The development of the inferior olivary complex in preweanling opossums

Summary Pre- and postsynaptic elements within the developing inferior olive (IO) of both control and experimental opossums were examined via electron microscopy. Electron dense boutons identified di-/mesencephalic, cerebellar and spinal afferents within the IO of 8–71 day old animals, which survived 4–48 hours following either midbrain hemisections or spinal transections.During its initial stage of development (3–22 days) the neuropil of the IO is segregated into fields of small diameter neurites or flocculent profiles. Within the fields of flocculent profiles, synaptic interactions are established, which are both infrequent and immature. Although some flocculent profiles are presynaptic, most are postsynaptic and emanate from olivary somata and dendrites. Synaptic contacts also occur with olivary somata, dendritic shafts, spines and dendritic varicosities. Clear round vesicles (crv's; 40 m) predominate within all boutons, normal ones as well as those which degenerate after di-/mesencephalic, cerebellar and spinal lesions; however, larger (70 m) dense cored vesicles (dcv's) are occasionally observed within some boutons. Degenerating terminals from all three sources primarily contact flocculent profiles and dendritic shafts.As the opossum matures (42 days) dramatic increases occur in the number and complexity of both pre- and postsynaptic elements. Marked variations are observed in the matrix density of dendritic shafts. Although all terminal boutons predominantly contain crv's, the number of dcv's within the population of presynaptic elements increases markedly. Concurently, olivary neurons are profusely studded with spines. Simple dendritic spines and spiny appendages as well as dendritic shafts are the most frequent postsynaptic structures within the principal nucleus (PO). Olivary somata and their spines, however, are postsynaptic to degenerating de-/mesencephalic afferents within the PO. Flocculent profiles, which persist within the accessory nuclei, and dendritic shafts are postsynaptic to degenerating spinal boutons.By 70 days of age synaptic contacts appear more mature and more nearly approximate those seen in the adult (King 1980). Few somatic contacts, opaque dendrites, dendritic varicosities, and flocculent profiles are evident within the PO. Dendritic shafts and spines are the principal postsynaptic structures. Many di-/mesencephalic and cerebellar afferents synapse within maturing synaptic clusters on spines between which a rare gap junction is observed. Other di-/mesencephalic and cerebellar endings in the PO as well as spinal endings in the accessory nuclei are presynaptic to dendritic shafts and spines external to synaptic clusters. This predilection for contacting more specific loci on olivary neurons provides good evidence for synaptic remodeling.As the olivary nuclei develop further, the incidence of gap junctions increases and pleomorphic vesicles appear within boutons. The glial investment of neuronal elements, including synaptic clusters, also becomes more extensive.In conclusion, early di-/mesencephalic, cerebellar and spinal synaptic contacts appear qualitatively uniform in their synaptic features and postsynaptic interactions. As olivary development proceeds, however, the distinguishing synaptic features of the nuclear complex become more apparent. Synaptic remodeling occurs as some midbrain and cerebellar terminals are localized within synaptic clusters. The ultrastructural features characteristic of the adult IO are finally achieved by 80 days of age.This research was supported by N.I.H. Research Grant NS-08798     

Three-dimensional analysis of dendritic spines

Summary The glial envelope of dendritic spines in the visual and cerebellar cortices was evaluated by analysis of serial sections. Three-dimensional reconstructions of the protoplasmic astrocyte processes were made and the quantitative proportions of the glial cover on dendritic spines on spiny branchlets of Purkinje cells are, with the exception of afferent axon terminals, completely covered by the glial sheath (74.44%), dendritic spines of pyramidal cells are only partially covered (28.89%), so that spine stalks and even synaptic clefts frequently lack glial isolation. A new, relatively frequent configuration of subsurface cistern-astrocyte process — dendritic spine is described. A possible functional significance of the differences in the glial ensheathment of dendritic spines in visual and cerebellar cortices is discussed.     

Development and fine structure of murine Purkinje cells in dissociated cerebellar cultures: neuronal polarity

 Cerebellar Purkinje cells (PC) display a highly distinctive form of polarity. We have cultured murine PCs from dissociated E16 cerebellar anlagen for 1 week to investigate the early stages of neuronal compartmentalization and synaptic interactions, features which are important for the establishment of neuronal polarity. To unequivocally identify the PCs we utilized light and electron microscopic immunocytochemistry with an antiserum to the cell class-specific marker L7/pcp2 gene product. The PCs typically show a single, long axon, numerous short appendages classified as filopodia and protospines, and a small number of protodendrites. The nucleus is positioned asymmetrically in both the horizontal and vertical axes of the soma. The Golgi apparatus, coated and uncoated vesicles, and mitochondria are prominent ultrastructural features, while the endoplasmic reticulum is highly fragmented. The cell body receives rudimentary synapses on its smooth surfaces and appendages and no consistent morphological differences were detected between these elementary contacts. The axon is clearly identifiable; it emanates from either the cell body or a protodendrite, bifurcates at predominantly right angles, forms beaded collaterals, and terminates with relatively large growth cones. The varicosities of the PC axon contain pleomorphic synaptic vesicles and form rudimentary synapses primarily with the dendritic shafts of immunonegative neurons. The protodendrites are short, quickly tapering and sparsely branched; they emit numerous filopodia and immature spines and terminate with small growth cones. Rudimentary synapses are received on the proximal dendritic shafts and filopodia, and more mature synapses occur frequently on protospines. With few exceptions, PCs lie atop an astrocytic bed layer and glial processes are apposed to the various aspects of the PC body left free by the afferent axons. By contrast, PC processes are largely free of glial sheaths. We conclude that the ”stellate stage” of PC development in situ is replicated rather faithfully in culture and that PCs have established polarity and have begun to form intercellular contacts by 1 week in vitro. Moreover, the PCs are already morphologically distinct from other cell types in the 1-week cultures, although they have yet to develop the differentiated features that distinguish mature PCs. Accepted: 30 June 1997     

The postsynaptic targets of substance P-immunoreactive terminals in the rat neostriatum with particular reference to identified spiny striatonigral neurons

Summary Substance P-immunoreactive boutons were examined in the electron microscope in sections of the rat neostriatum that contained retrogradely labelled striatonigral neurons and/or Golgi-impregnated medium-size densely spiny neurons. The postsynaptic targets of the immunoreactive boutons were characterized on the basis of ultrastructural features, their projection to the substantia nigra and/or their somato-dendritic morphology. Substance P-immunoreactive axonal boutons formed symmetrical synaptic specializations. Of a total of 233 randomly identified synaptic boutons 72.5% made contact with dendritic shafts, 15% with dendritic spines and 10.7% with perikarya. The ultrastructural characteristics of some of the postsynaptic neuronal perikarya were consistent with their identification as striatal interneurons. Similarly, the observation of some of the substance P-containing terminals in contact with spines, spine-bearing dendritic shafts and perikarya with the ultrastructural characteristics of medium-size densely spiny neurons suggested that one of the targets of substance P-positive terminals are striatal projection neurons. Direct evidence for this was obtained in sections from rats that had received injections of horseradish peroxidase conjugated with wheatgerm agglutinin in the substantia nigra. The perikarya of retrogradely labeled striatonigral neurons were found to receive symmetrical synaptic input from substance P-positive boutons. Ultrastructural analysis of Golgi-impregnated medium-size densely spiny neurons, some of which were also retrogradely labeled from the substantia nigra, demonstrated directly that this class of neuron was postsynaptic to the substance P-immunoreactive boutons. The combination of Golgi-impregnation with substance P-immunocytochemistry made it possible to study the pattern or topography of the substance P-positive input to medium size densely spiny neurons. The substance P-containing boutons made contact predominantly with perikarya and dendritic shafts. This pattern of input is markedly different from that of other identified inputs to medium-size densely spiny neurons.     

Dendritic arborization and spines of the neurons of the cat and human periaqueductal gray: A light,confocal laser scanning,and electron microscope study

Neurons of the periaqueductal gray (PAG) have an extensive dendritic tree which plays an important role in the neuronal circuits supporting the functional activities of this region. The complexity of the local circuits is increased by the occurrence of dendritic spines. We have compared the dendritic and spine organization in the cat with that of man in order to verify whether an inverse relationship exists between dendritic tree extension and spine density and complexity. Sections of cat and human PAG prepared according to the Golgi-Cox method were studied with the conventional light microscope (LM) and the confocal laser scanning microscope (CLSM). The cat PAG was also studied at the electron microscopic level. The light microscopic study provided the morphoquantitative characteristics of the dendritic arborization and spines of the multipolar and fusiform neurons of the human and cat PAG. The CLSM methodology, thanks to the three-dimensional reconstruction of the neurons and the rotation of the reconstructed images, brought into view dendritic branches and spines that could not have been observed at the LM, thereby showing a wider dendritic tree and more numerous spines. The data combined from LM and CLSM demonstrate that in both species most spiny neurons are multipolar and probably projection neurons. In man, the multipolar neurons show a more extensive dendritic tree due to a wider secondary ramification, which would seem to be balanced by more numerous spines in cat. At the electron microscopic level, axo-dendritic synapses are numerous and show symmetrical and asymmetrical junctions in equal proportions; furthermore, the great majority of the spines are in contact with synaptic boutons which contain round vesicles and make predominantly asymmetrical contacts features which indicate excitatory activity. The combined use of different techniques gave a complete picture of the dendritic tree and spines of the neurons of human and cat PAG and showed a wider dendritic surface available for the receipt of the synaptic contacts than had been reported previously. Furthermore, our findings demonstrate that the PAG dendritic spines are important and specific structures in the synaptic complex of the neuropil, suggesting that they might create a local device to modulate and integrate the afferent inputs, probably in an excitatory way. The differences observed in the two species suggest that afferent information might be handled in different ways in human and cat PAG. Anat. Rec. 251:316–325, 1998. © 1998 Wiley-Liss, Inc. © 1998 Wiley-Liss, Inc.     

Synaptic relationships of a type of GABA-immunoreactive neuron (clutch cell), spiny stellate cells and lateral geniculate nucleus afferents in layer IVC of the monkey striate cortex

The precise stimulus specificity of striate cortical neurons is strongly influenced by processes involving gamma-aminobutyric acid (GABA). In the visual cortex of the monkey most afferents from the lateral geniculate nucleus terminate in layer IVC. We identified a type of smooth dendritic neuron (clutch cell) that was immunoreactive for GABA, and whose Golgi-impregnated dendrites and axon were largely restricted to layer IVC beta. The slightly ovoid somata were 8-12 micron by 12-15 micron and the dendritic field was often elongated, extending 80-200 micron in one dimension. The axonal field was 100-150 micron in diameter and densely packed with large bulbous boutons. Although mainly located in IVC beta both the dendritic and axonal processes entered IVC alpha. Fine structural features of GABA-immunoreactive and-impregnated clutch cells and impregnated spiny stellate cells were compared. Clutch cells had more cytoplasm, more densely packed mitochondria and endoplasmic reticulum, and made type II as opposed to type I synapses. A random sample of 159 elements postsynaptic to three clutch cells showed that they mainly terminated on dendritic shafts (43.8-58.5%) and spines (20.8-46.3%), rather than somata (10-17%). The majority of the postsynaptic targets were characteristic of spiny stellate cells. This was directly demonstrated by studying synaptic contacts between an identified GABA positive clutch cell and the dendrites and soma of an identified spiny stellate cell. The termination of clutch cells mainly on dendrites and spines of spiny stellate cells suggests that they interact with other inputs to the same cells. Following an electrolytic lesion in the ipsilateral lateral geniculate nucleus we examined the distribution of degenerating terminals on three identified spiny stellate neurons in layer IVC beta. Out of eight synapses from the lateral geniculate nucleus one was on a dendritic shaft, the rest on spines. Only a small fraction of all synapses on the cells were from degenerating boutons. A clutch cell within the area of dense terminal degeneration was not contacted by terminals from the lateral geniculate nucleus. The results show that layer IVC in the monkey has a specialized GABAergic neuron that terminates on spiny stellate cells monosynaptically innervated from the lateral geniculate nucleus. Possible functions of clutch cells may include inhibitory gating of geniculate input to cortex; maintenance of the antagonistic subregions in the receptive fields; and the creation from single opponent of double colour opponent receptive fields.     

Ultrastructure of degenerating cerebellothalamic terminals in the ventral medial nucleus of the cat

Summary Terminal degeneration of cerebellar afferents in the ventral medial thalamic nucleus (VM) was studied in cats at the ultrastructural level after uni- or bilateral lesions in the brachium conjunctivum (BC). To achieve discrete lesions within the BC, a new very accurate stereotaxic technique was used. Numerous large terminals belonging to a population of so-called LR boutons were observed degenerating in the VM. The boutons displayed a wide variety of degenerative changes. Some revealed the features of the classical neurofilamentous type of degeneration. Others, although containing a slightly increased number of neurofilaments, featured much more prominently large numbers of coated vesicle shells and heavy accumulations of a flocculent electrondense material. Degeneration in a third group of boutons similar to some extent to the light type of degeneration was characterized by tight clumping of enormously swollen or distorted synaptic vesicles within a light matrix. At later stages, however, all these boutons were believed to become shrunken and electron-dense since intermediate stages between the light- and dark-appearing boutons were observed. The degenerating cerebellar boutons formed asymmetrical synaptic contacts. Groups of 3 or 4 boutons terminated upon dendrites of projection neurons synapsing more frequently on spines than on dendritic stems. The synaptic contacts between cerebellar boutons and the vesicle-containing dendrites of local circuit neurons were encountered as often if not more than the contacts on projection neuron dendrites. Triads consisting of cerebellar boutons and dendrites of both types of neurons were observed very regularly. This synaptic arrangement provides the anatomical basis for the modification of cerebellar input in the VM by interneurons.     

Tyrosine hydroxylase-immunoreactive boutons in synaptic contact with identified striatonigral neurons, with particular reference to dendritic spines

Tyrosine hydroxylase-immunoreactive fibres in the rat neostriatum were studied in the electron microscope in order to determine the nature of the contacts they make with other neural elements. The larger varicose parts of such fibres contained relatively few vesicles and rarely displayed synaptic membrane specializations; however, thinner parts of axons (0.1-0.4 micron) contained many vesicles and had symmetrical membrane specializations, indicative of en passant type synapses. By far the most common postsynaptic targets of tyrosine hydroxylase-immunoreactive boutons were dendritic spines and shafts, although neuronal cell bodies and axon initial segments also received such input. Six striatonigral neurons in the ventral striatum were identified by retrograde labelling with horseradish peroxidase and their dendritic processes were revealed by Golgi impregnation using the section-Golgi procedure. The same sections were also developed to reveal tyrosine hydroxylase immunoreactivity and so we were able to study immunoreactive boutons in contact with the Golgi-impregnated striatonigral neurons. Each of the 280 immunoreactive boutons examined in the electron microscope displayed symmetrical synaptic membrane specializations: 59% of the boutons were in synaptic contact with the dendritic spines, 35% with the dendritic shafts and 6% with the cell bodies of striatonigral neurons. The dendritic spines of striatonigral neurons that received input from immunoreactive boutons invariably also received input, usually more distally, from unstained boutons that formed asymmetrical synaptic specializations. A study of 87 spines along the dendrites of an identified striatonigral neuron showed that the most common type of synaptic input was from an individual unstained bouton making asymmetrical synaptic contact (53%), while 39% of the spines received one asymmetrical synapse and one symmetrical immunoreactive synapse. It is proposed that the spatial distribution of presumed dopaminergic terminals in synaptic contact with different parts of striatonigral neurons has important functional implications. Those synapses on the cell body and proximal dendritic shafts might mediate a relatively non-selective inhibition. In contrast, the major dopaminergic input that occurs on the necks of dendritic spines is likely to be highly selective since it could prevent the excitatory input to the same spines from reaching the dendritic shaft. One of the main functions of dopamine released from nigrostriatal fibres might thus be to alter the pattern of firing of striatal output neurons by regulating their input.     

Cerebellar macroneurons in microexplant cell culture: Ultrastructural morphology

Microexplant cell cultures of fetal rat cerebellum contain essentially monolayer networks of Purkinje cells, occasional granule cells and neurons from the deep nuclei. The neurons and occasional filament-packed glial cells develop on top of a sheet of flattened, non-neuronal cells. In the absence of extrinsic input to the cerebellum and greatly reduced numbers of granule cells, the Purkinje cells develop a stunted and non-oriented dendritic arbor similar to that observed in agranular cerebella. The Purkinje cell dendritic branches, however, are spine-covered. Although the spines are not enveloped by glia and are only rarely contacted by a presynaptic bouton, most spines display a patch of electron-dense material resembling a postsynaptic membrane specialization. The Purkinje cells develop synaptic interactions among themselves and with granule cells. The ultrastructural morphology of boutons derived from both Purkinje cells and large neurons of the deep nuclei, identified after intracellular injection of horseradish peroxidase, is consistent with that observed in vivo.The present study indicates that cerebellar Purkinje cells survive and differentiate in a culture system in which individual neurons are accessible for electrophysiological and morphological analyses.     

Morphology of intracellularly stained spiny neurons in rat striatal grafts.

Two to six months after implantation of fetal striatal primordia into the kainic acid-lesioned neostriatum of adult rats, spiny neurons in the grafts were stained intracellularly with biocytin. To determine whether the spiny neurons in the grafts differentiate morphologically as in the host neostriatum, the intracellularly stained spiny neurons in the grafts were studied with light and electron microscopy and compared with that of spiny neurons in the host neostriatum. The spiny neurons in the grafts had ovoid or polygonal cell bodies with dendrites radiating in all directions. The somata were smooth and the dendrites, except for their most proximal portions, were rich in spines. All these features resembled the appearance of spiny neurons in the intact neostriatum. However, quantitative studies showed that the somata of spiny neurons in the grafts were larger than those in the host neostriatum (projected cross-sectional areas of 230 +/- 64.6 microns 2 in the grafts and 158 +/- 28.9 microns 2 in the host) and the spine density of graft neurons was lower than that of host neurons. Cells near the border of the grafts had dendrites extending both into the graft and into the host neostriatum. In these cells, the dendrites in the grafts had fewer spines than the dendrites in the host tissue. The axons of spiny neurons in the grafts had very large and dense intrastriatal collateral arborizations, which occupied a much larger volume than that of the dendritic domain of the parent cells. The local axonal arborizations of each of these cells filled almost the entire graft. In some cells, axonal branches were traced outside the grafts and were seen to enter the internal capsule fascicles. Unlike spiny neurons in the normal adult neostriatum, the spiny cells of the graft could have nuclear indentations. With this exception, the ultrastructural features of spiny neurons in the grafts were very similar to those in the hosts. Many unlabeled boutons made synapses on identified spiny neurons in the grafts. Terminals with small round vesicles made synaptic contacts on dendritic shafts and dendritic spines, while terminals with flattened or pleomorphic vesicles contacted somata, dendrites, and dendritic spines. Labeled axon collaterals of graft neurons made symmetrical synapses on somata, dendrites and spines in the grafts and in the host neostriatum. In the grafts, more than 60% of the axon terminals contacted dendritic shafts. The proportion of axosomatic and axospinous synapses varied substantially from cell to cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiology, morphology and detailed passive models of guinea-pig cerebellar Purkinje cells.

1. Purkinje cells (PCs) from guinea-pig cerebellar slices were physiologically characterized using intracellular techniques. Extracellular caesium ions were used to linearize the membrane properties of PCs near the resting potential. Under these conditions the average input resistance, RN, was 29 M omega, the average system time constant, tau 0, was 82 ms and the average cable length, LN, was 0.59. 2. Three PCs were fully reconstructed following physiological measurements and staining with horseradish peroxidase. Assuming that each spine has an area of 1 micron 2 and that the spine density over the spiny dendrites is ten spines per micrometre length, the total membrane area of each PC is approximately 150,000 microns 2, of which approximately 100,000 microns 2 is in the spines. 3. Detailed passive cable and compartmental models were built for each of the three reconstructed PCs. Computational methods were devised to incorporate globally the huge number of spines into these models. In all three cells the models predict that the specific membrane resistivity, Rm, of the soma is much lower than the dendritic Rm (approximately 500 and approximately 100,000 omega cm2 respectively). The specific membrane capacitance, Cm, is estimated to be 1.5-2 muF cm-2 and the specific cytoplasm resistivity, Ri, is 250 omega cm. 4. The average cable length of the dendrites according to the model is 0.13 lambda, suggesting that under caesium conditions PCs are electrically very compact. Brief somatic spikes, however, are expected to attenuate 30-fold when spreading passively into the dendritic terminals. A simulated 200 Hz train of fast, 90 mV somatic spikes produced a smooth 12 mV steady depolarization at the dendritic terminals. 5. A transient synaptic conductance increase, with a 1 nS peak at 0.5 ms and a driving force of 60 mV, is expected to produce approximately 20 mV peak depolarization at the spine head membrane. This EPSP then attenuates between 200- and 900-fold into the soma. Approximately 800 randomly distributed and synchronously activated spiny inputs are required to fire the soma. 6. The passive model of the PC predicts a poor resolution of the spatio-temporal pattern of the parallel fibre input. An equally sized, randomly distributed group of approximately 1% of the parallel fibres, activated within a time window of a few milliseconds, would result in approximately the same composite EPSP at the soma.     

Membrane structure at synaptic junctions in area CA1 of the rat hippocampus

In tissue from area CA1 of the rat hippocampus prepared for electron microscopic study by thin-sectioning, asymmetric synaptic junctions were found on dendritic spines, spiny dendritic shafts, and non-spiny dendritic shafts. In freeze-fractured preparations, aggregates of large particles were found on the extracellular half of the postsynaptic membrane at each of these synaptic junctions. Particle aggregate areas were measured and particle packing densities were computed at dendritic spine synapses and dendritic shaft synapses in area CA1, and compared to similar measures of particle aggregates on dendritic spines of cerebellar Purkinje cells. All of these CA1 and cerebellar synapses are excitatory and are thought to use glutamate as a neurotransmitter. There was a tendency for the dispersion of particles within individual aggregates to be less uniform in larger aggregates in both area CA1 and cerebellar cortex. Distinct particle-free zones could be distinguished in the center of particle aggregates on large "mushroom-shaped" spines in area CA1. There was no statistically significant difference between the particle densities at CA1 dendritic spines (2848 +/- 863 particles/micron2) and CA1 dendritic shafts (2707 +/- 718 particles/micron2). However, the average density of particles at cerebellar dendritic spine synapses (3614 +/- 1081 particles/micron2) was significantly greater than at dendritic spine or shaft synapses found in area CA1. Symmetric synaptic junctions were observed on the CA1 pyramidal cell somas and dendritic shafts in thin-sectioned preparations. These synapses typically exert an inhibitory action mediated by gamma-aminobutyric acid. In freeze-fracture preparations, large varicosities were found apposed to the pyramidal somal and dendritic membranes, but there were no specializations of particle distribution on either the extracellular or the cytoplasmic half of the fractured postsynaptic membranes. This finding parallels observations from freeze-fracture preparations of other GABAergic synapses in the central nervous system.     

Does bouton morphology optimize axon length?

The total length of cortical axons could be reduced if the parent axons maintained straight trajectories and simply connected to dendritic shafts via spine-like terminaux boutons and to dendritic spines via bead-like en passant boutons. Cortical axons from cat area 17 were reconstructed from serial electron micrographs and their bouton morphology was correlated with their synaptic targets. En passant or terminaux boutons did not differ in the proportion of synapses they formed with dendritic spines and shafts, and thus, the two morphological variants of synaptic bouton do not contribute directly to optimizing axon length.     

Termination of cortical afferents on identified neurons in the caudate nucleus of the cat

Summary A combined Golgi/electron microscopic technique was used to investigate the fine structure and synaptology of Golgi-stained spiny neurons in the caudate nucleus of the cat. In order to study the termination sites of cortical afferents on Golgistained spiny neurons, cortical fibres were caused to degenerate by making extensive cortical lesions 3 days prior to fixation of the animals.When examined in the electron microscope, perikarya of labelled spiny neurons have a round nucleus, a few mitochondria and microtubules, and a poorly developed Golgi apparatus and rough endoplasmic reticulum. Only rarely are axo-somatic contacts seen. Labelled dendrites exhibit a moderate number of microtubules and sometimes elongated mitochondria. Numerous labelled spines are seen in the vicinity of their parent dendrites. They are contacted by smaller type I and type III boutons and larger type IV boutons (Hassler et al. 1978). Large boutons filled with clear round vesicles establish symmetric contacts with labelled dendritic shafts.Degenerating boutons of cortical afferents are seen in contact with spines and, more rarely, with dendritic shafts of Golgi-stained spiny neurons. All degenerating boutons synapsing with labelled structures are found some distance from the cell body. No contacts of degenerating cortical boutons with the soma or with stem dendrites of Golgi-stained spiny neurons are found.     

Ultrastructural organization of slice cultures from rat visual cortex

Summary We have been studying the fine structural organization of slice cultures prepared from the visual cortex of 6-day-old rats and cultured for 2 weeks using a roller culture technique. Neurons in culture exhibited the characteristic cytological differences between perikarya, axons and dendrites. Neuronal and glial processes formed a dense neuropil with minimal extracellular spaces, and within the neuropil there were numerous synaptic contacts. Both morphological types of cortical synapses, type I (asymmetrical) and type II (symmetrical) could be readily identified in slice cultures. The pattern of synaptic connections in culture was remarkably similar to that observed in normal cerebral cortex: asymmetrical synapses were usually found in contact with dendritic spines, less frequently with dendritic shafts, and never on perikarya, whereas symmetrical synapses were found mostly on perikarya, occasionally on dendritic shafts but never on dendritic spines. Synaptic morphology appeared mature after 2 weeksin vitro and did not show the immature features observed at the time of culture preparation. Taken together with our previous light microscopic studies, these results indicate that cortical slice cultures are organotypically organized and serve as a useful model to study mechanisms of cortical development and plasticity.     

Ultrastructure of Golgi-impregnated and gold-toned spiny and aspiny neurons in the monkey neostriatum

Summary Golgi-impregnated, gold-toned spiny and aspiny neurons in the monkey neostriatum were deimpregnated and examined at the electron microscope level.Spiny type I neurons have relatively large nuclei with few indentations and aggregates of chromatin under the nuclear membrane which in some regions give the appearance of a dark rim. The small quantity of surrounding cytoplasm is poor in organelles.Aspiny type I neurons have eccentric, highly indented nuclei. The relatively large proportion of cytoplasm is rich in organelles especially Golgi apparatus and rough endoplasmic reticulum which often appears in stacks.Synapses with symmetric membrane densities are common on the somata of spiny type I neurons. Those on the proximal and distal dendritic shafts are few in number and asymmetric, and those on spines more frequent and primarily asymmetric. Aspiny type I neurons have few synapses on their cell bodies. Proximal and distal dendrites, however, are contacted by numerous profiles which contain small round vesicles and make both symmetric and asymmetric synapses. The same axon terminals also synapse with dendritic spines of spiny neurons, indicating that an input, most likely of afferent origin, is shared by both cell types. Other less frequently occurring profiles forming symmetric membrane densities also contact the dendrites of aspiny and spiny neurons. The axon hillocks and initial segments of both neuronal types receive a synaptic input, which is more common on spiny cells.Results offer unequivocal evidence for the differences in the ultrastructure of these two most common categories of medium-size neostriatal neurons, which may help in their proper identification in standard material, as well as information on the types and distributions of synaptic inputs onto these neurons. Moreover, the findings clarify some controversies in the literature probably originating from observations on a mixed population of cells of medium size.     

The projection of the visual cortex on the Clare-Bishop area in the cat

Summary Following large lesions of the cat visual cortex, the distribution of degenerating terminal boutons in the Clare-Bishop area was studied electron microscopically. Degenerating boutons were found throughout the cortical layers but mostly in layer III (51% of the total number of degenerating boutons) and layer V (24%). A smaller number of boutons were found in layers II (12%) and IV (9%), and very few in layers VI (3%) and I (1%). No degenerating terminals were observed in the upper two-thirds of layer I. Seventy-six per cent of the total degenerating boutons terminated on dendritic spines, 22% on dendritic shafts, and 2% on somata. Some degenerating boutons made synaptic contacts with somata and dendrites of nonpyramidal neurons. For example, one degenerating bouton was observed in contact with an apical dendrite of a fusiform cell. Three examples of dendritic spines, with which degenerating boutons made synaptic contacts, were found to belong to spinous stellate cells. No degenerating boutons were observed making synaptic contacts with profiles that could conclusively be traced to pyramidal cell somata.     

Morphological changes in the rat neostriatum after unilateral 6-hydroxydopamine injections into the nigrostriatal pathway

Destruction of the dopamine-containing neurons in the rat substantia nigra results in morphological changes in the striatum which have been characterized at both the light and electron microscopic levels. After a unilateral 6-hydroxydopamine injection into the medial forebrain bundle, Golgi-impregnated medium-sized spiny neurons in the neostriatum ipsilateral to the injection had a lower density of spines on their dendrites than those on the contralateral side. A similar decrease in spine density was apparent from 12 days until at least 13.5 months after the lesion. A bilateral loss of spines occurred with increasing age regardless of the presence or absence of the nigrostriatal dopaminergic pathway. At the ultrastructural level, the general pattern of synaptic input to the Golgi-impregnated medium-sized spiny neurons was similar on both sides of the brain. The most obvious class of afferent boutons contacting these spiny neurons formed prominent asymmetrical synaptic specializations with the heads of the spines. The numbers of asymmetric synaptic profiles counted in random electron micrographs from the striata ipsilateral and contralateral to the lesion were not significantly different from each other. A small but significant increase in the length of asymmetric synaptic specialization profiles was, however, detected in the striata lacking a dopamine input.     

Reconstruction of the defective cerebellar circuitry in adult Purkinje cell degeneration mutant mice by Purkinje cell replacement through transplantation of solid embryonic implants

Solid pieces of cerebellar primordia taken from 12-day-old C57BL embryos were implanted into the cerebellar parenchyma of 3- to 4-month-old "Purkinje cell degeneration" mutant mice and analysed 2-3 months later. Purkinje cell replacement was followed by means of immunocytochemistry with antisera against either cyclic guanosine monophosphate-dependent protein kinase or vitamin D-dependent calcium-binding protein, which allows the complete staining of these neurons. Although all solid graft implants survived, their fate within the mutant cerebellum varied in three ways: Often, a more or less large fragment of the solid graft remained in the white matter, close to the cortex or even partially replacing it. These remnants contained a few distorted Purkinje cells and a region corresponding to the transplanted deep nuclei, composed of numerous immunostained axons and axon terminals surrounding immunonegative neurons. Less frequently remnants of the graft were extruded to an extracerebellar location, between two adjacent folia. They contained a few Purkinje cells intermixed with granule cells and other neurons. In a few cases corresponding to superficial deposition, the implants developed lobulated and trilaminated minicerebella which were located outside the mutant cerebellum but integrated into it. In all three situations, a large number of grafted Purkinje cells succeeded in moving out of the implants and in invading the host molecular layer. These Purkinje cells develop flattened dendritic trees perpendicular to host bundles of parallel fibres. Ultrastructural examination of the synaptic investment of Purkinje cells which have reached the host molecular layer revealed that they acquire normal synaptic inputs although complex pericellular baskets and pinceau formation do not develop. Axons from molecular layer interneurons synapse on perikaryal and smooth dendritic membranes, climbing fibres synapse on stubby spines emerging from thick dendritic branches, and parallel fibres contact almost exclusively the long-necked spines of the distal spiny branchlets. Finally, Purkinje cells which succeed in migrating to molecular layer regions no further than 0.6 mm from the host deep nuclei are able to grow axons which reach appropriate target areas and establish synaptic connections on nuclear neurons. The results obtained from this series of long-term survival cerebellar transplantations point to the possibility of fulfilling most of the conditions necessary for functional restoration of neural grafts in systems in which neurons are connected in a point-to-point manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of functional glia on the electrophysiology of Purkinje cells in organotypic cerebellar cultures

Previous studies have shown that exposure of organotypic cerebellar explants to cytosine arabinoside (Sigma) for the first five days in vitro drastically reduced the granule cell population and severely affected glial function. Myelination was absent and astrocytes failed to ensheath Purkinje cells. In the absence of astrocytic ensheathment, Purkinje cell somata became hyperinnervated by Purkinje cell recurrent axon collaterals. Recurrent axon collaterals also projected to Purkinje cell dendritic spines. In later studies, exposure of cerebellar cultures to a different formulation of cytosine arabinoside (Pfanstiehl) also affected granule cells and oligodendrocytes but did not compromise astrocyte function. The different susceptibility of astrocytes to the two preparations of cytosine arabinoside (Sigma and Pfanstiehl) has provided the opportunity to examine the electrophysiological properties of Purkinje cells in the presence and absence of functional glia. Ensheathed Purkinje cells in granuloprival cultures exhibit within two weeks in vitro similar passive membrane properties as Purkinje cells in control cultures. Their input resistance is significantly higher and their spontaneous single-unit discharge is significantly lower than that of unensheathed Purkinje cells. This effect suggests that ensheathed Purkinje cells in cytosine arabinoside (Pfanstiehl)-treated cultures are more responsive to the profuse Purkinje cell recurrent axon collateral inhibitory projection to dendritic spines. These studies also show that the presence of functional glia and/or astrocytic ensheathment can be correlated with the development of complex spike activity by Purkinje cells in vitro. Purkinje cells in cultures treated with cytosine arabinoside (Pfanstiehl), which does not compromise astrocytic ensheathment, display membrane conductances and spike activity similar to mature Purkinje cells in control cultures. By contrast, Purkinje cells in cultures treated with cytosine arabinoside (Sigma), and devoid of astrocytic ensheathment, display mainly simple spike activity reminiscent of the type of activity seen in less mature neurons.