No abstract

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 microm. 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.

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.

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 gamma-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.

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 microm2; lumbar motoneurons were slightly larger than brachial motoneurons. The mean length of the combined dendritic tree of brachial neurons was 29 408 microm and that of lumbar neurons 46 806 microm. The mean surface area was 127 335 microm2 in brachial neurons, and 168 063 microm2 in lumbar neurons. The soma - dendrite surface area ratio was 3 - 5% in most cases. Dendrites with a diameter of 600 microm from the soma. This suggests that about two-thirds of the synapses impinged upon distant dendrites >600 microm 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).