Purkinje Cell Dendritic Spines Research Articles

Cerebellar Purkinje cells from the lurcher mutant and wild-type mouse grown in vitro: a light and electron microscope study.

Lurcher is an autosomal semidominant murine mutation. Lurcher heterozygotes (+/Lc) lose all their cerebellar Purkinje cells by adulthood. Explants from 2 days postnatal (P2) wild-type (+/+) and +/Lc cerebellar cortex were grown in vitro to investigate the role of local neuronal environment and afferent input on the degenerating +/Lc Purkinje cell. In Lurcher explants, Purkinje cells were maintained for up to 25 days in vitro. No significant difference was observed between +/+ and +/Lc Purkinje cell numbers from 10 to 20 days in vitro, as revealed by calbindin-D immunoreactivity. Growing +/Lc explants in association with +/+ explants resulted in no significant difference in Purkinje cell survival (10-20 days in vitro). Image analysis of the gross morphology of calbindin-D-immunostained Purkinje cells from +/+ and +/Lc explants grown in vitro revealed a significant decrease in the total area and dendritic lengths of +/Lc Purkinje cells (15 and 20 days in vitro). The fine structure of +/Lc and +/+ Purkinje cells was examined under the electron microscope (10-25 days in vitro). No difference in ultrastructure was observed between +/Lc and +/+ Purkinje cells grown in vitro, and many features similar to normal Purkinje cell development in vivo were present. These included monosynaptic parallel fibre synapses with Purkinje cell dendritic spines, other interneuron synapses with Purkinje cell dendrites and soma, astroglial investment, and minimal extracellular space in the neuropil. Unusual features observed included a persistence of the perisomatic spines in some Purkinje cells, an absence of Nissl bodies in the Purkinje cell perikaryon, naked Purkinje cell dendritic spines, and occasional heterologous synapses. The results are discussed in the light of previous chimeric analysis of the Lurcher mutation, and a hypothesis is put forward to explain the survival of +/Lc Purkinje cells in vitro.

Circuit reorganization in granuloprival cerebellar cultures in the absence of neuronal activity.

Neonatal mouse cerebellar cultures exposed to cytosine arabinoside for the first 5 days in vitro to destroy granule cells and compromise glia undergo a circuit reorganization featured by profuse sprouting of Purkinje cell recurrent axon collaterals, which hyperinnervate the somata of other Purkinje cells and project to Purkinje cell dendritic spines. Such granuloprival cultures were exposed continuously from explanation to tetrodotoxin and elevated levels of magnesium to block neuronal activity. A similar circuit reorganization occurred, except that there was a reduction in the number of axospinous synapses and Purkinje cell axosomatic synapses, which in this case were all inhibitory. Functionally, after recovery from the blockade, granuloprival cultures developed sustained cortical hyperactivity, which was consistent with the reduction of inhibitory synapses. While the absence of neuronal activity did not prevent reorganizational changes following granule cell loss, the full development of the inhibitory circuitry was not attained. These results further support the concept that spontaneous neuronal activity is necessary for the complete development of inhibitory synapses.

Morphology of parallel fibres in the cerebellar cortex of the rat: an experimental light and electron microscopic study with biocytin.

Microinjections of biocytin have been made in the granular layer of the rat cerebellar cortex in order to label the axonal projections of a localised population of granule cells. Light microscopic techniques were used to determine the lengths of the parallel fibres and to measure the spacing and size of the fibre varicosities. Fibres were longest in the superficial one-third of the molecular layer, where mean overall length was 4.7 mm, and mean length decreased to 4.2 mm in the lower one-third of the molecular layer. We found no very short fibres but a small population deep in the molecular layer had a branch length of about one-half the average. Mean intervaricosity interval and varicosity size varied with distance from proximal to distal along the fibres. Mean intervaricosity interval was 3.7 microns within 250 microns of the fibre bifurcation points and progressively increased towards the distal ends, where the mean interval was 7.4 microns. Mean varicosity size was 0.82 microns 2 in this proximal region and decreased to 0.47 microns 2 about 1.2 mm distally. Mean intervaricosity interval on the ascending axons of the granule cells was 4.0 microns. Electron microscopy revealed that a high proportion (89%) of the parallel fibre varicosities formed synaptic junctions. The majority of the synapses (91%) were formed on Purkinje cell dendritic spines. Some varicosities also formed simultaneous synaptic contacts or double synapses with two spines. These double synapses occurred more frequently in the proximal region of the fibres (11%) than on the distal ends (2%). The length of the postsynaptic density also differed according to the location of the varicosities and the mean length at the proximal parallel fibre synapses was 0.59 microns compared with 0.38 microns at the distal synapses. It is concluded that a beam or bundle of parallel fibres originating from cells in a focal region of the granular layer will exert a graded synaptic influence on its target Purkinje cells, with the most powerful influence occurring on cells located around the proximal region of the fibres where they bifurcate and the weakest action being exerted on cells located at the distal end of the fibres.

Ontogeny of proenkephalin mRNA and enkephalin peptide expression in the cerebellar cortex of the rat: spatial and temporal patterns of expression follow maturational gradients in the external granular layer and in Purkinje cells

Proenkephalin mRNA and peptide products were examined in developing cells of the postnatal rat cerebellar cortex using in situ hybridization and immunocytochemistry. On day 7, proenkephalin mRNA was first detected as discrete cellular labeling in Golgi cells and as a diffuse hybridization signal over the Purkinje cell layer. On day 14, proenkephalin mRNA and peptide products primarily appeared in distinct subpopulations of Purkinje cells present in the posterior and lateral cerebellum. Similarly, in the external granular layer (EGL), enkephalin immunoreactivity was present only in the posterior and lateral portions of the cerebellum on day 14. However, proenkephalin mRNA was not detected in enkephalin-immunoreactive EGL cells. On day 21, the subset of Purkinje cells that expressed proenkephalin mRNA and peptides were distributed more uniformly throughout the cerebellum. On day 28, a few enkephalin-immunoreactive Purkinje cells were uniformly present throughout the cerebellum, but proenkephalin mRNA was not detected in most of these cells. The spatial gradients in proenkephalin mRNA expression evident in the Purkinje cells of younger rats were no longer present in 28-day-old rats. These findings are important, because endogenous opioids such as enkephalin have been previously shown to inhibit the growth of Purkinje cell dendrites and dendritic spines, and inhibit the rate of mitosis in EGL neuroblasts. Cells do not develop at uniform rates within the cerebellum. There are regional differences in the timing of the formation of the EGL, and in the morphogenesis of Purkinje cells. In conjunction with previous work, the present findings suggest that during development, the pattern of enkephalin immunoreactivity in Purkinje and EGL cells closely follows the spatial and temporal gradients of maturation in both these cell types. The emergence and disappearance of enkephalin immunoreactivity in Purkinje and EGL cells is spatially and temporally related, and coincides with proenkephalin mRNA expression in Purkinje cells. Thus, the transient and coordinated appearance of enkephalin in cerebellar Purkinje and EGL cells may contribute to regional differences in the rate of cerebellar maturation, and may help synchronize the developmental interactions between these two cell types.

Optical Imaging of Cytosolic Calcium, Electrophysiology, and Ultrastructure in Pyramidal Neurons of Organotypic Slice Cultures from Rat Hippocampus

Organotypic slice cultures from rat hippocampal cortex grown in an interface between culture medium and a CO2-enriched atmosphere maintained much of the morphological connectivity characteristic of the hippocampus in situ and thinned out considerably, facilitating optical measurements of fluorescent dyes sensitive to Ca2+ in individual neurons. Pyramidal neurons of the CA3 region presented morphological features of differentiated cells, including complex dendritic arborization and large numbers of dendritic spines. The fine cytoskeletal substructure at the postsynaptic density, below the plasma membrane, and within the core of the head and neck of dendritic spines in rapidly frozen slice cultures presents the characteristic morphology previously described for Purkinje cell dendritic spines in acutely dissected cerebellar cortex slices after rapid freezing. CA3 neurons responded to intracellular current injection with a train of action potentials, spike frequency adaptation, and a slow afterhyperpolarization. These spike trains caused rapid increases in dendritic [Ca2+]i that decayed to resting levels after termination of the current pulse. Dendritic spines were clearly observed in proximal dendrites of CA3 neurons in live preparations. [Ca2+]i transients in these dendritic spines closely followed the changes observed in the main dendritic shaft. Orthodromic synaptic stimulation from the dentate hilus generated long-lasting synaptic potentials accompanied by large [Ca2+]i transient in CA3 pyramidal neurons. The [Ca2+]i response was first observed in the proximal dendrites, after which the soma exhibited a [Ca2+]i increase, returning to resting levels within 10 s after the synaptic stimulus. Slice cultures thus provide a favorable opportunity to investigate [Ca2+]i responses in individual neurons maintained in an organotypic synaptic environment, taking advantage of high-resolution optical techniques.

Immunohistochemical visualization of a metabotropic glutamate receptor.

The immunocytochemical localization of the recently cloned metabotropic glutamate receptor 1 alpha (mGluR1 alpha) was demonstrated with a C-terminus specific antibody in rat cerebellar cortex. This antibody detects a 138-140 kDa major, and a 46 kDa minor band in membrane preparations of rat cortex and cerebellum. mGluR1 alpha immunoreactivity (mGRi) was present in Purkinje and basket cells. Purkinje cell dendritic spines and their postsynaptic membranes showed selective labelling. Presynaptic membranes, parallel fibres and glial processes were devoid of mGRi. It is suggested that the selective postsynaptic localization of this receptor at the dendritic spines of Purkinje cells serves as the morphological basis for long term depression processes in the molecular layer of the cerebellar cortex.

Cytosine arabinoside effects in mouse cerebellar cultures in the presence of astrocytes

Organotypic cerebellar cultures derived from neonatal mice were exposed to recent preparations of cytosine arabinoside that destroyed oligodendrocytes and drastically reduced granule cells, but did not reduce the astrocyte population. The cultures were analysed by light and electron microscopy and by extracellular electrophysiological recording. Purkinje cells survived in greater numbers than in untreated expiants and sprouted excess recurrent axon collaterals that formed heterotypical synapses with Purkinje cell dendritic spines. These changes were similar to those found in earlier studies with a cytosine arabinoside preparation that did reduce the astrocyte population, in addition to destroying oligodendrocytes and granule cells. Results with recent cytosine arabinoside preparations that differed from those obtained previously included astrocytic ensheathment of Purkinje cells and apposition of many unattached dendritic spines, encasement of heterotypical synapses by astroglial processes, a loss of Purkinje cell somatic spines and a lack of somatic hyperinnervation of Purkinje cells by sprouted recurrent axon collateral terminals. All of these differences were attributed to the presence of adequate numbers of competent astrocytes. Heterotypical synapses formed by sprouted recurrent axon collateral terminals and Purkinje cell dendritic spines were functional, as indicated by cortical inhibition in response to antidromic Purkinje cell activation in the absence of somatic hyperinnervation. These results give further definition to the role of astrocytes in cerebellar development and plasticity.

Induction of dendritic spine proliferation by an astrocyte secreted factor

A number of neuron-supporting functions have been ascribed to astrocytes. In this study we found that a proliferation of Purkinje cell dendritic spines, a target site for presynaptic axon terminals, was induced in cytosine arabinoside-treated cerebellar cultures by exposure to astrocyte-conditioned medium. This result suggests that astrocytes may instigate the elaboration of postsynaptic neuronal elements prior to the appearance of axons. This may be an important mechanism in neural development, postdevelopmental neuroplastic change, or regeneration.

F3/F11 cell surface molecule expression in the developing mouse cerebellum is polarized at synaptic sites and within granule cells

The distribution of the F3/F11 neuronal cell surface molecule was investigated in the developing and adult mouse cerebellum by immunocytochemistry at the light and electron microscopic levels. F3/F11 was confined to subsets of neuronal types, since the Purkinje cell body and dendritic arborization as well as the stellate cells were not immunoreactive. In the young developing cerebellum, the granule cell axons strongly express F3/F11 as soon as they begin to grow, consistent with a functional role in promoting directional outgrowth of neuronal processes. In 10-d-old and adult cerebella, the granule cell bodies and dendrites were not immunoreactive whereas the parallel fibers, which are the granule cell axons, were labeled including in their presynaptic varicosities. By contrast, dendrites, cell bodies, and axons of Golgi cells were labeled by anti-F3 antibodies. Hence, F3/F11 can either be expressed throughout the cell or be polarized to the axons. This raises the question of how segregation of the glypiated F3/F11 molecule between different subcellular compartments depending on the type of neuron is achieved. F3/F11 was found to be present at three types of synaptic sites, suggesting that it might play a role in the formation and maintenance of synapses. However, in each type of synpase, F3/F11 was present at only the pre- or postsynaptic site, never at both: the parallel fiber varicosities contained F3/F11 whereas the postsynaptic compartment in contact, that is, the Purkinje cell dendritic spines, did not. The granule cell dendrites were unlabeled while the mossy fiber terminals contacting them were immunoreactive, and finally, the Golgi cell dendrites and dendritic spines were labeled while the presynaptic compartment contacting them was not. If F3/F11 functions as an adhesion molecule in vivo as indicated by in vitro assays, F3/F11-mediated adhesion is likely to be heterophilic.

Reorganization of organotypic cultures of mouse cerebellum exposed to cytosine arabinoside: A timed ultrastructural study

This study was designed to examine the sequential changes in the developing granuloprival cerebellar culture. In this model of anomalous cerebellar development, organotypic cultures derived from newborn Swiss-Webster mice were exposed to the DNA synthesis inhibitor, cytosine arabinoside, at explantation and were fixed for electron microscopic examination on successive days in vitro. Similar developmental stages were compared in control explants. Granule cell destruction began early, and was widespread by 2 days in vitro, when oligodendrocyte destruction also began in treated cultures. A few granule cells survived, but no recognizable oligodendrocytes remained by 7 days in vitro, at a time when myelin was initially evident in control explants. Purkinje cell recurrent axon collateral sprouting began at 3 days in vitro in cultures exposed to cytosine arabinoside, and the sprouted terminals initially synapsed with Purkinje cell somata, somatic spines and dendritic shafts. Synapses with Purkinje cell dendritic spines developed later, at approximately the same time as parallel fiber-Purkinje cell dendritic spine synapses formed in control cultures. Astrocytic ensheathment of control Purkinje cells was well underway by 6 days in vitro and Purkinje cell somata were relatively rounded and almost completely ensheathed by 9 days in vitro. Glial ensheathment did not occur in cytosine arabinoside treated cultures, and Purkinje cell somata were scalloped at 7 days in vitro by excess impinging recurrent axon collateral terminals, and never developed the smooth contours characteristic of control Purkinje cells. Purkinje cell somatic spines persisted in treated explants, and reduction of excess extracellular space was delayed until 12 days in vitro, when most of the developmental changes had been completed. The earlier development of synapses by excess recurrent axon collateral terminals with Purkinje cell somata, somatic spines and dendritic shafts, followed by the later development of heterotypical synapses with dendritic spines, in parallel with synapse formation by normal presynaptic elements, suggests that the sequence of development of synapses is a function of the maturational state of the postsynaptic components.

Expression of proenkephalin mRNA in developing cerebellar cortex of the rat: expression levels coincide with maturational gradients in Purkinje cells

The cellular localization of proenkephalin (PE) mRNA expression was systematically examined in midsagittal (vermal) sections of the developing rat cerebellar cortex by in situ hybridization. PE mRNA was initially detected in Golgi cells of postnatal day 7 (PND 7) rats and in each group thereafter. Moreover, PND 7 rats also displayed an intense layer of PE mRNA hybridization signal over the Purkinje mell layer. By PND 14,mdistinct cellular labeling was observed in a subpopulation of Purkinje cells in all lobules of the vermis except lobule III. At PND 7 and 14, the area and level of intensity of Purkinje cell associated PE mRNA hybridization signal followed a gradient that was most intense caudally but then decreased rostrally. At PND 21, the proportion of labeled Purkinje cells and the intensity of PE hybridization signal was evenly dispersed between the anterior and posterior lobules of the cerebellar vermis. PE hybridization signal was not detected in the developing neural cells of the external granular layer or the interneurons of the molecular layer in the vermis. These results indicate that the ontogeny of PE mRNA expression in Purkinje cells is developmentally regulated since levels of expression closely follow the chronological order of settling and maturation of these neurons. Based on prior evidence that endogenous opioids inhibit the growth of Purkinje cell dendrites and dendritic spines, PE expression is likely to be important for Purkinje cell maturation.

Intraventricular administration of substance P induces unattached Purkinje cell dendritic spines in rats.

Substance P was infused in the lateral ventricles of twenty Lewis rats for twenty days. The animals under the influence of the substance P demonstrated grooming of the head, the body and the forepaws. On the twentieth day the animals were sacrificed and the cerebellar cortex was processed for electron microscopy. The ultrastructural analysis revealed that although the granule cells, the parallel fibers and the systems of the afferent fibers were intact, numerous unattached Purkinje cell dendritic spines were seen embedded in the soma of the astrocytes, demonstrating postsynaptic differentiation. Numerous unattached spines of the secondary and tertiary dendritic branches of the Purkinje cells were also seen in the molecular layer surrounded by astrocytic sheath. Free unattached spines were also seen not surrounded by any astrocytic process, which did not demonstrate any postsynaptic specialization. The development of unattached Purkinje cell dendritic spines, in an otherwise intact cerebellar cortex, following the intraventricular administration of substance P, suggests that it may act as local growth factor, enforcing the preprogrammed-capability of the Purkinje cells in developing new synaptic surfaces.

The arrangement of actin filaments in the postsynaptic cytoplasm of the cerebellar cortex revealed by quick-freeze deep-etch electron microscopy

The cytoskeletal architecture of the postsynaptic cytoplasm in the cerebellar cortex of mice and rats was observed by quick-freeze, deep-etch electron microscopy. The postsynaptic cytoplasm was mainly filled with a network of actin filaments (∼8 nm in width). The tips of the actin filaments were closely associated with the true inner side of the postsynaptic membranes. However, the organization of the actin filaments was distinct depending on the types of synapses. In axosomatic synapses the actin filaments tended to run randomly and form a network while in the postsynaptic spine, such as Purkinje cell dendritic spines, the actin filaments were mainly arranged parallel to the stalk of the spines. Only a few actin filaments were found in the postsynaptic cytoplasm of some axodendritic synapses such as mossy fiber-granule cell synapses. In most cases a mesh of fine strands (∼6 nm in width) and granular substances was observed just underneath the postsynaptic membranes which also associated with actin filaments. The arrangement of actin filaments in the spine does not support the possibility of constriction of spines as a basis for long-term depression (LTD).

Membrane and cytoplasmic structure at synaptic junctions in the mammalian central nervous system.

Application of rapid freezing, freeze substitution fixation, and freeze fracture techniques to the study of synaptic junctions in the mammalian central nervous system has revealed new aspects of synaptic structure that are consistent with and partially explicate advances in synaptic biochemistry and physiology. In the axoplasm adjacent to the presynaptic active zone, synaptic vesicles are linked to large spectrin-like filamentous proteins by shorter proteins that resemble synapsin I in morphology. This mesh of presynaptic filamentous proteins serves to concentrate synaptic vesicles in the vicinity of the active zone. The affinity with which the vesicles are bound by the mesh is probably modulated by the extent of phosphorylation at specific sites on the constituent filamentous proteins, and changes in the binding affinity result in changes in transmitter release. The structural organization of the postsynaptic density in Purkinje cell dendritic spines consists of very fine strands with adherent, heterogeneous globular proteins. Some of these globular proteins probably correspond to protein kinases and their substrates. The postsynaptic density, positioned at the site of the maximal depolarization caused by synaptic currents, apparently serves as a supporting framework for a variety of proteins, which respond to and transduce postsynaptic depolarization. At least two classes of filamentous protein fill the cytoplasm of spines with a complex mesh, which presumably contributes to maintenance of the spine shape. Membrane bound cisterns are a ubiquitous feature of Purkinje cell dendritic spines. Studies of rapidly frozen tissue with electron probe microanalysis and elemental imaging reveal that these cisterns take up and sequester calcium, which is derived from the extracellular space, and which probably enters the spine as part of the synaptic current.

Quantitative study of the Purkinje cell dendritic spines in the rat cerebellum.

The number of spines on an individual Purkinje cell in the cerebellar cortex of the rat was determined by stereological methods. Investigations were based on thin section electron micrographs, freeze fracture replicas, and horseradish peroxidase labeled cells. Purkinje cell dendritic spines in our embedded material had a mean length of 1.4 +/- 0.05 micron and mean neck and head diameters of 0.22 +/- 0.01 micron and 0.45 +/- 0.02 micron, respectively. From these dimensions, an estimate of spine volume in embedded material of 0.132 micron 3 was obtained. The density of dendritic spines in our fixed material was 8.15 x 10(8) or 7.24 x 10(8) per microliters of molecular layer from volume fraction and density per mm2, respectively. The number of spines per linear micron of Purkinje cell spiny branchlet was 17.2 from freeze fracture and 17.6 from horseradish peroxidase labeled dendrites. These all indicate that there are between 154,000 and 175,000 spines on the dendritic tree of each Purkinje cell, considerably more than previously reported for the rat.

Number of parallel fiber synapses on an individual Purkinje cell in the cerebellum of the rat.

In the present study, stereological techniques applied to electron micrographs of the molecular layer of the rat cerebellum have been used to estimate the number of parallel fiber synapses on the dendritic tree of a single Purkinje cell. Quantitative features of the parallel fiber to Purkinje cell dendritic spine synapses and of the parallel fibers were investigated as a preliminary to estimating the number of synapses. Parallel fiber to Purkinje cell synapses are flattened disclike structures with a mean axial ratio of 14.7 and a mean diameter of 319 microns in fixed tissue. The density of synapses in our fixed material was 8.17 x 10(8) per microliters of molecular layer. Determination of the length density of the synapses per unit area of micrograph indicated a synapse density of 8.03 x 10(8) per microliters. These densities give a total number of synapses per Purkinje cell of 1.74 x 10(5) and 1.71 x 10(5), respectively. Estimation of the number of parallel fiber varicosities and of varicosity length gave a density of 9.31 x 10(8) varicosities per microliters of molecular layer and determining the mean number of parallel fiber to Purkinje cell synapses per varicosity gave a synapse density of 9.82 x 10(8) per microliters, equivalent to 2.09 x 10(5) per Purkinje cell. The reasons why this estimate is likely to be too high are discussed. We conclude that there are some 175,000 parallel fiber synapses on an individual Purkinje cell dendritic tree in the cerebellar cortex of the rat, considerably more than previously reported.

Activity-dependent accumulation of calcium in Purkinje cell dendritic spines.

The calcium content of synapses of parallel fibers on Purkinje cell dendritic spines was determined by electron probe x-ray microanalysis of freeze-dried cryosections from directly frozen slices of mouse cerebellar cortex. In fresh slices frozen within 20-30 sec of excision, calcium concentrations ranging from 0.8 to 18.6 mmol/kg of dry weight were measured in cisterns of smooth endoplasmic reticulum within Purkinje cell dendritic spines. The average calcium content of spine cisterns in rapidly excised slices (6.7 +/- 0.6 mmol/kg of dry weight +/- SEM) was higher than the average calcium content of spine cisterns in brain slices incubated without stimulation for 1-2 hr before direct freezing (2.5 +/- 0.4 mmol/kg of dry weight). Depolarization of incubated cerebellar slices by isotonic 55 mM KCl resulted in the accumulation within spine cisterns of very high amounts of calcium or isotonically substituted strontium, both derived from the extracellular fluid. These results suggest that one function of spine cisterns is to sequester free calcium that enters the spine through ligand-gated or voltage-gated channels during synaptic transmission.

Transplanted astrocytes reduce synaptic density in the neuropil of cerebellar cultures

Cytosine arabinoside-treated neonatal mouse cerebellar cultures, devoid of granule cells and mature glia, demonstrate heterologous synapses between sprouted Purkinje cell recurrent axon collaterals and dendritic spines in the neuropil. Such cultures were transplanted with optic nerve as a source of glia, and the effect on neuropil synapses was investigated. There was a significant reduction in the number of synapses in the neuropil and an increase in the number of free dendritic spines. Many of these spines occurred in clusters, unapposed by glial processes. The effect on the synapse density was not due to a comparable increase in the area occupied by the added astrocytes or an increase in nerve terminal diameter. The results suggest that astrocytes alter the density of neuropil synapses and may also induce the sprouting of dendritic spines.

Substructure in the postsynaptic density of Purkinje cell dendritic spines revealed by rapid freezing and etching.

In tissue prepared by rapid freezing, freeze fracture, and shallow etching, the postsynaptic density of Purkinje cell dendritic spines has a substructure consisting of fine filaments and irregular, globular adherent proteins. The number and packing density of the globular proteins vary from region to region within a single density and are even more variable when different junctions are compared. Whereas actinlike microfilaments and spectrinlike filaments are juxtaposed to the postsynaptic density, they do not appear to be continuous with the constituent filaments of the density. We suggest that the postsynaptic density at this class of synapse is composed of fine filamentous proteins that insert on the postsynaptic membrane and serve as a supporting framework for a variety of globular proteins. The globular proteins may vary qualitatively and quantitatively from junction to junction, and are positioned in the region of the spine that has the greatest concentration of ionized calcium entering with the synaptic current, and the greatest extent of postsynaptic depolarization.

Quantification of immunogold labelling reveals enrichment of glutamate in mossy and parallel fibre terminals in cat cerebellum

The glutamate immunoreactivity of different cell populations was compared quantitatively in the cerehellar cortex of cat, using an antiserum raised against glutamate coupled to bovine serum albumin by glutaraldehyde. Neuronal and glial processes were identified on serial electron microscopic sections which were processed by a postembedding immunogold procedure. The surface density of colloidal gold particles was used for statistical comparison of the relative levels of glutamate in cell populations, or in different parts of the same population. The terminals of mossy and parallel fibres had significantly higher levels of glutamate immunoreactivity than Golgi cell terminals, granule cell dendritic digits. Purkinje cell dendrites or dendritic spines. Golgi cell terminals were identified by their position and GABA immunoreactivity as revealed by immunogold in serial sections. The dendritic digits of the putative glutamatergic granule cells had significantly higher glutamate immunoreactivity than did Purkinje cell dendrites and dendritic spines. Glial cell processes in the molecular layer had lower level of glutamate immunoreactivity than any of the neuronal processes. The results demonstrate that the highest levels of glutamate immunoreactivity occur in mossy and parallel fibre presynaptic terminals that are known to have an excitatory effect. This supports previous suggestions that glutamate may be a transmitter at these synapses. The measurement of the levels of putative amino acid transmitters in identified neuronal populations, or in different parts of the same population, could have wide applications in studies on the chemical neuroanatomy of the nervous system.