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Sleep deprivation (SD) is commonplace in the modern way of life and has a substantial social, medical, and human cost. Sleep deprivation induces cognitive impairment such as loss of executive attention, working memory decline, poor emotion regulation, increased reaction times, and higher cognitive functions are particularly vulnerable to sleep loss. Furthermore, SD is associated with obesity, diabetes, cardiovascular diseases, cancer, and a vast majority of psychiatric and neurodegenerative disorders are accompanied by sleep disturbances. Despite the widespread scientific interest in the effect of sleep loss on synaptic function, there is a lack of investigation focusing on synaptic transmission on the proteome level. In the present study, we report the effects of SD and recovery period (RP) on the cortical synaptic proteome in rats. Synaptosomes were isolated after 8 h of SD performed by gentle handling and after 16 h of RP. The purity of synaptosome fraction was validated with western blot and electron microscopy, and the protein abundance alterations were analyzed by mass spectrometry. We observed that SD and RP have a wide impact on neurotransmitter-related proteins at both the presynaptic and postsynaptic membranes. The abundance of synaptic proteins has changed to a greater extent in consequence of SD than during RP: we identified 78 proteins with altered abundance after SD and 39 proteins after the course of RP. Levels of most of the altered proteins were upregulated during SD, while RP showed the opposite tendency, and three proteins (Gabbr1, Anks1b, and Decr1) showed abundance changes with opposite direction after SD and RP. The functional cluster analysis revealed that a majority of the altered proteins is related to signal transduction and regulation, synaptic transmission and synaptic assembly, protein and ion transport, and lipid and fatty acid metabolism, while the interaction network analysis revealed several connections between the significantly altered proteins and the molecular processes of synaptic plasticity or sleep. Our proteomic data implies suppression of SNARE-mediated synaptic vesicle exocytosis and impaired endocytic processes after sleep deprivation. Both SD and RP altered GABA neurotransmission and affected protein synthesis, several regulatory processes and signaling pathways, energy homeostatic processes, and metabolic pathways.

Synaptosomes are frequently used research objects in neurobiology studies focusing on synaptic transmission as they mimic several aspects of the physiological synaptic functions. They contain the whole apparatus for neurotransmission, the presynaptic nerve ending with synaptic vesicles, synaptic mitochondria and often a segment of the postsynaptic membrane along with the postsynaptic density is attached to its outer surface. As being artificial functional organelles, synaptosomes are viable for several hours, retain their activity, membrane potential, and capable to store, release, and reuptake neurotransmitters. Synaptosomes are ideal subjects for proteomic analysis. The recently available separation and protein detection techniques can cope with the reduced complexity of the organelle and enable the simultaneous qualitative and quantitative analysis of thousands of proteins shaping the structural and functional characteristics of the synapse. Synaptosomes are formed during the homogenization of nervous tissue in the isoosmotic milieu and can be isolated from the homogenate by various approaches. Each enrichment method has its own benefits and drawbacks and there is not a single method that is optimal for all research purposes. For a proper proteomic experiment, it is desirable to preserve the native synaptic structure during the isolation procedure and keep the degree of contamination from other organelles or cell types as low as possible. In this article, we examined five synaptosome isolation methods from a proteomic point of view by the means of electron microscopy, Western blot, and liquid chromatography-mass spectrometry to compare their efficiency in the isolation of synaptosomes and depletion of contaminating subcellular structures. In our study, the different isolation procedures led to a largely overlapping pool of proteins with a fairly similar distribution of presynaptic, active zone, synaptic vesicle, and postsynaptic proteins; however, discrete differences were noticeable in individual postsynaptic proteins and in the number of identified transmembrane proteins. Much pronounced variance was observed in the degree of contamination with mitochondrial and glial structures. Therefore, we suggest that in selecting the appropriate isolation method for any neuroproteomics experiment carried out on synaptosomes, the degree and sort/source of contamination should be considered as a primary aspect. Electronic supplementary material The online version of this article (10.1007/s00726-020-02912-6) contains supplementary material, which is available to authorized users.

All neurodegenerative diseases involve a relatively long period of timeframe from the onset of the disease to complete loss of functions. Extending this timeframe, even at a reduced level of function, would improve the quality of life of patients with these devastating diseases. The retina, as the part of the central nervous system and a frequent site of many distressing neurodegenerative disease, provides an ideal model to investigate the feasibility of extending the functional timeframe through pharmacologic intervention. Retinitis Pigmentosa (RP) is a group of blinding diseases. Although the rate of progression and degree of visual loss varies, there is usually a prolonged time before patients totally lose their photoreceptors and vision. It is believed that inhibitory mechanisms are still intact and may become relatively strong after the gradual loss of photoreceptors in RP patients. Therefore, it is possible that light-evoked responses of retinal ganglion cells and visual information processes in retinal circuits could be "unmasked" by blocking these inhibitory mechanisms restoring some level of visual function. Our results indicate that if the inhibition in the inner retina was unmasked in the retina of the rd10 mouse (the well-characterized RP mimicking, clinically relevant mouse model), the light-evoked responses of many retinal ganglion cells can be induced and restore their normal light sensitivity. GABA A receptor plays a major role in this masking inhibition. ERG b-wave and behavioral tests of spatial vision partly recovered after the application of PTX. Hence, removing retinal inhibition unmasks signalling mediated by surviving cones, thereby restoring some degree of visual function. These results may offer a novel strategy to restore the visual function with the surviving cones in RP patients and other gradual and progressive neurodegenerative diseases.

The retinas of many species show regional specialisations that are evident in the differences in the processing of visual input from different parts of the visual field. Regional specialisation is thought to reflect an adaptation to the natural visual environment, optical constraints, and lifestyle of the species. Yet, little is known about regional differences in synaptic circuitry. Here, we were interested in the topographical distribution of connexin-36 (Cx36), the major constituent of electrical synapses in the retina. We compared the retinas of mice, rats, and cats to include species with different patterns of regional specialisations in the analysis. First, we used the density of Prox1-immunoreactive amacrine cells as a marker of any regional specialisation, with higher cell density signifying more central regions. Double-labelling experiments showed that Prox1 is expressed in AII amacrine cells in all three species. Interestingly, large Cx36 plaques were attached to about 8–10% of Prox1-positive amacrine cell somata, suggesting the strong electrical coupling of pairs or small clusters of cell bodies. When analysing the regional changes in the volumetric density of Cx36-immunoreactive plaques, we found a tight correlation with the density of Prox1-expressing amacrine cells in the ON, but not in the OFF sublamina in all three species. The results suggest that the relative contribution of electrical synapses to the ON- and OFF-pathways of the retina changes with retinal location, which may contribute to functional ON/OFF asymmetries across the visual field.

Vision is our primary sense as the human eye is the gateway for more than 65% of information reaching the human brain. Today’s increased exposure to different wavelengths and intensities of light from light emitting diode (LED) sources could induce retinal degeneration and accompanying neuronal cell death. Damage induced by chronic phototoxic reactions occurring in the retina accumulates over years and it has been suggested as being responsible for the etiology of many debilitating ocular conditions. In this work, we examined how LED stimulation affects vision by monitoring changes in the expression of death and survival factors as well as microglial activation in LED-induced damage (LID) of the retinal tissue. We found an LED-exposure-induced increase in the mRNA levels of major apoptosis-related markers BAX, Bcl-2, and Caspase-3 and accompanying widespread microglial and Caspase-3 activation. Everyday LED light exposure was accounted for in all the described changes in the retinal tissue of mice in this study, indicating that overuse of non-filtered direct LED light can have detrimental effects on the human retina as well.

Connexin-36 (Cx36) is the major constituent of mammalian retinal gap junctions positioned in key signal pathways. Here, we examined the laminar and large-scale topographical distribution of Cx36 punctate immunolabels in the retina of the cat, a classical model of the mammalian visual system. Calretinin-immunoreactive (CaR-IR) cell populations served to outline the nuclear and plexiform layers and to stain specific neuronal populations. CaR-IR cells included horizontal cells in the outer retina, numerous amacrine cells, and scattered cells in the ganglion cell layer. Cx36-IR plaques were found among horizontal cell dendrites albeit without systematic colocalization of the two labels. Diffuse Cx36 immunoreactivity was found in the cytoplasm of AII amacrine cells, but no colocalization of Cx36 plaques was observed with either the perikarya or the long varicose dendrites of the CaR-IR non-AII amacrine cells. Cx36 puncta were seen throughout the entire inner plexiform layer showing their highest density in the ON sublamina. The densities of AII amacrine cell bodies and Cx36 plaques in the ON sublamina were strongly correlated across a wide range of eccentricities suggesting their anatomical association. However, the high number of plaques per AII cell suggests that a considerable fraction of Cx36 gap junctions in the ON sublamina is formed by other cell types than AII amacrine cells drawing attention to extensive but less studied electrically coupled networks. Electronic supplementary material The online version of this article (10.1007/s00429-019-01876-y) contains supplementary material, which is available to authorized users.

The behavior of female rats changes profoundly as they become mothers. The brain region that plays a central role in this regulation is the preoptic area, and lesions in this area eliminates maternal behaviors in rodents. The molecular background of the behavioral changes has not been established yet; therefore, in the present study, we applied proteomics to compare protein level changes associated with maternal care in the rat preoptic area. Using 2-dimensional fluorescence gel electrophoresis followed by identification of altered spots with mass spectrometry, 12 proteins were found to be significantly increased, and 6 proteins showed a significantly reduced level in mothers. These results show some similarities with a previous proteomics study of the maternal medial prefrontal cortex and genomics approaches applied to the preoptic area. Gene ontological analysis suggested that most altered proteins are involved in glucose metabolism and neuroplasticity. These proteins may support the maintenance of increased neuronal activity in the preoptic area, and morphological changes in preoptic neuronal circuits are known to take place in mothers. An increase in the level of alpha-crystallin B chain (Cryab) was confirmed by Western blotting. This small heat shock protein may also contribute to maintaining the increased activity of preoptic neurons by stabilizing protein structures. Common regulator and target analysis of the altered proteins suggested a role of prolactin in the molecular changes in the preoptic area. These results first identified the protein level changes in the maternal preoptic area. The altered proteins contribute to the maintenance of maternal behaviors and may also be relevant to postpartum depression, which can occur as a molecular level maladaptation to motherhood.

The nervous system demands an adequate oxygen and metabolite exchange, making pericytes (PCs), the only vasoactive cells on the capillaries, essential to neural function. Loss of PCs is a hallmark of multiple diseases, including diabetes, Alzheimer&rsquo, s, amyotrophic lateral sclerosis (ALS) and Parkinson&rsquo, s. Platelet-derived growth factor receptors (PDGFRs) have been shown to be critical to PC function and survival. However, how PDGFR-mediated PC activity affects vascular homeostasis is not fully understood. Here, we tested the hypothesis that imatinib, a chemotherapeutic agent and a potent PDGFR inhibitor, alters PC distribution and thus induces vascular atrophy. We performed a morphometric analysis of the vascular elements in sham control and imatinib-treated NG2-DsRed mice. Vascular morphology and the integrity of the blood&ndash, retina barrier (BRB) were evaluated using blood albumin labeling. We found that imatinib decreased the number of PCs and blood vessel (BV) coverage in all retinal vascular layers, this was accompanied by a shrinkage of BV diameters. Surprisingly, the total length of capillaries was not altered, suggesting a preferential effect of imatinib on PCs. Furthermore, blood&ndash, retina barrier disruption was not evident. In conclusion, our data suggest that imatinib could help in treating neurovascular diseases and serve as a model for PC loss, without BRB disruption.

The most prevalent Ca2+-buffer proteins (CaBPs: parvalbumin&mdash, PV, calbindin&mdash, CaB, calretinin&mdash, CaR) are widely expressed by various neurons throughout the brain, including the retinal ganglion cells (RGCs). Even though their retinal expression has been extensively studied, a coherent assessment of topographical variations is missing. To examine this, we performed immunohistochemistry (IHC) in mouse retinas. We found variability in the expression levels and cell numbers for CaR, with stronger and more numerous labels in the dorso-central area. CaBP+ cells contributed to RGCs with all soma sizes, indicating heterogeneity. We separated four to nine RGC clusters in each area based on expression levels and soma sizes. Besides the overall high variety in cluster number and size, the peripheral half of the temporal retina showed the greatest cluster number, indicating a better separation of RGC subtypes there. Multiple labels showed that 39% of the RGCs showed positivity for a single CaBP, 30% expressed two CaBPs, 25% showed no CaBP expression, and 6% expressed all three proteins. Finally, we observed an inverse relation between CaB and CaR expression levels in CaB/CaR dual- and CaB/CaR/PV triple-labeled RGCs, suggesting a mutual complementary function.

Tissue clearing methods enable the imaging of biological specimens without sectioning. However, reliable and scalable analysis of large imaging datasets in three dimensions remains a challenge. Here we developed a deep learning-based framework to quantify and analyze brain vasculature, named Vessel Segmentation & Analysis Pipeline (VesSAP). Our pipeline uses a convolutional neural network (CNN) with a transfer learning approach for segmentation and achieves human-level accuracy. By using VesSAP, we analyzed the vascular features of whole C57BL/6J, CD1 and BALB/c mouse brains at the micrometer scale after registering them to the Allen mouse brain atlas. We report evidence of secondary intracranial collateral vascularization in CD1 mice and find reduced vascularization of the brainstem in comparison to the cerebrum. Thus, VesSAP enables unbiased and scalable quantifications of the angioarchitecture of cleared mouse brains and yields biological insights into the vascular function of the brain.

Retinal ganglion cells (RGCs) encrypt stimulus features of the visual scene in action potentials and convey them toward higher visual centers in the brain. Although there are many visual features to encode, our recent understanding is that the ~46 different functional subtypes of RGCs in the retina share this task. In this scheme, each RGC subtype establishes a separate, parallel signaling route for a specific visual feature (e.g., contrast, the direction of motion, luminosity), through which information is conveyed. The efficiency of encoding depends on several factors, including signal strength, adaptational levels, and the actual efficacy of the underlying retinal microcircuits. Upon collecting inputs across their respective receptive field, RGCs perform further analysis (e.g., summation, subtraction, weighting) before they generate the final output spike train, which itself is characterized by multiple different features, such as the number of spikes, the inter-spike intervals, response delay, and the rundown time (transience) of the response. These specific kinetic features are essential for target postsynaptic neurons in the brain in order to effectively decode and interpret signals, thereby forming visual perception. We review recent knowledge regarding circuit elements of the mammalian retina that participate in shaping RGC response transience for optimal visual signaling.

Intracellular β-amyloid (Aβ) accumulation is an early event in Alzheimer's disease (AD) progression. Recently, it has been uncovered that presenilins (PSs), the key components of the amyloid precursor protein (APP) processing and the β-amyloid producing γ-secretase complex, are highly enriched in a special sub-compartment of the endoplasmic reticulum (ER) functionally connected to mitochondria, called mitochondria-associated ER membrane (MAM). A current hypothesis of pathogenesis of Alzheimer's diseases (AD) suggests that MAM is involved in the initial phase of AD. Since MAM supplies mitochondria with essential proteins, the increasing level of PSs and β-amyloid could lead to metabolic dysfunction because of the impairment of ER-mitochondrion crosstalk. To reveal the early molecular changes of this subcellular compartment in AD development MAM fraction was isolated from the cerebral cortex of 3 months old APP/PS1 mouse model of AD and age-matched C57BL/6 control mice, then mass spectrometry-based quantitative proteome analysis was performed. The enrichment and purity of MAM preparations were validated with EM, LC-MS/MS and protein enrichment analysis. Label-free LC-MS/MS was used to reveal the differences between the proteome of the transgenic and control mice. We obtained 77 increased and 49 decreased protein level changes in the range of - 6.365 to + 2.988, which have mitochondrial, ER or ribosomal localization according to Gene Ontology database. The highest degree of difference between the two groups was shown by the ATP-binding cassette G1 (Abcg1) which plays a crucial role in cholesterol metabolism and suppresses Aβ accumulation. Most of the other protein changes were associated with increased protein synthesis, endoplasmic-reticulum-associated protein degradation (ERAD), oxidative stress response, decreased mitochondrial protein transport and ATP production. The interaction network analysis revealed a strong relationship between the detected MAM protein changes and AD. Moreover, it explored several MAM proteins with hub position suggesting their importance in Aβ induced early MAM dysregulation. Our identified MAM protein changes precede the onset of dementia-like symptoms in the APP/PS1 model, suggesting their importance in the development of AD.

To establish synaptic proteome changes associated with motherhood, we isolated synaptosome fractions from the hypothalamus of mother rats and non-maternal control females at the 11th postpartum day. Proteomic analysis by two-dimensional differential gel electrophoresis combined with mass spectrometric protein identification established 26 significant proteins, 7 increasing and 19 decreasing protein levels in the dams. The altered proteins are mainly involved in energy homeostasis, protein folding, and metabolic processes suggesting the involvement of these cellular processes in maternal adaptations. The decrease in a significantly altered protein, complement component 1q subcomponent-binding protein (C1qbp) was validated with Western blotting. Furthermore, immunohistochemistry showed its presence in hypothalamic fibers and terminals in agreement with its presence in synaptosomes. We also found the expression of C1qbp in different hypothalamic nuclei including the preoptic area and the paraventricular hypothalamic nucleus at the protein and at the mRNA level using immunohistochemistry and in situ hybridization histochemistry, respectively. Bioinformatical network analysis revealed that cytokines, growth factors, and protein kinases are common regulators, which indicates a complex regulation of the proteome change in mothers. The results suggest that maternal responsiveness is associated with synaptic proteins level changes in the hypothalamus, and that growth factors and cytokines may govern these alterations. Biological significance The period of motherhood is accompanied with several behavioral, neuroendocrine, emotional and metabolic adaptations in the brain. Although it is established that various hypothalamic networks participate in the maternal adaptations of the rodent brain, our knowledge on the molecular background of these alterations remains seriously limited. In the present study, we first determined that the functional alterations of the maternal brain can be detected at the level of the synaptic proteome in the hypothalamus. Independent confirmation of synaptic localization, and also the established decrease in the level of C1qbp protein suggest the validity of the data. Common regulators of altered proteins belonging to the growth factor and cytokine family suggest that the synaptic adaptation is governed by these extracellular signals and future studies should focus on their specific roles. Our study was also the first to describe the expression pattern of C1qbp in the hypothalamus, a protein potentially involved in mitochondrial and neuroimmunological regulations of synaptic plasticity. Its presence in the preoptic area responsible for maternal behaviors and also in the paraventricular hypothalamic and arcuate nuclei regulating hormonal levels suggests that the same proteins may be involved in different aspects of maternal adaptations. The conclusions of the present work contribute to establishing the molecular alterations that determine different maternal adaptations in the brain. Since maternal changes are models of neuronal plasticity in all social interactions, the reported results can affect a wide field of molecular and behavioral neuroscience.

Acute total sleep deprivation (SD) impairs memory consolidation, attention, working memory and perception. Structural, electrophysiological and molecular experimental approaches provided evidences for the involvement of sleep in synaptic functions. Despite the wide scientific interest on the effects of sleep on the synapse, there is a lack of systematic investigation of sleep-related changes in the synaptic proteome. We isolated parietal cortical and thalamic synaptosomes of rats after 8h of total SD by gentle handling and 16h after the end of deprivation to investigate the short- and longer-term effects of SD on the synaptic proteome, respectively. The SD efficiency was verified by electrophysiology. Protein abundance alterations of the synaptosomes were analyzed by fluorescent two-dimensional differential gel electrophoresis and by tandem mass spectrometry. As several altered proteins were found to be involved in synaptic strength regulation, our data can support the synaptic homeostasis hypothesis function of sleep and highlight the long-term influence of SD after the recovery sleep period, mostly on cortical synapses. Furthermore, the large-scale and brain area-specific protein network change in the synapses may support both ideas of sleep-related synaptogenesis and molecular maintenance and reorganization in normal rat brain.

Proteomic differences between rat dams and control mothers deprived of their pups immediately after delivery were investigated in the medial prefrontal cortex (mPFC). A 2-D DIGE minimal dye technique combined with LC–MS/MS identified 32 different proteins that showed significant changes in expression in the mPFC, of which, 25 were upregulated and 7 were downregulated in dams. The identity of one significantly increased protein, the small heat-shock protein alpha-crystallin B chain (Cryab), was confirmed via Western blot analysis. Alpha-crystallin B chain was distributed in scattered cells in the mPFC, as demonstrated by immunohistochemistry. Furthermore, it was found to be localized in parvalbumin-containing neurons using double labeling. The elevation of its mRNA level in rat dams was also demonstrated via RT-PCR. The functional classification of the altered proteins was conducted using the UniProt and Gene Ontology protein databases. The identified proteins predominantly participate in synaptic transport and plasticity, neuron development, oxidative stress and apoptosis, and cytoskeleton organization. A common regulator and target analysis of these proteins determined using the Elsevier Pathway Studio Platform suggests that protein level changes associated with pup nursing are driven by growth factors and cytokines, while the MAP kinase pathway was identified as a common target. A high proportion of the proteins that were found to be altered in the mPFC are associated with depression. Biological significance The behavior and emotional state of females change robustly when they become mothers. The brain, which governs these changes, may also undergo molecular alterations in mothers. As no proteomics approaches have been applied regarding maternal changes in the brain, we addressed this issue in the mPFC as this brain area is the uppermost cortical center of maternal control and the associated mood changes. The high number of protein-level alterations found between mothers taking care of their litter and those without pups indicates that pup nursing is associated with cortical protein-level changes. Alterations in proteins participating in synaptic transport, plasticity and neuron development suggest neuroplastic changes in the maternal brain. In turn, the relatively high number of altered proteins in the mPFC associated with depression suggests that the physiological effects of the protein-level alterations in the maternal mPFC could promote the incidence of postpartum depression. Cryab, a protein confirmed to be increased during maternal behaviors, was selectively found in parvalbumin cells, which, as fast-spiking interneurons, are associated with depression. The function of Cryab should be further investigated to establish whether it can be used to identify drug targets for future drug development.

In the visual system, retinal ganglion cells (RGCs) of various subtypes encode preprocessed photoreceptor signals into a spike output which is then transmitted towards the brain through parallel feature pathways. Spike timing determines how each feature signal contributes to the output of downstream neurons in visual brain centers, thereby influencing efficiency in visual perception. In this study, we demonstrate a marked population-wide variability in RGC response latency that is independent of trial-to-trial variability and recording approach. RGC response latencies to simple visual stimuli vary considerably in a heterogenous cell population but remain reliable when RGCs of a single subtype are compared. This subtype specificity, however, vanishes when the retinal circuitry is bypassed via direct RGC electrical stimulation. This suggests that latency is primarily determined by the signaling speed through retinal pathways that provide subtype specific inputs to RGCs. In addition, response latency is significantly altered when GABA inhibition or gap junction signaling is disturbed, which further supports the key role of retinal microcircuits in latency tuning. Finally, modulation of stimulus parameters affects individual RGC response delays considerably. Based on these findings, we hypothesize that retinal microcircuits fine-tune RGC response latency, which in turn determines the context-dependent weighing of each signal and its contribution to visual perception.

Ca2+-binding buffer proteins (CaBPs) are widely expressed by various neurons throughout the central nervous system (CNS), including the retina. While the expression of CaBPs by photoreceptors, retinal interneurons and the output ganglion cells in the mammalian retina has been extensively studied, a general description is still missing due to the differences between species, developmental expression patterns and study-to-study discrepancies. Furthermore, CaBPs are occasionally located in a compartment-specific manner and two or more CaBPs can be expressed by the same neuron, thereby sharing the labor of Ca2+ buffering in the intracellular milieu. This article reviews this topic by providing a framework on CaBP functional expression by neurons of the mammalian retina with an emphasis on human and mouse retinas and the three most abundant and extensively studied buffer proteins: parvalbumin, calretinin and calbindin.

An increasing number of studies have revealed associations between pre- and perinatal immune activation and the development of schizophrenia and autism spectrum disorders (ASDs). Accordingly, neuroimmune crosstalk has a considerably large impact on brain development during early ontogenesis. While a plethora of heterogeneous abnormalities have already been described in established maternal immune activation (MIA) rodent and primate animal models, which highly correlate to those found in human diseases, the underlying molecular background remains obscure. In the current study, we describe the long-term effects of MIA on the neocortical pre- and postsynaptic proteome of adolescent rat offspring in detail. Molecular differences were revealed in sub-synaptic fractions, which were first thoroughly characterized using independent methods. The widespread proteomic examination of cortical samples from offspring exposed to maternal lipopolysaccharide administration at embryonic day 13.5 was conducted via combinations of different gel-based proteomic techniques and tandem mass spectrometry. Our experimentally validated proteomic data revealed more pre- than postsynaptic protein level changes in the offspring. The results propose the relevance of altered synaptic vesicle recycling, cytoskeletal structure and energy metabolism in the presynaptic region in addition to alterations in vesicle trafficking, the cytoskeleton and signal transduction in the postsynaptic compartment in MIA offspring. Differing levels of the prominent signaling regulator molecule calcium/calmodulin-dependent protein kinase II in the postsynapse was validated and identified specifically in the prefrontal cortex. Finally, several potential common molecular regulators of these altered proteins, which are already known to be implicated in schizophrenia and ASD, were identified and assessed. In summary, unexpectedly widespread changes in the synaptic molecular machinery in MIA rats were demonstrated which might underlie the pathological cortical functions that are characteristic of schizophrenia and ASD.

Peripheral injection of bacterial lipopolysaccharide (LPS) facilitates 8–10 Hz spike-wave discharges (SWD) characterizing absence epilepsy in WAG/Rij rats. It is unknown however, whether peripherally administered LPS is able to alter the generator areas of epileptic activity at the molecular level. We injected 1 mg/kg dose of LPS intraperitoneally into WAG/Rij rats, recorded the body temperature and EEG, and examined the protein expression changes of the proteome 12 h after injection in the fronto-parietal cortex and thalamus. We used fluorescent two-dimensional differential gel electrophoresis to investigate the expression profile. We found 16 differentially expressed proteins in the fronto-parietal cortex and 35 proteins in the thalamus. It is known that SWD genesis correlates with the transitional state of sleep–wake cycle thus we performed meta-analysis of the altered proteins in relation to inflammation, epilepsy as well as sleep. The analysis revealed that all categories are highly represented by the altered proteins and these protein-sets have considerable overlap. Protein network modeling suggested that the alterations in the proteome were largely induced by the immune response, which invokes the NFkB signaling pathway. The proteomics and computational analysis verified the known functional interplay between inflammation, epilepsy and sleep and highlighted proteins that are involved in their common synaptic mechanisms. Our physiological findings support the phenomenon that high dose of peripheral LPS injection increases SWD-number, modifies its duration as well as the sleep–wake stages and decreases body temperature.

Alzheimer's disease (AD) is a multifactorial disease of wide clinical heterogenity. Overproduction of amyloid precursor protein (APP) and accumulation of β-amyloid (Aβ) and tau proteins are important hallmarks of AD. The identification of early pathomechanisms of AD is critically important for discovery of early diagnosis markers. Decreased brain metabolism is one of the earliest clinical symptoms of AD that indicate mitochondrial dysfunction in the brain. We performed the first comprehensive study integrating synaptic and non-synaptic mitochondrial proteome analysis (two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry) in correlation with Aβ progression in APP/PS1 mice (3, 6, and 9 months of age). We identified changes of 60 mitochondrial proteins that reflect the progressive effect of APP overproduction and Aβ accumulation on mitochondrial processes. Most of the significantly affected proteins play role in the mitochondrial electron transport chain, citric acid cycle, oxidative stress, or apoptosis. Altered expression levels of Htra2 and Ethe1, which showed parallel changes in different age groups, were confirmed also by Western blot. The common regulator bioinformatical analysis suggests the regulatory role of tumor necrosis factor (TNF) in Aβ-mediated mitochondrial protein changes. Our results are in accordance with the previous postmortem human brain proteomic studies in AD in the case of many proteins. Our results could open a new path of research aiming early mitochondrial molecular mechanisms of Aβ accumulation as a prodromal stage of human AD.

Dopaminergic neurons of the central nervous system are mainly found in nuclei of the midbrain and the hypothalamus that provide subcortical and cortical targets with a rich and divergent innervation. Disturbance of signaling through this system underlies a variety of deteriorating conditions such as Parkinson's disease and schizophrenia. Although retinal dopaminergic signaling is largely independent of the above circuitry, malfunction of the retinal dopaminergic system has been associated with anomalies in visual adaptation and a number of retinal disorders. Dopamine (DA) is released mainly in a paracrine manner by a population of tyrosine hydroxylase expressing (TH(+) ) amacrine cells (AC) of the mammalian retina; thus DA reaches virtually all retinal cell types by diffusion. Despite this paracrine release, however, the so called AII ACs have been considered as the main targets of DA signaling owing to a characteristic and robust ring-like TH(+) innervation to the soma/dendritic-stalk area of AII cells. This apparent selectivity of TH(+) innervation seems to contradict the divergent DAergic signaling scheme of other brain loci. In this study, however, we show evidence for intimate proximity between TH(+) rings and somata of neurochemically identified non-AII cells. We also show that this phenomenon is not species specific, as we observe it in popular mammalian animal models including the rabbit, the rat, and the mouse. Finally, our dataset suggests the existence of further, yet unidentified post-synaptic targets of TH(+) dendritic rings. Therefore, we hypothesize that TH(+) ring-like structures target the majority of ACs non-selectively and that such contacts are wide-spread among mammals. Therefore, this new view of inner retinal TH(+) innervation resembles the divergent DAergic innervation of other brain areas through the mesolimbic, mesocortical, and mesostriatal signaling streams. AII amacrine cells have been considered as the main targets of dopamine signaling in the mammalian retina owing to a characteristic ring-like innervation from dopaminergic (TH(+) ) amacrine cells (green) to somata of AII cells (red). In this study, we show the intimate proximity of TH(+) rings and somata of non-AII cells, including starburst-a amacrine cells (blue) and other unidentified amacrine cells (magenta). We find that this phenomenon is not species specific and it occurs in a number of popular mammalian animal models. We hypothesize that TH(+) ring-inputs target most amacrine cells non-selectively and thus it resembles the divergent dopaminergic innervation of other brain areas.

The synapse is a particularly important compartment of neurons. To reveal its molecular characteristics we isolated whole brain synaptic (sMito) and non-synaptic mitochondria (nsMito) from the mouse brain with purity validated by electron microscopy and fluorescence activated cell analysis and sorting. Two-dimensional differential gel electrophoresis and mass spectrometry based proteomics revealed 22 proteins with significantly higher and 34 proteins with significantly lower levels in sMito compared to nsMito. Expression differences in some oxidative stress related proteins, such as superoxide dismutase [Mn] (Sod2) and complement component 1Q subcomponent-binding protein (C1qbp), as well as some tricarboxylic acid cycle proteins, including isocitrate dehydrogenase subunit alpha (Idh3a) and ATP-forming β subunit of succinyl-CoA ligase (SuclA2), were verified by Western blot, the latter two also by immunohistochemistry. The data suggest altered tricarboxylic acid metabolism in energy supply of synapse while the marked differences in Sod2 and C1qbp support high sensitivity of synapses to oxidative stress. Further functional clustering demonstrated that proteins with higher synaptic levels are involved in synaptic transmission, lactate and glutathione metabolism. In contrast, mitochondrial proteins associated with glucose, lipid, ketone metabolism, signal transduction, morphogenesis, protein synthesis and transcription were enriched in nsMito. Altogether, the results suggest a specifically tuned composition of synaptic mitochondria. Biological significance Neurons communicate with each other through synapse, a compartment metabolically isolated from the cell body. Mitochondria are concentrated in presynaptic terminals by active transport to provide energy supply for information transfer. Mitochondrial composition in the synapse may be different than in the cell body as some examples have demonstrated altered mitochondrial composition with cell type and cellular function in the muscle, heart and liver. Therefore, we posed the question whether protein composition of synaptic mitochondria reflects its specific functions. The determined protein difference pattern was in accordance with known functional specialties of high demand synaptic mitochondria. The data also suggest specifically tuned metabolic fluxes for energy production by means of interaction with glial cells surrounding the synapse. These findings provide possible mechanisms for dynamically adapting synaptic mitochondrial output to actual demand. In turn, an increased vulnerability of synaptic mitochondria to oxidative stress is implied by the data. This is important from theoretical but potentially also from therapeutic aspects. Mitochondria are known to be affected in some neurodegenerative and psychiatric disorders, and proteins with elevated level in synaptic mitochondria, e.g. C1qbp represent targets for future drug development, by which synaptic and non-synaptic mitochondria can be differentially affected.

Chronic cerebral hypoperfusion (CCH) evokes mild cognitive impairment (MCI) and contributes to the progression of vascular dementia and Alzheimer's disease (AD). How CCH induces these neurodegenerative processes that may spread along the synaptic network and whether they are detectable at the synaptic proteome level of the cerebral cortex remains to be established. In the present study, we report the synaptic protein changes in the cerebral cortex after stepwise bilateral common carotid artery occlusion (BCCAO) induced CCH in the rat. The occlusions were confirmed with magnetic resonance angiography 5 weeks after the surgery. Synaptosome fractions were prepared using sucrose gradient centrifugation from cerebral cortex dissected 7 weeks after the occlusion. The synaptic protein differences between the sham operated and CCH groups were analyzed with label-free nanoUHPLC-MS/MS. We identified 46 proteins showing altered abundance due to CCH. In particular, synaptic protein and lipid metabolism, as well as GABA shunt-related proteins showed increased while neurotransmission and synaptic assembly-related proteins showed decreased protein level changes in CCH rats. Protein network analysis of CCH-induced protein alterations suggested the importance of increased synaptic apolipoprotein E (APOE) level as a consequence of CCH. Therefore, the change in APOE level was confirmed with Western blotting. The identified synaptic protein changes would precede the onset of dementia-like symptoms in the CCH model, suggesting their importance in the development of vascular dementia.

Retinal ganglion cells (RGC) have been described to react to light stimuli either by producing short bursts of spikes or by maintaining a longer, continuous train of action potentials. Fast, quickly decaying responses are considered to be transient in nature and encode information about movement and direction, while cell responses that show a slow, drawn-out response fall into the sustained category and are thought to be responsible for carrying information related to color and contrast. Multiple approaches have been introduced thus far to measure and determine response transiency. In this study, we adopted and slightly modified a method described by Zeck and Masland to characterize RGC response transiency values and compare them to those obtained by alternative methods. As the first step, RGC spike responses were elicited by light stimulation and peristimulus time histograms (PSTHs) were generated. PSTHs then were used to calculate the time constant (PSTHτ approach). We show that this method is comparable to or more reliable than alternative approaches to describe the temporal characteristics of RGC light responses. In addition, we also show that PSTHτ-s are compatible with time constants measured on RGC and/or bipolar cell graded potentials; thus they are suitable for studying signaling through parallel retinal pathways.

Tyrosine-hydroxylase-positive (TH(+)) amacrine cells release dopamine in a paracrine manner and also form GABA-ergic contact sites with inner retinal neurons. The best known sites are formed by TH(+) fibrous rings and AII amacrine cell somata in stratum 1 of the inner plexiform layer (IPL). An AII amacrine cell is a highly compartmentalized neuron with relatively large soma, a stout dendritic stalk and two sets of processes, one showing lobular appearance and extending horizontally in stratum 1 and a second transversally elongated group of fibers in strata 4 and 5. Although, all of these compartments have been reported as tic sites, it is uncertain if TH(+) amacrine cell inputs are homogeneously distributed or they rather target specific AII cell compartments. In this study we investigated the TH(+)/AII cell system by immunohistochemistry to map the potential synaptic contacts in the rabbit retina. We found numerous intimate contacts between the two amacrine cell populations throughout the IPL. However, TH(+) fibers favored the soma/main stalk region of AII amacrine cells and only contacted lobular appendages and transversal processes sporadically. In addition to the well-studied contacts between AII cell somata and TH(+) rings in stratum 1 we found that the main stalk region in stratum 3 serves as a secondary major target for TH(+) axons. These data thus clearly show that TH(+) contacts to AII amacrine cells are highly compartment specific.

Retinal ganglion cells (RGCs) in dark-adapted retinas show a range of threshold sensitivities spanning ∼3 log units of illuminance. Here, we show that the different threshold sensitivities of RGCs reflect an inhibitory mechanism that masks inputs from certain rod pathways. The masking inhibition is subserved by GABAThe responses of rod photoreceptors, which subserve dim light vision, are carried through the retina by three independent pathways. These pathways carry signals with largely different sensitivities. Retinal ganglion cells (RGCs), the output neurons of the retina, show a wide range of sensitivities in the same dark-adapted conditions, suggesting a divergence of the rod pathways. However, this organization is not supported by the known synaptic morphology of the retina. Here, we tested an alternative idea that the rod pathways converge onto single RGCs, but inhibitory circuits selectively mask signals so that one pathway predominates. Indeed, we found that application of GABA receptor blockers increased the sensitivity of most RGCs by unmasking rod signals, which were suppressed. Our results indicate that inhibition controls the threshold responses of RGCs under dim ambient light. This mechanism can ensure that appropriate signals cross the bottleneck of the optic nerve in changing stimulus conditions.

Besides the opioids the standard management of the World Health Organization suggests NSAIDs (non-steroidal anti-inflammatory drugs) alone or in combination to enhance analgesia in malignant and non-malignant pain therapy. The applicability of NSAIDs in a nasal formulation is a new approach in pharmaceutical technology. In order to enhance the nasal absorption of meloxicam (MX) as an NSAID, its salt form, meloxicam potassium monohydrate (MXP), registered by Egis Plc., was investigated in comparison with MX. The physico-chemical properties of the drugs (structural analysis, solubility and dissolution rate) and the mucoadhesivity of nasal formulations were controlled. In vitro and in vivo studies were carried out to determine the nasal applicability of MXP as a drug candidate in pain therapy. It can be concluded that MX and MXP demonstrated the same equilibrium solubility at the pH5.60 of the nasal mucosa (0.017mg/ml); nonetheless, MXP indicated faster dissolution and a higher permeability through the synthetic membrane. The animal studies justified the short Tmax value (15min) and the high AUC of MXP, which is important in acute pain therapy. It can be assumed that the low mucoadhesivity of MXP spray did not increase the residence time in the nasal cavity, and the elimination from the nasal mucosa was therefore faster than in the case of MX. Further experiments are necessary to prove the therapeutic relevance of this MXP-containing innovative intranasal formulation.

Converging evidence indicates that electrical synaptic transmission via gap junctions plays a crucial role in signal processing in the retina. In particular, amacrine and ganglion cells express numerous gap junctions, resulting in extensive electrical networks in the proximal retina. Both connexin36 (Cx36) and connexin45 (Cx45) subunits are widely distributed in the inner plexiform layer (IPL) and therefore are likely contribute to gap junctions formed by a number of ganglion cell subtypes. In the present study, we used the gap junction-permeant tracer Neurobiotin to compare the coupling pattern of different ganglion cell subtypes in wild-type (WT) and Cx36 knockout (KO) mouse retinas. We found that homologous ganglion-to-ganglion cell coupling was lost for two subtypes after deletion of Cx36, whereas two other ganglion cell subtypes retained homologous coupling in the KO mouse. In contrast, deletion of Cx36 resulted in a partial or complete loss of ganglion-to-amacrine cell heterologous coupling in 9 of 10 ganglion cell populations studied. Overall, our results indicate that Cx36 is the predominant subunit of gap junctions in the proximal mouse retina, expressed by most ganglion cell subtypes, and thereby likely plays a major role in the concerted activity generated by electrical synapses.

It is now clear that electrical coupling via gap junctions isprevalent across the retina, expressed by each of the fivemain neuronal types. With the introduction of mutants inwhich selective gap junction connexins are deleted, themouse has recently become an important model for studyingthe function of coupling between retinal neurons. In thisstudy we examined the tracer-coupling pattern of ganglioncells by injecting them with the gap junction-permanenttracer Neurobiotin to provide, for the first time, a comprehensivesurvey of ganglion cell coupling in the wildtypemouse retina. Murine ganglion cells were differentiated into22 morphologically distinct subtypes based on somadendriticparameters. Most (16/22) ganglion cell subtypes were tracer-coupled to neighboring ganglion and/or amacrinecells. The amacrine cells coupled to ganglion cellsdisplayed either polyaxonal or wide-field morphologies withextensive arbors. We found that different subtypes of ganglioncells were never coupled to one another, indicatingthat they subserved independent electrical networks. Finally, we found that the tracer-coupling patterns of the 22ganglion cell populations were largely stereotypic acrossthe 71 retinas studied. Our results indicate that electricalcoupling is extensive in the inner retina of the mouse, suggestingthat gap junctions play essential roles in visual information processing.

We studied the morphology and physiology of a unique wide-field amacrine cell in the rabbit retina. These cells displayed a stereotypic dendritic morphology consisting of a large, circular and monostratified arbor that often extended over 2 mm. Their responses contained both somatic and dendritic sodium spikes suggesting active propagation of synaptic signals within the dendritic arbor. This idea is supported by the enormous size of their ON-OFF receptive fields. Interestingly, these cells exhibited separate ON and OFF receptive fields that, while concentric, were vastly different in size. Whereas the ON receptive field of these cells extended nearly 2 mm, the OFF receptive field was typically 75% smaller. Blockade of voltage-gated sodium channels with QX-314 dramatically reduced the large ON receptive field, but had little effect on the smaller OFF receptive field. These results indicate a spatial disparity in the location of on- and off-center bipolar cell inputs to the dendritic arbor of wide-field amacrine cells. In addition, the active propagation of signals suggests that synaptic inputs are integrated both locally and globally within the dendritic arbor.

Accumulating evidence from recent literature underline the important roles of tissue non specific alkaline phosphatase (TNAP) in diverse functions as well as diseases of the nervous system. Exploration of TNAP in well characterized neural circuits such as the retina, might significantly advance our understanding regarding neural TNAP's roles. This chapter reviews the scarce literature as well as our findings on retinal TNAP. We found that retinal TNAP activity was preserved and followed diverse patterns throughout vertebrate evolution. We have consistently observed TNAP activity (1) in retinal vessels, (2) in photoreceptors and (3) in the majority of the studied species in the outer (OPL) and inner plexiform layers (IPL), where synaptic transmission occurs. Importantly, in some species the IPL exhibits several TNAP positive strata. These strata exactly corresponded those seen after quadruple immunohistochemistry with four canonical IPL markers (tyrosine hydroxylase, choline acetyltransferase, calretinin, protein kinase C α). Diabetes results in diminishing retinal TNAP activity before changes in canonical markers could be observed in a rat model. The presence of TNAP activity at critical sites of neurotransmission suggests its important and evolutionary conserved role in vision. In diabetes, the decreased TNAP activity indicates neurological alterations adding further evidence for the role of TNAP in brain diseases.

Using patch-clamp techniques, we investigated the characteristics of the spontaneous oscillatory activity displayed by starburst amacrine cells in the mouse retina. At a holding potential of -70 mV, oscillations appeared as spontaneous, rhythmic inward currents with a frequency of approximately 3.5 Hz and an average maximal amplitude of approximately 120 pA. Application of TEA, a potassium channel blocker, increased the amplitude of oscillatory currents by >70% but reduced their frequency by approximately 17%. The TEA effects did not appear to result from direct actions on starburst cells, but rather a modulation of their synaptic inputs. Oscillatory currents were inhibited by 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), an antagonist of AMPA/kainate receptors, indicating that they were dependent on a periodic glutamatergic input likely from presynaptic bipolar cells. The oscillations were also inhibited by the calcium channel blockers cadmium and nifedipine, suggesting that the glutamate release was calcium dependent. Application of AP4, an agonist of mGluR6 receptors on on-center bipolar cells, blocked the oscillatory currents in starburst cells. However, application of TEA overcame the AP4 blockade, suggesting that the periodic glutamate release from bipolar cells is intrinsic to the inner plexiform layer in that, under experimental conditions, it can occur independent of photoreceptor input. The GABA receptor antagonists picrotoxin and bicuculline enhanced the amplitude of oscillations in starburst cells prestimulated with TEA. Our results suggest that this enhancement was due to a reduction of a GABAergic feedback inhibition from amacrine cells to bipolar cells and the resultant increased glutamate release. Finally, we found that some ganglion cells and other types of amacrine cell also displayed rhythmic activity, suggesting that oscillatory behavior is expressed by a number of inner retinal neurons.

The AII amacrine cells are critical elements in the primary rod pathway of the mammalian retina, acting as an obligatory conduit of rod signals to both on- and off-center ganglion cells. In addition to the chemical synaptic circuitry they subserve, AII cells form two types of electrical synapses corresponding to gap junctions formed between neighboring AII cells as well as junctions formed between AII cells and on-center cone bipolar cells. Our recent results indicate that coupling between AII cells and cone bipolar cells forms an obligatory synapse for transmission of scotopic visual signals to on-center ganglion cells. In contrast, AII–AII cell coupling acts to maintain the sensitivity of the primary rod pathway by allowing for summation of synchronous activity and the attenuation of asynchronous background noise. Further, the conductance of AII–AII cell gap junctions is highly dynamic, regulated by ambient light conditions, thereby preserving the fidelity of rod signaling over the scotopic operating range from starlight to twilight.

Retinal bipolar cells are interneurons that transmit visual signals from photoreceptors to ganglion cells. Although the visual pathways mediated by bipolar cells have been well characterized, the genes that regulate their development and function are largely unknown. To determine the role in bipolar cell development of the homeobox gene Vsx1 , whose retinal expression is restricted to a major subset of differentiating and mature cone bipolar (CB) cells, we targeted the gene in mice. Bipolar cell fate was not altered in the absence of Vsx1 function, because the pan-bipolar markers Chx10 and Ret-B1 continued to be expressed in inner nuclear layer neurons labeled by the Vsx1 -targeting reporter gene, τ LacZ . The specification, number, and gross morphology of the subset of on-center and off-center (OFF)-CB cells defined by τ LacZ expression from the Vsx1 locus were also normal in Vsx1 τ LacZ / Vsx1 τ LacZ mice. However, the terminal differentiation of OFF-CB cells in the retina of Vsx1 τ LacZ / Vsx1 τ LacZ mice was incomplete, as demonstrated by a substantial reduction in the expression of at least four markers (recoverin, NK3R, Neto1, and CaB5) for these interneurons. These molecular abnormalities were associated with defects in retinal function and documented by electroretinography and in vitro ganglion cell recordings specific to cone visual signaling. In particular, there was a general reduction in the light-mediated activity of OFF, but not on-center, ganglion cells. Thus, Vsx1 is required for the late differentiation and function of OFF-CB cells and is associated with a heritable OFF visual pathway-specific retinal defect.

Diffusible oligomeric assemblies of the amyloid beta-protein (Abeta) could be the primary factor in the pathogenic pathway leading to Alzheimer's disease (AD). Converging lines of evidence support the notion that AD begins with subtle alterations in synaptic efficacy, prior to the occurrence of extensive neuronal degeneration. Recently, however, a shared or overlapping pathogenesis for AD and epileptic seizures occurred as aberrant neuronal hyperexcitability, as well as nonconvulsive seizure activity were found in several different APP transgenic mouse lines. This generated a renewed attention to the well-known comorbidity of AD and epilepsy and interest in how Abeta oligomers influence neuronal excitability. In this study therefore, we investigated the effect of various in vitro-aged Abeta(1-42) oligomer solutions on the perforant pathway-evoked field potentials in the ventral hippocampal dentate gyrus in vivo. Firstly, Abeta oligomer solutions (1 microl, 200 microM) which had been aggregated in vitro for 0, 24 or 72h were injected into the hippocampus of urethane-anesthetized rats, in parallel with in vitro physico-chemical characterization of Abeta oligomerization (atomic force microscopy, thioflavin-T fluorescence). We found a marked increase of hippocampal population spike (pSpike) after injection of the 24-h Abeta oligomer solution and a decrease of the pSpike amplitude after injection of the 72-h Abeta oligomer. Since urethane anesthesia affects the properties of hippocampal evoked potentials, we repeated the injection of these two Abeta oligomer solutions in awake, freely moving animals. Evoked responses to perforant pathway stimulation revealed a 70% increase of pSpike amplitude 50 min after the 24-h Abeta oligomer injection and a 55% decrease after the 72-h Abeta oligomer injection. Field potentials, that reflect synaptic potentials, were not affected by the Abeta injection. These results demonstrate that oligomeric Abeta aggregates elicit opposite electrophysiological effects on neuronal excitability which depend on their degree of oligomerization.

To examine the functions of electrical synapses in the transmission of signals from rod photoreceptors to ganglion cells, we generated connexin36 knockout mice. Reporter expression indicated that connexin36 was present in multiple retinal neurons including rod photoreceptors, cone bipolar cells, and AII amacrine cells. Disruption of electrical synapses between adjacent AIIs and between AIIs and ON cone bipolars was demonstrated by intracellular injection of Neurobiotin. In addition, extracellular recording in the knockout revealed the complete elimination of rod-mediated, on-center responses at the ganglion cell level. These data represent direct proof that electrical synapses are critical for the propagation of rod signals across the mammalian retina, and they demonstrate the existence of multiple rod pathways, each of which is dependent on electrical synapses.

Intracellular recordings were made from narrow-field, bistratified AII amacrine cells in the isolated, superfused retina-eyecup of the rabbit. Pharmacological agents were applied to neurons to dissect the synaptic pathways subserving AII cells so as to determine the circuitry generating their off-surround responses. Application of the GABA antagonists, picrotoxin, bicuculline and 1,2,5,6-tetrahydropyridine-4-yl methylphosphinic acid (TPMPA) all increased the on-centre responses of AII amacrine cells, but attenuated the off-surround activity. At equal concentrations, picrotoxin was approximately twice as effective as bicuculline or TPMPA in modifying the response activity of AII amacrine cells. These results indicate that the mechanism underlying surround inhibition of AII amacrine cells includes activation of both GABA(A) and GABA(C) receptors in an approximately equal ratio. Application of the GABA antagonists also increased the size of on-centre receptive fields of AII amacrine cells. Again, picrotoxin was most effective, producing, on average, a 54 % increase in the size of the receptive field, whereas bicuculline and TPMPA produced comparable 34 and 33 % increases, respectfully. Application of the voltage-gated sodium channel blocker TTX produced effects on AII amacrine cells qualitatively similar to those of the GABA blockers. Intracellular application of the chloride channel blocker 4,4'-dinitro-stilbene-2,2'-disulphonic acid (DNDS) abolished the direct effects of GABA on AII amacrine cells. Moreover, DNDS increased the amplitude of both the on-centre and off-surround responses. The failure of DNDS to block the off-surround activity indicates that it is not mediated by direct GABAergic inhibition. Taken together, our results suggest that surround receptive fields of AII amacrine cells are generated indirectly by the GABAergic, reciprocal feedback synapses from S1/S2 amacrine cells to the axon terminals of rod bipolar cells.

A common feature of neurodegenerative diseases is the formation of misfolded, mostly enzyme resistant proteins. These substances may form toxic assemblies according to the current concept of the neurodegenerative diseases. Overlapping of the misfolded proteins is typical in these disorders. The formation of misfolded proteins and toxic aggregates point to a common pathway of these disorders: failure in normal protein folding in the ER as a consequence of ER-stress and mitochondrial energy production. Alzheimer's disease (AD) is a rather heterogeneous, multifactorial disorder with wide clinical heterogeneity and is classified into several subtypes. In AD the processing of the amyloid precursor protein (APP) and formation of toxic β-amyloid (Aβ) structures occur intraneuronally. Aβ affects both ER and mitochondria and disturbs Ca(2+)-homeostasis of the cells. Mitochondrial dysfunction is one of the main pathological events in AD. Mitochondria accumulate Aβ derived from the ER/Golgi or from the mitochondria-associated ER-membranes (MAM). Free radicals, oxidative stress and increasing Ca(2+)-concentration in mitochondria cause decreased ATP production. Mitochondrial dynamic and trafficking are also altered as a result of Aβ toxicity. Synaptic mitochondria show a very high vulnerability. Depletion of Ca(2+) level in the ER results in dysfunction of protein folding and evokes unfolded protein response (UPR), and affects also mitochondria. MAM may play special role in the ER-mitochondria cross talk. Mitochondria themselves (using mitochondria-targeting antioxidants such as MitoQ) could be a special target for AD treatment. Another targets are the UPR cascade proteins (PERK, IRE1, ATF6) and receptors involved in Ca(2+) -level stabilization of the ER (Ryr, IP3R).

Secondary cell death via gap junctions (GJs) plays a role in the propagation of neuronal loss under a number of degenerative disorders. Here, we examined the role of GJs in neuronal death in the retina, which has arguably the most diverse expression of GJs in the CNS. Initially, we induced apoptotic death by injecting single retinal ganglion cells and glia with cytochrome C and found that this resulted in the loss of neighboring cells to which they were coupled via GJs. We next found that pharmacological blockade of GJs eradicated nearly all amacrine cell loss and reduced retinal ganglion cell loss by ∼70% after induction of either excitotoxic or ischemic insult conditions. These data indicate that the GJ-mediated secondary cell death was responsible for the death of most cells. Whereas genetic deletion of the GJ subunit Cx36 increased cell survivability by ∼50% under excitotoxic condition, cell loss in Cx45 knock-out mouse retinas was similar to that seen in wild-type mice. In contrast, ablation of Cx45 reduced neuronal loss by ∼50% under ischemic insult, but ablation of Cx36 offered no protection. Immunolabeling of the connexins showed differential changes in protein expression consistent with their differing roles in propagating death signals under the two insults. These data indicate that secondary cell death is mediated by different cohorts of GJs dependent on the connexins they express and the type of initial insult. Our results suggest that targeting specific connexins offers a novel therapeutic strategy to reduce progressive cell loss under different neurodegenerative conditions.

Tissue non-specific alkaline phosphatase (TNAP), an abundant ectophosphatase, is present in various organs including the brain and retina of several vertebrate species. Evidence is emerging that TNAP influences neural functions in multiple ways. In rat, strong TNAP activity has been found in retinal vessels, photoreceptors, and both synaptic layers. In the present study, we identified eleven strata of the inner plexiform layer (IPL) by using TNAP histochemistry alone. The TNAP strata corresponded exactly to the strata seen after combined immunohistochemistry with four canonical IPL markers (TH-ChAT-CR-PKCα). Therefore, as described in other mammalian species, our data support the existence of multiple morphologically and functionally discernible IPL strata in rats. Remarkably, the stratification pattern of the IPL was severely disrupted in a diabetic rat model, even before changes in the canonical IPL markers were detectable. These findings indicate that TNAP histochemistry offers a more straightforward, but also more sensitive, method for investigating retinal strata and their diabetes-induced degeneration.

Connexin36 (Cx36) is the major gap junction forming protein in the brain and the retina; thus, alterations in its expression indicate changes in the corresponding circuitry. Many structural changes occur in the early postnatal retina before functional neuronal circuits are finalized, including those that incorporate gap junctions. To reveal the time-lapse formation of inner retinal gap junctions, we examine the developing postnatal rat retina from birth (P0) to young adult age (P20) and follow the expression of Cx36 in the mRNA and protein levels. We found a continuous elevation in the expression of both the Cx36 transcript and protein between P0 and P20 and a somewhat delayed Cx36 plaque formation throughout the inner plexiform layer (IPL) starting at P10. By using tristratificated calretinin positive (CaR(+)) fibers in the IPL as a guide, we detected a clear preference of Cx36 plaques for the ON sublamina from the earliest time of detection. This distributional preference became more pronounced at P15 and P20 due to the emergence and widespread expression of large (0.1 μm(2)) Cx36 plaques in the ON sublamina. Finally, we showed that parvalbumin-positive (PV(+)) AII amacrine cell dendrites colocalize with Cx36 plaques as early as P10 in strata 3 and 4, whereas colocalizations in stratum 5 became characteristic only around P20. We conclude that Cx36 expression in the rat IPL displays a characteristic succession of changes during retinogenesis reflecting the formation of the underlying electrical synaptic circuitry. In particular, AII cell gap junctions, first formed with ON cone bipolar cells and later with other AII amacrine cells, accounted for the observed Cx36 expressional changes.

Evidence is emerging with regard to the role of tissue non-specific alkaline phosphatase (TNAP) in neural functions. As an ectophosphatase, this enzyme might influence neural activity and synaptic transmission in diverse ways. The localization of the enzyme in known neural circuits, such as the retina, might significantly advance an understanding of its role in normal and pathological functioning. However, the presence of TNAP in the retina is scarcely investigated. Our multispecies comparative study (zebrafish, cichlid, frog, chicken, mouse, rat, golden hamster, guinea pig, rabbit, sheep, cat, dog, ferret, squirrel monkey, human) using enzyme histochemistry and Western blots has shown the presence of TNAP activity in the retina of several mammalian species, including humans. Although the TNAP activity pattern varies across species, we have observed the following trends: (1) in all investigated species (except golden hamster), retinal vessels display TNAP activity; (2) TNAP activity consistently occurs in the photoreceptor layer; (3) in majority of the investigated species, marked TNAP activity is present in the outer and inner plexiform layers. In zebrafish, frog, chicken, guinea pig, and rat, TNAP histochemistry has revealed several sublayers of the inner plexiform layer. Frog, golden hamster, guinea pig, mouse, and human retinas possess a subpopulation of amacrine cells positively staining for TNAP activity. The expression of TNAP in critical sites of retinal signal transmission across a wide range of species suggests its fundamental, evolutionally conserved role in vision.

Calretinin-containing retinal amacrine cells are heterogeneous with regard to their neurochemical properties. In the rabbit retina, about 90% of them contain glycine, as evidenced in the present study by double-label immunocytochemistry. In a previous report, we showed that a small population of amacrine cells contains both γ-aminobutyric acid and calretinin. In this study, we further identified this cell population by means of known secondary markers. However, none of the markers we tested (choline acetyltransferase, serotonin accumulation, NADPH-diaphorase, vasoactive intestinal polypeptide) co-localized with calretinin. A small population (1%) of the cells in the ganglion cell layer contains both calretinin and glycine. Since calretinin-positive cells in the ganglion cell layer have been identified as ganglion cells based on soma size and presence of calretinin-positive axons in the optic nerve fiber layer, this population may represent a class of ganglion cell which contains glycine. Our results, together with those of other studies, suggest that calretinin is not a general marker of any of the well-known amacrine cell types in the mammalian retina. Rather, calretinin, just as other calcium-binding proteins, is distributed in a species-specific manner. At the same time it appears that, as shown for horizontal cells, one or more of the major buffer-type calcium-binding proteins of the EF-hand family is present in most of the retinal amacrine cells.

Twenty Holstein-Friesian breeding bulls (62-79 months of age) were examined 3 times, at 30-day intervals. Scrotal thermograms for assessment of scrotal surface temperature (SST) and blood samples for plasma testosterone concentrations were taken just before and then 45 and 90 min, respectively, after treatment with GnRH (50 micrograms, Gonavet, i.m. per bull). Following GnRH treatment, there generally were significant increases in mean values of both top SST (range, -0.1 to 1.4 degrees C) and bottom SST (range, 0.3 to 1.8 degrees C). Scrotal circumference was highly repeatable but SST and video-measurements of scrotal dimensions were less repeatable, because apparently they were affected by ambient temperature. Plasma testosterone concentrations before GnRH treatment were more repeatable than those after GnRH treatment. Correlations between examinations of 0.67 to 0.81 and -0.14 to 0.47, respectively, but the converse was true for SST measurements. Semen was collected with an artificial vagina 3 times per week for 12 weeks starting 2 weeks before the first examination. The total number of spermatozoa per ejaculate was highly repeatable and the percentage of motile and live spermatozoa were relatively consistent. Separate regressions for each variable and for each examination were conducted for these 3 semen characteristics as dependent variables. For the number of spermatozoa per ejaculate and for the percentage of motile spermatozoa, significant independent variables were plasma testosterone concentrations and difference between top and bottom SST, respectively. The slopes of these equations were nearly all negative and the R2 was from 0.15 to 0.42. For prediction of the percentage of live spermatozoa, both SST gradient and plasma testosterone concentrations were significant independent variables. For these regressions, the slopes were negative and the regression coefficients were generally lower than for the other 2 dependent variables (range, 0.16 to 0.25). Treatment with GnRH and assessment of SST and plasma testosterone concentrations have some correlation with the semen production in the mature bull.

The frog retina contains numerous 28 kDa calbindin positive elements in its every layer. At the same time, parvalbumin has been observed only in a few elements in the visual system of amphibian species, whilst calretinin immunoreactivity could be detected in salamander retina and the optic tectum of tench. However, the presence and distribution of calretinin have been described to date neither in the retina nor in the other parts of the visual system of anurans. Therefore, the aim of this study is to describe the calretinin immunoreactive elements in the retina and the optic tectum of the frog and to establish whether or not the expression of this calcium-binding protein is transmitter-related and/or cell type specific in these parts of the central nervous system. In the retina, numerous bipolar cells showed calretinin immunoreactivity. The axon terminals of the bipolar cells branched in both the OFF and ON sublayers of the inner plexiform layer. The few labeled amacrine cells were larger than 10 μ m in diameter. Over 50% of the cells in the ganglion cell layer contained calretinin. The labeled cells in the ganglion cell layer were of usually 16–22 μ m in diameter, although a few smaller cells were also seen. Accordingly, many optic fibers were also labeled. In colocalization experiments, gamma-aminobutyric acid and calretinin were found in partially overlapping amacrine cell populations, cells with the former marker being much more numerous. At the same time, all the gamma-aminobutyric acid positive bipolar cells also contained calretinin. Most of the calretinin positive neurons in the ganglion cell layer however were only single-labeled. Axons of ganglion cells terminated in B, C and F sublayers of layer 9 in the optic tectum. Local tectal neuron populations in layer 4, 6, 8 and 9 were also labeled and a few calretinin positive cells were detected also in layer 2. Approximately 10% of the tectal cells were found to be immunoreactive for calretinin. Layer 4 and 6 cells were mostly large pear-shaped neurons while cells in the 8th layer were small pear-shaped and ganglion cells labeled too. Coexistence of gamma-aminobutyric acid and calretinin was characteristic in cells of the upper tectal layers while they were not detected in neurons of deep layers of the tectum. After monocular enucleation, contralateral to the removed eye, calretinin-immunoreactivity disappeared almost completely from F sublayer and became less pronounced in sublayers B and C after 90 days. Calretinin-immunoreactivity remained mostly unchanged in local tectal cells. The results show that, although its function remains undetermined, calretinin is the major EF-hand calcium-binding protein in the frog retina and optic tectum.

In the rabbit retina, parvalbumin has been localized selectively to AII amacrine cells, while 28 kDa calbindin could be detected in horizontal cells, in one type of depolarizing cone bipolar cell and a population of wide-field amacrine cells. The distribution of the third neuronal calcium binding protein, calretinin, however, has not been studied to date in detail in the rabbit retina. Therefore in this study we aimed to describe the overall distribution of calretinin in the different retinal layers and the possible colocalization pattern with other neurochemical marker molecules. A few cone photoreceptor cells were found to be labeled, whereas the outer plexiform layer was free from immunoreactive elements. In the most proximal row of the inner nuclear layer amacrine cells were labeled, while more distally a few cells emitted beaded axon-like processes toward the outer retina. There were large (18-28 microm in diameter) cells labeled in the ganglion cell layer, of which many apparently had their axon stained. Some of the calretinin immunoreactive amacrine cells (the AII neurons) also contained parvalbumin. Colocalization of calretinin and 28 kDa calbindin could not be ascertained in the same amacrine cell populations, nor was tyrosine hydroxylase present in calretinin-containing cells. There was partial colocalization of calretinin in the gamma-aminobutyric acid-positive amacrine cell population. Parvalbumin containing ganglion cells were also positive for calretinin; however, the calretinin-positive ganglion cells were more numerous. gamma-Aminobutyric acid could be colocalized in some calretinin-positive neurons of the ganglion cell layer.

Neurons throughout the brain show spike activity that is temporally correlated to that expressed by their neighbors, yet the generating mechanism(s) remains unclear. In the retina, ganglion cells (GCs) show robust, concerted spiking that shapes the information transmitted to central targets. Here we report the synaptic circuits responsible for generating the different types of concerted spiking of GC neighbors in the mouse retina. The most precise concerted spiking was generated by reciprocal electrical coupling of GC neighbors via gap junctions, whereas indirect electrical coupling to a common cohort of amacrine cells generated the correlated activity with medium precision. In contrast, the correlated spiking with the lowest temporal precision was produced by shared synaptic inputs carrying photoreceptor noise. Overall, our results demonstrate that different synaptic circuits generate the discrete types of GC correlated activity. Moreover, our findings expand our understanding of the roles of gap junctions in the retina, showing that they are essential for generating all forms of concerted GC activity transmitted to central brain targets.

Gap junctions connect cells in the bodies of all multicellular organisms, forming either homologous or heterologous (i.e. established between identical or different cell types, respectively) cell-to-cell contacts by utilizing identical (homotypic) or different (heterotypic) connexin protein subunits. Gap junctions in the nervous system serve electrical signaling between neurons, thus they are also called electrical synapses. Such electrical synapses are particularly abundant in the vertebrate retina where they are specialized to form links between neurons as well as glial cells. In this article, we summarize recent findings on retinal cell-to-cell coupling in different vertebrates and identify general features in the light of the evergrowing body of data. In particular, we describe and discuss tracer coupling patterns, connexin proteins, junctional conductances and modulatory processes. This multispecies comparison serves to point out that most features are remarkably conserved across the vertebrate classes, including (i) the cell types connected via electrical synapses; (ii) the connexin makeup and the conductance of each cell-to-cell contact; (iii) the probable function of each gap junction in retinal circuitry; (iv) the fact that gap junctions underlie both electrical and/or tracer coupling between glial cells. These pan-vertebrate features thus demonstrate that retinal gap junctions have changed little during the over 500 million years of vertebrate evolution. Therefore, the fundamental architecture of electrically coupled retinal circuits seems as old as the retina itself, indicating that gap junctions deeply incorporated in retinal wiring from the very beginning of the eye formation of vertebrates. In addition to hard wiring provided by fast synaptic transmitter-releasing neurons and soft wiring contributed by peptidergic, aminergic and purinergic systems, electrical coupling may serve as the ‘skeleton’ of lateral processing, enabling important functions such as signal averaging and synchronization.

Our study investigated the effect of ischemic postconditioning (IPO) in intestinal warm ischemia/reperfusion (I/R) and autotransplantation models.Warm ischemia was performed by occlusion of superior mesenteric artery for 1, 3 and 6 hours in white domestic pigs (n = 15). Prior to 3 hours reperfusion the intestine was postconditioned by 3 cycles of 30-seconds ischemia and 30-seconds reperfusion (IPO protocol). In the cold ischemia group (n = 15) the bowel was preserved in University of Wisconsin solution for 1, 3, and 6 hours. Prior to 3 hours reperfusion IPO protocol was applied, too. Tissue samples were collected after laparotomy (control) and at the end of the reperfusion periods. As far as oxidative stress markers, malondialdehyde and reduced glutathione (GSH) levels and superoxide dismutase (SOD) activity were determined. Tissue damage was evaluated by qualitative (Park-classification) and quantitative (Scion Image) methods.As regards oxidative stress parameters, lipidperoxidation decreased and the protective effect of endogenous antioxidants (GSH, SOD) retained significantly by IPO procedure at the end of reperfusion. Tissue injury correlated significantly by the duration of warm ischemia and cold preservation. Quantitative analysis demonstrated that IPO ameliorated tissue injury in each group (p0.05).IPO significantly attenuated intestinal oxidative stress and morphological damages in warm and cold I/R models.

A fundamental organizing feature of the visual system is the segregation of ON and OFF responses into parallel streams to signal light increment and decrement. However, we found that blockade of GABAergic inhibition unmasks robust ON responses in OFF α-ganglion cells (α-GCs). These ON responses had the same centre-mediated structure as the classic OFF responses of OFF α-GCs, but were abolished following disruption of the ON pathway with L-AP4. Experiments showed that both GABA(A) and GABA(C) receptors are involved in the masking inhibition of this ON response, located at presynaptic inhibitory synapses on bipolar cell axon terminals and possibly amacrine cell dendrites. Since the dendrites of OFF α-GCs are not positioned to receive excitatory inputs from ON bipolar cell axon terminals in sublamina-b of the inner plexiform layer (IPL), we investigated the possibility that gap junction-mediated electrical synapses made with neighbouring amacrine cells form the avenue for reception of ON signals. We found that the application of gap junction blockers eliminated the unmasked ON responses in OFF α-GCs, while the classic OFF responses remained. Furthermore, we found that amacrine cells coupled to OFF α-GCs display processes in both sublaminae of the IPL, thus forming a plausible substrate for the reception and delivery of ON signals to OFF α-GCs. Finally, using a multielectrode array, we found that masked ON and OFF signals are displayed by over one-third of ganglion cells in the rabbit and mouse retinas, suggesting that masked crossover excitation is a widespread phenomenon in the inner mammalian retina.