MARCKS regulates neuritogenesis and interacts with a CDC42 signaling network.

Through the process of neuronal differentiation, newly born neurons change from simple, spherical cells to complex, sprawling cells with many highly branched processes. One of the first stages in this process is neurite initiation, wherein cytoskeletal modifications facilitate membrane protrusion and extension from the cell body. Hundreds of actin modulators and microtubule-binding proteins are known to be involved in this process, but relatively little is known about how upstream regulators bring these complex networks together at discrete locations to produce neurites. Here, we show that Myristoylated alanine-rich C kinase substrate (MARCKS) participates in this process. Marcks-/- cortical neurons extend fewer neurites and have less complex neurite arborization patterns. We use an in vitro proteomics screen to identify MARCKS interactors in developing neurites and characterize an interaction between MARCKS and a CDC42-centered network. While the presence of MARCKS does not affect whole brain levels of activated or total CDC42, we propose that MARCKS is uniquely positioned to regulate CDC42 localization and interactions within specialized cellular compartments, such as nascent neurites.

Neurotypic cell attachment and growth on III-nitride lateral polarity structures.

III-nitride materials have recently received increasing levels of attention for their potential to successfully interface with, and sense biochemical interactions in biological systems. Expanding on available sensing schemes (including transistor-based devices,) a III-N lateral polarity structure capable of introducing quasi-phase matching through a periodic polarity grating presents a novel platform for second harmonic generation. This platform constitutes a non-linear optical phenomenon with exquisite sensitivity to the chemical state of a surface or interface. To characterize the response of a biological system to the nanostructured lateral polarity structures, we cultured neurotypic PC12 cells on AlGaN with varying ratios of Al:Ga - 0, 0.4, 0.6, and 1 - and on surfaces of varying pitch to the III-polar vs. N-polar grating - 5, 10, 20 and 50 µm. While some toxicity associated with increasing Al is observed, we documented and quantified trends in cell responses to the local material polarity and nanoscale roughness. The nitrogen-polar material has a significantly higher nanoscale roughness than III-polar regions, and a 80-200 nm step height difference between the III-polar and N-polar materials in the lateral polarity configuration generates adequate changes in topography to influence cell growth, improves cell adhesion and promotes cell migration along the direction of the features. As the designed material configuration is further explored for biochemical sensing, the lateral polarity scheme may provide a route in assessing the non-specific protein adsorption to this varying nano-topography that drives the subsequent cell response.

Analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors.

Ongoing neurogenesis in discrete sectors of the adult central nervous system depends on the mitotic activity of an elusive population of adult stem cells. The existence of adult neural stem cells provides an alternative approach to transplantation of embryonic stem cells in cell-based therapies. Owing to the limited intrinsic fate of adult stem cells and inhibitory nature of the adult brain for neurogenesis, accommodation for circuit replacement in the brain will require genetic and epigenetic manipulation. Here, we show that a replication-incompetent Equine Infectious Anemia Virus (EIAV) is highly suitable for stable and persistent gene transfer to adult neural stem cells. The transduced regions were free of long-lasting neuroimmune responses to EIAV. Transduction in the subventricular zone was specific to the stem cell niche, but spared the progeny of adult neural stem cells that includes transit amplifying progenitors (TAPs) and migrating neuroblasts. With time, EIAV-transduced stem cells passed on the transgene to TAPs and migrating neuroblasts, which ultimately differentiated into neurons in the olfactory bulbs. We show that EIAV is highly suitable for discovery and assessment of mechanisms that regulate proliferation, migration and differentiation in the postnatal brain.

Reinduction of ErbB2 in astrocytes promotes radial glial progenitor identity in adult cerebral cortex.

Radial glial cells play a critical role in the construction of mammalian brain by functioning as a source of new neurons and by providing a scaffold for radial migration of new neurons to their target locations. Radial glia transform into astrocytes at the end of embryonic development. Strategies to promote functional recovery in the injured adult brain depend on the generation of new neurons and the appropriate guidance of these neurons to where they are needed, two critical functions of radial glia. Thus, the competence to regain radial glial identity in the adult brain is of significance for the ability to promote functional repair via neurogenesis and targeted neuronal migration in the mature brain. Here we show that the in vivo induction of the tyrosine kinase receptor, ErbB2, in mature astrocytes enables a subset of them to regain radial glial identity in the mature cerebral cortex. These new radial glial progenitors are capable of giving rise to new neurons and can support neuronal migration. These studies indicate that ErbB2 signaling critically modulates the functional state of radial glia, and induction of ErbB2 in distinct adult astrocytes can promote radial glial identity in the mature cerebral cortex.

Sequence of information processing for emotions based on the anatomic dialogue between prefrontal cortex and amygdala.

The prefrontal cortex and the amygdala have synergistic roles in regulating purposive behavior, effected through bidirectional pathways. Here we investigated the largely unknown extent and laminar relationship of prefrontal input-output zones linked with the amygdala using neural tracers injected in the amygdala in rhesus monkeys. Prefrontal areas varied vastly in their connections with the amygdala, with the densest connections found in posterior orbitofrontal and posterior medial cortices, and the sparsest in anterior lateral prefrontal areas, especially area 10. Prefrontal projection neurons directed to the amygdala originated in layer 5, but significant numbers were also found in layers 2 and 3 in posterior medial and orbitofrontal cortices. Amygdalar axonal terminations in prefrontal cortex were most frequently distributed in bilaminar bands in the superficial and deep layers, by columns spanning the entire cortical depth, and less frequently as small patches centered in the superficial or deep layers. Heavy terminations in layers 1-2 overlapped with calbindin-positive inhibitory neurons. A comparison of the relationship of input to output projections revealed that among the most heavily connected cortices, cingulate areas 25 and 24 issued comparatively more projections to the amygdala than they received, whereas caudal orbitofrontal areas were more receivers than senders. Further, there was a significant relationship between the proportion of 'feedforward' cortical projections from layers 2-3 to 'feedback' terminations innervating the superficial layers of prefrontal cortices. These findings indicate that the connections between prefrontal cortices and the amygdala follow similar patterns as corticocortical connections, and by analogy suggest pathways underlying the sequence of information processing for emotions.

The role of neuregulin-ErbB4 interactions on the proliferation and organization of cells in the subventricular zone.

Coordinated regulation of neuronal progenitor differentiation in the subventricular zone (SVZ) is a fundamental feature of adult neurogenesis. However, the molecular control of this process remains mostly undeciphered. Here, we investigate the role of neuregulins (NRGs) in this process and show that a NRG receptor, ErbB4, is primarily expressed by polysialylated neural cell adhesion molecule immature neuroblasts but is also detected in a subset of GFAP+ astroglial cells, ependymal cells, and Dlx2+ precursors in the SVZ. Of the NRG ligands, both NRG1 and -2 are expressed by immature polysialylated neural cell adhesion molecule neuroblasts in the SVZ. NRG2 is also expressed by some of the GFAP+ putative stem cells lining the ventricles. Infusion of exogenous NRG1 leads to rapid aggregation of Dlx2+ cells in the SVZ and affects the initiation and maintenance of organized neuroblast migration from the SVZ toward the olfactory bulb. In contrast, the infusion of NRG2 increased the number of Sox2 and GFAP+ precursors in the SVZ. An outcome of this NRG2 effect is an increase in the number of newly generated migrating neuroblasts in the rostral migratory stream and GABAergic interneurons in the olfactory bulb. The analysis of conditional null mice that lack NRG receptor, ErbB4, in the nervous system revealed that the observed activities of NRG2 require ErbB4 activation. These results indicate that different NRG ligands affect distinct populations of differentiating neural precursors in the neurogenic regions of the mature forebrain. Furthermore, these studies identify NRG2 as a factor capable of promoting SVZ proliferation, leading to the formation of new neurons in vivo.

Receptor tyrosine kinase ErbB4 modulates neuroblast migration and placement in the adult forebrain.

Neural progenitor proliferation, differentiation and migration are continually active in the rostral migratory stream of the adult brain. Here, we show that the receptor tyrosine kinase ErbB4 is expressed prominently by the neuroblasts present in the subventricular zone and the rostral migratory stream. The neuregulins (NRG1-NRG3), which have been identified as ErbB4 ligands, are detected either in the stream or in adjacent regions. Mice deficient in ErbB4 expressed under the control of either the nestin or the hGFAP promoter have altered neuroblast chain organization and migration and deficits in the placement and differentiation of olfactory interneurons. These findings suggest that ErbB4 activation helps to regulate the organization of neural chains that form the rostral migratory stream and influences the differentiation of olfactory interneuronal precursors.

Pathways for emotion: interactions of prefrontal and anterior temporal pathways in the amygdala of the rhesus monkey.

The amygdala has been implicated in processing information about the emotional significance of the environment and in the expression of emotions, through robust pathways with prefrontal, anterior temporal areas, and central autonomic structures. We investigated the anatomic organization and intersection of these pathways in the amygdala in rhesus monkeys with the aid of bidirectional, retrograde and anterograde tracers. Connections of the amygdala with orbitofrontal and medial prefrontal areas were robust and bidirectional, whereas connections with lateral prefrontal areas were sparse, unidirectional and ascending. Orbitofrontal axons terminated densely in a narrow band around the borders of the magnocellular basolateral nucleus, surrounded by projection neurons along a continuum through the nuclei of the basal complex. In contrast, the input and output zones of medial prefrontal areas were intermingled in the amygdala. Moreover, medial prefrontal axonal terminations were expansive, spreading into the parvicellular basolateral nucleus, which is robustly connected with hypothalamic autonomic structures, suggesting that they may influence the expressive emotional system of the amygdala. On the other hand, orbitofrontal axons heavily targeted the intercalated masses, which issue inhibitory projections to the central nucleus, at least in rats and cats. The central nucleus, in turn, issues a significant inhibitory projection to hypothalamic and brainstem autonomic structures. This evidence suggests that orbitofrontal areas exercise control on the internal processing of the amygdala. In addition, the results provided direct evidence that the connections of anterior temporal visual and auditory association cortices occupy overlapping territories with the orbitofrontal cortices particularly in the posterior half of the amygdala, and specifically within the intermediate sector of the basolateral nucleus and in the magnocellular part of the basomedial nucleus (also known as accessory basal), suggesting a closely linked triadic network. This intricate network may be recruited in cognitive tasks that are inextricably linked with emotional associations.

Neural interaction between the basal forebrain and functionally distinct prefrontal cortices in the rhesus monkey.

The prefrontal cortex in rhesus monkeys is a heterogeneous region by structure, connections and function. Caudal medial and orbitofrontal cortices receive input from cortical and subcortical structures associated with emotions, autonomic function and long-term memory, while lateral prefrontal cortices are linked with structures associated with working memory. With the aid of neural tracers we investigated whether functionally distinct orbitofrontal, medial and lateral prefrontal cortices have specific or common connections with an ascending modulatory system, the basal forebrain. Ascending projections originated in the diagonal band and the basalis nuclei of the basal forebrain in regions demarcated by choline acetyltransferase. Although the origin of projections from the basal forebrain to lateral, medial and orbitofrontal cortices partially overlapped, projections showed a general topography. The posterior part of the nucleus basalis projected preferentially to lateral prefrontal areas while its rostrally adjacent sectors projected to medial and orbitofrontal cortices. The diagonal band nuclei projected to orbitofrontal and medial prefrontal areas. Cortical and subcortical structures that are interconnected appear to have a similar pattern of connections with the basal forebrain. In comparison to the ascending projections, the descending projections were specific, originating mostly in the posterior (limbic) component of medial and orbitofrontal cortices and terminating in the diagonal band nuclei and in the anterior part of the nucleus basalis. In addition, prefrontal limbic areas projected to two other systems of the basal forebrain, the ventral pallidum and the extended amygdala, delineated with the striatal-related markers dopamine, adenosine 3':5'-monophosphate regulated phosphoprotein of M(r) 32kDa, and the related phosphoprotein Inhibitor-1. These basal forebrain systems project to autonomic nuclei in the hypothalamus and brainstem. We interpret these results to indicate that lateral prefrontal areas, which have a role in working memory, receive input from, but do not issue feedback projections to the basal forebrain. In contrast, orbitofrontal and medial prefrontal areas, which have a role in emotions and long-term memory, have robust bidirectional connections with the basal forebrain. Moreover, orbitofrontal and medial prefrontal cortices target the ventral pallidum and the extended amygdala, through which high-order association areas may activate motor autonomic structures for the expression of emotions.