Efficacy and mechanism of action of intrahippocampalD-cycloserine in an animal model of Posttraumatic Stress Disorder (PTSD)

Fairbairn, Lorren Rosli (2015-03)

Thesis (PhD)--Stellenbosch University, 2015.

Thesis

ENGLISH SUMMARY : Posttraumatic stress disorder (PTSD) has been described as a persistent (Bremner et al., 1996) and incapacitating (Zatzick et al., 1997; Mendlowicz &Stein 2000) psychiatric disorder which occurs after exposure to a potentially traumatic event (DSM-5, APA 2013). Exposure based therapy (EBT) is one of the most common and effective therapies for posttraumatic stress disorder (Mendes et al., 2008). The procedure involves controlled exposure to the feared stimulus in the absence of any overt danger. EBT in humans is procedurally very similar to fear extinction training in animal models of emotional learning, such as fear conditioning (Norton & Price, 2007). It has previously been shown that dysfunctional fear extinction underpins PTSD pathophysiology (Keane et al., 1985; Cohen et al., 2006; Amstadter et al., 2009). With recent studies demonstrating fear extinction as an essential process for studying putative pharmacotherapies for clinical use in PTSD treatment. One such novel pharmacological agent, D-cycloserine (DCS), has been investigated in both preclinical and clinical studies of anxiety and has been reported to facilitate extinction of learned fear in rats and to promote exposure-based therapies in humans (Walker et al., 2002; Ledgerwood et al., 2003; Davis et al., 2006; Bontempo et al., 2012). DCS is a partial agonist of the N-methyl-D-aspartate receptor (NMDAR) and exerts its effects by binding to the glycine regulatory site of the NMDA complex. In addition, these glutamatergic receptors, specifically the hippocampal NMDARs and their subsequent signalling pathways have been implicated in fear extinction. PTSD affects individuals in all sectors of society and is as much a concern with respect to children as to adults. Considering that adolescence is a period of heightened vulnerability for mood and anxiety disorders, it is crucial to observe the effects of trauma on this developmental stage. Therefore the main aim of this study was to determine whether intrahippocampal infusions of DCS could reverse the PTSD phenotype, as displayed by our animal model, in both adolescents and adults with special focus on fear extinction. In the current study, we used a fear conditioning paradigm consisting of a brief, intense electric footshock (1.5 mA) and a neutral tone (80 dB, 9 kHz) to represent the traumatic event and investigated the efficacy and molecular mechanism of action of DCS on the behaviour and neurochemistry of adolescent and adult rats. The present study was particularly interested in the effects of DCS on hippocampal brain-derived neurotrophic factor (BDNF), hippocampal NMDAR expression levels, factors downstream of the NMDA signalling pathway (i.e. neuronal nitric oxide synthase) and protein changes in the hippocampus (HC) of fear conditioned and DCS-treated animals. Our animal model generated the following key findings. Firstly, various behavioural tests demonstrated that fear conditioned rats exhibited a PTSD-like disorder as shown by their increased and sustained conditioned fear response and increased anxiety-like symptoms. These effects were reversed by intrahippocampal DCS infusions, as assessed by behavioural freezing. Secondly, an upregulation of hippocampal NMDARs was noted in fear conditioned rats, while repeated administration of intrahippocampal DCS reduced this effect. Thirdly, intrahippocampal DCS infusions enhanced dorsal hippocampal BDNF expression in DCS treated groups, with fear conditioned rats expressing the lowest BDNF levels. Fourthly, intrahippocampal DCS administration elicited similar patterns in adolescents and adults with regards to fear extinction i.e. a decreased fear response was noted in both age groups after DCS administration. Lastly, we observed that hippocampal protein expression differed between adolescent and adult rats. Most proteins were distinctly expressed in either of the two age groups. The protein, neurabin-2 was specifically expressed during the adolescent period. Furthermore, footshock led to an increase in adolescent protein expression, whereas DCS treatment led to a decrease in adolescent protein expression. Overall, this study supported and extended previous findings that DCS has a therapeutic effect on fear conditioning by enhancing extinction of anxiety-like symptoms in rodents. We were able to show that animals subjected to fear conditioning/trauma show signs of alterations in proteins involved in neuronal plasticity, calcium (Ca2+) homeostasis, cellular stress, cell cycle arrest, initiation of apoptotic mechanisms and cell signalling dysregulation. These proteins all have a role in one or more of the neurochemical parameters as examined in our PTSD model i.e. interact with the HC, BDNF, nNOS or NMDARs. Therefore, additional studies are needed to elucidate the relationship between epigenetic modifications and the resulting proteomic responses as demonstrated in our study. In addition, the role of BDNF in PTSD has to be further investigated, be it as a biomarker or as adjunctive therapy for PTSD.

AFRIKAANSE OPSOMMING : Posttraumatiesestresversteuring (PTSV) is ‘n ernstige en uitmergelendepsigiatrieseversteuring, watnablootstellingaan ‘n potensieeltraumatiesegebeurtenis,kanvoorkom(DSM-5, APA 2013). Ditblykdatterapiewatgebaseer is op blootstelling(blootstellinggebaseerdeterapie) die meesalgemene eneffektiesteterapievir PTSV is (Mendes et al., 2008). Die procedure behelsblootstellingaan ‘n stimulus watvreesinboesem en word ondergekontroleerde of beheerdetoestandeuitgevoer, dus in die afwesigheid van direktegevaar. Die prosedure in die behandeling van mensetoonsterkooreenkomste met die van dieremodelle met spesifiekeverwysingnavreeskondisioneringwaar die klem op emosioneleonderrig en vreesuitwissingval (Norton en Price, 2007). Voorheen is bewysdat PTSV op ‘n patofisiologiesewyseonderliggend is aandisfunksionelevreesuitwissing (Keane et al, 1985, Cohen et al, 2006, Amstadler et al, 2009). Onlangse studies toondie belangrikheid en fenomenaleimpakwatvreesuitwissing as ‘n noodsaaklike proses het op huidige en toekomstige studies virfarmakologieseterapiee en kliniesebehandeling van PTSV. So byvoorbeeld is die farmakologiesemiddel, D-cycloserine (DCS) getoets in prekliniese en kliniese studies waarangsvlakkeondersoek is. Daar is bevinddat DCS die uitwissing van aangeleerdeangs in rottefassiliteer en dit .bemarkookblootstellinggebaseerdeterapieewatvir die mens van belang is (Walker et al 2002, Ledgerwood et al 2003, Davis et al 2006, Bontempo et al 2012) DCS is ‘n gedeeltelike agonis van die N-metiel-D-aspartaat reseptor (NMDAR) en beoefen sy uitwerking deur binding met die glisien regulatoriese plek van die NMDA kompleks. Bykomend by vreesuitwissing word die glutamaat reseptore, met spesifieke verwysing na die hippocampus (HC) NMDARs en hul gevolglike sein paaie betrek. PTSV affekteer individue van alle vlakke van die samelewing en is ‘n groot kommer wat kinders sowel as volwassenes betref. Gegee die feit dat adollessensie ‘n tydperk van verhoogde/toenemende weerloosheid ten opsigte van buie en angs versteurings is, is dit noodsaaklik om die uitwerking van trauma op die ontwikkelingstadium waar te neem. Derhalwe was die hoofdoel van hierdie studie om vas te stel of intrahippokampus infusies van DCS die teenoorgestelde van die PTSV fenotipe kan wees soos uitgebeeld deur die dieremodel in beide adollessente en volwassene stadia met spesifieke fokus op vreesversteurings. In die huidige studie het ons ‘n vrees gekondisioneerde paradigma bestaande uit ‘n kort intense elektriese skok (1,5 mA) en ‘n neutrale toon (80 dB, 9kHz) gebruik om die traumatiese gebeurtenis te verteenwoordig en die doeltreffendheid en molekulere meganisme van DCS op die gedrag en neurochemie van die adollesent en volwasse rotte te ondersoek. Ons studie was veral gemik op die uitwerking van DCS op die HC, die brein-afkomstige neurotrofiese faktor, hippokampus NMDAR uitdrukkingsvlakke, faktore onderliggend aan die NMDA sein paaie (nNOS) en proteïenveranderinge in die HC van vrees gekondisioneerde en DCS behandelde diere. Die volgende bevindinge is gemaak op grond van ons dieremodel: Eerstens het verskeie gedragstoetse gedemonstreer dat angs gekondisioneerde rotte ‘n PTSV gewyse versteuring openbaar. Dit is bewys deur hul toenemende en volgehoue gekondisioneerde vrees respons en toenemende angsgewyse simptome. Hierdie effekte is weerspreek deur die intrahippokampus infusies van DCS, soos geassesseer deur die vriesgedrag van die diere. Tweedens is ‘n opgradering van HC NMDAR in vrees gekondisioneerde rotte waargeneem, terwyl herhaalde toediening van intrahippokampus DCS hierdie effek verlaag het. Derdens verbeter intrahippokampus DCS infusies die dorsal hippokampus BDNF uitdrukking in DCS behandelde groepe, met vrees gekondisioneerde rotte wat die laagste BDNF vlakke toon. Vierdens het intrahippokampus DCS toediening wat vreesuitwissing betref, soortgelyke patrone in adollessente en volwassenes meegebring; dit is ‘n afnemende vreesrespons wat in beide ouderdomsgropee na DCS toediening waargeneem is. Laastens het ons waargeneem dat die HC proteïene uitdrukking tussen adollessente en volwasse rotte verskil. Die meeste proteïene was onderskeidelik uitgedruk in enigeen van die twee ouderdomsgroepe. ‘n Unieke proteien, naamlik neurabin-2 was spesifiek uitgedruk gedurende die adollessente tydperk. Gevolglik het voetskok gelei tot ‘n toename in adollessente proteïene uitdrukking, teenoor DCS behandeling wat ‘n afname in adollessente proteïene uitdrukking tot gevolg gehad het. In oorsig, ondersteun hierdie studie ons alternatiewe hipoteses en uitgebreide vroeere bevindings dat DCS ‘n terapeutiese uitwerking op vreeskondisionering het en DCS verbeter die uitwissing van angsgewyse simptome in knaagdiere. Ons was in staat om te bewys dat diere wat aan vreeskondisionering/trauma onderwerp was, tekens getoon het van verandering in proteïene betrokke in neuronale plastisiteit, kalsium homeostase, sellulere stress, selsiklus arrestasie, aanvang van apoptotiese meganismes en sellulere sein disregulasie. Hierdie proteïene het almal ‘n rol gespeel in een of meer van die neurochemiese parameters so in ons PTSV model ondersoek is; genaamd die interaskie van die HC, BDNF, nNOS en NMDAR. Derhalwe is verdere addisionele studies nodig om die verwantskap tussen epigenetiese modifikasies en die gevolglike proteomiese response, soos in ons studie gedemonstreer, uit te brei. Dit is ook nodig dat die rol van BDNF in PTSV verder ondersoek moet word, hetsy as ‘n bio-merker of as bykomende terapie vir PTSV.

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