The p75 neurotrophin receptor is required for the survival of neuronal progenitors and normal formation of the basal forebrain, striatum, thalamus and neocortex.

During development, the p75 neurotrophin receptor (p75NTR) is widely expressed in the nervous system where it regulates neuronal differentiation, migration and axonal outgrowth. p75NTR also mediates the survival and death of newly born neurons, with functional outcomes being dependent on both timing and cellular context. Here, we show that knockout of p75NTR from embryonic day 10 (E10) in neural progenitors using a conditional Nestin-Cre p75NTR floxed mouse causes increased apoptosis of progenitor cells. By E14.5, the number of Tbr2-positive progenitor cells was significantly reduced and the rate of neurogenesis was halved. Furthermore, in adult knockout mice, there were fewer cortical pyramidal neurons, interneurons, cholinergic basal forebrain neurons and striatal neurons, corresponding to a relative reduction in volume of these structures. Thalamic midline fusion during early postnatal development was also impaired in Nestin-Cre p75NTR floxed mice, indicating a novel role for p75NTR in the formation of this structure. The phenotype of this strain demonstrates that p75NTR regulates multiple aspects of brain development, including cortical progenitor cell survival, and that expression during early neurogenesis is required for appropriate formation of telencephalic structures.

Recurrent network activity drives striatal synaptogenesis.

Neural activity during development critically shapes postnatal wiring of the mammalian brain. This is best illustrated by the sensory systems, in which the patterned feed-forward excitation provided by sensory organs and experience drives the formation of mature topographic circuits capable of extracting specific features of sensory stimuli. In contrast, little is known about the role of early activity in the development of the basal ganglia, a phylogenetically ancient group of nuclei fundamentally important for complex motor action and reward-based learning. These nuclei lack direct sensory input and are only loosely topographically organized, forming interlocking feed-forward and feed-back inhibitory circuits without laminar structure. Here we use transgenic mice and viral gene transfer methods to modulate neurotransmitter release and neuronal activity in vivo in the developing striatum. We find that the balance of activity between the two inhibitory and antagonist pathways in the striatum regulates excitatory innervation of the basal ganglia during development. These effects indicate that the propagation of activity through a multi-stage network regulates the wiring of the basal ganglia, revealing an important role of positive feedback in driving network maturation.

Mutant huntingtin is present in neuronal grafts in Huntington disease patients.

OBJECTIVE: Huntington disease (HD) is caused by a genetically encoded pathological protein (mutant huntingtin [mHtt]), which is thought to exert its effects in a cell-autonomous manner. Here, we tested the hypothesis that mHtt is capable of spreading within cerebral tissue by examining genetically unrelated fetal neural allografts within the brains of patients with advancing HD. METHODS: The presence of mHtt aggregates within the grafted tissue was confirmed using 3 different types of microscopy (bright-field, fluorescence, and electron), 2 additional techniques consisting of Western immunoblotting and infrared spectroscopy, and 4 distinct antibodies targeting different epitopes of mHtt aggregates. RESULTS: We describe the presence of mHtt aggregates within intracerebral allografts of striatal tissue in 3 HD patients who received their transplants approximately 1 decade earlier and then died secondary to the progression of their disease. The mHtt(+) aggregates were observed in the extracellular matrix of the transplanted tissue, whereas in the host brain they were seen in neurons, neuropil, extracellular matrix, and blood vessels. INTERPRETATION: This is the first demonstration of the presence of mHtt in genetically normal and unrelated allografted neural tissue transplanted into the brain of affected HD patients. These observations raise questions on protein spread in monogenic neurodegenerative disorders of the central nervous system characterized by the formation of mutant protein oligomers/aggregates.

Striatal development involves a switch in gene expression networks, followed by a myelination event: implications for neuropsychiatric disease.

Because abnormal development of striatal neurons is thought to be the part of pathology underlying major psychiatric illnesses, we studied the expression pattern of genes involved in striatal development and of genes comprising key striatal-specific pathways, during an active striatal maturation period, the first two postnatal weeks in rat. This period parallels human striatal development during the second trimester, when prenatal stress is though to lead to increased risk for neuropsychiatric disorders. To identify genes involved in this developmental process, we used subtractive hybridization, followed by quantitative real-time PCR, which allowed us to characterize the developmental expression of over 60 genes, many not previously known to play a role in neuromaturation. Of these 12 were novel transcripts, which did not match known genes, but which showed strict developmental expression and may play a role in striatal neurodevelopment. An additional 89 genes were identified as strong candidates for involvement in this neurodevelopmental process. We show that during the first two postnatal weeks in rat, an early gene expression network, still lacking key striatal-specific signaling pathways, is downregulated and replaced by a mature gene expression network, containing key striatal-specific genes including the dopamine D1 and D2 receptors, conferring to these neurons their functional identity. Therefore, before this developmental switch, striatal neurons lack many of their key phenotypic characteristics. This maturation process is followed by a striking rise in expression of myelination genes, indicating a striatal-specific myelination event. Such strictly controlled developmental program has the potential to be a point of susceptibility to disruption by external factors. Indeed, this period is known to be a susceptibility period in both humans and rats.

The mouse homeobox gene Gbx2 is required for the development of cholinergic interneurons in the striatum.

Mammalian forebrain cholinergic neurons are composed of local circuit neurons in the striatum and projection neurons in the basal forebrain. These neurons are known to arise from a common pool of progenitors that primarily resides in the medial ganglionic eminence (MGE). However, little is known about the genetic programs that differentiate these two types of cholinergic neurons. Using inducible genetic fate mapping, here we examined the developmental fate of cells that express the homeodomain transcription factor Gbx2 in the MGE. We show that the Gbx2 lineage-derived cells that undergo tangential migration exclusively give rise to almost all cholinergic interneurons in the striatum, whereas those undergoing radial migration mainly produce noncholinergic neurons in the basal forebrain. Deletion of Gbx2 throughout the mouse embryo or specifically in the MGE results in abnormal distribution and significant reduction of cholinergic neurons in the striatum. We show that early-born (before embryonic day 12.5) cholinergic interneurons preferentially populate the lateral aspect of the striatum and mature earlier than late-born (after embryonic day 12.5) neurons, which normally reside in the medial part of the striatum. In the absence of Gbx2, early-born striatal cholinergic precursors display abnormal neurite outgrowth and increased complexity, and abnormally contribute to the medial part of the caudate-putamen, whereas late-born striatal cholinergic interneurons are mostly missing. Together, our data demonstrate that Gbx2 is required for the development of striatal cholinergic interneurons, perhaps by regulating tangential migration of the striatal cholinergic precursors.

Tachykinin-immunoreactive neurons in developing feline neostriatum: somatodendritic morphogenesis demonstrated by combined immunohistochemistry/Golgi impregnation-gold toning.

This investigation was designed to survey and characterize the development of a key link between chemically mediated neurotransmission and neuronal cytoarchitecture in mammalian basal ganglia. Peroxidase immunohistochemical and Golgi impregnation/gold toning methods were combined to doubly label the tachykinin neuromodulator signature and somatodendritic structure of neostriatal neurons in late fetal, postnatal and adult cats. The results supported 3 conclusions of considerable significance. (1) Colocalization of immunohistochemical and Golgi impregnation/gold toning labels is a feasible, rational and productive means to identify and determine the somatodendritic morphogenesis of tachykinin neurons. (2) The application of this method to developing feline neostriatum demonstrates directly that the principal tachykinin cells are medium-sized spiny neurons, which undergo progressive growth and elaboration of cell bodies, dendritic arbors and dendritic spines during the late fetal and postnatal periods. (3) There is a strong but incomplete concordance between tachykinin and medium-sized spiny neuronal phenotypes, because a minor variant of medium-sized spiny neurons and rare subgroups of medium- and large-sized sparse spiny neurons also show the tachykinin neuromodulator signature. Taken together, these results suggest that neostriatal neurons show an early commitment to heterogeneous tachykinin phenotypes, although the full and final expression of their somatodendritic characteristics coincides with synaptogenesis.

Medium spiny neurons for transplantation in Huntington's disease.

Cell-replacement therapy for Huntington's disease is one of very few therapies that has reported positive outcomes in clinical trials. However, for cell transplantation to be made more readily available, logistical, standardization and ethical issues associated with the current methodology need to be resolved. To achieve these goals, it is imperative that an alternative cell source be identified. One of the key requirements of the cells is that they are capable of acquiring an MSN (medium spiny neuron) morphology, express MSN markers such as DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa), and function in vivo in a manner that replicates those that have been lost to the disease. Developmental biology has progressed in recent years to provide a vast array of information with regard to the key signalling events involved in the proliferation, specification and differentiation of striatal-specific neurons. In the present paper, we review the rationale for cell-replacement therapy in Huntington's disease, discuss some potential donor sources and consider the value of developmental markers in the identification of cells with the potential to develop an MSN phenotype.

Prenatal inflammatory effects on nigrostriatal development in organotypic cultures.

Maternal intrauterine infection, and the accompanying inflammation in the fetal brain, represent a significant risk to the developing fetus. Dopamine (DA) neurons have been shown to be particularly vulnerable to inflammation induced by injection of the bacterial endotoxin lipopolysaccharide (LPS). In order to further examine the nature of this vulnerability, we used a combination of in vivo prenatal LPS exposure, and in vitro analysis of nigrostriatal development in organotypic cultures prepared from LPS-exposed rat fetuses. Control co-cultures prepared from unexposed E14 substantia nigra (SN/VTA) and E21 striatum exhibited numerous DA neurons in the nigral piece and robust ingrowth into the striatal piece. When E14 SN/VTA was obtained from fetuses exposed to LPS (0.1 mg/kg) on E10, initial DA cell numbers and striatal innervation in co-cultures were normal, but at longer durations in vitro, a reduction in DA neurons was observed. When striatal tissue from fetuses exposed to LPS on E14 or E18 was used in combination with non-exposed SN/VTA, DA neurons initially exhibited a normal pattern of ingrowth into LPS-exposed striatum. However, with longer durations in vitro, DA neurons were lost more rapidly when co-cultured with LPS-exposed striatum. Despite the loss of DA neurons, striatal DA innervation was only reduced in cultures prepared with striatum exposed to LPS at E18, at the longest time period examined. Experiments in which unexposed SN/VTA was given the choice to grow toward control striatum or toward LPS-exposed striatum supported the idea that the tropic qualities of the striatum were not altered by LPS-induced inflammation. Thus, the inflammation induced by LPS not only affects the SN/VTA DA neurons, but also alters the neurotrophic--although not the neurotropic--characteristics of the striatum. Such alterations in nigrostriatal development may demonstrate how adverse perinatal events predispose the developing brain toward the later development of Parkinson's disease.

Anatomical and physiological evidence for D1 and D2 dopamine receptor colocalization in neostriatal neurons.

Despite the importance of dopamine signaling, it remains unknown if the two major subclasses of dopamine receptors exist on the same or distinct populations of neurons. Here we used confocal microscopy to demonstrate that virtually all striatal neurons, both in vitro and in vivo, contained dopamine receptors of both classes. We also provide functional evidence for such colocalization: in essentially all neurons examined, fenoldopam, an agonist of the D1 subclass of receptors, inhibited both the Na+/K+ pump and tetrodotoxin (TTX)-sensitive sodium channels, and quinpirole, an agonist of the D2 subclass of receptors, activated TTX-sensitive sodium channels. Thus D1 and D2 classes of ligands may functionally interact in virtually all dopamine-responsive neurons within the basal ganglia.

Active intermixing of indirect and direct neurons builds the striatal mosaic.

The striatum controls behaviors via the activity of direct and indirect pathway projection neurons (dSPN and iSPN) that are intermingled in all compartments. While such cellular mosaic ensures the balanced activity of the two pathways, its developmental origin and pattern remains largely unknown. Here, we show that both SPN populations are specified embryonically and intermix progressively through multidirectional iSPN migration. Using conditional mutant mice, we found that inactivation of the dSPN-specific transcription factor Ebf1 impairs selective dSPN properties, including axon pathfinding, while molecular and functional features of iSPN were preserved. Ebf1 mutation disrupted iSPN/dSPN intermixing, resulting in an uneven distribution. Such architectural defect was selective of the matrix compartment, highlighting that intermixing is a parallel process to compartment formation. Our study reveals while iSPN/dSPN specification is largely independent, their intermingling emerges from an active migration of iSPN, thereby providing a novel framework for the building of striatal architecture.

Nanosize titanium dioxide stimulates reactive oxygen species in brain microglia and damages neurons in vitro.

BACKGROUND: Titanium dioxide is a widely used nanomaterial whose photo-reactivity suggests that it could damage biological targets (e.g., brain) through oxidative stress (OS). OBJECTIVES: Brain cultures of immortalized mouse microglia (BV2), rat dopaminergic (DA) neurons (N27), and primary cultures of embryonic rat striatum, were exposed to Degussa P25, a commercially available TiO(2) nanomaterial. Physical properties of P25 were measured under conditions that paralleled biological measures. FINDINGS: P25 rapidly aggregated in physiological buffer (800-1,900 nm; 25 degrees C) and exposure media (approximately 330 nm; 37 degrees C), and maintained a negative zeta potential in both buffer (-12.2 +/- 1.6 mV) and media (-9.1 +/- 1.2 mV). BV2 microglia exposed to P25 (2.5-120 ppm) responded with an immediate and prolonged release of reactive oxygen species (ROS). Hoechst nuclear stain was reduced after 24-hr (>or=100 ppm) and 48-hr (>or=2.5 ppm) exposure. Microarray analysis on P25-exposed BV2 microglia indicated up-regulation of inflammatory, apoptotic, and cell cycling pathways and down-regulation of energy metabolism. P25 (2.5-120 ppm) stimulated increases of intracellular ATP and caspase 3/7 activity in isolated N27 neurons (24-48 hr) but did not produce cytotoxicity after 72-hr exposure. Primary cultures of rat striatum exposed to P25 (5 ppm) showed a reduction of immunohistochemically stained neurons and microscopic evidence of neuronal apoptosis after 6-hr exposure. These findings indicate that P25 stimulates ROS in BV2 microglia and is nontoxic to isolated N27 neurons. However, P25 rapidly damages neurons at low concentrations in complex brain cultures, plausibly though microglial generated ROS.

Neurosphere-derived neural cells show region-specific behaviour in vitro.

Neurosphere cultures provide a useful model to study neural stem/progenitor cells (NSC/NPCs). The degree to which neurospheres (NS) retain their regional identity in vitro has, however, been questioned. Here, NS obtained from mouse embryonic cortex, striatum or spinal cord were compared after differentiation. Neurons from cortical NS formed well ordered clusters containing astrocytes, those from striatal NS formed an external ring at the borderof the astrocyte layer, whereas those from spinal cord NS spread radially like the astrocytes. Such in-vitro neural behaviour was region-specific and persisted in clonal conditions, providing evidence of the maintenance of positional cues in NS cultures.

The corridor task: striatal lesion effects and graft-mediated recovery in a model of Huntington's disease.

Experimental validation of cell replacement therapy as a treatment of neurodegenerative diseases requires the demonstration of graft-mediated behavioural recovery. The Corridor task proved to be simple and efficient to conduct with a robust ipsilateral retrieval bias in our rodent Huntington's disease model. The Corridor task is a viable behavioural option, particularly to non-specialised laboratories, for the evaluation of lateralised striatal damage and the probing of alternative therapeutic strategies, including transplantation.

[Formation of structural and ultrastructural organization of striatum rat postnatal ontogenesis upon changes in their embryonal development].

By light microscopy (by Nissl and Golgi), electron microscopy, and immunohistochemistry methods, formation of structure of the brain striatum dorsolateral part from birth to the 3-month age was studied in rats submitted to acute hypoxia at the period of embryogenesis. It has been established that hypoxia at the 13.5th day (E13.5) leads to a delay of neuronogenesis for the first two weeks of postnatal development as compared with control animals, while the majority of large neurons at this period are degenerated by the type of chromatolysis with swelling cell body and processes and lysis of cytoplasmic organoids. By the end of the 3rd week, shrunk hyperchromic or picnomorphic neurons with the electron-dense cytoplasm and enlarged tubules of endoplasmic reticulum and Golgi complex were also observed. An increased number of swollen processes of glial cells was detected in neuropil around degenerating neurons. By the 30th day as well as in adult rats there was observed destruction of mitochondrial apparatus, an increase of the number of lysosomes, and the appearance of bladed nuclei - signs of apoptotic cell death, which was also confirmed by an increased expression of proapoptotic p53 protein and its colocalization with caspase-3 in a part of neurons. Morphometrical analysis has shown a decrease of density of striatum cell arrangement and a change of ratio of different cell types in the rats submitted to hypoxia as compared with control group. At early stages of postnatal ontogenesis there was the greatest decrease (42.3% at the 5th day, 14.2% at the 10th day, p < 0.01) of the number of large neurons with the area more than 80 microm2. After 3 weeks of postnatal development the number of middlesize neurons (30-95 microm2) decreased (by 11.8-19.2%) as compared with control. The obtained data show that a change of conditions of embryogenesis (hypoxia) at the period of the most intensive proliferation of the forebrain neuroblasts leads to disturbances of the process of formation of the striatum nervous tissue. This can be the cause of delay of development and disturbances of behavior and learning observed in rats submitted to prenatal hypoxia.

Formation of the structural and ultrastructural organization of the striatum in early postnatal ontogenesis of rats in altered conditions of embryonic development.

Light (Nissl and Golgi methods) and electron microscopy methods were used to study the formation of the structure of the striatum during the first two weeks after birth in rats subjected to acute hypoxia at different times of embryogenesis. The dynamics of the physiological development of the same population of rats were studied in parallel. Hypoxia at day 13.5 of embryogenesis was found to lead to delayed neurogenesis (delayed establishment of elements of the neuropil and differentiation of cells) and abnormalities in the structure of the striatum (degeneration, particularly chromatolysis, of neurons and the appearance of glial nodes). Morphometric analysis demonstrated a decrease in the total number of cells in the striatum; small changes in large neurons were seen. Hypoxia at day 18.5 of embryogenesis produced no significant changes. Structural abnormalities were accompanied by changes in the process of the animals' physiological development. The data obtained here show that changes in the conditions of embryogenesis (hypoxia) during the period of the most intense proliferation of neuroblasts in the forebrain lead to impairment of the process of formation of striatal nervous tissue and the body as a whole in the period of early postnatal ontogenesis.

Methylation QTLs in the developing brain and their enrichment in schizophrenia risk loci.

We characterized DNA methylation quantitative trait loci (mQTLs) in a large collection (n = 166) of human fetal brain samples spanning 56-166 d post-conception, identifying >16,000 fetal brain mQTLs. Fetal brain mQTLs were primarily cis-acting, enriched in regulatory chromatin domains and transcription factor binding sites, and showed substantial overlap with genetic variants that were also associated with gene expression in the brain. Using tissue from three distinct regions of the adult brain (prefrontal cortex, striatum and cerebellum), we found that most fetal brain mQTLs were developmentally stable, although a subset was characterized by fetal-specific effects. Fetal brain mQTLs were enriched amongst risk loci identified in a recent large-scale genome-wide association study (GWAS) of schizophrenia, a severe psychiatric disorder with a hypothesized neurodevelopmental component. Finally, we found that mQTLs can be used to refine GWAS loci through the identification of discrete sites of variable fetal brain methylation associated with schizophrenia risk variants.

Parallel FoxP1 and FoxP2 expression in songbird and human brain predicts functional interaction.

Humans and songbirds are two of the rare animal groups that modify their innate vocalizations. The identification of FOXP2 as the monogenetic locus of a human speech disorder exhibited by members of the family referred to as KE enables the first examination of whether molecular mechanisms for vocal learning are shared between humans and songbirds. Here, in situ hybridization analyses for FoxP1 and FoxP2 in a songbird reveal a corticostriatal expression pattern congruent with the abnormalities in brain structures of affected KE family members. The overlap in FoxP1 and FoxP2 expression observed in the songbird suggests that combinatorial regulation by these molecules during neural development and within vocal control structures may occur. In support of this idea, we find that FOXP1 and FOXP2 expression patterns in human fetal brain are strikingly similar to those in the songbird, including localization to subcortical structures that function in sensorimotor integration and the control of skilled, coordinated movement. The specific colocalization of FoxP1 and FoxP2 found in several structures in the bird and human brain predicts that mutations in FOXP1 could also be related to speech disorders.

Dopamine modulates cell cycle in the lateral ganglionic eminence.

Dopamine is a neuromodulator the functions of which in the regulation of complex behaviors such as mood, motivation, and attention are well known. Dopamine appears in the brain early in the embryonic period when none of those behaviors is robust, raising the possibility that dopamine may influence brain development. The effects of dopamine on specific developmental processes such as neurogenesis are not fully characterized. The neostriatum is a dopamine-rich region of the developing and mature brain. If dopamine influenced neurogenesis, the effects would likely be pronounced in the neostriatum. Therefore, we examined whether dopamine influenced neostriatal neurogenesis by influencing the cell cycle of progenitor cells in the lateral ganglionic eminence (LGE), the neuroepithelial precursor of the neostriatum. We show that dopamine arrives in the LGE via the nigrostriatal pathway early in the embryonic period and that neostriatal neurogenesis progresses in a dopamine-rich milieu. Dopamine D1-like receptor activation reduces entry of progenitor cells from the G(1)- to S-phase of the cell cycle, whereas D2-like receptor activation produces the opposite effects by promoting G(1)- to S-phase entry. D1-like effects are prominent in the ventricular zone, and D2-like effects are prominent in the subventricular zone. The overall effects of dopamine on the cell cycle are D1-like effects, most likely because of the preponderance of D1-like binding sites in the embryonic neostriatum. These data reveal a novel developmental role for dopamine and underscore the relevance of dopaminergic signaling in brain development.

[Formation of striatum structural and ultrastructural organization in the early postnatal ontogenesis of rats subjected to altered conditions of their embryonic development].

Formation of the structure of striatum during two postnatal weeks in rats subjected to acute hypoxia during various periods of their embryonic development was studied using light microscopic (Nissl's stain and Golgi's silver nitrate impregnation) methods and electron microscopy. This study was supplemented by a simultaneous investigation of physiological development of the same population of rats. The data obtained demonstrated that prenatal hypoxia on day 13.5 of embryonic development (E13.5) led to a delayed neurogenesis (retardation in the development of neuropil elements and cell differentiation) as well as to the malformation of the structure of striatum (degeneration, in particular, chromatolysis of neurons and glial nodule formation). Morphometric analysis demonstrated that prenatal hypoxia on E13.5 resulted in a statistically significant decrease in cell number in the striatum, these changes being especially pronounced in large neurons. Prenatal hypoxia on E18.5, however, caused no significant changes in striatum. Structural changes in the striatum were shown to be accompanied by significant changes in the physiological development of animals. The data obtained demonstrated that the alteration of the conditions of embryogenesis (hypoxia) during the period of most intensive proliferation of forebrain neuroblasts resulted in the disturbances of the formation of both striatum nervous tissue of the organism as a whole during early postnatal ontogenesis.

The neostriatal mosaic: basis for the changing distribution of neurokinin-1 receptor immunoreactivity during development.

The pattern of neurokinin-1 receptor-like immunoreactivity (NK-1Rir) was mapped in perinatal and adult mouse striatum by using a new polyclonal antiserum. NK-1Rir was detected in the differentiating regions of the ganglionic eminences on embryonic day 12.5 (E12.5). NK-1Rir structures were enriched in the striatal patch compartment between E16.5 and approximately postnatal day 3 (P3); distributed more uniformly, within portions of both the patch and matrix compartments on P7; and enriched in the matrix compartment in the adult. Analysis of the phenotype of NK-1Rir cells on P2, P7, and in the adult suggested that cholinergic cells accounted for the majority of NK-1Rir cells early postnatally, with increasing contributions from somatostatinergic cells later postnatally. In the adult, approximately half of NK-1Rir cells were cholinergic and half were somatostatinergic. The transient enrichment of NK-1R-bearing cells and processes in the patch compartment which contains cells that express substance P (SP), a putative ligand for the NK-1R, may be a consequence of compartment formation or may be functionally important for compartment development.