Functional expression and subcellular localization of the Cl- cotransporters KCC2 and NKCC1 in rodent hippocampal and neocortical neurons

The work presented here has focused on the role of cation-chloride cotransporters (CCCs) in (1) the regulation of intracellular chloride concentration within postsynaptic neurons and (2) on the consequent effects on the actions of the neurotransmitter gamma-aminobutyric acid (GABA) mediated by GABAA receptors (GABAARs) during development and in pathophysiological conditions such as epilepsy. In addition, (3) we found that a member of the CCC family, the K-Cl cotransporter isoform 2 (KCC2), has a structural role in the development of dendritic spines during the differentiation of pyramidal neurons. Despite the large number of publications dedicated to regulation of intracellular Cl-, our understanding of the underlying mechanisms is not complete. Experiments on GABA actions under resting steady-state have shown that the effect of GABA shifts from depolarizing to hyperpolarizing during maturation of cortical neurons. However, it remains unclear, whether conclusions from these steady-state measurements can be extrapolated to the highly dynamic situation within an intact and active neuronal network. Indeed, GABAergic signaling in active neuronal networks results in a continuous Cl- load, which must be constantly removed by efficient Cl- extrusion mechanisms. Therefore, it seems plausible to suggest that key parameters are the efficacy and subcellular distribution of Cl- transporters rather than the polarity of steady-state GABA actions. A further related question is: what are the mechanisms of Cl- regulation and homeostasis during pathophysiological conditions such as epilepsy in adults and neonates? Here I present results that were obtained by means of a newly developed method of measurements of the efficacy of a K-Cl cotransport. In Study I, the developmental profile of KCC2 functionality during development was analyzed both in dissociated neuronal cultures and in acute hippocampal slices. A novel method of photolysis of caged GABA in combination with Cl- loading to the somata was used in this study to assess the extrusion efficacy of KCC2. We demonstrated that these two preparations exhibit a different temporal profile of functional KCC2 upregulation. In Study II, we reported an observation of highly distorted dendritic spines in neurons cultured from KCC2-/- embryos. During their development in the culture dish, KCC2-lacking neurons failed to develop mature, mushroom-shaped dendritic spines but instead maintained an immature phenotype of long, branching and extremely motile protrusions. It was shown that the role of KCC2 in spine maturation is not based on its transport activity, but is mediated by interactions with cytoskeletal proteins. Another important player in Cl- regulation, NKCC1 and its role in the induction and maintenance of native Cl- gradients between the axon initial segment (AIS) and soma was the subject of Study III. There we demonstrated that this transporter mediates accumulation of Cl- in the axon initial segment of neocortical and hippocampal principal neurons. The results suggest that the reversal potential of the GABAA response triggered by distinct populations of interneurons show large subcellular variations. Finally, a novel mechanism of fast post-translational upregulation of the membrane-inserted, functionally active KCC2 pool during in-vivo neonatal seizures and epileptiform-like activity in vitro was identified and characterized in Study IV. The seizure-induced KCC2 upregulation may act as an intrinsic antiepileptogenic mechanism.Gamma-aminovoihappo (GABA) on tärkein ehkäisevä välittäjäaine nisäkkäiden aivoissa. GABA:n vaikutukset perustuvat kloridia läpäisevien kanavien avautumiseen, ja solunsisäinen kloridipitoisuus vaikuttaa siten kanavien kautta kulkevan sähkövirran suuntaan eli polariteettiin.Vaikka solunsisäisen kloridipitoisuuden säätelyä on tutkittu sadoissa julkaisuissa, sen taustalla olevia mekanismeja ei tunneta vielä täysin. Kokeet soluviljelmillä ja aivoileikkeillä osoittivat jo kauan sitten että GABA:n vaikutus kortikaalisten hermosolujen solukalvoon muuttuu depolarisoivasta hyperpolarisoivaksi aivojen maturoituessa. On kuitenkin kyseenalaista, voidaanko näitä tuloksia suoraan yleistää ehjän ja aktiivisen hermoverkon dynaamiseen toimintaan. Aktiivisissa hermoverkoissa GABA-välitteinen signalointi aiheuttaa jatkuvaa kloridin kerääntymistä. Siksi kloriditransportterien ajalliset ja paikalliset tehokkuuden muutokset ovat fysiologisesti tärkeämpiä tekijöitä kuin GABA-vaikutuksen polaariteetti lepäävässä hermosolussa. Väitöskirjani ensimmäisessä kokeellisessa työssä KCC2-kotransportterin funktionaalisuuden kehitysprofiilia analysoitiin hermosoluviljelmissä ja hippokampusleikkeissä. Osoitimme, että näissä kähdessa preparaatissa KCC2:n ilmentymisen ajallinen profiili on erilainen, mikä johtunee tiettyjen ns. troofisten tekijöiden puutteesta viljemissä. Toisessa tutkimuksessa havaitsimme että dendriittiset okaset (jotka ovat keskeisiä rakenteita ärsyttäviä signaaleja välittävissä synapseissa) eivät kehity hermosoluissa joilta puuttu KCC2:ta koodaava geeni. Osoitimme, että KCC2:lla on keskeinen rakenteellinen rooli dendriittisten okasten kypsymisessä. Tämä siis ei perustu KCC2-molekyylin kuljettajatoimintaan vaan KCC2:n vuorovaikutuksiin solun tukirankaan liittyvien proteiinien kanssa. Kolmannen tutkimuksen kohteena oli NKCC1, joka on toinen tärkeä kuljettajamolekyyli kloridin säätelyssä. Osoitimme että NKCC1 pitää yllä kloridigradienttia hermosolujen aksonin initiaalisegmentin (AIS) ja soluosan välillä. Nämä kokeet tehtiin neokorteksin ja hippokampuksen neuroneilla aivoleikkeissä. Näiden tulosten perusteella voi olettaa että GABA:n vaikutuksia säätelee hermosolun eri osissa ehkäisevien synapsien läheisyydessä toimivien KCC2- ja NKCC1-molekyylien jakauma. GABA saattaa jopa toimia ärsyttävänä välittäjäaineena AIS:ssa. Neljännessä tutkimuksessa käsiteltiin uutta mekanismia jolla KCC2-molekyylin toiminta aktivoituu. Havaitsimme että epileptinen hermoverkkotoiminta immatuureissa aivoissa johtaa nopeaan KCC2-molekyylien insertioon hermosolukalvoihin. Tällainen epileptisten kohtausten aiheuttama KCC2:n säätely saattaa toimia hermosoluja suojelevana mekanismina

Implications of the N-terminal heterogeneity for the neuronal K-Cl cotransporter KCC2 function

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BDNF is required for seizure-induced but not developmental up-regulation of KCC2 in the neonatal hippocampus

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Transcranial magnetic stimulation set-up for small animals

Transcranial magnetic stimulation (TMS) is widely applied on humans for research and clinical purposes. TMS studies on small animals, e.g., rodents, can provide valuable knowledge of the underlying neurophysiological mechanisms. Administering TMS on small animals is, however, prone to technical difficulties, mainly due to their small head size. In this study, we aimed to develop an energy-efficient coil and a compatible experimental set-up for administering TMS on rodents. We applied a convex optimization process to develop a minimum-energy coil for TMS on rats. As the coil windings of the optimized coil extend to a wide region, we designed and manufactured a holder on which the rat lies upside down, with its head supported by the coil. We used the set-up to record TMS-electromyography, with electromyography recorded from limb muscles with intramuscular electrodes. The upside-down placement of the rat allowed the operator to easily navigate the TMS without the coil blocking their field of view. With this paradigm, we obtained consistent motor evoked potentials from all tested animals.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

In vivo Calcium Imaging of Evoked Calcium Waves in the Embryonic Cortex

The dynamics of intracellular calcium fluxes are instrumental in the proliferation, differentiation, and migration of neuronal cells. Knowledge thus far of the relationship between these calcium changes and physiological processes in the developing brain has derived principally from ex vivo and in vitro experiments. Here, we present a new method to image intracellular calcium flux in the cerebral cortex of live rodent embryos, whilst attached to the dam through the umbilical cord. Using this approach we demonstrate induction of calcium waves by laser stimulation. These waves are sensitive to ATP-receptor blockade and are significantly increased by pharmacological facilitation of intracellular-calcium release. This approach is the closest to physiological conditions yet achieved for imaging of calcium in the embryonic brain and as such opens new avenues for the study of prenatal brain development. Furthermore, the developed method could open the possibilities of preclinical translational studies in embryos particularly important for developmentally related diseases such as schizophrenia and autism.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

Of Mice and Men, and Chandeliers

How does the human neocortex reliably propagate information through neural circuits? One mechanism appears to involve relying on strong connections from pyramidal neurons to interneurons and a depolarizing action of cortical chandelier cells

Complex Events Initiated by Individual Spikes in the Human Cerebral Cortex

Synaptic interactions between neurons of the human cerebral cortex were not directly studied to date. We recorded the first dataset, to our knowledge, on the synaptic effect of identified human pyramidal cells on various types of postsynaptic neurons and reveal complex events triggered by individual action potentials in the human neocortical network. Brain slices were prepared from nonpathological samples of cortex that had to be removed for the surgical treatment of brain areas beneath association cortices of 58 patients aged 18 to 73 y. Simultaneous triple and quadruple whole-cell patch clamp recordings were performed testing mono- and polysynaptic potentials in target neurons following a single action potential fired by layer 2/3 pyramidal cells, and the temporal structure of events and underlying mechanisms were analyzed. In addition to monosynaptic postsynaptic potentials, individual action potentials in presynaptic pyramidal cells initiated long-lasting (37 ± 17 ms) sequences of events in the network lasting an order of magnitude longer than detected previously in other species. These event series were composed of specifically alternating glutamatergic and GABAergic postsynaptic potentials and required selective spike-to-spike coupling from pyramidal cells to GABAergic interneurons producing concomitant inhibitory as well as excitatory feed-forward action of GABA. Single action potentials of human neurons are sufficient to recruit Hebbian-like neuronal assemblies that are proposed to participate in cognitive processes

HB-GAM (pleiotrophin) reverses inhibition of neural regeneration by the CNS extracellular matrix

Chondroitin sulfate (CS) glycosaminoglycans inhibit regeneration in the adult central nervous system (CNS). We report here that HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin), a CS-binding protein expressed at high levels in the developing CNS, reverses the role of the CS chains in neurite growth of CNS neurons in vitro from inhibition to activation. The CS-bound HB-GAM promotes neurite growth through binding to the cell surface proteoglycan glypican-2; furthermore, HB-GAM abrogates the CS ligand binding to the inhibitory receptor PTPs (protein tyrosine phosphatase sigma). Our in vivo studies using two-photon imaging of CNS injuries support the in vitro studies and show that HB-GAM increases dendrite regeneration in the adult cerebral cortex and axonal regeneration in the adult spinal cord. Our findings may enable the development of novel therapies for CNS injuries.Peer reviewe