D-Pinitol attenuates postmenopausal symptoms in ovariectomized mice.

AIMS: Menopause is a natural process in women that can lead to post-menopausal syndrome with symptoms such as hot flushes, weight gain, anxiety, cognitive decline, and depression. Hormonal replacement therapy is commonly prescribed. However, it has serious adverse effects. Herbal medicinal products and isoflavones are used as alternatives. D-Pinitol found in Pinaceae and Fabaceae families has anti-inflammatory and antioxidant effects. However, it has not received as much attention as isoflavones. In this study, we investigated whether D-pinitol could alleviate post-menopausal symptoms using an ovariectomized (OVX) mouse model. MAIN METHODS: Female ICR mice were divided into six groups: sham (vehicle), OVX (vehicle), OVX + D-pinitol (10, 30, 100 mg/kg, p.o.), and OVX + estradiol (0.5 mg/kg, s.c.). Treatment with vehicle, D-pinitol, and estradiol began at seven weeks post ovariectomy. We employed several behavioral tests, hot-flush test, and Western blot analysis. KEY FINDINGS: We found that D-pinitol treatment (30, 100 mg/kg, p.o.) reversed cognitive dysfunction in OVX mice (novel object recognition and Y-maze test). Additionally, D-pinitol alleviated anxiety-like behaviors (elevated plus-maze) and reversed depressive-like behaviors (splash test, tail suspension test). It also normalized increased basal tail skin temperature in OVX mice. Moreover, D-pinitol administration reversed decreased expression of ERß and synaptophysin and phosphorylation of ERK and PI3K-Akt-GSK-3ß induced by OVX in the hippocampus and prefrontal cortex. SIGNIFICANCE: These findings indicate that D-pinitol might be a promising candidate for treating post-menopausal symptoms by increasing ERß and synaptophysin expression levels and activation of ERK or PI3K-Akt-GSK-3ß signaling pathway, at least in part.

Organoids of patient-derived medullary thyroid carcinoma: The first milestone towards a new in vitro model in dogs.

Organoid cultures could constitute a valuable in vitro model to explore new treatments for canine (c) medullary thyroid carcinoma (MTC). The study's objectives were to establish and characterize 3D organoid cultures of cMTC using histology and immunohistochemistry (IHC) and to evaluate the effect of antitumor drugs on organoids' viability. Five cMTC tissue samples were used to develop organoid cultures of which one organoid line, named cMTC N°2, could be passaged for an extended period. This cMTC N°2 organoid line was further compared to the primary tumour regarding morphology and IHC expression of thyroid transcription factor-1 (TTF-1), thyroglobulin, calcitonin, synaptophysin, vimentin, Ki-67, cyclooxygenase-2 (COX-2), P-glycoprotein and vascular endothelial growth factor (VEGF). Quality control of the cMTC N°2 organoid line was achieved by a single nucleotide polymorphism (SNP) array of the organoids, primary tumour and healthy blood cells of the same dog. The effect of carboplatin, meloxicam and toceranib phosphate (TOC) on cMTC N°2 organoids' viability was evaluated. The cMTC N°2 organoid line was cultured for 94 days and showed similar histological features with the primary tumour. Immunolabelling for TTF-1, thyroglobulin, calcitonin and VEGF was similar between the primary tumour and cMTC N°2 organoids. Compared to the primary tumour, organoids showed higher immunolabelling for vimentin and Ki-67, and lower immunolabelling for synaptophysin, COX-2 and P-glycoprotein. The SNP genotype was similar for each chromosome between healthy blood cells, primary tumour and cMTC N°2 organoids. Carboplatin, meloxicam and TOC had no effect on cMTC N°2 organoid cell viability within achievable in vivo concentration range. In conclusion, the cMTC N°2 organoid line is a promising first milestone towards an established in vitro organoid model to explore pathophysiology and new treatment modalities in cMTC.

Paternal stress alters synaptic density and expression of GAP-43, GRIN1, M1 and SYP genes in the hippocampus and cortex of offspring of stress-induced male rats.

Adverse experiences during pregnancy have a negative impact on the neuronal structure and behavior of offspring, but the effects of a father's life events on the outcome of progeny are scarce. The present study is intended to investigate whether paternal stress affects the offspring brain structure, especially those regions concerned with learning and formation of memory, namely the hippocampus (HC) and prefrontal cortex (PFC), and also the expression of certain genes linked to learning and memory in the offspring. Induced stress to male rats by five stressors, one per day followed by allowing them to mate with the normal, unstressed female. Synaptophysin immunoreactivity was assessed in the tissue sections of the HC and PFC as well as expression of genes concerned with learning and memory was evaluated by RT-PCR in the progeny of stress-received males. The progeny of stressed rats had reduced antisynaptophysin immunoreactivity in the HC and PFC. The synaptic density in HC was less in the A-S (Offspring of male rats who received stress during adulthood) and PA-S (offspring of male rats who received stress during both adolescence and adulthood) than in P-S (offspring of male rats who received stress during adolescence) and C-C (offspring of control) groups. Similar results were observed even in the PFC. The results of post hoc tests proved that the HC and PFC of the progeny of stress-exposed rats exhibited considerably less synaptic density than control (P<0.05), and the levels of expression of GAP-43, GRIN1, M1, and SYP genes in HC and PFC were down-regulated. This study concludes that paternal adverse experiences can affect the offspring's synaptic plasticity and also the genes, which can regulate learning and formation of memory.

Miracle Tree (Moringa oleifera) Attuned GFAP and Synaptophysin Levels, Oxidative Stress and Biomarkers in Cerebellar Fluorosis of Pregnant Rats.

<b>Background and Objective:</b> Cerebellar fluorosis is a health issue associated with excessive exposure to fluoride (F) either in direct or indirect ways as pesticides, drinking water and caries preventing prescriptions. It is characterized by elevation in oxidative stress, inflammation, demyelination and Purkinje cell loss. <i>Moringa oleifera</i> (M), is a widely cultivated plant used as a health-booster agent in modulating various disorders because of its high content of vitamins and minerals. The beneficial effect of moringa against fluoride-induced cerebellar toxicity in pregnant rats was investigated in this study. <b>Materials and Methods:</b> Twenty pregnant rats were administered daily 300 mg kg¹ <i>M. oleifera</i> aqueous extract incorporated with 10 mg kg¹ of F intoxication from the 1st day of gestation until the 20th day. Following the termination of the trial, sera were collected and cerebellar tissue was removed for further examinations, along with the assessment of maternity. <b>Results:</b> The <i>M. oleifera</i> significantly normalized serum FSH, LH, progesterone, dopamine and serotonin levels of F-intoxicated mothers. Additionally, <i>M. oleifera</i> markedly prevented the lipid peroxidation and DNA fragmentation indicated by the tail length and moment in comet assay (-34.4 and -75.3%, respectively, when compared to the fluoride intoxicated group), while sustaining the levels of SOD and CAT revealing its antioxidant activity. The <i>M. oleifera</i> regressed the cerebellar α-amylase (-25.4%) and acetylcholinesterase activity (-40.6%), also attenuated GFAP (-73.4%, p<0.0001), synaptophysin level (216.6%, p<0.0001) and IL-6 expression (-91.2%) comparing to fluoride only treated mothers. <b>Conclusion:</b> Histological and ultrastructural examinations confirmed the recuperating effects of <i>M. oleifera</i> on mothers' cerebellar tissue intoxicated with fluoride indicated by intact folia and restored Purkinje cells number and architecture. The maternal study emphasized the anti-abortifacient activity of moringa against fluoride induced-fetotoxicity.

Interferon-beta induces major histocompatibility complex of class I (MHC-I) expression and a proinflammatory phenotype in cultivated human astrocytes.

Major histocompatibility complex class I (MHC-I) has been implicated in several types of neuroplasticity phenomena. Interferon beta-1b (IFN-ß) increases MHC-I expression by motoneurons after sciatic nerve crush in mice, improving axonal growth and functional recovery. Additionally, IFN-ß induces glial hypertrophy associated with upregulation of glial fibrillary acidic protein (GFAP) and MHC-I in murine astrocytes in vitro. As knowledge about MHC-I and its role in synaptic plasticity in human astrocytes (HAs) is scarce, we investigated these aspects in mature HAs obtained from the neocortex of patients undergoing surgery due to hippocampal sclerosis. Cells were exposed to media in the absence (0 IU/ml) or presence of IFN-ß for 5 days (500 IU/ml). Beta-2 microglobulin (ß2m), a component of the MHC-I, GFAP and vimentin proteins, was quantified by flow cytometry (FC) and increased by 100%, 60% and 46%, respectively, after IFN-ß exposure. We also performed qRT-PCR gene expression analyses for ß2m, GFAP, vimentin, and pro- and anti-inflammatory cytokines. Our data showed that IFN-ß-treated astrocytes displayed ß2m and GFAP gene upregulation. Additionally, they presented a proinflammatory profile with increase in the IL-6 and IL-1ß genes and a tendency to upregulate TNF-α. Moreover, we evaluated the effect of HAs conditioned medium (CM) on the formation/maintenance of neurites/synapses by the PC12 lineage. Synaptophysin protein expression was quantified by FC. The CM of IFN-ß-activated astrocytes was not harmful to PC12 neurites, and there was no change in synaptophysin protein expression. Therefore, IFN-ß activated HAs by increasing GFAP, vimentin and MHC-I protein expression. Like MHC-I modulation and astrocyte activation may be protective after peripheral nerve damage and in some neurodegenerative conditions, this study opens perspectives on the pathophysiological roles of astroglial MHC-I in the human CNS.

Korean Red Pine (Pinus densiflora) Bark Extract Attenuates Aß-Induced Cognitive Impairment by Regulating Cholinergic Dysfunction and Neuroinflammation.

In this study, we investigated the anti-amnesic effect of Korean red pine (Pinus densiflora) bark extract (KRPBE) against amyloid beta1-42 (Aß1-42)-induced neurotoxicity. We found that treatment with KRPBE improved the behavioral function in Aß-induced mice, and also boosted the antioxidant system in mice by decreasing malondialdehyde (MDA) content, increasing superoxide dismutase (SOD) activities, and reducing glutathione (GSH) levels. In addition, KRPBE improved the cholinergic system by suppressing reduced acetylcholine (ACh) content while also activating acetylcholinesterase (AChE), regulating the expression of choline acetyltransferase (ChAT), postsynaptic density protein-95 (PSD-95), and synaptophysin. KRPBE also showed an ameliorating effect on cerebral mitochondrial deficit by regulating reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and ATP levels. Moreover, KRPBE modulated the expression levels of neurotoxicity indicators Aß and phosphorylated tau (p-tau) and inflammatory cytokines TNF-α, p-IκB-α, and IL-1ß. Furthermore, we found that KRPBE improved the expression levels of neuronal apoptosis-related markers BAX and BCl-2 and increased the expression levels of BDNF and p-CREB. Therefore, this study suggests that KRPBE treatment has an anti-amnestic effect by modulating cholinergic system dysfunction and neuroinflammation in Aß1-42-induced cognitive impairment in mice.

Corticosterone and Adrenocorticotrophic Hormone Secretion Is Recovered after Immune Challenge or Acute Restraint Stress in Sepsis Survivor Animals.

BACKGROUND: Clinical and experimental studies report a dysregulation of hypothalamus-pituitary-adrenal (HPA) axis during sepsis that causes impairment in hormone secretion in the late phase contributing for the pathophysiology of the disease. However, it is unclear whether this alteration persists even after the disease remission. METHODS: We evaluated the effect of an immune challenge or restraint stress on the hormone secretion of HPA axis in sepsis survivor rats. Sepsis was induced by cecal ligation-puncture (CLP) surgery. Naive or animals that survive 5 or 10 days after CLP were submitted to lipopolysaccharide (LPS) injection or restraint stress. After 60 min, blood was collected for plasma nitrate, cytokines, adrenocorticotropic hormone (ACTH), and corticosterone (CORT) and brain for synaptophysin and hypothalamic cytokines. RESULTS: Five days survivor animals showed increased plasma nitrate (p < 0.001) and interleukin (IL)-1ß levels (p < 0.05) that were abolished in the 10 days survivors. In the hypothalamus of both survivors, the reverse was seen with IL-6 increased (p < 0.01), while IL-1ß did not show any alteration. Synaptophysin expression was reduced in both survivors and did not change after any stimuli. Only the LPS administration increased plasma and/or inflammatory mediators levels in both groups (survivors and naive) being apparently lower in the survivors. There was no difference in the increased secretion pattern of ACTH and CORT observed in the naive and sepsis survivor animals submitted to immune challenge or restraint stress. CONCLUSION: We conclude that the HPA axis is already recovered soon after 5 days of sepsis induction responding with normal secretion of ACTH and CORT when required.

Grape seed extract effects on hippocampal neurogenesis, synaptogenesis and dark neurons production in old mice. Can this extract improve learning and memory in aged animals?

BACKGROUND: During the elderly, hippocampal neurogenesis and synaptogenesis reduce and dark neurons (DNs) increase, leading to cognitive impairment. It is believed that natural products can protect the neural cells and system by protecting from damages or promoting regeneration. Therefore, the effects of grape seed extract (GSE) on the hippocampus of aged mice were investigated in this study. METHODS: twelve old mice were divided into two groups of control and GSE. Animals in the GSE group received 300 mg/kg of GSE for eight weeks via gavage. At the end of treatment, cognition performance was evaluated by Morris water maze (MWM) and passive avoidance tests. Hippocampal neurogenesis, synaptogenesis and DNs production were evaluated with immunohistochemistry and histological evaluations on 5-micron coronal tissue sections. RESULTS: The hippocampal mean number of double cortin positive cells (DCX+) per unit area, as well as synaptophysin expression in the GSE group, were significantly higher than the control group (p < 0.01). The frequency of DNs in the GSE group was lower than the control group (p < 0.05). Behavioral tests showed that GSE improves memory and learning performance. CONCLUSION: Consuming GSE in the elderly can potentially alleviate the age-related reduction of hippocampal neurogenesis and synaptogenesis. It is also able to decrease hippocampal DNs production and increase memory and learning.

Effects of 5.8 GHz microwave on hippocampal synaptic plasticity of rats.

OBJECTIVE: 5.8 GHz spectrum is gaining more attention in wireless technology. To explore the potential hazards, we investigated the effect of exposure to 5.8 GHz microwave on learning and memory ability of rats. Methods: Morris Water maze (MWM), Novel object recognition (NOR) and Fear conditioning test (FCT) were used to evaluate the ability of spatial and non-spatial memory of rats. The hippocampal morphology, the level of brain injury factors in serum and the mitochondrial membrane potential of hippocampal neurons was examined to evaluate the damage of hippocampal neurons. The density of dendritic spines, the ultrastructure of synapses and the level of PSD95, Synaptophysin, p-CREB and CREB were detected to evaluate the hippocampal synaptic plasticity. RESULTS: Compared with Sham group, there was no significant difference in the performance of ethology (in MWM, NOR, FCT) in Microwave 2 h group or Microwave 4 h group. The hippocampal morphology, the serum level of brain injury factors and the content of mitochondrial JC-1 monomer in Microwave 2 h group or Microwave 4 h group did not change obviously, compared with Sham group. The density of dendritic spines, the ultrastructure of synapse and the level of PSD95, Synaptophysin, p-CREB and CREB in hippocampus in Microwave 2 h group or Microwave 4 h group did not significantly change, compared with Sham group. CONCLUSION: Under this experimental condition, exposure to 5.8 GHz microwave could not affect the hippocampal synaptic plasticity of rats.

The contribution of microglia to early synaptic compensatory responses that precede ß-amyloid-induced neuronal death.

Glial-neuronal cross-talk has a critical role in the development of neurodegenerative conditions, including Alzheimer's Disease, where it affects neuronal responses to ß-amyloid peptide (Aß)-induced toxicity. We set out to identify factors regulating synaptic responses to Aß, dissecting the specific role of glial signaling. A low concentration of aggregated Aß42 induced selective up-regulation of mature brain-derived neurotrophic factor (BDNF) expression and release in rat organotypic hippocampal cultures as well as in cortical pure microglia. Conditioned media from resting (CMC) or Aß42-treated (CMA) microglia were tested for their effects on synaptophysin expression in SH-SY5Y neuronal-like cells during challenge with Aß42. Both CMC and CMA prevented Aß-induced synaptophysin loss. In the presence of Aß + CMA, synaptophysin was over-expressed, although it appeared partly clumped in cell bodies. Synaptophysin over-expression was not directly dependent on BDNF signaling on neuronal-like cells, but relied on autocrine BDNF action on microglia. FM1-43 labeling experiments revealed compromised synaptic vesicle recycling in Aß42-treated neuronal-like cells, rescued by microglial conditioned medium. In these conditions, significant and prolonged neuroprotection was observed. Our results point to microglia as a target for early intervention, given its positive role in supporting neuronal compensatory responses to Aß synaptotoxicity, which potentially lead to their extended survival.

Synaptopodin is upregulated by IL-13 in eosinophilic esophagitis and regulates esophageal epithelial cell motility and barrier integrity.

Eosinophilic esophagitis (EoE) is an allergic inflammatory disease of the esophagus mediated by an IL-13-driven epithelial cell transcriptional program. Herein, we show that the cytoskeletal protein synaptopodin (SYNPO), previously associated with podocytes, is constitutively expressed in esophageal epithelium and induced during allergic inflammation. In addition, we show that the SYNPO gene is transcriptionally and epigenetically regulated by IL-13 in esophageal epithelial cells. SYNPO was expressed in the basal layer of homeostatic esophageal epithelium, colocalized with actin filaments, and expanded into the suprabasal epithelium in EoE patients, where expression was elevated 25-fold compared with control individuals. The expression level of SYNPO in esophageal biopsies correlated with esophageal eosinophil density and was improved following anti-IL-13 treatment in EoE patients. In esophageal epithelial cells, SYNPO gene silencing reduced epithelial motility in a wound healing model, whereas SYNPO overexpression impaired epithelial barrier integrity and reduced esophageal differentiation. Taken together, we demonstrate that SYNPO is induced by IL-13 in vitro and in vivo, is a nonredundant regulator of epithelial cell barrier function and motility, and is likely involved in EoE pathogenesis.

Activation of the ileal neuroendocrine tumor cell line P-STS by acetylcholine is amplified by histamine: role of H3R and H4R.

Neuroendocrine tumors may present with pseudoallergic reactions like diarrhea and idiopathic anaphylaxis. Here we present the P-STS human ileal neuroendocrine cell line as a model cell line for these tumors. Neuroendocrine markers and changes in cytoplasmic calcium concentration ([Ca2+]i) in response to several possible activators of 5-hydroxytryptamine (5-HT) release were analyzed. P-STS cells still expressed chromogranin A and synaptophysin after 2 years of culture. Tryptophan hydroxylase 1 mRNA and a low amount of 5-HT were also detected. Acetylcholine (ACh) caused a rise in [Ca2+]i. Somatostatin inhibited, whereas histamine (HA) but not the HA receptor ligand betahistine enhanced activation by ACh. The [Ca2+]i response to ACh/HA was inhibited by the HA receptor H3 (H3R) agonist methimepip and by the antidepressant imipramine. Further [Ca2+]i response studies indicated the presence of H4Rs and of a functional calcium sensing receptor. High or low affinity IgE receptor protein or mRNA were not detected. Taken together, neuroendocrine markers and response to intestinal neurotransmitters approve the P-STS cell line as a valuable model for enterochromaffin cells. Enhancement of their ACh-induced pro-secretory response by HA, with a role for H3R and H4R, suggests an amplifying role of neuroendocrine cells in allergen-induced diarrhea or anaphylaxis.

Protective effects of testosterone on cognitive dysfunction in Alzheimer's disease model rats induced by oligomeric beta amyloid peptide 1-42.

Cognitive dysfunction is known to be influenced by circulating sex steroidal hormones. The aim of this study was to examine the protective effect and possible protective mechanism of testosterone (T) on cognitive performance in male rats induced by intrahippocampal injections of beta amyloid 1-42 oligomers (Aß1-42). Treatment with T as evidenced by the Morris water maze (MWM) test significantly shortened escape latency and reduced path length to reach the platform compared to the control (C). During probe trials, the T group displayed a significantly greater percent of time in the target quadrant and improved the number of platform crossings compared with C, flutamide (F), an antiandrogen, and a combined F and T group. Flutamide markedly inhibited the influence of T on cognitive performance. Following Nissl staining, the number of intact pyramidal cells was significantly elevated in the T group, and the effect of T was blocked by F. Immunohistochemisty and Western blot analysis showed that the protein expression level of Aß 1-42 was markedly decreased and expression levels of synaptophysin (SYN) significantly increased with T, while F inhibited all T-mediated effects. Our data suggest that the influence of T on cognitive performance was mediated via androgen receptors (AR) to remove beta amyloid, which leads to enhanced synaptic plasticity.

Rewiring neuronal microcircuits of the brain via spine head protrusions--a role for synaptopodin and intracellular calcium stores.

Neurological diseases associated with neuronal death are also accompanied by axonal denervation of connected brain regions. In these areas, denervation leads to a decrease in afferent drive, which may in turn trigger active central nervous system (CNS) circuitry rearrangement. This rewiring process is important therapeutically, since it can partially recover functions and can be further enhanced using modern rehabilitation strategies. Nevertheless, the cellular mechanisms of brain rewiring are not fully understood. We recently reported a mechanism by which neurons remodel their local connectivity under conditions of network-perturbance: hippocampal pyramidal cells can extend spine head protrusions (SHPs), which reach out toward neighboring terminals and form new synapses. Since this form of activity-dependent rewiring is observed only on some spines, we investigated the required conditions. We speculated, that the actin-associated protein synaptopodin, which is involved in several synaptic plasticity mechanisms, could play a role in the formation and/or stabilization of SHPs. Using hippocampal slice cultures, we found that ~70 % of spines with protrusions in CA1 pyramidal neurons contained synaptopodin. Analysis of synaptopodin-deficient neurons revealed that synaptopodin is required for the stability but not the formation of SHPs. The effects of synaptopodin could be linked to its role in Ca(2+) homeostasis, since spines with protrusions often contained ryanodine receptors and synaptopodin. Furthermore, disrupting Ca(2+) signaling shortened protrusion lifetime. By transgenically reintroducing synaptopodin on a synaptopodin-deficient background, SHP stability could be rescued. Overall, we show that synaptopodin increases the stability of SHPs, and could potentially modulate the rewiring of microcircuitries by making synaptic reorganization more efficient.

Enhanced resistance to fatigue and altered calcium handling properties of sarcalumenin knockout mice.

Sarcalumenin is a Ca2+-binding protein located in the sarcoplasmic reticulum of striated muscle cells, the physiological function of which has not been fully determined yet. Using sarcalumenin knockout (sar(-/-)) mice, we showed that sar ablation altered store-operated Ca2+ entry (SOCE) and enhanced muscle fatigue resistance. Sar(-/-) mice fatigued less with treadmill exercise, and intact isolated soleus and extensor digitorum longus muscles from sar(-/-) mice were more resistant to intermittent fatiguing stimulation than those from wild-type mice. Enhanced SOCE was observed in the sar(-/-) muscles. Biochemical analysis revealed that sar(-/-) muscles contained significantly elevated expression of mitsugumin 29 (MG29), a synaptophysin-related membrane protein located in the triad junction of skeletal muscle. Because the ablation of mg29 has been shown to cause increased fatigability and dysfunction of SOCE, the enhanced SOCE activity seen in sar(-/-) muscle may be correlated with the increased expression of MG29. Our data suggest that systemic ablation of sarcalumenin caused enhanced resistance to muscle fatigue by compensatory changes in Ca2+ regulatory proteins that effect SOCE.

Activación de la calcio calmodulina quinasa II, CaMKII, por el uridin nucleósido difosfato, UDP, en neuronas granulares de cerebelo / Calcium Calmoduline Kinase II, CaMKII, activation by the uridine nucleoside diphosphate, UPD, in cerebellar granule neurons

Las neuronas granulares de cerebelo en cultivo presentan receptores metabotrópicos de nucleótidos de tipo P2Y6, cuyo agonista fisiológico específico es el nucleótido, uridina difosfato, UDP. Estudios de PCR muestran la presencia de este receptor y el incremento de la expresión con el tiempo. Los estudios de respuesta en célula individual mediante microfluorimetría muestran un incremento del calcio citosólico al estimular con UDP, siendo los incrementos mas significativos en el soma. El incremento del calcio citosólico produce la activación de diversos enzimas dependientes de calcio y concretamente de la calcio-calmodulina quinasa II, CAMKII, enzima que se encuentra ampliamente distribuida en toda la topografía de la neurona granular. Este enzima al activarse se auto-fosforila y mediante anticuerpos contra la forma fosforilada se pueden detectar las zonas mas activas en la célula. Cuando se estimula con UDP, la CaMKII fosforilada aparece fundamentalmente asociada al soma neural, con mucha menor actividad en las prolongaciones axodendríticas, lo que se corresponde con la distribución de los receptores P2Y6 funcionales

Cultured granule cells from cerebellum exhibit nucleotide metabotropic receptors such as the P2Y6 subtype, which physiological agonist is the uridine diphosphate, UDP. The PCR analysis show the presence of P2Y6 messenger RNA, increasing with the days in culture. Single cell microfluorimetric studies show citosolic calcium increase in response to UDP, this being more significant at the soma level. The cytosolic calcium increase triggers cellular responses mediated by calcium dependent enzymes. This is the case for calcium-calmodulin kinase II, CaMKII, which is extensively distributed through the granule neuron, according the immunocytochemical studies. This enzyme once activated is able to autophosphorylate and by using antibodies against the phosphorylated form the active zones can be detected. After UDP stimulation, the location of the phosphorylated form of CaMKII appears to be mainly at the neural soma, with lower presence at the axodendritic prolongations, which correlates with the functional P2Y6 subtype receptor distribution

Influence of afferent synaptic innervation on the discharge variability of cat abducens motoneurones.

The discharge variability of abducens motoneurones was studied after blocking inhibitory synaptic inputs or both excitatory and inhibitory inputs by means of an intramuscular (lateral rectus) injection of either a low (0.5 ng kg(-1)) or a high dose (5 ng kg(-1)) of tetanus neurotoxin (TeNT), respectively. Motoneuronal firing increased after low-dose TeNT. High-dose treatment, however, produced a firing depression, and in some cells, a total lack of modulation in relation to eye movements. Firing became increasingly more regular with larger TeNT doses as shown by significant reductions in the coefficient of variation after low- and high-dose treatments. Similarly, autocorrelation histograms of interspike intervals increased the number of resolvable peaks twofold in low-dose-treated motoneurones and sevenfold in high-dose-treated motoneurones. The plots of standard deviation versus the mean instantaneous firing frequency showed an upward deflexion with low firing frequencies. The upward deflexion occurred in controls at 39.9 +/- 4.9 ms, an interval similar to the mean afterhyperpolarisation (AHP) duration (48.4 +/- 8.8 ms). Low-dose TeNT treatment shifted the deflexion point to 20.9 +/- 3.9 ms, whereas the high dose increased it to 60.7 +/- 6.1 ms, in spite of the fact that no differences in AHP parameters between groups were found. The density of synaptophysin-immunoreactive boutons decreased by 14 % after the low-dose treatment and 40.5 % after the high-dose treatment, indicating that protracted synaptic blockade produces elimination of synaptic boutons. It is concluded that abducens motoneurone spike variability during spontaneous ocular fixations depends largely on the balance between inhibitory and excitatory synaptic innervation.

Nectin: an adhesion molecule involved in formation of synapses.

The nectin-afadin system is a novel cell-cell adhesion system that organizes adherens junctions cooperatively with the cadherin-catenin system in epithelial cells. Nectin is an immunoglobulin-like adhesion molecule, and afadin is an actin filament-binding protein that connects nectin to the actin cytoskeleton. Nectin has four isoforms (-1, -2, -3, and -4). Each nectin forms a homo-cis-dimer followed by formation of a homo-trans-dimer, but nectin-3 furthermore forms a hetero-trans-dimer with nectin-1 or -2, and the formation of each hetero-trans-dimer is stronger than that of each homo-trans-dimer. We show here that at the synapses between the mossy fiber terminals and dendrites of pyramidal cells in the CA3 area of adult mouse hippocampus, the nectin-afadin system colocalizes with the cadherin-catenin system, and nectin-1 and -3 asymmetrically localize at the pre- and postsynaptic sides of puncta adherentia junctions, respectively. During development, nectin-1 and -3 asymmetrically localize not only at puncta adherentia junctions but also at synaptic junctions. Inhibition of the nectin-based adhesion by an inhibitor of nectin-1 in cultured rat hippocampal neurons results in a decrease in synapse size and a concomitant increase in synapse number. These results indicate an important role of the nectin-afadin system in the formation of synapses.

Synaptogyrins regulate Ca2+-dependent exocytosis in PC12 cells.

Synaptogyrins constitute a family of synaptic vesicle proteins of unknown function. With the full-length structure of a new brain synaptogyrin isoform, we now show that the synaptogyrin family in vertebrates includes two neuronal and one ubiquitous isoform. All of these synaptogyrins are composed of a short conserved N-terminal cytoplasmic sequence, four homologous transmembrane regions, and a variable cytoplasmic C-terminal tail that is tyrosine-phosphorylated. The localization, abundance, and conservation of synaptogyrins suggest a function in exocytosis. To test this, we employed a secretion assay in PC12 cells expressing transfected human growth hormone (hGH) as a reporter protein. When Ca2+-dependent hGH secretion from PC12 cells was triggered by high K+ or alpha-latrotoxin, co-transfection of all synaptogyrins with hGH inhibited hGH exocytosis as strongly as co-transfection of tetanus toxin light chain. Synaptophysin I, which is distantly related to synaptogyrins, was also inhibitory but less active. Inhibition was independent of the amount of hGH expressed but correlated with the amount of synaptogyrin transfected. Inhibition of exocytosis was not observed with several other synaptic proteins, suggesting specificity. Analysis of the regions of synaptogyrin required for inhibition revealed that the conserved N-terminal domain of synaptogyrin is essential for inhibition, whereas the long C-terminal cytoplasmic tail is largely dispensable. Our results suggest that synaptogyrins are conserved components of the exocytotic apparatus, which function as regulators of Ca2+-dependent exocytosis.