Key Roles of CACNA1C/Cav1.2 and CALB1/Calbindin in Prefrontal Neurons Altered in Cognitive Disorders.

Importance: The risk of mental disorders is consistently associated with variants in CACNA1C (L-type calcium channel Cav1.2) but it is not known why these channels are critical to cognition, and whether they affect the layer III pyramidal cells in the dorsolateral prefrontal cortex that are especially vulnerable in cognitive disorders. Objective: To examine the molecular mechanisms expressed in layer III pyramidal cells in primate dorsolateral prefrontal cortices. Design, Setting, and Participants: The design included transcriptomic analyses from human and macaque dorsolateral prefrontal cortex, and connectivity, protein expression, physiology, and cognitive behavior in macaques. The research was performed in academic laboratories at Yale, Harvard, Princeton, and the University of Pittsburgh. As dorsolateral prefrontal cortex only exists in primates, the work evaluated humans and macaques. Main Outcomes and Measures: Outcome measures included transcriptomic signatures of human and macaque pyramidal cells, protein expression and interactions in layer III macaque pyramidal cells using light and electron microscopy, changes in neuronal firing during spatial working memory, and working memory performance following pharmacological treatments. Results: Layer III pyramidal cells in dorsolateral prefrontal cortex coexpress a constellation of calcium-related proteins, delineated by CALB1 (calbindin), and high levels of CACNA1C (Cav1.2), GRIN2B (NMDA receptor GluN2B), and KCNN3 (SK3 potassium channel), concentrated in dendritic spines near the calcium-storing smooth endoplasmic reticulum. L-type calcium channels influenced neuronal firing needed for working memory, where either blockade or increased drive by ß1-adrenoceptors, reduced neuronal firing by a mean (SD) 37.3% (5.5%) or 40% (6.3%), respectively, the latter via SK potassium channel opening. An L-type calcium channel blocker or ß1-adrenoceptor antagonist protected working memory from stress. Conclusions and Relevance: The layer III pyramidal cells in the dorsolateral prefrontal cortex especially vulnerable in cognitive disorders differentially express calbindin and a constellation of calcium-related proteins including L-type calcium channels Cav1.2 (CACNA1C), GluN2B-NMDA receptors (GRIN2B), and SK3 potassium channels (KCNN3), which influence memory-related neuronal firing. The finding that either inadequate or excessive L-type calcium channel activation reduced neuronal firing explains why either loss- or gain-of-function variants in CACNA1C were associated with increased risk of cognitive disorders. The selective expression of calbindin in these pyramidal cells highlights the importance of regulatory mechanisms in neurons with high calcium signaling, consistent with Alzheimer tau pathology emerging when calbindin is lost with age and/or inflammation.

A coming-of-age story: adult neurogenesis or adolescent neurogenesis in rodents?

It is surprising that after more than a century using rodents for scientific research, there are no clear, consensual, or consistent definitions for when a mouse or a rat becomes adult. Specifically, in the field of adult hippocampal neurogenesis, where this concept is central, there is a trend to consider that puberty marks the start of adulthood and is not uncommon to find 30-day-old mice being described as adults. However, as others discussed earlier, this implies an important bias in the perceived importance of this trait because functional studies are normally done at very young ages, when neurogenesis is at its peak, disregarding middle aged and old animals that exhibit very little generation of new neurons. In this feature article we elaborate on those issues and argue that research on the postnatal development of mice and rats in the last 3 decades allows to establish an adolescence period that marks the transition to adulthood, as occurs in other mammals. Adolescence in both rat and mice ends around postnatal day 60 and therefore this age can be considered the onset of adulthood in both species. Nonetheless, to account for inter-individual, inter-strain differences in maturation and for possible delays due to environmental and social conditions, 3 months of age might be a safer option to consider mice and rats bona fide adults, as suggested by The Jackson Labs.

Nanoscale imaging of pT217-tau in aged rhesus macaque entorhinal and dorsolateral prefrontal cortex: Evidence of interneuronal trafficking and early-stage neurodegeneration.

INTRODUCTION: Tau phosphorylated at threonine-217 (pT217-tau) is a novel fluid-based biomarker that predicts onset of Alzheimer's disease (AD) symptoms, but little is known about how pT217-tau arises in the brain, as soluble pT217-tau is dephosphorylated post mortem in humans. METHODS: We used multilabel immunofluorescence and immunoelectron microscopy to examine the subcellular localization of early-stage pT217-tau in entorhinal and prefrontal cortices of aged macaques with naturally occurring tau pathology and assayed pT217-tau levels in plasma. RESULTS: pT217-tau was aggregated on microtubules within dendrites exhibiting early signs of degeneration, including autophagic vacuoles. It was also seen trafficking between excitatory neurons within synapses on spines, where it was exposed to the extracellular space, and thus accessible to cerebrospinal fluid (CSF)/blood. Plasma pT217-tau levels increased across the age span and thus can serve as a biomarker in macaques. DISCUSSION: These data help to explain why pT217-tau predicts degeneration in AD and how it gains access to CSF and plasma to serve as a fluid biomarker.

Nanoscale imaging of pT217-tau in aged rhesus macaque entorhinal and dorsolateral prefrontal cortex: Evidence of interneuronal trafficking and early-stage neurodegeneration.

INTRODUCTION: pT217-tau is a novel fluid-based biomarker that predicts onset of Alzheimer's disease (AD) symptoms, but little is known about how pT217-tau arises in brain, as soluble pT217-tau is dephosphorylated postmortem in humans. METHODS: We utilized multi-label immunofluorescence and immunoelectron-microscopy to examine the subcellular localization of early-stage pT217-tau in entorhinal and prefrontal cortices of aged macaques with naturally-occurring tau pathology and assayed pT217-tau levels in plasma. RESULTS: pT217-tau was aggregated on microtubules within dendrites exhibiting early signs of degeneration, including autophagic vacuoles. It was also seen trafficking between excitatory neurons within synapses on spines, where it was exposed to the extracellular space, and thus accessible to CSF/blood. Plasma pT217-tau levels increased across the age-span and thus can serve as a biomarker in macaques. DISCUSSION: These data help to explain why pT217-tau predicts degeneration in AD and how it gains access to CSF and plasma to serve as a fluid biomarker.

Chronic GCPII (glutamate-carboxypeptidase-II) inhibition reduces pT217Tau levels in the entorhinal and dorsolateral prefrontal cortices of aged macaques.

Introduction: Current approaches for treating sporadic Alzheimer's disease (sAD) focus on removal of amyloid beta 1-42 (Aß1-42) or phosphorylated tau, but additional strategies are needed to reduce neuropathology at earlier stages prior to neuronal damage. Longstanding data show that calcium dysregulation is a key etiological factor in sAD, and the cortical neurons most vulnerable to tau pathology show magnified calcium signaling, for example in dorsolateral prefrontal cortex (dlPFC) and entorhinal cortex (ERC). In primate dlPFC and ERC, type 3 metabotropic glutamate receptors (mGluR3s) are predominately post-synaptic, on spines, where they regulate cAMP-calcium signaling, a process eroded by inflammatory glutamate carboxypeptidase II (GCPII) actions. The current study tested whether enhancing mGluR3 regulation of calcium via chronic inhibition of GCPII would reduce tau hyperphosphorylation in aged macaques with naturally-occurring tau pathology. Methods: Aged rhesus macaques were treated daily with the GCPII inhibitor, 2-MPPA (2-3-mercaptopropyl-penanedioic acid (2-MPPA)),Aged rhesus macaques were treated daily with the GCPII inhibitor, 2-MPPA (2-3-mercaptopropyl-penanedioic acid (2-MPPA)). Results: Aged macaques that received 2-MPPA had significantly lower pT217Tau levels in dlPFC and ERC, and had lowered plasma pT217Tau levels from baseline. pT217Tau levels correlated significantly with GCPII activity in dlPFC. Both 2-MPPA- and vehicle-treated monkeys showed cognitive improvement; 2-MPPA had no apparent side effects. Exploratory CSF analyses indicated reduced pS202Tau with 2-MPPA administration, confirmed in dlPFC samples. Discussion: These data provide proof-of-concept support that GCPII inhibition can reduce tau hyperphosphorylation in the primate cortices most vulnerable in sAD. GCPII inhibition may be particularly helpful in reducing the risk of sAD caused by inflammation. These data in nonhuman primates should encourage future research on this promising mechanism. Highlights: Inflammation is a key driver of sporadic Alzheimer's disease.GCPII inflammatory signaling in brain decreases mGluR3 regulation of calcium.Chronic inhibition of GCPII inflammatory signaling reduced pT217Tau in aged monkeys.GCPII inhibition is a novel strategy to help prevent tau pathology at early stages.

Localization of PDE4D, HCN1 channels, and mGluR3 in rhesus macaque entorhinal cortex may confer vulnerability in Alzheimer's disease.

Alzheimer's disease cortical tau pathology initiates in the layer II cell clusters of entorhinal cortex, but it is not known why these specific neurons are so vulnerable. Aging macaques exhibit the same qualitative pattern of tau pathology as humans, including initial pathology in layer II entorhinal cortex clusters, and thus can inform etiological factors driving selective vulnerability. Macaque data have already shown that susceptible neurons in dorsolateral prefrontal cortex express a "signature of flexibility" near glutamate synapses on spines, where cAMP-PKA magnification of calcium signaling opens nearby potassium and hyperpolarization-activated cyclic nucleotide-gated channels to dynamically alter synapse strength. This process is regulated by PDE4A/D, mGluR3, and calbindin, to prevent toxic calcium actions; regulatory actions that are lost with age/inflammation, leading to tau phosphorylation. The current study examined whether a similar "signature of flexibility" expresses in layer II entorhinal cortex, investigating the localization of PDE4D, mGluR3, and HCN1 channels. Results showed a similar pattern to dorsolateral prefrontal cortex, with PDE4D and mGluR3 positioned to regulate internal calcium release near glutamate synapses, and HCN1 channels concentrated on spines. As layer II entorhinal cortex stellate cells do not express calbindin, even when young, they may be particularly vulnerable to magnified calcium actions and ensuing tau pathology.

Molecular programs of regional specification and neural stem cell fate progression in macaque telencephalon.

During early telencephalic development, intricate processes of regional patterning and neural stem cell (NSC) fate specification take place. However, our understanding of these processes in primates, including both conserved and species-specific features, remains limited. Here, we profiled 761,529 single-cell transcriptomes from multiple regions of the prenatal macaque telencephalon. We deciphered the molecular programs of the early organizing centers and their cross-talk with NSCs, revealing primate-biased galanin-like peptide (GALP) signaling in the anteroventral telencephalon. Regional transcriptomic variations were observed along the frontotemporal axis during early stages of neocortical NSC progression and in neurons and astrocytes. Additionally, we found that genes associated with neuropsychiatric disorders and brain cancer risk might play critical roles in the early telencephalic organizers and during NSC progression.

Role of intracortical neuropil growth in the gyrification of the primate cerebral cortex.

The convolutions of the mammalian cerebral cortex allow the enlargement of its surface and addition of novel functional areas during evolution while minimizing expansion of the cranium. Cognitive neurodevelopmental disorders in humans, including microcephaly and lissencephaly, are often associated with impaired gyrification. In the classical model of gyrification, surface area is initially set by the number of radial units, and the forces driving cortical folding include neuronal growth, formation of neuropil, glial cell intercalation, and the patterned growth of subcortical white matter. An alternative model proposes that specified neurogenic hotspots in the outer subventricular zone (oSVZ) produce larger numbers of neurons that generate convexities in the cortex. This directly contradicts reports showing that cortical neurogenesis and settling of neurons into the cortical plate in primates, including humans, are completed well prior to the formation of secondary and tertiary gyri and indeed most primary gyri. In addition, during the main period of gyrification, the oSVZ produces mainly astrocytes and oligodendrocytes. Here we describe how rapid growth of intracortical neuropil, addition of glial cells, and enlargement of subcortical white matter in primates are the primary forces responsible for the post-neurogenic expansion of the cortical surface and formation of gyri during fetal development. Using immunohistochemistry for markers of proliferation and glial and neuronal progenitors combined with transcriptomic analysis, we show that neurogenesis in the ventricular zone and oSVZ is phased out and transitions to gliogenesis prior to gyral development. In summary, our data support the classical model of gyrification and provide insight into the pathogenesis of congenital cortical malformations.

Inhibition of glutamate-carboxypeptidase-II in dorsolateral prefrontal cortex: potential therapeutic target for neuroinflammatory cognitive disorders.

Glutamate carboxypeptidase-II (GCPII) expression in brain is increased by inflammation, e.g. by COVID19 infection, where it reduces NAAG stimulation of metabotropic glutamate receptor type 3 (mGluR3). GCPII-mGluR3 signaling is increasingly linked to higher cognition, as genetic alterations that weaken mGluR3 or increase GCPII signaling are associated with impaired cognition in humans. Recent evidence from macaque dorsolateral prefrontal cortex (dlPFC) shows that mGluR3 are expressed on dendritic spines, where they regulate cAMP-PKA opening of potassium (K+) channels to enhance neuronal firing during working memory. However, little is known about GCPII expression and function in the primate dlPFC, despite its relevance to inflammatory disorders. The present study used multiple label immunofluorescence and immunoelectron microscopy to localize GCPII in aging macaque dlPFC, and examined the effects of GCPII inhibition on dlPFC neuronal physiology and working memory function. GCPII was observed in astrocytes as expected, but also on neurons, including extensive expression in dendritic spines. Recordings in dlPFC from aged monkeys performing a working memory task found that iontophoresis of the GCPII inhibitors 2-MPPA or 2-PMPA markedly increased working memory-related neuronal firing and spatial tuning, enhancing neural representations. These beneficial effects were reversed by an mGluR2/3 antagonist, or by a cAMP-PKA activator, consistent with mGluR3 inhibition of cAMP-PKA-K+ channel signaling. Systemic administration of the brain penetrant inhibitor, 2-MPPA, significantly improved working memory performance without apparent side effects, with largest effects in the oldest monkeys. Taken together, these data endorse GCPII inhibition as a potential strategy for treating cognitive disorders associated with aging and/or neuroinflammation.

Neuroanatomical and psychological considerations in temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy and is associated with a variety of structural and psychological alterations. Recently, there has been renewed interest in using brain tissue resected during epilepsy surgery, in particular 'non-epileptic' brain samples with normal histology that can be found alongside epileptic tissue in the same epileptic patients - with the aim being to study the normal human brain organization using a variety of methods. An important limitation is that different medical characteristics of the patients may modify the brain tissue. Thus, to better determine how 'normal' the resected tissue is, it is fundamental to know certain clinical, anatomical and psychological characteristics of the patients. Unfortunately, this information is frequently not fully available for the patient from which the resected tissue has been obtained - or is not fully appreciated by the neuroscientists analyzing the brain samples, who are not necessarily experts in epilepsy. In order to present the full picture of TLE in a way that would be accessible to multiple communities (e.g., basic researchers in neuroscience, neurologists, neurosurgeons and psychologists), we have reviewed 34 TLE patients, who were selected due to the availability of detailed clinical, anatomical, and psychological information for each of the patients. Our aim was to convey the full complexity of the disorder, its putative anatomical substrates, and the wide range of individual variability, with a view toward: (1) emphasizing the importance of considering critical patient information when using brain samples for basic research and (2) gaining a better understanding of normal and abnormal brain functioning. In agreement with a large number of previous reports, this study (1) reinforces the notion of substantial individual variability among epileptic patients, and (2) highlights the common but overlooked psychopathological alterations that occur even in patients who become "seizure-free" after surgery. The first point is based on pre- and post-surgical comparisons of patients with hippocampal sclerosis and patients with normal-looking hippocampus in neuropsychological evaluations. The second emerges from our extensive battery of personality and projective tests, in a two-way comparison of these two types of patients with regard to pre- and post-surgical performance.

An assessment of the existence of adult neurogenesis in humans and value of its rodent models for neuropsychiatric diseases.

In sub-mammalian vertebrates like fishes, amphibians, and reptiles, new neurons are produced during the entire lifespan. This capacity diminishes considerably in birds and even more in mammals where it persists only in the olfactory system and hippocampal dentate gyrus. Adult neurogenesis declines even more drastically in nonhuman primates and recent evidence shows that this is basically extinct in humans. Why should such seemingly useful capacity diminish during primate evolution? It has been proposed that this occurs because of the need to retain acquired complex knowledge in stable populations of neurons and their synaptic connections during many decades of human life. In this review, we will assess critically the claim of significant adult neurogenesis in humans and show how current evidence strongly indicates that humans lack this trait. In addition, we will discuss the allegation of many rodent studies that adult neurogenesis is involved in psychiatric diseases and that it is a potential mechanism for human neuron replacement and regeneration. We argue that these reports, which usually neglect significant structural and functional species-specific differences, mislead the general population into believing that there might be a cure for a variety of neuropsychiatric diseases as well as stroke and brain trauma by genesis of new neurons and their incorporation into existing synaptic circuitry.

Transcriptomic taxonomy and neurogenic trajectories of adult human, macaque, and pig hippocampal and entorhinal cells.

The hippocampal-entorhinal system supports cognitive functions, has lifelong neurogenic capabilities in many species, and is selectively vulnerable to Alzheimer's disease. To investigate neurogenic potential and cellular diversity, we profiled single-nucleus transcriptomes in five hippocampal-entorhinal subregions in humans, macaques, and pigs. Integrated cross-species analysis revealed robust transcriptomic and histologic signatures of neurogenesis in the adult mouse, pig, and macaque but not humans. Doublecortin (DCX), a widely accepted marker of newly generated granule cells, was detected in diverse human neurons, but it did not define immature neuron populations. To explore species differences in cellular diversity and implications for disease, we characterized subregion-specific, transcriptomically defined cell types and transitional changes from the three-layered archicortex to the six-layered neocortex. Notably, METTL7B defined subregion-specific excitatory neurons and astrocytes in primates, associated with endoplasmic reticulum and lipid droplet proteins, including Alzheimer's disease-related proteins. This resource reveals cell-type- and species-specific properties shaping hippocampal-entorhinal neurogenesis and function.

Radial Glial Cells: New Views on Old Questions.

Radial glial cells (RGC) are at the center of brain development in vertebrates, acting as progenitors for neurons and macroglia (oligodendrocytes and astrocytes) and as guides for migration of neurons from the ventricular surface to their final positions in the brain. These cells originate from neuroepithelial cells (NEC) from which they inherit their epithelial features and polarized morphology, with processes extending from the ventricular to the pial surface of the embryonic cerebrum. We have learnt a great deal since the first descriptions of these cells at the end of the nineteenth century. However, there are still questions regarding how and when NEC transform into RGC or about the function of intermediate filaments such as glial fibrillary acidic protein (GFAP) in RGCs and their dynamics during neurogenesis. For example, it is not clear why RGCs in primates, including humans, express GFAP at the onset of cortical neurogenesis while in rodents it is expressed when it is essentially complete. Based on an ultrastructural analysis of GFAP expression and cell morphology of dividing progenitors in the developing neocortex of the macaque monkey, we show that RGCs become the main progenitor in the developing cerebrum by the start of neurogenesis, as all dividing cells show glial features such as GFAP expression and lack of tight junctions. Also, our data suggest that RGCs retract their apical process during mitosis. We discuss our findings in the context of the role and molecular characteristics of RGCs in the vertebrate brain, their differences with NECs and their dynamic behavior during the process of neurogenesis.

Variation of Human Neural Stem Cells Generating Organizer States In Vitro before Committing to Cortical Excitatory or Inhibitory Neuronal Fates.

Better understanding of the progression of neural stem cells (NSCs) in the developing cerebral cortex is important for modeling neurogenesis and defining the pathogenesis of neuropsychiatric disorders. Here, we use RNA sequencing, cell imaging, and lineage tracing of mouse and human in vitro NSCs and monkey brain sections to model the generation of cortical neuronal fates. We show that conserved signaling mechanisms regulate the acute transition from proliferative NSCs to committed glutamatergic excitatory neurons. As human telencephalic NSCs develop from pluripotency in vitro, they transition through organizer states that spatially pattern the cortex before generating glutamatergic precursor fates. NSCs derived from multiple human pluripotent lines vary in these early patterning states, leading differentially to dorsal or ventral telencephalic fates. This work furthers systematic analyses of the earliest patterning events that generate the major neuronal trajectories of the human telencephalon.

Gliogenesis in the outer subventricular zone promotes enlargement and gyrification of the primate cerebrum.

The primate cerebrum is characterized by a large expansion of cortical surface area, the formation of convolutions, and extraordinarily voluminous subcortical white matter. It was recently proposed that this expansion is primarily driven by increased production of superficial neurons in the dramatically enlarged outer subventricular zone (oSVZ). Here, we examined the development of the parietal cerebrum in macaque monkey and found that, indeed, the oSVZ initially adds neurons to the superficial layers II and III, increasing their thickness. However, as the oSVZ grows in size, its output changes to production of astrocytes and oligodendrocytes, which in primates outnumber cerebral neurons by a factor of three. After the completion of neurogenesis around embryonic day (E) 90, when the cerebrum is still lissencephalic, the oSVZ enlarges and contains Pax6+/Hopx+ outer (basal) radial glial cells producing astrocytes and oligodendrocytes until after E125. Our data indicate that oSVZ gliogenesis, rather than neurogenesis, correlates with rapid enlargement of the cerebrum and development of convolutions, which occur concomitantly with the formation of cortical connections via the underlying white matter, in addition to neuronal growth, elaboration of dendrites, and amplification of neuropil in the cortex, which are primary factors in the formation of cerebral convolutions in primates.

Noradrenergic α1-Adrenoceptor Actions in the Primate Dorsolateral Prefrontal Cortex.

Noradrenergic (NE) α1-adrenoceptors (α1-ARs) contribute to arousal mechanisms and play an important role in therapeutic medications such as those for the treatment of posttraumatic stress disorder (PTSD). However, little is known about how α1-AR stimulation influences neuronal firing in the dorsolateral prefrontal cortex (dlPFC), a newly evolved region that is dysfunctional in PTSD and other mental illnesses. The current study examined the effects of α1-AR manipulation on neuronal firing in dlPFC of rhesus monkeys performing a visuospatial working memory task, focusing on the "delay cells" that maintain spatially tuned information across the delay period. Iontophoresis of the α1-AR antagonist HEAT (2-{[ß-(4-hydroxyphenyl)ethyl]aminomethyl}-1-tetralone) had mixed effects, reducing firing in a majority of neurons but having nonsignificant excitatory effects or no effect in remaining delay cells. These data suggest that endogenous NE has excitatory effects in some delay cells under basal conditions. In contrast, the α1-AR agonists phenylephrine and cirazoline suppressed delay cell firing and this was blocked by coadministration of HEAT. These results indicate an inverted-U dose response for α1-AR actions, with mixed excitatory actions under basal conditions and suppressed firing with high levels of α1-AR stimulation such as with stress exposure. Immunoelectron microscopy revealed α1-AR expression presynaptically in axons and axon terminals and postsynaptically in spines, dendrites, and astrocytes. It is possible that α1-AR excitatory effects arise from presynaptic excitation of glutamate release, whereas postsynaptic actions suppress firing through calcium-protein kinase C opening of potassium channels on spines. The latter may predominate under stressful conditions, leading to loss of dlPFC regulation during uncontrollable stress.SIGNIFICANCE STATEMENT Noradrenergic stimulation of α1-adrenoceptors (α1-ARs) is implicated in posttraumatic stress disorder (PTSD) and other mental disorders that involve dysfunction of the prefrontal cortex, a brain region that provides top-down control. However, the location and contribution of α1-ARs to prefrontal cortical physiology in primates has received little attention. This study found that α1-ARs are located near prefrontal synapses and that α1-AR stimulation has mixed effects under basal conditions. However, high levels of α1-AR stimulation, as occur with stress, suppress neuronal firing. These findings help to explain why we lose top-down control under conditions of uncontrollable stress when there are high levels of noradrenergic release in brain and why blocking α1-AR, such as with prazosin, may be helpful in the treatment of PTSD.

Adult Human Hippocampus: No New Neurons in Sight.

In this issue of Cerebral Cortex, Cipriani et al. are following up on the recent report of Sorrels et al. to add novel immunohistological observations indicating that, unlike rodents, adult and aging humans do not acquire new neurons in the hippocampus. The common finding emerging from these 2 different, but almost simultaneous studies is highly significant because the dentate gyrus of the hippocampus was, until recently, considered as the only structure in the human brain that may continue neurogenesis throughout the full life span.

Species-dependent posttranscriptional regulation of NOS1 by FMRP in the developing cerebral cortex.

Fragile X syndrome (FXS), the leading monogenic cause of intellectual disability and autism, results from loss of function of the RNA-binding protein FMRP. Here, we show that FMRP regulates translation of neuronal nitric oxide synthase 1 (NOS1) in the developing human neocortex. Whereas NOS1 mRNA is widely expressed, NOS1 protein is transiently coexpressed with FMRP during early synaptogenesis in layer- and region-specific pyramidal neurons. These include midfetal layer 5 subcortically projecting neurons arranged into alternating columns in the prospective Broca's area and orofacial motor cortex. Human NOS1 translation is activated by FMRP via interactions with coding region binding motifs absent from mouse Nos1 mRNA, which is expressed in mouse pyramidal neurons, but not efficiently translated. Correspondingly, neocortical NOS1 protein levels are severely reduced in developing human FXS cases, but not FMRP-deficient mice. Thus, alterations in FMRP posttranscriptional regulation of NOS1 in developing neocortical circuits may contribute to cognitive dysfunction in FXS.

Development and distribution of neuronal cilia in mouse neocortex.

Neuronal primary cilia are not generally recognized, but they are considered to extend from most, if not all, neurons in the neocortex. However, when and how cilia develop in neurons are not known. This study used immunohistochemistry for adenylyl cyclase III (ACIII), a marker of primary cilia, and electron microscopic analysis to describe the development and maturation of cilia in mouse neocortical neurons. Our results indicate that ciliogenesis is initiated in late fetal stages after neuroblast migration, when the mother centriole docks with the plasma membrane, becomes a basal body, and grows a cilia bud that we call a procilium. This procilium consists of a membranous protrusion extending from the basal body but lacking axonemal structure and remains undifferentiated until development of the axoneme and cilia elongation starts at about postnatal day 4. Neuronal cilia elongation and final cilia length depend on layer position, and the process extends for a long time, lasting 8-12 weeks. We show that, in addition to pyramidal neurons, inhibitory interneurons also grow cilia of comparable length, suggesting that cilia are indeed present in all neocortical neuron subtypes. Furthermore, the study of mice with defective ciliogenesis suggested that failed elongation of cilia is not essential for proper neuronal migration and laminar organization or establishment of neuronal polarity. Thus, the function of this organelle in neocortical neurons remains elusive.