Guy Doucet

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]DA 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 dead 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.

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 of 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.

Serotonin innervation in adult rat neostriatum. I. Quantified regional distribution

The distributional features of the serotonin (5-HT) innervation in adult rat neostriatum were examined and quantified using two complementary chemoanatomical methods: 5-HT-immunohistochemistry on serial histological sections and radioautography after [3H]5-HT uptake in whole cerebral hemisphere slices. As visualized and measured after peroxidase-antiperoxidase immunostaining, the neostriatal 5-HT fiber network pervading the entire neostriatum was 2-3 times denser in its ventral than dorsal parts, and showed a slight rostrocaudal increase in density. Its axonal length ranged from 1.06 to 4.18 m per mm3 of striatal tissue. Radioautographic counts of the [3H]5-HT-labeled axon varicosities within comparable sectors of the neostriatum showed good correlation with this distribution pattern. As extrapolated after appropriate corrections for incomplete detection at the chosen exposure time and from the thickness of sections examined, the number of neostriatal 5-HT varicosities (innervation density) ranged from 1.5 to 4.8 millions and averaged 2.6 millions per mm3 of tissue. These quantitative results provided new insights into the topographical organization of the dorsal raphe-neostriatal 5-HT projection system. They already allow for meaningful correlations with currently available microchemical data on intrastriatal 5-HT levels and should also be of considerable significance when as precise information becomes available on the number and localization of different 5-HT receptors and uptake carriers within rat neostriatum.

EphA4 Signaling Regulates Phospholipase Cγ1 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 has 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 Cγ1 (PLCγ1) in heterologous cells and in hippocampal slices. This interaction occurs through an Src homology 2 domain of PLCγ1 and requires the EphA4 juxtamembrane tyrosines. In the brain, PLCγ1 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.

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 influence 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. J. Comp. Neurol. 501:691–702, 2007.

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 and 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.

DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain

The transmembrane protein deleted in colorectal cancer (DCC) and its ligand, netrin-1, regulate synaptogenesis during development, but their function in the mature central nervous system is unknown. Given that DCC promotes cell-cell adhesion, is expressed by neurons, and activates proteins that signal at synapses, we hypothesized that DCC expression by neurons regulates synaptic function and plasticity in the adult brain. We report that DCC is enriched in dendritic spines of pyramidal neurons in wild-type mice, and we demonstrate that selective deletion of DCC from neurons in the adult forebrain results in the loss of long-term potentiation (LTP), intact long-term depression, shorter dendritic spines, and impaired spatial and recognition memory. LTP induction requires Src activation of NMDA receptor (NMDAR) function. DCC deletion severely reduced Src activation. We demonstrate that enhancing NMDAR function or activating Src rescues LTP in the absence of DCC. We conclude that DCC activation of Src is required for NMDAR-dependent LTP and certain forms of learning and memory.

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 in 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. J. Comp. Neurol. 512:798–813, 2009.

Comparative evaluation of [3H]WIN 35428 and [3H]GBR 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 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°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°C showed no correlation with the number of DA varicosities. A linear correlation could be obtained after incubation with [3H]GBR 12935 at 4°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. Synapse 25:163–175, 1997.

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. We 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.