Tonically Active Kainate Receptors (tKARs) : A Novel Mechanism Regulating Neuronal Function in the Brain

Fast excitatory transmission between neurons in the central nervous system is mainly mediated by L-glutamate acting on ligand gated (ionotropic) receptors. These are further categorized according to their pharmacological properties to AMPA (2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid), NMDA (N-Methyl-D-aspartic acid) and kainate (KAR) subclasses. In the rat and the mouse hippocampus, development of glutamatergic transmission is most dynamic during the first postnatal weeks. This coincides with the declining developmental expression of the GluK1 subunit-containing KARs. However, the function of KARs during early development of the brain is poorly understood. The present study reveals novel types of tonically active KARs (hereafter referred to as tKARs) which play a central role in functional development of the hippocampal CA3-CA1 network. The study shows for the first time how concomitant pre- and postsynaptic KAR function contributes to development of CA3-CA1 circuitry by regulating transmitter release and interneuron excitability. Moreover, the tKAR-dependent regulation of transmitter release provides a novel mechanism for silencing and unsilencing early synapses and thus shaping the early synaptic connectivity. The role of GluK1-containing KARs was studied in area CA3 of the neonatal hippocampus. The data demonstrate that presynaptic KARs in excitatory synapses to both pyramidal cells and interneurons are tonically activated by ambient glutamate and that they regulate glutamate release differentially, depending on target cell type. At synapses to pyramidal cells these tKARs inhibit glutamate release in a G-protein dependent manner but in contrast, at synapses to interneurons, tKARs facilitate glutamate release. On the network level these mechanisms act together upregulating activity of GABAergic microcircuits and promoting endogenous hippocampal network oscillations. By virtue of this, tKARs are likely to have an instrumental role in the functional development of the hippocampal circuitry. The next step was to investigate the role of GluK1 -containing receptors in the regulation of interneuron excitability. The spontaneous firing of interneurons in the CA3 stratum lucidum is markedly decreased during development. The shift involves tKARs that inhibit medium-duration afterhyperpolarization (mAHP) in these neurons during the first postnatal week. This promotes burst spiking of interneurons and thereby increases GABAergic activity in the network synergistically with the tKAR-mediated facilitation of their excitatory drive. During development the amplitude of evoked medium afterhyperpolarizing current (ImAHP) is dramatically increased due to decoupling tKAR activation and ImAHP modulation. These changes take place at the same time when the endogeneous network oscillations disappear. These tKAR-driven mechanisms in the CA3 area regulate both GABAergic and glutamatergic transmission and thus gate the feedforward excitatory drive to the area CA1. Here presynaptic tKARs to CA1 pyramidal cells suppress glutamate release and enable strong facilitation in response to high-frequency input. Therefore, CA1 synapses are finely tuned to high-frequency transmission; an activity pattern that is common in neonatal CA3-CA1 circuitry both in vivo and in vitro. The tKAR-regulated release probability acts as a novel presynaptic silencing mechanism that can be unsilenced in response to Hebbian activity. The present results shed new light on the mechanisms modulating the early network activity that paves the way for oscillations lying behind cognitive tasks such as learning and memory. Kainate receptor antagonists are already being developed for therapeutic use for instance against pain and migraine. Because of these modulatory actions, tKARs also represent an attractive candidate for therapeutic treatment of developmentally related complications such as learning disabilities.Tässä väitöskirjatyössä on löydetty kokonaan uusi, toonisesti aktiivinen kainaattireseptorityyppi (tKAR), sekä tutkittu sen fysiologista merkitystä hippokampuksen hermosoluissa varhaisen postnataalisen kehityksen aikana. Hippokampuksen CA3 alueen glutamaattivälitteisissä synapseissa nämä GluK1-alayksikön sisältävät kainaattireseptorit reseptorit jarruttavat glutamaatin vapautumista pyramidisoluihin, ja lisäävät sen vapautumista inhibitorisiin interneuroneihin. tKAR:it tarjoavatkin uudenlaisen presynaptisen säätelymekanismin nopean glutamaattisingnaloinnin säätelyyn aivoissa. Lisäksi CA3 alueen interneuroneissa on myös postsynaptisia tKAR:eja, joiden aktivaatio pienentää hyperpolaroivaa K+ -virtaa. Tämä mahdollistaa spontaanit, korkeataajuiset aktiopotentiaaliryöpyt kehittyvissä interneuroneissa, millä puolestaan on keskeinen merkitys hippokampuksen pyramidisolujen ärtyvyydelle. Sekä pre- että postsynaptisille tKAR:eille on yhteistä paitsi jatkuva aktivaatio, niin myös G-proteiiniaktivaatioon liittyvä signalointi, joka niin ikään on uusi piirre kainaattireseptoreille. tKAR-välitteiset säätelymekanismit häviävät toisen postnataaliviikon aikana, samalla kun hippokampuksen toiminnassa tapahtuu huomattavia muutoksia liittyen esim. moniin kognitiivisiin toimintoihin liittyvien synkronisten hermoverkko-oskillaatioiden ilmenemiseen. Onkin ilmeistä, että nyt löydetyt mekanismit ovat tärkeitä tekijöitä hippokampuksen kehityksen säätelyssä

Tropomyosin Tpm3.1 is required to maintain the structure and function of the axon initial segment

The axon initial segment (AIS) is the site of action potential initiation and serves as a cargo transport filter and diffusion barrier that helps maintain neuronal polarity. The AIS actin cytoskeleton comprises actin patches and periodic sub-membranous actin rings. We demonstrate that tropomyosin isoform Tpm3.1 co-localizes with actin patches and that the inhibition of Tpm3.1 led to a reduction in the density of actin patches. Furthermore, Tpm3.1 showed a periodic distribution similar to sub-membranous actin rings but Tpm3.1 was only partially congruent with sub-membranous actin rings. Nevertheless, the inhibition of Tpm3.1 affected the uniformity of the periodicity of actin rings. Furthermore, Tpm3.1 inhibition led to reduced accumulation of AIS structural and functional proteins, disruption in sorting somatodendritic and axonal proteins, and a reduction in firing frequency. These results show that Tpm3.1 is necessary for the structural and functional maintenance of the AIS.Peer reviewe

Tropomyosin Tpm3.1 Is Required to Maintain the Structure and Function of the Axon Initial Segment

The axon initial segment (AIS) is the site of action potential initiation and serves as a cargo transport filter and diffusion barrier that helps maintain neuronal polarity. The AIS actin cytoskeleton comprises actin patches and periodic sub-membranous actin rings. We demonstrate that tropomyosin isoform Tpm3.1 co-localizes with actin patches and that the inhibition of Tpm3.1 led to a reduction in the density of actin patches. Furthermore, Tpm3.1 showed a periodic distribution similar to sub-membranous actin rings but Tpm3.1 was only partially congruent with sub-membranous actin rings. Nevertheless, the inhibition of Tpm3.1 affected the uniformity of the periodicity of actin rings. Furthermore, Tpm3.1 inhibition led to reduced accumulation of AIS structural and functional proteins, disruption in sorting somatodendritic and axonal proteins, and a reduction in firing frequency. These results show that Tpm3.1 is necessary for the structural and functional maintenance of the AIS

The GO terms of biological processes, molecular functions and cellular components in P7 <i>Cstb^−/−</i> cerebellum.

<p>The GO terms of biological processes, molecular functions and cellular components in P7 <i>Cstb^−/−</i> cerebellum.</p

Spontaneous postsynaptic currents in Purkinje cells.

<p><b>(A)</b> Representative traces of spontaneous EPSCs and IPSCs in control and <i>Cstb^−/−</i> Purkinje cells. EPSCs are seen as inward currents (downward deflections) and IPSCs are seen as outward currents (upward deflections). Insets show single IPSCs and EPSCs taken from the time points indicated by asterisks. An arrow indicates a synchronous burst of IPSCs seen in control but not in <i>Cstb^−/−</i> Purkinje cells. <b>(B)</b> The occurrence of IPSCs and synchronous burst of IPSCs on control and <i>Cstb^−/−</i> Purkinje cells. Individual cells measured are shown as spheres. <b>(C)</b> The frequency of EPSCs was significantly higher in <i>Cstb^−/−</i> cells compared to controls (p = 0.034). The data are expressed as mean frequency (Hz) ± SE. *, p<0.05; **, p<0.01.</p

The GO terms of biological processes, molecular functions and cellular components in P30 <i>Cstb^−/−</i> cerebellum.

<p>The GO terms of biological processes, molecular functions and cellular components in P30 <i>Cstb^−/−</i> cerebellum.</p

The GO terms of biological processes, molecular functions and cellular components in P5+2 DIV <i>Cstb^−/−</i> cerebellar granule cells.

<p>The GO terms of biological processes, molecular functions and cellular components in P5+2 DIV <i>Cstb^−/−</i> cerebellar granule cells.</p

Autoradiography of GABA_A receptors.

<p><b>(A)</b> Binding of [³H]muscimol was significantly reduced in P30 <i>Cstb^−/−</i> cerebellum (p = 0.037). No change was seen at P7. <b>(B)</b> Binding of [³H]Ro15-4513 did not show significant changes at P7 or at P30. However, when diazepam (DZ) was added to reveal the diazepam-insensitive α6 subunit-dependent GABA_A receptor subtype, decreased binding for [³H]Ro15-4513 (p = 0.012) was seen. The data are expressed as mean radioactivity levels (nCi/mg) ± SE. *, p<0.05. <b>(C)</b> Representative images of [³H]muscimol binding to P30 <i>Cstb^−/−</i> and control brain. <b>(D)</b> Representative images of [³H]Ro15-4513 binding to P30 <i>Cstb^−/−</i> and control brain without and <b>(E)</b> in presence of diazepam.</p