Glial Sheath Research Articles

The Internal Structure of the Nucleus geniculatus lateralis ventralis in the Avian Brain: A Golgi Study and Electron Microscopic Investigation

Different types of neurons in the ventral geniculate nucleus of the thalamus of chicks were visualised by Golgi impregnation. The dendritic tree of projection neurons branched in a sphere-like territory in both the ventral and middle areas of the lamina externa. The dendrites of projection neurons in the lamina interna descended into the lamina externa and entered both the ventral and middle dendritic areas. One or two dendrites of the lamina interna neurons also emitted branches that developed a dorsal sphere-like dendritic territory. Optic terminals labelled by Golgi impregnation or injection of biotinylated dextran amine were found in these dendritic territories gathered into groups. They established synapses in these areas (synaptic islands or fields without a glial sheath) with different dendritic profiles, and a few gamma-aminobutyric acid (GABA)-positive terminals synapsed with them. No glomerulus-like synaptic complexes ensheathed by glial processes were found. Optic terminals also contacted the stem dendrites of projection neurons and GABA-positive neuron cell bodies and dendrites. Numerous synapses established by both optic and GABA-positive terminals were found on the proximal dendritic stems of the lamina interna projection neurons.

Determinants of slowed diffusion in the complex space of the cerebellar glomerulus

Using analytical solutions for two- and three-dimensional (2D and 3D, respectively) and fractal 2D/3D geometry with an absorbing boundary, we modeled glutamate diffusion in a glomerulus, the structure situated around the mossy fiber (MF) terminal in the cerebellum and surrounded by a glial sheath. The model with fractal geometry gave the best fit of experimental AMPA and NMDA receptor-mediated evoked postsynaptic currents (EPSC) at the MF-granule cell synapse. A comparison of the numerically integrated glutamate concentration in an idealized model of glomerulus morphology with analytical solutions reveals that the peculiarities of glomerulus geometry can explain the better fit by the solution with fractal dimension only of experimental EPSC arising from local release, but not from spillover of glutamate. An asynchronous vesicle release only slightly influences the shape of the spillover waveform. Anomalously slow diffusion of glutamate can be an explanation of the observed discrepancy between experimental results and simulations with the 3D model. A good fit of spillover-induced EPSC obtained in the simulations that use a solution for fractional Brownian motion and a match of experimental estimations and theoretical parameters of the diffusion model confirms this assumption.

Immunohistochemical classification and functional morphology of human choroidal ganglion cells.

To characterize human choroidal ganglion cells (CGCs) further, regarding their immunohistochemical and ultrastructural appearance and their pre- and postsynaptic connections. . Choroidal wholemounts and serial sections of human donor eyes were stained with antibodies against neuronal nitric oxide synthase (nNOS), vasoactive intestinal peptide (VIP), tyrosine hydroxylase (TH), vesicular monoaminergic transporter (VMAT)-2, vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), calretinin, galanin, synaptophysin, and alpha-smooth muscle actin. Ultrathin sections of glutaraldehyde-fixed eyes were studied with an electron microscope. All CGCs stained for nNOS, most for VIP, approximately 45% for calretinin, and only single neurons for NPY and galanin. Ultrastructurally, the CGCs showed an incomplete glial sheath and, in places, showed close contact to surrounding collagen fibrils. The CGCs were in close contact with numerous boutons staining for the different neuronal markers including synaptophysin, nNOS, VIP, NPY, TH, VMAT-2, VAChT, calretinin, and NPY. The data indicate a complex integrative function of CGCs. The immunohistochemical and ultrastructural characteristics also indicate that the CGCs may have mechanosensory properties. The complex synaptic information points to a specific regulative CGC function in parallel with ciliary muscle contraction (accommodation). Axons originating from CGCs mainly supply the choroidal vasculature, thus implicating the CGCs as vasodilative neurons, but single CGCs may also innervate other structures such as nonvascular choroidal smooth muscle cells.

Early development of the Drosophila brain: IV. Larval neuropile compartments defined by glial septa.

In this study, we have analyzed the architecture of the brain neuropile of the Drosophila larva, which is formed by two main structural elements: long axon tracts and terminal axonal/dendritic arborizations carrying synapses. By using several molecular markers expressed in neurons and glial cells, we show that the early larval neuropile is subdivided by glial sheaths into numerous compartments. The three-dimensional layout of these compartments and their relationship to the pattern of long axon tracts described in the accompanying article (Nassif et al. [2003] J. Comp. Neurol 417-434) was modeled by using a three-dimensional illustration computer software. On the basis of their location relative to each other and to long axon tracts, larval brain compartments can be identified with compartments defined by structural and functional criteria for the adult fly brain. We find that small precursors of most of the compartments of the adult central brain can be identified in the early larva. Changes in brain compartmental organization occurring during larval growth are described. Neuropile compartments, representing easily identifiable landmark structures, will assist in future analyses of Drosophila brain development in which the exact location of neurons and their axonal trajectories is of importance.

Anatomical aspects of glia–synapse interaction: the perisynaptic glial sheath consists of a specialized astrocyte compartment

Amongst several forms of glia–neuronal communication, glia–synaptic interaction appears particularly interesting in the light of the well-known examples of two-way signaling between neurons and astrocytes. We review recent structural and physiological evidence showing that the structural correlate of glia–synaptic interaction is the peripheral astrocyte process (PAP) positioned next to the synaptic cleft. The structural and functional properties of these processes suggest that the PAP represents a separate astroglial compartment, in particular since it is characterized by the restricted localization of the actin-binding ERM protein ezrin. The structural properties of PAPs and this protein form the basis of rapid morphological changes of PAPs. The physiological relevance of PAP plasticity is illustrated by the example of the suprachiasmatic nucleus, where astrocytes display a high degree of activity-dependent plasticity reflecting circadian time.

A role for non-neuronal cells in synchronization of intracellular calcium oscillations in primate LHRH neurons

A study using multiunit activity recording from the medial basal hypothalamus indicates that pulsatile luteinizing hormone-releasing hormone (LHRH) release is a consequence of synchronized activity of individual LHRH neurons. An increase in single unit spike components extracted from the multiunit “LHRH pulse-generator” activity by cluster analysis occurs as a simultaneous increase in the firing rate of many individual hypothalamic neurons rather than the recruitment of new bursting neurons. The mechanism underlying LHRH synchronization is currently unknown. This is because, unlike oxytocin and vasopressin neurons, LHRH neurons do not form a nucleus in the hypothalamus and there is little evidence for physical contact between LHRH neurons—such as synapses or syncytium formation—that allow the synchronization of LHRH neurons. The perikarya of LHRH neurons are covered by glial sheaths and LHRH neuroterminals are intimately associated with glial end feet. The chapter also explains synchronization of [Ca 2 +] i oscillations and LHRH neurosecretion.

Peripheral astrocyte processes: monitoring by selective immunostaining for the actin-binding ERM proteins.

Astrocytes extend thin lamellate processes in the neuropil, in particular around synapses, where they can modulate synaptic function or mediate glial-neuronal communication. Previous studies have shown that these lamellate perisynaptic processes change their shape in response to neuronal activity, but the underlying mechanisms have remained unclear. Similarly, the molecular composition of these fine, sheet-like astrocytic processes (often 50-100 nm wide) is not understood but has to be related to their dynamic properties. To this end, we have studied the presence of ezrin, radixin, and moesin (ERM proteins) in the rat hippocampus and in primary cultured astrocytes, applying immunoperoxidase, immunofluorescence, and immunogold techniques. These three ERM proteins are known as actin-binding proteins that link the cell membrane to the actin cytoskeleton, particularly in microvillus-bearing epithelial cells. In cell culture, anti-ezrin and antiradixin, but not antimoesin, antibodies were specific for astrocytes, which often displayed selective staining of filopodia and microvilli. Nonoverlapping visualization of astrocytic peripheral and stem processes was obtained by immunocytochemical double labeling for ezrin and GFAP, respectively. In sections of rat hippocampus, homogeneous labeling of the neuropil, but not of cell layers, resulted from immunostaining of fine, peripheral astrocyte processes, as confirmed ultrastructurally. Our data show that the fine peripheral processes of astrocytes, which also constitute the perisynaptic glial sheath, are specialized in that they contain characteristic actin-associated molecules, likely to contribute to their dynamic properties. Applying anti-ezrin and anti-radixin as selective markers, plasticity of these perisynaptic glial processes can be analyzed.

The Role of Perisynaptic Glial Sheaths in Glutamate Spillover and Extracellular Ca 2+ Depletion

Recent findings suggest that rapid activation of extrasynaptic receptors and transient depletion of extracellular Ca 2+ may represent an important component of glutamatergic synaptic transmission. These phenomena imply a previously unrecognized role for synaptic glial sheaths: to retard extracellular diffusion in the synaptic vicinity. The present study is an attempt to assess the extent and physiological implications of this retardation using a detailed compartmental model of the typical synaptic environment. The model allows reconstruction of a partial (asymmetric) glial sheath covered with transporter molecules, which gives a more realistic representation of the vicinity of central synapses. Simulations show to what extent, in conditions compatible with physiology, the occupancy of synaptic receptors and the depletion of Ca 2+ in the cleft increase with increased glial coverage. The impact of glial sheaths on synaptic transmission is shown to become greater with smaller synapses and with slower kinetics of perisynaptic ion transients. At a calyceal synapse, a profound temporal filtering of fast Ca 2+ influx is found, and similar phenomena are predicted to occur following simultaneous activation of multiple synapses in the neuropil. The results provide a quantitative guidance for interpretation of physiological experiments that address fast transients of neurotransmitters and small ions in the brain tissue.

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.

Neuroligin 3 is a vertebrate gliotactin expressed in the olfactory ensheathing glia, a growth-promoting class of macroglia.

The molecular mechanisms that drive glia-glial interactions and glia-neuronal interactions during the development of the nervous system are poorly understood. A number of membrane-bound cell adhesion molecules have been shown to play a role, although the precise nature of their involvement is unknown. One class of molecules with cell adhesive properties used in the nervous system is the serine-esterase-like family of transmembrane proteins. A member of this class, a glia-specific protein called gliotactin, has been shown to be necessary for the development of the glial sheath in the peripheral nervous system of Drosophila melanogaster. Gliotactin is essential for the development of septate junctions in the glial sheath of individual and neighboring glia. Mutations that remove this protein result in paralysis and eventually death due to a breakdown in the glial-based blood-nerve barrier. To study the role of gliotactin during vertebrate nervous system development, we have isolated a potential vertebrate gliotactin homologue from mice and rat and found that it corresponds to neuroligin 3. Using a combination of RT-PCR and immunohistochemistry, we have found that neuroligin 3 is expressed during the development of the nervous system in many classes of glia. In particular neuroligin 3 is expressed in the olfactory ensheathing glia, retinal astrocytes, Schwann cells, and spinal cord astrocytes in the developing embryo. This expression is developmentally controlled such that in postnatal and adult stages, neuroligin 3 continues to be expressed at high levels in the olfactory ensheathing glia, a highly plastic class of glia that retain many of their developmental characteristics throughout life.

Atlas of olfactory organs of Drosophila melanogaster: 2. Internal organization and cellular architecture of olfactory sensilla

Antennae and maxillary palps of Drosophila melanogaster were studied with the electron microscope on serial sections of cryofixed specimens. The number of epidermal cells roughly equals the number of sensilla, except for regions where the latter are scarce or absent. Each epidermal cell forms about two non-innervated spinules, a prominent subcuticular space and a conspicuous basal labyrinth, suggesting a high rate of fluid transport through the sensory epithelium. The internal organization and fine structure of trichoid, intermediate and basiconic sensilla is very similar. Receptor cell somata are invested by thin glial sheaths extending distad to the inner dendritic segments. Further distally, the thecogen cell forms a sleeve around the dendrites, but an extracellular dendrite sheath is absent. At the base of the cuticular apparatus, the inner sensillum-lymph space around the ciliary and outer dendritic segments is confluent with the large outer sensillum-lymph space formed by the trichogen and tormogen cells. All three auxiliary cells exhibit many features of secretory and transport cells but extend only thin basal processes towards the haemolymph sinus. The bauplan and fine structure of coeloconic sensilla differs in the following aspects: (1) the ciliary segment of the dendrites is located deeper below the base of the cuticular apparatus than in the other sensillum types; (2) a prominent dendrite sheath is always present, separating inner and outer sensillum-lymph spaces completely; (3) the apical microlamellae of the auxiliary cells are more elaborate, but free sensillum-lymph spaces are almost absent; (4) there are always four not three auxiliary cells. Morphometric data are presented on the diameter of inner and outer dendritic segments and on the size of receptor cells, as well as of the receptor and auxiliary cell nuclei. The special fine structural features of Drosophila olfactory sensilla are discussed under the aspects of sensillar function and the localization of proteins relevant for stimulus transduction.

Uptake and metabolism of glutamate at non-synaptic regions of crayfish central nerve fibers: implications for axon–glia signaling

In crayfish and squid giant nerve fibers, glutamate appears to be an axon–glia signaling agent. We have investigated glutamate transport and metabolism by crayfish central nerve fibers in order to identify possible mechanisms by which glutamate could subserve this non-synaptic signaling function. Accumulation of radiolabeled l-glutamate by desheathed cephalothoracic nerve bundles was temperature and Na + dependent, linear with time for at least 8 h and saturable at about 0.5–1 mM l-glutamate. Most accumulated radiotracer was associated with the periaxonal glial sheath and remained as glutamate. Compounds known to block glutamate transport in invertebrate peripheral nerves or mammalian brain slices or cell cultures were also effective on crayfish central nerve fibers. Tissue radiotracer levels were only 3% of control levels when 1 mM p-chloromercuriphenylsulfonate was present, and 13%, 20%, 26%, 38% and 42% of control levels, respectively, when l-cysteate, l-cysteine sulfinate, l-aspartate, d-aspartate or dl- threo-β-hydroxyaspartate was present. l-Glutamine, GABA, N-methyl- dl-aspartate, α-aminoadipate and d-glutamate were without inhibitory effect on tissue tracer accumulation. Radiolabeled d-aspartate was an equivalent non-metabolized substitute for radiolabeled l-glutamate. d-Aspartate, p-chloromercuriphenylsulfonate and GABA had comparable effects on isolated medial giant nerve fibers. These studies indicate that l-glutamate is taken up primarily by the periaxonal glia of crayfish central nerve fibers by a low-affinity, saturable, Na +-dependent transport system and is retained by the fibers primarily in that form. Our results suggest that the glia are not only the target of the glutamate signal released from non-synaptic regions of the crayfish medial giant axon during high-frequency stimulation, but that they are also the primary site of its inactivation.

Distribution and synaptology of vasoactive intestinal polypeptide (VIP) immunoreactive structures in the rat periaqueductal grey.

Light microscopic analysis of the rat midbrain periaqueductal grey (PAG) showed vasoactive intestinal polypeptide immunoreactive (VIP-ir) neurons localized at the lateral and ventral walls of the aqueduct. Some varicose VIP-ir elements were detected closely associated with the ependyma. While several VIP-ir elements were encountered immediately under the ependyma, in a few cases, VIP-ir cell bodies were seen on the luminal surface of the ependymal cells lining the aqueduct. Electron microscopy revealed that most of these cells possessed the characteristics of a local circuit neuron. All VIP-ir cells had indented nuclei. Two types were distinguished: one with rounded cell body receiving numerous axo-somatic synapses established by VIP-negative axons. The other cell type was fusiform and its surface was almost fully isolated from axonal contacts by a glial sheath. The VIP-ir processes were interconnected with other periaqueductal cells by a variety of synaptic contacts. VIP-ir axon terminals formed asymmetric synapses with immunonegative dendritic shafts often in glomerulus-like assemblies. The postsynaptic immunonegative dendrites were of the aspinous, beaded type. We suggest that VIP-ir cells and processes in the midbrain PAG establish connections between the longitudinal functional columns of this region. On the basis of their morphology, VIP-ir cells in the PAG appear to be excitatory, terminating on inhibitory interneurons. Thus, a VIP-stimulated inhibition may be instrumental in the coordination of responses evoked by the stimulation of PAG columns.

Peripheral synapses at identifiable mechanosensory neurons in the spider Cupiennius salei: synapsin-like immunoreactivity.

Indirect immunocytochemical tests were used at the light- and electron-microscopic levels to investigate peripheral chemical synapses in identified sensory neurons of two types of cuticular mechanosensors in the spider Cupiennius salei Keys.: (1) in the lyriform slit-sense organ VS-3 (comprising 7-8 cuticular slits, each innervated by 2 bipolar sensory neurons) and (2) in tactile hair sensilla (each supplied with 3 bipolar sensory cells). All these neurons are mechanosensitive. Application of a monoclonal antibody against Drosophila synapsin revealed clear punctate immunofluorescence in whole-mount preparations of both mechanoreceptor types. The size and overall distribution of immunoreactive puncta suggested that these were labeled presynaptic sites. Immunofluorescent puncta were 0.5-6.8 micrometer long and located 0.5-6.6 micrometer apart from each other. They were concentrated at the initial axon segments of the sensory neurons, while the somata and the dendritic regions showed fewer puncta. Western blot analysis with the same synapsin antibody against samples of spider sensory hypodermis and against samples from the central nervous system revealed a characteristic doublet band at 72 kDa and 75 kDa, corresponding to the apparent molecular mass of synapsin in Drosophila and in mammals. Conventional transmissionelectron-microscopic staining demonstrated that numerous chemical synapses (with at least 2 vesicle types) were present at these mechanosensory neurons and their surrounding glial sheath. The distribution of these synapses corresponded to our immunofluorescence results. Ultrastructural examination of anti-synapsin-stained neurons confirmed that reaction product was associated with synaptic vesicles. We assume that the peripheral synaptic contacts originate from efferents that could exert a complex modulatory influence on mechanosensory activity.

Anomalies associated with dye exclusion as a measure of axolemmal repair in vertebrate axons

After axonal injury, dye exclusion is often used as a measure of the re-establishment of a structural barrier. We now report that this use of dye exclusion is equivocal in two situations. (1) When a negatively-charged hydrophilic fluorescent dye (HFD) was placed in the physiological saline (PS) surrounding a crayfish medial giant axon (CMGA) before transection, this dye did not readily diffuse into the cut ends after transection whereas uncharged or neutralized dyes did do so. (2) When axoplasm flowed out of the cut ends of a transected squid giant axon (SGA), this outflow markedly slowed hydrophilic fluorescent dyes from diffusing into the cut ends. These anomalies suggest that dye exclusion by an injured axon does not always indicate that a structural barrier has formed. Therefore, dye assessments of axonal repair require control experiments that rule out anomalous exclusion due to dye interactions (biochemical and fluid dynamics) with components (axoplasm, axolemma, glial sheath, etc.) of the particular axon under study.

Heat-shock proteins in axoplasm: high constitutive levels and transfer of inducible isoforms from glia.

To characterize heat-shock proteins (HSPs) of the 70-kDa family in the crayfish medial giant axon (MGA), we analyzed axoplasmic proteins separately from proteins of the glial sheath. Several different molecular weight isoforms of constitutive HSP 70s that were detected on immunoblots were approximately 1-3% of the total protein in the axoplasm of MGAs. To investigate inducible HSPs, MGAs were heat shocked in vitro or in vivo, then the axon was bathed in radiolabeled amino acid for 4 hours. After either heat-shock treatment, protein synthesis in the glial sheath was decreased compared with that of control axons, and newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa appeared in both the axoplasm and the sheath. Because these radiolabeled proteins were present in MGAs only after heat-shock treatments, we interpreted the newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa to be inducible HSPs. Furthermore, the 72-kDa radiolabeled band in heat-shocked axoplasm and glial sheath samples comigrated with a band possessing HSP 70 immunoreactivity. The amount of heat-induced proteins in axoplasm samples was greater after a 2-hour heat shock than after a 1-hour heat shock. These data indicate that MGA axoplasm contains relatively high levels of constitutive HSP 70s and that, after heat shock, MGA axoplasm obtains inducible HSPs of 72 kDa, 84 kDa, and 87 kDa from the glial sheath. These constitutive and inducible HSPs may help MGAs maintain essential structures and functions following acute heat shock.

Development and fine structure of murine Purkinje cells in dissociated cerebellar cultures: dendritic differentiation, synaptic maturation, and formation of cell-class specific features

The morphological differentiation of E16 murine Purkinje cells (PCs) in dissociated cerebellar cultures was analyzed by light and electron microscopic immunocytochemistry after 2-5 weeks in vitro (wiv), with particular emphasis on dendritic differentiation, synaptic maturation, and formation of stereotypical fine structural features. This study complements a companion paper on the features of PCs after 1 wiv. After 2 wiv, the PCs have an eccentric nucleus and the cytoplasmic organelles appear immature; the axon has a distinct initial segment and beaded axon collaterals but its boutons still contain sparse synaptic vesicles; dendrites show few bifurcations and tufts of spiny branchlets. After 3 wiv, the PCs display a centered nucleus, an extensive hypolemmal cisternal system, and stacks of up to four cisterns of granular endoplasmic reticulum; there is an increased number of dendritic bifurcations, spiny branchlets, mature spines, and axonal branches; dendritic tips still contain vesicle clusters, suggesting growth, and many synapses and afferent boutons continue to display immature features. After 4 wiv, elaborate perinucleolar coiled body rosettes, subsurface cistern-mitochondrion complexes and large stacks of granular endoplasmic reticulum finally appear within the soma; dendrites show a further increase in the numbers of bifurcations, segments and spines; most spines are synaptic and show mature features; afferent synapses are differentially distributed; PC boutons consistently display mature features and show a considerable degree of target specificity, although naked spines and reduced glial sheaths persist. After 5 wiv, PCs do not show further maturation and some dystrophic features appear. We conclude that under standard conditions and despite the disruption of normal tissue organization, PCs in dissociated cultures differentiate maximally after 4 wiv, at which stage they display many of the light and electron microscopic features that characterize mature PCs in situ. This prolonged developmental time-frame resembles that in the normal cerebellum. In view of the increasing usage of dissociated cerebellar cultures to study aspects of neuronal differentiation, synaptic activation and neuronal-glial interactions, an elucidation of the neurocytology of dissociated cerebellar cultures as presented in this study provides important clues for the interpretation of experimental data.

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 anti-serum 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.

Delaminating myelin membranes help seal the cut ends of severed earthworm giant axons.

Transected axons are often assumed to seal by collapse and fusion of the axolemmal leaflets at their cut ends. Using photomicroscopy and electronmicroscopy of fixed tissues and differential interference contrast and confocal fluorescence imaging of living tissues, we examined the proximal and distal cut ends of the pseudomyelinated medial giant axon of the earthworm, Lumbricus terrestris, at 5-60 min post-transection in physiological salines and Ca2+-free salines. In physiological salines, the axolemmal leaflets at the cut ends do not completely collapse, much less fuse, for at least 60 min post-transection. In fact, the axolemma is disrupted for 20-100 microm from the cut end at 5-60 min post-transection. However, a barrier to dye diffusion is observed when hydrophilic or styryl dyes are placed in the bath at 15-30 min post-transection. At 30-60 min post-transection, this barrier to dye diffusion near the cut end is formed amid an accumulation of some single-layered and many multilayered vesicles and other membranous material, much of which resembles delaminated pseudomyelin of the glial sheath. In Ca2+-free salines, this single and multilayered membranous material does not accumulate, and a dye diffusion barrier is not observed. These and other data are consistent with the hypothesis that plasmalemmal damage in eukaryotic cells is repaired by Ca2+-induced vesicles arising from invaginations or evaginations of membranes of various origin which form junctional contacts or fuse with each other and/or the plasmalemma.

Morphological correlates of neural regeneration in the feeding system ofAplysia californica after central nervous system lesions

Morphological techniques were used to study regeneration of central neural pathways involved in feeding behavior following bilateral crushes of the cerebral-buccal connectives (CBCs). Electron microscopic analysis revealed that CBC crushes completely transect axons within the nerve core while leaving a remnant of the nerve sheath intact. Changes in the ultrastructure of the CBCs at the crush site were determined for 1, 7, 14, 21, and 50 days postlesion. At 1 day postlesion, the crush site was no longer compressed, and the nerve core had assumed a circular shape. In addition, several small axon profiles were evident, and large areas of tissue debris and prominent microglial cells were observed. Membranous debris and hemocytes were also present in sinuses that appeared in the sheath adjacent to the crush site. From 7 to 50 days postlesion, the core of the nerve at the crush site increased in size due to the addition of small diameter axons. Initially, the sheath surrounding the crush site exhibited hyperplasia and contained a few small bundles of processes, apparently due to newly sprouted axons that had strayed from the nerve core. By 50 days postlesion, the crush site appeared nearly normal; the nerve core was reacquiring the normal radial pattern of axon profiles with some medium-sized axon profiles covered with glial sheath and exhibiting invaginations typical of the intact CBC. However, there was still a distinct lack of large diameter axons. Cobalt backfills across the crush site revealed neurons in the cerebral ganglion by postlesion day 9. Positions of stained cell bodies were consistent with those observed in controls, although the numbers of stained neurons did not recover to control levels even by postlesion day 63. The changes in the crush site and return of cell body staining with time postlesion are correlated with the recovery of consummatory feeding.