Melanopsin (Opn4) is the opsin photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs). It has a conserved opsin structure and activation mechanism, yet demonstrates unusual functional properties that suggest it will possess unique structure-function relationships. The aim of this thesis was to characterise key OPN4 residues by examining the impact of non-synonymous mutations on melanopsin function. A genotype-driven screen of a chemically-mutagenized mouse archive led to the identification of a novel Opn4 mutant, S310A, located at a known opsin spectral tuning site. Action spectra from ipRGC and pupil light responses (PLR) of Opn4^S310A mice revealed no change in wavelength of peak sensitivity. However, Opn4^S310A PLR was significantly less sensitive at longer wavelengths, consistent with a short-wavelength shift in spectral sensitivity. This suggests S310A acts as a spectral tuning site in melanopsin. Next, the impact of naturally-occurring missense variants in human melanopsin (hOPN4) was examined in vitro. Fluorescent calcium imaging of 16 hOPN4 variants expressed in HEK293 cells revealed four hOPN4 variants abolished or attenuated responses to light (Y146C, R168C, G208S and S308F). These variants were located in conserved opsin motifs for chromophore binding or hydrogen-bond networks, functional roles apparently shared by melanopsin. Finally, two hOPN4 single nucleotide polymorphisms (SNPs) P10L and T394I, associated with abnormal non-image forming behaviour in humans, were explored in vivo. Using targeted viral-delivery of hOPN4 SNPs to mouse ipRGCs, a range of OPN4-driven behaviours, such as circadian photoentrainment and pupil light responses, were found to be comparable with hOPN4 WT control. Multi-electrode array recordings of ipRGCs transduced with hOPN4 T394I virus had significantly attenuated sensitivity and faster response offset, indicating this site may be functionally important for melanopsin activity but compensatory rod and cone input limits changes to non-image forming behaviour.

Parkinson’s Disease affects 10 million people worldwide, with 40% of patients developing an associated psychosis which has been identified by studies as the number one source of caretaker distress and is related to increased mortality. This is further complicated by the fact that typical antipsychotic drugs worsen many of the motor symptoms implicated in Parkinson’s Disease, with only one commercially available drug able to ameliorate both symptoms. This problem ushers the development of novel drugs to treat these symptoms, as first tested on research animals. Complicating matters, drug effectiveness on the degree of psychosis is hard to obtain in animals without a reliable biomarker. However, a hallmark of psychotic states is thought to be the reduced coordination between brain structures, through neuronal synchronization, as demonstrated by steady-state responses and is suggested to be a potential biomarker of psychosis. By building a MATLAB software we were able to analyze the degree of neural synchronization between structures, during an auditory steady-state response, in rats that had been unilaterally lesioned by the 6-Hydroxydopamine model of Parkinson’s Disease, before and after administration of the psychotomimetic drug MK801. These rats had been chronically implanted with 128-channel multi electrode array, enabling us to measure the strength of coherence between several limbic structures, associated with auditory processing, from the sampled local field potential, identifying the degree of synchronization in the animal brain. As our data demonstrate that coherence levels dropped in the psychotic drug state, for structures in both the healthy and the Parkinsonian hemisphere, we are able to further demonstrate the validity of coherence measures as a biomarker for psychosis. These results demonstrate that our software can be used as a tool to assess the therapeutic response of drugs developed, aimed at treating Parkinson’s associated psychosis. / Parkinsons sjukdom drabbar 10 miljoner världen över, där 40% av patienterna utvecklar en associerad psykos vilket har visats vara en av de största stressfaktorerna för deras vårdgivare och är även förknippat med en högre dödlighetsgrad. Denna situation förvärras av det faktum att de vanliga antipsykotiska drogerna kan förvärra många av de motoriska symptom som utgörs av Parkinsons sjukdom och det finns i dagsläget enbart en enda kommersiell drog som kan dämpa bägge symptom samtidigt. Detta problem frammanar vidare utveckling av nya läkemedel som kan behandla dessa symptom, som innebär att de först måste testas på försöksdjur. En komplikation som uppstår i relation till detta är svårigheten att utvärdera om läkemedel har någon terapeutisk effekt på de psykotiska tillstånden, enbart genom att observera försöksdjurens beteenden, och en pålitlig biomarkör krävs istället. En lösning kan dock finnas i det faktum att psykotiska tillstånd karaktäriseras av en reducerad förmåga för olika hjärnområden att koordinera genom neural synkronisering vilket demonstreras av ‘steady- state’ responser. Detta föreslår att ett mått på graden av koordineringsförmåga kan agera som en möjlig biomarkör för psykotiska tillstånd. Genom att konstruera ett MATLAB-program kunde vi analysera graden av synkronicitet mellan hjärnstrukturer, under den auditiva steady- state responsen i råttor som hade blivit ensidigt lesionerade genom 6-Hydroxiddopamin modellen av Parkinsons sjukdom, före och efter administration av den psykotomimetiska drogen MK801. Dessa råttor hade blivit kroniskt implanterade med 128 elektroder vilket möjliggjorde att vi kunde mäta styrkan i koherens i den lokala fält potentialen mellan limbiska strukturer, som är associerade med auditiv processering, vilket möjliggjorde identifiering av3dessa strukturers synkronicitet. Vår data demonstrerar att koherensen minskade under det psykotiska drogtillståndet för limbiska strukturer både i den intakta och den lesionerade hjärnhalvan. Detta är en vidare demonstration av att koherensnivåer kan agera som en biomarkör för det psykotiska tillståndet, liksom att vår mjukvara kan nyttjas som ett verktyg för att utvärdera nya läkemedels behandlingsförmåga på Parkinsons psykos.