Positive regulation of neocortical synapse formation by the Plexin-D1 receptor.

Synapse formation is a critical process during neural development and is coordinated by multiple signals. Several lines of evidence implicate the Plexin-D1 receptor in synaptogenesis. Studies have shown that Plexin-D1 signaling is involved in synaptic specificity and synapse formation in spinal cord and striatum. Expression of Plexin-D1 and its principal neural ligand, Sema3E, by neocortical neurons is temporally and spatially regulated, reaching the highest level at the time of synaptogenesis in mice. In this study, we examined the function of Plexin-D1 in synapse formation by primary neocortical neurons in vitro. A novel, automated image analysis method was developed to quantitate synapse formation under baseline conditions and with manipulation of Plexin-D1 levels. shRNA and overexpression manipulations caused opposite changes, with reduction resulting in less synapse formation, an effect distinct from that reported in the striatum. The data indicate that Plexin-D1 operates in a cell context-specific fashion, mediating different synaptogenic outcomes depending upon neuron type.

Genetic disruption of the autism spectrum disorder risk gene PLAUR induces GABAA receptor subunit changes.

Disruption of the GABAergic system has been implicated in multiple developmental disorders, including epilepsy, autism spectrum disorder and schizophrenia. The human gene encoding uPAR (PLAUR) has been shown recently to be associated with the risk of autism. The uPAR(-/-) mouse exhibits a regionally-selective reduction in GABAergic interneurons in frontal and parietal regions of the cerebral cortex as well as in the CA1 and dentate gyrus subfields of the hippocampus. Behaviorally, these mice exhibit increased sensitivity to pharmacologically-induced seizures, heightened anxiety, and atypical social behavior. Here, we explore potential alterations in GABAergic circuitry that may occur in the context of altered interneuron development. Analysis of gene expression for 13 GABA(A) receptor subunits using quantitative real-time polymerase chain reaction (PCR) indicates seven subunit mRNAs (alpha(1), alpha(2), alpha(3), beta(2), beta(3), gamma(2S) and gamma(2L)) of interest. Semi-quantitative in situ hybridization analysis focusing on these subunit mRNAs reveals a complex pattern of potential gene regulatory adaptations. The levels of alpha(2) subunit mRNAs increase in frontal cortex, CA1 and CA3, while those of alpha3 decrease in frontal cortex and CA1. In contrast, alpha(1) subunit mRNAs are unaltered in any region examined. beta(2) subunit mRNAs are increased in frontal cortex whereas beta(3) subunit mRNAs are decreased in parietal cortex. Finally, gamma(2S) subunit mRNAs are increased in parietal cortex while gamma(2L) subunit mRNAs are increased in the dentate gyrus, potentially altering the gamma(2S):gamma(2L) ratio in these two regions. For all subunits, no changes were observed in forebrain regions where GABAergic interneuron numbers are normal. We propose that disrupted differentiation of GABAergic neurons specifically in frontal and parietal cortices leads to regionally-selective alterations in local circuitry and subsequent adaptive changes in receptor subunit composition. Future electrophysiological studies will be useful in determining how alterations in network activity in the cortex and hippocampus relate to the observed behavioral phenotype.

Disruption of Foxg1 expression by knock-in of cre recombinase: effects on the development of the mouse telencephalon.

The cre/loxP system is used routinely to manipulate gene expression in the mouse nervous system. In order to delete genes specifically from the telencephalon, the Foxg1-cre line was created previously by replacing the intron-less Foxg1 coding region with cre, resulting in a Foxg1 heterozygous mouse. As the telencephalon of heterozygous Foxg1 mice was reported to be normal, this genotype often has been used as the control in subsequent analyses. Here we describe substantial disruption of forebrain development of heterozygous mice in the Foxg1-cre line, maintained on the C57BL/6J background. High resolution magnetic resonance microscopy reveals a significant reduction in the volume of the neocortex, hippocampus and striatum. The alteration in the neocortex results, in part, from a decrease in its tangential dimension, although gross patterning of the cortical sheet appears normal. This decrease is observed in three different Foxg1 heterozygous mouse lines, independent of the method of achieving deletion of the Foxg1 gene. Although Foxg1 is not expressed in the diencephalon, three-dimensional magnetic resonance microscopy revealed that thalamic volume in the adult is reduced. In contrast, at postnatal day 4, thalamic volume is normal, suggesting that interactions between cortex and dorsal thalamus postnatally produce the final adult thalamic phenotype. In the Foxg1-cre line maintained on the C57BL/6J background, the radial domain of the cerebral cortex also is disrupted substantially, particularly in supragranular layers. However, neither Foxg1 heterozygous mice of the Foxg1-tet (tetracycline transactivator) line, nor those of the Foxg1-lacZ and Foxg1-cre lines maintained on a mixed background, displayed a reduced cortical thickness. Thus Cre recombinase contributes to the radial phenotype, although only in the context of the congenic C57BL/6J background. These observations highlight an important role for Foxg1 in cortical development, reveal noteworthy complexity in the invocation of specific mechanisms underlying phenotypes expressed following genetic manipulations and stress the importance of including appropriate controls of all genotypes.

Regional differences in neurotrophin availability regulate selective expression of VGF in the developing limbic cortex.

Gene and protein expression patterns in the cerebral cortex are complex and often change spatially and temporally through development. The signals that regulate these patterns are primarily unknown. In the present study, we focus on the regulation of VGF expression, which is limited to limbic cortical areas early in development but later expands into sensory and motor areas. We isolated neurons from embryonic day 17 rat cortex and demonstrate that the profile of VGF expression in perirhinal (expressing) and occipital (nonexpressing) populations in vitro is similar to that in the perinatal cortex in vivo. The addition of neutralizing neurotrophin antibodies indicates that endogenous brain-derived neurotrophic factor (BDNF) is necessary for the normal complement of VGF-expressing neurons in the perirhinal cortex, although endogenous neurotrophin-3 (NT-3) regulates the expression of VGF in a subpopulation of cells. ELISA analysis demonstrates that there is significantly more BDNF present in the perirhinal cortex compared with the occipital cortex in the perinatal period. However, the total amount of NT-3 is similar between the two regions and, moreover, there is considerably more NT-3 than BDNF in both areas, a finding seemingly in conflict with regional VGF expression. Quantification of the extracellular levels of neurotrophins in perirhinal and occipital cultures using ELISA in situ analysis indicates that perirhinal neurons release significantly more BDNF than the occipital population. Furthermore, the amount of NT-3 released by the perirhinal neurons is significantly less than the amount of BDNF. Local injection of BDNF in vivo into a normally negative VGF region results in robust ectopic expression of VGF. These data suggest that the local availability of specific neurotrophins for receptor occupation, rather than the total amount of neurotrophin, is a critical parameter in determining the selective expression of VGF in the developing limbic cortex.

Regionalization of the cerebral cortex: developmental mechanisms and models.

The cerebral cortex is composed of functionally specialized areas that have unique connections with other cortical targets and subcortical nuclei. The developmental mechanisms responsible for the formation of discrete regions must include the regulation of the expression of genes encoding proteins that control axon guidance and targeting. New data on patterns of gene expression demonstrate the early appearance of such guidance molecules, thus reflecting the early emergence of regional specification within the cortex. Transplant and cell culture studies suggest that the decisions made by neuronal progenitor cells to express region-appropriate phenotypes is controlled by the capacity of the cells to respond to and have access to specific signals. The key to understanding cortical specification may lie in determining the factors that control receptor diversity on progenitors and the temporal and spatial distribution of inductive signals within the forebrain.

Complex signaling responsible for molecular regionalization of the cerebral cortex.

The formation of discrete functional areas is a key event in the development of the cerebral cortex. The expression patterns of several molecules associated with axon guidance reveal specification of regional identity during fetal development within the cortex, with different area-specific features acquired at very early to later stages of corticogenesis. Cell culture experiments suggest that complex mechanisms regulate the differentiation of region-appropriate phenotypes. In all instances that we have examined thus far, however, the final phenotype adopted by cortical neurons is governed by the capacity of the cell to respond to regionalizing signals; this is reflected in the heterogeneity of receptor expression by progenitors, and the temporal and spatial distribution of such signals within the forebrain.

The role of ErbB receptor signaling in cell fate decisions by cortical progenitors: evidence for a biased, lineage-based responsiveness to different ligands.

We recently identified the required collaborative signaling of TGFalpha and collagen type IV to regulate cell fate choice in the cerebral cortex, measured by the expression of the limbic system associated membrane protein (LAMP) by nonlimbic, sensorimotor progenitors. We show that activation of different members of the erbB receptor family can similarly modulate the specification of cortical area fate. The region of the cerebral wall from which progenitor cells arise does not influence the response to the neuregulin-1 or TGFalpha, but a subpopulation of progenitors is not competent to express LAMP in response to neuregulin-1. The heterogeneity in the responsiveness by progenitors to the two growth factors is reflected in the expression of different repertoires of erbB receptors. Using clonal analysis, we demonstrate that there may be a lineage-dependent mechanism regulating the ability of neuronal progenitors to respond to specific inductive cues that control cell fate.

Mechanisms specifying area fate in cortex include cell-cycle-dependent decisions and the capacity of progenitors to express phenotype memory.

Progenitor cells in the early developing cerebral cortex produce neurons destined for discrete functional areas in response to specific inductive signals. Using lineage analysis, we show that cortical progenitor cells at different fetal ages retain the memory of an area-specific inductive signal received in vivo, even though they may pass through as many as two cell cycles in the absence of the signal in culture. When exposed to inductive signals in vitro, only those progenitors that progress through at least one complete cell cycle alter their areal phenotype. Our findings suggest that induction of an areal phenotype is linealy inherited, with the phenotype specified prior to the final cell cycle.

Patterning and specification of the cerebral cortex.

Regionalization of the cerebral cortex occurs during development by the formation of anatomically and functionally discrete areas of the brain. Descriptive evidence based on expression of molecules and structural features suggests that an early parcelation of the cerebral wall may occur during fetal development. Experimental strategies using tissue transplants and cell culture models have explored the nature of the timing of areal specification. New signaling systems displaying the sensitivity of precursor cells to environmental cues that define the fate of neurons destined for specific areas of the cortex have been discovered. Studies in the field now suggest mechanisms of regulating cell phenotype in the cortex that are common to all parts of the neuraxis.

Signaling pathways that regulate specification of neurons in developing cerebral cortex.

The expression of the limbic system-associated membrane protein (LAMP), a marker of specific functional regions of the cerebral cortex, has been used to determine the environmental signals that regulate cortical regionalization. Transplant and cell culture studies have shown previously that the fate of precursor cells, based on LAMP expression, is amenable to regulation by exposure to novel environmental stimuli. This has been demonstrated in vitro to be dependent upon exposure to transforming factor-alpha and collagen type IV. Results following exposure to the inductive signals for a specific duration indicate a cell cycle dependence on the decision to become a limbic or nonlimbic cortical neuron. It appears, therefore, that areal and laminar fates are both influenced by mechanisms that specify commitment early in cortical development.

Complementary distribution of collagen type IV and the epidermal growth factor receptor in the rat embryonic telencephalon.

We previously identified an interaction between collagen type IV and the EGF receptor that regulates the differentiation of a limbic cortical phenotype in vitro (Ferri and Levitt, 1995). In the present study, we map the expression of the EGF receptor and collagen type IV in the embryonic telencephalon of the rat. At embryonic day (E) II, the earliest age examined, both proteins are coexpressed throughout the ventricular zone in the cerebral wall; this zone remains immunoreactive throughout corticogenesis (E14-E19). The cells comprising the subventricular zone are the most intensely immunoreactive for the EGF receptor, although little collage type IV is detected in this region. In contrast, postmitotic neurons that leave the proliferative zones are negative for the receptor. Moreover, during the peak of neuronal migration, the intermediate zone lacks collagen type IV immunoreactivity. Neurons that settle in the cortical plate once again exhibit EGF receptor immunoreactivity; this same zone is devoid of collagen type IV. By E19, coexpression of both proteins is evident only in the rostral extension of the subventricular zone, the pathway of migrating cells leading to the olfactory bulb. The temporal and spatial overlap of the EGF receptor and collage type IV in the cortical progenitor pool in vivo indicates that these molecules may participate in the initial decisions of neuronal differentiation. Their modified distribution during cortical maturation suggests a changing role for both proteins.

Environmental signals influence expression of a cortical areal phenotype in vitro independent of effects on progenitor cell proliferation.

We have shown previously that, in vitro, cortical progenitor cells isolated from specific locations of the cerebral wall can adopt area-specific fates, assayed by expression of the limbic system-associated membrane protein (LAMP; R. T. Ferri and P. Levitt, Cereb. Cortex 3, 187-198, 1993). Progenitors destined to produce LAMP neurons, however, will differentiate to express the limbic molecular phenotype if grown with TGFalpha and collagen type IV (R. T. Ferri and P. Levitt, Development 121, 1151-1160, 1995), while other signals fail to induce LAMP. The present study used BrdU labeling of progenitor cells to examine whether modulation of LAMP expression was paralleled by predictable changes in cell proliferation. The general pattern of proliferation is similar under a variety of culture conditions: approximately half the cells are mitotic, and activity is always highest during the first 24 hr in vitro, with little cell division occurring by the third day. Moreover, the rate of proliferation, in the presence or absence of TGFalpha, is the same on all substrates tested, with the exception of laminin. The TGFalpha/collagen type IV signaling system that induces LAMP expression by the precursors has no modulating effect on their proliferative kinetics. Nonetheless, only progenitors that are mitotically active respond to LAMP-inducing signals; only 60% of the neurons, representing those that have divided at least once in culture, can be induced to express LAMP. The data suggest that while specific signals affect choice of area phenotype during the cell cycle, they do so in the absence of major changes in proliferative behavior.

Rescue of both rapidly and slowly degenerating neurons in the dorsal lateral geniculate nucleus of adult rats by a cortically derived neuron survival factor.

We investigated the death of dorsal lateral geniculate nucleus (dLGN) neurons after lesions to the visual cortex of adult rats and the effects of supplying target-derived neurotrophic molecules to the lesion cavity. The neurotrophic factor is retrieved from cocultures of the embryonic primordia of the geniculocortical pathway and its survival promoting properties for different populations of dLGN neurons (based on their time of origin) have been documented in previous studies of neonatal rats with occipital cortex lesions. In the present study, rats were exposed to [3H]thymidine on E14 or E15/16 to label either earlier or later generated dLGN neurons. When animals were at least 45 days old we made discrete lesions to the principal projection zones in area 17 of these two dLGN populations. Counts of surviving labeled cells show a relatively rapid death of E15/16 dLGN neurons in control animals, with a maximal loss by 2 weeks postlesion. The death of E14 dLGN neurons is more protracted, with a maximal loss by 2 months postlesion. A 2-week infusion of the CM fraction rescues the majority of the neurons that would otherwise die in both populations compared to the controls which receive a similarly prepared fraction of unconditioned medium. Moreover, this CM fraction can sustain E14-generated dLGN neurons up to 6 weeks after the neurotrophic factor(s) is no longer being supplied exogenously. Thus the rescue of axotomized adult dLGN neurons appears to be permanent, at least for the early generated population. These findings are consistent with the idea that target-derived molecules have a role in the survival of mature neurons, as they are known to have for developing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Different populations of dorsal lateral geniculate nucleus neurons have concentration-specific requirements for a cortically derived neuron survival factor.

A macromolecular fraction of conditioned culture medium (CM) derived from explant cocultures of embryonic rat posterior cortex and caudal thalamus is able to support the survival of neurons in the dorsal lateral geniculate nucleus (dLGN) of newborn rats following ablation of dLGN cortical target areas. In the present study we tested whether the survival-promoting activity of this target-derived neurotrophic agent was concentration dependent and whether different subpopulations of dLGN neurons were equally responsive. With the starting concentration of the CM fraction designated X, increasing concentration results in a progressive falloff in trophic activity so that at 200X overall dLGN survival is similar to that seen in unconditioned medium (UM) controls. In contrast, diluting the fraction produces an increase in activity until maximal survival is achieved at 0.2X. Further dilutions result in a decline in trophic activity until control values are reached at 0.001X. Two populations of neurons within the dLGN, defined by their time of origin, respond in a specific manner to the different concentrations. Neurons generated during the early stages of neurogenesis (E14) have maximal survival (25.8%) at 0.05X, whereas those neurons generated later (E15/16) are maximally supported (30.7% survival) at 10X, a 200-fold difference in concentration. While it is possible that separate neurotrophic and neurotoxic molecules exist for each of these populations of dLGN neurons, the most parsimonious interpretation of the data is that a single cortically derived neurotrophic factor exists whose production is strictly controlled during development to achieve maximal effect on different populations of thalamic neurons that may be functionally distinct.

Motoneurone survival requirements during development: the change from immature astrocyte dependence to myotube dependence.

The survival requirements of motoneurones obtained from differently aged avian embryos was analysed, in both heterogeneous cultures of motoneurones with spinal cord cells and homogeneous cultures of motoneurones obtained by cell sorting. It was found that medium conditioned by contact with immature astrocytes could maintain more than 75% of the motoneurones plated from 5-day embryos for two days; however, this astrocyte medium could not maintain motoneurones plated from 8-day embryos above control levels at two days. In contrast, medium conditioned by contact with myotubes could not maintain motoneurones plated from 5-day embryos above control levels for two days; this myotube medium could maintain more than 70% of the motoneurones plated from 8-day embryos for two days. The change in the receptivity of motoneurones to astrocyte-conditioned medium may be due to their ageing. Thus, motoneurones from 6-day embryos could not be sustained above control numbers in culture for 4 days with astrocyte media, in the same way as motoneurones from 8-day embryos degenerate by two days. In contrast, more than 70% of motoneurones plated from 6-day embryos could be maintained in culture for 4 days with myotube media in the same way as motoneurones from 8-day embryos for two days. The results indicate that motoneurones from 5-day embryos are dependent for their survival on immature astrocytes but that this switches to a dependence on myotubes during the normal motoneurone death period from 6 days to 10 days of embryonic age.

Motoneurone survival is induced by immature astrocytes from developing avian spinal cord.

Dissociated spinal cords of 6-day chick embryos were grown on monolayers consisting primarily of either flat (relatively immature) or process-bearing (relatively mature) astrocytes. Cultures rich in flat astrocytes maintained about 80% of the motoneurones originally plated for 48 h in vitro. However, process-bearing astrocytes were unable to support motoneurone survival. Medium conditioned by contact with the monolayers of flat astrocytes also promoted motoneurone survival for 48 h. Maximal activity occurred over the concentration range 55-110 micrograms/ml protein. After 48 h, the number of motoneurones dropped to control levels both in the conditioned medium and on the monolayers. This effect could not be reversed by the introduction of fresh conditioned media at 48 h. This indicated a decrease in the requirements of more mature motoneurones for this media as muscle-conditioned medium could support 80% of the motoneurones initially plated for 96 h. Thus, relatively immature astrocytes were capable of supporting the survival of 6-day montoneurones in vitro for up to 48 h and this effect is mediated through the release of a soluble substance.

Motor neuron survival and neuritic extension from spinal cord explants induced by factors released from denervated muscle.

Extracts prepared from denervated adult skeletal muscle contain increased amounts of neurotrophic activity which promotes both survival of dissociated motor neurons and the outgrowth of neurites from explants of spinal cord maintained in serum-free defined media. The trophic activity is specific for motor neurons and reaches a peak within the first week post-denervation. In these most potent extracts the neurite outgrowth enhancement is a linearly increasing function of protein concentration at low concentrations; at higher concentrations the neurite activity-concentration relationship saturates and in the milligram range the relationship becomes inhibitory. When media containing active denervated muscle extract was preincubated over polycationic substrata, it lost the ability to promote neuritic growth; this could be restored if fresh extract was added to the cultures. Thus it was demonstrated that within the denervated muscle extract there are physically separable agents responsible for neuron survival and neurite expression. It is possible that the release of neurotrophic factors may be in part responsible for the in vivo phenomenon of nerve sprouting.

Survival of purified motor neurones in vitro: effects of skeletal muscle-conditioned medium.

Spinal motor neurones in the adult mouse were labelled retrogradely with both True Blue and horseradish peroxidase (HRP). Using the cell sorter, motor neurones were separated on the basis of cell size and the intensity of True Blue fluorescence. Cultures of the sorted cells were then prepared and the motor neurones were identified by their HRP labelling. There was found to be a 4.8-fold increase in motor neurones over the unsorted population such that 40% of the cells present in culture was labelled. Medium conditioned over skeletal muscle was shown to enhance the survival of motor neurones in these cultures.