The astroglial cell that guides nerve fibers from growth cone to synapse in organotypic cultures of the fetal mouse spinal cord

We present electron microscopic and autoradiographic studies done using organotypic cultures of spinal cord explants excised from 15 days of gestation mouse embryos. Nerve fibers growing from the spinal cord explant carry at their tips immature mitotic astrocytic cells that lead their growth cones. These glial cells divide only during the active phase of neuronal growth, and correspond ultrastructurally to radial glia. They provide a specific cellular substrate for neuronal growth. Some growth cones form axoglial synapses with smooth membranes of immature glial cells. In contrast, maturing glial cells sprout cytoplasmic processes that tightly wrap individual growth cones and effectively arrest their growth. Next, the processes gather nerve endings into islets and nerve fibers into bundles. After internalizing nerve endings, the glial processes withdraw, bringing the endings into contact with each other. The direct neuronal appositions lead to the transformation of growth cones into presynaptic endings, signaled by their collection of presynaptic vesicles. Clustering of the vesicles at presynaptic axoglial or axodendritic membranes indicates the onset of synaptogenesis-completed by differentiation of spinous and compound synapses. Concomitant with the progress of synaptogenesis, astrocytic investment within the neuropil progressively diminishes. The differentiating astrocytic processes show secretory and tethering activity toward nerve fibers and their endings. Our observations demonstrate that astroglial cells-depending on their developmental stage-first promote and then arrest neuronal growth, and induce synaptogenesis. Thus, at any time, the growing nerve fibers are not only supported but also controlled by the astroglial cells.     

Intracellular localization of heat shock mRNAs (hsc70 and hsp70) to neural cell bodies and processes in the control and hyperthermic rabbit brain

Heat shock proteins are essential cellular proteins that may play important roles in cellular repair and/or protection. This report focuses on the expression of two members of the hsp70 multigene family, namely, constitutive hsc70 mRNA and stress-inducible hsp70 mRNA in the control and hyperthermic rabbit brain. The intracellular localization of these heat shock mRNAs was examined using high-resolution nonradioactive in situ hybridization. The distribution of hsc70 mRNA and hsp70 mRNA was examined in (1) neuronal cell bodies and their dendritic processes and (2) oligodendrocytes and their cellular processes. In control animals, hsc70 mRNA was detected in the apical dendritic processes and cell bodies of cortical layer II and V neurons, CA3 and CA4 neurons, deep cerebellar neurons, and brainstem neurons. A time course analysis of hsc70 mRNA, after a physiologically relevant increase in body temperature of 2.6°C, revealed more distal transport of this constitutive message into dendrites of these neuronal populations. In the same neuronal populations, basal levels of hsp70 mRNA were observed in the cell body; however, this mRNA was not detected in dendritic processes in control or hyperthermic animals. After hyperthermia, hsp70 mRNA was strongly induced in oligodendrocytes and transported to the processes of these glial cells. The localization of heat shock messages in the processes of these neural cell types could provide a mechanism for local control of synthesis of heat shock proteins in cellular compartments that are remote from the cell body. © 1996 Wiley-Liss, Inc.     

Neuron–Glia Interaction in the Suprachiasmatic Nucleus: A Double Labeling Light and Electron Microscopic Immunocytochemical Study in the Rat

The morphological interactions between astroglial and neuronal elements were elucidated in the rat suprachiasmatic nucleus (SCN) by light and electron microscopic immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP), vasoactive intestinal peptide (VIP) and arginine-vasopressin (AVP). Throughout the SCN, particularly in its ventral portion, GFAP-like-immunoreactive (GFAP-LI) astroglial elements were found. These astrocytes displaying GFAP-like immunoreactivity occasionally contained fairly well-developed organelles. Some of these astrocytes were found as satellite cells in close contact with non-immunoreactive neuronal perikarya and processes. Around the neurons, GFAP-LI astroglial processes were also observed to cover some portions of presynaptic and postsynaptic elements. In addition, these astroglial elements were seen between two neuronal somata and pericytes of blood capillaries as glial endfeet. By double labeling immunoelectron microscopy using antibodies against GFAP/VIP and GFAP/AVP, some portions of VIP-like-immunoreactive or AVP-like-immunoreactive neuronal somata and processes were found to be engulfed by GFAP-LI astroglial processes. The possible functional roles of the morphological interactions between astroglial and neuronal elements are discussed.     

Nerve terminals of squid photoreceptor neurons contain a heterogeneous population of mRNAs and translate a transfected reporter mRNA

It is now well established that the distal structural/functional domains of the neuron contain 2a diverse population of mRNAs that program the local synthesis of protein. However, there is still a paucity of information on the composition and function of these mRNA populations in the adult nervous system. To generate empirically, hypotheses regarding the function of the local protein synthetic system, we have compared the mRNAs present in the squid giant axon and its parental cell bodies using differential mRNA display as an unbiased screen. The results of this screen facilitated the identification of 31 mRNAs that encoded cytoskeletal proteins, translation factors, ribosomal proteins, molecular motors, metabolic enzymes, nuclear-encoded mitochondrial mRNAs, and a molecular chaperone. Results of cell fractionation and RT-PCR analyses established that several of these mRNAs were present in polysomes present in the presynaptic nerve terminal of photoreceptor neurons, indicating that these mRNAs were being actively translated. Findings derived from in vitro transfection studies established that these isolated nerve terminals had the ability to translate a heterologous reporter mRNA. Based upon these data, it is hypothesized that the local protein synthetic system plays an important role in the maintenance/remodelling of the cytoarchitecture of the axon and nerve terminal, maintenance of the axon transport and mRNA translation systems, as well as contributing to the viability and function of the local mitochondria.     

Taurine immunoreactivity in the rat supraoptic nucleus: Prominent localization in glial cells

Taurine is an inhibitory amino acid that hyperpolarizes magnocellular neurosecretory neurons. To determine which cell types in the rat supraoptic nucleus contain taurine, we used a monoclonal antibody raised against a taurine conjugate. Preembedding immunocytochemistry was carried out at the light and electron microscopic levels using diaminobenzidine and gold-substituted silver-intensified peroxidase as markers. We report the presence of taurine in all cellular compartments of the supraoptic nucleus, except axons, with variable labeling intensities among the different compartments. Few cell bodies of magnocellular neurons were immunoreactive, but many distal dendrites and some proximal ones showed weak-to-moderate levels of immunoreactivity. Strong immunoreactivity was found over glial cell bodies and their processes, in particular in the ventral glial lamina of the supraoptic nucleus. Large astrocytic processes labeled with the taurine antibody included the endfeet participating in the glial limitans around capillaries and at the ventral surface of the hypothalamus. Other types of immunoreactive astropytic profiles were found scattered within the neuropil where these processes participated in different interactions with the neuronal elements of the supraoptic nucleus. Immunoreactive glial expansions, sometimes even the main process of the glial cell, engulfed axonal boutons. Other labeled glial processes were found between two magnocellular perikarya or closely apposed to the membrane of axonal boutons contacting the neuronal cell bodies. The frequent finding of closely apposed glial and dendritic elements bearing different levels of taurine-like immunoreactivity suggests that exchange of taurine between those two compartments may occur. We propose that taurine could be released from supraoptic glia by a small decrease in osmolarity or by receptor-mediated mechanisms during conditions of low hormonal (vasopressin and/or oxytocin) needs. Such released taurine could then act on presynaptic or postsynaptic sites, or both, to exert its neuromodulatory actions. © 1995 Wiley-Liss, Inc.     

Structural Compartments within Neurons: Developmentally Regulated Organization of Microfilament Isoform mRNA and Protein

The microfilament system is thought to be a crucial cytoskeletal component regulating development and mature function of neurons. The intracellular distribution of the microfilament isoform components, actin and tropomyosin (Tm), in neurons primarilyin vivo,has been investigated at both the mRNA and the protein level using isoform specific riboprobes and antibodies. Ourin vivoandin vitrostudies have identified at least six neuronal compartments based on microfilament isoform mRNA localization: the developing soma, the mature soma, growth cone, developing axon hillock/proximal axon, mature somatodendritic and mature axonal pole soma. Protein localization patterns revealed that the isoforms were frequently distributed over a wider area than their respective mRNAs, suggesting that isoform specific patterns of mRNA targeting may influence, but do not absolutely determine, microfilament isoform location. Tm4 and Tm5 showed identical mRNA targeting in the developing neuron but distinct protein localization patterns. We suggest that in this instance mRNA location may best be viewed as a regulated site of synthesis and assembly, rather than a regulator of protein localization per se. In addition, Tm5 and β-actin mRNA and protein locations were developmentally regulated, suggesting the possibility that environmental signals modulate targeting of specific mRNAs and their proteins. Thus, developmentally regulated mRNA localization and positional translation may act in concert with protein transport to regulate neuronal microfilament composition and consequently neuronal structure.     

Glial modulation of synaptic transmission in culture

Accumulating evidence has demonstrated the existence of bidirectional communication between glial cells and neurons, indicating an important active role of glia in the physiology of the nervous system. Neurotransmitters released by presynaptic terminals during synaptic activity increase intracellular Ca(2+) concentration in adjacent glial cells. In turn, activated glia may release different transmitters that can feed back to neuronal synaptic elements, regulating the postsynaptic neuronal excitability and modulating neurotransmitter release from presynaptic terminals. As a consequence of this evidence, a new concept of the synaptic physiology, the tripartite synapse, has been proposed, in which glial cells play an active role as dynamic regulatory elements in neurotransmission. In the present article we review evidence showing the ability of astrocytes to modulate synaptic transmission directly, with the focus on studies performed on cell culture preparations, which have been proved extremely useful in the characterization of molecular and cellular processes involved in astrocyte-mediated neuromodulation.     

Decrease in striatal enkephalin mRNA in mouse models of Huntington's disease

Huntington's disease is a devastating progressive neurodegenerative illness characterized by massive neuronal loss in the striatum. It is caused by the presence of an expanded CAG repeat in the gene encoding huntingtin, a protein of unknown function. We have examined the expression of neurotransmitters and other antigens present in striatal neurons with immunohistochemistry, and the level of expression of mRNAs encoding enkephalin, substance P, and glutamic acid decarboxylases with quantitative in situ hybridization histochemistry, in the striatum of two mouse models of Huntington's disease: transgenic animals expressing exon 1 of the human huntingtin gene with 144 CAG repeats and "knock-in" mice containing a chimeric mouse/human exon 1 with 71 or 94 CAG repeats inserted by homologous targeting. Although the transgenic (but not the knock-in) mice were previously shown to display prominent huntingtin- and ubiquitin-containing nuclear inclusions in striatal neurons, in situ nick translation followed by emulsion autoradiography did not reveal any DNA damage in striatum or cortex in these mice. Immunolabeling for calbindin D 28K, enkephalin, substance P, glutamic acid decarboxylases (M(r) 65,000 or 67,000, GAD65 and GAD67), somatostatin, choline acetyltransferase, parvalbumin, and glial fibrillary acidic protein were remarkably similar in transgenic, knock-in, and wild-type mice. Both transgenic and knock-in mice, however, showed a marked decrease in the level of expression of enkephalin mRNA in striatal neurons without significant decreases in mRNAs encoding substance P, GAD65, or GAD67. The data indicate that decreased expression of enkephalin mRNA may be an early sign of neuronal dysfunction due to the Huntington's disease mutation.     

Chronic maintenance of presynaptic terminals in gliotic hippocampus following ischemia

Following brief cerebral ischemia, neurons are selectively damaged and die, whereas glial cells and blood vessels survive. This phenomenon of selective vulnerability is well illustrated in the hippocampal CA1 region. Five min of forebrain ischemia in the Mongolian gerbil produced selective neuronal necrosis in the hippocampal CA1 sector. After destruction and loss of CA1 neurons, a remarkable glial reaction (gliosis) was seen. The thickness of the CA1 subfield remained unchanged until 1 month after ischemia and then gradually shrank over several months. Ultrastructural observation of this region revealed persistent maintenance of presynaptic structures. Numerous presynaptic terminals containing synaptic vesicles were scattered throughout the gliotic scar tissue. These presynaptic terminals were apposed to degenerative structures which seemed most likely to be remnants of dendrites. In another group of animals, at one month following ischemic damage in the CA1 sector, the CA3 neurons were destroyed by kainic acid injection. In these animals, numerous degenerating presynaptic boutons were seen in the CA1 sector when fixed 4 days following kainate injection. These results indicate that even in gliotic tissue, presynaptic terminals can survive and maintain their structural characteristics although neuronal cell bodies are almost absent.     

Specific interaction between Sam68 and neuronal mRNAs: implication for the activity-dependent biosynthesis of elongation factor eEF1A

In cultured hippocampal neurons and in adult brain, the splicing regulatory protein Sam68 is partially relocated to the somatodendritic domain and associates with dendritic polysomes. Transfer to the dendrites is activity-dependent. We have investigated the repertoire of neuronal mRNAs to which Sam68 binds in vivo. By using coimmunoprecipitation and microarray screening techniques, Sam68 was found to associate with a number of plasticity-related mRNA species, including Eef1a1, an activity-responsive mRNA coding for translation elongation factor eEF1A. In cortical neuronal cultures, translation of the Eef1a1 mRNA was strongly induced by neuronal depolarisation and correlated with enhanced association of Sam68 with polysomal mRNAs. The possible function of Sam68 in Eef1a1 mRNA utilization was studied by expressing a dominant-negative, cytoplasmic Sam68 mutant (GFP-Sam68DeltaC) in cultured hippocampal neurons. The level of eEF1A was lower in neurons expressing GFP-Sam68DeltaC than in control neurons, supporting the proposal that endogenous Sam68 may contribute to the translational efficiency of the Eef1a1 mRNA. These findings are discussed in the light of the complex, potentially crucial regulation of eEF1A biosynthesis during long-term synaptic change.     

mRNA expression patterns of the cGMP-hydrolyzing phosphodiesterases types 2, 5, and 9 during development of the rat brain

Recent evidence indicates that cGMP plays an important role in neural development and neurotransmission. Since cGMP levels depend critically on the activities of phosphodiesterase (PDE) enzymes, mRNA expression patterns were examined for several key cGMP-hydrolyzing PDEs (type 2 [PDE2], 5 [PDE5], and 9 [PDE9]) in rat brain at defined developmental stages. Riboprobes were used for nonradioactive in situ hybridization on sections derived from embryonic animals at 15 days gestation (E15) and several postnatal stages (P0, P5, P10, P21) until adulthood (3 months). At all stages PDE9 mRNA was present throughout the whole central nervous system, with highest levels observed in cerebellar Purkinje cells, whereas PDE2 and PDE5 mRNA expression was more restricted. Like PDE9, PDE5 mRNA was abundant in cerebellar Purkinje cells, although it was observed only on and after postnatal day 10 in these cells. In other brain regions, PDE5 mRNA expression was minimal, detected in olfactory bulb, cortical layers, and in hippocampus. PDE2 mRNA was distributed more widely, with highest levels in medial habenula, and abundant expression in olfactory bulb, olfactory tubercle, cortex, amygdala, striatum, and hippocampus. Double immunostaining of PDE2, PDE5, or PDE9 mRNAs with the neuronal marker NeuN and the glial cell marker glial fibrillary acidic protein revealed that these mRNAs were predominantly expressed in neuronal cell bodies. Our data indicate that three cGMP-hydrolyzing PDE families have distinct expression patterns, although specific cell types coexpress mRNAs for all three enzymes. Thus, it appears that differential expression of PDE isoforms may provide a mechanism to match cGMP hydrolysis to the functional demands of individual brain regions.     

Local synthesis of axonal and presynaptic RNA in squid model systems

The presence of active systems of protein synthesis in axons and nerve endings raises the question of the cellular origin of the corresponding RNAs. Our present experiments demonstrate that, besides a possible derivation from neuronal cell bodies, axoplasmic RNAs originate in periaxonal glial cells and presynaptic RNAs derive from nearby cells, presumably glial cells. Indeed, in perfused squid giant axons, delivery of newly synthesized RNA to the axon perfusate is strongly stimulated by axonal depolarization or agonists of glial glutamate and acetylcholine receptors. Likewise, incubation of squid optic lobe slices with [3H]uridine leads to a marked accumulation of [3H]RNA in the large synaptosomes derived from the nerve terminals of retinal photoreceptor neurons. As the cell bodies of these neurons lie outside the optic lobe, the data demonstrate that presynaptic RNA is locally synthesized, presumably by perisynaptic glial cells. Overall, our results support the view that axons and presynaptic regions are endowed with local systems of gene expression which may prove essential for the maintenance and plasticity of these extrasomatic neuronal domains.     

Developmental changes in KCC1, KCC2 and NKCC1 mRNAs in the rat cerebellum

Cation chloride cotransporters are considered to play pivotal roles in controlling the intracellular and extracellular ionic environments of neurons, hence controlling neuronal function. To establish how these cotransporters are involved in cerebellum development, we investigated the expression of KCC1, KCC2 and NKCC1 mRNAs in the developing rat cerebellum using in situ hybridization histochemistry. In the external germinal layer, where premature cells exist, we found substantial KCC1 and NKCC1 mRNA expression on P7 and P14, while KCC2 mRNA was not detected. In contrast, KCC2 mRNA was already expressed in Purkinje cells on P1. We also observed KCC2 mRNA expression in postmigratory granule cells after P7. The expression of KCC1, KCC2, and NKCC1 mRNAs reached adult patterns by P21. In the adult cerebellum, KCC2 mRNA was expressed in most neurons, including Purkinje cells, granule cells, and stella/basket cells, while KCC1 and NKCC1 mRNAs were only detected in granule cells and glial cells. These findings suggest that in the rat cerebellum KCC2 mRNA expression is induced when neurons arrive their final destinations.