Protracted postnatal development of sparse, specific dentate granule cell activation in the mouse hippocampus.

The dentate gyrus (DG) is a critical entry point regulating function of the hippocampus. Integral to this role are the sparse, selective activation characteristics of the principal cells of the DG, dentate granule cells (DGCs). This sparse activation is important both in cognitive processing and in regulation of pathological activity in disease states. Using a novel, combined dynamic imaging approach capable of resolving sequentially both synaptic potentials and action potential firing in large populations of DGCs, we characterized the postnatal development of firing properties of DG neurons in response to afferent activation in mouse hippocampal-entorhinal cortical slices. During postnatal development, there was a protracted, progressive sparsification of responses, accompanied by increased temporal precision of activation. Both of these phenomena were primarily mediated by changes in local circuit inhibition, and not by alterations in afferent innervation of DGCs because GABA(A) antagonists normalized developmental differences. There was significant θ and γ frequency-dependent synaptic recruitment of DGC activation in adult, but not developing, animals. Finally, we found that the decision to fire or not fire by individual DGCs was robust and repeatable at all stages of development. The protracted postnatal development of sparse, selective firing properties, increased temporal precision and frequency dependence of activation, and the fidelity with which the decision to fire is made are all fundamental circuit determinants of DGC excitation, critical in both normal and pathological function of the DG.

Synaptic targeting and functional modulation of GluK1 kainate receptors by the auxiliary neuropilin and tolloid-like (NETO) proteins.

Auxiliary proteins modify the biophysical function and pharmacological properties of ionotropic glutamate receptors and likely are important components of receptor signaling complexes in vivo. The neuropilin and tolloid-like proteins (NETO) 1 and NETO2, two closely related CUB domain-containing integral membrane proteins, were identified recently as auxiliary proteins that slowed GluK2a kainate receptor current kinetics without impacting receptor membrane localization. Here we demonstrate that NETO2 profoundly slows the desensitization rate of GluK1 kainate receptors, promotes plasma membrane localization of transfected receptors in heterologous cells and rat hippocampal neurons, and targets GluK1-containing receptors to synapses. Conversely, the closely related protein NETO1 increases the rate of GluK1 receptor desensitization. Incorporation of NETO proteins into kainate receptor-signaling complexes therefore extends the temporal range of receptor gating by over an order of magnitude. The presence of these auxiliary proteins could underlie some of the unusual aspects of kainate receptor function in the mammalian CNS.

A novel form of low-frequency hippocampal mossy fiber plasticity induced by bimodal mGlu1 receptor signaling.

Mossy fiber synapses act as the critical mediators of highly dynamic communication between hippocampal granule cells in the dentate gyrus and CA3 pyramidal neurons. Excitatory synaptic strength at mossy fiber to CA3 pyramidal cell synapses is potentiated rapidly and reversibly by brief trains of low-frequency stimulation of mossy fiber axons. We show that slight modifications to the pattern of stimulation convert this short-term potentiation into prolonged synaptic strengthening lasting tens of minutes in rodent hippocampal slices. This low-frequency potentiation of mossy fiber EPSCs requires postsynaptic mGlu1 receptors for induction but is expressed presynaptically as an increased release probability and therefore impacts both AMPA and NMDA components of the mossy fiber EPSC. A nonconventional signaling pathway initiated by mGlu1 receptors contributes to induction of plasticity, because EPSC potentiation was prevented by a tyrosine kinase inhibitor and only partially reduced by guanosine 5'-O-(2-thiodiphosphate). A slowly reversible state of enhanced synaptic efficacy could serve as a mechanism for altering the integrative properties of this synapse within a relatively broad temporal window.

IQGAP1 regulates NR2A signaling, spine density, and cognitive processes.

General or brain-region-specific decreases in spine number or morphology accompany major neuropsychiatric disorders. It is unclear, however, whether changes in spine density are specific for an individual mental process or disorder and, if so, which molecules confer such specificity. Here we identify the scaffolding protein IQGAP1 as a key regulator of dendritic spine number with a specific role in cognitive but not emotional or motivational processes. We show that IQGAP1 is an important component of NMDAR multiprotein complexes and functionally interacts with the NR2A subunits and the extracellular signal-regulated kinase 1 (ERK1) and ERK2 signaling pathway. Mice lacking the IQGAP1 gene exhibited significantly lower levels of surface NR2A and impaired ERK activity compared to their wild-type littermates. Accordingly, primary hippocampal cultures of IQGAP1(-/-) neurons exhibited reduced surface expression of NR2A and disrupted ERK signaling in response to NR2A-dependent NMDAR stimulation. These molecular changes were accompanied by region-specific reductions of dendritic spine density in key brain areas involved in cognition, emotion, and motivation. IQGAP1 knock-outs exhibited marked long-term memory deficits accompanied by impaired hippocampal long-term potentiation (LTP) in a weak cellular learning model; in contrast, LTP was unaffected when induced with stronger stimulation paradigms. Anxiety- and depression-like behavior remained intact. On the basis of these findings, we propose that a dysfunctional IQGAP1 gene contributes to the cognitive deficits in brain disorders characterized by fewer dendritic spines.

Tau protein is cross-linked by transglutaminase in P301L tau transgenic mice.

The microtubule-associated protein tau is highly soluble under physiological conditions. However, in tauopathies, tau protein aggregates into insoluble filaments and neurofibrillary tangles (NFTs). The mechanisms underlying the formation of tau filaments and NFTs in tauopathies remain unclear. Several lines of evidence suggest that transglutaminase may cross-link tau into stable, insoluble aggregates, leading to the formation of NFTs in Alzheimer's disease and progressive supranuclear palsy. To further determine the contribution of transglutaminase in the formation of NFTs, we compared the levels of cross-linked tau protein from P301L tau transgenic mice that develop NFTs to four-repeat wild-type (4RWT) tau transgenic and nontransgenic mice that do not develop NFT pathology. Immunoprecipitation and immunoblotting experiments show that transglutaminase cross-links phosphorylated tau in the hindbrain of P301L tau transgenic mice but not in mice overexpressing 4RWT tau and nontransgenic mice. Cross-linked, phosphorylated tau from P301L tau transgenic mice runs as high-molecular mass aggregates on Western blots, similar to cross-linked tau from paired helical filaments of Alzheimer's disease. We also used double-label immunofluorescence to demonstrate colocalization of PHF-1-immunoreactive tau and the transglutaminase-catalyzed cross-link in the hindbrain, spinal cord, and cortex of P301L tau transgenic mice. In the spinal cord, 87% of PHF-1-labeled cells colocalize with the transglutaminase-catalyzed cross-link. Additionally, transglutaminase enzymatic activity is significantly elevated in the spinal cord of P301L tau transgenic mice. These studies further implicate transglutaminase in the formation and/or stabilization of NFT and paired helical filaments and provide a model system to investigate the therapeutic potential of transglutaminase inhibitors in tauopathies.

Novel N-methylated 8-oxoisoguanines from Pacific sponges with diverse neuroactivities.

Marine organisms have yielded a variety of metabolites with neuropharmacological applications. Here we describe the isolation and pharmacological characterization of four novel, neurologically active purines 1-4, isolated from Haplosclerida sponges collected in the Republic of Palau. The structures were determined by analyses of spectral and X-ray data. Compound 1 induced convulsions upon intracerebroventricular injection into mice, with a CD50 value of 2.4 nmol/mouse. Purines 2-4 were active in mouse bioassays at higher doses. The seizurogenic activity of 1 was correlated with inhibition of neuronal GABAergic transmission, with only a modest impact on excitatory signaling, in electrophysiological recordings from hippocampal neurons. Despite having a purine template structure, the inhibitory activity of 1 was not prevented by a nonselective adenosine receptor antagonist. Thus, 1 represents a novel substituted purine that elicits convulsions through its actions on inhibitory neurotransmission. These 8-oxoisoguanine analogs comprise a new family of compounds closely related in structure to endogenous neurosignaling molecules and commonly used CNS stimulants.

Prenatal ethanol exposure reduces mGluR5 receptor number and function in the dentate gyrus of adult offspring.

BACKGROUND: Previous studies in our laboratory indicated that metabotropic glutamate receptor (mGluR)-stimulated phosphoinositide hydrolysis is markedly reduced in the hippocampal formation of adult rat offspring whose mothers drank moderate amounts of ethanol during pregnancy. In the present study, we extended these observations by measuring the impact of prenatal ethanol exposure on proteins associated with the mGluR5 receptor-effector system along with two mGluR5 agonist-mediated responses in dentate gyrus of adult offspring. METHODS: Sprague-Dawley rat dams consumed one of three diets throughout gestation: (1) a BioServ liquid diet that contained 5% ethanol (v/v), (2) pair-fed an isocalorically equivalent amount of 0% ethanol liquid diet, or (3) lab chow ad libitum. Microdissected slices of dentate gyrus were prepared from adult female offspring from each diet group and used for (1) Western blot analyses of mGluR5, the G-proteins Galphaq and Galpha11, and phospholipase C-beta1; (2) 2-chloro-5-hydroxyphenylglycine (CHPG)-stimulated growth associated protein 43 (GAP-43) phosphorylation; or (3) CHPG potentiation of electrically evoked [H]-D-aspartate (D-ASP) release from dentate gyrus slices. RESULTS: In tissue prepared from untreated control rats, CHPG produced a dose-dependent increase in phosphate incorporation into GAP-43, with maximal agonist stimulation occurring at 20 microM of CHPG. CHPG produced a quantitatively similar dose-dependent increase in the potentiation of electrically evoked D-ASP release from dentate gyrus slices from untreated controls. Fetal ethanol exposure reduced the amount of dentate gyrus mGluR5 receptor protein by 36% compared with the diet control groups. There were no significant differences between diet groups in the two G-proteins or phospholipase C-beta1 protein. Fetal ethanol exposure reduced CHPG-stimulated GAP-43 phosphorylation to approximately one half the amount of CHPG stimulation observed in the control diet groups. Prenatal ethanol exposure also reduced CHPG potentiation of D-ASP release to a similar degree compared with control. CONCLUSIONS: These results indicate that prenatal exposure to moderate quantities of ethanol reduces mGluR5 expression in the dentate gyrus of adult offspring. Although the subcellular site(s) for reduced mGluR5 expression cannot be discerned from Western blot data, the quantitatively similar effects of prenatal ethanol exposure on mGluR5 agonist stimulation of presynaptically localized GAP-43 phosphorylation and CHPG potentiation of evoked D-ASP release suggest that the presynaptic nerve terminal is one site where prenatal ethanol exposure has reduced mGluR5 receptor number and function. Furthermore, these data implicate these neurochemical alterations as one factor contributing to the hippocampal synaptic plasticity and behavioral deficits that we have observed previously in prenatal ethanol-exposed offspring.