Morpho‐physiological criteria divide dentate gyrus interneurons into classes

GABAergic inhibitory interneurons control fundamental aspects of neuronal network function. Their functional roles are assumed to be defined by the identity of their input synapses, the architecture of their dendritic tree, the passive and active membrane properties and finally the nature of their postsynaptic targets. Indeed, interneurons display a high degree of morphological and physiological heterogeneity. However, whether their morphological and physiological characteristics are correlated and whether interneuron diversity can be described by a continuum of GABAergic cell types or by distinct classes has remained unclear. Here we perform a detailed morphological and physiological characterization of GABAergic cells in the dentate gyrus, the input region of the hippocampus. To achieve an unbiased and efficient sampling and classification we used knock‐in mice expressing the enhanced green fluorescent protein (eGFP) in glutamate decarboxylase 67 (GAD67)‐positive neurons and performed cluster analysis. We identified five interneuron classes, each of them characterized by a distinct set of anatomical and physiological parameters. Cross‐correlation analysis further revealed a direct relation between morphological and physiological properties indicating that dentate gyrus interneurons fall into functionally distinct classes which may differentially control neuronal network activity. © 2013 The Authors. Hippocampus Published by Wiley Periodicals, Inc.     

Transient expression of fluorescent tau proteins promotes process formation in PC12 cells: contributions of the tau C-terminus to this process

The neuronal microtubule-associated protein tau promotes microtubule assembly and has been implicated in the development of axonal morphology. In this study, PC12 cells were transiently transfected with constructs coding fusion proteins of human tau with green fluorescent protein (GFP). Expression of tau constructs actively stabilized microtubules. Expression of the C-terminus of tau can mimic this effect in living cells, though to a lesser extent because of the absence of the tau N-terminus. However, tau colocalization with microtubules did not require the presence of the tau N-terminus. Transient expression of tau (including tau24, a four-repeat human tau isoform encoded in 383 residues, and tau23, human fetal tau isoform encoded in 352 residues) stimulated process formation in PC12 cells, and this occurred faster with tau24 than with tau23. The residues (residues 154-172 in tau23) that confer microtubule nucleation activity of tau in vitro are not required for tau-directed process formation. However, when tau induces the formation of cellular processes in response to cortical breakdown by cytochalasin B, residues 154-172 must be present. Thus, it appears that tau may serve to promote cellular process outgrowth in cultured neuronal cells and that C-terminus of tau is essential to this process.     

Region- and age-specific deficits in gamma-aminobutyric acidergic neuron development in the telencephalon of the uPAR(-/-) mouse

We have previously shown that in adult mice with a null mutation in the urokinase-type plasminogen activator receptor (uPAR) gene, maintained on a C57BL/6J/129Sv background, there is a selective loss of GABAergic interneurons in anterior cingulate and parietal cortex, with the parvalbumin-expressing subpopulation preferentially affected. Here, we performed a more detailed anatomical analysis of uPAR(-/-) mutation on the congenic C57BL/6J background. With glutamic acid decarboxylase-67 and gamma-aminobutyric acid (GABA) immunostaining, there is a similar region-selective loss of cortical interneurons in the congenic uPAR(-/-) mice from the earliest age examined (P21). In contrast, the loss of parvalbumin-immunoreactive cells is observed only in adult cortex, and the extent of this loss is less than in the mixed background. Moreover, earlier in development, although there are normal numbers of parvalbumin cells in the uPAR(-/-) cortex, fewer cells coexpress GABA, suggesting that the parvalbumin subpopulation migrates appropriately to the cortex, but does not differentiate normally. Among the other forebrain regions examined, only the adult hippocampus shows a loss of GABAergic interneurons, although the somatostatin, rather than the parvalbumin, subpopulation contributes to this loss. The data suggest that uPAR function is necessary for the normal development of a subpopulation of GABAergic neurons in the telencephalon. It is likely that the late-onset parvalbumin phenotype is due to the effects of an altered local environment on selectively vulnerable neurons and that the extent of this loss is strain dependent. Thus, an interplay between complex genetic factors and the environment may influence the phenotypic impact of the uPAR mutation both pre- and postnatally.     

Expression pattern of activation and adhesion molecules on peripheral blood CD4 T-lymphocytes in relapsing-remitting multiple sclerosis patients: a serial analysis

We studied the expression of various cell surface molecules (CD25, CD28, CD29, CD45RO, CD56, LFA-1, VLA-4) on peripheral blood CD4⁺ T-cells in 6 relapsing-remitting multiple sclerosis (RR-MS) patients. Furthermore, changes in the expression pattern of these surface markers during intervals of increased disease activity, which was measured by gadolinium (Gd-DTPA) magnetic resonance imaging (MRI) were examined. Although several patients showed signs of increased disease activity during the observation period, this was not paralleled by a relevant change in the expression of these activation and adhesion molecules.     

Differential gene expression profiling in the mouse brain during motor skill learning: focus on the striatum structure

Much research has implicated the striatum in motor learning, but the underlying mechanism is still under extensive investigation. In this study, genome-wide analysis of gene expression was conducted in mice that have learned a complex motor task. It is well recognized that successful learning requires repetitive training and is learned slowly over several training sessions. We therefore used mice that have fully learned the accelerating rotarod task that discriminates the faster and slower phases of motor learning. As important modulators of movement behavior, the striatum was the target of this analysis along with the cerebellum and anterior cortex. To identify potential genes implicated in long memorization process, we compared the lists of genes modulated in the striatum to those modulated in the cerebellum and cortex. As a second approach, we also determined which gene ontology categories were enriched in modulated striatal genes and identified genes with the highest numbers of annotation throughout categories. Although only some of these changes were further confirmed by RT-PCR, these two complementary analyses allowed the identification of highly relevant genes like calcium/calmodulin-dependent protein kinase 2, protein kinase C zeta and N-methyl-d-aspartate receptors. Notably, these genes are all associated with synaptic plasticity, suggesting that stabilized neuronal connections in the striatum are the foundation of durable motor memory. Our study provides the first report of a whole genome analysis of gene expression in mice that have memorized a new complex motor task, and expands our knowledge on striatal gene expression changes associated with motor skill learning.     

Developmental characterization of Zswim5 expression in the progenitor domains and tangential migration pathways of cortical interneurons in the mouse forebrain

GABAergic interneurons play an essential role in modulating cortical networks. The progenitor domains of cortical interneurons are localized in developing ventral forebrain, including the medial ganglionic eminence (MGE), caudal ganglionic eminence (CGE), preoptic area (POA), and preoptic hypothalamic border domain (POH). Here, we characterized the expression pattern of Zswim5, an MGE-enriched gene in the mouse forebrain. At E11.5–E13.5, prominent Zswim5 expression was detected in the subventricular zone (SVZ) of MGE, POA, and POH, but not CGE of ventral telencephalon where progenitors of cortical interneurons resided. At E15.5 and E17.5, Zswim5 expression remained in the MGE/pallidum primordium and ventral germinal zone. Zswim5 mRNA was markedly decreased after birth and was absent in the adult forebrain. Interestingly, the Zswim5 expression pattern resembled the tangential migration pathways of cortical interneurons. Zswim5-positive cells in the MGE appeared to migrate from the MGE through the SVZ of LGE to overlying neocortex. Indeed, Zswim5 was co-localized with Nkx2.1 and Lhx6, markers of progenitors and migratory cortical interneurons. Double labeling showed that Ascl1/Mash1-positive cells co-expressed Zswim5. Zswim5 expressing cells contained none or at most low levels of Ki67 but co-expressed Tuj1 in the SVZ of MGE. These results suggest that Zswim5 is immediately upregulated as progenitors exiting cell cycle become postmitotic. Given that recent studies have elucidated that the cell fate of cortical interneurons is determined shortly after becoming postmitotic, the timing of Zswim5 expression in early postmitotic interneurons suggests a potential role of Zswim5 in regulation of neurogenesis and tangential migration of cortical interneurons.     

Novel interneuronal network in the mouse posterior piriform cortex

The neural circuits of the piriform cortex mediate field potential oscillations and complex functions related to integrating odor cues with behavior, affective states, and multisensory processing. Previous anatomical studies have established major neural pathways linking the piriform cortex to other cortical and subcortical regions and major glutamatergic and GABAergic neuronal subtypes within the piriform circuits. However, the quantitative properties of diverse piriform interneurons are unknown. Using quantitative neural anatomical analysis and electrophysiological recording applied to a GAD65-EGFP transgenic mouse expressing GFP (green fluorescent protein) under the control of the GAD65 promoter, here we report a novel inhibitory network that is composed of neurons positive for GAD65-EGFP in the posterior piriform cortex (PPC). These interneurons had stereotyped dendritic and axonal properties that were distinct from basket cells or interneurons expressing various calcium-binding proteins (parvalbumin, calbindin, and calretinin) within the PPC. The GAD65-GFP neurons are GABAergic and outnumbered any other interneurons (expressing parvalbumin, calbindin, and calretinin) we studied. The firing pattern of these interneurons was highly homogenous and is similar to the regular-spiking nonpyramidal (RSNP) interneurons reported in primary sensory and other neocortical regions. Robust dye coupling among these interneurons and expression of connexin 36 suggested that they form electrically coupled networks. The predominant targets of descending axons of these interneurons were the dendrites of Layer III principal cells. Additionally, synapses were found on dendrites and somata of deep Layer II principal neurons and Layer III basket cells. A similar interneuronal subtype was also found in GAD65-EGFP-negative mouse. The extensive dendritic bifurcation at superficial lamina IA among horizontal afferent fibers and unique axonal targeting pattern suggests that these interneurons may play a role in direct feedforward inhibitory and disinhibitory olfactory processing. We conclude that the GAD65-GFP neurons may play distinct roles in regulating information flow and olfactory-related oscillation within the PPC in vivo.     

A novel dopamine transporter transgenic mouse line for identification and purification of midbrain dopaminergic neurons reveals midbrain heterogeneity

Midbrain dopaminergic (DAergic) neurons are a heterogeneous cell group, composed of functionally distinct cell populations projecting to the basal ganglia, prefrontal cortex and limbic system. Despite their functional significance, the midbrain population of DAergic neurons is sparse, constituting only 20 000–30 000 neurons in mice, and development of novel tools to identify these cells is warranted. Here, a bacterial artificial chromosome mouse line [Dat1‐enhanced green fluorescent protein (eGFP)] from the Gene Expression Nervous System Atlas (GENSAT) that expresses eGFP under control of the dopamine transporter (DAT) promoter was characterized. Confocal microscopy analysis of brain sections showed strong eGFP signal reporter in midbrain regions and striatal terminals that co‐localized with the DAergic markers DAT and tyrosine hydroxylase (TH). Thorough quantification of co‐localization of the eGFP reporter signal with DAT and TH in the ventral midbrain showed that a vast majority of eGFP‐expressing neurons are DAergic. Importantly, expression profiles also revealed DAergic heterogeneity when comparing substantia nigra and ventral tegmental area. Dat1‐eGFP mice showed neither change in synaptosomal DA uptake nor altered levels of DAT and TH in both striatum and midbrain. No behavioural difference between Dat1‐eGFP and wild‐type was found, suggesting that the strain is not aberrant. Finally, cell populations highly enriched in DAergic neurons can be obtained from postnatal mice by fluorescence‐activated cell sorting and the sorted neurons can be cultured in vitro. The current investigation demonstrates that eGFP expression in this mouse line is selective for DAergic neurons, suggesting that the Dat1‐eGFP mouse strain constitutes a promising tool for delineating new aspects of DA biology.     

Acute and chronic caffeine administration differentially alters striatal gene expression in wild-type and adenosine A(2A) receptor-deficient mice

In order to assess for the respective involvement of adenosine A(1) and A(2A) receptors (A(2A)-R) in the consequences of short- and long-term caffeine exposure on gene expression, the effects of acute caffeine administration on striatal, cortical, and hippocampal expression of immediate early genes (IEG), zif-268 and arc, and the effects of long-term caffeine or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) exposure (once daily for 15 days) on striatal gene expression of substance P, enkephalin, and glutamic acid decarboxylase isoforms, GAD65 and GAD67, were evaluated in wild-type and A(2A)-R-deficient (A(2A)-R(-/-)) mice. In situ hybridization histochemistry was performed using oligonucleotides followed by quantitative image analysis. Our results demonstrated that a biphasic response of IEG expression to acute caffeine observed in the wild-type striatum was resumed in a monophasic response in the mutant striatum. In the cerebral cortex and hippocampus, the effect of caffeine was weak in wild-type, whereas in mutant mice it induced a 2-3-fold increase in the IEG expression to restore a level similar to the wild-type basal expression. Chronic caffeine and DPCPX-mediated regulation in neuropeptide and GADs striatal gene expression typically showed the mimicking of alterations resulting from the A(2A)-R genetic deficiency in 25 mg/kg caffeine-treated wild-type mice as well as the dose-dependent normalization of substance P and enkephalin expression in A(2A)-R(-/-) mice. These results indicate that, depending on the dose, the blockade of A(2A)-R or A(1) receptors by caffeine is preferentially revealed leading to highly differential alterations in striatal gene expression and they also suggested the central role of these two receptors on the control of dopaminergic functions.     

Glutamic acid decar☐ylase activity decreases in mouse neocortex after lesions of the basal forebrain

Glutamic acid decar☐ylase (GAD) activity was measured in the cerebral cortex of animals after acute and chronic lesions to basal forebrain cholinergic nuclei. Such lesions were shown to result in an extensive depletion of cholinergic markers in parietal cerebral cortex. A statistically significant 30% decrease in GAD activity was first detected at 6 weeks postlesion and was still measurable 8 months after the lesion. These results suggest that cholinergic inputs to cortex indirectly or directly influence GABAergic transmission in cortex.     

Differential expression of CPD1 during postnatal development in the mouse cerebellum

Several regulated mRNAs were detected by applying differential display to the mouse cerebellum during postnatal development. One cDNA fragment, referred to as CPD1 (GenBank U89345), was characterized and cloned. Northern blots showed maximum mRNA expression at postnatal day seven (P7). The mRNA encodes a protein of 260 amino acids. In situ RT-PCR showed that CPD1 is expressed mainly in granule cells and faintly in Purkinje cells. Polyclonal rabbit antibodies and oligobodies (oligonucleotide-based synthetic antibodies) revealed a protein of 34 kDa in Western blots. Immunohistochemistry showed not only marked nuclear staining but also mild cytoplasmic localization. Granule cells undergoing active division (P4) showed very little expression of CPD1 protein, which increases from P7 to P17. CPD1, affinity-purified using a chemically synthesized oligobody inhibits the activity of protein phosphatase PP2A but not protein phosphatase PP1. Differentiated PC12 cells also showed nuclear and cytoplasmic localization. Interestingly, maximal cytoplasmic CPD1/PP2A colocalization was observed near cell membrane regions that are far from growing neurites, and on growing cones. These results suggest that CPD1 might have an important role in cerebellar development.     

Cobalt chloride induces neurite outgrowth in rat pheochromocytoma PC-12 cells through regulation of endothelin-2/vasoactive intestinal contractor

We investigated whether endothelin-2/vasoactive intestinal contractor (ET-2/VIC) gene expression, upregulated by hypoxia in cancer cells, was associated with differentiation in neuronal cells. RT-PCR analysis, morphological observations, and immunostaining revealed that CoCl2, a hypoxic mimetic agent, at 200 microM increased expression of the ET-2/VIC gene, decreased expression of the ET-1 gene, and induced neurite outgrowth in PC-12 rat pheochromocytoma cells. These effects induced by 200 microM CoCl2 were completely inhibited by the antioxidant N-acetyl cysteine at 20 mM. In addition, CoCl2 increased the level of intracellular reactive oxygen species (ROS) at an early stage. Furthermore, interleukin (IL)-6 gene expression was upregulated upon the differentiation induced by CoCl2. These results suggest that expression of ET-2/VIC and ET-1 mediated by ROS may be associated with neuronal differentiation through the regulation of IL-6. When the cells were treated with 500 microM CoCl2 for 24 hr, however, ET-2/VIC gene expression disappeared, IL-6 gene expression was downregulated, and necrosis was subsequently induced in the PC-12 cells.     

Ectopic expression of the neural cell adhesion molecule L1 in astrocytes leads to changes in the development of the corticospinal tract

The cell recognition molecule L1, of the immunoglobulin superfamily, participates in the formation of the nervous system and has been shown to enhance cell migration and neurite outgrowth in vitro. To test whether ectopic expression of L1 would influence axonal outgrowth in vivo, we studied the development of the corticospinal tract in transgenic mice expressing L1 in astrocytes under the control of the GFAP-promoter. Corticospinal axons innervate their targets by extending collateral branches interstitially along the axon shaft following a precise spatio-temporal pattern. Using DiI as an anterograde tracer, we found that in the transgenic animals, corticospinal axons appear to be defasciculated, reach their targets sooner and form collateral branches innervating the basilar pons at earlier developmental stages and more diffusely than in wild type littermates. Collateral branches in the transgenic mice did not start out as distinct rostral and caudal sets, but they branched from the axon segments in a continuous rostrocaudal direction across the entire region of the corticospinal tract overlying the basilar pons. The ectopic branches are transient and no longer present at postnatal day 22. The earlier outgrowth and altered branching pattern of corticospinal axons in the transgenics is accompanied by an earlier differentiation of astrocytes. Taken together, our observations provide evidence that the ectopic expression of L1 on astrocytes causes an earlier differentiation of these cells, results in faster progression of corticospinal axons and influences the branching pattern of corticospinal axons innervating the basilar pons.     

Moderate lead exposure elicits neurotrophic effects in cerebral cortical precursor cells in culture

Lead (Pb) persists as an environmental toxicant despite aggressive environmental and occupational regulation. Neurotoxicological effects of acute Pb poisoning range from subtle cognitive deficits, to clumsiness and ataxia, to coma and seizures. In adult neurotoxicity, reductions of blood Pb levels are often associated with reversal of clinical signs. In children, however, the effects are more likely to endure, with even low levels of chronic Pb exposure correlating with decreasing IQ. These persistent effects likely result from neurodevelopmental insults, such as altered cell survival or maturation, although the mechanisms have not been fully defined. In the present study we define the effects of moderate-level Pb exposure on mammalian neurogenesis using a well-characterized cortical precursor model. Gestational day 14.5 rat cerebral cortical precursor cells were cultured in defined media and cell number, precursor proliferation, apoptosis, and neuritic process outgrowth were assessed following exposure to a range of Pb acetate concentrations. Surprisingly, whereas a concentration of 30 microg/ml Pb acetate was acutely toxic to neurons, concentrations between 1 and 10 microg/ml Pb acetate (approximately 3 microM and 30 microM Pb, respectively) increased cell number: 10 times as many cells exposed to 10 microg/ml Pb were present on day 4 as compared to control. The increase in cell number was not a result of increased proliferation, however, as DNA synthesis did not increase. Rather, Pb exposure maintained the survival of cortical precursors, as the progressive apoptosis occurring under control conditions was markedly reduced by the metal. Additionally, neuritic process initiation and outgrowth increased in a concentration-dependent manner, with processes four times as abundant on day 1 and twice as long on day 2. These results suggest that brief exposure to lead during neurogenesis directly affects cell survival and process development, potentially altering cortical arrangement. Consequently, alterations in neural circuitry may underlie some of the neurological effects of Pb exposure during brain development.     

Specificity of the second messenger pathways involved in basic fibroblast growth factor‐induced survival and neurite growth in chick ciliary ganglion neurons

Basic fibroblast growth factor (bFGF) exerts multiple neurotrophic actions on cultured neurons from the ciliary ganglion of chick embryo, among them promotion of neuronal survival and of neurite outgrowth. To understand the specificity of the signal transduction cascades involved in the control of these processes, we used pharmacological inhibitors of the three main effectors known to act downstream of the bFGF receptor (FGFR): phospholipase Cγ (PLCγ), mitogen‐activated protein kinase (MAPK), and phosphatidylinositol 3‐kinase (PI3‐K). Neuronal survival was assessed at 24 and 48 hr; neurite growth was analyzed both on dissociated neurons and on explants of whole ganglia. Our data show that only the PI3‐K pathway is involved in the survival‐promoting effect of bFGF; on the other hand, all three effectors converge on the enhancement of neurite outgrowth, both on isolated neurons and in whole ganglia. © 2009 Wiley‐Liss, Inc.