András Birinyi

Zinc co-localizes with GABA and glycine in synapses in the lamprey spinal cord

The presence of zinc in synaptic terminals in the lamprey spinal cord was examined utilizing a modification of the Timm′s sulfide silver method and with the fluorescent marker 6‐methoxy‐8‐quinolyl‐p‐toluenesulfonamide (TSQ). Axons labeled with a Timm′s staining method were predominantly located in the lateral region of the dorsal column. This correlated with a maximum of TSQ fluorescence in this region of the spinal cord. Single labeled terminals accumulating Timm reaction product were also found throughout the gray matter and fiber tracts. At the ultrastructural level, zinc was located in a population of synaptic terminals that co‐localized γ‐aminobutyric acid (GABA) and glycine. Possible effects of Zn2+ on neuronal activity were examined. In spinobulbar interneurons, which receive GABAergic input in the dorsal column, zinc potentiated responses to GABA application, but it did not affect responses to GABA in motoneurons. Responses in motoneurons to pressure application of glycine were also not affected by Zn2+. Zinc, however, potentiated monosynaptic glycinergic inhibitory postsynaptic potentials (IPSPs) evoked in motoneurons by inhibitory locomotor network interneurons and increased frequency, but not amplitude of spontaneous miniature IPSPs recorded in the presence of tetrodotoxin (TTX), suggesting presynaptic effects. Glutamate responses and the amplitude of monosynaptic excitatory postsynaptic potentials (EPSPs) in motoneurons were reduced by zinc. These effects appeared to be mediated largely postsynaptically through an effect on the N‐methyl‐D‐aspartate (NMDA) component of the glutamatergic input. Our results thus show that free zinc is present in inhibitory synaptic terminals in the lamprey spinal cord, and that it may function as a modulator of inhibitory synaptic transmission. J. Comp. Neurol. 433:208–221, 2001.

Synaptic Targets of Commissural Interneurons in the Lumbar Spinal Cord of Neonatal Rats

There is strong evidence that commissural interneurons, neurons with axons that extend to the contralateral side of the spinal cord, play an important role in the coordination of left/right alternation during locomotion. In this study we investigated the projections of commissural interneurons to motor neurons and other commissural interneurons on the other side of the spinal cord in neonatal rats. To establish whether there are direct contacts between axons of commissural interneurons and motor neurons, we carried out two series of experiments. In the first experiment we injected biotinylated dextran amine (BDA) into the lateral motor column to retrogradely label commissural interneurons that may have direct projections to motor neurons. Stained neurons were recovered in the ventromedial areas of the contralateral gray matter in substantial numbers. In the second experiment BDA was injected into the ventromedial gray matter on one side of the lumbar spinal cord, whereas motor neurons were simultaneously labeled on the opposite side by applying biocytin onto the ventral roots. BDA injections into the ventromedial gray matter labeled a strong axon bundle that arose from the site of injection, crossed the midline in the ventral commissure, and extensively arborized in the contralateral ventral gray matter. Many of these axons made close appositions with dendrites and somata of motor neurons and also with commissural interneurons retrogradely labeled with BDA. The results suggest that commissural interneurons may establish monosynaptic contacts with motor neurons on the opposite side of the spinal cord. Our findings also indicate that direct reciprocal connections between commissural interneurons on the two sides of the spinal cord may also exist. J. Comp. Neurol. 461:429–440, 2003.

Location of dye-coupled second order and of efferent vestibular neurons labeled from individual semicircular canal or otolith organs in the frog

Vestibular nerve branches innervating the sensory epithelia of the three semicircular canals or of the three otolith organs of frogs were selectively labeled in-vitro with biocytin. Labeled afferent fibers from the semicircular canals, utricle, and lagena were encountered in each of the four vestibular nuclei and their projections overlapped considerably. Saccular afferent fibers projected to the dorsal (acoustic) nuclei and smaller projections to the vestibular nuclei were regionally restricted. Per semicircular canal or otolith organ about equal numbers (11-14) of medium sized vestibular neurons (between 7.5 and 17 microm in diameter) were dye-coupled to afferent fibers. Most of these dye-coupled vestibular neurons were located in the lateral and descending vestibular nuclei between the VIIIth and IXth nerves. The superior vestibular nucleus was relatively free of dye-coupled vestibular neurons. The location of this subpopulation of central vestibular neurons supports the notion that these neurons are part of a particular vestibulospinal pathway. In addition, from each of the canal and/or otolith organs about 3-4 efferent vestibular neurons were labeled retrogradely. These neurons (between 15 and 26 microm in diameter) were located ventral to the vestibular nuclear complex. The branching of efferent vestibular neurons was shown by the presence of neurons that were double labeled by two different fluorescent dyes applied in the same experiment to the anterior and posterior ramus of the same VIIIth nerve, respectively. The branching of these efferent neuron axons explained the presence of collaterals and terminals in the sensory epithelia of a number of untreated ipsilateral endorgans.

Ultrastructural organization of lamprey reticulospinal synapses in three dimensions

The giant reticulospinal synapse in lamprey provides a unique model to study synaptic vesicle traffic. The axon permits microinjections, and the active zones are often separated from each other, which makes it possible to track vesicle cycling at individual release sites. However, the proportion of reticulospinal synapses with individual active zones (“simple synapses”) is unknown and a quantitative description of their organization is lacking. Here, we report such data obtained by serial section analysis, intermediate‐voltage electron microscopy, and electron tomography. The simple synapse was the most common type (78%). It consisted of one active zone contacting one dendritic process. The remaining synapses were “complex,” mostly containing one vesicle cluster and two to three active zones synapsing with distinct dendritic shafts. Occasional axosomatic synapses with multiple active zones forming synapses with the same cell were also observed. The vast majority of active zones in all synapse types contained both chemical and electrotonic synaptic specializations. Quantitative analysis of simple synapses showed that the majority had active zones with a diameter of 0.8–1.8 μm. The number of synaptic vesicles and the height of the vesicle cluster in middle sections of serially cut synapses correlated with the active zone length within, but not above, this size range. Electron tomography of simple synapses revealed small filaments between the clustered synaptic vesicles. A single vesicle could be in contact with up to 12 filaments. Another type of filament, also associated with synaptic vesicles, emerged from dense projections. Up to six filaments could be traced from one dense projection. J. Comp. Neurol. 450:167–182, 2002.

The Extent of the Dendritic Tree and the Number of Synapses in the Frog Motoneuron.

Frog motoneurons were intracellularly labelled with cobaltic lysine in the brachial and the lumbar segments of the spinal cord, and the material was processed for light microscopy in serial sections. With the aid of the neuron reconstruction system NEUTRACE, the dendritic tree of neurons was reconstructed and the length and surface area of dendrites measured. The surface of somata was determined with the prolate – oblate average ellipsoid calculation. Corrections were made for shrinkage and for optical distortion. The mean surface area of somata was 6710 μm2; lumbar motoneurons were slightly larger than brachial motoneurons. The mean length of the combined dendritic tree of brachial neurons was 29 408 μm and that of lumbar neurons 46 806 μm. The mean surface area was 127 335 μm2 in brachial neurons, and 168 063 μm2 in lumbar neurons. The soma – dendrite surface area ratio was 3 – 5% in most cases. Dendrites with a diameter of ≤ 1.0 μm constituted ∼ 75% of the combined dendritic length in most of the neurons. Unlike in the cat, there was no correlation between the size of stem dendrites and the extent of daughter branches. From the synaptic density estimated in earlier electron microscope investigations of frog motoneuron dendrites (Antal et al., J. Neurocytol., 15, 303–310, 1986; 21, 34–49, 1992), and from the present data, the number of synapses on the dendritic tree was calculated. The calculations indicated 26 949 synapses on the smallest and 61 519 synapses on the largest neuron if the synaptic density was multiplied by the length of the dendritic tree. If the synaptic density was multiplied by the surface area of the dendritic tree the calculation yielded 23 337 synapses for the smallest and 60 682 synapses for the largest neuron. More than 60% of the combined surface area of dendrites was >600 μm from the soma. This suggests that about two‐thirds of the synapses impinged upon distant dendrites >600 μm from the soma. The efficacy of synapses at these large distances is investigated on model neurons in the accompanying paper (Wolf et al., Eur. J. Neurosci., 4, 1013–1021, 1992).

Investigation of the dendritic geometry of brain stem motoneurons with different functions using multivariant statistical techniques in the frog

We give an account of an effort to make quantitative morphological distinctions between motoneurons innervating functionally different muscles in the trigeminal and facial motor nuclei of the frog. Six groups of neurons were considered in the two nuclei on the basis of their peripheral targets. One group consisted of neurons (n = 7) innervating the levator bulbi muscle, which separates the orbital cavity from the oral cavity. In the second, third and fourth groups, motoneurons (n = 27) innervating jaw closer muscles (temporalis, masseter, pterygoideus) were studied. Neurons (n = 6) innervating the submaxillary muscle comprised the fifth group. This muscle forms the muscular floor of the mouth. It is active in deglutition and contributes to the opening of the mouth. The sixth group is formed by neurons of the facial nucleus (n = 7), which innervate the depressor mandibulae muscle. This is the main opener of the mouth. Neurons were selectively stained by cobalt labelling through the muscle nerves and the morphometric values of successfully labelled neurons were fed into a IBM AT 386 computer through a digitizing tablet for three-dimensional reconstruction. Four neurons labelled directly through the motor root of the trigeminal nerve but innervating unidentified muscles were added to the investigation. Two sets of quantitative measurements were taken from the neurons. In the first set (neurometric data), 17 quantitative variables were measured in the perikaryon and the dendritic arbor. In the second set, 15 variables concerned with the orientation and shape of the dendritic tree, the relation of the perikaryon to the dendritic tree and the spatial expansion of dendrites were measured in the three dimensions of Cartesian space (product-moment data). The data were subjected to multivariant statistical analysis. First, they were partitioned with cluster analysis. The average linkage between groups algorithm and the cosine of vectors of variables, or the Pearson correlation similarity coefficients were used. Neurometric data and product-moment data were analysed separately and in combination, and six to seven clusters were considered. In each case, the majority of neurons innervating jaw closer muscles were grouped into clusters different from neurons innervating jaw opener muscles. The best separation of functionally different neurons was achieved with the neurometric data set. The groups of neurons obtained from cluster analysis were subjected to non-parametric discriminant analysis with the eight nearest-neighbour classification criterion, and the results were checked with a cross-validation technique.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurotransmitter systems of commissural interneurons in the lumbar spinal cord of neonatal rats

The circuits that generate rhythmic locomotor activities are located in the ventromedial area of the lumbar spinal cord and comprise commissural interneurons necessary for left-right alternation during walking movements. In this study we injected biotinylated dextran amine (BDA) into the ventromedial gray matter of the lumbar spinal cord of neonatal rats to label commissural interneurons. Anterogradely labeled axons arose from the site of injection, crossed the midline in the anterior commissure and arborized extensively in the contralateral ventral horn of the spinal cord. The presence of neurotransmitter systems in labeled axon terminals of commissural interneurons was investigated by using antibodies raised against specific transmitter-related proteins. Boutons potentially containing inhibitory amino acids were identified by applying glutamic acid decarboxylase (GAD65/67) and glycine transporter 2 antibodies. Out of 1146 BDA-labeled axon terminals, 663 boutons were assumed on this basis to be inhibitory; 76% of these terminals were immunoreactive for glycine transporter, 53% were immunoreactive for GAD and about 30% of inhibitory boutons might contain both inhibitory amino acids. Boutons potentially containing putative excitatory neurotransmitter were revealed with antibodies raised against vesicular glutamate transporters 1 and 2. Out of 590 BDA-labeled boutons about one fourth (158) were immunoreactive for glutamate transporters. These mammalian commissural interneurons are compared to the glycinergic commissural interneurons in the swimming CPGs of lamprey and the Xenopus tadpole. Our results show that commissural interneurons in the mammalian spinal cord form a heterogeneous group including glutamatergic excitatory and GABAergic and glycinergic inhibitory neurons.

A fast 3-dimensional neuronal tree reconstruction system that uses cubic polynomials to estimate dendritic curvature.

The main goal of this work was to develop and test the accuracy of our 3DARBOR neuronal tree reconstruction system by comparing it with a very precise but time-consuming method of reconstruction (NEUTRACE). Comparison was performed by reconstructing 18 dendritic trees of frog spinal motoneurons from serial sections with both methods and comparing several morphological summaries of the two reconstructions. In 3DARBOR the planar projection of the dendritic trees was drawn and fed into an IBM-compatible PC through a graphic tablet. Dendritic coordinates along the perpendicular (focus) axis on the plane of drawing were estimated by an interpolation method. The interpolation was based on the lengths of projected dendrites and the coordinates of points where dendrites entered the next section. Focus axis coordinates of these points could automatically be calculated from the serial numbers and thicknesses of sections. 3DARBOR was tested by comparison of the distributions of characteristic points of dendritic trees, segment lengths and branching angles. Product moment analysis on dendritic trees was also performed. It was concluded that 3DARBOR is a fast enough reconstruction system without any systematical error of interpolation that can correctly supply the most morphological parameters and visualize the natural arborizations.

Simulation of the Effect of Synapses: the Significance of the Dendritic Diameter in Impulse Propagation

The effectiveness of synapses at various sites of the dendritic tree was studied using a segmental cable model with a program developed by Hines (Int. J. Biomed. Comput., 24, 55–68, 1989). The model rendered possible a high‐fidelity simulation of the dendritic geometry of a frog motoneuron described in the accompanying paper (Birinyi et al., Eur. J. Neurosci., 1003–1012, 1992). The model was used in the passive membrane mode and the synaptic activity was simulated with current injections into large and small diameter dendrites at proximal and distal locations. Synaptic efficiency was defined by the charge transfer ratio expressed as the proportion of the injected current which appeared at the soma. The charge transfer ratio was determined with uniform and non‐uniform distribution of specific membrane resistance over the soma–dendrite surface while the diameter of selected dendrite segments changed. The best charge transfer ratio was found with the largest dendrite membrane resistance, and the maximum efficiency of synaptic activity appeared at the original size of the dendrite segment stimulated. The amount of current that flowed in the proximal and distal directions from the segment stimulated depended on the diameter of that segment. The increase in diameter of proximal dendrites increased synaptic efficiency on distal dendrites, whereas the reverse caused a decline in synaptic efficiency on proximal dendrites. In addition to the diameter of dendrites, the arborization pattern also played a significant role in this mechanism. It is concluded that the cellulipetal increase in dendrite diameter greatly increases synaptic efficiency.

Extracellular matrix molecules exhibit unique expression pattern in the climbing fiber-generating precerebellar nucleus, the inferior olive.

Extracellular matrix (ECM) accumulates around different neuronal compartments of the central nervous system (CNS) or appears in diffuse reticular form throughout the neuropil. In the adult CNS, the perineuronal net (PNN) surrounds the perikarya and dendrites of various neuron types, whereas the axonal coats are aggregations of ECM around the individual synapses, and the nodal ECM is localized at the nodes of Ranvier. Previous studies in our laboratory demonstrated on rats that the heterogeneous distribution and molecular composition of ECM is associated with the variable cytoarchitecture and hodological organization of the vestibular nuclei and may also be related to their specific functions in gaze and posture control as well as in the compensatory mechanisms following vestibular lesion. Here, we investigated the ECM expression pattern in the climbing fiber-generating inferior olive (IO), which is functionally related to the vestibular nuclei. By using histochemical and immunohistochemical methods, the most characteristic finding was the lack of PNNs, presumably due to the absence of synapses on the perikarya and proximal dendrites of IO neurons. On the other hand, the darkly stained dots or ring-like structures in the neuropil might represent the periaxonal coats around the axon terminals of olivary synaptic glomeruli. We have observed positive ECM reaction for the hyaluronan, tenascin-R, hyaluronan and proteoglycan link protein 1 (HAPLN1) and various chondroitin sulfate proteoglycans. The staining intensity and distribution of ECM molecules revealed a number of differences between the functionally different subnuclei of IO. We hypothesized that the different molecular composition and intensity differences of ECM reaction is associated with different control mechanisms of gaze and posture control executed by the visuomotor-vestibular, somatosensory and integrative subnuclei of the IO.