Ectopic Purkinje-like cells are GABAergic: Immunohistochemistry with an immune serum against glutamic acid decarboxylase

Summary Intensely stained cells are found in the cerebellar white matter of the vermis and paravermis in adult rats after immunoreaction with an immune serum raised against glutamic acid decarboxylase (GAD). The cells are similar in size to cortical Purkinje cells and three times the size of Golgi cells of the internal granule layer, and have a thick immunopositive cell process emerging from a welldefined cytoplasmic cone. In the cytoplasm, immunoprecipitates are more dense around the nucleus as in normally located Purkinje cells. The morphological appearance of the immunopositive cells suggests that they may be ectopically located Purkinje cells. The soma of the ectopic Purkinje cells is contacted by a few darkly stained terminal boutons. Data indicate that, in spite of the different cellular environment, ectopic Purkinje cells can develop not only the typical morphological pattern already described but also other intrinsic features, such as their typical inhibitory neurotransmitter.     

Interactions between cerebellar Purkinje cells and their associated astrocytes

Some neurons, including cerebellar Purkinje cells, are completely ensheathed by astrocytes. When granule cell neurons and functional glia were eliminated from newborn mouse cerebellar cultures by initial exposure to a DNA synthesis inhibitor, Purkinje cells lacked glial sheaths and there was a tremendous sprouting of Purkinje cell recurrent axon collaterals, terminals of which hyperinnervated Purkinje cell somata, including persistent somatic spines, and formed heterotypical synapses with Purkinje cell dendritic spines, sites usually occupied by parallel fiber (granule cell axon) terminals. Purkinje cells in such preparations failed to develop complex spikes when recorded from intracellularly, and their membrane input resistances were low, making them less sensitive to inhibitory input. If granule cells and oligodendrocytes were eliminated, but astrocytes were not compromised, sprouting of recurrent axon collaterals occurred and their terminals projected to Purkinje cell dendritic spines, but the Purkinje cells had astrocytic sheaths, their somata were not hyperinnervated, the somatic spines had disappeared, complex spike discharges predominated, and membrane input resistance was like that of Purkinje cells in untreated control cultures. When cerebellar cultures without granule cells and glia were transplanted with granule cells and/or glia from another source, a series of changes occurred that included stripping of excess Purkinje cell axosomatic synapses by astrocytic processes, reduction of heterotypical axospinous synapses in the presence of astrocytes, disappearance of Purkinje cell somatic spines with astrocytic ensheathment, and proliferation of Purkinje cell dendritic spines after the introduction of astrocytes. Dendritic spine proliferation was followed by formation of homotypical axospinous synapses when granule cells were present or persistence as unattached spines in the absence of granule cells. The results of these studies indicate that astrocytes regulate the numbers of Purkinje cell axosomatic and axospinous synapses, induce Purkinje cell dendritic spine proliferation, and promote the structural and functional maturation of Purkinje cells.     

Effects of long-term hypothyroidism in the morphology and synaptic organization of cerebellar ectopic granule cells

Abundant ectopic granule cells scattered in the cerebellar molecular layer have been observed in 30-day-old hypothyroid rats. Their morphological features indicate that they must be regarded as mature heterotopic cells arrested during their migration towards the granular layer. As their impoverished dendritic trees are identical to those seen in controls, it is unlikely that the lack of thyroid hormones played a major role in the deficient dendritic outgrowth. The study of 180-day-old hypothyroid rats revealed that although ectopic granule cells remained quite numerous, their number per unit surface was lesser than in the 30-day-old hypothyroid group. This finding may be related to the capacity displayed by heterotopic neurons to establish synaptic contacts with the components of the molecular layer. This was inferred by the presence of a peculiar synaptic cell investment formed by axosomatic and somatodendritic contacts in 180-day-old hypothyroid rats which shows that the surviving ectopic granule cells manage to adapt to an adverse milieu.     

Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development

We report that the alternatively spliced isoforms of nonmuscle myosin heavy chain II-B (NHMC II-B) play distinct roles during mouse brain development. The B1-inserted isoform of NMHC II-B, which contains an insert of 10 amino acids near the ATP-binding region (loop 1) of the myosin heavy chain, is involved in normal migration of facial neurons. In contrast, the B2-inserted isoform, which contains an insert of 21 amino acids near the actin-binding region (loop 2), is important for postnatal development of cerebellar Purkinje cells. Deletion of the B1 alternative exon, together with reduced expression of myosin II-B, results in abnormal migration and consequent protrusion of facial neurons into the fourth ventricle. This protrusion is associated with the development of hydrocephalus. Restoring the amount of myosin II-B expression to wild-type levels prevents these defects, showing the importance of total myosin activity in facial neuron migration. In contrast, deletion of the B2 alternative exon results in abnormal development of cerebellar Purkinje cells. Cells lacking the B2-inserted isoform show reduced numbers of dendritic spines and branches. Some of the B2-ablated Purkinje cells are misplaced in the cerebellar molecular layer. All of the B2-ablated mice demonstrated impaired motor coordination.     

Remodeling of retinal ganglion cell dendrites in the absence of action potential activity

The dendrites of ganglion cells in the retina have an excess number of spines and branches that are normally lost during the first postnatal month of development. We investigated whether this dendritic remodeling can be prevented when the action potential activity of ganglion cells is abolished by chronic intraocular injections of tetrodotoxin (TTX) during the first 4 or 5 postnatal weeks in the cat. Dendritic tree morphologies of alpha and beta ganglion cells from TTX-treated, non-TTX-treated (contralateral eye), and normal control retinae were compared after intracellular filling with Lucifer yellow. Qualitative observations and quantitative measurements indicate that TTX treatment does not prevent the normally occurring loss of spines and dendritic branches. Indeed, the dendritic trees of both alpha and beta cells in TTX injected eyes actually have even fewer spines and branches than normal cells at equivalent ages. However, because the total dendritic lengths of these cells are also reduced after TTX blockade, spine density is indistinguishable from untreated animals at the same age. In addition, although dendritic field areas are not altered with treatment, the complexity of the dendritic trees is reduced. These observations suggest that dendritic remodeling can occur in the absence of ganglion cell action potential activity. Thus, the factors that influence the dendritic and axonal development of retinal ganglion cells must differ, because similar TTX treatment during the period of axonal remodeling does have profound effects on the final pattern of terminal arborizations.     

Remodeling of retinal ganglion cell dendrites in the absence of action potential activity.

The dendrites of ganglion cells in the retina have an excess number of spines and branches that are normally lost during the first postnatal month of development. We investigated whether this dendritic remodeling can be prevented when the action potential activity of ganglion cells is abolished by chronic intraocular injections of tetrodotoxin (TTX) during the first 4 or 5 postnatal weeks in the cat. Dendritic tree morphologies of alpha and beta ganglion cells from TTX-treated, non-TTX-treated (contralateral eye), and normal control retinae were compared after intracellular filling with Lucifer yellow. Qualitative observations and quantitative measurements indicate that TTX treatment does not prevent the normally occurring loss of spines and dendritic branches. Indeed, the dendritic trees of both alpha and beta cells in TTX injected eyes actually have even fewer spines and branches than normal cells at equivalent ages. However, because the total dendritic lengths of these cells are also reduced after TTX blockade, spine density is indistinguishable from untreated animals at the same age. In addition, although dendritic field areas are not altered with treatment, the complexity of the dendritic trees is reduced. These observations suggest that dendritic remodeling can occur in the absence of ganglion cell action potential activity. Thus, the factors that influence the dendritic and axonal development of retinal ganglion cells must differ, because similar TTX treatment during the period of axonal remodeling does have profound effects on the final pattern of terminal arborizations.     

CEREBELLAR ALTERATIONS IN THE WEAVER MOUSE

The fine structure of the cerebellum of weaver mouse was examined and the paucity of granule cells and their axons, the parallel fibers, was confirmed. Unexpectedly, however, the dendritic spines of the Purkinje cells which, in normal animals, are the postsynaptic mates of the parallel fibers, were present. Furthermore, their essential morphology and their staining reactions were indistinguishable from those of the Purkinje cell dendritic spines in normal animals. Possible mechanisms of development are discussed.     

A golgi study of cell types in the dentate gyrus of the adult human brain

Summary 1. The morphology of neurons in the dentate gyrus of the adult human brain was analyzed with two variants of Golgi technique.2. About 20 neuronal types and subtypes were observed in the dentate gyrus of the adult human, several of which had not previously been described in the human. The human dentate gyrus harbors 4 types of neurons in the molecular layer, 3 types within the granule cell layer, and at least 10 types in the hilus.3. Compared to the granule neurons in the rat brain, human granule neurons show a much greater variability. Many of these human neurons have basal dendrites and/or axonal spines. Also, there are significant differences among these neurons regarding the density of their dendritic trees and dendritic spines. In contrast to the rat, human hilar neurons with complex spines have complex spines not only on their dendrites but also on their cell bodies.4. This study opens the door for further morphological studies involving specific diseases such as Alzheimer's disease and epilepsy.     

Inductive role of mossy fibers of hippocampus in the development of dendritic spines in aberrant synaptogenesis at neurotransplantation

The dentate fascia of the hippocampal formation isolated from 20-day-old Wistar rat fetuses was subjected to heterotopic transplantation into the somatosensory area of the neocortex of adult rats of the same strain. Five months after surgery, neurotransplantates, together with neighboring area of the neocortex, were studied using light and electron microscopy. We carried out a detailed study of the ultrastructure of the ectopic synaptic endings formed by the axons of granular neurons of the dentate fascia (mossy fibers) with neurons of the neocortex unusual for them in a normal state. Ultrastructural analysis revealed that most ectopic synaptic endings produce its determinant morphological features: giant sizes of presynaptic knobs, active zones with branched dendritic spines, and adherens junctions with the surface of dendrites. The data indicate that the mossy fibers growing from neurotransplantates induce structural and chemical reorganization of dendrites of the neocortex using transmembrane adherens junctions, such as puncta adherentia junctions. This results in the differentiation of active zones and development of dendritic spines typical for giant synaptic endings that are invaginated into presynaptic endings. Thus, the ability of neurons of the dentate fascia to form aberrant synaptic connections at transplantation results from the inductive synaptogenic properties of mossy fibers.     

Targeting of proteins of the striatin family to dendritic spines: role of the coiled-coil domain

Striatin, SG2NA and zinedin, the three mammalian members of the striatin family are multimodular WD-repeat, calmodulin and calveolin-binding proteins. These scaffolding proteins, involved in both signaling and trafficking, are highly expressed in neurons. Using ultrastructural immunolabeling, we showed that, in Purkinje cells and hippocampal neurons, SG2NA is confined to the somatodendritic compartment with the highest density in dendritic spines. In cultured hippocampal neurons, SG2NA is also highly concentrated in dendritic spines. By expressing truncated forms of HA-tagged SG2NAbeta, we demonstrated that the coiled-coil domain plays an essential role in the targeting of SG2NA within spines. Furthermore, co-immunoprecipitation experiments indicate that this coiled-coil domain is also crucial for the homo- and hetero-oligomerization of these proteins. Thus, oligomerization of the striatin family proteins is probably an obligatory step for their routing to the dendritic spines, and hetero-oligomerization explains why all these proteins are often co-expressed in the neurons of the rat brain and spinal cord.     

Destabilization of cortical dendrites and spines by BDNF.

Particle-mediated gene transfer and two-photon microscopy were used to monitor the behavior of dendrites of individual cortical pyramidal neurons coexpressing green fluorescent protein (GFP) and brain-derived neurotrophic factor (BDNF). While the dendrites and spines of neurons expressing GFP alone grew modestly over 24-48 hr, coexpressing BDNF elicited dramatic sprouting of basal dendrites, accompanied by a regression of dendritic spines. Compared to GFP-transfected controls, the newly formed dendrites and spines were highly unstable. Experiments utilizing Trk receptor bodies, K252a, and overexpression of nerve growth factor (NGF) demonstrated that these effects were mediated by secreted BDNF interacting with extracellular TrkB receptors. Thus, BDNF induces structural instability in dendrites and spines, which, when restricted to particular portions of a dendritic arbor, may help translate activity patterns into specific morphological changes.     

The rat olivocerebellar system visualized in detail with anterograde PHA-L tracing technique, and sprouting of climbing fibers demonstrated after subtotal olivary lesions

The rat olivocerebellar climbing fiber system has been investigated at the light and electron microscopic level with anterograde Phaseolus vulgaris leucoagglutinin (PHA-L) tracing. From PHA-L Injections in different parts of the inferior olive labelled axons could be traced to the contralateral cerebellum. Arriving in the deep cerebellar white matter, the olivocerebellar axons ran around and through the cerebellar nuclei. Plexuses of labelled terminal fibers appeared in the cerebellar nuclei, and the density of this innervation was estimated to 1-4 million varicosities per mm3. Ultrastructurally, these boutons engaged in asymmetric synapses with small dendrites. Bundles of labelled fibers continued into the folial white matter, and terminated as climbing fibers in sagittal zones of the cerebellar cortex. Both the cortical and nuclear terminations of the olivocerebellar system are strictly topographically organized. The plasticity of climbing fibers was studied after partial lesions of the inferior olive induced by 3-acetylpyridine. One to 6 months after the lesion, surviving climbing fibers demonstrated extensive sprouting. The newly formed axons originated from parent climbing fiber plexuses, grew in the direction of parallel fibers, and formed terminal plexuses around several neighbouring Purkinje cells. As normal climbing fiber terminals, these terminals formed asymmetric synapses with spines of proximal Purkinje cell dendrites, and evidence by Benedetti et al. (1983) shows that the regenerated innervation is electrophysiologically functional. It is suggested that denervated Purkinje cells release a trophic substance, which stimulate surviving climbing fibers to sprouting, axonal growth and synapse formation.     

Fast extraction of neuron morphologies from large-scale SBFSEM image stacks

Neuron morphology is frequently used to classify cell-types in the mammalian cortex. Apart from the shape of the soma and the axonal projections, morphological classification is largely defined by the dendrites of a neuron and their subcellular compartments, referred to as dendritic spines. The dimensions of a neuron’s dendritic compartment, including its spines, is also a major determinant of the passive and active electrical excitability of dendrites. Furthermore, the dimensions of dendritic branches and spines change during postnatal development and, possibly, following some types of neuronal activity patterns, changes depending on the activity of a neuron. Due to their small size, accurate quantitation of spine number and structure is difficult to achieve (Larkman, J Comp Neurol 306:332, 1991). Here we follow an analysis approach using high-resolution EM techniques. Serial block-face scanning electron microscopy (SBFSEM) enables automated imaging of large specimen volumes at high resolution. The large data sets generated by this technique make manual reconstruction of neuronal structure laborious. Here we present NeuroStruct, a reconstruction environment developed for fast and automated analysis of large SBFSEM data sets containing individual stained neurons using optimized algorithms for CPU and GPU hardware. NeuroStruct is based on 3D operators and integrates image information from image stacks of individual neurons filled with biocytin and stained with osmium tetroxide. The focus of the presented work is the reconstruction of dendritic branches with detailed representation of spines. NeuroStruct delivers both a 3D surface model of the reconstructed structures and a 1D geometrical model corresponding to the skeleton of the reconstructed structures. Both representations are a prerequisite for analysis of morphological characteristics and simulation signalling within a neuron that capture the influence of spines.     

Morphological characteristics of various segments of local connections in the rat somatosensory cortex

Pyramidal, aspinous, sparsely-spinous bipolar and multipolar neurons of the rat sensomotor cerebral cortex, impregnated after Golgi method, have been studied at an electron microscopical level. The ultrastructural characteristics of the pyramidal neurons differs from that of the nonpyramidal cells. Distribution of various synaptic contacts on the cellular surface and cortical postsynaptic targets of the axonal arborizations of the neurons are revealed. On the body of the pyramidal cells only symmetrical synapses exist, on large dendritic trunks symmetrical synapses prevail, on the spines and the terminal dendritic branches assymetrical synapses mainly predominate. Axonal collateralies of the pyramidal cells form asymmetrical synapses on the spines, small and middle dendrites. There are more axo-somatic synapses on the bodies of the nonpyramidal neurons than on the pyramidal cells, among them both symmetrical and asymmetrical types of the synapses occur. On the trunks and small dendrites of the nonpyramidal cells both types of synaptic contacts are revealed. In the distal direction of the dendrites the number of the asymmetrical synapses becomes predominating. Axons of the bipolar cells form asymmetrical synapses on the spines, small and middle dendrites. Axons of the multipolar cells form symmetrical synapses on the dendrites and the dendritic trunks of the nondifferentiated cells. Differences in the distribution character of the synaptic inlets and various postsynaptic targets of the axonal systems in the cells assume various functional role of the identified neurons.     

Golgi-type I and Golgi-type II Neurons in the Ventral Anterior Thalamic Nucleus of the Adult Human: Morphological Features and Quantitative Analysis

The morphological and quantitative features of neurons in the adult human ventral anterior thalamic nucleus were studied in Golgi preparations. Two neuronal types were found and their quantitative features were studied. Golgi-type I neurons were medium to large cells with dense dendritic trees and dendritic protrusions and short hair-like appendages. They have somatic mean diameter of 30.8 μm (±9.4, n = 85). They have an average 100.3 dendritic branches, 48.97 dendritic branching points, and 58.85 dendritic tips. The mean diameters of their primary, secondary, and tertiary dendrites were 3.1 μm (±1, n = 80), 1.85 μm (±0.8, n = 145), and 1.5 μm (±0.4, n = 160), respectively. Golgi-type II neurons were small to medium cells with few sparsely branching dendrites and dendritic stalked appendages with or without terminal swellings. They have somatic mean diameters of 22.2 μm (±5.8, n = 120). They have an average 33.76 dendritic branches, 16.49 dendritic branching points, and 21.97 dendritic tips. The mean diameters of their primary, secondary, and tertiary dendrites were 1.6 μm (±0.86, n = 70), 1.15 μm (±0.55, n = 118), and 1 μm (±0.70, n = 95), respectively. These quantitative data may form the basis for further quantitative studies involving aging or some degenerative diseases that may affect cell bodies and/or dendritic trees of the Golgi-type I and/or Golgi-type II thalamic neurons.     

Fluorescent Labeling of Newborn Dentate Granule Cells in GAD67-GFP Transgenic Mice: A Genetic Tool for the Study of Adult Neurogenesis

Neurogenesis in the adult hippocampus is an important form of structural plasticity in the brain. Here we report a line of BAC transgenic mice (GAD67-GFP mice) that selectively and transitorily express GFP in newborn dentate granule cells of the adult hippocampus. These GFP⁺ cells show a high degree of colocalization with BrdU-labeled nuclei one week after BrdU injection and express the newborn neuron marker doublecortin and PSA-NCAM. Compared to mature dentate granule cells, these newborn neurons show immature morphological features: dendritic beading, fewer dendritic branches and spines. These GFP⁺ newborn neurons also show immature electrophysiological properties: higher input resistance, more depolarized resting membrane potentials, small and non-typical action potentials. The bright labeling of newborn neurons with GFP makes it possible to visualize the details of dendrites, which reach the outer edge of the molecular layer, and their axon (mossy fiber) terminals, which project to the CA3 region where they form synaptic boutons. GFP expression covers the whole developmental stage of newborn neurons, beginning within the first week of cell division and disappearing as newborn neurons mature, about 4 weeks postmitotic. Thus, the GAD67-GFP transgenic mice provide a useful genetic tool for studying the development and regulation of newborn dentate granule cells.     

Scanning electron microscopy of human cerebellar cortex

Summary Scanning electron microscopy and cryofracture technique were applied to study neuronal architecture and synaptic connections of the human cerebellum. Samples were processed according to the technique of Humphreys et al. (1975) with minor modifications. The granule cells exhibit unbranched filiform axons and coniform dendritic processes. The latter show typical claw-like endings making gearing type synaptic contacts with mossy fiber rosettes. The unattached mossy rosettes appear as solid club-like structures. Some fractographs show individual granule cells, Golgi neurons and glomerular islands. The climbing fibers and their Scheibel's collaterals were also characterized. In the Purkinje layer the surface fracture was produced at the level of the Bergmann glial cells, which are selectively removed, allowing us to visualize the rough surface of Purkinje cells and the supra- and infraganglionic plexuses of basket cell axons which appeared as entangled threads. In the molecular layer the three-dimensional configuration of the Purkinje secondary and tertiary dendritic branches was obtained. The filiform parallel fibers make cruciform synaptic contacts with the Purkinje dendritic spines. The appearance of stellate neuronal somata closely resembled that of the granule cells. The subpial terminals of Bergmann fibers appeared attached to the exterior of the folia forming the rough surfaced external glial limiting membrane.     

The actin binding domain of βI-spectrin regulates the morphological and functional dynamics of dendritic spines

Actin microfilaments regulate the size, shape and mobility of dendritic spines and are in turn regulated by actin binding proteins and small GTPases. The βI isoform of spectrin, a protein that links the actin cytoskeleton to membrane proteins, is present in spines. To understand its function, we expressed its actin-binding domain (ABD) in CA1 pyramidal neurons in hippocampal slice cultures. The ABD of βI-spectrin bundled actin in principal dendrites and was concentrated in dendritic spines, where it significantly increased the size of the spine head. These effects were not observed after expression of homologous ABDs of utrophin, dystrophin, and α-actinin. Treatment of slice cultures with latrunculin-B significantly decreased spine head size and decreased actin-GFP fluorescence in cells expressing the ABD of α-actinin, but not the ABD of βI-spectrin, suggesting that its presence inhibits actin depolymerization. We also observed an increase in the area of GFP-tagged PSD-95 in the spine head and an increase in the amplitude of mEPSCs at spines expressing the ABD of βI-spectrin. The effects of the βI-spectrin ABD on spine size and mEPSC amplitude were mimicked by expressing wild-type Rac3, a small GTPase that co-immunoprecipitates specifically with βI-spectrin in extracts of cultured cortical neurons. Spine size was normal in cells co-expressing a dominant negative Rac3 construct with the βI-spectrin ABD. We suggest that βI-spectrin is a synaptic protein that can modulate both the morphological and functional dynamics of dendritic spines, perhaps via interaction with actin and Rac3.     

Morphological characteristics of different types of neurons in the ventrooralis anterior,ventrooralis internus,and ventrooralis posterior nuclei in the human thalamus

1. Golgi-Kopsch preparations of the oral ventral nuclei of human thalamus were analyzed in an attempt to classify the neuronal types. 2. Three types of neurons are described for the first time in humans. Type I neurons are large or medium in size and bear dendrites with protrusions, spines, and short hair-like appendages. Some have a radiate dendritic arbor and others have dendrites grouped in tufts. The dendritic trees of these neurons are dense. 3. Type II neurons are medium or small in size with less dense dendritic trees. These cells have somatic as well as dendritic appendages of different forms. 4. Relatively rare is a type of very small neurons, type III, with few and sparsely branching dendrites.