Suzanne Chen

Colocalization of neurotransmitters in the deep cerebellar nuclei.

SummaryAn abundance of glycine and glycine receptor immunoreactivities was found in all three parts of the deep cerebellar nuclei. Glycine immunoreactivity was restricted to small neurons throughout most of the deep cerebellar nuclei except for a few large positive neurons in the ventral part of the fastigial nuclei. In addition, glycine immunoreactivity was found in boutons outlining somata of large glycine negative neurons. Complementary to the glycine positive boutons was an intense glycine receptor immunoreactivity on large deep cerebellar nuclei neurons. Comparisons of immunoreactivities for glycine, GABA and aspartate in consecutive one micron sections revealed that many small neurons colocalized glycine and GABA, while some large neurons in the fastigal region colocalized glycine and aspartate.Ultrastructural investigations revealed glycine receptors on postsynaptic sites of dendrites and somata. Most boutons, which were presynaptic to glycine receptor sites, were filled with small flattened vesicles; however, a small percentage of boutons had round clear or dense core vesicles. Frequently, each bouton apposed multiple active zones on the dendrite or soma. One of these active zones was positive for glycine receptor and another was negative.This study supports: (1) glycine as a neurotransmitter in deep cerebellar nuclei, and (2) glycine and GABA colocalization in the same cell and bouton, but releasing to different receptor sites on the target neuron. Furthermore, the coexistence of glycine with GABA in the same deep cerebellar neuron may play an important role in controlling the conset and duration of signal transmission.

Plasticity of the parallel Fiber-Purkinje cell synapse by spine takeover and new synapse formation in the adult rat

Alteration in synaptic connectivity between Purkinje cell spines and parallel fibers of the cerebellum were studied following partial deafferentation of Purkinje cells in the the adult rat. Transection of parallel fibers by two lesions placed at a 1 mm interval on the folial crest were used to produce degeneration of these afferents. Ultrastructural analysis of synapses on Purkinje cell spines revealed degeneration with vacating of postsynaptic sites within 6 h. Reactive synaptogenesis as takeover of Purkinje cell spines by formation of new synapses from remaining parallel fibers occurred even before degenerating parallel fibers had vacated postsynaptic sites. This was accompanied by a marked increase in the number of dual innervations by reactive parallel fibers within one day. Some vacated postsynaptic sites were lost as indicated by a reduction in the number of synapses and others may have been taken over by newly formed synapses on spines. In addition, new synapses formed between the shafts of Purkinje cell branchlets and parallel fibers. Sprouting of parallel fibers occurred as small extensions without tubules while Purkinje cell spines reacted by forming elongated and multiple heads which contacted different parallel fibers. After 5 days degenerating boutons were rarely found. Enlarged spine heads were each capped by a proportionally enlarged parallel fiber bouton and joined by an elongated synaptic junction to parallel fibers. Some parallel fiber boutons were greatly enlarged and capped numerous profiles of spines. This study shows that formation of new pre- and postsynaptic sites takes precedence over reoccupation of original contacts and that multiple synapses on individual spines are being eliminated to give rise to single contacts with boutons. This elimination resulted in enlargement of synaptic contact areas between Purkinje cell spines and parallel fibers by taking over postsynaptic sites from some vacated and eliminated boutons.

Selective ablation of neurons by methylazoxymethanol during pre- and postnatal brain development

Neonatal or pregnant albino rats were injected with either single or double doses of methylazoxymethanol (20 mg/kg) to test the temporal specificity of its effect on clearly definable regions of the brain. A single dose, to dams from gestational day 11 to 21 (G11-G21) and to neonatal rats from birth to postnatal day 5 (P0-P5), produced differential weight reductions among various brain regions. Two prominent peaks of reduction were found: one occurring between G13 and G15 for the cerebrum and hippocampus and one occurring between P0 and P1 for the cerebellum and olfactory bulbs. Dual injections of the drug on G14 and G15 produced 60% weight reduction in the cerebrum, and slightly earlier injections on G13 and G14 reduced the weight of the cerebellum by about 23%. This weight reduction was accompanied by narrowing of the cerebellar width, which we believe was due to fewer Purkinje cells. Dual injections of methylazoxymethanol at P0 and P1 reduced the weight of the olfactory bulb by 65%, the cerebellum by 62%, and the hippocampus by 18%. These results show that its short action is within the window of cell division for various neurons and becomes additive on two successive days. This precise toxic effect on brain development can be used to disproportionally reduce the number of neuroblasts in specific regions of the brain. A differential ablation allows analysis of plasticity on pyramidal and nonpyramidal cells of the neocortex and hippocampus, Purkinje and granule cells of the cerebellum, and the granule and mitral cells of the olfactory bulbs.

Vulnerability of cerebellar development in malnutrition—I. Quantitation of layer volume and neuron numbers

Abstract The volumes of the cerebellar layers and numerical relationships, between granule cells and Purkinje cells, were analyzed following developmental malnutrition for differences in cerebellar changes between male and female rats. Control and protein-deficient diets were administered during pregestation, gestation, lactation and to offspring from weaning until death at 60 days of age. Volume of the granular, molecular and myelinated layers and the area of the Purkinje cell layer were determined from area and length measurements on serial sections. Density of granule and Purkinje cells was quantitated and used with granular layer volume and area of the Purkinje cell layer, respectively, for defining total cell numbers of each cell type. Although the wet weight of the cerebellum was not different between male or female deficient groups, the volume of the entire cerebellum, as well as its layers, was reduced more in the deficient female group than the deficient males. This difference between the two types of preparations was attributed to larger interfolial spaces in the deficient female group, presumably due to overall brain volume displacement within a cranial cavity space having the same size. The most striking difference between sexes was seen in the volume of the molecular layer where deprived females had a nearly 30% reduction as compared to their controls. The granular layer was decreased twice as much in the deficient females as in the deficient males. The largest volume change but least difference between sexes was in the myelinated layer. The reduction in granule cell number was reflected in a decreased granular layer volume rather than granular cell density. Granule cells were reduced by 20% in deficient females and 10% in deficient males but the number of Purkinje cells was constant between all groups. The result was a major shift in the ratio of granule cells to Purkinje cells for the deficient female group and a small change in ratio for the deficient male group. The reason for the more marked effect of malnutrition on the female could not be determined from this experimental paradigm, although much of the difference may be the result of behavior in competitive feeding during lactation.

Regulation of granule cell number by a predetermined number of Purkinje cells in development

Development dysgenesis of Purkinje cells or granule cells was analyzed for the reciprocal effect of reduced number of each cell type on the other. A single pre- or postnatal injection of methylazoxymethanol acetate (MAM) in the rat reduces either the number of Purkinje cells or the number of granule cells when administered at the time of their respective genesis. The total number of these two types of neurons was obtained from cell density values of each layer and the total volume of the granular layer and the area of the Purkinje cell layer. The results show that Purkinje cells (targets) strictly determine the maximum number of granule cells (afferent neurons) following deficits in the number of Purkinje cells produced by prenatal MAM administration. Deficits in Purkinje cells were accompanied by a proportionally smaller number of granule cells so that the ratio remained constant. On the other hand, the reduction in the number of granule cells of the postnatal MAM model did not affect the number of Purkinje cells. These results indicate that the maximum number of these afferent neurons is constrained unidirectionally through a property defined by the number of their target neurons which develop earlier. Furthermore the number of afferent cells had no effect on the number of target cells.

Vulnerability of cerebellar development in malnutrition-II. Intrinsic determination of total synaptic area on purkinje cell spines.

Abstract In a previous investigation, we demonstrated a greater reduction in the ratio of granule cells to Purkinje cells in malnourished female rats than male rats that were reared under the same conditions ( Hillman & Chen , 1981 ). In this study, the synaptic relationships between these two types of cells were analyzed to determine how this difference in ratio was accommodated in cerebellar circuitry. Control diets (25% casein) and experimental diets (8% casein), having the same calories, were administered pregestationally and through lactation with continuation of the respective diets to offspring until death at 60 days. Molecular layer volume and synaptic density were determined and the relative number of synapses on each Purkinje cell was estimated from the number of synapses in the molecular layer and total number of Purkinje cells. The average length of synaptic profiles on Purkinje cell spines was measured and the average synaptic contact area on each Purkinje cell was calculated. We found that the average number of synapses on individual Purkinje cells decreased in the deficient female group but not in the deficient male group and there was an increase in the average length of synaptic profiles in the malnourished female group but not in the deficient male group. In some cerebella of females, elongated synaptic profiles on giant spines were seen on Purkinje cells. Comparison of the average number of synapses on each Purkinje cell with average length of synaptic profiles revealed an inverse relationship between size and number of synapses for two control and two experimental groups and a subgroup of females with giant spines. After conversion of average profile length to average contact area and taking into account the number of synapses on each Purkinje cell, we found that Purkinje cells for all five groups had the same total synaptic area. This inverse relationship was believed to reflect a constant total contact area for parallel fiber synapses on Purkinje cells. It is suggested that a constancy in total synaptic area means that the amounts of macromolecules that are incorporated into postsynaptic membrane specializations are determined intrinsically by the Purkinje cell. However, the distribution of these molecules to synaptic sites on dendritic trees can be shifted by interaction of parallel fiber synapses with the Purkinje cell.

Robust synaptic plasticity of striatal cells following partial deafferentation

Partial ablation of the cerebral cortical input to the neostriatum generates a rapid lasting effect on the size of remaining synaptic sites. The neocortex was lesioned in adult rats and the neostriatum was analyzed for effects on remaining spines of principal cells during the period from 2 to 40 days. There was an increase in the size of spine heads, boutons and synaptic contact sites. The spine heads became very complex and a corresponding bouton enlargement was accompanied by an increase in the number of synaptic vesicles. By two days, the average profile length of postsynaptic membrane densities (PSDs) had increased by 25% representing an equivalent 50% increase in synaptic contact area. The number of synaptic sites was reduced on each principal neuron of the lesioned group. Comparison of the number of sites per unit volume to their average contact area revealed a reciprocal relationship indicating a conservation in the total synaptic contact area on each neuron. This effect was consistent for all postsurgical days. The lack of a significant return of synaptic number by 40 days indicates that axonal sprouting is not a major factor in neuronal plasticity in the adult striatum. The rapid increase in the size of spines, boutons and synaptic sites at remaining connections suggests that dendrites are the first to initiate the plasticity response in adult neurons through postsynaptic attachments and their corresponding receptor structure. The underlying mechanism of this plasticity may be through a conservation of macromolecules forming postsynaptic membrane specializations on target neurons. Remaining axons appear to follow the dendritic response with a plasticity generating presynaptic appositional specializations to match the contact area of the postsynaptic site.

Compartmentation of the cerebellar cortex by protein kinase C delta

Six isozymes of protein kinase C were analysed in the rat cerebellum using immunohistochemistry. The results revealed a non-uniform distribution of protein kinase C delta among Purkinje, basket, and stellate cells. Serial-section mapping of the delta immunoreactivity revealed that (i) the number and intensity of labeled Purkinje cells increased from rostral to caudal while labeled basket-stellate cells decreased caudally; (ii) the majority of Purkinje cells were labeled in the nodulus, flocculus, and paraflocculus while the anterior lobules were mostly negative; and (iii) labeled Purkinje cells formed distinct parasagittal bands in lobules 6-9 of the paravermis and vermis. The banding of protein kinase C delta within subsets of Purkinje cells suggests units of cerebellar circuitry with specific signaling properties through protein phosphorylation. The visual-vestibular regions of the cerebellum contained the highest amount of the isozyme.

Ultrastructural localization of the plasmalemmal calcium pump in cerebellar neurons

In a previous study, fluorescence labeling of a plasmalemmal ATPase protein with the 5F10 monoclonal antibody revealed prominent antigen in the cerebellar molecular layer surrounding the somata and dendrites of Purkinje cells. In the present study, this antibody labeled with silver enhanced nano-sized gold particles on semithin plastic sections revealed a clearly demarcated plasma membrane outlining the somata and entire dendritic arbors of Purkinje cells including their spines. Ultrastructural analysis of horseradish peroxidase preparations showed reaction product along the plasmalemma and extending on to the sub-plasmalemmal endoplasmic reticulum. In the granular layer, somata of granule cells were reactive, as were their dendritic extensions into glomeruli where reactive claws surrounded voids formed by mossy fiber rosettes. Somata and dendrites of cerebellar nuclear cells also had reactive zones that were limited to the plasma membrane and a narrow zone of the sub-plasmalemmal endoplasmic reticulum. Comparative labeling of this protein and P channel protein revealed similar plasmalemmal locations. This study shows that a specific calcium ATPase pump protein is located on the plasmalemma of certain types of cerebellar neurons. The ultrastructural distribution of calcium pump and P channel antibodies occurred in punctate sites along the plasma membrane of dendrites and spines of Purkinje cells. The close association between P-type calcium channels and the plasma membrane calcium pump is consistent with rapid extrusion of intracellular calcium from neurons endowed with large numbers of voltage-gated calcium channels.

Dying-back of Purkinje cell dendrites with synapse loss in aging rats

Qualitative and quantitative changes were found in the cerebellar circuitry of old as compared to young rats. The old group had a reduced number of synapses (at least 30%), however, there was an increase in the size of remaining synaptic components (13.5% for spine head volume, 66% for bouton volume, and 17% for the area of synaptic contact zones). Furthermore, there were pronounced morphological changes in the older group appearing as: 1) prominent lipofuscin bodies in Purkinje cell somata, 2) numerous myelinated fibers in the lower part of the molecular layer, 3) tortuous Purkinje cell dendrites in a thinned molecular layer, and 4) abundant vacuolar profiles and membrane swirls in small and intermediate-sized dendrites. Our findings suggest that Purkinje cell dendrites are dying-back reducing the target field for granule cells and that remaining synaptic sites compensate by increasing synaptic contact area as well as the size of pre- and postsynaptic structures.