III metabotropic glutamate receptors in modulation of miniature synaptic activity in frog spinal cord motoneurons].

The results of present work demonstrate significant modulating effects mediated by group II and III mGluRs on miniature postsynaptic potentials (mPSP) of the frog spinal motoneurons. The mode of group II and III mGluRs ligands influences, i. e. the changes in the mPSPs average frequency without significant changes in their average amplitude, suggests the presynaptic mechanism of modulation by the change in transmitter release. Selective antagonists of group II and III mGluRs (EGLU and MAP4) increased the average frequency of mPSPs by 52.8 +/- 30.2% (in 4 of 6 motoneurons) and by 54.7 +/- 23.7% (in all 7 motoneurons), respectively. Application of the group III mGluRs agonist LAP4 decreased the mPSPs frequency by 21.8 +/- 5.2% in 3 of 5 motoneurons. The efficiency of the antagonist usage and comparative low efficiency of the agonist suggest that presynaptic mGluRs at motoneuronal synapses under normal condition possess some level of tonic activity. The lack of group II mGluR antagonist effect on some motoneurons appears to be explained by specific localization of the group II mGluRs in preterminal area which is distant from the transmitter release site. The hetero-receptor modulation of pharmacologically isolated inhibitory miniature activity and its glycine- and GABAergic fractions by group III mGluRs was investigated. MAP4 application has been shown to increase the glycine-mediated mlPSPs frequency more than GABA-mediated mlPSPs frequency: in average by 97.6 +/- 20.7% (n = 7) and 54.6 +/- 20.8% (n = 5), respectively. This difference may be due to the segregation of the postsynaptic glycine- and GABA-receptors. The preliminary examination of the convergence of the presynaptic mGluRs and metabotropic GABA(B) receptors influences on GABA-mediated IPSPs was undertaken. It has been shown that presynaptic GABA(B) receptors are tonically active under normal condition. Under condition of GABA(B) receptor blockage by phaclofen, the application of group III mGluR agonist L-AP4 elicited typical effect which was completely taken off by subsequent application of the group III mGluRs antagonist MAP4. This result is in accordance with the assumption that the effects mediated by GABA(B) receptors and mGluRs are independent.

Three types of inhibitory miniature potentials in frog spinal cord motoneurons: possible GABA and glycine cotransmission.

Miniature inhibitory postsynaptic potentials (mIPSP) of motoneurons in isolated frog spinal cord were recorded in conditions of blockade of the conduction of nerve spikes and ionotropic glutamate receptors (TTX, 1 microM, CNQX, 25 microM, D-AP5, 50 microM). Three types of mIPSP were identified: those with fast and slow time characteristics and mIPSP with two-component decays. Two-component mIPSP accounted for 8.7% of all selected responses, fast mIPSP for 64.5%, and slow mIPSP for 26.8%. Blockade of GABA(A) receptors with bicuculline (20 microM) led to decreases in the numbers of slow and two-component mIPSP and an increase in the number of mIPSP with fast kinetics. Strychnine (1 microM), a blocker of glycine receptors, led to a reduction in the number of fast receptors and an increase in the number of slow potentials. These data suggest that frog spinal cord motoneurons have three types of inhibitory mIPSP, mediated by GABA, glycine, and simultaneous release of these two transmitters from the same presynaptic terminals.

Comparative study of spinal motoneuron axon collaterals.

Numerous spinal motoneurons in mammals possess recurrent axon collaterals included in a feedback loop for controlling motoneuron activity. For nonmammalian vertebrates, the data concerning the existence of collaterals and their intraspinal branching are fragmentary and contradictory. We focused on axonal branching of motoneurons in lampreys, frogs, turtles and young rats, using light microscopic analysis of HRP- or neurobiotin-labeled motoneurons. In lampreys, only a restricted portion of spinal motoneurons, related to the dorsal fins, showed recurrent collaterals. In frogs, a great complexity and high total length of collateral branches as well as a great number of axon swellings were found. In turtles, axon collateralization of spinal motoneurons was much more restricted, and present in particular in lumbar motoneurons innervating proximal hindlimb muscles. Young rat spinal motoneurons have rather abundant recurrent axon collaterals. It is likely that the presence of axon collaterals from spinal motoneurons is related to the level of complexity of locomotion.

[Modulation of miniature inhibitory potentials in motoneurons of the turtle spinal cord by group II metabotropic glutamate receptors]. / Moduliatsiia miniatiurnykh tormoznykh potentsialov motoneironov spinnogo mozga cherepakhi metabotropnymi glutamatnymi retseptorami gruppy II.

The role of group II metabotropic glutamate receptors (mGluRs) in modulation of inhibitory synaptic activity was studied by intracellular recording of motoneuron miniature inhibitory spontaneous postsynaptic potentials (mIPSPs) in isolated lumbar segments of the turtle spinal cord in the medium containing TTX, CNQX, AP-5. The ratio of mIPSPs with fast and slow kinetics (83% vs 17%) is in accordance with the ratio shown for glycine- and GABA-mediated IPSP or IPSCs (Jones et al., 1988; Gao et al., 2001). In the majority of investigated motoneurons, the selective group II mGluRs antagonist EGLU (100-250 microM) increased the frequency of mIPSPs by 106.6 +/- 74.4% (n = 9) without affecting average amplitude, suggesting a presynaptic site of mGluRs action providing for the transmitter release reduction. The analysis of EGLU action on mIPSPs with different time courses (selection by half-width) showed that the frequency of inhancement of miniature inhibitory activity is caused by predominantly short-duration mIPSPs (ba 84.0 +/- 18.2%; n = 9), which are probably glycineergic. However, EGLU did not influence the mIPSPs frequency under condition of GABA-receptor blockade by bicuculline (20 microM). This fact suggest that group II mGluRs could modulate glycinergic transmission to the turtle spinal motoneurons on the necessary condition that GABergic system is active.

[Recurrent axon collaterals of lumbar motor neurons in turtles]. / Vozvratnye aksonnye kollaterali poiasnichnykh motoneironov cherepakhi.

A combined morphophysiological study was made of connections between motoneurons on the superfused isolated lumbar spinal cord of Testudo horsfieldi. Postsynaptic potentials of motoneurons, followed by antidromic stimulations of ventral root filaments (VR-PSPs), were recorded intracellularly. Depolarizing VP-PSPs had short latencies (1.0-1.5 mc) and amplitudes in the range of 0.3-3.0 mV. At the constant stimulus intensity, the fluctuations of amplitudes were recorded. In some motoneurons, hyperpolarizing VP-PSRs with the latencies 2.5-3.0 mc were observed. A possible structural basis of VR-PSPs was studied by the horseradish peroxidase (HRP) method. After HRP application on thin ventral root filaments the retrograde staining of motoneurons revealed recurrent axon collaterals of labeled motoneurons. Three-dimensional computer reconstructions showed one to three collaterals given off by motoneuron axons. There were up to 19 points of branching in a single collateral. In some cases the full length of collateral trees reached 4.0 mm. The collateral branches had up to 72 "en passant" and terminal axon swellings. The swellings (presumed contacting boutons) were distributed in the ventral and intermedial gray matter and in the ventromedial while matter and revealed on motoneurons and inerneurons. These data suggest the participation of the motor axon collaterals in the motoneuron--motoneuron communication in the turtle spinal cord whereas only dendro-dendritic contacts had been discussed earlier.

[The effect of metabotropic glutamate receptors on longitude of posttetanic reaction in spinal motoneurons of frog]. / Vliianie metabotropnykh glutamatnykh retseptorov na dlitel'nost' posttetanicheskikh reaktsii v motoneironakh spinnogo mozga liagushki.

Effects of metabotropic glutamate receptors of the duration of posttetanic changes in monosynaptic excitatory postsynaptic potentials (mEPSP), evoked by afferent and reticulospinal input stimulation, were investigated in lumbar motoneurons of the frog isolated spinal cord. It was found that application of MAP4 (25 microM), a selective antagonist of group III of these receptors, prolonged posttetanic potentiation and depression of synaptic transmission, whereas activation of this group of metabotropic glutamate receptors by L-AP4 (1 mM), a selective agonist of these receptors, suppressed the amplitude of synaptic responses, but did not affect the dynamics of development of posttetanic changes. The NMDA receptor antagonist AP5 (50 microM), added to the perfusing solution, blocked completely the effects produced by MAP4. Neither selective antagonist MCCG (400 microM), nor agonist tACPD (50 microM) of group II metabotropic glutamate receptors affected the terms of mEPSP posttetanic potentiation and depression, although the latter, in contrast to the antagonist, in most cases increased the synaptic potential amplitude. The data obtained permit to suggest that group III metabotropic receptors may control the duration of posttetanic changes of synaptic transmission in the frog spinal motoneurons. The long-term changes in the investigated synapses seem to be mediated by activation of postsynaptic metabotropic glutamate receptors (most likely, of group I receptors), which is normally masked with activation of group III presynaptic autoreceptors. The mechanism of such an induction essentially depends on activation of NMDA type of inotropic glutamate receptors.

Comparison of the topology and growth rules of motoneuronal dendrites.

The complexity, shape, and branching modes of the dendrites of spinal motoneurons were compared in cat, rat, and frog using topological analysis and growth models. The complexity of motoneuronal dendrites, measured as the mean number of terminal segments, varied significantly among samples and was related to contractile properties of innervated motor units. Despite this variation, all mature motoneurons having a mean number of terminal segments per dendrite greater than ten (up to 24.3) exhibited a narrow range of values of coefficients describing the symmetry of tree shapes (0.42-0.47). This implies low variability in the topological shape of motoneuronal dendrites of different animals. This similarity of tree shapes proved to be a result of the similarity of growth rules. The growth of the dendrites could be described to a first approximation by a two-parameter (Q and S) model called the QS model and by a multitype Markovian model. The estimation of parameters of the QS model, in which parameter Q is related to the probability of branching of intermediate segments, revealed that Q was equal or close to 0, implying that branching of dendrites is restricted to terminal segments. The estimates of the parameter S, which describes whether the probability of branching increases (S < 0) or decreases (S > 0) exponentially with segment order, were positive. This was in agreement with the results of estimation of probabilities of branching provided by the Markovian model, which showed that the branching probabilities decreased with segment order in an exponential manner in most of the neurons studied. The QS and Markovian models involve different assumptions about the sequence and timing of branching events, and selection of the best model can provide insight into details of dendritic outgrowth. Extensive simulation of tree outgrowth using a Markovian model revealed significant differences between stimulated trees and real dendrites, particularly with regard to variability of the number of terminals and to symmetry. In contrast, the QS model provided a good fit to the mean values and standard deviations of basic topological parameters. This model is adequate to describe the shape of mature motoneuronal dendrites. It implies that dendritic branches have many opportunities to bifurcate during the whole time of development and that bifurcating potency of a branch is a function of the number and position of other branches of that dendrite. Combined with analysis of metrical properties such as lengths of segments, the QS model can assist in a quantitative analysis of development and plasticity.

Quantal analysis based on spectral methods.

A method for calculating quantal size based on spectral analysis of postsynaptic potential (PSP) amplitude distributions was developed and tested by Monte-Carlo techniques. It was combined with a test to reveal the bias of the estimate of quantal size and to determine whether the peaks in amplitude distributions reflect quantal release or a sampling artifact. Spectral density was computed using fast Fourier transformation after subtraction of a fitted polynomial from the probability density function. The method overestimated quantal size for values less than two standard deviations of noise, indicating that those estimates as well as estimates of quantal size computed for examples of non-quantal distributions are not reliable. This was the case for 34 of 36 sets of sensorimotor excitatory PSPs of the frog, suggesting that most values of the quanta in synapses between primary fibres and lumbar motoneurons are smaller than 70-90 microV.

Modeling of the quantal release at interneuronal synapses: analysis of permissible values of model moments.

A theoretical study of effects of the different factors on fluctuation of post-synaptic potential (PSP) amplitudes was undertaken, using computation of regions of permissible values (RPV) of the ratio between the variance and the mean number of the quanta released (R1) and the ratio between the third moment and the variance (R2). The RPVs of these indexes for the binomial model were compared with regions determined for a number of models incorporating several factors. It has been shown that the involvement of temporal non-uniformity of transmitter release probability, decremental spreading of potentials along dendrites, and failure of spike propagation give the values of skewness index R2 less, compared to the binomial model. Simultaneously, a number of other factors, especially spatial non-uniformity of release probabilities in single release sites, would give amplitude histograms with high positive values of the index. The values of R1 and R2, calculated for 21 samples of sensorimotor EPSP amplitudes, were biased from RPV of these parameters constructed for the binomial model. The scattergram of R1 and R2 can be explained by the presence of two kinds of contacts which release quantum with different probabilities. The same was true for the beta-model based on the assumption that probabilities of quantal release are a sample of values of random variable that has beta-distribution. From analysis of the distribution of individual release probabilities, obtained from evaluation of beta-model parameters, is concluded that a greater part of boutons in the sensorimotor synapses release transmitter with very low probabilities, there being, however, a few boutons with probabilities close to 1.

[The pathways of the stabilization of the amplitude of postsynaptic potentials in the interneuronal synapses of vertebrates]. / Puti stabilizatsii amplitudy postsinapticheskikh potentsialov v mezhneironnykh sinapsakh pozvonochnikh.

The influence of various factors on the degree of stabilization of postsynaptic potential amplitude has been studied by mathematical modelling. Increasing of transmitter release probability in single boutons, spatial non-uniformity of these probabilities, interaction of release sites, non-linear summation of potentials lead to amplitude stabilization. Temporal fluctuations of the release probability, failures of responses from contact groups and activation of a variable number of the fibers have the opposite influence. Comparison of synaptic transmission parameters in sensorimotor synapses of Cyclostomata, amphibians and mammals showed that during evolution of these synapses two different pathways of amplitude stabilization had been realized. Formation of highly effective chemical contacts is probably the most progressive pathway among them.

Relation between structural and release parameters at the young rat sensorimotor connection.

The present work was carried out on isolated spinal cords of young rats. The aim of this study was the combined morphological and electrophysiological investigation of sensorimotor connections labelled with horseradish peroxidase and the evaluation of the relationship between their structural and functional properties. Sensorimotor contacts were widely distributed along the postsynaptic cell: from the soma and juxtasomatic dendrites to distal dendrites. The number of contacting boutons in the connection of a single afferent fibre and an individual motoneuron was about 10. The amplitude fluctuation patterns of the unitary and the minimal excitatory postsynaptic potentials of the motoneurons fitted with predictions based on a binomial model. A close correspondence was found between the estimated number of binomial release sites, n, and the number of contacting boutons. The calculated size of the quantal potential was about 100 microV. The difference in the organization of sensorimotor connections of the young rat and the frog is discussed.

Sensorimotor connections in the lumbar spinal cord of the young rat: a morphological study.

A morphological investigation of sensorimotor connections was performed on the isolated lumbar spinal cord of 8-15-day-old rats using horseradish peroxidase labelling techniques. Horseradish peroxidase was applied to the filaments of dorsal and ventral roots and injected intracellularly into motoneurons. The labelled afferent fibres and their contacts on motoneurons were examined under a light microscope. Numerous afferent collaterals entered the lateral motor nuclei. In the medial motor nuclei a few afferent collaterals were found. Some fibres were visible passing through the ventral commissure. The number of boutons per afferent collateral in the motor nuclei was 40-60. A single terminal branch contained one to five boutons (average 1.5). Predominating axodendritic and apparent axosomatic contacts were found between afferent fibres and motoneurons belonging to the lateral motor nuclei. The contacting boutons were both terminaux and en passant. As a rule, the sensorimotor connection involved dorsally and rostrocaudally directed dendrites of the first to sixth orders.

[Morphologic and quantum characteristics of sensomotor neuron synapses in isolated rat spinal cord]. / Morfologicheskie i kvantovye kharakteristiki sensomotoneironnykh sinapsov v izolirovannom spinnom mozge krysiat.

The structural-functional relationships of sensorimotor connections were studied in isolated lumbar cord segments of 7-14 day-old rats. Individual motoneurons were found to have synaptic contacts only with a single collateral of the afferent fibre. The number of contacting boutons gained 10. The n parameter of the binomial model was established to reflect the number of contacts at the sensorimotor connection. The analysis of sensorimotor EPSPs by convolution with two binomial distributions has shown that sites of the transmitter release were different in their probability (efficiency) of response to nerve impulse.