Synaptic Endings Research Articles

Solubility of α-synuclein species in the L62 mouse model of synucleinopathy

The accumulation of α-synuclein (α-Syn) into Lewy bodies is a hallmark of synucleinopathies, a group of neurological disorders that include Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Small oligomers as well as larger fibrils of α-Syn have been suggested to induce cell toxicity leading to a degenerative loss of neurones. A richer understanding of α-Syn aggregation in disease, however, requires the identification of the different α-Syn species and the characterisation of their biochemical properties. We here aimed at a more in-depth characterisation of the α-Syn transgenic mice, Line 62 (L62), and examined the deposition pattern and solubility of human and murine α-Syn in these mice using immunohistochemical and biochemical methods. Application of multiple antibodies confirmed mAb syn204 as the most discriminatory antibody for human α-Syn in L62. Syn204 revealed an intense and widespread immunohistochemical α-Syn labelling in parietal cortex and hippocampus, and to a lower level in basal forebrain and hindbrain regions. The labelled α-Syn represented somatic inclusions as well as processes and synaptic endings. Biochemical analysis revealed a Triton-resistant human α-Syn pool of large oligomers, a second pool of small oligomers that was not resistant to solubilization with urea/Triton. A third SDS-soluble pool of intermediate sized aggregates containing a mixture of both, human and mouse α-Syn was also present. These data suggest that several pools of α-Syn can exist in neurones, most likely in different cellular compartments. Information about these different pools is important for the development of novel disease modifying therapies aimed at α-Syn.

Representing connectivity circuits in autonomic physiology: the SPARC map of organ innervation

The Stimulating Peripheral Activity to Relieve Conditions (SPARC) program is a NIH-funded consortium to improve the understanding of how the autonomic nervous system (ANS) interacts with end organs and the central nervous system. A major goal of SPARC is to use this knowledge to develop the next generation of neuromodulator devices as effective disease therapies. To support neuromodulation planning, the routes of ANS neuron populations (as well as those populations conveying visceral sensing) are to be mapped in terms of the anatomical structures where they pass through or terminate. Such a map would also serve as an organizing scaffold for (i) a growing SPARC repository of electrophysiology recordings and molecular assay data, as well as (ii) the computational simulation of the effect of neural stimulation on any relevant point on the CNS or ANS. The resulting SPARC map represents connectivity as a network of conduits, and is known as the SPARC Connectivity Knowledge Base of the Autonomic Nervous System (SCKAN). SCKAN contains explicit knowledge about CNS-ANS-end organ circuitry derived from SPARC data and scientific literature, in a form that supports computational reasoning. All connections are annotated with standard reference SPARC vocabularies allowing us to integrate datasets that are annotated to the same annotation standard. Circuits on the map represent details of ANS connectivity associated with a particular organ, e.g., bladder control, defensive breathing, modulation of peristalsis or cardiac inotropy/chronotropy. These circuits are created through interviews with SPARC investigators, anatomical experts and the scientific literature. They contain detailed representations of neuron populations giving rise to ANS connections, including mappings of the locations of cell bodies, dendrites, axon segments and synaptic endings. Circuits are modeled using ApiNATOMY, a knowledge model and tool suite specifically created to represent biological connectivity. Furthermore, these detailed circuits are supplemented with general knowledge on connectivity between CNS nuclei, ANS ganglia, nerves, and end organs derived from the scientific literature. To facilitate extracting this knowledge from the literature and maintaining relevance over time, we developed a Natural Language Processing (NLP) pipeline, which extracts connectivity relationships between unique anatomical structures within a sentence from the scientific literature. SCKAN is being used to create a queryable visual atlas of ANS circuitry through the SPARC portal. SPARC Funding: NIH Common Fund, NIH Office of the Director, Award OT2 OD030541 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ultrastructural Immunocytochemistry of GABAergic Cells in Neocortical Neurotransplants.

Neural transplantation is a promising regenerative therapy in the treatment of several neurological diseases. Importantly, transplanted tissue should not become a source of pathological functional activity. To assess the possibility of maintaining the balance between excitatory and inhibitory processes, an electron microscopic immunochemical study of the GABAergic system in rat neocortical transplants was performed. Accumulation of GABA-positive label in astrocytes and a relatively insignificant immune reaction to GABA in neurons and synaptic endings were found. These findings suggest that under conditions of impaired differentiation of GABA-containing neurons that generate phasic inhibition through inhibitory synapses, tonic inhibitory influences predominate in neurotransplants due to GABA released from astrocytes.

Embryonic medial ganglionic eminence cells survive and integrate into the inferior colliculus of adult mice

Acoustic overexposure can lead to decreased inhibition in auditory centers, including the inferior colliculus (IC), and has been implicated in the development of central auditory pathologies. While systemic drugs that increase GABAergic transmission have been shown to provide symptomatic relief, their side effect profiles impose an upper-limit on the dose and duration of use. A treatment that locally increases inhibition in auditory nuclei could mitigate these side effects. One such approach could be transplantation of inhibitory precursor neurons derived from the medial ganglionic eminence (MGE). The present study investigated whether transplanted MGE cells can survive and integrate into the IC of non-noise exposed and noise exposed mice. MGE cells were harvested on embryonic days 12-14 and injected bilaterally into the IC of adult mice, with or without previous noise exposure. At one-week post transplantation, MGE cells possessed small, elongated soma and bipolar processes, characteristic of migrating cells. By 5 weeks, MGE cells exhibited a more mature morphology, with multiple branching processes and axons with boutons that stain positive for the vesicular GABA transporter (VGAT). The MGE survival rate after 14 weeks post transplantation was 1.7% in non-noise exposed subjects. MGE survival rate was not significantly affected by noise exposure (1.2%). In both groups the vast majority of transplanted MGE cells (>97%) expressed the vesicular GABA transporter. Furthermore, electronmicroscopic analysis indicated that transplanted MGE cells formed synapses with and received synaptic endings from host IC neurons. Acoustic stimulation lead to a significant increase in the percentage of endogenous inhibitory cells that express c-fos but had no effect on the percentage of c-fos expressing transplanted MGE cells. MGE cells were observed in the IC up to 22 weeks post transplantation, the longest time point investigated, suggesting long term survival and integration. These data provide the first evidence that transplantation of MGE cells is viable in the IC and provides a new strategy to explore treatment options for central hearing dysfunction following noise exposure.

Topographical Mapping of Catecholaminergic Axon Distribution and Morphology in the Flat‐Mounts of the Mouse Heart

The sympathetic nervous system plays a crucial role in the control of heart functions. However, the distribution and morphology of sympathetic postganglionic innervation of the heart has not been well elucidated, which has significantly impeded the understanding of the sympathetic neuroanatomical organization and control of the heart. Our goal is to provide a map of the topographical innervation of sympathetic postganglionic neurons in the whole heart of mice. In this study, we used Tyrosine hydroxylase (TH) as a marker for sympathetic axons and applied a combination of state‐of‐the‐art techniques, including confocal microscopy, Zeiss Imager microscopy, flat‐mount tissue processing and immunohistochemistry, Neurolucida 360 Tracing and integration of the tracing data onto a 3D heart scaffold. We found that 1) Multiple large extrinsic TH‐IR nerve bundles entered the heart and branched out into smaller axons which covered the whole left and right atria and ventricles, 2) These TH‐IR bundles branched out into smaller axon bundles and ramified into individual axons covering the whole left and right atria and ventricles. 3) The TH‐IR axons formed an extensive neural network in the epicardium and axons formed varicose synaptic endings within the cardiac muscle. 4) TH‐IR axons and terminals were densest in the sinoatrial (SA) and atrioventricular (AV) node regions. 5) TH‐IR axons innervated blood vessels and fat cells. 6) Many principal neurons in intrinsic cardiac ganglia and small intensely fluorescent (SIF) cells were TH‐IR. 7) However, TH‐IR axons do not appear to innervate any cardiac principal neurons and SIF cells. 8) TH‐IR axon innervation will be integrated into a 3D scaffold. Our work will provide a comprehensive topographical map of the catecholaminergic efferent axon distribution, innervation, and morphology of the whole heart at single cell/axon/synapse scale that will be used to create a cardiac sympathetic‐brain atlas. It will also provide an anatomical reference to evaluate the altered sympathetic cardiac control in pathological conditions. The first two authors contributed equally to this work.

Distribution of Aquaporin-4 channels in hippocampus and prefrontal cortex in mk-801-treated balb/c mice

ABSTRACT Functional disorders of the glymphatic system and Aquaporin-4 (AQP-4) channels take part in the pathophysiology of neurodegenerative disease. The aim of this study was to describe the distribution of AQP-4 channels in the prefrontal cortex and hippocampus in a mouse model of NMDA receptor blocking agent-induced schizophrenia-like behavior model. NMDA receptor antagonist MK-801 was used to produce the experimental schizophrenia model. MK-801 injections were administered for eleven days to Balb/c mice intraperitoneally. Beginning from the sixth day of injection, the spatial learning and memory of the mice were tested by the Morris water maze (MWM) task. A group of mice was injected with MK-801 for ten days without the MWM task. Hippocampus and prefrontal specimens were collected from this group. Tissue samples were stained immunohistochemically and AQP-4 channels were examined by electron microscope. Time to find the platform was significantly longer at MK-801 injected group than the control group at the MWM task. Also, time spent at the target quadrant by the MK-801 group was shorter compared to the control group. AQP-4 expression increased significantly at MK-801 group glial cells, neuronal perikaryon, perineuronal and pericapillary spaces. In the MK-801 group, there was remarkable damage in neurons and glial cells. Increased AQP-4 channel expression and neurodegeneration at the MK-801 group induced with schizophrenia-like behavior model. MK-801 induced NMDA receptor blockade causes a decline in cognitive and memory functions. Increased AQP-4 expression at the prefrontal cortex and hippocampus to elicit and transport products of synaptic neurotransmitters and end metabolites is suggested.

Expression of Ectonucleoside Triphosphate Diphosphohydrolase 2 (NTPDase2) Is Negatively Regulated Under Neuroinflammatory Conditions In Vivo and In Vitro.

Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) hydrolyzes extracellular ATP to ADP, which is the ligand for P2Y1,12,13 receptors. The present study describes the distribution of NTPDase2 in adult rat brains in physiological conditions, and in hippocampal neurodegeneration induced by trimethyltin (TMT). The study also describes the regulation of NTPDase2 by inflammatory mediators in primary astrocytes and oligodendroglial cell line OLN93. In physiological conditions, NTPDase2 protein was most abundant in the hippocampus, where it was found in fibrous astrocytes and synaptic endings in the synaptic-rich hippocampal layers. In TMT-induced neurodegeneration, NTPDase2-mRNA acutely decreased at 2-dpi and then gradually recovered to the control level at 7-dpi and 21-dpi. As determined by immunohistochemistry and double immunofluorescence, the decrease was most pronounced in the dentate gyrus (DG), where NTPDase2 withdrew from the synaptic boutons in the polymorphic layer of DG, whereas the recovery of the expression was most profound in the subgranular layer. Concerning the regulation of NTPDase2 gene expression, proinflammatory cytokines IL-6, IL-1β, TNFα, and IFNγ negatively regulated the expression of NTPDase2 in OLN93 cells, while did not altering the expression in primary astrocytes. Different cell-intrinsic stressors, such as depletion of intracellular energy store, oxidative stress, endoplasmic reticulum stress, and activation of protein kinase C, also massively disturbed the expression of the NTPDase2 gene. Together, our results suggest that the expression and the activity of NTPDase2 transiently cease in neurodegeneration and brain injury, most likely as a part of the acute adaptive response designed to promote cell defense, survival, and recovery.

Influence of Topiramate on the Synaptic Endings of the Temporal Lobe Neocortex in an Experimental Model of Hyperthermia-Induced Seizures: An Ultrastructural Study.

The objective of this pioneering study was to assess potentially neuroprotective properties of topiramate (TPM), a broad spectrum and newer-generation antiepileptic used against damage to synaptic endings of the temporal lobe neocortex in experimental hyperthermia-induced seizures (HS). TPM (80 mg/kg b.m.) was administered in young male Wistar rats with an intragastric tube before and immediately after HS. Specimens (1 mm3) collected from the neocortex, fixed via transcardial perfusion with paraformaldehyde and glutaraldehyde solution, were routinely processed for transmission-electron microscopic study, i.e., for descriptive and morphometric analysis. The ultrastructure of neocortical neuropil components affected by hyperthermic stress showed distinct swelling of pre and post-synaptic axodendritic and axospinal endings, including total disintegration. Mitochondria were markedly damaged in synaptic structures. Axoplasm of presynaptic boutons contained a decreased number of synaptic vesicles. Synaptic junctions showed active zone-shortening. Preventive administration of TPM before HS induction demonstrated neuroprotective effects against synaptic damage in approximately 1/4 of these structures. Interestingly, beneficial effects on synapsis morphology were more common in perivascular zones close to well-preserved capillaries. They were demonstrated by smaller swelling of both presynaptic and postsynaptic parts, well-preserved mitochondria, an increased number and regular distribution of synaptic vesicles within axoplasm, and a significantly increased synaptic active zones. However, topiramate used directly after HS was ineffective in the prevention of hyperthermia-evoked synaptic injury. Our findings support the hypothesis that topiramate applied before HS can protect some neocortical synapses via the vascular factor by enhancing blood–brain barrier components and improving the blood supply of gray matter in the temporal lobe, which may be significant in febrile seizure-prevention in children.

Multiple Pathways of LRRK2-G2019S/Rab10 Interaction in Dopaminergic Neurons.

Background:Inherited mutations in the LRRK2 protein are common causes of Parkinson’s disease, but the mechanisms by which increased kinase activity of mutant LRRK2 leads to pathological events remain to be determined. In vitro assays (heterologous cell culture, phospho-protein mass spectrometry) suggest that several Rab proteins might be directly phosphorylated by LRRK2-G2019S. An in vivo screen of Rab expression in dopaminergic neurons in young adult Drosophila demonstrated a strong genetic interaction between LRRK2-G2019S and Rab10.Objective:To determine if Rab10 is necessary for LRRK2-induced pathophysiological responses in the neurons that control movement, vision, circadian activity, and memory. These four systems were chosen because they are modulated by dopaminergic neurons in both humans and flies.Methods:LRRK2-G2019S was expressed in Drosophila dopaminergic neurons and the effects of Rab10 depletion on Proboscis Extension, retinal neurophysiology, circadian activity pattern (‘sleep’), and courtship memory determined in aged flies.Results:Rab10 loss-of-function rescued LRRK2-G2019S induced bradykinesia and retinal signaling deficits. Rab10 knock-down, however, did not rescue the marked sleep phenotype which results from dopaminergic LRRK2-G2019S. Courtship memory is not affected by LRRK2, but is markedly improved by Rab10 depletion. Anatomically, both LRRK2-G2019S and Rab10 are seen in the cytoplasm and at the synaptic endings of dopaminergic neurons.Conclusion:We conclude that, in Drosophila dopaminergic neurons, Rab10 is involved in some, but not all, LRRK2-induced behavioral deficits. Therefore, variations in Rab expression may contribute to susceptibility of different dopaminergic nuclei to neurodegeneration seen in people with Parkinson’s disease.

Limits of temperature adaptation and thermopreferendum

BackgroundManaging the limits of temperature adaptation is relevant both in medicine and in biotechnology. There are numerous scattered publications on the identification of the temperature limits of existence for various organisms and using different methods. Dmitry Petrovich Kharakoz gave a general explanation for many of these experimental results. The hypothesis implied that each cycle of synaptic exocytosis includes reversible phase transitions of lipids of the presynaptic membrane due to the entry and subsequent removal of calcium ions from the synaptic terminal. The correspondence of the times of phase transitions has previously been experimentally shown on isolated lipids in vitro. In order to test the hypothesis of D.P. Kharakoz in vivo, we investigated the influence of the temperature of long-term acclimatization on the temperature of heat and cold shock, as well as on the kinetics of temperature adaptation in zebrafish. Testing the hypothesis included a comparison of our experimental results with the results of other authors obtained on various models from invertebrates to humans.ResultsThe viability polygon for Danio rerio was determined by the minimum temperature of cold shock (about 6 °C), maximum temperature of heat shock (about 43 °C), and thermopreferendum temperature (about 27 °C). The ratio of the temperature range of cold shock to the temperature range of heat shock was about 1.3. These parameters obtained for Danio rerio describe with good accuracy those for the planarian Girardia tigrina, the ground squirrel Sermophilus undulatus, and for Homo sapiens.ConclusionsThe experimental values of the temperatures of cold shock and heat shock and the temperature of the thermal preferendum correspond to the temperatures of phase transitions of the lipid-protein composition of the synaptic membrane between the liquid and solid states. The viability range for zebrafish coincides with the temperature range, over which enzymes function effectively and also coincides with the viability polygons for the vast majority of organisms. The boundaries of the viability polygon are characteristic biological constants. The viability polygon of a particular organism is determined not only by the genome, but also by the physicochemical properties of lipids that make up the membrane structures of synaptic endings. The limits of temperature adaptation of any biological species are determined by the temperature range of the functioning of its nervous system.

The association of C789A polymorphism in the dopamine beta-hydroxylase gene (DBH) and aggressive behaviour in dogs

The genetic basis of aggressive behaviour has been examined extensively, including analysis of genes encoding neurotransmitters, signalling molecules and regulatory enzymes, as well as their synthesis and degradation. Dopamine beta-hydroxylase, an enzyme catalysing the conversion of dopamine into norepinephrine in synaptic endings, significantly affects the modulation of emotional states and behaviour. The aim of this study was to determine the association of C789A polymorphism in the canine dopamine beta-hydroxylase gene (DBH) and aggressive behaviour in dogs. A total of 110 dogs of different breeds were analysed. All animals were classified according to their individual behavioural characteristics, defined by a veterinary interview and observation. Polymorphism was analysed using ACRS-PCR (amplification created restriction site-polymerase chain reaction) method. Significant differences in DBH genotypes and allele frequency between aggressive and non-aggressive dogs were observed (χ2 = 16,232, P = 0.0003). In aggressive dogs, the CC genotype (0.788) and C allele (0.815) were most frequent while in non-aggressive dogs, their frequencies were significantly lower (0.361 and 0.404, respectively). The obtained results indicate that DBH is a promising candidate gene for canine behavioural study.

BIOCHEMISTRY OF SHORT-TERM AND LONG-TERM MEMORY

Memory is provided by changes in synapses in neural circuits: short-term memory - by functional changes in a separate sensory neuron and a separate motor neuron, long-term memory - by structural changes (regrowth of new synapses).During the formation of short-term memory in synapses, cAMP, protein kinase A, are used, which act inside the cell and transmit a signal that cause the release of large amounts of the neurotransmitter glutamate. Two independent mechanisms are involved in the formation of long-term memory: - one triggers a long-term strengthening of synaptic connections, directing protein kinase A to the nucleus, which activates the CREB protein, thereby turning on the structural genes encoding proteins necessary for the growth of new synaptic connections; - the other reinforces the already formed memory, supporting the newly formed synaptic endings, which requires local synthesis of proteins.

Spatiotemporal Analysis of Cochlear Nucleus Innervation by Spiral Ganglion Neurons that Serve Distinct Regions of the Cochlea

Cochlear neurons innervate the brainstem cochlear nucleus in a tonotopic fashion according to their sensitivity to different sound frequencies (known as the neuron’s characteristic frequency). It is unclear whether these neurons with distinct characteristic frequencies use different strategies to innervate the cochlear nucleus. Here, we use genetic approaches to differentially label spiral ganglion neurons (SGNs) and their auditory nerve fibers (ANFs) that relay different characteristic frequencies in mice. We found that SGN populations that supply distinct regions of the cochlea employ different cellular strategies to target and innervate neurons in the cochlear nucleus during tonotopic map formation. ANFs that will exhibit high-characteristic frequencies initially overshoot and sample a large area of targets before refining their connections to correct targets, while fibers that will exhibit low-characteristic frequencies are more accurate in initial targeting and undergo minimal target sampling. Moreover, similar to their peripheral projections, the central projections of ANFs show a gradient of development along the tonotopic axis, with outgrowth and branching of prospective high-frequency ANFs initiated about two days earlier than those of prospective low-frequency ANFs. The processes of synaptogenesis are similar between high- and low-frequency ANFs, but a higher proportion of low-frequency ANFs form smaller endbulb synaptic endings. These observations reveal the diversity of cellular mechanisms that auditory neurons that will become functionally distinct use to innervate their targets during tonotopic map formation.

Analgesia with Gabapentin and Pregabalin May Involve N-Methyl-d-Aspartate Receptors, Neurexins, and Thrombospondins.

The gabapentinoid drugs gabapentin and pregabalin (Neurontin and Lyrica) are mainstay treatments for neuropathic pain and preventing focal seizures. Both drugs have similar effects to each other in animal models and clinically. Studies have shown that a protein first identified as an auxiliary subunit of voltage-gated calcium channels (the α 2 δ-subunit type 1 [α 2 δ-1], or Ca V a2d1) is the high-affinity binding site for gabapentin and pregabalin and is required for the efficacy of these drugs. The α 2 δ-1 protein is required for the ability of gabapentin and pregabalin to reduce neurotransmitter release in neuronal tissue, consistent with a therapeutic mechanism of action via voltage-gated calcium channels. However, recent studies have revealed that α 2 δ-1 interacts with several proteins in addition to voltage-gated calcium channels, and these additional proteins could be involved in gabapentinoid pharmacology. Furthermore, gabapentin and pregabalin have been shown to modify the action of a subset of N-methyl-d-aspartate-sensitive glutamate receptors, neurexin-1α, and thrombospondin proteins by binding to α 2 δ-1. Thus, these effects may contribute substantially to gabapentinoid therapeutic mechanism of action. SIGNIFICANCE STATEMENT: It is widely believed that gabapentin and pregabalin act by modestly reducing the membrane localization and activation of voltage-gated calcium channels at synaptic endings in spinal cord and neocortex via binding to the α 2 δ-1 protein. However, recent findings show that the α 2 δ-1 protein also interacts with N-methyl-d-aspartate-sensitive glutamate receptors, neurexin-1α, thrombospondins (adhesion molecules), and other presynaptic proteins. These newly discovered interactions, in addition to actions at calcium channels, may be important mediators of gabapentin and pregabalin therapeutic effects.

Pre‐hypertensive molecular alterations in the paraventricular nucleus of the hypothalamus during angiotensin‐II hypertension

The paraventricular nucleus of the hypothalamus (PVN) plays a critical role in the development of hypertension in response to elevated levels of angiotensin‐II (Ang‐II). Upstream circumventricular organs, regions lacking a blood‐brain‐barrier, sense circulating Ang‐II and in turn influence endocrine and autonomic control via efferent projections to the PVN. Despite progress in unraveling the function of the PVN in hypertension development, the underlying signaling mechanisms remain unclear. Notably, the majority of investigations have examined alterations in the PVN during established hypertension, which limits our understanding of temporal changes that may occur prior to elevations in arterial blood pressure. Based on this, we investigated key molecular targets in the PVN during the pre‐hypertensive phase of Ang‐II hypertension. C57Bl/6J male mice were implanted with subcutaneous osmotic minipumps for chronic delivery of low dose Ang‐II (600 ng/kg/min). Brains were harvested for immunohistochemistry analysis of target molecules (n=3–4/group) at baseline (day 0) and pre‐hypertensive (days 3, 5, 7) timepoints. Histological analysis of synaptophysin, a marker for synaptic terminal innervation, indicated a progressive increase in immunoreactivity that peaked by ~10% within the first week of Ang‐II infusion (day 7 fold change: 1.1± 0.03 density/area, p<0.05); indicative of increased synaptic input to the PVN prior to elevations in arterial blood pressure. Previous findings have demonstrated that circulating arginine vasopressin protein (AVP) is elevated during chronic Ang‐II hypertension. Consistent with this, AVP immunoreactivity in the PVN was elevated as early as day 3 of Ang‐II infusion (day 3 fold change: 1.7 ± 0.09 density/area, p<0.05), and this response was sustained for up to 7 days (day 5 fold change: 1.4 ± 0.02; day 7 fold change: 1.3 ± 0.03 density/area, both p<0.05). Interestingly, we also observed synaptic end AVP immunoreactivity in the circumventricular subfornical organ very early during Ang‐II infusion (e.g. day 3 fold change: 1.2 ± 0.02 density/area, p<0.05), suggesting a possible feedback loop between the PVN and subfornical organ in AVP regulation. Unlike AVP, PVN oxytocin immunoreactivity was not changed during Ang‐II hypertension (e.g. day 7 fold change: 1.01 ± 0.08 density/area, p>0.05). Collectively, these findings indicate early molecular changes within the PVN prior to the development of Ang‐II hypertension including: 1) Gradual synaptic innervation of the PVN during the first week of Ang‐II infusion; 2) Very early Ang‐II associated elevations in PVN AVP expression, but not oxytocin; and 3) Potential alterations in the innervation of hypothalamic AVP fibers to the circumventricular subfornical organ in response to Ang‐II.Support or Funding Information19CDA34630010 (to Jin Kwon Jeong) and 1R01HL141393 (to Colin N. Young)

ЛОКАЛИЗАЦИЯ 5-HT 2C - И 5-HT 1B -СЕРОТОНИНОВЫХ РЕЦЕПТОРОВ В ОРЕКСИНЕРГИЧЕСКИХ НЕЙРОНАХ ПЕРИФОРНИКАЛЬНОЙ ОБЛАСТИ ГИПОТАЛАМУСА ГРЫЗУНОВ

Both the orexin peptides produced from prepro-orexin in orexinergic neurons of the lateral hypothalamus and hypothalamic perifornical area (PFA) and serotonin coming to these hypothalamic areas from synaptic endings of serotonergic neurons of the midbrain raphe nuclei play an important role in the regulation both of feeding behavior and circadian rhythms. This indicates a close relationship between the serotonin and orexin systems in the lateral hypothalamus and PFA which, however, remain poorly studied. The aim of this study was to identify immunohistochemically and quantify the 2C-subtype (5-HT2CR) and the 1B-subtype (5-HT1BR) serotonin receptors in orexinergic neurons of the hypothalamic PFA in mice and rats. An immunohistochemical reaction for orexin-A was used to identify orexinergic neurons, and the double fluorescence immunolabeling was applied to assess the colocalization of serotonin receptors and orexin-A. Using double immunolabeling, the colocalization of orexin-A and 5-HT2C R and 5-HT1BR in the hypothalamic PFA of different rodent species, such as C57Bl/6J mice and Wistar rats, was demonstrated. It was shown that all orexinergic neurons expressed both types of serotonin receptors. At the same time, only 32–35% of 5-HT2CR and 5-HT1BR were localized in the orexinergic neurons, while the remaining part of them was expressed in the other types of PFA neurons. Thus, the immunohistochemical evidence was obtained for the serotonin-mediated regulation of orexinergic neurons’ functional activity in the rodent hypothalamic PFA, and it was established that these effects of serotonin can be realized through 5-HT2CR and 5-HT1BR.

Disturbed neurotransmitter homeostasis in ether lipid deficiency.

Plasmalogens, the most prominent ether (phospho)lipids in mammals, are structural components of most cellular membranes. Due to their physicochemical properties and abundance in the central nervous system, a role of plasmalogens in neurotransmission has been proposed, but conclusive data are lacking. Here, we targeted this issue in the glyceronephosphate O-acyltransferase (Gnpat) KO mouse, a model of complete deficiency in ether lipid biosynthesis. Throughout the study, focusing on adult male animals, we found reduced brain levels of various neurotransmitters. In the dopaminergic nigrostriatal tract, synaptic endings but not neuronal cell bodies were affected. Neurotransmitter turnover was altered in ether lipid-deficient murine as well as human post-mortem brain tissue. A generalized loss of synapses did not account for the neurotransmitter deficits, since the levels of several presynaptic proteins appeared unchanged. However, reduced amounts of vesicular monoamine transporter indicate a compromised vesicular uptake of neurotransmitters. As exemplified by norepinephrine, the release of neurotransmitters from Gnpat KO brain slices was diminished in response to strong electrical and chemical stimuli. Finally, addressing potential phenotypic correlates of the disturbed neurotransmitter homeostasis, we show that ether lipid deficiency manifests as hyperactivity and impaired social interaction. We propose that the lack of ether lipids alters the properties of synaptic vesicles leading to reduced amounts and release of neurotransmitters. These features likely contribute to the behavioral phenotype of Gnpat KO mice, potentially modeling some human neurodevelopmental disorders like autism or attention deficit hyperactivity disorder.

Изменение взаимодействий между астроцитарными отростками и синаптическими окончаниями при генерации эпилептиформной активности

The aim of the present work was to compare the structural organization of excitatory synaptic contacts in intraocular septal transplants exhibiting normal and epileptiform activity. The work was performed on Wistar rats. The donor material for transplantation was the septal area of the brain isolated from 17-day-old rat fetuses. Three months after the grafting, the electrophysiological properties of neurotransplants were tested using short-term stimulation by single electrical impulses. According to the results of tests, the transplants were divided into two groups, with normal and epileptiform activity. A microscopic study of neurotransplants from both groups showed that nerve and glial cells, as well as the neuropil consisting of axons, dendrites, synaptic endings, and astrocytic processes were well differentiated. Axodendritic and axospinous synaptic endings were considered as three-part structural complexes (tripartite synapses), which included not only pre- and postsynaptic components, but also astrocytic processes surrounding them. Most of them had the morphological features of exciting contacts with strongly expressed postsynaptic densities whose size is a correlate of the efficiency of nervous transmission. A morphometric analysis of these synapses from functionally different types of transplants revealed no significant difference in the length of postsynaptic densities, as well as in the cross-section area and the perimeter of presynaptic boutons. At the same time, significant differences in the degree of the surrounding of synapses by astrocytic processes were found. In grafts characterized by epileptiform activity, the proportion of perisynaptic astrocytic processes was 1.8 times less than in the control ones. The data obtained indicate that perisynaptic astrocytic processes are the first to react to electrical stimulation and initiate the generation of epileptiform activity. It is assumed that the reduction of the astrocytic coverage of excitatory synapses facilitates the spread of neurotransmitters within extracellular spaces and the involvement of neighboring neurons in the synchronized neuronal activity.

Effects of topiramate on the ultrastructure of synaptic endings in the hippocampal CA1 and CA3 sectors in the rat experimental model of febrile seizures: the first electron microscopy report.

The present study aimed at exploring a potentially neuroprotective effect of topiramate (TPM), one of the most commonly used newer-generation, broad-spectrum, antiepileptic drugs against ultrastructural damage of hippocampal synaptic endings in the experimental model of febrile seizures (FS). The study used male young Wistar rats aged 22-30 days, divided into three experimental groups and the control group. Brain maturity in such animals corresponds to that of 1- or 2-year-old children. Hyperthermic stress was evoked by placing animals in a 45°C water bath for four consecutive days. TPM at a dose of 80 mg/kg b.m. was administered with an intragastric tube before and immediately after FS. Specimens (1 mm3) collected from the hippocampal CA1 and CA3 sectors, fixed via transcardial perfusion with a solution of paraformaldehyde and glutaraldehyde, were routinely processed for transmission-electron microscopic analysis. Advanced ultrastructural changes induced by hyperthermic stress were manifested by distinct swelling of hippocampal pre- and post-synaptic axodendritic and axospinal endings, including their vacuolization and disintegration. The axoplasm of the presynaptic boutons contained a markedly decreased number of synaptic vesicles and their abnormal accumulation in the active synaptic region. The synaptic junctions showed a dilated synaptic cleft and a decreased synaptic active zone. TPM used directly after FS was ineffective in the prevention of hyperthermia-induced injury of synaptic endings in hippocampal CA1 and CA3 sectors. However, "prophylactic" administration of TPM, prior to FS induction, demonstrated a neuroprotective effect against synaptic damage in approximately 25% of the synaptic endings in the hippocampal sectors, more frequently located in perivascular zones. It was manifested by smaller oedema of both presynaptic and postsynaptic parts, containing well-preserved mitochondria, increased number and regular distribution of synaptic vesicles within the axoplasm, and increased synaptic active zone. Our current and previous findings suggest that TPM administered "prophylactically", before FS, could exert a favourable effect on some synapses, indirectly, via the vascular factor, i.e. protecting blood-brain barrier components and through better blood supply of the hippocampal CA1 and CA3 sectors, which may have practical implications.

Perfidious synaptic transmission in the guinea-pig auditory brainstem.

The presence of ‘giant’ synapses in the auditory brainstem is thought to be a specialization designed to encode temporal information to support perception of pitch, frequency, and sound-source localisation. These ‘giant’ synapses have been found in the ventral cochlear nucleus, the medial nucleus of the trapezoid body and the ventral nucleus of the lateral lemniscus. An interpretation of these synapses as simple relays has, however, been challenged by the observation in the gerbil that the action potential frequently fails in the ventral cochlear nucleus. Given the prominence of these synapses it is important to establish whether this phenomenon is unique to the gerbil or can be observed in other species. Here we examine the responses of units, thought to be the output of neurons in receipt of ‘giant’ synaptic endings, in the ventral cochlear nucleus and the medial nucleus of the trapezoid body in the guinea pig. We found that failure of the action-potential component, recorded from cells in the ventral cochlear nucleus, occurred in ~60% of spike waveforms when recording spontaneous activity. In the medial nucleus of the trapezoid body, we did not find evidence for action-potential failure. In the ventral cochlear nucleus action-potential failures transform the receptive field between input and output of bushy cells. Additionally, the action-potential failures result in “non-primary-like” temporal-adaptation patterns. This is important for computational models of the auditory system, which commonly assume the responses of ventral cochlear nucleus bushy cells are very similar to their “primary like” auditory-nerve-fibre inputs.