Axonal Kainate Receptors Modulate the Strength of Efferent Connectivity by Regulating Presynaptic Differentiation

Kainate type of glutamate receptors (KARs) are highly expressed during early brain development and may influence refinement of the circuitry, via modulating synaptic transmission and plasticity. KARs are also localized to axons, however, their exact roles in regulating presynaptic processes remain controversial. Here, we have used a microfluidic chamber system allowing specific manipulation of KARs in presynaptic neurons to study their functions in synaptic development and function in vitro. Silencing expression of endogenous KARs resulted in lower density of synaptophysin immunopositive puncta in microfluidically isolated axons. Various recombinant KAR subunits and pharmacological compounds were used to dissect the mechanisms behind this effect. The calcium permeable (Q) variants of the low-affinity (GluK1-3) subunits robustly increased synaptophysin puncta in axons in a manner that was dependent on receptor activity and PKA and PKC dependent signaling. Further, an associated increase in the mean active zone length was observed in electron micrographs. Selective presynaptic expression of these subunits resulted in higher success rate of evoked EPSCs consistent with higher probability of glutamate release. In contrast, the calcium-impermeable (R) variant of GluK1 or the high-affinity subunits (GluK4,5) had no effect on synaptic density or transmission efficacy. These data suggest that calcium permeable axonal KARs promote efferent connectivity by increasing the density of functional presynaptic release sites.Peer reviewe

The Kainate Receptor Subunit GluK2 Interacts With KCC2 to Promote Maturation of Dendritic Spines

Kainate receptors (KAR) play a crucial role in the plasticity and functional maturation of glutamatergic synapses. However, how they regulate structural plasticity of dendritic spines is not known. The GluK2 subunit was recently shown to coexist in a functional complex with the neuronal K-Cl cotransporter KCC2. Apart from having a crucial role in the maturation of GABAergic transmission, KCC2 has a morphogenic role in the maturation of dendritic spines. Here, we show thatin vivolocal inactivation of GluK2 expression in CA3 hippocampal neurons induces altered morphology of dendritic spines and reduction in mEPSC frequency. GluK2 deficiency also resulted in a strong change in the subcellular distribution of KCC2 as well as a smaller somatodendritic gradient in the reversal potential of GABA(A). Strikingly, the aberrant morphology of dendritic spines in GluK2-deficient CA3 pyramidal neurons was restored by overexpression of KCC2. GluK2 silencing in hippocampal neurons significantly reduced the expression of 4.1N and functional form of the actin filament severing protein cofilin. Consistently, assessment of actin dynamics using fluorescence recovery after photobleaching (FRAP) of beta-actin showed a significant increase in the stability of F-actin filaments in dendritic spines. In conclusion, our results demonstrate that GluK2-KCC2 interaction plays an important role in the structural maturation of dendritic spines. This also provides novel insights into the connection between KAR dysfunction, structural plasticity, and developmental disorders.Peer reviewe

3 '-UTR poly(T/U) repeat of EWSR1 is altered in microsatellite unstable colorectal cancer with nearly perfect sensitivity

Approximately 15 % of colorectal cancers exhibit instability of short nucleotide repeat regions, microsatellites. These tumors display a unique clinicopathologic profile and the microsatellite instability status is increasingly used to guide clinical management as it is known to predict better prognosis as well as resistance to certain chemotherapeutics. A panel of five repeats determined by the National Cancer Institute, the Bethesda panel, is currently the standard for determining the microsatellite instability status in colorectal cancer. Recently, a quasimonomorphic mononucleotide repeat 16T/U at the 3' untranslated region of the Ewing sarcoma breakpoint region 1 gene was reported to show perfect sensitivity and specificity in detecting mismatch repair deficient colorectal, endometrial, and gastric cancers in two independent populations. To confirm this finding, we replicated the analysis in 213 microsatellite unstable colorectal cancers from two independent populations, 148 microsatellite stable colorectal cancers, and the respective normal samples by PCR and fragment analysis. The repeat showed nearly perfect sensitivity for microsatellite unstable colorectal cancer as it was altered in 212 of the 213 microsatellite unstable (99.5 %) and none of the microsatellite stable colorectal tumors. This repeat thus represents the first potential single marker for detecting microsatellite instability.Peer reviewe

Rapid and Differential Regulation of AMPA and Kainate Receptors at Hippocampal Mossy Fibre Synapses by PICK1 and GRIP

AbstractWe identified four PDZ domain-containing proteins, syntenin, PICK1, GRIP, and PSD95, as interactors with the kainate receptor (KAR) subunits GluR52b, GluR52c, and GluR6. Of these, we show that both GRIP and PICK1 interactions are required to maintain KAR-mediated synaptic function at mossy fiber-CA3 synapses. In addition, PKCα can phosphorylate ct-GluR52b at residues S880 and S886, and PKC activity is required to maintain KAR-mediated synaptic responses. We propose that PICK1 targets PKCα to phosphorylate KARs, causing their stabilization at the synapse by an interaction with GRIP. Importantly, this mechanism is not involved in the constitutive recycling of AMPA receptors since blockade of PDZ interactions can simultaneously increase AMPAR- and decrease KAR-mediated synaptic transmission at the same population of synapses

Plasma etched carbon microelectrode arrays for bioelectrical measurements

Carbon-based materials have attracted much attention in biological applications like interfacing electrodes with neurons and cell growth platforms due to their natural biocompatibility and tailorable material properties. Here we have fabricated sputtered carbon thin film electrodes for bioelectrical measurements. Reactive ion etching (RIE) recipes were optimized with Taguchi method to etch the close field unbalanced magnetron sputtered carbon thin film (nanocarbon, nC) consisting of nanoscale crystalline sp(2)-domains in amorphous sp(3)-bonded backbone. Plasma etching processes used gas mixtures of Ar/O-2/SF6/CHF3 for RIE and O-2/SF6 for ICP-RIE. The highest achieved etch rate for nanocarbon was >> 389 nm/min and best chromium etch mask selectivity was 135:1. Biocompatibility of the material was tested with rat neuronal cultures. Next, we fabricated multielectrode arrays (MEA) with carbon recording electrodes and metal wiring. Organotypic brain slices grown on the MEAs were viable and showed characteristic spontaneous electrical network activity. The results demonstrate that interactions with nanocarbon substrate support neuronal survival and maturation of functional neuronal networks. Thus the material can have wide applications in biomedical research.Peer reviewe

Pharmacological and optical activation of TrkB in Parvalbumin interneurons regulate intrinsic states to orchestrate cortical plasticity

Elevated states of brain plasticity typical for critical periods of early postnatal life can be reinstated in the adult brain through interventions, such as antidepressant treatment and environmental enrichment, and induced plasticity may be critical for the antidepressant action. Parvalbumin-positive (PV) interneurons regulate the closure of developmental critical periods and can alternate between high and low plasticity states in response to experience in adulthood. We now show that PV plasticity states and cortical networks are regulated through the activation of TrkB neurotrophin receptors. Visual cortical plasticity induced by fluoxetine, a widely prescribed selective serotonin reuptake inhibitor (SSRI) antidepressant, was lost in mice with reduced expression of TrkB in PV interneurons. Conversely, optogenetic gain-of-function studies revealed that activation of an optically activatable TrkB (optoTrkB) specifically in PV interneurons switches adult cortical networks into a state of elevated plasticity within minutes by decreasing the intrinsic excitability of PV interneurons, recapitulating the effects of fluoxetine. TrkB activation shifted cortical networks towards a low PV configuration, promoting oscillatory synchrony, increased excitatory-inhibitory balance, and ocular dominance plasticity. OptoTrkB activation promotes the phosphorylation of Kv3.1 channels and reduces the expression of Kv3.2 mRNA providing a mechanism for the lower excitability. In addition, decreased expression and puncta of Synaptotagmin2 (Syt2), a presynaptic marker of PV interneurons involved in Ca2+-dependent neurotransmitter release, suggests lower inputs onto pyramidal neurons suppressing feed-forward inhibition. Together, the results provide mechanistic insights into how TrkB activation in PV interneurons orchestrates the activity of cortical networks and mediating antidepressant responses in the adult brain.Peer reviewe

Expression of GluK1c underlies the developmental switch in presynaptic kainate receptor function

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Molecular mechanisms controlling synaptic recruitment of GluA4 subunit-containing AMPAreceptors

Synaptic recruitment of AMPA receptors (AMPARs) represents a key postsynaptic mechanism driving functional development and maturation of glutamatergic synapses. At immature hippocampal synapses, PKA-driven synaptic insertion of GluA4 is the predominant mechanism for synaptic reinforcement. However, the physiological significance and molecular determinants of this developmentally restricted form of plasticity are not known. Here we show that PKA activation leads to insertion of GluA4 to synaptic sites with initially weak or silent AMPAR-mediated transmission. This effect depends on a novel mechanism involving the extreme C-terminal end of GluA4, which interacts with the membrane proximal region of the C-terminal domain to control GluA4 trafficking. In the absence of GluA4, strengthening of AMPAR-mediated transmission during postnatal development was significantly delayed. These data suggest that the GluA4-mediated activation of silent synapses is a critical mechanism facilitating the functional maturation of glutamatergic circuitry during the critical period of experience-dependent fine-tuning

Eleven Candidate Susceptibility Genes for Common Familial Colorectal Cancer

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Allelic Variation at the 8q23.3 Colorectal Cancer Risk Locus Functions as a Cis-Acting Regulator of EIF3H

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