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Urogvanilin (UGN) pripada porodici gvanilinskih peptida (GP) koji u probavnom sustavu reguliraju transport elektrolita i vode aktivacijom gvanilat-ciklaze C, do danas jedinog poznatog receptora za GP-e. Ovim radom pokazujemo izražaj UGN-a na mRNA i proteinskoj razini u pojedinim regijama mozga koji je ovisan o hranjenju iako se do sada smatralo da se UGN ne stvara u mozgu. Uz do sada poznati izražaj gvanilat-ciklaze C (GC-C) u srednjem mozgu i hipotalamusu, otkrili smo nova mjesta izražaja GC-C u neuronima (korteks velikog mozga, Purkinjeove stanice i duboke jezgre malog mozga), ali ne i u astrocitima. UGN hiperpolarizira staničnu membranu Purkinjeovih stanica i smanjuje frekvenciju izbijanja dok u astrocitima dovodi do povećanja unutarstanične koncentracije Ca2+ putem signalnog puta neovisnog o GC-C/cGMP-u. Učinak UGN-a u hipotalamusu na termogenezu uzrokovanu hranom te aktivaciju smeđeg masnog tkiva (BAT) nakon obroka postoji kod mužjaka, ali ne i ženki divljeg tipa životinja ni kod oba spola životinja kojima nedostaje GC-C. Istraživanje je provedeno i na zdravim dobrovoljcima i pokazuje kako je aktivacija BAT-a nakon obroka ovisna o spolu, dobi i indeksu tjelesne mase. Rad ukazuje na ulogu UGN-a u regulaciji BAT-a koja je od neizmjerne važnosti za razvoj novih terapijskih pristupa u terapiji pretilosti, metaboličkog sindroma i šećerne bolesti., Uroguanylin (UGN) belongs to the family of guanylin peptides. They regulate electrolyte and water transport in intestine via guanylate cyclase C, the only known receptor for guanylin peptides. In this thesis we are showing expression of UGN in different brain regions which is regulated by feeding, although it was believed that brain is not expressing UGN. Guanylate cyclase C (GC-C) is expressed in midbrain and hypothalamus, while we discovered additional brain regions expressing GC-C in neurons (cerebral cortex, Purkinje cells and deep nuclei of the cerebellum) but not in astrocytes. UGN hyperpolarized Purkinje cells and decreased firing rate while in astrocytes lead to increase in intracellular Ca2+ concentration via GC-C/cGMP independent signalling pathway. Effects of UGN in hypothalamus on diet-induced thermogenesis and brown adipose tissue (BAT) activation after a meal exist in male but not female WT animals and GC-C KO animals of both genders. Similar research was performed on healthy volunteers which showed that activation of BAT after a meal is gender, age, and body mass index dependent. Role of UGN in BAT regulations is of immeasurable importance for development of the new therapeutic approaches in treatment of obesity, metabolic syndrome, and diabetes mellitus.

Activation of guanylate cyclase (GC) A after ischemic stroke is neuroprotective. The aim is to investigate the effect of GC-C activation by uroguanylin (UGN) on ischemic stroke. MCAO was performed on GC-C knock out (GC-C KO), UGN KO mice and their WT littermates. MR images were acquired before and 24h after stroke. Ca2+ response was recorded in astrocytes 48h after MCAO. Systolic (SP), diastolic (DP) and mean arterial blood pressure (MAP) were recorded. GC-C KO develop significantly smaller ischemic lesions. Though they have higher SP, DP and MAP, blood flow or its reduction during MCAO did not differ between KO and WT. Unlike GC-C KO, UGN KO and WT have stronger Ca2+ response to UGN stimulation in peri- ischemic astrocytes and this difference may result in smaller lesions in GC-C KO. Stronger activation was explained when immunohistochemical staining showed GC-C expression in peri-ischemic astrocytes, while under normoxic conditions it is limited to neurons. GC-C activation leads to development of larger lesions. Its expression in peri-ischemic astrocytes causes stronger activation of the Ca2+-dependent signalling pathway which could stimulate the Na+/H+ exchanger causing tissue acidification and neuronal death. ACKNOWLEDGEMENTS: Research was funded by the Scientific Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic- ischemic damage in perinatal and adult brain” ; GAKK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund), “Young researchers' career development project – training of doctoral students” and BRADISCHEMIA (UIP-2017-05-8082) of Croatian Science Foundation funded by European Union’s European Social Fund.

INTRODUCTION: Stroke is one of the leading causes of mortality and disability in industrialized countries. Guanylate cyclase (GC) A activation after ischemic stroke is neuroprotective1. The aim of this study is to investigate what effect GC-C activation by uroguanylin (UGN) has on ischemic stroke. MATERIALS AND METHODS: Middle cerebral artery occlusion (MCAO) was performed on GC-C knock out (GC-C KO), UGN KO mice and their WT littermates. MR images were acquired before and 24h after stroke induction. Ca2+ response was recorded in astrocytes of peri-ischemic and contralateral cortices 48h after MCAO. Systolic (SP), diastolic (DP) and mean arterial blood pressure (MAP), heart rate, tail blood volume and flow were recorded with a non-invasive tail-cuff method. RESULTS: Animals lacking GC-C develop significantly smaller ischemic lesions compared to their WT littermates. Though GC-C KO mice have higher SP, DP and MAP, blood flow or the reduction of blood flow during MCAO did not differ between KO and WT animals. UGN KO and WT mice have a stronger Ca2+ response to UGN stimulation in cortical peri-ischemic astrocytes compared to the healthy hemisphere while this stronger activation is absent in GC-C KO mice. The observed difference in response may result in smaller ischemic lesions observed in GC-C KO mice. This was explained when immunohistochemical staining showed GC-C expression in peri-ischemic astrocytes, while under normoxic conditions GC-C expression is present only in neurons2. CONCLUSION: Contrary to GC-A, GC-C activation is not neuroprotective but instead leads to the development of larger ischemic lesions. Its expression in peri-ischemic astrocytes causes a stronger activation of the Ca2+-dependent signalling pathway which could stimulate the Na+/H+ exchanger causing tissue acidification and neuronal death. ACKNOWLEDGEMENTS: Research was funded by the Scientific Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic- ischemic damage in perinatal and adult brain” ; GA KK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund). The work of doctoral student Anja Barić has been fully supported by the “Young researchers' career development project – training of doctoral students” and project BRADISCHEMIA (UIP-2017-05- 8082) of the Croatian Science Foundation funded by the European Union from the European Social Fund.

INTRODUCTION: Stroke is one of the leading causes of mortality and disability the world. Guanylate cyclase (GC) A activation after ischemic stroke is neuroprotective. The aim of this study is to investigate what effect GC-C activation by uroguanylin (UGN) has on ischemic stroke. MATERIALS AND METHODS: Middle cerebral artery occlusion (MCAO) was performed on GC-C knock out (GC-C KO), UGN KO mice and their WT littermates. MR images were acquired before and 24h after stroke induction. Ca2+ response was recorded in astrocytes of peri-ischemic and contralateral cortices 48h after MCAO. Systolic (SP), diastolic (DP) and mean arterial blood pressure (MAP) were recorded with a non-invasive tail-cuff method. Immunohistochemical staining and in situ hybridization were used to confirm GC-C expression in neurons and astrocytes before any after stroke induction. RESULTS: GC-C KO mice develop 35% smaller ischemic lesions compared with their WT littermates. Although higher SP, DP and MAP were recorded in GC-C KO animals when compared with WT mice, blood flow or the reduction of blood flow during MCAO did not differ between those test groups. A stronger Ca2+ response to UGN stimulation was recorded in in cortical peri-ischemic astrocytes of UGN KO and WT mice but this stronger activation is absent in GC-C KO mice. The observed difference in response may result in smaller ischemic lesions observed in GC-C KO mice. This difference in Ca2+ response could be explained by the shift in GC-C expression that occurs after stroke – under normoxic conditions GC-C is expressed only in neurons, but after ischemic injury GC-C becomes strongly expressed in peri-ischemic astrocytes and is rarely found in neurons. CONCLUSION: Contrary to GC-A, GC-C activation is not neuroprotective but instead leads to the development of larger ischemic lesions. Its expression in peri-ischemic astrocytes causes a stronger activation of the Ca2+-dependent signalling pathway which likely stimulates the Na+/H+ exchanger, leading to tissue acidification and neuronal death. ACKNOWLEDGEMENTS: Research was funded by the Scientific Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic- ischemic damage in perinatal and adult brain” ; GA KK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund). The work of doctoral student Anja Barić has been fully supported by the “Young researchers' career development project – training of doctoral students” and project BRADISCHEMIA (UIP-2017-05- 8082) of the Croatian Science Foundation funded by the European Union from the European Social Fund.

Stroke is one of the leading causes of mortality and disability in industrialized countries. Since guanylate cyclase (GC) A activation has a neuroprotective effect after ischemic stroke, the aim of this study is to determine if uroguanylin (UGN), an agonist of GC-C, has a similar effect. In this study, middle cerebral artery occlusion (MCAO) was performed on GC-C KO and UGN KO mice and their WT littermates. Before and 24h after MCAO MR images were taken. 48h following MCAO brain slices were isolated and Ca2+ response to UGN stimulation was recorded. Immunohistochemical staining was performed with GC-C, NeuN, and GFAP antibodies. Both groups of WT animals and UGN KO animals exhibit a stronger Ca2+ response to UGN stimulation in astrocytes of the ischemic penumbra in cerebral cortex but not in the unaffected hemisphere. This stronger activation is gone in GC-C KO animals which might lead to development of smaller ischemic lesions in GC-C KO mice compared to their WT littermates (GC-C WT: 111.3±13.8 mm3, n = 5 ; GC-C KO: 71.5±10.6 mm3, n=6, p=0.039). Although mean arterial pressure was significantly higher in GC-C KO mice compared to their WT littermates (GC-C WT: 68.5±1.8 mmHg, GC-C KO: 80.6±3.5 mmHg, n=6, p=0.011), the blood flow measured before and during MCAO did not differ significantly (Flow before MCAO: GC-C WT: 310±8 LDFU (laser Doppler flux unit), n=5 ; GC-C KO: 336±11 LDFU, n=6, p=0.07 ; Flow during MCAO: GC-C WT: 70±6 LDFU, n=5 ; GC-C KO: 70±4 LDFU, n=6, p=0.999), suggesting no effects of the blood pressure on the blood flow during MCAO or size of the stroke lesion. Since GC-C becomes expressed on penumbral astrocytes following ischemia, while in normoxic conditions it is expressed only in cortical neurons, effects of GC-C on intracellular Ca2+ concentration could be due to activation of cGMP-dependent Ca2+ channels in penumbral astrocytes. Stronger activation of the Ca2+- dependent signaling pathway could lead to the development of larger ischemic lesions in GC-C WT compared to GC-C KO animals, possibly through upregulation of Na+/H+ exchanger followed by tissue acidification and neuronal death. Research was funded by the Scientific Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic-ischemic damage in perinatal and adult brain” ; GA KK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund). The work of doctoral student Anja Barić has been fully supported by the “Young researchers' career development project – training of doctoral students” and project BRADISCHEMIA (UIP-2017-05-8082) of the Croatian Science Foundation funded by the European Union from the European Social Fund.

The interface of excitable cells and stimulation or recording electrodes is essential for bioelectronic applications. Parameters such as the electrode impedance and capacitance, interface electrochemistry, surface structuring and long term in vivo stability have been thoroughly studied. However, in many applications, especially clinical ones, only trivial electrode geometries are used, resulting in increased charge density thresholds. Using optimized electrode geometries and stimulation protocols may overall be more effective, especially in the case of implanted bioelectronic devices with limited current generation capabilities. For highly localized target specific electrostimulation, electrode design and stimulation protocol are crucial parameters to consider. We consider multiple planar and 3D electrode configurations for stimulating excitable cells and tissues, and different stimulation protocols using pulsed and modulated current. A comprehensive finite element method (FEM) model encompassing realistic capacitive photo-electrode (organic electrolytic photocapacitor – OEPC) and tissue model is made in COMSOL Multiphysics® software. Electrodes are characterized by their contact electrical properties, contact capacitance and contact resistance, while the OEPC is characterized by its equivalent circuit model. Realistic cell membranes and action potential propagation are implemented using the Hodgkin–Huxley model which can be tailored to a specific cell type. We show that using the optimized electrode configuration enables multifold current density enhancement at the targeted stimulation area which enables effective cell and tissue excitation while minimising the residual effect on the surrounding tissue. Our numerical findings are validated in vitro using cortical neuron cell cultures and mouse brain slices.

Schizophrenia is one of the most wide-spread mental brain disorders with complex and largely unknown etiology. To characterize the impact of schizophrenia at a cellular level, we performed single nucleus RNA sequencing of gt;190,000 neurons from the dorsolateral prefrontal cortex of patients with schizophrenia and matched controls (7 vs 11, respectively). In addition, to correlate data with cortical anatomy, gt;100,000 neurons were analyzed topographically by immunohistochemistry in an extended cohort of cases with schizophrenia and controls (10 vs 10). Compositional analysis of RNA sequencing data revealed reduction in relative abundance across all families of GABAergic neurons and a concomitant increase in principal neurons, which was most pronounced for supragranular subtypes (layers 2-3). Moreover, supragranular subtypes of GABAergic interneurons showed most dramatic transcriptomic changes. These results were substantiated by histological analysis, which revealed a reduction in the density of calretinin, calbindin and parvalbumin GABAergic interneurons particularly in layer 2. Common effect of schizophrenia on supragranular neuronal networks was underlined by downregulation of protein processing genes and upregulation of neuronal development/plasticity genes across supragranular subtypes of principal neurons and GABAergic interneurons. In situ hybridization and spatial transcriptomics further confirmed supragranular layer neuron vulnerability, revealing complexity of schizophrenia-affected cortical circuits. These point towards general network impairment within supragranular layers being a core substrate associated with schizophrenia symptomatology.Competing Interest StatementThe authors have declared no competing interest.

Guanylin peptides (GPs) and their receptor, guanylate cyclase C (GC-C), have recently become a topic of great interest in metabolic research. Guanylin and uroguanylin are the most investigated GPs and they belong to a larger family of natriuretic peptides. GPs play a physiological role in regulation of electrolyte balance via the intestine and the kidney by regulating the energy balance via their action in the brain. In addition to well-known cGMP signaling pathway, GPs activate a GC-C independent signaling pathway in the intestine, kidneys, as well as the brain. Even though the existence of two separate signaling pathways for other natriuretic peptides is well investigated, the GC-C independent signaling pathway is still a mystery. In this review, we summarize the recent discoveries related to the actions of GPs with special attention to the GC-C independent signaling pathway. We also discuss the main controversies in the field. Sex differences in GPs action via GC-C dependent and independent signaling pathway could address some of the discrepancies in literature. Here, we overview the role of GPs and their signaling pathways in the most common diseases of the modern world.

Stroke is one of the leading causes of mortality and disability in industrialized countries. Guanylate cyclase (GC) A activation has a neuroprotective effect after ischemic stroke [1] therefore the aim of this study is to determine if agonists of GC-C have similar effects. Uroguanylin (UGN) activates guanylate cyclase C (GC-C) and a Ca2+-dependent signaling pathway [2]. In this study, middle cerebral artery occlusion (MCAO) was performed on wild type (WT), GC-C KO and UGN KO mice. Before and 24h after MCAO MR images were taken. 48h following MCAO brain slices were isolated and Ca2+ response to UGN stimulation was recorded. Immunohistochemical staining was performed with GC-C, NeuN, and GFAP antibodies. WT and UGN KO animals exhibit a stronger Ca2+ response to UGN stimulation in astrocytes of the ischemic penumbra in cerebral cortex but not in the unaffected hemisphere. This stronger activation is gone in GC-C KO animals which results in development of smaller ischemic le¬sions in GC-C KO mice compared to their WT littermates. Considering the fact that GC-C becomes expressed on penumbral astrocytes following ischemia, while in normoxic conditions it is expressed only in cortical neurons, effects of GC-C on intracellular Ca2+ concentration could be due to activation of cGMP-dependent Ca2+ channels in penumbral astrocytes [3]. Stronger activation of the Ca2+-dependent signaling pathway could lead to the development of larger ischemic lesions, possibly through upregula¬tion of Na+/H+ exchanger, tissue acidification, and neuronal death. [1] Dobrivojević M, Špiranec K, Gorup D, Erjavec I, Habek N, Radmilović M, Unfirer S, Ćosić A, Drenjančević I, Gajović S, Sinđić A (2016) Urodilatin reverses the detrimental influence of bradykinin in acute ischemic stroke. Exp Neurol 284:1-10. [2] Habek N, Ratko M, Dugandžić A (2021) Uroguanylin Increases Ca2+ Concentration in Astrocytes via Guanylate Cyclase C-independent Signalling Pathway. Croat Med J. In press. [3] Rappaport JA, Waldman SA (2018) The Guanylate Cyclase C-cGMP Signaling Axis Opposes Intestinal Epithe¬lial Injury and Neoplasia. Front Oncol 8:299. Funding: Research was funded by the Scientific Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic- ischemic damage in perinatal and adult brain” ; GA KK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund). The work of doctoral student Anja Barić has been fully supported by the “Young researchers’ career development project – training of doctoral students” and project BRADISCHEMIA (UIP-2017-05- 8082) of the Croatian Science Foundation funded by the European Union from the European Social Fund.

Introduction/Objectives: Stroke is one of the leading causes of mortality and disability in industrialized countries. Guanylate cyclase (GC) A activation has a neuroprotective effect after ischemic stroke. Therefore, the aim of this study is to determine if agonists of GC-C have similar effects. Uroguanylin (UGN) activates guanylate cyclase C (GC-C) and a Ca2+-dependent signaling pathway. Participants, Materials/Methods: In this study, middle cerebral artery occlusion (MCAO) was performed on wild type (WT), GC-C KO and UGN KO mice. Before and 24h after MCAO MR images were taken. 48h following MCAO brain slices were isolated and Ca2+ response to UGN stimulation was recorded. Immunohistochemical staining was performed with GC-C, NeuN, and GFAP antibodies. Results: WT and UGN KO animals exhibit a stronger Ca2+ response to UGN stimulation in astrocytes of the ischemic penumbra in cerebral cortex but not in the unaffected hemisphere. This stronger activation is gone in GC-C KO animals which results in development of smaller ischemic lesions in GC-C KO mice compared to their WT littermates. Considering the fact that GC-C becomes expressed on penumbral astrocytes following ischemia, while in normoxic conditions it is expressed only in cortical neurons, effects of GC-C on intracellular Ca2+ concentration could be due to activation of cGMP-dependent Ca2+ channels in penumbral astrocytes. Conclusions: Stronger activation of the Ca2+-dependent signaling pathway could lead to the development of larger ischemic lesions, possibly through upregulation of Na+/H+ exchanger, tissue acidification, and neuronal death.

Anxiety-like disorders are the most common mental disorders in the modern world with an incidence two times higher in women than in men. Amygdala, the brain region involved in emotional processing and fear conditioning, shows distinctive structural and physiological sexual dimorphism. Agonists of membrane-bound guanylate cyclase (GC) A and B have been shown to possess dose-dependent anxiolytic properties. Therefore, the aim of this study is to determine if activation of guanylate cyclase C (GC-C) in amygdala could affect anxiety- like behaviour differently in female than in male mice. In this study we used immunohistochemical staining in male and female wild-type (WT) animals, with GC-C knock-out animals (GC-C KO) as controls. GC-C mRNA levels in amygdala and hypothalamus were evaluated using qPCR. Anxiety levels were tested with two behavioural tests (home cage escape, elevated plus maze). Vaginal swabs were stained with 0.1% cresyl violet stain and analysed using a stereomicroscope to determine the phase of the oestrus cycle. GC-C is expressed in the neurons of basolateral nucleus and cortical area of amygdala. During the oestrous cycle, GC-C expression changes differently in amygdala compared to hypothalamus. Therefore, only female mice in diestrus showed different anxiety levels compared to male mice, which is even more pronounced in GC-C KO mice. As expected, no difference in anxiety levels between genotype was present in male animals. Female mice demonstrate different anxiety levels during the diestrus phase compared to male animals. GC-C is present in amygdala, and its inhibition during diestrus could be responsible for the difference in anxiety levels between genders and during different phases of the oestrous cycle. Our results indicate that GC-C activation may have anxiolytic properties similar to activation of other membrane-bound GCs. FUNDING: This work has been supported by Croatian Science Foundation under the project FURNACE (IP- 2018-01-7416) and co-financed by the European Union through the European Regional Development Fund, Operational Programme Competitiveness and Cohesion, grant Agreement No. KK.01.1.1.01.0007, CoRE - Neuro.

INTRODUCTION: Recent research has shown that the activation of guanylate cyclase (GC)-A, but not guanylate cyclase B (GC-B), leads to a decrease of brain lesion size following middle cerebral artery occlusion (MCAO). AIM: Our aim was to investigate the potential role of uroguanylin (UGN)-dependent signalling pathways (GC-C or Ca2+-dependent) in the development of ischemic stroke. METHODS: Lesion volumes were measured in WT, GC-C and UGN KO mice by MR imaging 1 day after MCAO. Intracellular Ca2+ concentrations were measured in slices of ipsilateral and contralateral hemispheres. RESULTS: GC-C KO animals exhibited significantly smaller lesion volumes 1 day after MCAO than WT mice. This difference was not present between UGN KO and WT littermates. The hemisphere with stroke shows a greater increase in intracellular Ca2+ concentrations following UGN stimulation compared to the healthy hemisphere of the same animal. This difference is absent in GC-C KO animals, while UGN KO animals exhibit a prolonged Ca2+ response compared to their WT littermates. CONCLUSION: The reduction in stroke volume present in GC-C KO but not UGN KO mice indicates that the activation of the Ca2+-dependent signalling pathway may have potentially harming effects. The possible mechanism could be increased UGN activity on pH regulation in astrocytes via Ca2+ signalling pathway and worsening of existing interstitial post ischemic acidosis. ACKNOWLEDGEMENTS Research was funded by the Scientific Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic-ischemic damage in perinatal and adult brain” ; GA KK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund). The work of doctoral student Anja Barić has been fully supported by the “Young researchers' career development project – training of doctoral students” and project BRADISCHEMIA (UIP-2017-05-8082) of the Croatian Science Foundation funded by the European Union from the European Social Fund.

AIM: Recent research has shown that the activation of guanylate cyclase (GC)-A leads to a decrease of brain lesion size following middle cerebral artery occlusion (MCAO). Our aim was to investigate the potential role of uroguanylin (UGN)-dependent signalling pathways in the development of ischemic stroke. METHODS: Lesion volumes were measured in WT, GC-C and UGN-KO mice by MR imaging 1 and 7 days after MCAO. Intracellular Ca2+ concentrations were measured in slices of ipsilateral and contralateral hemispheres. RESULTS: GC-C-KO animals exhibited significantly smaller lesion volumes 1 day after MCAO than WT mice, with no difference 7 days later. This difference was not present between UGN-KO and WT littermates, although there was significant difference in UGN- dependent activation of Ca2+ signalling pathway in penumbral zone of ischemic lesion. CONCLUSION: The reduction in stroke volume present in GC-C-KO but not UGN-KO mice indicates that the activation of GC-C may cause an increase in ischemic lesion volumes, while GC-C-independent (Ca2+-dependent) signalling pathway may have protective effect. ACKNOWLEDGEMENTS Research was funded by the Scientific Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic- ischemic damage in perinatal and adult brain” ; GA KK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund). The work of doctoral student Anja Barić has been fully supported by the “Young researchers' career development project – training of doctoral students” and project BRADISCHEMIA (UIP-2017-05- 8082) of the Croatian Science Foundation funded by the European Union from the European Social Fund.

INTRODUCTION Anxiety-like disorders are the most common mental disorders in the modern world with an incidence two times higher in women than men. Amygdala, the brain region involved in emotional processing and fear conditioning, shows distinctive structural and physiological sex differences. AIM Since agonists of membrane-bound guanylate cyclase (GC) A and B have been shown to possess anxiolytic properties, the aim of this study is to determine if activation of guanylate cyclase C (GC-C) in amygdala affects anxiety-like behaviour differently in female than in male mice. MATERIALS AND METHODS In this study we used immunohistochemical staining in male and female wild-type (WT) animals, with GC-C knock-out animals (GC-C KO) as controls. GC-C mRNA levels in amygdala and hypothalamus were evaluated using qPCR. Anxiety levels were tested with two behavioural tests (home cage escape, elevated plus maze). Phases of the oestrus cycle were determined by staining vaginal swabs with 0.1% cresyl violet stain and analysis using a stereomicroscope. RESULTS GC-C is expressed in the neurons of basolateral amygdaloid nucleus and the cortical amygdaloidal area. During the oestrous cycle, GC-C expression changes differently in amygdala compared to hypothalamus. Therefore, only female mice in diestrus showed different anxiety levels compared to male mice, which is even more pronounced in GC- C KO mice. As expected, no difference in anxiety levels between genotype was present in male animals. CONCLUSION Female mice demonstrate different anxiety levels during the diestrus phase compared to male animals. GC-C is present in amygdala, and its inhibition during diestrus could be responsible for the difference in anxiety levels between genders and during different phases of the estrous cycle. Our results indicate that GC-C activation may have anxiolytic properties similar to activation of other membrane-bound GCs. ACKNOWLEDGEMENTS This work has been fully supported by Croatian Science Foundation under the project FURNACE (IP- 2018-01-7416).

We aim to introduce a non-invasive perinatal mild hypoxic brain lesion in rats. Nineteen Wistar Han® (RccHan®: WIST) rats, (9 females and 10 males) were randomly divided into hypoxic and control group on postnatal day 1 (P1) when hypoxia was induced in a hypobaric chamber (Atm350mmHg, pO273mmHg, temperature ≈ 25°C) during 2 hours, while controls were kept in normal housing conditions. Behavioral tests were performed at P30 and P70 using the open field, hole board, social choice, and T-maze tests. Samples of brain tissue from adult animals (P105) were used for histochemical examination of the cytoarchitectonics (Nissl staining), interneurons (parvalbumin immunohistochemistry) and perineuronal nets (Wisteria floribunda agglutinin, histochemistry). After short-term perinatal rat brain injury, there were no disturbances in the brain macro-morphology or any other pathoanatomical consequence of the treatment. Also, treated animals had intact exploratory, anxiety-like and social behavior. Still, distinct changes in morphology, number, and distribution of the parvalbumin-immunoreactive neurons and perineuronal nets in different brain regions were observed. Moreover, treated animals displayed significantly impaired learning. In conclusion, the proposed rat model of non-invasive hypoxic brain lesion has indicated consistent disturbances in brain connectivity related to cognitive processes, mimicking perinatal mild post-hypoxia condition in humans. Further characterization and evaluation of the model, on molecular, cytological, and connectivity levels is needed. The study was co-financed by the European Union through the European Regional Development Fund, Operational Programme Competitiveness, and Cohesion, grant agreement No. KK.01.1.1.01.0007, CoRE – Neuro, University of Zagreb Support BM054 and Croatian Science Foundation.

This study investigates changes in neuronal connectivity and cognitive abilities after mild perinatal hypoxic brain lesion in a new non- invasive model of brain injury in rats. For the purpose of this research, nineteen Wistar Han® (RccHan®:WIST) rats, (9 females and 10 males) were randomly divided into hypoxic and control group on postnatal day 1 (P1) when hypoxia was induced in a warm (≈ 25°C) hypobaric chamber (Atm350mmHg, pO273mmHg) during 2 hours, while controls were kept in normal housing conditions. Behavioral tests were performed at P30 and P70 using the open field, hole board, social choice, and T-maze tests. Samples of brain tissue from adult animals (P105) were used for histochemical examination of cytoarchitectonics (Nissl staining), interneurons (parvalbumin immunohistochemistry) and perineuronal nets (Wisteria floribunda agglutinin, histochemistry). After this non- invasive hypoxic brain injury, histo- architectonics and the laminar and structural organization of the rat telencephalon, as well as motor and socialization patterns were preserved. However, distinct changes in morphology, number, and distribution of the parvalbumin-immunoreactive neurons and perineuronal nets in the midcingulate cortex, hippocampus, and somatosensory cortex were observed. Injured animals also show significantly impaired learning behavior. Thus, the mild perinatal hypoxic brain lesion in the rat leads to disturbances in brain connectivity responsible for learning and cognitive processes that are not compensated after the provoked hypoxia. Further characterization and evaluation of this brain injury model, on molecular, cytological and connectivity levels, is needed. Acknowledgments Research was funded by the Scientific Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic ischemic damage in perinatal and adult brain” GA KK 01 1 1 01 0007 funded by the European Union through the European Regional Development Fund) and Croatian Science Foundation.

In the study, fifty-two Wistar Han® (RccHan®:WIST) rats, (26 females and 26 males) were randomly divided into hypoxic and control group on postnatal day 1 (P1) when hypoxia was induced in a warm (≈ 25°C) hypobaric chamber (Atm 350mmHg, pO2 73mmHg) during 2 hours, while controls were kept in normal housing conditions. Behavioral testing were performed at P30 and P70 using open field, hole board, social choice, and T-maze. Samples of brain tissue from adult animals (P105-120) were used for histochemical examination of cytoarchitectonics (Nissl staining), interneurons (parvalbumin immunohistochemistry) and perineuronal nets (Wisteria floribunda agglutinin, histochemistry). Twelve Wistar pups (P1), 6 females and 6 males, was sacrificed at 8, 12 and 24h after hypoxic treatment and samples of their brain tissue were used for examination of microglia (CD68, immunohistochemistry). After mild perinatal hypoxic brain lesion, structural cerebral cytoarchitectonics, as well as the laminar and structural organization of the telencephalon, were preserved. Although, distinct changes in morphology, number, and distribution of the parvalbumin-immunoreactive neurons and perineuronal nets in the midcingulate cortex and hippocampus were observed. Microglial reactivity in lateral ventricle was most prominent at 24h after hypoxia. Compared to controls, motor and socialization patterns were preserved, while treated rats had better performance in open field, especially treated females. Moreover, treated animals also shown impaired learning behavior. Thus, the mild perinatal hypoxic brain lesion in rats leads to consistent disturbances in brain connectivity related to cognitive processes that mimic perinatal mild post-hypoxia condition in humans. Further characterization and evaluation of this non-invasive brain lesion model, on molecular, cytological and connectivity levels, is needed to disclose developmental disturbances that are not compensated after the provoked hypoxia and therefore lead to cognitive deficits. Research was funded by the Scientific Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic ischemic damage in perinatal and adult brain” GA KK 01 1 1 01 0007 funded by the European Union through the European Regional Development Fund) and Croatian Science Foundation.

Aims: Agonists of guanylate cyclase (GC) A but not GC-B are reducing the size of the brain lesion after an ischemic stroke. The aim of this study is to show if GC-C is involved in development of ischemic stroke. Methods: Middle cerebral artery occlusion (MCAO) was performed in wild type (WT) and GC-C knock-out (GC-C KO) mice. The volumes of brain lesions were determined 24h and 7 days after MCAO by MRI. In addition, patch clamp experiments and immunohistochemistry were performed. Results: Twenty-four hours after MCAO, GC-C KO animals have decreased lesion volumes compared to WT mice (WT:121±12 mm3, GC-C KO:79±14 mm3). Even though we showed a reduction in brain lesion 7 days after MCAO in WT mice, there was no change in GC-C KO mice and differences between mice were not present. Furthermore, GC-C agonist, uroguanylin, hyperpolarized cerebellar Purkinje neurons, (-7.2±1.4mV, n=5) while this effect was abolished in GC-C KO mice (1.6±1.7mV, n=4). Since most of the ischemic lesion of our mouse model is located in the cerebral cortex, it is not surprising that GC-C is present in neurons of cerebral cortex. Conclusion: Effects of GC-C activation on ischemic stroke are opposite to effects of GC-A agonists. GC-C KO mice develop smaller lesion volume 24h but not 7 days after an ischemic stroke compared to WT mice suggesting the possible benefit of specific GC-C inhibitors in treatment of ischemic stroke. Research was funded by the Scientific Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic-ischemic damage in perinatal and adult brain” ; GA KK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund) and Croatian Science Foundation project BRADISCHEMIA (UIP-2017-05-8082).

INTRODUCTION: Stroke has been identified as one of the leading causes of mortality in industrializes countries. Natriuretic peptides are involved in different physiological and patophophysiological conditions in the brain. Agonists of guanylate cyclase A but not B lead to decrease of brain lesion size after middle cerebral occlusion (MCAO). Therefore, uroguanylin (UGN) and its receptor guanylate cyclase C (GC-C), could also play a role in development of brain lesions and edema after ischemic injury. AIM: The purpose of this study is to describe the role of GC-C and its agonist, UGN, in the development of ischemic stroke in the murine model of ischemia. METHODS: MCAO was performed in wild type (WT), GC- C and UGN knock-out (GC-C -/-, UGN -/-) mice. Lesion volumes were measured 1 and 7 days after stroke and correlated to neurological impairment test scores. RESULTS: GC-C -/- animals show a significant reduction in lesion volumes 1 day after MCAO compared to their WT counterparts, whose lesion volume diminishes 7 days after MCAO. However, UGN -/- animals developed similar lesion volumes and oedema size 1 day after MCAO as their wild-type littermates (UGN +/+). CONCLUSION: Our results show that the activation of GC-C could lead to an increase in lesion volume following ischemic stroke. The difference in stroke volume is visible 1 day after MCAO, and is only present in GC-C-/- and not in UGN -/- animals. This difference could indicate GC-C activation by another agonist or the existence of an GC-C independent signalling pathway. ACKNOWLEDGEMENTS Research was funded by the Scientific Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic-ischemic damage in perinatal and adult brain” ; GA KK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund) and Croatian Science Foundation project BRADISCHEMIA (UIP-2017-05-8082).

Uroguanylin (UGN) belongs to the family of guanylin peptides. They regulate electrolyte and water transport in intestine via guanylate cyclase C, the only known receptor for guanylin peptides. In this thesis we are showing expression of UGN in different brain regions which is regulated by feeding, although it was believed that brain is not expressing UGN. Guanylate cyclase C (GC-C) is expressed in midbrain and hypothalamus, while we discovered additional brain regions expressing GC-C in neurons (cerebral cortex, Purkinje cells and deep nuclei of the cerebellum) but not in astrocytes. UGN hyperpolarized Purkinje cells and decreased firing rate while in astrocytes lead to increase in intracellular Ca2+ concentration via GC-C/cGMP independent signalling pathway. Effects of UGN in hypothalamus on diet-induced thermogenesis and brown adipose tissue (BAT) activation after a meal exist in male but not female WT animals and GC-C KO animals of both genders. Similar research was performed on healthy volunteers which showed that activation of BAT after a meal is gender, age, and body mass index dependent. Role of UGN in BAT regulations is of immeasurable importance for development of the new therapeutic approaches in treatment of obesity, metabolic syndrome, and diabetes mellitus.

INTRODUCTION: Guanylin peptides (GP), guanylin and uroguanylin (UGN), are intestinal peptides which activate guanylate cyclase C (GC-C) and increase cGMP concentration. GC-C is present not only in the intestine, but also in the Substantia nigra and Ventral tegmental areas of the brain where GP neuromodulate neuron activity. GC-C KO mice are the new animal model for ADHD. GC-C has also been reported in the hypothalamus where it plays an important role in the regulation of feeding and satiation. AIMS: The existence of cGMP/GC-C independent signaling pathway was suggested two decades ago ; however, the exact nature of the signaling pathway was never discovered. Therefore the aim of this study is to investigate the cGMP/GC-C independent signaling pathway in astrocytes which are a suitable model because they do not express GC-C. METHODS: In this study we performed patch clamp experiments and measured intracellular Ca2+ concentrations and pH in primary cultures of astrocytes and brain slices of wild type and GC-C KO mice. RESULTS: This study shows that GP hyperpolarized astrocyte membranes, which is opposite to depolarization caused by cGMP. By measuring intracellular concentration of Ca2+ we showed, for the first time, that UGN induces changes in intracellular Ca2+ levels in primary astrocyte cultures and in different regions of the brain, such as the hypothalamus, cerebral and cerebellar cortex. Furthermore, regulation of pH by astrocytes is important for neuronal function. GP regulate pH by changing transport of hydrogen and bicarbonate in astrocytes. CONCLUTION: The results of this study suggest that the effects of UGN on astrocytes via a Ca2+- dependent signaling pathway could be involved in modulation of neuronal activity. Grants: European Regional Development Fund, Operational Programme Competitiveness and Cohesion (K.K.01.1.1.01.0007). „Young researchers' career development project – training of doctoral students“ of the Croatian Science Foundation funded by the European Union from the European Social Fund.

The Rice-Vannuci hypoxic-ischemic-brain lesion in the rat, and its various modifications, used as a standard model for hypoxic brain lesions, are invasive surgical treatments classified as severe procedure (EU Directive 63/10, Article 15). We aim to introduce a non-invasive hypoxic brain lesion rat model of mild to moderate severity, to serve for the research targeting the fetal hypoxic brain lesions occurring during midgestation (23-32 weeks post- conception) in humans. In the present study, Wistar rats (9 females and 10 males) were randomly divided into hypoxic and control group on postnatal day 1 (P1) when hypoxia was induced in a warm (≈ 25°C) hypobaric chamber (Atm 350mmHg, pO273mmHg) during 2 hours, while controls were kept in normal housing conditions. Possible behavioral deficits were examined in a battery of tests: open field, hole board, the T-maze and social choice at P30 and P70. Samples of brain tissue from adult animals (P105-120) were used for histochemical examination of cytoarchitectonics (Nissl staining), interneurons (parvalbumin immunohistochemistry) and perineuronal nets (Wisteria floribunda agglutinin, histochemistry). Compared to controls, hypoxic animals had intact exploratory, anxiety-like and social behavior, but displayed significantly impaired learning. There were no disturbances in the brain macro-morphology or any other pathoanatomical consequence of the treatment, and the cytoarchitecture, as well as the laminar and structural organization of the telencephalon, were preserved. However, changes in morphology, number, and distribution of the parvalbumin-immunoreactive neurons and perineuronal nets, distinct in different regions of the telencephalon were observed. In conclusion, the proposed rat model of non- invasive hypoxic brain injury has indicated consistent disturbances in brain connectivity related to cognitive processes, that mimic perinatal mild post-hypoxia condition in humans. Further characterization and evaluation of the model, on molecular, cytological and connectivity levels, is needed to disclose developmental disturbances that are not compensated after the provoked hypoxia and therefore lead to cognitive deficits. (Fund: KK.01.1.1.01.0007, CoRE – Neuro).

Cell membranes are not uniform in their protein and lipid composition but organized in microdomains termed lipid rafts with higher concentration of (glyco)sphingolipids, cholesterol, and specific membrane proteins. The investigation of lipid rafts is thriving since the disturbed lipid microenvironment is recognized as highly important in pathogenesis of numerous human disorders. It is challenging to isolate lipid rafts from the bulk membrane in a way that would accurately reflect their composition and organization in living cells. Most often, detergent-resistant membranes enriched in cholesterol and sphingomyelin are extracted using the non-ionic detergent Triton X-100 (Tx-100). However, Tx-100 causes a redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins, which makes it an unacceptable choice for investigating the exact relationship between membrane gangliosides and proteins. The aim of this work was to develop a method for lipid raft isolation that would retain ganglioside and protein distribution for the purposes of a larger study investigating the crosstalk between gangliosides and specific membrane proteins. Since no-detergent methods of lipid raft isolation were not sufficiently effective or reproducible in our hands, the detergent Brij O20 was used in extraction followed by optimized sucrose density ultracentrifugation. Successful raft isolation was confirmed by Western blotting of non-lipid raft membrane protein transferrin receptor and lipid raft markers protein flotilin and ganglioside GM1 detected by cholera toxin subunit B immuno-overlay. The distribution of these markers, as well as gangliosides GT1b, GD1a, and specific membrane proteins, was compared in lipid rafts and the bulk membrane isolated with Tx-100 and Brij O20. Ganglioside distribution was found to be different as a result of Tx-100 vs. Brij020 isolation. Using Tx-100, the majority of ganglioside GD1a appears in raft fractions with almost no staining in bulk membrane, while Brij O20 isolation reveals a higher proportion of GD1a in the bulk membrane. Striking evidence for protein redistribution in Tx-100 isolation was found for transmembrane protein neuroplastin (Np) which is known to be affected by ganglioside environment. In Tx-100 isolation, the vast majority of Np is segregated in non- raft fractions (90% of all Np), compared to Brij O20 isolation where around 70% of Np is distributed in non-raft fractions. We show that isolating lipid rafts using Tx-100 leads to redistribution of gangliosides and specific proteins. This work will enable more accurate lipid raft analysis in respect to ganglioside and membrane proteins composition and lead to improved resolution of lipid- protein relations within lipid rafts.

Cessation of glucose and oxygen supply after ischemic brain injury leads to apoptotic and nectotic processes, which results in breakdown of the blood-brain barrier and consequent cerebral edema formation. Bradykinin is involved in the formation of cerebral edema increasing the size of brain damage. The aim of our research was clarifying the possible mechanisms of action of natriuretic peptides on bradykinin signaling, and their interaction after ischemic brain injury, since natriuretic peptides decrease brain edema via still unknown mechanisms. The results of this research indicate the existence of an endogenous antagonist of the bradykinin type 2 receptor and the possible protective role of natriuretic peptides in treatment of stroke.

Bradykinin is involved in the formation of cerebral edema after ischemic brain injury increasing the size of brain. Furthmore, it is known that natriuretic peptides are involved in decreasing cerebral edema via still unknown mechanisms. First, we examined the effects of natriuretic peptides and bradykinin in vitro in HEK-293 cells, primary isolated neurons and astrocytes using the whole cell patch clamp technique and by measuring the intracellular calcium concentration. In a mouse model of ischemic brain injury, we measured the size of the ischemic lesion and edema using microCT. In HEK- 293 cells, ligands of GC-A, but not GC-B, inhibit the bradykinin signaling pathway by activating RGS protein which is responsible for inactivation of G coupled protein. Our preliminary results show that the same inhibition exists in primary isolated mouse neurons. In in vivo experiments, when urodilatin, agonist of GC-A was applied, no brain lesion was detected by microCT scanning. When only bradykinin was applied, brain damage increased. Applying a combination of natriuretic peptides with bradykinin the size of the lesion and the brain edema decreased. The results indicated the existence of an endogenous antagonist of the bradykinin signaling pathway and a possible protective role of natriuretic peptides during stroke.

Among the consequences of a stroke are brain edemas, which additionally increase brain damage. This study is focused on cerebral edemas and bradykinin as their direct cause. It is known that natriuretic peptides decrease cerebral edema after ischemic brain injury through mechanisms that are still unknown. We examined the effects of natriuretic peptides and the bradykinin signaling pathway in vitro in HEK293 cells, primary isolated neurons, and astrocytes using the whole cell patch clamp technique and by measuring the intracellular calcium concentration. In a mouse model of ischemic brain injury (MCAO), we determined the degree of neurological damage in the mouse brain in vivo after applying only bradykinin or a combination of bradykinin with natriuretic peptides. We measured the size of the ischemic lesion and edema by micro CT and performed histological staining by Nissl. In HEK293 cells, ligands of guanylat cycalse A, but not guanylat cyclase B, inhibited the bradykinin signaling pathway (bradykinin receptor type 2-dependent). Our preliminary results showed that the same inhibition exists in primary isolated mouse neurons. When natriuretic peptides were applied, no brain damage was detected. As shown in our previous study, when only bradykinin was applied, brain damage increased in comparison to control animals through an increase in the brain edema. However, the combination with natriuretic peptides decreased the size of the lesion and the brain edema. The results indicate the existence of an endogenous antagonist of the bradykinin signaling pathway and a possible protective role for natriuretic peptides in humans during strokes.