Direct activation of zebrafish neurons by ultrasonic stimulation revealed by whole CNS calcium imaging.

OBJECTIVE: Ultrasounds (US) use in neural engineering is so far mainly limited to ablation through high intensity focused ultrasound, but interesting preliminary results show that low intensity low frequency ultrasound could be used instead to modulate neural activity. However, the extent of this modulatory ability of US is still unclear, as in in vivo studies it is hard to disentangle the contribution to neural responses of direct activation of the neuron by US stimulation and indirect activation due either to sensory response to mechanical stimulation associated to US, or to propagation of activity from neighboring areas. Here, we aim to show how to separate the three effects and assess the presence of direct response to US stimulation in zebrafish. APPROACH: We observed in zebrafish larvae brain-wide US-induced activity patterns through calcium imaging microscopy. Sensory response to mechanical stimulation was assessed with a US shield. Activity propagation was assessed with inter-area latency evaluation. MAIN RESULTS: We prove that in selected brain regions the zebrafish's neural response is mainly due to direct activation, later spreading to the other regions. Shielding the neurons from direct US stimulation resulted in a significantly attenuated response, showing that sensory stimulation does not play a prominent role. SIGNIFICANCE: US non-invasive neuromodulatory approach might lead to novel ways to test and control neural activity, and hence to novel neuromodulatory therapies. Future studies will focus on the biophysical structure of directly responsive neurons to capture the mechanisms of US induced activity.

Optical mapping of neuronal activity during seizures in zebrafish.

Mapping neuronal activity during the onset and propagation of epileptic seizures can provide a better understanding of the mechanisms underlying this pathology and improve our approaches to the development of new drugs. Recently, zebrafish has become an important model for studying epilepsy both in basic research and in drug discovery. Here, we employed a transgenic line with pan-neuronal expression of the genetically-encoded calcium indicator GCaMP6s to measure neuronal activity in zebrafish larvae during seizures induced by pentylenetretrazole (PTZ). With this approach, we mapped neuronal activity in different areas of the larval brain, demonstrating the high sensitivity of this method to different levels of alteration, as induced by increasing PTZ concentrations, and the rescuing effect of an anti-epileptic drug. We also present simultaneous measurements of brain and locomotor activity, as well as a high-throughput assay, demonstrating that GCaMP measurements can complement behavioural assays for the detection of subclinical epileptic seizures, thus enabling future investigations on human hypomorphic mutations and more effective drug screening methods. Notably, the methodology described here can be easily applied to the study of many human neuropathologies modelled in zebrafish, allowing a simple and yet detailed investigation of brain activity alterations associated with the pathological phenotype.

Immunohistochemistry for BRAF(V600E) antibody VE1 performed in core needle biopsy samples identifies mutated papillary thyroid cancers.

BRAF(V600E) is the most frequent genetic mutation in papillary thyroid cancer (PTC) and has been reported as an independent predictor of poor prognosis of these patients. Current guidelines do not recommend the use of BRAF(V600E) mutational analysis on cytologic specimens from fine needle aspiration due to several reasons. Recently, immunohistochemistry using VE1, a mouse anti-human BRAF(V600E) antibody, has been reported as a highly reliable technique in detecting BRAF-mutated thyroid and nonthyroid cancers. The aim of this study was to test the reliability of VE1 immunohistochemistry on microhistologic samples from core needle biopsy (CNB) in identifying BRAF-mutated PTC. A series of 30 nodules (size ranging from 7 to 22 mm) from 30 patients who underwent surgery following CNB were included in the study. All these lesions had had inconclusive cytology. In all cases, both VE1 and BRAF(V600E) genotypes were evaluated. After surgery, final histology demonstrated 21 cancers and 9 benign lesions. CNB correctly diagnosed 20/20 PTC and 5/5 adenomatous nodules. One follicular thyroid cancer and 4 benign lesions were assessed at CNB as uncertain follicular neoplasm. VE1 immunohistochemistry revealed 8 mutated PTC and 22 negative cases. A 100% agreement was found when positive and negative VE1 results were compared with BRAF mutational status. These data are the first demonstration that VE1 immunohistochemistry performed on thyroid CNB samples perfectly matches with genetic analysis of BRAF status. Thus, VE1 antibody can be used on thyroid microhistologic specimens to detect BRAF(V600E)-mutated PTC before surgery.

Phenotypic changes of the thyrocyte membrane in papillary thyroid carcinoma. A three-dimensional study.

Aim of the study was to assess the presence of structural changes in the complex carbohydrate chains of thyroid epithelia undergoing neoplastic transformation. We investigated thyroid cells from neoplastic lesions using a panel of lectins with specific affinity for distinct carbohydrate residues. Sixty samples of thyroid tissue, including normal, hyperplastic and neoplastic lesions were obtained from surgical specimens and blindly evaluated with lectin stains. Confocal microscopy was used to obtain three-dimensional (3-D) images of the samples with a positive reaction. Wheat germ agglutinin (WGA) was consistently positive on the apical membrane of papillary thyroid carcinomas (PTC), was weakly expressed in follicular carcinomas (FC) and resulted negative in normal thyrocytes and in benign conditions. The 3-D microscopy model showed that the WGA staining pattern in light microscopy corresponds to a continuous layer on the luminal surface of both papillary and tubular structures of PTC cells. The other lectins under evaluation did not provide any significant result. In conclusion, in PTC the apical border of thyrocytes showed a strong, specific and consistent staining with WGA. These findings may be related to a modified interaction of thyroglobulin molecule with thyroid cell membrane and with the expression of molecules that are involved in the process of tumorigenesis and tumor progression.