GABAergic Signaling and Neuronal Chloride Regulation in the Control of Network Events in the Immature Hippocampus

Spontaneously arising network events are a characteristic feature of all developing neural networks. This activity is crucial for normal neuronal development and the establishment of appropriate synaptic connectivity. In the developing hippocampus, depolarizing GABAergic drive is essential in generation of early network events, known as giant depolarizing potentials (GDPs). Blockade of GABAergic signaling leads to hypersynchronization of the network and emergence of ictal-like events, pointing to dual, both excitatory and inhibitory roles for GABA, in regulation of these events. In Studies I-III of this thesis, we examined the role of GABAA receptor (GABAAR) -mediated neurotransmission with some parallel work on glycinergic signaling as well as neuronal Cl- regulation in modulation of GDPs in the developing rodent hippocampus. In Study I, we demonstrate that low levels of GABA and glycine suppress GDPs by activating extrasynaptic receptors. This implies that regardless of the depolarizing drive for Cl- currents at this developmental stage, a low conductance via Cl- -permeable GABAARs and glycine receptors (GlyRs) can cause efficient shunting and inhibition of the network events. In Study II, we discovered that sustained activation of a subset of hippocampal interneurons, caused by the neuropeptide arginine vasopressin (AVP), silences the network events in the perinatal hippocampus, regardless of the maturational level of the GABAergic system as compared across species. This is attributed to decreased synchronous interneuronal input that is essential for the GDP generation. In Study III, we demonstrate that transport-functional K-Cl cotransporter 2 (KCC2) is present in the CA3 pyramidal neurons already in the perinatal stages in mice and rats. Cl- extrusion by KCC2 counteracts the dominant Na-K-2Cl cotransporter 1 (NKCC1) -mediated Cl- uptake and restrains the depolarizing GABAergic drive onto the CA3 pyramidal cells. Thereby, function of KCC2 limits pyramidal neuron spiking and synchronization during GDPs and participates in the modulation of GDPs from their developmental onset. This work describes novel physiological GABAergic mechanisms that control GDPs in the perinatal rodents and establishes a role for KCC2 in regulation of pyramidal neuron excitability and synchronization during GDPs starting from their developmental onset.Lukemattomat tutkimukset ovat osoittaneet, että spontaanisti syntyvä synkroninen aktiivisuus on kaikille kehittyville hermoverkoille ominainen piirre. Aktiivisuus ja sen laukaisemat signalointikaskadit tukevat hermosolujen normaalia kehitystä ja ohjaavat oikeiden hermoyhteyksien muodostumista. Kehittyvän hippokampuksen alueella solujen korkeasta kloriditasosta johtuva depolaarinen GABA ajaa GDP:inä (giant depolarizing potentials) tunnettuja synkronisia verkkotason aktiivisuusryöppyjä. GABAergisen signaloinnin hiljentäminen johtaa kuitenkin epileptisen aktiivisuuden muodostumiseen hermoverkossa, viitaten siihen, että GABAn rooli kehittyvässä hippokampuksessa on kaksisuuntainen: sekä hillitsevä, että kiihdyttävä. Tämän väitöskirjan osatöissä I-III tutkin sähköfysiologisin menetelmin GABA A-reseptorivälitteisen viestinnän ja solujen kloridisäätelyn roolia GDP:iden säätelyssä perinataaleissa hiirissä ja rotissa. Työssäni esittelen uusia fysiologisia mekanismeja GABA-välitteisessä GDP:iden säätelyssä ja osoitan ensimmäistä kertaa kloridikuljettaja KCC2:n keskeisen roolin pyramidisolujen ärtyvyyden ja verkkotason aktiivisuuden säätelyssä kehittyvässä hippokampuksessa.

KCC2-Mediated Cl- Extrusion Modulates Spontaneous Hippocampal Network Events in Perinatal Rats and Mice

It is generally thought that hippocampal neurons of perinatal rats and mice lack transport-functional K-Cl cotransporter KCC2, and that Cl- regulation is dominated by Cl- uptake via the Na-K-2Cl cotransporter NKCC1. Here, we demonstrate a robust enhancement of spontaneous hippocampal network events (giant depolarizing potentials [GDPs]) by the KCC2 inhibitor VU0463271 in neonatal rats and late-gestation, wildtype mouse embryos, but not in their KCC2-null littermates. VU0463271 increased the depolarizing GABAergic synaptic drive onto neonatal CA3 pyramidal neurons, increasing their spiking probability and synchrony during the rising phase of a GDP. Our data indicate that Cl- extrusion by KCC2 is involved in modulation of GDPs already at their developmental onset during the perinatal period in mice and rats.Peer reviewe

Vasopressin excites interneurons to suppress hippocampal network activity across a broad span of brain maturity at birth

During birth in mammals, a pronounced surge of fetal peripheral stress hormones takes place to promote survival in the transition to the extrauterine environment. However, it is not known whether the hormonal signaling involves central pathways with direct protective effects on the perinatal brain. Here, we show that arginine vasopressin specifically activates interneurons to suppress spontaneous network events in the perinatal hippocampus. Experiments done on the altricial rat and precocial guinea pig neonate demonstrated that the effect of vasopressin is not dependent on the level of maturation (depolarizing vs. hyperpolarizing) of postsynaptic GABA(A) receptor actions. Thus, the fetal mammalian brain is equipped with an evolutionarily conserved mechanism well-suited to suppress energetically expensive correlated network events under conditions of reduced oxygen supply at birth.Peer reviewe

Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

Intracellular pH is a potent modulator of neuronal functions. By catalyzing (de)hydration of CO2, intracellular carbonic anhydrase (CA(i)) isoforms CA2 and CA7 contribute to neuronal pH buffering and dynamics. The presence of two highly active isoforms in neurons suggests that they may serve isozyme-specific functions unrelated to CO2-(de)hydration. Here, we show that CA7, unlike CA2, binds to filamentous actin, and its overexpression induces formation of thick actin bundles and membrane protrusions in fibroblasts. In CA7-overexpressing neurons, CA7 is enriched in dendritic spines, which leads to aberrant spine morphology. We identified amino acids unique to CA7 that are required for direct actin interactions, promoting actin filament bundling and spine targeting. Disruption of CA7 expression in neocortical neurons leads to higher spine density due to increased proportion of small spines. Thus, our work demonstrates highly distinct subcellular expression patterns of CA7 and CA2, and a novel, structural role of CA7.Peer reviewe

Bumepamine, a brain-permeant benzylamine derivative of bumetanide, does not inhibit NKCC1 but is more potent to enhance phenobarbital's anti seizure efficacy

Correction Volume: 143 Pages: 349-350 DOI: 10.1016/j.neuropharm.2018.10.012Based on the potential role of Na-K-Cl cotransporters (NKCCs) in epileptic seizures, the loop diuretic bumetanide, which blocks the NKCC1 isoforms NKCC1 and NKCC2, has been tested as an adjunct with phenobarbital to suppress seizures. However, because of its physicochemical properties, bumetanide only poorly penetrates through the blood-brain barrier. Thus, concentrations needed to inhibit NKCC1 in hippocampal and neocortical neurons are not reached when using doses (0.1-0.5 mg/kg) in the range of those approved for use as a diuretic in humans. This prompted us to search for a bumetanide derivative that more easily penetrates into the brain. Here we show that bumepamine, a lipophilic benzylamine derivative of bumetanide, exhibits much higher brain penetration than bumetanide and is more potent than the parent drug to potentiate phenobarbital's anticonvulsant effect in two rodent models of chronic difficult-to-treat epilepsy, amygdala kindling in rats and the pilocarpine model in mice. However, bumepamine suppressed NKCC1-dependent giant depolarizing potentials (GDPs) in neonatal rat hippocampal slices much less effectively than bumetanide and did not inhibit GABA-induced Ca2+ transients in the slices, indicating that bumepamine does not inhibit NKCC1. This was substantiated by an oocyte assay, in which bumepamine did not block NKCC1a and NKCC1b after either extra- or intracellular application, whereas bumetanide potently blocked both variants of NKCC1. Experiments with equilibrium dialysis showed high unspecific tissue binding of bumetanide in the brain, which, in addition to its poor brain penetration, further reduces functionally relevant brain concentrations of this drug. These data show that CNS effects of bumetanide previously thought to be mediated by NKCC1 inhibition can also be achieved by a close derivative that does not share this mechanism. Bumepamine has several advantages over bumetanide for CNS targeting, including lower diuretic potency, much higher brain permeability, and higher efficacy to potentiate the anti-seizure effect of phenobarbital.Peer reviewe