Embryonic Origin of the Islet1 and Pax6 Neurons of the Chicken Central Extended Amygdala Using Cell Migration Assays and Relation to Different Neuropeptide-Containing Cells.

In a recent study, we tentatively identified different subdivisions of the central extended amygdala (EAce) in chicken based on the expression of region-specific transcription factors (including Pax6 and Islet1) and several phenotypic markers during embryonic development. Such a proposal was partially based on the suggestion that, similarly to the subdivisions of the EAce of mammals, the Pax6 and Islet1 neurons of the comparable chicken subdivisions derive from the dorsal (Std) or ventral striatal embryonic domains (Stv), respectively. To investigate whether this is true, in the present study, we carried out cell migration assays from chicken Std or Stv combined with immunofluorescence for Pax6 or Islet1. Our results showed that the cells of the proposed chicken EAce truly originate in either Std (expressing Pax6) or Stv (expressing Islet1). This includes lateral subdivisions previously compared to the intercalated amygdalar cells and the central amygdala of mammals, also rich in Std-derived Pax6 cells and/or Stv-derived Islet1 cells. In the medial region of the chicken EAce, the dorsal part of the lateral bed nucleus of the stria terminalis (BSTL) contains numerous cells expressing Nkx2.1 (mostly derived from the pallidal domain), but our migration assays showed that it also contains neuron subpopulations from the Stv (expressing Islet1) and Std (expressing Pax6), resembling the mouse BSTL. These findings, together with those previously published in different species of mammals, birds and reptiles, support the homology of the chicken EAce to that of other vertebrates, and reinforce the existence of several cell subcorridors inside the EAce. In addition, together with previously published data on neuropeptidergic cells, these results led us to propose the existence of at least seventeen neuron subtypes in the EAce in rodents and/or some birds (chicken and pigeon). The functional significance and the evolutionary origin of each subtype needs to be analyzed separately, and such studies are mandatory in order to understand the multifaceted modulation by the EAce of fear responses, ingestion, motivation and pain in different vertebrates.

Pallial origin of basal forebrain cholinergic neurons in the nucleus basalis of Meynert and horizontal limb of the diagonal band nucleus.

The majority of the cortical cholinergic innervation implicated in attention and memory originates in the nucleus basalis of Meynert and in the horizontal limb of the diagonal band nucleus of the basal prosencephalon. Functional alterations in this system give rise to neuropsychiatric disorders as well as to the cognitive alterations described in Parkinson and Alzheimer's diseases. Despite the functional importance of these basal forebrain cholinergic neurons very little is known about their origin and development. Previous studies suggest that they originate in the medial ganglionic eminence of the telencephalic subpallium; however, our results identified Tbr1-expressing, reelin-positive neurons migrating from the ventral pallium to the subpallium that differentiate into cholinergic neurons in the basal forebrain nuclei projecting to the cortex. Experiments with Tbr1 knockout mice, which lack ventropallial structures, confirmed the pallial origin of cholinergic neurons in Meynert and horizontal diagonal band nuclei. Also, we demonstrate that Fgf8 signaling in the telencephalic midline attracts these neurons from the pallium to follow a tangential migratory route towards the basal forebrain.

Expression of cLhx6 and cLhx7/8 suggests a pallido-pedunculo-preoptic origin for the lateral and medial parts of the avian bed nucleus of the stria terminalis.

We investigated the origin of the avian bed nucleus of the stria terminalis (BST) and other parts of the avian subpallial amygdala, by studying the expression of the LIM-homeobox chick genes Lhx6 (cLhx6) and Lhx7/8 (cLhx7/8) in the embryonic chicken telencephalon. Our results indicate that these genes are expressed in a subpallial subdomain partially overlapping the expression of Nkx2.1, which includes pallidal, peduncular, commissural preoptic and pallidoseptal subdivisions comparable to those of mammals. The lateral and medial parts of the avian BST express cLhx6 and/or cLhx7/8, suggesting that they derive from the Nkx2.1-expressing subpallial domain. Our results indicate that the avian lateral BST (BSTL) contains two components, a dorsal part rich in cLhx6 and lacking cLhx7/8 expression that may derive from the pallidal subdivision, and a ventral part showing moderate or light expression of cLhx6 and cLhx7/8, which may derive from the peduncular subdivision. Moreover, the medial BST (BSTM1 and BSTM2) shows moderate to strong expression of cLhx6 and very strong expression of cLhx7/8 throughout development, and appears to derive from both the peduncular and the commissural preoptic subdivisions. Based on this, the avian dorsal BSTL appears comparable to the mammalian BSTL, whereas the avian ventral BSTL and at least part of BSTM may be comparable to the anterior and posteromedial parts of the mammalian BSTM. We also identified a ventrolateral portion of BSTM (BSTM3) and other cell corridors expressing cLhx6 and/or cLhx7/8 in chicken and propose their homology with specific parts of the extended amygdala of mammals.

Development and adult organization of the lateral part of the bed nucleus of the stria terminalis in the chicken.

The lateral part of the bed nucleus of the stria terminalis (BSTL) is a component of the subpallial amygdala located near the ventral sulcus of the lateral ventricle, but its limits have not been well defined in birds. In this study, we analyzed the expression patterns of a number of neurochemical markers: GABA, calbindin (CB), calretinin (CR), or neuronal nitric oxide synthase (nNOS), in the embryonic and adult chicken brain, to further characterize the organization of the avian BSTL. From embryonic day 16, it was possible to distinguish three different regions within BSTL on the basis of cytoarchitectonic and immunohistochemical features. A central region, referred to as lateral bed nucleus of the stria terminalis pars densocellularis (BSTLdc), is characterized by numerous tightly packed cell bodies, most of which are GABA-immunoreactive (ir), and two peripheral regions with lower cellular density displaying a moderate GABA expression, referred to as lateral bed nucleus of the stria terminalis, plexiform part 1 (BSTLp1) and plexiform part 2 (BSTLp2), respectively. In contrast to BSTLdc, both plexiform parts are characterized by the presence of many fibers and terminals immunoreactive for nNOS and CR, as well as some CR-ir scattered cells. A distinctive feature of BSTLp2 is a population of CB-ir cells embedded in a slightly CB-ir neuropil. Comparison of our immunohistochemical data with gene expression data suggests that BSTLdc and BSTLp1 are pallidal in nature, whereas BSTLp2 receives important contributions from the entopeduncular/preoptic area.

Anxiety-like behavior and structural changes of the bed nucleus of the stria terminalis (BNST) in gestational protein-restricted male offspring.

Animal evidence has suggested that maternal emotional and nutritional stress during pregnancy is associated with behavioral outcomes in offspring. The nature of the stresses applied may differ, but it is often assumed that the mother's hippocampus-hypothalamic-pituitary-adrenal (HHPA) axis response releases higher levels of glucocorticoid hormones. The bed nucleus of the stria terminalis (BNST) is in a pivotal position to regulate the HHPA axis and the stress response, and it has been implicated in anxiety behavior. In the current study, to search whether BNST structural changes and neurochemical alterations are associated with anxiety-related behavior in adult gestational protein-restricted offspring relative to an age-matched normal protein diet (NP) rats, we conduct behavioral tests and, BNST dendritic tree analysis by Sholl analysis, associated to immunoblotting-protein quantification [11ß-HSD2, GR, MR, AT1R, 5HT1A and 5HT2A, corticotrophin-releasing factor (CRH) and CRH1]. Dams were maintained either on isocaloric standard rodent chow [with NP content, 17% casein or low protein content (LP), 6% casein] chow throughout their entire pregnancy. Here, in rats subjected to gestational protein restriction, we found: (a) a significant reduction in dendritic length and impoverished dendritic arborization in BNST neurons; (b) an elevated plasmatic corticosterone levels; and (c) associated with enhanced anxiety-like behavior when compared with age-matched NP offspring. Moreover, altered protein (11ß-HSD2, GR, MR and type 1 CRH receptors) expressions may underlie the increase in anxiety-like behavior in LP offspring. This work represents the first demonstration that BNST developmental plasticity by maternal protein restriction, resulting in fine structural changes and neurochemical alterations that are associated with modified behavioral states.

Comparative aspects of p73 and Reelin expression in Cajal-Retzius cells and the cortical hem in lizard, mouse and human.

Cajal-Retzius (CR) cells of the mammalian neocortex co-express the extracellular matrix protein Reelin and p73, a transcription factor involved in cell death and survival. Most neocortical CR cells derive from the cortical hem, with minor additional sources. We analyzed the distribution of Reelin and p73 immunoreactive (ir) neurons in the telencephalon of Lacerta galloti from early embryonic stages to hatching. Numerous Reelin-ir cells appeared in the pallial MZ from the preplate stage onward. Conversely, p73-ir cells were rare in the pallial preplate and not observed in the cortical plate. Subpallial p73-ir cells spread from the septum and the telencephalic-diencephalic boundary to the pial surface of the basal forebrain and amygdala, respectively, where they co-expressed Reelin and p73. A small group of Reelin/p73-ir CR cells appeared in a rudimentary cortical hem at the interface of the medial cortex and choroid plexus. Comparison with early embryonic stages of mice and humans showed similar foci of p73-ir cells in the septum and at the telencephalic-diencephalic boundary and revealed an increasing prominence of the cortical hem, in parallel with increasing numbers of neocortical Reelin/p73 positive CR cells, which attain highest differentiation in the human brain. Our data show that Reelin-expression in the pallium is evolutionarily conserved and independent of a cortical hem, and suggest that p73 in the cortical hem may be involved in the evolutionary increase in number and complexity of the mammalian neocortical CR cells.

Polychlorinated biphenyls exert selective effects on cellular composition of white matter in a manner inconsistent with thyroid hormone insufficiency.

Developmental exposure to polychlorinated biphenyls (PCBs) is associated with a variety of cognitive deficits in humans, and recent evidence implicates white matter development as a potential target of PCBs. Because PCBs are suspected of interfering with thyroid hormone (TH) signaling in the developing brain, and because TH is important in oligodendrocyte development, we tested the hypothesis that PCB exposure affects the development of white matter tracts by disrupting TH signaling. Pregnant Sprague Dawley rats were exposed to the PCB mixture Aroclor 1254 (5 mg/kg), with or without cotreatment of goitrogens from gestational d 7 until postnatal d 15. Treatment effects on white matter development were determined by separately measuring the cellular density and proportion of myelin-associated glycoprotein (MAG)-positive, O4-positive, and glial fibrillary acidic protein (GFAP)-positive cells in the genu of the corpus callosum (CC) and in the anterior commissure (AC). Hypothyroidism decreased the total cell density of the CC and AC as measured by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining and produced a disproportionate decrease in MAG-positive oligodendrocyte density with a simultaneous increase in GFAP-positive astrocyte density. These data indicate that hypothyroidism reduces cellular density of CC and AC and fosters astrocyte development at the expense of oligodendrocyte density. In contrast, PCB exposure significantly reduced total cell density but did not disproportionately alter MAG-positive oligodendrocyte density or change the ratio of MAG-positive oligodendrocytes to GFAP-positive astrocytes. Thus, PCB exposure mimicked some, but not all, of the effects of hypothyroidism on white matter composition.

Evidence of altered brain sexual differentiation in mice exposed perinatally to low, environmentally relevant levels of bisphenol A.

Humans are routinely exposed to bisphenol A (BPA), an estrogenic chemical present in food and beverage containers, dental composites, and many products in the home and workplace. BPA binds both classical nuclear estrogen receptors and facilitates membrane-initiated estrogenic effects. Here we explore the ability of environmentally relevant exposure to BPA to affect anatomical and functional measures of brain development and sexual differentiation. Anatomical evidence of alterations in brain sexual differentiation were examined in male and female offspring born to mouse dams exposed to 0, 25, or 250 ng BPA/kg body weight per day from the evening of d 8 of gestation through d 16 of lactation. These studies examined the sexually dimorphic population of tyrosine hydroxylase (TH) neurons in the rostral periventricular preoptic area, an important brain region for estrous cyclicity and estrogen-positive feedback. The significant sex differences in TH neuron number observed in control offspring were diminished or obliterated in offspring exposed to BPA primarily because of a decline in TH neuron number in BPA-exposed females. As a functional endpoint of BPA action on brain sexual differentiation, we examined the effects of perinatal BPA exposure on sexually dimorphic behaviors in the open field. Data from these studies revealed significant sex differences in the vehicle-exposed offspring that were not observed in the BPA-exposed offspring. These data indicate that BPA may be capable of altering important events during critical periods of brain development.

Ontogeny of the projections from the anteroventral periventricular nucleus of the hypothalamus in the female rat.

Neurons in the anteroventral periventricular nucleus of the hypothalamus (AVPV) mediate a variety of autonomic functions. In adults they primarily innervate neuroendocrine nuclei in the periventricular zone of the hypothalamus, including the paraventricular and arcuate nuclei (PVH, ARH). Ascending projections from the AVPV also provide inputs to the ventrolateral septum (LSv) and the principal division of the bed nuclei of the stria terminalis (BSTp). Consistent with a role in regulating preovulatory luteinizing hormone secretion, rostral projections from the AVPV contact gonadotropin-releasing hormone (GnRH) neurons surrounding the vascular organ of the lamina terminalis (OVLT). To study the development of these pathways, we placed implants of the lipophilic tracers DiI and CMDiI into the AVPV of female rats ranging in age from embryonic day 19 (E19) through adulthood. The earliest projections targeted a population of GnRH neurons, with apparent contacts from labeled fibers observed as early as E19. These connections appeared to be fully developed before birth, as similar numbers of appositions from AVPV projections onto the GnRH-immunoreactive cells were observed at all ages examined. Caudal projections were delayed relative to projections to the OVLT. Labeled AVPV fibers reached the PVH during the first postnatal week, and fibers targeting the BSTp and LSv were not observed until the second and third postnatal weeks, respectively. Labeled AVPV fibers were not seen in the ARH of animals at any age. Our results demonstrate that projections from the AVPV develop with both spatial and temporal specificity, innervating each target with a unique developmental profile.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse.

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable. Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Repulsion and attraction of axons by semaphorin3D are mediated by different neuropilins in vivo.

Class 3 semaphorins are known to repel and/or sometimes attract axons; however, their role in guiding developing axons in the CNS in vivo is still essentially unknown. We investigated the role of Semaphorin3D (Sema3D) in the formation of the early axon pathways in the zebrafish CNS. Morpholino knock-down shows that Sema3D is essential for the correct formation of two early axon pathways. Sema3D appears to guide axons of the nucleus of the medial longitudinal fasciculus (nucMLF) by repulsion and modulation of fasciculation. In contrast, Sema3D appears to be attractive to telencephalic neurons that form the anterior commissure (AC). Knock-down of Neuropilin-1A (Npn-1A) phenocopied the effects of Sema3D knock-down on the nucMLF axons, and knock-down of either Npn-1A or Npn-2B phenocopied the defects of the AC. Furthermore, simultaneous partial knock-down experiments demonstrated genetic interactions among Sema3D, Npn-1A, and Npn-2B. Together, these data support the hypothesis that Sema3D may act as a repellent through receptors containing Npn-1A and as an attractant via receptors containing Npn-1A and Npn-2B.

Sexual differentiation of the bed nucleus of the stria terminalis in humans may extend into adulthood.

Gonadal steroids have remarkable developmental effects on sex-dependent brain organization and behavior in animals. Presumably, fetal or neonatal gonadal steroids are also responsible for sexual differentiation of the human brain. A limbic structure of special interest in this regard is the sexually dimorphic central subdivision of the bed nucleus of the stria terminalis (BSTc), because its size has been related to the gender identity disorder transsexuality. To determine at what age the BSTc becomes sexually dimorphic, the BSTc volume in males and females was studied from midgestation into adulthood. Using vasoactive intestinal polypeptide and somatostatin immunocytochemical staining as markers, we found that the BSTc was larger and contains more neurons in men than in women. However, this difference became significant only in adulthood, showing that sexual differentiation of the human brain may extend into the adulthood. The unexpectedly late sexual differentiation of the BSTc is discussed in relation to sex differences in developmental, adolescent, and adult gonadal steroid levels.

Target-dependent sexual differentiation of a limbic-hypothalamic neural pathway.

Neural pathways between sexually dimorphic forebrain regions develop under the influence of sex steroid hormones during the perinatal period, but how these hormones specify precise sex-specific patterns of connectivity is unknown. A heterochronic coculture system was used to demonstrate that sex steroid hormones direct development of a sexually dimorphic limbic-hypothalamic neural pathway through a target-dependent mechanism. Explants of the principal nucleus of the bed nuclei of the stria terminalis (BSTp) extend neurites toward explants of the anteroventral periventricular nucleus (AVPV) derived from male but not female rats. Coculture of BSTp explants from male rats with AVPV explants derived from females treated in vivo with testosterone for 9 d resulted in a high density of neurites extending from the BSTp to the AVPV explant, as was the case when the BSTp explants were derived from females and the AVPV explants were derived from males or androgen-treated females. These in vitro findings suggest that during the postnatal period testosterone induces a target-derived, diffusible chemotropic activity that results in a sexually dimorphic pattern of connectivity.

Abnormalities in neuronal process extension, hippocampal development, and the ventricular system of L1 knockout mice.

In humans, mutations in the L1 cell adhesion molecule are associated with a neurological syndrome termed CRASH, which includes corpus callosum agenesis, mental retardation, adducted thumbs, spasticity, and hydrocephalus. A mouse model with a null mutation in the L1 gene (Cohen et al., 1997) was analyzed for brain abnormalities by Nissl and Golgi staining and immunocytochemistry. In the motor, somatosensory, and visual cortex, many pyramidal neurons in layer V exhibited undulating apical dendrites that did not reach layer I. The hippocampus of L1 mutant mice was smaller than normal, with fewer pyramidal and granule cells. The corpus callosum of L1-minus mice was reduced in size because of the failure of many callosal axons to cross the midline. Enlarged ventricles and septal abnormalities were also features of the mutant mouse brain. Immunoperoxidase staining showed that L1 was abundant in developing neurons at embryonic day 18 (E18) in wild-type cerebral cortex, hippocampus, and corpus callosum and then declined to low levels with maturation. In the E18 cortex, L1 colocalized with microtubule-associated protein 2, a marker of dendrites and somata. These new findings suggest new roles for L1 in the mechanism of cortical dendrite differentiation, as well as in guidance of callosal axons and regulation of hippocampal development. The phenotype of the L1 mutant mouse indicates that it is a potentially valuable model for the human CRASH syndrome.

Early embryonic administration of xenoestrogens alters vasotocin system and male sexual behavior of the Japanese quail.

The copulatory behavior and the parvocellular vasotocin (VT) system of the nucleus of the stria terminalis (BST) are sexually dimorphic in the Japanese quail. Embryonic administration of estradiol benzoate (EB) induces an organizational effect determining the disappearance of such a dimorphism (male shows behavior and cerebral phenotype of the female). The VT parvocellular system can therefore be considered an accurate marker of the sexual differentiation of brain circuits and a very sensitive indicator of the activity of estrogen-like substances on neural circuits. To test this hypothesis we administered diethylstilbestrol (DES), a powerful synthetic xenoestrogen, genistein (GEN), a phytoestrogen produced by soy, and bisphenol A (BPA). After 3 days of incubation, quail eggs were injected with vehicle, EB, DES, GEN or BPA. Administration of BPA caused an early blockage of development and no further analyses were done on the BPA groups. At puberty, the copulatory behavior of EB- or DES-treated male quail was totally abolished, whereas only the highest doses of GEN determined a significant decrease of the behavior. After the tests, the animals were sacrificed and perfused. The fractional area (FA) covered by VT immunoreactivity was analyzed in BST, medial preoptic nucleus, and lateral septum by computerized image analysis. The FA was significantly reduced after treatment with EB, DES and GEN at high doses. These results confirm that the sexually dimorphic VT system of the Japanese quail is a sensible indicator of the effects of xenoestrogens at the level of the central nervous system.

Sex chromosome complement determines sex differences in aromatase expression and regulation in the stria terminalis and anterior amygdala of the developing mouse brain.

Aromatase, which converts testosterone in estradiol, is involved in the generation of brain sex dimorphisms. Here we used the "four core genotypes" mouse model, in which the effect of gonadal sex and sex chromosome complement is dissociated, to determine if sex chromosomes influence the expression of brain aromatase. The brain of 16 days old XY mouse embryos showed higher aromatase expression in the stria terminalis and the anterior amygdaloid area than the brain of XX embryos, independent of gonadal sex. Furthermore, estradiol or dihydrotestosterone increased aromatase expression in cultures of anterior amygdala neurons derived from XX embryos, but not in those derived from XY embryos. This effect was also independent of gonadal sex. The expression of other steroidogenic molecules, estrogen receptor-α and androgen receptor was not influenced by sex chromosomes. In conclusion, sex chromosomes determine sex dimorphisms in aromatase expression and regulation in the developing mouse brain.

Early prenatal development of the human precommissural septum.

The development of the septum was studied in human embryos and fetuses ranging from 8 to 24.5 weeks of menstrual age (22.2 to 216 mm crown-rump length). Neuroblasts migrating from the ventricular layer of the ventromedial hemispheric wall form a narrow intermediate layer that constitutes the primordial septum (8 weeks). Only a primordial nucleus of the diagonal band is identifiable within the gradually enlarging primordial septum at early stages. By 10 weeks the primordial septum is subdivided into medial and lateral zones. At 11.5 weeks well-defined medial nuclei and the nucleus of the diagonal band are evident within the medial zone. Differentiation within the lateral zone occurs by 12.5 weeks with the appearance of nucleus lateralis pars interna. Nucleus dorsalis is developing in the lateral zone by 14.5 weeks and, by 15.5 weeks, well-defined nuclei are present throughout the lateral zone. Further neuronal maturation and conformational changes result in the nearly adult appearance of the septum in older fetuses. Although a definite mediolateral differentiation-gradient occurs, individual nuclei appear to differentiate along their own longitudinal gradient. Evidence presented suggests that the earliest fibers within the primordial septum are related to the tuberculum olfactorium and the medial forebrain bundle, that septohippocampal fibers appear at 10 weeks, hippocamposeptal fibers by 11.5 weeks, and that, later, stria terminalis fibers develop. The suggested developmental relationships of the septum with the hypothalamus (and brainstem), tuberculum, hippocampus, and amygdala emphasizes its role as an internode in the limbic system.

Regulation of axonal ingrowth into area dentata as studied by sequential, double intraocular brain tissue transplantation.

The anterior eye chamber was used as a model environment to study, in isolation, the interaction of embryonic area dentata transplants with transplants of one of three important sources of in situ innervation: entorhinal cortex, locus coeruleus or septal nuclei. None of these brain regions significantly affected the morphogenesis or in oculo growth of area dentata transplants. All three brain regions innervated the area dentata transplant. Entorhinal cortical transplants sent nerve fibers into a limited, and apparently specific, region of area dentata that was adjacent to the entorhinal transplant. This light innervation contrasts to the predominant innervation of area dentata by entorhinal cortex in situ. The fluorescent, noradrenergic neurons of locus coeruleus provided the area dentata transplant with an abundance of fine varicose nerve fibers. Given about 100 noradrenergic neurons in the locus coeruleus transplant and 4 to 6 months joint survival, the area dentata transplant was noradrenergically hyperinnervated. The cholinergic neurons of the septal nuclei transplant had a prolific ingrowth of acetylcholinesterase (AChE)-positive nerve fibers to the area dentata transplant. There appeared to be a mutual exclusion between the extrinsic AChE-positive fibers and the intrinsic Timm's-positive granule cell mossy fibers in the area dentata transplant. We conclude that isolated replicas of the coeruleo-, septo-, and entorhinal cortico-dentate pathways can be made through sequential intraocular double grafting. The nature of the in oculo connectivity between these replicates offers clues as to the mechanisms that might account for the regulation of nerve growth.

Multiple ephrins regulate hippocampal neurite outgrowth.

Increasing evidence indicates that the eph family of ligands and receptors guides the formation of topographic maps in the brain through repulsive interactions. For example, we have recently found that in the hippocamposeptal system, the ligand ephrin-A2, which is expressed in an increasing gradient from dorsal to ventral septum, selectively induces pruning of topographically inappropriate medial hippocampal axons. The recent detection of ephrins A3 and A5, as well as A2, in the septum raised critical functional questions. Do the ligands act combinatorially, ensuring appropriate three-dimensional spatiotemporal projection, or do they exert entirely distinct actions in addition to guidance mechanisms? To approach these alternatives, we cloned mouse ephrin-A2 and compared the activities of the three ligands. Here, we show that these ligands reduce the number of hippocampal neurites in a similar fashion. The effect was regionally specific; medial hippocampal neurites were reduced 1.5- to 1.8-fold, whereas lateral hippocampal neurites were not significantly affected, conforming to topographic projection in vivo. Furthermore, we found that ephrins regulated neurite number in a stage-specific fashion, affecting E19 hippocampal neurites more than E16 neurites. Our observations suggest that all three septal ephrins, A2, A3, and A5, play spatiotemporally specific roles in guiding topographic projections from the hippocampus.

A role for cingulate pioneering axons in the development of the corpus callosum.

In many vertebrate and invertebrate systems, pioneering axons play a crucial role in establishing large axon tracts. Previous studies have addressed whether the first axons to cross the midline to from the corpus callosum arise from neurons in either the cingulate cortex (Koester and O'Leary [1994] J. Neurosci. 11:6608-6620) or the rostrolateral neocortex (Ozaki and Wahlsten [1998] J. Comp. Neurol. 400:197-206). However, these studies have not provided a consensus on which populations pioneer the corpus callosum. We have found that neurons within the cingulate cortex project axons that cross the midline and enter the contralateral hemisphere at E15.5. By using different carbocyanine dyes injected into either the cingulate cortex or the neocortex of the same brain, we found that cingulate axons crossed the midline before neocortical axons and projected into the contralateral cortex. Furthermore, the first neocortical axons to reach the midline crossed within the tract formed by these cingulate callosal axons, and appeared to fasciculate with them as they crossed the midline. These data indicate that axons from the cingulate cortex might pioneer a pathway for later arriving neocortical axons that form the corpus callosum. We also found that a small number of cingulate axons project to the septum as well as to the ipsilateral hippocampus via the fornix. In addition, we found that neurons in the cingulate cortex projected laterally to the rostrolateral neocortex at least 1 day before the neocortical axons reach the midline. Because the rostrolateral neocortex is the first neocortical region to develop, it sends the first neocortical axons to the midline to form the corpus callosum. We postulate that, together, both laterally and medially projecting cingulate axons may pioneer a path for the medially directed neocortical axons, thus helping to guide these axons toward and across the midline during the formation of the corpus callosum.