Kainate receptor function in rodent subcortical visual processing.

Glutamate is found throughout the central nervous system and has been shown to be an important excitatory neurotransmitter in the visual system. There are two subdivisions of receptor on which this ubiquitous neurotransmitter acts, metabotropic (mGluR) and ionotropic (GluR) glutamate receptors. There are eight sub types of mGluR falling into three groups, and fifteen GluR subunits also divided into three groups. Kainate receptors (KARs) comprise one group of the ionotropic glutamate receptor subdivision. Relay cells of the lateral geniculate nucleus (LGN) are driven and modulated by a variety of NMDA, AMPA and metabotropic receptors. In addition, investigation into the involvement of mGluR, AMPA and NMDA receptor function in the synaptic processing of the superior colliculus (SC) has been well documented. It has been difficult, however, to establish specific KAR function in these brain structures due to lack of pharmacological agents acting solely at kainate receptors. In recent years such agents have become available, thus enabling the present study of GluR5 involvement in visual processing within the SC and LGN. The purpose of this body of work has been to assess the involvement of GluR5-containing Kainate receptors (KARs) in synaptic transmission between retinal ganglion cells (RGCs) and subcortical brain structures involved in the processing of visual information namely the superficial superior colliculus (SSC) and the lateral geniculate nucleus (LGN). The majority of the work focused on the function of KARs in the SSC. To elucidate the involvement of KARs in visual processing, both in vivo and in vitro methods were utilised. In vivo electrophysiology was used for extracellular recording of evoked activity of both SSC and LGN neurons in response to visual stimuli. This was carried out during intravenous injection of GluR5 antagonist. In vivo recording twinned with iontophoretic administration of GluR5-specific pharmacological compounds was also employed to investigate KAR participation in direct synaptic transmission between RGCs and the SSC neurons. The same technique was used to study KAR involvement in the phenomenon of response habituation exhibited by these neurons. To parallel in vivo protocols, in vitro SSC slice experiments were performed to study the effect of GluR5 agonists and antagonists on evoked postsynaptic currents. This enabled the administration of drugs at concentrations specific for GluR5 subunits whilst investigating GluR5 involvement in direct synaptic transmission between RGC input and SSC neurons. In addition, a paired pulse protocol was employed to propose a presynaptic location of GluR5-containing KARs at retinal input into the SSC. Furthermore, the use of GluR5- specific and GABAR-specific compounds during evoked current recording indicated the involvement of GluR5-containing receptors in the direct modulation of excitatory but not inhibitory input into the SSC. In summary, therefore, both in vivo and in vitro electrophysiology techniques were used to indicate a location and function for GluR5 KARs in the subcortical visual system of the rat. GluR5-containing receptors were found to modulate visual processing of both the LGN and SSC. It was unclear whether these receptors were located in the LGN itself due to the use of systemic injection protocols, however, iontophoresis of GluR5-selective drugs demonstrated a role in modulating visual responses within the SSC. The mechanism by which GluR5 receptors modulated responses in the SSC was further elucidated by a series of whole cell patch-clamp experiments which revealed that GluR5-containing receptors reduced synaptic transmission at excitatory inputs directly onto recorded cells and those connections with the intrinsic inhibitory circuitry of the SSC. In addition a paired-pulse protocol was used to determine that the decrease in excitatory transmission was caused by the presynaptic reduction of glutamatergic transmission

INVESTIGATIONS OF THE STRUCTURE-FUNCTION RELATIONSHIP IN KAINATE RECEPTORS USING FÖRSTER RESONANCE ENERGY TRANSFER

Kainate receptors belong to the family of ion channels known as the ionotropic glutamate receptors. Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission, modulate the release of presynaptic glutamate, and facilitate dendrite formation. Kainate receptors are unique among the ionotropic glutamate receptors in being modulated by sodium ions. They have also been implicated in the development of higher learning and epilepsy. In recent years a wealth of structural data has become available for the AMPA and NMDA classes; however, the structural characterization of kainate receptors has been limited. The work in this dissertation utilizes luminescence resonance energy transfer (LRET) and single-molecule Förster Resonance Energy Transfer (smFRET) in order to address this gap in the knowledge. We have characterized the structural arrangement and dynamics of the homomeric (GluK2) receptors and identified structural changes involved in the functional modulation by ions and auxiliary proteins. Additionally, we have characterized the arrangement and dynamics of the heteromeric (GluK2/GluK5). These data will build a foundation for the full biophysical characterization of kainate receptors; and contribute to the development of subunit-specific modulatory compounds to be used for disease therapies, and for more detailed characterization of brain function at the molecular level

Structure-function Relationships of the Pore-forming Subunits of the Ampa Receptor and the Auxiliary Subunit Stargazin

AMPA receptors mediate the majority of neurotransmission in the CNS. One of the most important properties of AMPA receptors is their quick and profound desensitization in the presence of glutamate. Although, the mechanism of AMPA receptor desensitization onset has been elucidated and involves breaking of the protein interactions on the dimer interface in response to agonist binding, the mechanism of recovery from desensitization remains unknown. When co-expressed with the auxiliary subunit stargazin AMPA receptor functional properties are affected: steady-state desensitization is decreased, recovery from desensitization is faster, kainate efficacy is increased and deactivation is slowed. The mechanism of stargazin's action on AMPA receptor and their sites of association remain unknown. Previous studies suggested that stargazin decreases AMPA receptor steady-state desensitization by stabilizing the dimer interface. However, in our studies we were able to demonstrate that stargazin's effect on steady-state desensitization in mutations that stabilize the dimer interface is additive, while its effects on steady-state desensitization in a mutant that destabilized the dimer interface is occluded, indicating that stargazin does not act by stabilizing the intra-dimer interface, but instead destabilizes the desensitized state and speeds the recovery from desensitization. To identify residues that may be part of association between the AMPA pore-forming subunits and stargazin we were guided by the homology between AMPA and kainate receptors and the crystal structure of the LBD. We identified residues downstream of helix H (K693 and K695) and in the M4 linker (S784 and A789) that when co-expressed with stargazin presented with loss of it effects, suggesting that these residues may participate in the association with stargazin. Based on our data we were also able to propose a mechanism for recovery from desensitization that includes residues in helix B (K434) and the M1 linker (K501 and K502). Utilizing a similar approach in stargazin we were able to identify areas that are necessary for its association with the AMPA receptors, including polar residues proximal to the plasma membrane in the 1st and 2nd extracellular loops and others that are crucial for its function, the conserved motif GLWXXC and conserved cysteine residues at positions 67 and 77.Department of Biochemistry and Molecular Biolog

MODULATION OF THE RECEPTOR GATING MECHANISM AND ALLOSTERIC COMMUNICATION IN IONOTROPIC GLUTAMATE RECEPTORS

Ionotropic glutamate receptors (iGluRs) found in mammalian brain are primarily known to mediate excitatory synaptic transmission crucial for learning and memory formation. The family of iGluRs consists of AMPA receptors, NMDA receptors and kainate receptors with each member having distinct physiological role. In the recent years, significant progress has been made in understanding the biophysical, and functional properties of iGluRs. The development of Cryo-EM and X-Ray crystallography techniques have further facilitated in the structural understanding of these receptors. However, the multidomain nature, large size of the protein, complex gating mechanism and inadequate knowledge regarding the conformational dynamics of the receptors during channel gating mechanism have been some of the limiting factors in elucidating the structure-function relation of iGluRs. Thus, to understand the conformational dynamics of iGluR family and correlate to its functional behavior, I have utilized single molecule Forster Resonance Energy Transfer (smFRET) and molecular dynamics simulation and specifically investigated the factors influencing gating mechanism and allosteric communication in heteromeric kainate receptor GluK2/K5 and NMDA receptor GluN1/N2A. Some of the major finding in this dissertation includes subunit arrangement of GluK2/K5 and its dynamics involved in resting and desensitized conditions. For the first time we have identified the conformational changes induced at GluK2 and GluK5 subunits in a heteromer GluK2/K5 when bound to different agonists. Utilizing MD simulations in GluN1/N2A NMDA receptors we have identified the structural pathway regarding the mechanism underlying negative cooperativity and how mutation in the receptor leads to abnormal functional behavior. These findings will allow us to understand the conformational control regarding modulation of receptor function and will serve as a basis for developing subunit and conformation-specific therapeutic drugs that can potentially control the abnormal activity of the receptors linked to several neurological diseases

Lääkekehitysmallin testaaminen hyperaktiivisille psykoottisille sairaustiloille

Bipolar disorder, schizophrenia and schizoaffective disorder are extremely debilitating illnesses that encompass affective and/or psychotic symptoms. Not only is there common symptomatology and genetic susceptibility, but the pharmacotherapy approaches are also similar. Nonetheless, molecular mechanisms underpinning these diseases are not yet fully understood. The theory that there is a dopaminergic dysfunction cannot account for all of the symptoms. Nor can the compounds that act on dopaminergic mechanisms successfully alleviate the symptoms. There is evidence to suggest that there are imbalances in other neurotransmitter systems, particularly the main excitatory pathway - the glutamatergic system. Glutamatergic transmission is essential for development,learning and memory and many other physiological functions of the brain. Glutamatergic receptors of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type mediate the majority of the fast synaptic neurotransmission. Here, Gria1−/− mice, lacking the GluA1 subunit of AMPA receptors, with concurrent schizophrenia-like and affective symptoms were used. The predictive validity was addressed using the standard and novel glutamate-modulating pharmacotherapeutics. The hyperactivity, the most robust feature of Gria1−/− mice and a hallmark of psychotic disorders, was attenuated by drug-treatments. Importantly, chronic treatments with lithium, valproate, topiramate, lamotrigine and perampanel were effective, evidence of their pharmacological efficacy after the acute, often sedative, treatment phase. In addition, excessive novelty-induced activation of the dorsal hippocampus of Gria1−/− mice as measured by c-Fos expression was blunted by the drug-treatments of which all are known to reduce the activity of the glutamatergic transmission. Other behaviours relevant to the schizoaffective symptomatology such as disinhibited risk-taking, less despair-like behaviour and highly exploratory phenotype as well as social deficits were partially responsive to treatment with mood-stabilisers. Moreover, Gria1−/− mice exhibited a slightly higher preference for sucrose and made more impulsive choices towards sucrose. The Gria1−/− mice may represent a suitable model for the screening of the preclinical efficacy of novel drugs on the hyperactive behaviour linked to manic episode of bipolar disorder, schizophrenia and schizoaffective disorder.Neuropsykiatriset sairaudet kuten maanis-depressiivinen sairaus ja skitsofrenia tuottavat runsaasti harmia sairastuneille, heidän lähipiirilleen ja yhteiskunnalle. Nykyinen lääkehoito ei ole optimaalista, ja näiden sairauksien perussyyt tunnetaan vielä huonosti. Tässä työssä keskityttiin hyperaktiiviseen oireeseen, mikä on yleistä näissä sairaustiloissa, ja käytettiin kokeellista poistogeenistä (yhden glutamaattireseptorin inaktivaatio) mallia, jonka tyypillinen vaste uuteen ympäristöön joutumisesta oli liikeaktiivisuuden kaksinkertaistuminen. Sen ei kuitenkaan katsottu johtuvan dopamiinin aktiivisuuden lisääntymisestä vaan aivojen tärkeimmän kiihoittavan välittäjäaineen glutamaatin mekanismien voimistumisesta. Eri tavoilla vaikuttavat lääkeaineet, jotka ovat kliinisessä käytössä maanis-depressiivisen sairauden (ja/tai epilepsian) hoidossa, selvästi vähensivät kokeellisen mallin liikeaktiivisuutta. Näin tapahtui myös pitkäaikaisen lääkeaineannon jälkeen, joten vaikutus ei johtunut akuutin annoksen väsyttävästä ja liikeaktiivisuutta lamaavasta vaikutuksesta. Tarkasti vain yhtä glutamaattireseptorityyppiä estävä epilepsian hoidossa käytetty lääke toimi myös tehokkaasti normaalistaen käyttäytymisen poikkeavuuden. Kaikille muillekin testatuille lääkkeille oli yhteistä glutamaattimekanismien estäminen. Samalla mallilla tutkittiin mitkä aivoalueet aktivoituivat kokeellisessa mallissa ja estivätkö testatut lääkkeet tämän aktivaation. Kaikki käyttäymistä normaalistaneet lääkeaineet myös estivät dorsaalisen hippokampuksen kohonnutta aktiivisuutta, jonka tulkittiin siten liittyvän korkeaan glutamaattiaktiivisuuteen. Poistogeenisen mallin monipuolinen käyttäytymisen tutkimus paljasti muitakin lääkeaineille herkkiä poikkeavuuksia, jotka voisivat liittyä maanis-depressiivinen sairauden mekanismeihin. Näin mallin katsottiin tulleen validoituksi prekliinisiä lääketestauksia varten etsittäessä uusia molekyylejä neuropsykiatristen sairauksien hoitoon

Biogenic Amines in Neurotransmission and Human Disease

Drawing on the expertise of experienced researchers in neurotransmission and catecholamines, this book provides a brief overview of the latest knowledge in the field. The book contains an introductory chapter that aims to explain the subsequent four chapters for researchers who are new to the field

Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors.

The NMDA type of glutamate receptor requires both glycine and glutamate to activate it efficiently. The receptor is thought to be an oligomer of two types of subunit, NR1 and NR2. Site-directed mutagenesis has shown that glycine potency is controlled by residues located in two areas on the NR1 subunit, one N-terminal of Ml and the other C-terminal of M3 (termed the S1 and S2 domains respectively). To test the hypothesis that the glutamate site exists on the NR2 subunits, the role of amino acid residues in similar areas on the NR2A subunit were investigated. These areas show homology with the ligand binding domains of bacterial periplasmic amino acid binding proteins. Two mutations caused an increase of glutamate EC50 by at least two orders of magnitude, (H466A and G669A) and one (T671A) by three orders, compared with wild-type. In contrast, glycine EC50s did not differ by more than 2-fold compared with the wild-type. Schild analysis was used to measure the binding of the competitive NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoic acid (APV) on the most 'shifted' mutant (T671 A). The slope of the Schild plot for the mutant receptor did not differ greatly from unity, 0.94 ± 0.16 (mean ± S.D.M), which is consistent with APV being a competitive antagonist on the mutant receptor. However the affinity of the receptor for APV (KB = 321 ±30 μM) was 255-fold less than wild-type (KB = 1-26 ± 0.07 μM) (means ± S.D.M). The large reduction of glutamate potency, together with unchanged Hill slope, and no gross reduction in maximum response, suggests that residues contributing to glutamate binding are located on the NR2A subunit. This is supported by the much- reduced affinity for APV in the T671A mutant. Such evidence suggests that this residue is important for the binding of glutamate to NR1/NR2A receptors. In a second project, transgenic founder mice were generated by pronuclear microinjection containing a 8 kb 5'UT fragment from the mouse NR1 gene driving the expression of the tetracycline-sensitive transactivator (tTA). This mouse may be useful for the study of neuronal-specific inducible gene expression in the future

The Opposing Roles of GluN2C and GluN2D NMDA Receptor Subunits in Modulating Neuronal Oscillations

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ion channels consisting of two GluN1 subunits and two other subunits from among GluN2A-2D and GluN3A-3B subunits. NMDARs play critical roles in synaptic plasticity, learning and memory, and higher brain function such as cognition and perception. Dysfunction of NMDARs (hyper-function and hypo-function of NMDARs) are related to various diseases, including stroke, schizophrenia, Alzheimer’s disease, and others. However, to date, NMDARs antagonists have mostly failed in clinical trials due to adverse effects. NMDARs antagonists replicate the core symptoms of schizophrenia which may underlie its ability to alter neuronal oscillations in the neural circuitry of different brain regions. Recent evidence has shown that GluN2C subunits of NMDAR are expressed in astrocytes in the cortex, and that GluN2D NMDAR subunits are enriched in the parvalbumin-containing GABAergic inhibitory interneurons in the cortex and midbrain structures. Other studies have shown that both astrocytes and parvalbumin-containing interneurons play an essential role in generating and maintaining neuronal oscillations. These findings imply that GluN2C and GluN2D subunits may be involved in the distinct neural circuitry which regulates neuronal oscillations and thus influence the brain function and contribute to various diseases states. The initial aims of this dissertation are to determine if GluN2C and GluN2D subunits have a role in regulating neuronal oscillations. We also measured the auditory evoked responses in wildtype and GluN2C- and GluN2D-KO mice. Lastly, we use ketamine as the tool drug to determine the role of NMDARs in neuronal oscillations in a CDKL5-KO mouse model. We found that spontaneous basal neuronal oscillations were elevated in GluN2C- and GluN2D-KO mice compared to WT mice. NMDARs antagonists increased the power of neuronal oscillations in WT mice; we found drug-induced power increase is abolished in GluN2D-KO mice and is augmented in GluN2C-KO mice. Furthermore, we also found GluN2D-KO mice displayed abnormal auditory evoked responses. Lastly, we test subunit-selective NMDARs drug and NMDARs allosteric modulators with distinct subunits selectivity developed by our lab, including PAMs and NAMs on these KO models

Development and characterization of a model of glutamate and domoate toxicity in cultured rat cerebellar granule neurons

A model of acute glutamate- and domoate-induced toxicity was developed and characterized in cultured rat cerebellar granule cells (CGCs) using experimental conditions which preserve the voltage-dependency of NMDA receptor function. Glutamate, which is normally non-toxic to CGCs in physiologic media (pH 7.4), was shown to induce a cytotoxic response after 2 hours when the exposure temperature was reduced from 37° to 22°. Pharmacological characterization of this response demonstrated that cytotoxicity is mediated by the activation of NMDA receptors, while non-NMDA receptors produce a depolarizing stimulus that enhances release of the voltage-dependent Mg²⁺ blockade of NMDA receptor ion channels. Reduced temperature was shown to facilitate NMDA receptor activation by compromising the ability of CGCs to maintain normal electrochemical gradients during glutamate-induced ion flux. When compared to glutamate, the non-NMDA receptor agonist, domoate, demonstrated an acute cytotoxic response in CGCs that was also mediated predominantly by NMDA receptors. NMDA receptor activation was produced secondary to a domoateinduced release of glutamate and aspartate from CGCs; therefore, domoate synergistically potentiates glutamate/aspartate-mediated neurotoxicity. Domoate-induced excitatory amino acid (EAA) release was investigated and found to occur almost exclusively through reversal of the high affinity Na+-coupled glutamate transporter and by osmoregulatory mechanisms. CGCs also responded to domoate-induced depolarization by releasing adenosine which suppresses exocytotic EAA release through A1 receptor activation. The functional and pharmacological characteristics of NMDA receptors were characterized in 12 DIC CGCs using the channel blocking compound [³H]MK-801 (dizocilpine). Kinetic analysis of [³H]MK-801 binding indicated the possible existence of at least two NMDA receptor populations on 12 DIC CGC membranes, and the equilibrium competition binding of MK-801 and other channel blocking compounds was consistent with the presence of high and low affinity binding sites. The neuroprotective potencies of NMDA receptor channel blockers correlated significantly with their affinities for the NMDA receptor derived from equilibrium competition analysis of [³H]MK-801 high-affinity binding. Thus, whereas 12 DIC CGCs express a pharmacologically heterogeneous population of NMDA receptors, it is the high-affinity component of [³H]MK-801 binding that mediates glutamate toxicity