Functional role of a polymorphism in the Pannexin1 gene in collagen-induced platelet aggregation.

Pannexin1 (Panx1) forms ATP channels that play a critical role in the immune response by reinforcing purinergic signal amplification in the immune synapse. Platelets express Panx1 and given the importance of ATP release in platelets, we investigated Panx1 function in platelet aggregation and the potential impact of genetic polymorphisms on Panx1 channels. We show here that Panx1 forms ATP release channels in human platelets and that inhibiting Panx1 channel function with probenecid, mefloquine or specific (10)Panx1 peptides reduces collagen-induced platelet aggregation but not the response induced by arachidonic acid or ADP. These results were confirmed using Panx1-/- platelets. Natural variations have been described in the human Panx1 gene, which are predicted to induce non-conservative amino acid substitutions in its coding sequence. Healthy subjects homozygous for Panx1-400C, display enhanced platelet reactivity in response to collagen compared with those bearing the Panx1-400A allele. Conversely, the frequency of Panx1-400C homozygotes was increased among cardiovascular patients with hyper-reactive platelets compared with patients with hypo-reactive platelets. Exogenous expression of polymorphic Panx1 channels in a Panx-deficient cell line revealed increased basal and stimulated ATP release from cells transfected with Panx1-400C channels compared with Panx1-400A expressing transfectants. In conclusion, we demonstrate a specific role for Panx1 channels in the signalling pathway leading to collagen-induced platelet aggregation. Our study further identifies for the first time an association between a Panx1-400A>C genetic polymorphism and collagen-induced platelet reactivity. The Panx1-400C variant encodes for a gain-of-function channel that may adversely affect atherothrombosis by specifically enhancing collagen-induced ATP release and platelet aggregation.

Brain-specific Foxp1 deletion impairs neuronal development and causes autistic-like behaviour.

Neurodevelopmental disorders are multi-faceted and can lead to intellectual disability, autism spectrum disorder and language impairment. Mutations in the Forkhead box FOXP1 gene have been linked to all these disorders, suggesting that it may play a central role in various cognitive and social processes. To understand the role of Foxp1 in the context of neurodevelopment leading to alterations in cognition and behaviour, we generated mice with a brain-specific Foxp1 deletion (Nestin-Cre(Foxp1-/-)mice). The mutant mice were viable and allowed for the first time the analysis of pre- and postnatal neurodevelopmental phenotypes, which included a pronounced disruption of the developing striatum and more subtle alterations in the hippocampus. More detailed analysis in the CA1 region revealed abnormal neuronal morphogenesis that was associated with reduced excitability and an imbalance of excitatory to inhibitory input in CA1 hippocampal neurons in Nestin-Cre(Foxp1-/-) mice. Foxp1 ablation was also associated with various cognitive and social deficits, providing new insights into its behavioural importance.

Altered phosphorylation but no neurodegeneration in a mouse model of tau hyperphosphorylation.

The role of hyperphosphorylation of tau in Alzheimer's disease is still unsolved. Here we describe a novel transgenic mouse model, expressing a pseudohyperphosphorylated (PHP) variant of the longest human CNS tau isoform in forebrain neurons. We report that pseudohyperphosphorylation decreases phosphorylation at T205 while other sites (T212, S262) are less or not affected compared to mice expressing wildtype tau. Despite the differences in phosphorylation, the subcellular distribution of tau is not affected and mice do not develop highly aggregated states of tau. PHP tau expressing mice do not show any evidence for neurodegeneration as determined from morphometric measurements of neocortical regions, caspase activation, analysis of mitochondrial dysfunction, or determination of spine densities. In agreement, no differences in learning and memory are observed. The data indicates that moderate levels of modified tau alone are not sufficient to induce tau aggregation or neurodegeneration in transgenic mice. With our model it becomes possible to study the effects of hyperphosphorylation at conditions which may prevail in an early preaggregation state of the disease.

Expression analysis of green fluorescent protein in retinal neurons of four transgenic mouse lines.

Transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of a cell-specific promoter have been used with great success to identify and label specific cell types of the retina. We studied the expression of EGFP in the retina of mice making use of four transgenic mouse lines. Expression of EGFP driven by the calretinin promoter was found in amacrine, displaced amacrine and ganglion cells. Comparison of the EGFP expression and calretinin immunolabeling showed that many but not all cells appear to be double labeled. Expression of EGFP under the control of the choline acetyltransferase promoter was found in amacrine cells; however, the cells did not correspond to the well known cholinergic (starburst) cells of the mouse retina. The expression of EGFP under the control of the parvalbumin promoter was restricted to amacrine cells of the inner nuclear layer and to cells of the ganglion cell layer (displaced amacrine cells and ganglion cells). Most of the cells were also immunoreactive for parvalbumin, however, differences in labeling intensity were observed. The expression of EGFP driven by the promoter for the 5-HT3 A receptor (5-HTR3A) was restricted to type 5 bipolar cells. In contrast, immunostaining for 5-HTR3A was found in synaptic hot spots in sublamina 1 of the inner plexiform layer and was not related to type 5 bipolar cells. The results show that these transgenic mice are very useful for future electrophysiological studies of specific types of amacrine and bipolar cells that express EGFP and thus permit directed microelectrode targeting under microscopic control.

Sweat gland innervation is pioneered by sympathetic neurons expressing a cholinergic/noradrenergic co-phenotype in the mouse.

Classic neurotransmitter phenotypes are generally predetermined and develop as a consequence of target-independent lineage decisions. A unique mode of target-dependent phenotype instruction is the acquisition of the cholinergic phenotype in the peripheral sympathetic nervous system. A body of work suggests that the sweat gland plays an important role to determine the cholinergic phenotype at this target site. A key issue is whether neurons destined to innervate the sweat glands express cholinergic markers before or only after their terminals make target contact. We employed cholinergic-specific over-expression of the vesicular acetylcholine transporter (VAChT) in transgenic mice to overcome sensitivity limits in the detection of initial cholinergic sweat gland innervation. We found that VAChT immunoreactive nerve terminals were present around the sweat gland anlage already from the earliest postnatal stages on, coincident selectively at this sympathetic target with tyrosine hydroxylase-positive fibers. Our results provide a new mechanistic model for sympathetic neuron-target interaction during development, with initial selection by the target of pioneering nerve terminals expressing a cholinergic phenotype, and subsequent stabilization of this phenotype during development.

Cerebellar granule cell Cre recombinase expression.

The cerebellum maintains balance and orientation, refines motor action, stores motor memories, and contributes to the timing aspects of cognition. We generated two mouse lines for making Cre recombinase-mediated gene disruptions largely confined to adult cerebellar granule cells. For this purpose we chose the GABA(A) receptor alpha6 subunit gene, whose expression marks this cell type. Here we describe mouse lines expressing Cre recombinase generated by 1) Cre knocked into the native alpha6 subunit gene by homologous recombination in embryonic stem cells; and 2) Cre recombined into an alpha6 subunit gene carried on a bacterial artificial chromosome (BAC) genomic clone. The fidelity of Cre expression was tested by crossing the mouse lines with the ROSA26 reporter mice. The particular alpha6BAC clone we identified will be valuable for delivering other gene products to cerebellar granule cells.

Insulin-like growth factor 1 and a cytosolic tyrosine kinase activate chloride outward transport during maturation of hippocampal neurons.

The development of hyperpolarizing inhibition is an important step in the maturation of neuronal networks. Hyperpolarizing inhibition requires Cl(-) outward transport that is accomplished by KCC2, a K(+)/Cl(-) cotransporter. We show that cultured hippocampal neurons initially contain an inactive form of the KCC2 protein, which becomes activated during subsequent maturation of the neurons. We also show that this process is accelerated by transient stimulation of IGF-1 receptors. Because the transporter can be rapidly activated by coapplication of IGF-1 and an Src kinase and can be deactivated by membrane-permeable protein tyrosine kinase inhibitors, we suggest that activation of K(+)/Cl(-) cotransporter function by endogenous protein tyrosine kinases mediates the developmental switch of GABAergic responses to hyperpolarizing inhibition.

Impaired electrical signaling disrupts gamma frequency oscillations in connexin 36-deficient mice.

Neural processing occurs in parallel in distant cortical areas even for simple perceptual tasks. Associated cognitive binding is believed to occur through the interareal synchronization of rhythmic activity in the gamma (30-80 Hz) range. Such oscillations arise as an emergent property of the neuronal network and require conventional chemical neurotransmission. To test the potential role of gap junction-mediated electrical signaling in this network property, we generated mice lacking connexin 36, the major neuronal connexin. Here we show that the loss of this protein disrupts gamma frequency network oscillations in vitro but leaves high frequency (150 Hz) rhythms, which may involve gap junctions between principal cells (Schmitz et al., 2001), unaffected. Thus, specific connexins differentially deployed throughout cortical networks are likely to regulate different functional aspects of neuronal information processing in the mature brain.

Genetically altered AMPA-type glutamate receptor kinetics in interneurons disrupt long-range synchrony of gamma oscillation.

Gamma oscillations synchronized between distant neuronal populations may be critical for binding together brain regions devoted to common processing tasks. Network modeling predicts that such synchrony depends in part on the fast time course of excitatory postsynaptic potentials (EPSPs) in interneurons, and that even moderate slowing of this time course will disrupt synchrony. We generated mice with slowed interneuron EPSPs by gene targeting, in which the gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67) was altered to drive expression of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunit GluR-B. GluR-B is a determinant of the relatively slow EPSPs in excitatory neurons and is normally expressed at low levels in gamma-aminobutyric acid (GABA)ergic interneurons, but at high levels in the GAD-GluR-B mice. In both wild-type and GAD-GluR-B mice, tetanic stimuli evoked gamma oscillations that were indistinguishable in local field potential recordings. Remarkably, however, oscillation synchrony between spatially separated sites was severely disrupted in the mutant, in association with changes in interneuron firing patterns. The congruence between mouse and model suggests that the rapid time course of AMPA receptor-mediated EPSPs in interneurons might serve to allow gamma oscillations to synchronize over distance.

Connexin expression in electrically coupled postnatal rat brain neurons.

Electrical coupling by gap junctions is an important form of cell-to-cell communication in early brain development. Whereas glial cells remain electrically coupled at postnatal stages, adult vertebrate neurons were thought to communicate mainly via chemical synapses. There is now accumulating evidence that in certain neuronal cell populations the capacity for electrical signaling by gap junction channels is still present in the adult. Here we identified electrically coupled pairs of neurons between postnatal days 12 and 18 in rat visual cortex, somatosensory cortex, and hippocampus. Notably, coupling was found both between pairs of inhibitory neurons and between inhibitory and excitatory neurons. Molecular analysis by single-cell reverse transcription-PCR revealed a differential expression pattern of connexins in these identified neurons.

Differential expression of group I metabotropic glutamate receptors in functionally distinct hippocampal interneurons.

Metabotropic glutamate receptors (mGluRs) have been proposed to be involved in oscillatory rhythmic activity in the hippocampus. However, the subtypes of mGluRs involved and their precise distribution in different populations of interneurons is unclear. In this study, we combined functional analysis of mGluR-mediated inward currents in CA1 oriens-alveus interneurons with anatomical and immunocytochemical identification of these interneurons and expression analysis of group I mGluR using single-cell reverse transcription-PCR (RT-PCR). Four major interneuron subtypes could be distinguished based on the mGluR-mediated inward current induced by the application of 100 microm trans-(1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (ACPD) under voltage-clamp conditions and the action potential firing pattern under current-clamp conditions. Type I interneurons responded with a large inward current of approximately 224 pA, were positive for somatostatin, and the majority expressed both mGluR1 and mGluR5. Type II interneurons responded with an inward current of approximately 80 pA, contained calbindin, and expressed mainly mGluR1. Type III interneurons responded with an inward current of approximately 60 pA. These interneurons were fast-spiking, contained parvalbumin, and expressed mainly mGluR5. Type IV interneurons did not respond with an inward current upon application of ACPD, yet they expressed group I mGluRs. Activation of group I mGluRs under current-clamp conditions increased spike frequency and resulted in rhythmic firing activity in type I and II, but not in type III and IV, interneurons. RT-PCR results suggest that activation of mGluR1 in the subsets of GABAergic interneurons, classified here as type I and II, may play an important role in mediating synchronous activity.

The electrogenic sodium bicarbonate cotransporter: developmental expression in rat brain and possible role in acid vulnerability.

The electrogenic sodium bicarbonate cotransporter (NBC) is expressed in glial cells in the brain and plays an important role in the regulation of both intracellular and extracellular pH. Differential vulnerability to acidosis between neurons and glia has been noted and may contribute to infarction after cerebral ischemia. Ionic substitution studies and inhibition of injury by 4, 4'-di-isothiocyanostilbene-2,2'-disulfonic acid suggest that NBC is involved in astrocyte vulnerability to acidic injury. Recently two NBC cDNAs differing in 5'-untranslated and N-terminal coding sequence have been cloned from kidney and pancreas. We cloned one of these cDNAs from rat brain and demonstrate here that the clone is functional by expression in Xenopus oocytes. We determined the developmental and regional expression of NBC in the brain by in situ hybridization. Expression was observed in the spinal cord at embryonic day 17, whereas expression in brain was first seen at approximately postnatal day 0 (P0), increased at P15, and persisted in the adult brain. Expression was widespread throughout the cerebellum, cortex, olfactory bulb, and subcortical structures. Cellular resolution of the in situ hybridization signal and double labeling for glial fibrillary acidic protein were consistent with a glial localization for NBC. Expression of NBC in 3T3 cells that do not normally express this transporter rendered them vulnerable to acid injury. The expression profile suggests that this transporter is critical during the later stages of brain development and could be one of the factors contributing to the different patterns of injury seen in perinatal versus adult cerebral ischemia.

Genetic and epigenetic regulation of NMDA receptor expression in the rat visual cortex.

The susceptibility of cortical networks to use-dependent modifications declines with age (critical period) and this decline of neuronal plasticity during development is paralleled by the shortening of NMDA receptor EPSCs. We showed previously in the somatosensory cortex that the shortening of NMDA receptor kinetics correlates with a developmentally-regulated increase in the NR2A subunit expression. Here we examine whether this developmental regulation of NR2A expression is related to the duration of critical periods and whether it is influenced by experience. Functional NMDA receptors and their molecular characteristics are studied in identified layer IV neurons of rat visual cortex. In this structure the time course of the critical period differs from that in the somatosensory cortex and can be changed by sensory deprivation, thus permitting examination of correlations between the time course of receptor expression and the duration of the critical period. We find that the developmental expression of the NR2A subunit is delayed compared with the somatosensory cortex, in agreement with the prolonged critical period in the visual cortex. Moreover, sensory deprivation further delays the developmental change in the NMDA receptor subunit composition, demonstrating the activity dependence of this process and strengthening the correlation between changes in subunit composition and the time course of the critical period.

Functional and molecular differences between voltage-gated K+ channels of fast-spiking interneurons and pyramidal neurons of rat hippocampus.

We have examined gating and pharmacological characteristics of somatic K+ channels in fast-spiking interneurons and regularly spiking principal neurons of hippocampal slices. In nucleated patches isolated from basket cells of the dentate gyrus, a fast delayed rectifier K+ current component that was highly sensitive to tetraethylammonium (TEA) and 4-aminopyridine (4-AP) (half-maximal inhibitory concentrations <0.1 mM) predominated, contributing an average of 58% to the total K+ current in these cells. By contrast, in pyramidal neurons of the CA1 region a rapidly inactivating A-type K+ current component that was TEA-resistant prevailed, contributing 61% to the total K+ current. Both types of neurons also showed small amounts of the K+ current component mainly found in the other type of neuron and, in addition, a slow delayed rectifier K+ current component with intermediate properties (slow inactivation, intermediate sensitivity to TEA). Single-cell RT-PCR analysis of mRNA revealed that Kv3 (Kv3.1, Kv3.2) subunit transcripts were expressed in almost all (89%) of the interneurons but only in 17% of the pyramidal neurons. In contrast, Kv4 (Kv4.2, Kv4.3) subunit mRNAs were present in 87% of pyramidal neurons but only in 55% of interneurons. Selective block of fast delayed rectifier K+ channels, presumably assembled from Kv3 subunits, by 4-AP reduced substantially the action potential frequency in interneurons. These results indicate that the differential expression of Kv3 and Kv4 subunits shapes the action potential phenotypes of principal neurons and interneurons in the cortex.

Differentiation of glycine antagonist sites of N-methyl-D-aspartate receptor subtypes. Preferential interaction of CGP 61594 with NR1/2B receptors.

The binding site for the co-agonist glycine on N-methyl-D-aspartate (NMDA) receptors has been mapped to the NR1 subunit whereas binding of the principal agonist glutamate is mediated by the NR2 subunits. Using the novel glycine site antagonist and photoaffinity label CGP 61594, distinct contributions of the NR2 subunit variants to the glycine antagonist binding domains of NMDA receptor subtypes are demonstrated. High affinity sites for CGP 61594 were exclusively displayed by NR1/2B receptors, as shown by their co-distribution with the NR2B subunit, by subunit-selective immunoprecipitation and by functional analysis of NR1/2B receptors expressed in Xenopus oocytes (inhibitory potency, IC50 = 45 +/- 11 nM). Other NMDA receptor subtypes are clearly distinguished by reduced inhibitory potencies for CGP 61594, being low for NR1/2A and NR1/2D receptors (IC50 = 430 +/- 105 nM and 340 +/- 61 nM, respectively) and intermediate for NR1/2C receptors (IC50 = 164 +/- 27 nM). Glycine antagonist sites with low and intermediate affinity for [3H]CGP 61594 were detected also in situ by radioligand binding in brain areas predominantly expressing the NR2A and NR2C subunits, respectively. Thus, [3H]CGP 61594 is the first antagonist radioligand that reliably distinguishes the glycine site of NMDA receptor subtypes. [3H]CGP 61594 is a promising tool to identify the NR2 subunit domains that contribute to differential glycine antagonist sites of NMDA receptor subtypes.

Dentate gyrus basket cell GABAA receptors are blocked by Zn2+ via changes of their desensitization kinetics: an in situ patch-clamp and single-cell PCR study.

Although GABA type A receptors (GABAARs) in principal cells have been studied in detail, there is only limited information about GABAARs in interneurons. We have used the patch-clamp technique in acute rat hippocampal slices in combination with single-cell PCR to determine kinetic, pharmacological, and structural properties of dentate gyrus basket cell GABAARs. Application of 1 mM GABA (100 msec) to nucleated patches via a piezo-driven fast application device resulted in a current with a fast rise and a marked biexponential decay (time constants 2.4 and 61.8 msec). This decay could be attributed to strong receptor desensitization. Dose-response curves for the peak and the slow component yielded EC50 values of 139 and 24 microM, respectively. Zn2+ caused a marked blocking effect on both the peak and the slow component via a noncompetitive mechanism (IC50 values of 8 and 16 microM). This led to an acceleration of the slow component as well as a prolongation of recovery from desensitization. Zn2+ sensitivity was suggested to depend on the absence of gamma-subunits in GABAARs. To test this hypothesis we performed single-cell reverse transcription PCR that revealed primarily the presence of alpha2-, beta2-, beta3-, gamma1-, and gamma2-subunit mRNAs. In addition, flunitrazepam increased the receptor affinity for its agonist, indicating the presence of functional benzodiazepine binding sites, i.e., gamma-subunits. Thus, additional factors seem to co-determine the Zn2+ sensitivity of native GABAARs. The modulatory effects of Zn2+ on GABAAR desensitization suggest direct influences on synaptic integration via changes in inhibition and shunting at GABAergic synapses.

A novel type of GABAergic interneuron connecting the input and the output regions of the hippocampus.

The main excitatory pathway of the hippocampal formation is controlled by a network of morphologically distinct populations of GABAergic interneurons. Here we describe a novel type of GABAergic interneuron located in the outer molecular layer (OML) of the rat dentate gyrus with a long-range forward projection from the dentate gyrus to the subiculum across the hippocampal fissure. OML interneurons were recorded in hippocampal slices by using the whole-cell patch-clamp configuration. During recording, cells were filled with biocytin for subsequent light and electron microscopic analysis. Neurons projecting to the subiculum were distributed throughout the entire OML. They had round or ovoid somata and a multipolar dendritic morphology. Two axonal domains could be distinguished: an extensive, tangential distribution within the OML and a long-range vertical and tangential projection to layer 1 and stratum pyramidale of the subiculum. Symmetric synaptic contacts were established by these interneurons on dendritic shafts in the OML and subiculum. OML interneurons were characterized physiologically by short action potential duration and marked afterhyperpolarization that followed the spike. On sustained current injection, they generated high-frequency (up to 130 Hz, 34 degrees C) trains of action potentials with only little adaptation. In situ hybridization and single-cell RT-PCR analysis for GAD67 mRNA confirmed the GABAergic nature of OML interneurons. GABAergic interneurons in the OML projecting to the subiculum connect the input and output regions of the hippocampus. Hence, they could mediate long-range feed-forward inhibition and may participate in an oscillating cross-regional interneuron network that may synchronize the activity of spatially distributed principal neurons in the dentate gyrus and the subiculum.