Expression of functional GABAc receptors in the brain

γ-aminobutyric acid (GABA) is the main inhibitory transmitter in the nervous system and acts via three distinct receptor classes: A, B, and C. GABAC receptors are ionotropic receptors comprising ρ subunits. In this work, we aimed to elucidate the expression of ρ subunits in the postnatal brain, the characteristics of ρ2 homo-oligomeric receptors, and the function of GABAC receptors in the hippocampus. In situ hybridization on rat brain slices showed ρ2 mRNA expression from the newborn in the superficial grey layer of the superior colliculus, from the first postnatal week in the hippocampal CA1 region and the pretectal nucleus of the optic tract, and in the adult dorsal lateral geniculate nucleus. Quantitative RT-PCR revealed expression of all three ρ subunits in the hippocampus and superior colliculus from the first postnatal day. In the hippocampus, ρ2 mRNA expression clearly dominated over ρ1 and ρ3. GABAC receptor protein expression was confirmed in the adult hippocampus, superior colliculus, and dorsal lateral geniculate nucleus by immunohistochemistry. From the selective distribution of ρ subunits, GABAC receptors may be hypothesized to be specifically involved in aspects of visual image motion processing in the rat brain. Although previous data had indicated a much higher expression level for ρ2 subunit transcripts than for ρ1 or ρ3 in the brain, previous work done on Xenopus oocytes had suggested that rat ρ2 subunits do not form functional homo-oligomeric GABAC receptors but need ρ1 or ρ3 subunits to form hetero-oligomers. Our results demonstrated, for the first time, that HEK 293 cells transfected with ρ2 cDNA displayed currents in whole-cell patch-clamp recordings. Homomeric rat ρ2 receptors had a decreased sensitivity to, but a high affinity for picrotoxin and a marked sensitivity to the GABAC receptor agonist CACA. Our results suggest that ρ2 subunits may contribute to brain function, also in areas not expressing other ρ subunits. Using extracellular electrophysiological recordings, we aimed to study the effects of the GABAC receptor agonists and antagonists on responses of the hippocampal neurons to electrical stimulation. Activation of GABAC receptors with CACA suppressed postsynaptic excitability and the GABAC receptor antagonist TPMPA inhibited the effects of CACA. Next, we aimed to display the activation of the GABAC receptors by synaptically released GABA using intracellular recordings. GABA-mediated long-lasting depolarizing responses evoked by high-frequency stimulation were prolonged by TPMPA. For weaker stimulation, the effect of TPMPA was enhanced after GABA uptake was inhibited. Our data demonstrate that GABAC receptors can be activated by endogenous synaptic transmitter release following strong stimulation or under conditions of reduced GABA uptake. The lack of GABAC receptor activation by less intensive stimulation under control conditions suggests that these receptors are extrasynaptic and activated via spillover of synaptically released GABA. Taken together with the restricted expression pattern of GABAC receptors in the brain and their distinctive pharmacological and biophysical properties, our findings supporting extrasynaptic localization of these receptors raise interesting possibilities for novel pharmacological therapies in the treatment of, for example, epilepsy and sleep disorders.Gamma-aminovoihappo (GABA) on keskushermoston tärkein ehkäisevä välittäjäaine. GABA-reseptorit jaetaan rakenteen ja farmakologisten ominaisuuksien perusteella kolmeen ryhmään. GABAA- ja GABAC-reseptorit ovat ionotrooppisia kloridikanavareseptoreita, kun taas GABAB-reseptorit ovat metabotrooppisia. Ionotrooppiset GABA-reseptorit koostuvat viidestä alayksiköstä, joita tunnetaan GABAC-reseptoreille kolme erilaista: ρ1, ρ2 ja ρ3. Väitöskirjatyön tarkoituksena oli selvittää GABAC-reseptoreiden ilmentymistä ja toimintaa keskushermostossa, erityisesti hippokampuksessa. Ensimmäisessä osatyössä GABAC-reseptoreiden ρ-alayksikköjä löytyi varhaisemmassa yksilönkehityksen vaiheessa ja useammalta keskushermoston alueelta kuin aiemmin oli havaittu. In situ -hybridisaatiomenetelmällä havaittiin ρ2-alayksikön lähetti-RNA:ta verkkokalvon, hippokampuksen, ylemmän nelikukkulalevyn ja muiden näköjärjestelmän tumakkeiden lisäksi myös näköaivokuorelta kolmesta eri kerroksesta. Kvantitatiivisella RT-PCR-menetelmällä todettiin kaikkien kolmen alayksikön lähetti-RNA:ta hippokampuksesta jo vastasyntyneeltä. Immunosytokemiallisilla vasta-ainevärjäyksillä voitiin vahvistaa, että ρ-proteiinia löytyi kaikilta niiltä aivoalueilta, joilla oli todettu ρ2-alayksikön mRNA:ta. Toisessa osatyössä pystyttiin ensimmäistä kertaa ekspressoimaan toimivia rotan ρ2-alayksikön muodostamia homomeerisiä reseptoreita HEK-293-soluissa. Ne olivat herkempiä sekä GABAlle että pelkästään GABAC-reseptoreihin vaikuttavalle CACAlle kuin ρ1ρ2-heteromeerit, mutta huomattavan epäherkkiä pikrotoksiinille, joka pystyi suurillakin pitoisuuksilla aiheuttamaan vain osittaisen antagonismin. Toisaalta kuitenkin tällä homomeerillä oli hyvin korkea-affiininen pikrotoksiinin sitoutumiskohta. Kolmannessa osatyössä voitiin osoittaa, että hippokampuksen CA1-alueen pyramidaalisoluissa on toimivia GABAC-reseptoreita. Niiden aktivaatio hyvin pienellä, toisen osatyön perusteella ρ2-alayksikköspesifisellä CACA-pitoisuudella pienensi hermosoluryhmän vasteita. Hyvin voimakas stimulaatiosarja aktivoi sekä GABAA- että GABAC-reseptorit CA1-alueella ja saadaan kaksiosainen vaste, joka koostuu tavanomaisesta hyperpolarisaatiosta ja pitkäkestoisesta depolarisaatiosta. GABAC-reseptoriantagonisti TPMPA sai aikaan oleellisia muutoksia erityisesti depolarisaatiovaiheen kestossa. Samantyyppinen vaste TPMPA:lle saatiin myös ilman erittäin voimakasta stimulaatiota silloin, kun välittäjäaineen poisto synapsiraosta oli estetty ja GABAa diffundoitui enemmän synapsiraon ulkopuolelle. Tulokset tukevat hypoteesia, että hippokampuksen GABAC-reseptorit saattavat olla ekstrasynaptisia eli sijaita välittömästi synapsin ulkopuolella ja aktivoitua silloin, kun välittäjäainetta läikkyy yli synapsiraosta. Ekstrasynaptisia GABAA-reseptoreja on aiemmin löydetty useilta alueilta, myös hippokampuksesta. GABAC-reseptorit ja nämä δ-alayksikön sisältävät ekstrasynaptiset GABAA-reseptorit muistuttavat toisiaan erityisesti herkkyydessään GABAlle ja desensitisoitumattomuudessaan. Väitöskirjatyön perusteella voidaan yhteenvetona olettaa, että GABAC-reseptorit osallistuvat liikkuvan kuvan prosessointiin aivoissa ja että hippokampuksen GABAC-reseptorit ovat ekstrasynaptisia ja optimoivat hippokampuksen tietojenkäsittelykapasiteettia läikkymisinhibition avulla. Hippokampuksessa GABAC-reseptoreiden aktivaatio pystyi hillitsemään epileptistyyppisiä, patologisen voimakkaita neuronipopulaation vasteita, jotka GABAA-reseptorien estäminen bikukulliinilla oli saanut aikaan. Kun lisäksi huomioidaan GABAC-reseptoreiden sijainti vain osassa keskushermostoa ja niiden erityinen farmakologia ja kinetiikka, väitöskirjatyön löydökset voivat tulevaisuudessa olla tärkeitä kehitettäessä uusia lääkkeitä toisaalta epilepsiaan, toisaalta unihäiriöihin, erityisesti narkolepsiaan ja unettomuuteen

Ullanlinna Narcolepsy Scale in diagnosis of narcolepsy

Study objectives To validate Ullanlinna Narcolepsy Scale (UNS) as a screening tool for narcolepsy in a clinical population and to compare it with Swiss Narcolepsy Scale (SNS) and Epworth Sleepiness Scale (ESS). Methods UNS questionnaires of 267 participants visiting Helsinki Sleep Clinic were analyzed. The diagnoses of the participants were narcolepsy type 1 (NT1, n = 89), narcolepsy type 2 (NT2, n = 10), other hypersomnias (n = 24), sleep apnea (n = 37), restless legs syndrome or periodic limb movement disorder (n = 56), and other sleep-related disorders (n = 51). In addition, ESS and SNS scores in a subset of sample (total N = 167) were analyzed and compared to UNS. Results Mean UNS score in NT1 was 22.0 (95% confidence interval [CI] = 20.4 to 23.6, range 9-43), which was significantly higher than in other disorders, including NT2 (mean 13.7, 95% CI = 10.3 to 17.1, range 7-21, p = .0013). Sensitivity and specificity of UNS in separating NT1 from other disorders were 83.5% and 84.1%, respectively. Positive and negative predictive values were 82.5% and 85.1%, respectively. Sensitivities of SNS and ESS in NT1 were 77.2% and 88.6%, and specificities 88.6% and 45.5%, respectively. There were no differences in receiver operating characteristic curves between UNS and SNS. UNS had moderate negative correlation with hypocretin-1 levels (r(s) = -.564, p <.001), and mean sleep latency in multiple sleep latency test (r(s)= -.608, p <.001). Conclusions UNS has high specificity and sensitivity for NT1 in a sleep clinic setting. UNS scores below 9 strongly suggest against the diagnosis of narcolepsy.Peer reviewe

Heart Rate Variability in Head-Up Tilt Tests in Adolescent Postural Tachycardia Syndrome Patients

Introduction:Postural tachycardia syndrome (POTS) is a suspected dysautonomia with symptoms of orthostatic intolerance and abnormally increased heart rate while standing. We aimed to study cardiac autonomic nervous system functioning in head-up tilt (HUT) in adolescents with POTS to find out if parasympathetic tone is attenuated in the upright position. Methods:We compared characteristics of a group of 25 (females 14/25; 56%) adolescents with POTS and 12 (females 4/12; 34%) without POTS aged 9-17 years. We compared heart rate variability with high- and low-frequency oscillations, and their temporal changes in HUT. Results:The high-frequency oscillations, i.e., HF, attenuated in both groups during HUT (p<0.05), but the attenuation was bigger in POTS (p= 0.04). In the beginning of HUT, low-frequency oscillations, i.e., LF, increased more in POTS (p= 0.01), but in the end of HUT, an attenuation in LF was seen in the POTS group (p<0.05), but not in the subjects without POTS. There were no associations of previous infections or vaccinations with POTS. Subjects with POTS were sleepier and their overall quality of life was very low. Conclusion:The results imply to an impaired autonomic regulation while standing in POTS, presenting as a lower HF and higher LF in the beginning of HUT and an attenuated LF in the prolonged standing position.Peer reviewe

Accuracy of Actigraphy Compared to Concomitant Ambulatory Polysomnography in Narcolepsy and Other Sleep Disorders

Actigraphy provides longitudinal sleep data over multiple nights. It is a less expensive and less cumbersome method for measuring sleep than polysomnography. Studies assessing accuracy of actigraphy compared to ambulatory polysomnography in different sleep-disordered patients are rare. We aimed to compare the concordance between these methods in clinical setting. We included 290 clinical measurements of 281 sleep laboratory patients (mean age 37.9 years, 182 female). Concomitant ambulatory polysomnography and actigraphy were analyzed to determine the agreement in patients with obstructive sleep apnea, narcolepsy, periodic leg movement disorder, hypersomnia, other rarer sleep disorders, or no organic sleep disorder. Bland-Altman plots showed excellent accuracy, but poor precision in single night results between the two methods in the measurement of sleep time, sleep efficiency, and sleep latency. On average, actigraphy tended to overestimate sleep time by a negligible amount, -0.13 min, 95% confidence interval [-5.9, 5.6] min in the whole sample. Overestimation was largest, -12.8 [-25.1, -0.9] min, in patients with obstructive sleep apnea. By contrast, in patients with narcolepsy, actigraphy tended to underestimate sleep time by 24.3 [12.4, 36.1] min. As for sleep efficiency, actigraphy underestimated it by 0.18 [-0.99, 1.35] % and sleep latency by 11.0 [8.5, 13.6] min compared to polysomnography. We conclude that, in measuring sleep time, actigraphy is reasonably reliable and helpful to be used for a week or two to exclude insufficient sleep in patients with the suspicion of narcolepsy. However, the effectiveness of actigraphy in determining sleep seems to decrease in subjects with low sleep efficiencies.Peer reviewe

Narcolepsy Associated with Pandemrix Vaccine

After the connection between AS03-adjuvanted pandemic H1N1 vaccine Pandemrix and narcolepsy was recognized in 2010, research on narcolepsy has been more intensive than ever before. The purpose of this review is to provide the reader with current concepts and recent findings on the Pandemrix-associated narcolepsy. After the Pandemrix vaccination campaign in 2009-2010, the risk of narcolepsy was increased 5- to 14-fold in children and adolescents and 2- to 7-fold in adults. According to observational studies, the risk of narcolepsy was elevated for 2 years after the Pandemrix vaccination. Some confounding factors and potential diagnostic biases may influence the observed narcolepsy risk in some studies, but it is unlikely that they would explain the clearly increased incidence in all the countries where Pandemrix was used. An increased risk of narcolepsy after natural H1N1 infection was reported from China, where pandemic influenza vaccination was not used. There is more and more evidence that narcolepsy is an autoimmune disease. All Pandemrix-associated narcolepsy cases have been positive for HLA class II DQB1*06:02 and novel predisposing genetic factors directly linking to the immune system have been identified. Even though recent studies have identified autoantibodies against multiple neuronal structures and other host proteins and peptides, no specific autoantigens that would explain the disease mechanism in narcolepsy have been identified thus far. There was a marked increase in the incidence of narcolepsy after Pandemrix vaccination, especially in adolescents, but also in young adults and younger children. All vaccine-related cases were of narcolepsy type 1 characterized by hypocretin deficiency in the central nervous system. The disease phenotype and the severity of symptoms varied considerably in children and adolescents suffering from Pandemrix-associated narcolepsy, but they were indistinguishable from the symptoms of idiopathic narcolepsy. Narcolepsy type 1 is most likely an autoimmune disease, but the mechanisms have remained elusive.Peer reviewe

Liikaunisuuden kelpo hoito

•Liikaunisuus tarkoittaa poikkeavaa päiväaikaista väsymystä, johon yhdistyy pakonomainen nukahtamistarve. •Tavallisin päiväväsymyksen syy on riittämätön tai rikkonainen ja virkistämätön yöuni. Keskushermostoperäiset liikaunisuussairaudet sen sijaan ovat harvinaisia. •Liikaunisuuden syy tulee selvittää ja hoito kohdentaa siihen aina kun mahdollista. •Lääkkeettömistä hoitomuodoista on hyötyä taustasyystä riippumatta. •Keskushermostoperäisissä liikaunisuussairauksissa vireyttä parantava lääkehoito on usein tarpeen, mutta tilanne on aina arvioitava yksilöllisesti. Lääkehoitovaihtoehtojen keskeinen ongelma on näytön puute.Peer reviewe

Narkolepsian moninainen oirekuva ja käypä diagnostiikka

English summaryPeer reviewe

Incidence of narcolepsy after H1N1 influenza and vaccinations : Systematic review and meta-analysis

An increased incidence of narcolepsy was seen in many countries after the pandemic H1N1 influenza vaccination campaign in 2009-2010. The H1N1 vaccine - narcolepsy connection is based on observational studies that are prone to various biases, e.g., confounding by H1N1 infection, and ascertainment, recall and selection biases. A direct pathogenic link has, however, remained elusive. We conducted a systematic review and meta-analysis to analyze the magnitude of H1N1 vaccination related risk and to examine if there was any association with H1N1 infection itself. We searched all articles from PubMed, Web of Science and Scopus, and other relevant sources reporting the incidence and risk of post-vaccine narcolepsy. In our paper, we show that the risk appears to be limited to only one vaccine (Pandemrix (R)). During the first year after vaccination, the relative risk of narcolepsy was increased 5 to 14-fold in children and adolescents and 2 to 7-fold in adults. The vaccine attributable risk in children and adolescents was around 1 per 18,400 vaccine doses. Studies from Finland and Sweden also appear to demonstrate an extended risk of narcolepsy into the second year following vaccination, but such conclusions should be interpreted with a word of caution due to possible biases. Benefits of immunization outweigh the risk of vaccination-associated narcolepsy, which remains a rare disease. (C) 2017 Elsevier Ltd. All rights reserved.Peer reviewe

Misdiagnosis of narcolepsy caused by a false-positive orexin-A/hypocretin-1 enzyme immune assay

The diagnosis of narcolepsy is based on clinical history, sleep studies, and, in some cases, cerebrospinal fluid orexin-A/hypocretin-1 measurement. The gold standard for orexin measurement is the radioimmunoassay but other commercial kits are also available, such as the enzyme immune assay (EIA). The specificity of orexin EIA in humans is unknown. We report four cases where orexin levels were measured by EIA and resulted in false positives and the misdiagnosis of narcolepsy. Therefore, orexin EIA measurement should be strongly discouraged in a clinical setting. CITATION: Sarkanen T, Sved G, Juujärvi M, Alakuijala A, Partinen M. Misdiagnosis of narcolepsy caused by a false-positive orexin-A/hypocretin-1 enzyme immune assay. J Clin Sleep Med. 2022;18(8):2075-2078.acceptedVersionPeer reviewe

Chronotype as self-regulation: morning preference is associated with better working memory strategy independent of sleep

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