Regional expression and ultrastructural localization of EphA7 in the hippocampus and cerebellum of adult rat.

EphA7 is expressed in the adult central nervous system (CNS), where its roles are yet poorly defined. We mapped its distribution using in situ hybridization (ISH) and immunohistochemistry (IHC) combined with light (LM) and electron microscopy (EM) in adult rat and mouse brain. The strongest ISH signal was in the hippocampal pyramidal and granule cell layers. Moderate levels were detected in habenula, striatum, amygdala, the cingulate, piriform and entorhinal cortex, and in cerebellum, notably the Purkinje cell layer. The IHC signal distribution was consistent with ISH results, with transport of the protein to processes, as exemplified in the hippocampal neuropil layers and weakly stained pyramidal cell layers. In contrast, in the cerebellum, the Purkinje cell bodies were the most strongly immunolabeled elements. EM localized the cell surface-expression of EphA7 essentially in postsynaptic densities (PSDs) of dendritic spines and shafts, and on some astrocytic leaflets, in both hippocampus and cerebellum. Perikaryal and dendritic labeling was mostly intracellular, associated with the synthetic and trafficking machineries. Immunopositive vesicles were also observed in axons and axon terminals. Quantitative analysis in EM showed significant differences in the frequency of labeled elements between regions. Notably, labeled dendrites were ∼3-5 times less frequent in cerebellum than in hippocampus, but they were individually endowed with ∼10-40 times higher frequencies of PSDs, on their shafts and spines. The cell surface localization of EphA7, being preferentially in PSDs, and in perisynaptic astrocytic leaflets, provides morphologic evidence that EphA7 plays key roles in adult CNS synaptic maintenance, plasticity, or function. J. Comp. Neurol. 524:2462-2478, 2016. © 2016 Wiley Periodicals, Inc.

EphA4 is Involved in Sleep Regulation but Not in the Electrophysiological Response to Sleep Deprivation.

STUDY OBJECTIVES: Optimal sleep is ensured by the interaction of circadian and homeostatic processes. Although synaptic plasticity seems to contribute to both processes, the specific players involved are not well understood. The EphA4 tyrosine kinase receptor is a cell adhesion protein regulating synaptic plasticity. We investigated the role of EphA4 in sleep regulation using electrocorticography in mice lacking EphA4 and gene expression measurements. METHODS: EphA4 knockout (KO) mice, Clock(Δ19/Δ19) mutant mice and littermates, C57BL/6J and CD-1 mice, and Sprague-Dawley rats were studied under a 12 h light: 12 h dark cycle, under undisturbed conditions or 6 h sleep deprivation (SLD), and submitted to a 48 h electrophysiological recording and/or brain sampling at different time of day. RESULTS: EphA4 KO mice showed less rapid eye movement sleep (REMS), enhanced duration of individual bouts of wakefulness and nonrapid eye movement sleep (NREMS) during the light period, and a blunted daily rhythm of NREMS sigma activity. The NREMS delta activity response to SLD was unchanged in EphA4 KO mice. However, SLD increased EphA4 expression in the thalamic/hypothalamic region in C57BL/6J mice. We further show the presence of E-boxes in the promoter region of EphA4, a lower expression of EphA4 in Clock mutant mice, a rhythmic expression of EphA4 ligands in several brain areas, expression of EphA4 in the suprachiasmatic nuclei of the hypothalamus (SCN), and finally an unchanged number of cells expressing Vip, Grp and Avp in the SCN of EphA4 KO mice. CONCLUSIONS: Our results suggest that EphA4 is involved in circadian sleep regulation.

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.

The HEADS-ED: a rapid mental health screening tool for pediatric patients in the emergency department.

BACKGROUND AND OBJECTIVE: The American Academy of Pediatrics called for action for improved screening of mental health issues in the emergency department (ED). We developed the rapid screening tool home, education, activities/peers, drugs/alcohol, suicidality, emotions/behavior, discharge resources (HEADS-ED), which is a modification of "HEADS," a mnemonic widely used to obtain a psychosocial history in adolescents. The reliability and validity of the tool and its potential for use as a screening measure are presented. METHODS: ED patients presenting with mental health concerns from March 1 to May 30, 2011 were included. Crisis intervention workers completed the HEADS-ED and the Child and Adolescent Needs and Strengths-Mental Health tool (CANS MH) and patients completed the Children's Depression Inventory (CDI). Interrater reliability was assessed by using a second HEADS-ED rater for 20% of the sample. RESULTS: A total of 313 patients were included, mean age was 14.3 (SD 2.63), and there were 182 females (58.1%). Interrater reliability was 0.785 (P < .001). Correlations were computed for each HEADS-ED category and items from the CANS MH and the CDI. Correlations ranged from r = 0.17, P < .05 to r = 0.89, P < .000. The HEADS-ED also predicted psychiatric consult and admission to inpatient psychiatry (sensitivity of 82% and a specificity of 87%; area under the receiver operator characteristic curve of 0.82, P < .01). CONCLUSIONS: The results provide evidence to support the psychometric properties of the HEADS-ED. The study shows promising results for use in ED decision-making for pediatric patients with mental health concerns.

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 partner 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.

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 expression 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.

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.

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.

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 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 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.

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.

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 collaterals 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.

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 axons 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.