Transient up-regulation of retinal EphA3 and EphA5, but not ephrin-A2, coincides with re-establishment of a topographic map during optic nerve regeneration in goldfish

Eph tyrosine kinase receptors and their ligands, the ephrins, play a key role in the establishment of retinotectal topography during development. Tectal up-regulation of ephrin-A2 in goldfish, coincident with the reestablishment of a retinotectal map, suggests a similar role during optic nerve regeneration. Here we report a complementary study of EphA3, EphA5 and ephrin-A2 expression in the retina. EphA3 and EphA5 are transiently up-regulated as ascending naso-temporal gradients, whereas ephrin-A2 remains uniform. The expression profiles differ from those in developing chick and mouse, suggesting that different combinations of retinal Eph receptors and ligands can generate topographic guidance information.     

EphA/ephrin-A interactions during optic nerve regeneration: restoration of topography and regulation of ephrin-A2 expression

During visual system development, interactions between Eph tyrosine kinase receptors and their ligands, the ephrins, guide retinal ganglion cell (RGC) axons to their topographic targets in the optic tectum. Here we show that Eph/ephrin interactions are also involved in restoring topography during RGC axon regeneration in goldfish. Following optic nerve crush, EphA/ephrin-A interactions were blocked by intracranial injections of recombinant Eph receptor (EphA3-AP) or phospho-inositol phospholipase-C. Topographic errors with multiple inputs to some tectal loci were detected electrophysiologically and increased projections to caudal tectum demonstrated by RT-97 immunohistochemistry. In EphA3-AP-injected fish, ephrin-A2-expressing cells in the retino-recipient tectal layers were reduced in number compared to controls and their distribution was no longer graded. The findings, supported by in vitro studies, implicate EphA/ephrin-A interactions in restoring precise topography and in regulating ephrin-A2 expression during regeneration.     

Development of the hippocamposeptal projection in the rat

We analyzed the development of the hippocamposeptal projection and the morphology of the neurons giving rise to this projection. The fluorescent tracer Dil was injected into the septal region or the hippocampus in fixed brains of embryonic and early postnatal rats. Anterogradely labeled hippocampal axons first reached the septal region at E16. They ran along the midline of the brain, thereby approaching the medial septum. Axons to the lateral septum were first observed around E18/19. The lateral septum is partly innervated by collaterals of axons that travel to the medial septum. The projection to the lateral septal nuclei becomes more massive during early postnatal stages, whereas that to the medial septum becomes smaller. Cells in the medial septum retrogradely labeled by injection into the hippocampus were first observed at E18. Thus, the hippocamposeptal projection is established earlier than the septohippocampal projection. The first hippocampal projection neurons are nonpyramidal neurons that appear to pioneer the pathway to the septum. Pyramidal cell axons follow this first cohort of axons into the medial septum. Pyramidal cells could be retrogadely labeled from the medial septum during the perinatal period but then diminished in number. At P10, only nonpyramidal cells were labeled by medial septal injections. This indicates that the pyramidal component of this projection is transient and is removed shortly after birth. However, as is known from ther studies, hippocampal pyramidal cells give rise to a powerful projection to the lateral septum in adult animals. Our results show that there is a considerable remodeling of the projection from the hippocampus to the septum during ontogenetic development. © 1995 Willy-Liss, Inc.     

EphA receptors and ephrin-A ligands exhibit highly regulated spatial and temporal expression patterns in the developing olfactory system

The spatiotemporal expression patterns of the chemorepulsive EphA receptors, EphA4 and EphA7, and three ephrins-A2, A4 and A5, were examined in the developing rat primary olfactory system. Unlike the visual system that has simple and stable gradients of Ephs and ephrins, the olfactory system demonstrates complex spatiotemporal expression patterns of these molecules. Using immunohistochemistry, we demonstrate that expression of these molecules is dynamic and tightly regulated both within and between different cell types. We reveal restricted targeting of these proteins within subcellular compartments of some neurons. EphA4, ephrin-A2 and ephrin-A5 were expressed by primary olfactory axons during the embryonic formation of the olfactory nerve. There were no gradients in expression along the rostrocaudal or ventrodorsal axes in the nasal cavity and olfactory bulb. However, during the early neonatal period, axons expressing different levels of ephrin-A5 sorted out and terminated in a subpopulation of glomeruli that were mosaically dispersed throughout the bulb. The expression of EphA4 and ephrin-A2 was dramatically down-regulated on all axons during the early neonatal period of glomerular formation. The uniform co-expression of receptors and ligands before glomerular formation suggests they play a generic role in axon-axon interactions in the olfactory nerve and nerve fibre layer. In contrast, loss of EphA4 from axons during glomerular formation may facilitate the interaction of ephrin-A5 with Eph receptors on target cells in the bulb. While EphA4, EphA5 and EphA7 are not mosaically expressed by bulbar neurons, other Eph receptors may have expression patterns complementary to the ephrin-A5-positive subpopulation of glomeruli.     

EphA7-ephrin-A5 signaling in mouse somatosensory cortex: developmental restriction of molecular domains and postnatal maintenance of functional compartments

Members of the Eph family of receptor tyrosine kinases and their ligands, the ephrins, are expressed in distinct patterns in the forming cortex. EphA7 is expressed early in cortical development, becoming concentrated in anterior and posterior domains, whereas ephrin-A5 is expressed later in corticogenesis, highest in the middle region that has low levels of EphA7. The EphA7 gene produces full-length and truncated isoforms, which are repulsive and adhesive, respectively. Analysis of cortical RNA expression demonstrates that proportions of these isoforms change with time, from a more repulsive mix during embryogenesis to a more permissive mix postnatally. To examine how EphA7 and ephrin-A5 influence the formation of cortical regions, EphA7-/- mice were analyzed. Within the cortex of EphA7-/- mice, the distribution of ephrin-A5 was more extensive, encompassing its usual medial domain but also extending more posteriorly toward the occipital pole. Moreover, relative levels of ephrin-A5 along the cortex's anatomical axes changed in EphA7-/- animals, creating less striking shifts in ligand abundance. Furthermore, in vivo functional studies revealed that EphA7 exerts a repulsive influence on ephrin-A5-expressing cells during corticogenesis. In contrast, EphA7 appears to mediate permissive interactions in the postnatal cortex: the area of somatosensory cortex was significantly reduced in EphA7-/- mice. A similar reduction was present in ephrin-A5-/- animals and a more pronounced decrease was observed in EphA7/ephrin-A5-/- cortex. Taken together, this study supports a role for EphA7 and ephrin-A5 in the establishment and maintenance of certain cortical domains and suggests that the nature of their interactions changes with cortical maturity.     

Expression of EphA4, ephrin-A2 and ephrin-A5 during axon outgrowth to the hindlimb indicates potential roles in pathfinding

During neural development, spinal motor axons extend in a precise manner from the ventral portion of the developing spinal cord to innervate muscle targets in the limb. Although classical studies in avians have characterized the cellular interactions that influence motor axon pathfinding to the limb, less is known about the molecular mechanisms that mediate this developmental event. Here, we examine the spatiotemporal distributions of the EphA4 receptor tyrosine kinase (RTK) and its cognate ligands, ephrin-A2 and ephrin-A5, on motor neurons, their axons and their pathways to the avian hindlimb to determine whether these molecules may influence axonal projections. The expression patterns of EphA4, ephrin-A2 and ephrin-A5 mRNAs and proteins are highly complex and appear to exhibit some overlap during motor axon outgrowth and pathfinding to the hindlimb, reminiscent of the co-expression of Eph RTKs and ephrins in the retinotectal system. EphA4, similar to the carbohydrate moiety polysialic acid, strikingly marks the main dorsal, but not ventral, nerve trunk after axon sorting at the limb plexus region. Our results suggest that EphA4 RTK and its ligands may influence axon fasciculation and the sorting of axons at the limb plexus, contributing to the correct dorsoventral organization of nerve branches in the hindlimb.     

EphA/ephrin-A interactions regulate epileptogenesis and activity-dependent axonal sprouting in adult rats

The Eph family of tyrosine kinase receptors and their ligands, ephrins, are distributed in gradients and serve as molecular guidance cues for axonal patterning during neuronal development. Most of these molecules are also expressed in mature brain. Thus, we examine here the potential roles of such molecules in plasticity and activity-dependent mossy fiber sprouting of adult CNS. We show that the ligand ephrin-A3 and the receptor EphA5 are expressed in complementary gradients in the adult rat mossy fiber system. Using the kindling model, we demonstrate that exogenous immunoadhesins that affect the interaction of endogenous EphA receptors and ephrin-A ligands modulate the development of kindling, one type of long-term plasticity, in mature rat brain. These immunoadhesins, combined with epileptogenic stimulations, alter both the extent and the pattern of collateral axonal sprouting in the mossy fiber pathway. Our results suggest that EphA receptors and ephrin-A ligands modify neuronal plasticity and may serve as spatial cues that modulate the development and pattern of activation-dependent axonal growth in adult CNS.     

Ephrin-A2 and -A5 influence patterning of normal and novel retinal projections to the thalamus: conserved mapping mechanisms in visual and auditory thalamic targets

Sensory axons are targeted to modality-specific nuclei in the thalamus. Retinal ganglion cell axons project retinotopically to their principal thalamic target, the dorsal lateral geniculate nucleus (LGd), in a pattern likely dictated by the expression of molecular gradients in the LGd. Deafferenting the auditory thalamus induces retinal axons to innervate the medial geniculate nucleus (MGN). These retino-MGN projections also show retinotopic organization. Here we show that ephrin-A2 and -A5, which are expressed in similar gradients in the MGN and LGd, can be used to pattern novel retinal projections in the MGN. As in the LGd, retinal axons from each eye terminate in discrete eye-specific zones in the MGN of rewired wild-type and ephrin-A2/A5 knockout mice. However, ipsilateral eye axons, which arise from retinal regions of high EphA5 receptor expression and represent central visual field, terminate in markedly different ways in the two mice. In rewired wild-type mice, ipsilateral axons specifically avoid areas of high ephrin expression in the MGN. In rewired ephrin knockout mice, ipsilateral projections shift in location and spread more broadly, leading to an expanded representation of the ipsilateral eye in the MGN. Similarly, ipsilateral projections to the LGd in ephrin knockout mice are shifted and are more widespread than in the LGd of wild-type mice. In the MGN, as in the LGd, terminations from the two eyes show little overlap even in the knockout mice, suggesting that local interocular segregation occurs regardless of other patterning determinants. Our data demonstrate that graded topographic labels, such as the ephrins, can serve to shape multiple related aspects of afferent patterning, including topographic mapping and the extent and spread of eye-specific projections. Furthermore, when mapping labels and other cues are expressed in multiple target zones, novel projections are patterned according to rules that operate in their canonical targets.     

Ephrin/Eph receptor expression in brain of adult nonhuman primates: implications for neuroadaptation

In developing brain, Eph receptors and their ephrin ligands (Ephs/ephrins) are implicated in facilitating topographic guidance of a number of pathways, including the nigrostriatal and mesolimbic dopamine (DA) pathways. In adult rodent brain, these molecules are implicated in neuronal plasticity associated with learning and memory. Cocaine significantly alters the expression of select members of this family of axonal guidance molecules, implicating Ephs, ephrins in drug-induced neuroadaptation. The potential contribution of Ephs, ephrins to cocaine-induced reorganization of striatal circuitry brain in primates [Saka, E., Goodrich, C., Harlan, P., Madras, B.K., Graybiel, A.M., 2004. Repetitive behaviors in monkeys are linked to specific striatal activation patterns. J. Neurosci. 24, 7557-7565] is unknown because there are no documented reports of Eph/ephrin expression or function in adult primate brain. We now report that brains of adult old and new world monkeys express mRNA encoding EphA4 receptor and ephrin-B2 ligand, implicated in topographic guidance of dopamine and striatal neurons during development. Their encoded proteins distributed highly selectively in regions of adult monkey brain. EphA4 mRNA levels were prominent in the DA-rich caudate/putamen, nucleus accumbens and globus pallidus, as well as the medial and orbitofrontal cortices, hippocampus, amygdala, thalamus and cerebellum. Immunocytochemical localization of EphA4 protein revealed discrete expression in caudate/putamen, globus pallidus, substantia nigra, cerebellar Purkinje cells, pyramidal cells of frontal cortices (layers II, III and V) and the subgranular zone of the hippocampus. Evidence for EphA4 expression in dopamine neurons emerged from colocalization with tyrosine-hydroxylase-positive terminals in striatum and substantia nigra and ventral tegmental area cell bodies. The association of axonal guidance molecules with drug-induced reorganization of adult primate brain circuitry warrants investigation.     

Ephrins stimulate or inhibit neurite outgrowth and survival as a function of neuronal cell type

The Eph family of tyrosine kinase receptors and ligands play key roles in cell segregation and axon targeting in the developing nervous system. Interactions between the ligands and receptors cause repulsion or degeneration of receptor-positive axons from several brain regions including the retina, hippocampus, thalamus, and midbrain dopaminergic system. We extend these previous observations by showing that three A-ephrins also negatively regulate the growth of neurites from striatal and olfactory neurons. In addition to negative effects, however, we also report a trophic activity of the A-ephrins: Ephrin-A2 and A5 promote survival and neurite outgrowth of sympathetic neurons. These observations provide support to the notion that ephrins may function as either negative or positive signals in the developing nervous system.     

Septohippocampal properties of N-methyl-D-aspartate-induced theta-band oscillation and synchrony

Microinfusion of N-methyl-D-aspartate (NMDA) into apical dendrites of hippocampal CA1 pyramidal cells of urethane-anesthetized rats resulted in long lasting (20-30 min) induction of hippocampal synchrony at the field and cellular level. Power but not frequency of NMDA-induced theta was significantly greater than tail pinch-induced theta activity. This effect was antagonized by intrahippocampal infusion of AP5, but unaffected by i.v. atropine sulfate. During AP5 blockade tail pinch theta frequency and power were significantly reduced. Microinfusion of NMDA into the medial septum also resulted in long lasting induction of hippocampal theta field activity. Contrary to the results of hippocampal NMDA microinfusions, frequency but not power of NMDA-induced theta was significantly greater than tail pinch- induced theta activity. Microinfusion of AP5 into the medial septum significantly lowered power of tail pinch-induced theta but did not affect frequency. Wheel running behavior of rats induced by low levels of electrical stimulation of the posterior hypothalamic nucleus (PH) was completely abolished by microinfusion of AP5 into the medial septum, accompanied by a significant reduction in theta power and frequency. Wheel running and theta were maintained at control levels with high intensity PH stimulation. We propose that: (1) the glutamatergic septohippocampal projection represents a third pathway capable of generating hippocampal field and cellular synchrony, independent of that generated by the septohippocampal cholinergic and GABAergic projections, and (2) the septohippocampal glutamatergic projection serves to function as an interface between cholinergic and GABAergic modulated sensory processing Type 2 theta and movement related Type 1 theta.     

Hippocampal sympathetic ingrowth occurs following 192-IgG-Saporin administration

Electrolytic lesions of the medial septal region leads to an unusual neuronal reorganization in which peripheral sympathetic fibers, originating from the superior cervical ganglia, grow into the cholinergically denervated areas of the hippocampus. Since these lesions disrupt cells and fibers of passage which are non-cholinergic, there has been a debate whether Hippocampal Sympathetic Ingrowth is due only to cholinergic denervation of the hippocampus. Using the intraseptal administration of 192-IgG-Saporin, a specific cholinergic neurotoxin, we have found that hippocampal sympathetic ingrowth occurs in the cholinergically denervated hippocampus at 4, 8 and 12 weeks post Saporin injection. These results clearly suggest that hippocampal sympathetic ingrowth is due to the specific loss of the cholinergic projection from the medial septum.     

Single unit analysis of the hippocampal projections to the septum in the cat

The purpose of this study was to map the hippocampal efferent projections to the septum and to determine the synaptic organization of the hippocampal-septal system in the cat. Single unit responses were recorded in the septum with tungsten microelectrodes following electrical stimulation of the dorsal or ventral hippocampus in the anesthetized cat. Dorsal hippocampal stimulation produced excitatory unit driving at short latencies in the medial septum while ventral hippocampal stimulation produced short latency excitation in the lateral septum. The excitatory phase was invariably followed by a period of inhibition. Inhibition without a prior excitatory phase was seen in widespread regions of the septum upon either dorsal or ventral hippocampal stimulation. It was concluded that efferents from the dorsal and ventral hippocampus terminate in a topographic manner in the medial and lateral septum, respectively, and have excitatory effects upon these neurons as well. An interneuronal inhibitory network capable of influencing large regions of the septum was suggested from the data.     

Characterization of medial septal glutamatergic neurons and their projection to the hippocampus

The two neuronal populations that have been typically investigated in the septum use acetylcholine and GABA as neurotransmitters. The existence of noncholinergic, non-GABAergic, most likely glutamatergic septal neurons has recently been reported. However, their morphological characteristics, numbers, distribution, and connectivity have not been determined. Furthermore, the projection of septal glutamatergic neurons to the hippocampus has not been characterized. To address these issues, subpopulations of cholinergic and GABAergic neurons were identified by immunohistochemistry. In addition, the retrograde tracer fluorogold was injected into the hippocampus to determine the characteristics of a glutamatergic septo-hippocampal projection. Our work revealed that although glutamatergic neurons are found throughout the septum, they concentrate in medial septal regions. Using stereological probes, approximately 16,000 glutamatergic neurons were estimated in the medial septal region. Triple immunostaining showed that most glutamatergic neurons do not immunoreact with cholinergic or GABAergic neuronal markers (anti-ChAT or anti-GAD67 antibodies, respectively). Fluorogold injections into CA1, CA3, and dentate gyrus of the hippocampus showed that septal glutamatergic neurons project to each of these hippocampal regions, forming approximately 23% of the septo-hippocampal projection. Most cell bodies of septo-hippocampal glutamatergic neurons were located in the medial septum. The remaining cell bodies were found in the diagonal band. This data shows that glutamatergic neurons constitute a significant neuronal population in the septum and that a subpopulation of these neurons projects to hippocampal regions. Thus, the septo-hippocampal projection needs to be reconsidered as a three neurotransmitter pathway.     

Serotonergic and nonserotonergic projections from the rat interpeduncular nucleus to the septum, hippocampal formation and raphe: a combined immunocytochemical and fluorescent retrograde labelling study of neurons in the apical subnucleus

This study examined the subnuclear distribution and transmitter content of neurons in the interpeduncular nucleus (IPN) that projected to the septum, dorsal hippocampal formation, and/or raphe. Following the injection of fast blue into the medial septum/diagonal band nucleus and rhodamine-conjugated microspheres into the dorsal hippocampal formation (or vice versa), retrogradely-labelled cells were found throughout the apical subnucleus of the IPN. Incubation of these sections with 5-hydroxytryptamine antiserum indicated that a small number of fast blue- or rhodamine-positive cells also contained serotonin. Occasional apical cells contained both fast blue and rhodamine, indicating a dual projection via collaterals to both the septum and hippocampus. Injection of either dye into the raphe also retrogradely labelled cells in the apical subnucleus, none of which contained serotonin. These results suggest that the IPN may function to integrate the activity within subcortical limbic nuclei via widespread serotonergic and non-serotonergic projections.     

Septohippocampal adaptive GABAergic responses by AF64A treatment

A cholinergically disrupted laboratory animal has been produced by administration of the cholinotoxin ethylcholine aziridinium mustard (AF64A), which produced a dysfunction in the cholinergic forebrain system. After AF64A treatment, a reduction of choline acetyl transferase (ChAT) activity was measured in the hippocampal regions. ChAT activity was preferentially reduced in tissue samples of the dorsal with respect to the ventral hippocampus, and concomitantly with this reduction, a compensatory increase in ChAT activity in the medial septum was found. Tissue gamma‐aminobutyric acid (GABA) content in the hippocampal and septal brain areas was not affected by AF64A, indicating a specific effect on the cholinergic septohippocampal projection. The rate of GABA accumulation induced by aminooxyacetic acid administration was higher in the dorsal hippocampus and medial septum of AF64A‐treated animals, but not in their ventral hippocampus and lateral septum, where significant changes occurred in ChAT activity. Concomitantly with the changes in GABA metabolism, a significant B_max increase and K_d reduction of ³H‐flunitrazepam binding in the hippocampus of AF64A‐treated animals were associated with changes in the ChAT activity. This finding suggests an increase of GABA input on the cholinergic somas of the medial septum and an uncompensated GABAergic interneuron activity in the hippocampus. In this study, we present an adaptive mechanism of homotypic compensatory metabolism by cholinergic somas, and a heterotypic response of the GABAergic septohippocampal projection system, which was elicited by AF64A administration. J. Neurosci. Res. 55:178–186, 1999. © 1999 Wiley‐Liss, Inc.