Distinct subclasses of medium spiny neurons differentially regulate striatal motor behaviors

The direct and indirect pathways of the basal ganglia have been proposed to oppositely regulate locomotion and differentially contribute to pathological behaviors. Analysis of the distinct contributions of each pathway to behavior has been a challenge, however, due to the difficulty of selectively investigating the neurons comprising the two pathways using conventional techniques. Here we present two mouse models in which the function of striatonigral or striatopallidal neurons is selectively disrupted due to cell type–specific deletion of the striatal signaling protein dopamine- and cAMP-regulated phosphoprotein Mr 32kDa (DARPP-32). Using these mice, we found that the loss of DARPP-32 in striatonigral neurons decreased basal and cocaine-induced locomotion and abolished dyskinetic behaviors in response to the Parkinson''s disease drug L-DOPA. Conversely, the loss of DARPP-32 in striatopallidal neurons produced a robust increase in locomotor activity and a strongly reduced cataleptic response to the antipsychotic drug haloperidol. These findings provide insight into the selective contributions of the direct and indirect pathways to striatal motor behaviors.     

兴奋毒作用下皮层神经细胞Ca^2＋变化机制

目的：检测谷氨酸兴奋毒损伤时,有胞外有Ca^2 或无Ca^2 ,,或有尼莫地平和Ca^2 条件下,神经细胞内[(Ca^2 )]i的变化,方法：用Fura-2/AM体外检测新生大鼠皮层神经细胞内游离钙离子浓度的方法。结果：发现谷氨酸兴奋毒可使皮层神经细胞[Ca^2 ]i上升并有剂量依赖性,胞外无Ca^2 或加入尼莫地平时神经细胞[Ca^2 ]i虽也上升,但幅度降低。三种条件下神经细胞在兴奋毒作用下[Ca^2=]i变化各不相同。结论：表明谷氨酸活化神经细胞膜上受体,使胞内钙库动员和细胞膜Ca^2 通道开放,导致[Ca^2_]i在短时间内总体表现为持续上升,存在兴奋毒损伤过程中有膦脂酶A2活化过度参与的信使基础。     

Histaminergic responses by hypothalamic neurons that regulate lordosis and their modulation by estradiol

How do fluctuations in the level of generalized arousal of the brain affect the performance of specific motivated behaviors, such as sexual behaviors that depend on sexual arousal? A great deal of previous work has provided us with two important starting points in answering this question: (i) that histamine (HA) serves generalized CNS arousal and (ii) that heightened electrical activity of neurons in the ventromedial nucleus of the hypothalamus (VMN) is necessary and sufficient for facilitating the primary female sex behavior in laboratory animals, lordosis behavior. Here we used patch clamp recording technology to analyze HA effects on VMN neuronal activity. The results show that HA acting through H1 receptors (H1R) depolarizes these neurons. Further, acute administration of estradiol, an estrogen necessary for lordosis behavior to occur, heightens this effect. Hyperpolarization, which tends to decrease excitability and enhance inhibition, was not affected by acute estradiol or mediated by H1R but was mediated by other HA receptor subtypes, H2 and H3. Sampling of mRNA from individual VMN neurons showed colocalization of expression of H1 receptor mRNA with estrogen receptor (ER)-α mRNA but also revealed ER colocalization with the other HA receptor subtypes and colocalization of different subtypes with each other. The latter finding provides the molecular basis for complex “push-pull” regulation of VMN neuronal excitability by HA. Thus, in the simplest causal route, HA, acting on VMN neurons through H1R provides a mechanism by which elevated states of generalized CNS arousal can foster a specific estrogen-dependent, aroused behavior, sexual behavior.     

Depolarizing stimuli regulate nerve growth factor gene expression in cultured hippocampal neurons.

Although trophic factors and neuronal activity have been implicated in regulating functional synaptic circuits, the relationship of trophic interaction to impulse activity in synaptogenesis remains unclear. Using cultured hippocampus as a model system, we provide direct evidence that depolarization and impulse activity specifically increase nerve growth factor gene expression in neurons. Depolarizing stimuli, such as a high K+ concentration or the Na+ channel agonist veratridine, elicited a 3-fold increase of nerve growth factor mRNA levels in both explant and dissociated cultures. Blockade of depolarization by tetrodotoxin prevented the increase of neuronal nerve growth factor mRNA. Further, nerve growth factor gene expression was stimulated by picrotoxin, a gamma-aminobutyric acid antagonist frequently used to enhance hippocampal neuronal activity. Impulse regulation of trophic gene function may be relevant to developmental synaptogenesis and synaptic strengthening in learning and memory.     

Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain.

We used in situ hybridization histochemistry with synthetic oligodeoxyribonucleotide probes to identify cells that synthesize mRNAs encoding tyrosine hydroxylase in the mesencephalon and substance P, enkephalin, and dynorphin in the rat forebrain. Dopaminergic cells in the mesencephalon project to the forebrain and influence neuropeptide levels. We examined the effect of unilateral 6-hydroxydopamine lesions (which eliminated tyrosine hydroxylase mRNA-containing cells in the mesencephalon) on substance P, enkephalin, and dynorphin mRNA levels. Substance P mRNA levels were depressed, whereas enkephalin mRNA levels were elevated in consecutive sections from striatal areas in all animals. The effects of the lesions on dynorphin mRNA levels were less robust, and considerable variation between animals was observed. Changes were evident in the levels of message in individual cells but not in the numbers of labeled cells. These effects were not uniform throughout the dopamine-innervated areas, suggesting degrees of control not apparent with RNA blot-hybridization or dot-blot analyses.     

H2-Kb and H2-Db regulate cerebellar long-term depression and limit motor learning

There are more than 50 class I MHC (MHCI) molecules in the mouse genome, some of which are now known to be expressed in neurons; however, the role of classical MHCI molecules in synaptic plasticity is unknown. We report that the classical MHCI molecules, H2-K^b and H2-D^b, are co-expressed by Purkinje cells (PCs). In the cerebellum of mice deficient for both H2-K^b and H2-D^b (K^bD^b−/−), there is a lower threshold for induction of long-term depression (LTD) at parallel fiber to PC synapses. This change may be a result of additional glutamate release observed at K^bD^b−/− CF to PC synapses, which are thought to “train” the cerebellar circuit. A behavioral correlate of cerebellar LTD is motor learning; acquisition and retention of a Rotarod behavioral task is significantly better in K^bD^b−/− mice than in WT cohorts. These physiological and behavioral phenotypes in K^bD^b−/− mice reveal a surprising role for classical MHCI molecules in synaptic plasticity and motor learning.     

Local circuit neurons in the striatum regulate neural and behavioral responses to dopaminergic stimulation

Interneurons are critical for shaping neuronal circuit activity in many parts of the central nervous system. To study interneuron function in the basal ganglia, we tested and characterized an NK-1 receptor-based method for targeted ablation of specific classes of interneuron in the striatum. Our findings demonstrate that the neurotoxin SP-PE35, a substance P-Pseudomonas exotoxin conjugate, selectively targets striatal cholinergic and nitric oxide synthase/somatostatinergic interneurons when injected locally into the striatum. The effects of this selective cell targeting encompassed alterations in both behavioral and neural responses to dopaminergic stimulation, including altered patterns of early-gene response in striosomes and matrix. We conclude that NK-1-bearing local circuit neurons of the striatum regulate the differential responses of striatal projection neurons to dopamine-mediated signaling.     

S-nitrosylated SHP-2 contributes to NMDA receptor-mediated excitotoxicity in acute ischemic stroke

Overproduction of nitric oxide (NO) can cause neuronal damage, contributing to the pathogenesis of several neurodegenerative diseases and stroke (i.e., focal cerebral ischemia). NO can mediate neurotoxic effects at least in part via protein S-nitrosylation, a reaction that covalently attaches NO to a cysteine thiol (or thiolate anion) to form an S-nitrosothiol. Recently, the tyrosine phosphatase Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) and its downstream pathways have emerged as important mediators of cell survival. Here we report that in neurons and brain tissue NO can S-nitrosylate SHP-2 at its active site cysteine, forming S-nitrosylated SHP-2 (SNO–SHP-2). We found that NMDA exposure in vitro and transient focal cerebral ischemia in vivo resulted in increased levels of SNO–SHP-2. S-Nitrosylation of SHP-2 inhibited its phosphatase activity, blocking downstream activation of the neuroprotective physiological ERK1/2 pathway, thus increasing susceptibility to NMDA receptor-mediated excitotoxicity. These findings suggest that formation of SNO–SHP-2 represents a key chemical reaction contributing to excitotoxic damage in stroke and potentially other neurological disorders.     

Conditioned medium of human adipose-derived mesenchymal stem cells mediates protection in neurons following glutamate excitotoxicity by regulating energy metabolism and GAP-43 expression

Glutamate excitotoxicity has been implicated as one of the pathological mechanisms contributing to neuronal cell death and is involved in many neurological disorders. Stem cell transplantation is a promising approach for the treatment of nervous system damage or diseases. Previous studies have shown that mesenchymal stem cells (MSCs) have important therapeutic effects in experimental animal and preclinical disease model of central nervous system pathology. However, it is not well understood whether neurogenesis of MSCs or MSC conditioned-medium (CM) containing microparticles mediates therapeutic effects. Here, we investigated the neuroprotective effects of human adipose-derived MSCs (AMSCs) on cortical neurons using models of glutamate excitotoxicity. Following exposure to glutamate (100 μM, 15 min), cortical neurons were co-cultured with either AMSCs separated by a semiporous membrane (prohibiting direct cell-cell contact) or with AMSC-CM for 18 h. Compared to untreated control groups, AMSCs and AMSC-CM partially and similarly reduced neuronal cell damages, as indicated by reduced LDH release, a decreased number of trypan-positive cells and a decline in the number of apoptotic nuclei. Protection by CM was associated with increased GAP-43 expression and an elevated number of GAP-43-positive neurites. Furthermore, CM increased levels of ATP, NAD⁺ and NADH and the ratio of NAD⁺/NADH, while preventing a glutamate-induced decline in mitochondrial membrane potential. These results demonstrate that AMSC-CM mediates direct neuroprotection by inhibiting neuronal cell damage/apoptosis, promoting nerve regeneration and repair, and restoring bioenergy following energy depletion caused by glutamate excitotoxicity.     

Depolarizing influences regulate preprotachykinin mRNA in sympathetic neurons.

We have been studying mechanisms regulating neurotransmitter plasticity in sympathetic neurons. Neurons of the rat superior cervical ganglion (SCG) synthesize multiple putative transmitters, including the peptide substance P (SP). We have now examined steady-state levels of the mRNA encoding preprotachykinin (PPT), the SP precursor. A cloned cDNA probe was used to examine regulation mRNA levels in culture and in vivo. In RNA gel blot experiments, a single band (1.1 kilobases long) was observed in all cases in which an RNA was detected. A low level of PPT mRNA was detected by RNase protection assay in uncultured ganglia, suggesting that the low levels of SP previously observed in the normal ganglion in vivo are synthesized locally. When ganglia were maintained in culture, with consequent denervation, the steady-state level of PPT mRNA increased by 25-fold over the first 24 hr, and the high level was maintained for at least 7 days. RNase protection experiments indicated that the major message in the SCG is the beta-PPT mRNA, encoding both SP and neurokinin A peptide regions. Accumulation of the PPT mRNA in cultured ganglia was sharply inhibited by the depolarizing agent veratridine, and this effect was blocked by tetrodotoxin. Therefore, one form of neuronal plasticity, change in neurotransmitter metabolism, is regulated at least in part by altering steady-state levels of specific mRNA. More generally, extracellular signals may contribute to neuronal plasticity through changes in gene expression.     

Intricate interplay between astrocytes and motor neurons in ALS

ALS results from the selective and progressive degeneration of motor neurons. Although the underlying disease mechanisms remain unknown, glial cells have been implicated in ALS disease progression. Here, we examine the effects of glial cell/motor neuron interactions on gene expression using the hSOD1^G93A (the G93A allele of the human superoxide dismutase gene) mouse model of ALS. We detect striking cell autonomous and nonautonomous changes in gene expression in cocultured motor neurons and glia, revealing that the two cell types profoundly affect each other. In addition, we found a remarkable concordance between the cell culture data and expression profiles of whole spinal cords and acutely isolated spinal cord cells during disease progression in the G93A mouse model, providing validation of the cell culture approach. Bioinformatics analyses identified changes in the expression of specific genes and signaling pathways that may contribute to motor neuron degeneration in ALS, among which are TGF-β signaling pathways.ALS is a late-onset, fatal neurodegenerative disease caused by the selective loss of upper and lower motor neurons in the brain and spinal cord and progressive paralysis of voluntary muscles; death ultimately results from respiratory failure (reviewed in ref. 1). Most ALS cases (∼90%) are sporadic, with an unknown cause, whereas the remaining cases are of familial origin (reviewed in ref. 2), among which ∼20–25% are caused by dominantly inherited mutations in the SOD1 gene; this gene encodes a cytosolic Cu/Zn superoxide dismutase (3). Overproducing pathogenic alleles of human SOD1 in mice and rats leads to late-onset progressive motor neuron degeneration, strikingly similar to the human disease (4–7). Because the pathological progression in both sporadic and familial ALS is indistinguishable (8, 9), insights derived from studies of the SOD1 mouse model are thought to be informative for both sporadic and familial ALS pathology.The fundamental pathological basis for ALS remains to be determined along with the specific insults that target motor neurons for death. Mutant SOD1 genes are expressed ubiquitously in humans and mice and when expressed exclusively in mouse motor neurons, are not sufficient to cause disease (10–12). An important insight into this enigma was provided by the observation that the presence of mutant SOD1 within neighboring nonneuronal cells contributes to motor neuron toxicity and thereby, disease onset and progression (reviewed in ref. 13). The principal nonneuronal cell types implicated in motor neuron death in ALS are astrocytes, microglia, and oligodendrocytes; in vivo approaches focused on excising the mutant transgene from microglia and astrocytes in SOD1-based ALS mouse models have shown that disease onset and/or progression are affected (reviewed in ref. 13). There is increasing evidence that the presence of the mutant SOD1 protein in these nonneuronal cell types contributes significantly to ALS disease progression in the ALS mouse model. Evidence that astrocytes also play a negative role in human ALS was provided by a recent study showing that astrocytes generated from postmortem spinal cords from SOD1 or sporadic ALS patients adversely affect the viability of cultured ES cell-derived mouse motor neurons (14).The question of extrinsic vs. intrinsic effects on gene expression in motor neurons in ALS in vivo has been difficult to address using laser capture microdissection (LCMD), because only the cell soma is captured, excluding the dendritic arbor as well as the axon. Moreover, LCMD is limited to neuronal cells, because it is not possible to cleanly capture glial cell bodies from among the neuropil in the spinal cord. Other approaches involve studies of entire spinal cords, which are heterogeneous and do not provide cell type-specific information. Thus, either approach alone yields an incomplete picture. Consequently, it has thus far not been possible to relate progressive gene expression changes in motor neurons to changes in gene expression in the surrounding glial cells in whole-animal studies. A potential solution to this problem is to make use of cell culture models to study how glial cells adversely affect motor neuron viability.In previous studies, we (15) and others (6) established a cell culture system to study astrocyte/motor neuron interactions. This approach involves the generation of motor neurons by in vitro differentiation of ES cells derived from mice harboring the human SOD1^G93A transgene. These ES cell-derived motor neurons are cocultured with either ES cell-derived or primary glial cells. Remarkably, motor neurons in this coculture system recapitulate aspects of the abnormal pathology characteristic of ALS in humans as well as in transgenic mice (15). In addition, SOD1^G93A mutant glia can adversely affect the viability of mutant as well as WT motor neurons in vitro and in vivo (14, 16).Although each approach to the analysis of ALS disease mechanisms (postmortem ALS patient samples, mouse models, and cell culture) has its limitations, an integrated approach that combines whole-animal and cell culture analyses could provide novel insights into the pathways leading to motor neuron-specific degeneration. Here, we describe an adaptation of this in vitro model to capture both cell autonomous and nonautonomous changes in neuronal and glial cell gene expression. We studied FACS-purified ES cell-derived motor neurons in sandwich cultures (17), in which motor neurons plated on glass coverslips are cultured over primary glial cells as a function of time. We then used separate RNA sequencing (RNA-Seq) analysis of the two cell types to identify changes in gene expression profiles intrinsic to each cell type (cell autonomous effects) or mediated by the cocultured cell (cell nonautonomous effects).In parallel, we used RNA-Seq to determine expression profiles of a longitudinal series of whole spinal cords from the same G93A mouse model of ALS over the course of the disease. These data, combined with gene expression data from acutely isolated glia, microglia, and oligodendrocytes from control and mutant SOD1 mouse spinal cords and ALS patient postmortem human spinal cord samples, may lead to the identification of common features that will provide an ALS disease signature and thereby, identify potential ALS drug targets.     

Calreticulin chaperones regulate functional expression of vomeronasal type 2 pheromone receptors

A variety of social behaviors like intermale aggression, fear, and mating rituals are important for sustenance of a species. In mice, these behaviors have been implicated to be mediated by peptide pheromones that are sensed by a class of G protein-coupled receptors, vomeronasal receptor type 2 (V2Rs), expressed in the pheromone detecting vomeronasal organ. Matching V2Rs with their cognate ligands is required to learn what receptors the biologically relevant pheromones are acting on. However, this feat has been greatly limited by the unavailability of appropriate heterologous tools commonly used to study ligand receptor specificity, because this family of receptors fails to traffic to the surface of heterologous cells. Here we show that calreticulin, a housekeeping chaperone commonly expressed in most eukaryotic cells, is sparsely expressed in the vomeronasal sensory neurons (VSNs). Correspondingly, knockdown of calreticulin in commonly available cell lines enables V2Rs to efficiently target to the cell membrane. Using this knowledge, we have now been able to successfully surface express receptors and functionally identify cognate ligands. Additionally, calreticulin4, a homolog of calreticulin shows restricted and enriched expression in the VSNs. Interestingly, in heterologous cells, calreticulin4 does not inhibit surface expression of V2Rs and can in part carry out functions of calreticulin. On the basis of our data, we postulate that V2Rs may use a unique trafficking mechanism whereby an important and more commonly expressed chaperone is deleterious for membrane export and is replaced by a functionally equivalent homolog that does not inhibit export while carrying out its functions.     

Incarcerated drug-abusing mothers: their characteristics and vulnerability

Although the number of mothers with histories of drug addiction who are incarcerated has grown substantially in recent years, there is little information on their unique characteristics and vulnerability. Undertaken to address this issue, this study examined data on 167 incarcerated drug-abusing mothers from Baltimore City who had volunteered for a parenting program offered at a Maryland correctional facility. Prior to entering this program, mothers who consented to participate completed a battery of assessment measures, which included an extensive interview covering their early developmental and current experiences, along with standardized instruments measuring psychological adjustment and parenting satisfaction. Analyses of these data focused on the link between risk/protective factor information drawn from the early development experiences of the mothers and their current adjustment status. Results revealed significant relationships between higher risk levels and less favorable current adjustment. Implications of the findings of the study for both prevention and clinical intervention efforts targeting both mothers and their children are discussed.