Detection of zoonotic enteropathogens in captive large felids in Italy.

AIMS: Within the One Health paradigm, infectious disease surveillance have been developed for domestic and wild animals, leaving the role of captive non-domestic populations, especially felids in zoos and circuses, less explored. This study addresses the proximity of these captive animals to urban areas, necessitating focused monitoring for potential zoonotic enteropathogens. The present work aimed to investigate the presence of such zoonotic enteropathogens in faecal samples from captive large felid populations. METHODS AND RESULTS: A total of 108 faecal samples were collected in three circuses, five zoos and one rescue centre across Italy. Salmonella spp. isolation, serotyping and antimicrobial susceptibility testing were conducted on all samples. Additionally, 60 samples were also examined for gastrointestinal parasites using standard coprological techniques. Giardia spp. detection employed direct immunofluorescent staining and specific PCR, while Toxoplasma gondii was detected using PCR targeting B1 gene. A total of 51 Salmonella enterica subsp. enterica were isolated, with predominant serovariants including Infantis (43.1%), Coeln (11.8%) and Newport (11.8%). The captive felids likely act as asymptomatic carriers of foodborne Salmonella, with notable resistance ampicillin and trimethoprim-sulfamethoxazole, no resistance to enrofloxacin was noted. Microscopic analysis revealed Toxascaris leonina eggs in 11 faecal samples (18.3%) and Giardia duodenalis cysts in one animal (1.7%). CONCLUSIONS: Captive animals in public settings may act as sources of Salmonella infection and enteroparasitosis for both occupational and general exposure. The study emphasizes the role of captive animals in antimicrobial resistance dynamics, highlighting the need for routine pathogen screening in the management practices of zoological structures.

A live mammalian cells electroporation array for on-chip immunofluorescence.

The detection of intracellular proteins in vitro is commonly realized with immunofluorescence techniques, through which antibodies or markers are delivered into fixed cells and recognize specific proteins. Many innovative techniques, however, avoid cells fixation by chemical compounds and, among the others, electroporation is widely used. Here we demonstrate that in situ electroporation on thin film SiO2 capacitive microelectrodes can be realized with high efficiency to deliver fluorescent markers and antibodies into mammalian cell lines and primary neuronal cells to detect intracellular proteins, like actin. The results presented in this work open the way to the use of this technique for the detection of potentially any target protein, even through subsequent electroporations.

Intranasal Oxytocin and Vasopressin Modulate Divergent Brainwide Functional Substrates.

The neuropeptides oxytocin (OXT) and vasopressin (AVP) have been identified as modulators of emotional social behaviors and associated with neuropsychiatric disorders characterized by social dysfunction. Experimental and therapeutic use of OXT and AVP via the intranasal route is the subject of extensive clinical research. However, the large-scale functional substrates directly engaged by these peptides and their functional dynamics remain elusive. By using cerebral blood volume (CBV) weighted fMRI in the mouse, we show that intranasal administration of OXT rapidly elicits the transient activation of cortical regions and a sustained activation of hippocampal and forebrain areas characterized by high oxytocin receptor density. By contrast, intranasal administration of AVP produced a robust and sustained deactivation in cortico-parietal, thalamic and mesolimbic regions. Importantly, intravenous administration of OXT and AVP did not recapitulate the patterns of modulation produced by intranasal dosing, supporting a central origin of the observed functional changes. In keeping with this notion, hippocampal local field potential recordings revealed multi-band power increases upon intranasal OXT administration. We also show that the selective OXT-derivative TGOT reproduced the pattern of activation elicited by OXT and that the deletion of OXT receptors does not affect AVP-mediated deactivation. Collectively, our data document divergent modulation of brainwide neural systems by intranasal administration of OXT and AVP, an effect that involves key substrates of social and emotional behavior. The observed divergence calls for a deeper investigation of the systems-level mechanisms by which exogenous OXT and AVP modulate brain function and exert their putative therapeutic effects.

Sodium channel ß2 subunit promotes filopodia-like processes and expansion of the dendritic tree in developing rat hippocampal neurons.

The ß2 auxiliary subunit of voltage-gated sodium channels (VGSCs) appears at early stages of brain development. It is abundantly expressed in the mammalian central nervous system where it forms complexes with different channel isoforms, including Na(v)1.2. From the structural point of view, ß2 is a transmembrane protein: at its extracellular N-terminus an Ig-like type C2 domain mediates the binding to the pore-forming alpha subunit with disulfide bonds and the interactions with the extracellular matrix. Given this structural versatility, ß2 has been suggested to play multiple functions ranging from channel targeting to the plasma membrane and gating modulation to control of cell adhesion. We report that, when expressed in Chinese Hamster Ovary cells CHO-K1, the subunit accumulates at the perimetral region of adhesion and particularly in large lamellipodia-like membrane processes where it induces formation of filopodia-like structures. When overexpressed in developing embryonic rat hippocampal neurons in vitro, ß2 specifically promotes formation of filopodia-like processes in dendrites leading to expansion of the arborization tree, while axonal branching remains unaltered. In contrast to this striking and highly specific effect on dendritic morphology, the targeting of functional sodium channels to the plasma membrane, including the preferential localization of Na(v)1.2 at the axon, and their gating properties are only minimally affected. From these and previously reported observations it is suggested that ß2, among its multiple functions, may contribute to promote dendritic outgrowth and to regulate neuronal wiring at specific stages of neuronal development.

SigMate: a Matlab-based automated tool for extracellular neuronal signal processing and analysis.

Rapid advances in neuronal probe technology for multisite recording of brain activity have posed a significant challenge to neuroscientists for processing and analyzing the recorded signals. To be able to infer meaningful conclusions quickly and accurately from large datasets, automated and sophisticated signal processing and analysis tools are required. This paper presents a Matlab-based novel tool, "SigMate", incorporating standard methods to analyze spikes and EEG signals, and in-house solutions for local field potentials (LFPs) analysis. Available modules at present are - 1. In-house developed algorithms for: data display (2D and 3D), file operations (file splitting, file concatenation, and file column rearranging), baseline correction, slow stimulus artifact removal, noise characterization and signal quality assessment, current source density (CSD) analysis, latency estimation from LFPs and CSDs, determination of cortical layer activation order using LFPs and CSDs, and single LFP clustering; 2. Existing modules: spike detection, sorting and spike train analysis, and EEG signal analysis. SigMate has the flexibility of analyzing multichannel signals as well as signals from multiple recording sources. The in-house developed tools for LFP analysis have been extensively tested with signals recorded using standard extracellular recording electrode, and planar and implantable multi transistor array (MTA) based neural probes. SigMate will be disseminated shortly to the neuroscience community under the open-source GNU-General Public License.

An automated method for detection of layer activation order in information processing pathway of rat barrel cortex under mechanical whisker stimulation.

Rodents perform object localization, texture and shape discrimination very precisely through whisking. During whisking, microcircuits in corresponding barrel columns get activated to segregate and integrate tactile information through the information processing pathway. Sensory signals are projected through the brainstem and thalamus to the corresponding 'barrel columns' where different cortical layers are activated during signal projection. Therefore, having precise information about the layer activation order is desirable to better understand this signal processing pathway. This work proposes an automated, computationally efficient and easy to implement method to determine the cortical layer activation from intracortically recorded local field potentials (LFPs) and derived current source density (CSD) profiles: 1. Barrel cortex LFPs are represented by a template of four subsequent events: small positive/negative (E1) → large negative (E2) → slow positive (E3)→ slow long negative (E4). The method exploits the layer specific characteristics of LFPs to obtain latencies of the individual events (E1­E4), then taking the latency of E2 for calculating the layer activation order. 2. The corresponding CSD profile is calculated from the LFPs and the first sink's peak is considered as a reference point to calculate latencies and evaluate the layer activation order. Other reference points require manual calculation. Similar results of layer activation sequence are found using LFPs and CSDs. Extensive tests on LFPs recorded using standard borosilicate micropipettes demonstrated the method's workability. An interpretation of layer activation order and CSD profiles on the basis of a simplified interacortical barrel column architecture is also provided.

An automated method to determine angular preferentiality using LFPs recorded from rat barrel cortex by brain-chip interface under mechanical whisker stimulation.

The sensory information processing in the rodents is mainly done by whisking, through which they explore the environment, perform object localization, texture and shape discrimination very precisely. During whisking, microcircuits in the corresponding barrel columns get activated to segregate and integrate the tactile information through the information processing pathway. To primarily understand the whisking mechanism angular preferentiality determination is very important. In this work we propose an automated method to determine different events present in the local field potentials (LFPs), calculate latencies and amplitudes related to those events and use them along with the stimulation angle information to determine the angular preferentiality. The method is extensively tested on LFPs recorded from S1 barrel cortex of anesthetized rats using EOSFET (Electrolyte-Oxide-Semiconductor Field Effect Transistor) based neuronal probes.

SigMate: A MATLAB-based neuronal signal processing tool.

Advances in neuronal probe technology to record brain activity have posed a significant challenge in performing necessary processing and analysis of the recorded data. To be able to infer meaningful conclusions from the recorded signals through these probes, sophisticated signal processing and analysis tools are required. This paper presents a MATLAB-based novel tool, 'SigMate', capable of performing various processing and analysis incorporating the available standard tools and our in-house custom tools. The present features include, data display (2D and 3D), baseline correction, stimulus artifact removal, noise characterization, file operations (file splitter, file concatenator, and file column rearranger), latency estimation, determination of cortical layer activation order, spike detection, spike sorting, and are gradually growing. This tool has been tested extensively for the recordings using the standard micropipettes as well as implantable neural probes based on EOSFETs (Electrolyte-Oxide-Semiconductor Field Effect Transistors) and will be made available to the community shortly.

Automatic detection of layer activation order in information processing pathways of rat barrel cortex under mechanical whisker stimulation.

Whisking is the natural way by which rodents explore the environment. During whisking, microcircuits in the corresponding barrel columns get activated to segregate and integrate the tactile information through the information processing pathway. The local field potentials (LFPs) recorded from the barrel columns provide important information about this pathway. Different layers of the cortex get activated during this information processing, thus having precise information about the order of layer activation is desired. This work proposes an automated, computationally efficient and easy to implement method to determine the cortical layer activation for the signals recorded from barrel cortex of anesthetized rats upon mechanical whisker stimulation.

Increased spontaneous activity of a network of hippocampal neurons in culture caused by suppression of inhibitory potentials mediated by anti-gad antibodies.

INTRODUCTION: Anti-glutamic acid decarboxylase autoantibodies (GAD-Ab) are commonly considered the marker of autoimmune diabetes; they were first described in patients affected by stiff-person syndrome and recently, in ataxic or epileptic patients. The pathogenetic role of GAD-Ab remains unclear but inhibition of GABA synthesis or interference with GABA exocytosis are hypothesized. The aim of the study was to assess whether GAD-Ab interfere with neuronal transmission. PATIENTS AND METHODS: Serum from a GAD-Ab positive epileptic patient (by IHC and RIA), serum from a GAD-positive (only by RIA) diabetic case, sera from two epileptic GAD-Ab negative patients and a normal control were selected. Post-synaptic inhibitory potentials (IPSPs) were registered on hippocampal neurons in culture before and after the application of diluted sera in a patch clamp study. RESULTS: A significant increase in the frequency of IPSPs was observed after application of GAD-positive epileptic serum, while no effect was noted using sera from negative controls. CONCLUSION: The inhibition in neuronal transmission only after application of GAD-positive epileptic serum, suggests an interference with GABA function and consequently with neuronal inhibition supporting a pathogenetic role of GAD-Ab in the development of epilepsy.