Orwik

EphA4 is localized in clathrin-coated and synaptic vesicles in adult mouse brain.

EphA4, a receptor tyrosine kinase, is expressed in various pre-, post- and peri-synaptic organelles and implicated in the regulation of morphological and physiological properties of synapses. It regulates synaptic plasticity by acting as a binding pa... expand abstractrtner for glial ephrin-A3 and possibly other pre- or post-synaptic ephrins. Now, its trafficking mechanisms remain unknown. In this study, we examine the association of EphA4 with transport, clathrin-coated and synaptic vesicles using cell fractionation, vesicle immunoisolation and electron microscopy. EphA4 was found in highly purified fractions of clathrin-coated or synaptic vesicles. It was also detected in vesicles immuno-isolated with antibodies anti-synaptophysin, anti-vesicular glutamate transporter or anti-vesicular GABA transporter; demonstrating its presence in synaptic vesicles. However, it was not detected in immuno-isolated piccolo-bassoon transport vesicles. In vivo and in dissociated cultures, EphA4 was localized by immunoelectron microscopy in vesicular glutamate transporter 1-positive terminals of hippocampal neurons. Remarkably, the cell surface immunofluorescence of EphA4 increased markedly in cultured hippocampal neurons following KCl depolarization. These observations indicate that EphA4 is present in subsets of synaptic vesicles, can be externalized during depolarization, and internalized within clathrin-coated vesicles. This trafficking itinerary may serve to regulate the levels of EphA4 in the synaptic plasma membrane and thereby modulate signaling events that contribute to synaptic plasticity. collapse abstract

Long-term potentiation in isolated dendritic spines.

BACKGROUND: In brain, N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation can induce long-lasting changes in synaptic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor (AMPAR) levels. These changes are believed to underlie the ... expand abstractexpression of several forms of synaptic plasticity, including long-term potentiation (LTP). Such plasticity is generally believed to reflect the regulated trafficking of AMPARs within dendritic spines. However, recent work suggests that the movement of molecules and organelles between the spine and the adjacent dendritic shaft can critically influence synaptic plasticity. To determine whether such movement is strictly required for plasticity, we have developed a novel system to examine AMPAR trafficking in brain synaptosomes, consisting of isolated and apposed pre- and postsynaptic elements. METHODOLOGY/PRINCIPAL FINDINGS: We report here that synaptosomes can undergo LTP-like plasticity in response to stimuli that mimic synaptic NMDAR activation. Indeed, KCl-evoked release of endogenous glutamate from presynaptic terminals, in the presence of the NMDAR co-agonist glycine, leads to a long-lasting increase in surface AMPAR levels, as measured by [(3)H]-AMPA binding; the increase is prevented by an NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5). Importantly, we observe an increase in the levels of GluR1 and GluR2 AMPAR subunits in the postsynaptic density (PSD) fraction, without changes in total AMPAR levels, consistent with the trafficking of AMPARs from internal synaptosomal compartments into synaptic sites. This plasticity is reversible, as the application of AMPA after LTP depotentiates synaptosomes. Moreover, depotentiation requires proteasome-dependent protein degradation. CONCLUSIONS/SIGNIFICANCE: Together, the results indicate that the minimal machinery required for LTP is present and functions locally within isolated dendritic spines. collapse abstract

Developmental course of EphA4 cellular and subcellular localization in the postnatal rat hippocampus.

From embryonic development to adulthood, the EphA4 receptor and several of its ephrin-A or -B ligands are expressed in the hippocampus, where they presumably play distinct roles at different developmental stages. To help clarify these diverse roles i... expand abstractn the assembly and function of the hippocampus, we examined the cellular and subcellular localization of EphA4 in postnatal rat hippocampus by light and electron microscopic immunocytochemistry. On postnatal day (P) 1, the EphA4 immunostaining was robust in most layers of CA1, CA3, and dentate gyrus and then decreased gradually, until P21, especially in the cell body layers. At the ultrastructural level, focal spots of EphA4 immunoreactivity were detected all over the plasma membrane of pyramidal and granule cells, between P1 and P14, from the perikarya to the dendritic and axonal extremities, including growth cones and filopodia. This cell surface immunoreactivity then became restricted to the synapse-associated dendritic spines and axon terminals by P21. In astrocytes, the EphA4 immunolabeling showed a similar cell surface redistribution, from the perikarya and large processes at P1-P7, to small perisynaptic processes at P14-P21. In both cell types, spots of EphA4 immunoreactivity were also detected, with an incidence decreasing with maturation, on the endoplasmic reticulum, Golgi apparatus, and vesicles, organelles involved in protein synthesis, posttranslational modifications, and transport. The cell surface evolution of EphA4 localization in neuronal and glial cells is consistent with successive involvements in the developmental movements of cell bodies first, followed by process outgrowth and guidance, synaptogenesis, and finally synaptic maintenance and plasticity. collapse abstract

Pre-synaptic and post-synaptic localization of EphA4 and EphB2 in adult mouse forebrain.

The ephrin receptors EphA4 and EphB2 have been implicated in synaptogenesis and long-term potentiation in the cerebral cortex and hippocampus, where they are generally viewed as post-synaptic receptors. To determine the precise distribution of EphA4 ... expand abstractand EphB2 in mature brain synapses, we used subcellular fractionation and electron microscopy to examine the adult mouse forebrain/midbrain. EphA4 and EphB2 were both enriched in microsomes and synaptosomes. In synaptosomes, they were present in the membrane and the synaptic vesicle fractions. While EphA4 was tightly associated with PSD-95-enriched post-synaptic density fractions, EphB2 was easily extracted with detergents. In contrast, both receptors were found in the pre-synaptic active zone fraction. By electron microscopy, EphA4 was mainly detected in axon terminals, whereas EphB2 was more frequently detected in large dendritic shafts, in the hippocampus and cerebral cortex. However, in the ventrobasal thalamus, EphB2 was detected most frequently in axon terminals and thin dendritic shafts. The localization of EphA4 and EphB2 in multiple compartments of neurons and synaptic junctions suggests that they interact with several distinct scaffolding proteins and play diverse roles at synapses. collapse abstract

EphA4 signaling regulates phospholipase Cgamma1 activation, cofilin membrane association, and dendritic spine morphology.

Specialized postsynaptic structures known as dendritic spines are the primary sites of glutamatergic innervation at synapses of the CNS. Previous studies have shown that spines rapidly remodel their actin cytoskeleton to modify their shape and this h... expand abstractas been associated with changes in synaptic physiology. However, the receptors and signaling intermediates that restructure the actin network in spines are only beginning to be identified. We reported previously that the EphA4 receptor tyrosine kinase regulates spine morphology. However, the signaling pathways downstream of EphA4 that induce spine retraction on ephrin ligand binding remain poorly understood. Here, we demonstrate that ephrin stimulation of EphA4 leads to the recruitment and activation of phospholipase Cgamma1 (PLCgamma1) in heterologous cells and in hippocampal slices. This interaction occurs through an Src homology 2 domain of PLCgamma1 and requires the EphA4 juxtamembrane tyrosines. In the brain, PLCgamma1 is found in multiple compartments of synaptosomes and is readily found in postsynaptic density fractions. Consistent with this, PLC activity is required for the maintenance of spine morphology and ephrin-induced spine retraction. Remarkably, EphA4 and PLC activity modulate the association of the actin depolymerizing/severing factor cofilin with the plasma membrane. Because cofilin has been implicated previously in the structural plasticity of spines, this signaling may enable cofilin to depolymerize actin filaments and restructure spines at sites of ephrin-EphA4 contact. collapse abstract

Localization of EphA4 in axon terminals and dendritic spines of adult rat hippocampus.

Eph receptors and their ephrin ligands assume various roles during central nervous system development. Several of these proteins are also expressed in the mature brain, and notably in the hippocampus, where EphA4 and ephrins have been shown to influe... expand abstractnce dendritic spine morphology and long-term potentiation (LTP). To examine the cellular and subcellular localization of EphA4 in adult rat ventral hippocampus, we used light and electron microscopic immunocytochemistry with a specific polyclonal antibody against EphA4. After immunoperoxidase labeling, EphA4 immunoreactivity was found to be enriched in the neuropil layers of CA1, CA3, and dentate gyrus. In all examined layers of these regions, myelinated axons, small astrocytic leaflets, unmyelinated axons, dendritic spines, and axon terminals were immunolabeled in increasing order of frequency. Neuronal cell bodies and dendritic branches were immunonegative. EphA4-labeled dendritic spines and axon terminals corresponded to 9-19% and 25-40% of the total number of spines and axon terminals, respectively. Most labeled spines were innervated by unlabeled terminals, but synaptic contacts between two labeled elements were seen. The vast majority of synaptic junctions made by labeled elements was asymmetrical and displayed features of excitatory synapses. Immunogold labeling of EphA4 was located mostly on the plasma membrane of axons, dendritic spines, and axon terminals, supporting its availability for surface interactions with ephrins. The dual preferential labeling of EphA4 on pre- or postsynaptic specializations of excitatory synapses in adult rat hippocampus is consistent with roles for this receptor in synaptic plasticity and LTP. collapse abstract

Membrane-associated guidance cues direct the innervation of forebrain and midbrain by dorsal raphe-derived serotonergic axons.

Unlike many neurons that extend an axon precisely to a single target, individual dorsal raphe 5-HT neurons project to multiple brain regions and their axon terminals often lack classical synaptic specializations. It is not known how 5-HT axon collate... expand abstractrals select between multiple target fields, or even if 5-HT axons require specific guidance cues to innervate their targets. Nor is it known how these axon collaterals are restrained within specific innervation target regions. To investigate this, we challenged explants of dorsal raphe with co-explants, or cell membrane preparations of ventral midbrain, striatum or cerebral cortex. We provide evidence for membrane-associated cues that promote 5-HT axon growth into each of these three target regions. The axon growth-promoting activity was heat-, protease- and phosphatidylinositol-phospholipase-C (PI-PLC)-sensitive. Interestingly, 5-HT axons specifically lost the ability to grow in heterotypic explants, or membrane carpets, following contact with ventral midbrain or striatal, but not cortical, explants or membranes. This inductive activity associated with striatal and ventral midbrain membranes was sensitive to both high salt extraction and PI-PLC treatment. By contrast, the activity that inhibited 5-HT axon growth onto heterotypic membranes was sensitive only to high salt extraction. These results provide evidence that a glycosylphosphatidylinositol (GPI)-linked membrane protein promotes 5-HT axon growth, and that short-range membrane-bound, as well as GPI-linked, molecules contribute to the guidance of 5-HT axon collaterals. These findings suggest that 5-HT axon collaterals acquire a target-induced growth-inhibitory response to alternative targets, increasing their selectivity for the newly innervated field. collapse abstract

Seeking axon guidance molecules in the adult rat CNS.

Differences in host serotonin innervation of intrastriatal grafts are not determined by a glial scar or chondroitin sulfate proteoglycans.

Serotoninergic (5-HT) neurons of adult recipients provide a much denser innervation of striatal than ventral mesencephalic grafts implanted into the neostriatum of the rat. Moreover, grafts from both brain regions are more innervated by host 5-HT axo... expand abstractns after implantation in neonatal than adult hosts. To test the hypothesis that differences in glial scarring or expression of the growth inhibitory molecules, chondroitin sulfate proteoglycans (CSPG), be responsible for these differences in 5-HT innervation of neural grafts, we examined the 5-HT innervation, the astroglial reaction and the expression of CSPG in ventral mesencephalic grafts implanted into newborn (1-5 days old), juvenile (15 days old), or adult rats and in striatal grafts implanted in adult rats, using immunohistochemistry against 5-HT, glial fibrillary acidic protein (GFAP) and CSPG. Immunostaining for GFAP showed a stronger initial gliosis (1-10 days after grafting) in neonatal than adult recipients of mesencephalic grafts, but this gliosis subsided gradually at later time points. Nevertheless, a glial scar formed at the graft-host interface in both neonatal and adult recipients, 5-10 days after transplantation, although it decreased over a longer time course--up to 60 days--in adults. Immunostained astrocytes appeared first in the host brain tissue around the graft and then immunoreactive processes and perikarya gradually invaded the graft. Immunoreactivity for CSPG was similar in neonatal and adult hosts: it was strongly expressed inside the graft early after transplantation, and almost completely down-regulated at 60 days. The reaction of adult hosts to striatal and mesencephalic grafts was similar, although GFAP was more heterogeneously distributed and CSPG immunoreactivity remained in patches inside striatal grafts, even after 60 days. The 5-HT innervation of mesencephalic grafts was much denser after implantation in newborns than in adults. It was also stronger in striatal than in mesencephalic grafts implanted in adults. Thus, the presence of a glial scar or the expression of CSPG cannot totally account for the different degrees of 5-HT innervation in the various types of neural grafts. collapse abstract

Astrocytes from cerebral cortex or striatum attract adult host serotoninergic axons into intrastriatal ventral mesencephalic co-grafts.

The identification of axon growth inhibitory molecules offers new hopes for repair of the injured CNS. However, the navigational ability of adult CNS axons and the guidance cues they can recognize are still essentially unknown. Astrocytes may express... expand abstract guidance molecules and are known to have different regional phenotypes. To evaluate their influence on the affinity of adult serotoninergic (5-HT) axons for a projection target, we co-implanted astrocytes from the neonatal striatum, cortex, or ventral mesencephalon together with fetal ventral mesencephalic tissue into the striatum of adult rats. Two months after surgery, quantification after in vitro 5-[1,2-(3)H]serotonin ([(3)H]5-HT) uptake and autoradiography showed that ventral mesencephalic grafts with co-grafted cortical or striatal astrocytes were four times and three times, respectively, more densely innervated by host 5-HT axons than control ventral mesencephalic grafts with or without co-grafted ventral mesencephalic astrocytes. Immunohistochemistry for glial fibrillary acidic protein, vimentin, or chondroitin-sulfate proteoglycans revealed no qualitative or quantitative differences in host astroglial scar or production of inhibitory molecules that could explain these differences in 5-HT innervation. These results demonstrate that astrocytes grown in culture from different brain regions have the potential to influence the growth and maintenance of adult 5-HT axons in a graft of neural tissue from another brain region. It should now be feasible to identify the molecules expressed by cultured cortical or striatal, but not by ventral mesencephalic, astrocytes that have these tropic actions on 5-HT axons of the neostriatum. collapse abstract

Severe neuronal losses with age in the parietal cortex and ventrobasal thalamus of mice transgenic for the human NF-L neurofilament protein.

Transgenic mice expressing human light neurofilament protein (NF-L) display early perikaryal accumulations of disarrayed neurofilaments in layers II/III of the parietal cortex and in the ventrobasal complex of thalamus. This cytoskeletal abnormality,... expand abstract reflected by strong NF-L immunoreactivity, is transient in the developing cortex but persists until old age in the thalamus. To investigate whether it leads to neuronal death, the unbiased cell counting method of the dissector was applied to the parietal cortex and the thalamus of normal and transgenic mice at various postnatal (P10, P20, P90) and advanced ages (14-18 months). Similar data were also obtained from the primary visual cortex free of NF-L accumulation. Compared with normal, the total number of neurons in the parietal (but not occipital) cortex of transgenic mice showed little change during the postnatal period, but decreased markedly with old age, particularly in layers II/III. Severe neuronal loss was also documented in the thalamic ventrobasal complex of aged transgenic mice. The delayed neuronal death in the parietal cortex, occurring long after recovery from the NF-L accumulations, was suggestive of a combination of deleterious factors, including the early overproduction of neurofilament protein and subsequent loss of afferent input from the affected somatosensory thalamic nuclei. Furthermore, strong accumulation of lipofuscin in the neurons of aged transgenic mice suggested that oxidative stress partakes in the mechanisms through which NF-L overproduction compromises neuronal viability. collapse abstract

Serotonin axons of the neostriatum show a higher affinity for striatal than for ventral mesencephalic transplants: a quantitative study in adult and immature recipient rats.

We previously showed that grafts of fetal ventral mesencephalic tissue are practically not innervated by host serotonin (5-HT) axons after implantation into the striatum of rats aged more than 14 days, at variance with transplants of cortical or stri... expand abstractatal tissue into the adult striatum, which are well innervated by these axons. Using 5-HT immunohistochemistry and in vitro [3H]5-HT uptake/autoradiography, we have examined and quantified the innervation of ventral mesencephalic versus striatal grafts several months after implantation into the striatum of neonatal (postnatal day 5 or P5), juvenile (P15), and adult rats. Ventral mesencephalic grafts implanted in P5 rats received a moderate 5-HT innervation, while similar grafts implanted in P15 or adult recipients were almost free of any 5-HT fibers (-80%, compared to P5). The density of 5-HT innervation showed a tendency toward higher values in striatal than in ventral mesencephalic grafts (1.6-2 times higher in P5 and adult recipients; 4 times higher in P15 recipients). The difference was more striking, and significant, when only the true striatal portions of the striatal grafts were considered, i.e., DARPP-32-immunopositive areas (4-5 times higher in P5 and adult recipients; 10 times higher in P15 recipients). Accordingly, these DARPP-32-positive areas were also more densely innervated than the DARPP-32-negative zones of the same grafts (3 times higher at any age). The 5-HT innervation density also decreased with increasing age of the recipients in DARPP-32-positive, as well as DARPP-32-negative compartments of the striatal grafts (-75% in adults), but this decrease appeared more gradual (-50% in juveniles) than with mesencephalic grafts. It is concluded that the 5-HT axons innervating the neostriatum have a better affinity for striatal grafts than for ventral mesencephalic grafts or the nonstriatal portions of striatal grafts. In adulthood, the relative affinity of these axons for the different types of grafts is maintained, even though their growth capacity decreases irrespective of the target tissue considered. This experimental model may prove useful for the identification of the receptors and ligands that are responsible for target recognition by 5-HT axons and to test the possibility that the progressive decrease of axonal growth capacity from neonatal age to adulthood be related to a downregulation of such molecules. collapse abstract

Trophic and tropic effects of striatal astrocytes on cografted mesencephalic dopamine neurons and their axons.

Astrocytes from the ventral mesencephalon and from the striatum respectively promote the dendritic and axonal arborization of dopamine (DA) neurons in vitro. To test this response in vivo, astrocytes in primary cultures from the neonatal cerebral cor... expand abstracttex, ventral mesencephalon, or striatum were coimplanted with fetal ventral mesencephalic tissue into the intact or DA-denervated striatum of adult rats and these cografts examined after 3-6 months by tyrosine hydroxylase (TH) immunohistochemistry (intact recipients) or after 5-6 months by in vitro [3H]DA-uptake autoradiography (DA-denervated recipients). In contrast with single ventral mesencephalic grafts, all types of cograft displayed a rather uniform distribution of TH-immunoreactive perikarya. The average size of TH-immunoreactive cell bodies was not significantly different in cografts containing cortical or mesencephalic astrocytes and in single ventral mesencephalic grafts, but it was significantly larger in cografts containing striatal astrocytes. Nevertheless, the number of [3H]DA-labeled terminals in the DA-lesioned host striatum was clearly smaller with cografts of striatal astrocytes than with single mesencephalic grafts or with cografts containing cortical astrocytes. On the other hand, cografts of striatal astrocytes contained much higher numbers of [3H]DA-labeled terminals than the other types of graft or cograft. Thus, while cografted astrocytes in general influence the distribution of DA neurons within the graft, astrocytes from the neonatal striatum have a trophic effect on DA perikarya and a tropic effect on DA axons, keeping the latter within the graft. collapse abstract

Comparative evaluation of 3HWIN 35428 and 3HGBR 12935 as markers of dopamine innervation density in brain.

WIN 35428 and GBR 12935, two uptake blocker ligands of the membrane transporter for dopamine (DA), were evaluated as quantitative markers of DA innervation density in CNS tissue. From alternate rat brain slices respectively processed for either light... expand abstract microscope or film autoradiography, counts of DA axon terminals (varicosities) labeled by uptake/storage of [3H]DA were matched with densitometric measurements of the specific binding of [3H]WIN 35428 and [3H]GBR 12935 in the same anatomical areas. The relation between the two parameters was examined in 1) the normal cingulate cortex; 2) the neostriatum severely DA-denervated by unilateral intramesencephalic injections of 6-hydroxydopamine; and 3) the neostriatum, partly DA-reinnervated by an intrastriatal graft of fetal mesencephalic neurons after prior 6-hydroxydopamine lesion. For technical reasons, the hyperdense DA innervation of normal striatum was not amenable to such correlative testing. Data were subjected to multilevel analysis. Specific [3H]WIN binding at 37 degrees C was tightly and linearly correlated with the number of DA varicosities over the full range of DA innervation densities tested. The regression lines for intact cortex and for DA-denervated as well as DA-reinnervated neostriatum had the same slope and crossed the ordinate near zero. In contrast, [3H]GBR 12935 binding at 37 degrees C showed no correlation with the number of DA varicosities. A linear correlation could be obtained after incubation with [3H]GBR 12935 at 4 degrees C in the presence of ZnSO4, but the intercept of this regression line remained significantly above zero at origin, indicating extraneous binding to non-DA transporter sites. Providing that the hyperdense DA innervation of the normal neostriatum does not generate a particular problem in vivo as it does in vitro. WIN 35428, but not GBR 12935, might satisfy the selectivity and sensitivity requirements of a quantitative marker of DA innervation density for eventual use in positron emission tomographic studies. collapse abstract

Quantitative and morphometric data indicate precise cellular interactions between serotonin terminals and postsynaptic targets in rat substantia nigra.

We have quantified the density of serotonin axonal varicosities, their synaptic incidence and their distribution among potential targets in the pars reticulata and pars compacta of the rat substantia nigra. Serotonin axonal varicosities, counted at t... expand abstracthe light microscopic level following in vitro [3H]serotonin uptake and autoradiography, amounted to 9 x 10(6)/mm3 in the pars reticulata and 6 x 10(6)/mm3 in the pars compacta, among the densest serotonin innervations in brain. As determined at the electron microscopic level following immunolabelling for serotonin, virtually all serotonin varicosities in the pars reticulata and 50% of those in the pars compacta formed a synapse, essentially with dendrites. The combination of serotonin immunocytochemistry with tyrosine hydroxylase immunolabelling of dopamine neurons reveals that 20% of the serotonin synaptic contacts in the pars reticulata are on dopamine dendrites and 6% are on a type of unlabelled dendrite characterized by its peculiarly high cytoplasmic content of microtubules. The comparison of the diameter of the dendritic profiles that were in synaptic contact with serotonin-immunoreactive varicosities with the diameter of all other dendritic profiles of the same type suggests that serotoninergic varicosities innervate dopamine dendrites uniformly along their length, whereas they tend to contact microtubule-filled dendrites in more proximal regions and the other, unidentified dendrites in more distal regions. Furthermore, the size of the serotonin-immunoreactive varicosities and of their synaptic junctions is significantly smaller on dopamine dendrites and larger on microtubule-filled dendrites than on other, unidentified dendrites, indicating that the nature of the postsynaptic target is an important determinant of synaptic dimensions. These data should help to clarify the role of serotonin in the nigral control of motor functions. They indicate that this dense serotonin input to the substantia nigra is very precisely organized, acting through both "non-junctional" and "junctional" modes of neurotransmission in the pars compacta, which projects to the neostriatum and the limbic system, whereas the predominant mode of serotonin transmission appears to be of the "junctional" type in the pars reticulata, where serotonin can finely control the motor output of the basal ganglia by acting on the GABA projection neurons either directly or through the local release of dopamine by dopaminergic dendrites. The data also raise the possibility that the postsynaptic targets have trophic retrograde influences on serotoninergic terminals. collapse abstract

Dual character, asynaptic and synaptic, of the dopamine innervation in adult rat neostriatum: a quantitative autoradiographic and immunocytochemical analysis.

Dopamine (DA) axon terminals (varicosities) in the neostriatum of adult rats were examined for shape, size, content, synaptic incidence, type of junction, synaptic targets, and microenvironment after electron microscopic identification either by [3H]... expand abstractDA uptake autoradiography or by immunocytochemistry with monoclonal antibodies against DA-glutaraldehyde-protein conjugate. Both approaches yielded comparable results. Whether they were from the paraventricular or the mediodorsal neostriatum, respectively, the [3H]DA-labeled and DA-immunostained varicosities were generally oblong and relatively small; more than 60% contained one or more mitochondria. Sixty to seventy percent were asynaptic, and 30-40% were endowed with a synaptic membrane differentiation (junctional complex), as inferred by stereological extrapolation from single thin sections (both approaches) or observed directly in long, uninterrupted series of thin sections (immunocytochemistry). The synaptic DA varicosities always displayed symmetrical junctions: 67% with dendritic branches, 30% with dendritic spines, and 2-3% with neuronal cell bodies. DA varicosities juxtaposed to one another were frequent. Other axonal varicosities were more numerous in the immediate vicinity of DA varicosities than around randomly selected, unlabeled terminals. The respective microenvironments of DA and unlabeled varicosities also showed enrichment in the preferred synaptic targets of both groups of varicosities, with dendritic branches for DA and dendritic spines for the unlabeled ones. These data suggest a dual mode of operation that is diffuse as well as synaptic for the nigrostriatal DA system. In such a densely DA-innervated brain region, they also lead to the hypothesis that a basal level of extracellular DA might be maintained permanently around every tissue constituent and, thus, contribute to the mechanisms of action, properties, and functions (or dysfunctions) of DA within the neostriatum itself and as part of the basal ganglia circuitry. collapse abstract

Efficient immunodetection of various protein antigens in glutaraldehyde-fixed brain tissue.

Optimal ultrastructural preservation of brain tissue for electron microscopy is best achieved with fixatives containing high concentrations of glutaraldehyde, which is generally considered detrimental to the immunogenicity of most protein antigens. W... expand abstracte tested seventeen mono- or polyclonal antibodies against peptide or protein antigens, including a majority for which immunoreactivity had previously been reported to be sensitive to glutaraldehyde fixation. Forebrain sections of rats or mice fixed by perfusion with 3.5% glutaraldehyde were processed for pre-embedding immunocytochemistry by the avidin-biotin method. The resulting immunostaining was in most cases at least similar to that obtained in sections fixed with paraformaldehyde. Immunoreactivity against the mouse or human neurofilament protein NF-L was even improved, being similar to that previously reported for unfixed brain tissue. Of all antigens tested, only choline acetyltransferase, phenylethanolamine-N-methyl transferase, and neuropeptide Y were detected with lower sensitivity than after paraformaldehyde fixation, which was attributed to a rather restricted penetration of the primary antibody into glutaraldehyde-fixed tissue sections. These results indicate that glutaraldehyde may be envisaged as a possible fixative for optimal immunocytochemical detection of any tissue antigen at the electron microscopic level, including antigens which, on the basis of results obtained after fixation with paraformaldehyde-glutaraldehyde mixtures, were considered highly sensitive to glutaraldehyde fixation. collapse abstract

Neuronal expression of human immunodeficiency virus type 1 env proteins in transgenic mice: distribution in the central nervous system and pathological alterations.

It is now well documented that human immunodeficiency virus type 1 (HIV-1) induces encephalopathy in patients with AIDS. In vitro studies have implicated the envelope protein (gp120) as a factor which causes neuronal death. To better evaluate the rol... expand abstracte and elucidate the mechanisms of gp120 neurotoxicity, we have developed transgenic mice carrying a segment of the HIV-1 genome that expresses the viral gp160 protein under the control of the human neurofilament light gene promoter. In two separate lines of transgenic mice, the Env protein was found to be expressed in several nuclei of the brain stem and in the anterior horns of the spinal cord. The two lines showed identical patterns of Env expression. Neuropathological evaluation revealed numerous abnormal dendritic swellings in the immunostained motor neuron structures. Large and numerous neuritic swellings were also prominent in the nucleus gracilis and in the gracilis and cuneate fascicles. In addition, reactive astrocytosis was observed in several immunoreactive areas of the central nervous system. These transgenic mice offer a unique model to further investigate the role of HIV-1 Env protein in neuronal toxicity and to help elucidate the mechanisms that are involved. collapse abstract

Abnormal perikaryal accumulation of neurofilament light protein in the brain of mice transgenic for the human protein: sequence of postnatal development.

Adult mice transgenic for the human form of neurofilament light protein display abnormal perikaryal immunoreactivity for this protein in many regions of the CNS and notably the thalamus. To determine the sequence of development of these anomalies, we... expand abstract have compared normal and transgenic mice of different postnatal ages (P0-P70), using immunocytochemistry with primary antibodies recognizing both murine and human sequence of neurofilament light protein (NR-4) or the human form only (DP5-1-12). In normal mouse brainstem, several nuclei displayed immunoreactive perikarya at P0. The number of these perikarya culminated at P10, followed by a general decrease, some nuclei having lost all perikaryal immunostaining in adults. In transgenic mouse brainstem, the distribution of perikaryal immunoreactivity already resembled at P0 that of P10 in normal mouse, and remained unchanged in adults. Differences between normal and transgenic mice were even more pronounced in the forebrain. Some nuclei of normal mouse basal forebrain that were weakly immunopositive at P10 or P20, but no longer in adults, were already labeled at P0 and remained so or became more intense at later stages in transgenic mice. In the thalamus of normal mouse, perikaryal labeling was faint, confined to a few nuclei, and detected only transiently at P10, whereas in transgenics, it was already observed in some nuclei at P0, increased in intensity and extended to other nuclei at P10, and persisted thereafter. Strongly immunoreactive, inflated perikarya with excentric nuclei were prominent in these thalamic nuclei at P20, and even larger in size at P70. In the cerebral cortex of normal mice, layers II-III and layer V of many cytoarchitectonic areas showed immunoreactive cell bodies at P10, a distribution which became gradually restricted to the parietal cortex in adults. In transgenic mice, immunopositive cortical cell bodies were first detected at P3, filled layers II-III of numerous cortical areas at P10, and then rapidly decreased in number to approach the adult pattern at P20. In the cortex as well as thalamus of P10 transgenic mice, differences between the patterns of cellular staining with clones NR4 and DP5-1-12 antibodies indicated that both the murine and human proteins were accumulated in these neurons. Thus, neurofilament light protein accumulation in the transgenic mouse brain generally involved neurons displaying perikaryal immunoreactivity for the protein at least at some point during normal postnatal development.(ABSTRACT TRUNCATED AT 400 WORDS) collapse abstract

CNS distribution and overexpression of neurofilament light proteins (NF-L) in mice transgenic for the human NF-L: aberrant accumulation in thalamic perikarya.

Light microscopic immunocytochemistry with monoclonal antibodies recognizing both murine and human light neurofilament proteins (mNF-L and hNF-L) or hNF-L only was used to examine the distribution of NF-L in the CNS of adult mice, normal or transgeni... expand abstractc for the human gene. In normal mice, major fiber bundles were immunoreactive to the first antibody, with few exceptions such as the internal capsule, anterior commissure, and corpus callosum. Strong immunoreactivity was also present in the perikarya of motoneurons in the spinal cord and brainstem, as well as in other brainstem nuclei. Faint cell body staining was visible in layers II, III, and V of the parietal cortex and layers V and VI of the retrosplenial cingulate cortex. In transgenic mice, all forebrain as well as brainstem fiber tracts were intensely immunoreactive to both antibodies. Cell body staining was more intense than in normal mouse and involved additional forebrain and brainstem regions, including extended areas of cerebral cortex. Abnormal cell body labeling was particularly striking in several thalamic nuclei, where numerous darkly stained perikarya were considerably enlarged by accumulated immunoreactive material and exhibited eccentric and fragmented nuclei. At the electron microscopic level, these perikarya were filled with disarrayed filaments displacing all other organelles against the cytoplasmic membrane. Such aberrant accumulation of NF-L was presumably the result of an overexpression in selective subpopulations of CNS neurons. It was compatible with prolonged survival of the animal and could provide a new experimental model of neurodegenerative disease. collapse abstract

Host serotonin axons innervate intrastriatal ventral mesencephalic grafts after implantation in newborn rats.

This study investigated the potential of immature and adult serotonin (5-HT) neurons for axonal growth into intrastriatal grafts of ventral mesencephalic tissue. Implantation of dissociated fetal (embryonic days 14-15) ventral mesencephalic tissue wa... expand abstracts carried out in immature [postnatal days (P) 5-14] and adult rat neostriatum. The brains were processed 2-6 months later for dopamine and 5-HT immunocytochemistry. A few grafts implanted into adult and P7 recipients contained small numbers of cotransplanted 5-HT cell bodies. These also displayed a rich network of 5-HT axons, even in adult rats prelesioned with 5,7-dihydroxytryptamine, indicating the graft origin of these axons. All other grafts were totally devoid of 5-HT cell bodies. After implantation in adults, such grafts contained rare 5-HT axons. In contrast, in P5-P7 recipients, they displayed many 5-HT fibres, which were uniformly distributed. Such was no longer the case after implantation in P14 recipients, which showed minimal 5-HT innervation, as in adult recipients. Processing of naïve rat brain at different ages for 5-HT immunocytochemistry showed that 5-HT axons were still clearly less numerous in the neostriatum at P21 than in adults, whereas in the substantia nigra the 5-HT innervation developed more rapidly and was comparable, at P21, to that of adults. It was concluded that 5-HT axons are able to grow into ventral mesencephalic grafts, but mainly at the fetal stage and with decreasing capacity after birth.(ABSTRACT TRUNCATED AT 250 WORDS) collapse abstract

Dopamine neoinnervation in the substantia nigra and hyperinnervation in the interpeduncular nucleus of adult rat following neonatal cerebroventricular administration of 6-hydroxydopamine.

An aberrant network of dopamine axons was found to pervade the rat substantia nigra following neonatal destruction of its dopamine nerve cell bodies and dendrites by cerebroventricular administration of 6-hydroxydopamine. Light-microscopic immunocyto... expand abstractchemistry with a primary monoclonal antibody directed against dopamine-glutaraldehyde-protein was used to investigate the time-course of development and the critical period of induction of this ectopic dopamine innervation (neoinnervation). In rats 6-hydroxydopamine-lesioned at postnatal day 3 (P3) and examined at P7, P10, P15, P30 or later, some dopamine fibers were already present in the substantia nigra at P7; their number increased sharply until P15 and only slightly thereafter, assuming a topographic distribution reminiscent of the missing dopamine nerve cell bodies and dendrites. A similar growth of dopamine fibers took place in the substantia nigra after lesions made at P6, P9 and P12, but was less pronounced after lesion at P15 and absent after lesion at P21 or later. Excessive innervation by dopamine axons (hyperinnervation) was concomitantly observed in the nearby interpeduncular nucleus. The sprouting of dopamine axons in both regions was therefore rapid and coincided in time and space with the developmental redistribution of mesencephalic dopamine neurons in normal rat. It is conceivable that these aberrant dopamine innervations play a role in the peculiar behavior and responsiveness to dopaminergic agents manifested by neonatally 6-hydroxydopamine-lesioned rats. It will be of particular interest to investigate the functional consequences of the dopamine neoinnervation in the substantia nigra, where an eventual axonal release might thus be replacing the normal somatodendritic release of this amine. collapse abstract

Host striatal projections into fetal ventral mesencephalic tissue grafted to the striatum of immature or adult rat.

We have previously reported that few striatal axons from adult host brain innervate intrastriatal grafts of fetal ventral mesencephalic tissue. To see whether the immature rat brain would favor striatal innervation of the graft, unilateral implantati... expand abstracton of fetal ventral mesencephalic tissue was carried out at 7 (P7), 14 (P14), or 60 (adults) days of age in neonatally dopamine-(DA)-lesioned and nonlesioned rats. Immunocytochemistry for tyrosine hydroxylase (TH), and/or dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein-32 (DARPP-32) was performed 2-6 months later. In the great majority of immature and in all adult recipients, the resulting graft consisted of a distinct intrastriatal mass of tissue surrounded by the host parenchyma. Most TH-immunopositive neurons were found within the confines of such grafts, although some were lying at short distances into the host striatal tissue, particularly in immature recipients. In a few immature recipients, there was, however, extensive intermingling of TH-positive neurons with the adjacent host brain tissue. In all recipients grafted at P7, P14, or as adults, the distinct, intraparenchymal grafts contained moderate numbers of DARPP-32-positive processes, mainly at their periphery. These results indicate that the limited capacity of host striatal neurons to grow axons into transplanted fetal ventral mesencephalic tissue is not markedly different in young versus adult rats. A better integration of the ventral mesencephalic graft into the striatal circuitry of immature--as opposed to adult--recipients should therefore rely more on the higher tendency of DA neurons to become located into the host tissue following transplantation in young rats. collapse abstract

Ultrastructure and synaptic targets of the raphe-nigral projection in the rat.

A major input to the substantia nigra is from the 5-hydroxytryptamine-containing neurons in the dorsal raphe nucleus. In order to examine the morphology and distribution of this projection, rats were given injections of the anterograde tracers, Phase... expand abstractolus vulgaris-leucoagglutinin or biocytin, in the dorsal raphe nucleus and the substantia nigra was examined at both the light and electron microscopic levels. In addition, sections of the substantia nigra were immunostained for 5-hydroxytryptamine and examined in both the light and electron microscopes. Since dopaminergic neurons of the substantia nigra are known to be responsive to stimulation of the raphe and to applied 5-hydroxytryptamine, sections that contained anterogradely labelled terminals were further processed to reveal tyrosine hydroxylase immunoreactivity to determine whether the raphe input makes direct synaptic contact with dopaminergic neurons. Light microscopic analysis revealed that all divisions of the substantia nigra received input from the dorsal raphe which, in agreement with previous observations, showed a topographical organization. In that formed asymmetrical synaptic contact with dendritic shafts and spines. The synaptic boutons were often associated with subjunctional dense bodies. Terminals that displayed immunoreactivity for 5-hydroxytryptamine had a similar morphology, synaptic specialisations and postsynaptic targets to the anterogradely labelled terminals. In those sections that were stained for both anterogradely labelled terminals and tyrosine hydroxylase, the raphe-nigral terminals were seen to form asymmetrical synaptic contact with the dendrites of the dopaminergic neurons. It is concluded that dendrites of dopaminergic neurons in the substantia nigra pars reticulata represent at least one of the synaptic targets of the raphe-nigral projection and that these contacts provide an anatomical substrate for the effects of the dorsal raphe, and presumably 5-hydroxytryptamine, on dopaminergic systems in the substantia nigra. collapse abstract

Ultrastructural analysis of the serotonin hyperinnervation in adult rat neostriatum following neonatal dopamine denervation with 6-hydroxydopamine.

Serotonin (5-HT) immunocytochemistry was used at the electron microscopic level to characterize the ultrastructural features of 5-HT axon terminals (varicosities) hyperinnervating the neostriatum of adult rats, 3 months after a neonatal destruction o... expand abstractf the nigrostriatal dopamine system by intraventricular 6-hydroxydopamine. 5-HT-immunostained terminals from the anterior half of the hyperinnervated neostriatum were examined in single thin sections, and compared to their counterparts in vehicle-injected controls with respect to shape, size, organelle content, presence of a synaptic membrane differentiation and composition of the microenvironment. The intrinsic and relational features of the 5-HT-immunostained varicosities were essentially the same in 5-HT-hyperinnervated and control tissue. In particular, the frequency with which these varicosities made synaptic contacts was similarly low in both conditions (6-8% for whole varicosities), as already described in normal adult rat neostriatum. The distributional frequency of elements juxtaposed to the 5-HT-immunostained varicosities was also comparable in control and 5-HT-hyperinnervated tissue. However, in both conditions, there were much fewer dendritic spines in the microenvironment of 5-HT varicosities than around unlabeled terminals randomly selected from the same thin sections. This difference seemed entirely due to the numerous axo-spinous synaptic contacts made by the randomly selected, unlabeled varicosities. Together with recent observations on the 5-HT-hyperinnervation of adult rat hippocampus after grafts of fetal neurons, these data lead to the suggestion that mostly non-junctional neostriatal 5-HT terminals are not committed to a specific intratissular microenvironment. This might in part explain why they grow in excess when reinnervating adult tissue after a lesion or a graft. collapse abstract