Functional Properties of Dendritic Gap Junctions in Cerebellar Golgi Cells.

The strength and variability of electrical synaptic connections between GABAergic interneurons are key determinants of spike synchrony within neuronal networks. However, little is known about how electrical coupling strength is determined due to the inaccessibility of gap junctions on the dendritic tree. We investigated the properties of gap junctions in cerebellar interneurons by combining paired somato-somatic and somato-dendritic recordings, anatomical reconstructions, immunohistochemistry, electron microscopy, and modeling. By fitting detailed compartmental models of Golgi cells to their somato-dendritic voltage responses, we determined their passive electrical properties and the mean gap junction conductance (0.9 nS). Connexin36 immunofluorescence and freeze-fracture replica immunogold labeling revealed a large variability in gap junction size and that only 18% of the 340 channels are open in each plaque. Our results establish that the number of gap junctions per connection is the main determinant of both the strength and variability in electrical coupling between Golgi cells

Improved spike inference accuracy by estimating the peak amplitude of unitary [Ca 2+ ] transients in weakly GCaMP6f expressing hippocampal pyramidal cells

Investigating neuronal activity using genetically encoded Ca2+ indicators in behaving animals is hampered by inaccuracies in spike inference from fluorescent tracers. Here we combine two‐photon [Ca2+] imaging with cell‐attached recordings, followed by post hoc determination of the expression level of GCaMP6f, to explore how it affects the amplitude, kinetics and temporal summation of somatic [Ca2+] transients in mouse hippocampal pyramidal cells (PCs). The amplitude of unitary [Ca2+] transients (evoked by a single action potential) negatively correlates with GCaMP6f expression, but displays large variability even among PCs with similarly low expression levels. The summation of fluorescence signals is frequency‐dependent, supralinear and also shows remarkable cell‐to‐cell variability. We performed experimental data‐based simulations and found that spike inference error rates using MLspike depend strongly on unitary peak amplitudes and GCaMP6f expression levels. We provide simple methods for estimating the unitary [Ca2+] transients in individual weakly GCaMP6f‐expressing PCs, with which we achieve spike inference error rates of ∼5%

Distinct Nanoscale Calcium Channel and Synaptic Vesicle Topographies Contribute to the Diversity of Synaptic Function

The nanoscale topographical arrangement of voltage-gated calcium channels (VGCC) and synaptic vesicles (SVs) determines synaptic strength and plasticity, but whether distinct spatial distributions underpin diversity of synaptic function is unknown. We performed single bouton Ca2+ imaging, Ca2+ chelator competition, immunogold electron microscopic (EM) localization of VGCCs and the active zone (AZ) protein Munc13-1, at two cerebellar synapses. Unexpectedly, we found that weak synapses exhibited 3-fold more VGCCs than strong synapses, while the coupling distance was 5-fold longer. Reaction-diffusion modeling could explain both functional and structural data with two strikingly different nanotopographical motifs: strong synapses are composed of SVs that are tightly coupled (similar to 10 nm) to VGCC clusters, whereas at weak synapses VGCCs were excluded from the vicinity (similar to 50 nm) of docked vesicles. The distinct VGCC-SV topographical motifs also confer differential sensitivity to neuromodulation. Thus, VGCC-SV arrangements are not canonical, and their diversity could underlie functional heterogeneity across CNS synapses

A New Ochratoxin A Biodegradation Strategy Using <i>Cupriavidus basilensis</i> Őr16 Strain

<div><p>Ochratoxin-A (OTA) is a mycotoxin with possibly carcinogenic and nephrotoxic effects in humans and animals. OTA is often found as a contaminant in agricultural commodities. The aim of the present work was to evaluate OTA-degrading and detoxifying potential of <i>Cupriavidus basilensis</i> ŐR16 strain. <i>In vivo</i> administration of OTA in CD1 male mice (1 or 10 mg/kg body weight for 72 hours or 0.5 mg/kg body weight for 21 days) resulted in significant elevation of OTA levels in the blood, histopathological alterations- and transcriptional changes in OTA-dependent genes (<i>annexinA2</i>, <i>clusterin</i>, <i>sulphotransferase</i> and <i>gadd45</i> and <i>gadd153</i>) in the renal cortex. These OTA-induced changes were not seen in animals that have been treated with culture supernatants in which OTA was incubated with <i>Cupriavidus basilensis</i> ŐR16 strain for 5 days. HPLC and ELISA methods identified ochratoxin α as the major metabolite of OTA in <i>Cupriavidus basilensis</i> ŐR16 cultures, which is not toxic <i>in vivo</i>. This study has demonstrated that <i>Cupriavidus basilensis</i> ŐR16 efficiently degrade OTA without producing toxic adventitious metabolites.</p></div

Effect of acute (72 hours) OTA treatment on <i>gadd45</i> mRNA expression

<p>(<b>A</b>). MMS and OTA 10 elevated the <i>gadd45</i> mRNA level. The metabolised OTA by <i>Cupriavidus basilensis</i> ŐR16 (OTA 1 deg and OTA 10 deg groups) not influenced the mRNA level (One way ANOVA followed by the Tukey's <i>post hoc</i> test were used). Effect of acute (72 hours) OTA treatment on <i>gadd153</i> mRNA expression (<b>B</b>). OTA 10 elevated the <i>gadd153</i> mRNA level. The metabolised OTA by <i>Cupriavidus basilensis</i> ŐR16 (OTA 1 deg and OTA 10 deg groups) not influenced the mRNA level. (Kruskal-Wallis test was used) Abbreviations: MMS – Group treated with methyl methanesulfonate, OTA 1 and OTA 10 – Groups treated with 1 and 10 mg/kg body weight Ochratoxin A, OTA 1 deg and OTA 10 deg – Groups treated with 1 and 10 mg/kg body weight Ochratoxin A + <i>Cupriavidus basilensis</i> ŐR16 in modified Luria-Bertani medium, LB bact- <i>Cupriavidus basilensis</i> ŐR16 in modified Luria-Bertani medium. Data are presented as mean ± SD (n = 7–10, * p<0.05, ***p<0.001)</p

Ochratoxin-A biodegradation by <i>Cupriavidus basilensis</i> ŐR16 during 5 day incubation.

<p>Continuous decrease of the OTA concentration is detected in the supernatant and pellet, while OTα concentration is increasing. Abbreviations: HPLC (supernatant OTA) – OTA concentration in the supernatant originated from the degradation experiment measured by HPLC, HPLC (supernatant OTα) – OTα concentration in the supernatant originated from the degradation experiment measured by HPLC, ELISA (supernatant) OTA – OTA concentration in the supernatant originated from the degradation experiment measured by ELISA, ELISA (pellet) OTA – OTA concentration in the pellet originated from the degradation experiment measured by ELISA. Measurements were carried out in triplicate, SD>3%.</p

Effect of acute (72 hours) OTA treatment on <i>annexin2</i> mRNA expression

<p>(<b>A</b>). OTA 10 elevated the <i>annexin2</i> mRNA level. The metabolised OTA by <i>Cupriavidus basilensis</i> ŐR16 (OTA 1 deg and OTA 10 deg groups) did not influence the mRNA level (Kruskal-Wallis test was used). Effect of acute (72 hours) OTA treatment on <i>clusterin</i> mRNA expression (<b>B</b>). OTA 10 elevated the <i>clusterin</i> mRNA level. The metabolised OTA by <i>Cupriavidus basilensis</i> ŐR16 (OTA 1 deg and OTA 10 deg groups) did not influence the mRNA level. (One way ANOVA followed by the Tukey's <i>post hoc</i> test were used) Abbreviations: MMS – Group treated with methyl methanesulfonate, OTA 1 and OTA 10 – Groups treated with 1 and 10 mg/kg body weight Ochratoxin A, OTA 1 deg and OTA 10 deg- Groups treated with 1 and 10 mg/kg body weight Ochratoxin A + <i>Cupriavidus basilensis</i> ŐR16 in modified Luria-Bertani medium, LB bact – <i>Cupriavidus basilensis</i> ŐR16 in modified Luria-Bertani medium. Data are presented as mean ± SD (n = 7–10, ** p<0.01, *** p<0.001).</p

Effect of chronic (21 days) OTA treatment on <i>sult1c2</i> mRNA expression.

<p>The OTA 0.5 mg/kg bw chronic administration elevated the <i>sult1c2</i> mRNA level. The metabolised OTA by <i>Cupriavidus basilensis</i> ŐR16 (OTA 0.5 deg group) did not influence the mRNA level. (One way ANOVA followed by the Tukey's <i>post hoc</i> test were used) Abbreviations: MMS – Group treated with methyl methanesulfonate, OTA 0.5 – Group treated with 0.5 mg/kg body weight Ochratoxin A, OTA 0.5 deg- Group treated with 0.5 mg/kg body weight Ochratoxin A + <i>Cupriavidus basilensis</i> ŐR16 in modified Luria-Bertani medium, LB bact- <i>Cupriavidus basilensis</i> ŐR16 in modified Luria-Bertani medium. Data are presented as mean ± SD (n = 6–9, **p<0.01)</p