The influence of castration on intramural neurons of the urinary bladder trigone in male pigs.

The present study investigated the influence of castration performed at neonatal age on neuronal elements in the intramural ganglia of the urinary bladder trigone (UBT) in male pigs using double-labeling immunohistochemistry. The ganglia were examined in intact (IP) 7-day-old (castration day) pigs, and at 3 and 6 months after surgery. In IP and control (3- and 6-month-old noncastrated pigs) groups, virtually, all neurons were adrenergic (68%) or cholinergic (32%) in nature. Many of them (32%, 51%, and 81%, respectively; 56%, 75%, and 85% adrenergic; and 32%, 52%, and 65% cholinergic, respectively) stained for the androgen receptor (AR), and only a small number of nerve cells were caspase-3 (CASP-3)-positive. In 3- and 6-month-old castrated pigs, an excessive loss (87.6% and 87.5%, respectively) of neurons and intraganglionic nerve fibers was observed. The majority of the surviving adrenergic (61% and 72%, respectively) and many cholinergic (41% and 31%, respectively) neurons expressed CASP-3 and were also AR-positive (61% and 66%, and 40% and 36%, respectively). This study revealed for the first time the excessive loss of intramural UBT neurons following castration, which could have resulted from apoptosis induced by androgen deprivation.

Effect of castration on pelvic neurons in the male pig.

The present study investigated the influence of castration performed at neonatal age on neuronal elements in the anterior pelvic ganglion of the male pig with immunohistochemistry and quantitative real-time PCR (qPCR). The ganglia were examined 3 and 6 months after surgery. In 3-month-old castrated pigs (3MCP) 74% of adrenergic and 31% of cholinergic neurons stained for caspase-3 (CASP-3), and much greater numbers of perikarya than in the control animals expressed CGRP, galanin (GAL) and VIP (peptides known to have neuroprotective properties). In 6-months-old castrated pigs (6MCP), an excessive loss (90%) of neurons and intraganglionic nerve fibres was found. The survived adrenergic and cholinergic neurons also expressed CASP-3, CGRP, GAL or VIP. The qPCR results corresponded with immunofluorescence findings. In 3MCP, genes for CASP-3 and CGRP were up-regulated, while the expression of those for DßH, VAChT, GAL, VIP and SP displayed statistically insignificant variations. In 6MCP, distinctly up-regulated were genes for CGRP, GAL, VIP, SP, DßH and VAChT, while the expression of casp3 gene was down-regulated. The study revealed for the first time the excessive loss of pelvic neurons following castration, and a realistic assumption is proposed, that the neurons died due to apoptosis triggered by androgen deprivation.

Evaluation of zebrafish (Danio rerio) as an animal model for the viral infections of fish.

Zebrafish (Danio rerio) is a laboratory model organism used in different areas of biological research including studies of immune response and host-pathogen interactions. Thanks to many biological tools available, zebrafish becomes also an important model in aquaculture research since several fish viral infection models have been developed for zebrafish. Here, we have evaluated the possible use of zebrafish to study infections with fish viruses that have not yet been tested on this model organism. In vitro studies demonstrated that chum salmon reovirus (CSV; aquareovirus A) and two alloherpesviruses cyprinid herpesvirus 1 (CyHV-1) and cyprinid herpesvirus 3 (CyHV-3) are able to replicate in zebrafish cell lines ZF4 and SJD.1. Moreover, CSV induced a clear cytopathic effect and up-regulated the expression of antiviral genes vig-1 and mxa in both cell lines. In vivo studies demonstrated that both CSV and CyHV-3 induce up-regulation of vig-1 and mxa expression in kidney and spleen of adult zebrafish after infection by i.p. injection but not in larvae after infection by immersion. CyHV-3 is eliminated quickly from fish; therefore, virus clearing process could be evaluated, and in CSV-infected fish, a prolonged confrontation of the host with the pathogen could be studied.

Neuroanatomical Localization of Galanin in Zebrafish Telencephalon and Anticonvulsant Effect of Galanin Overexpression.

Galanin is a neuropeptide widely expressed in the nervous system, but it is also present in non-neuronal locations. In the brain, galanin may function as an inhibitory neurotransmitter. Several studies have shown that galanin is involved in seizure regulation and can modulate epileptic activity in the brain. The overall goal of the study was to establish zebrafish as a model to study the antiepileptic effect of galanin. The goal of this study was achieved by (1) determining neuroanatomical localization of galanin in zebrafish lateral pallium, which is considered to be the zebrafish homologue of the mammalian hippocampus, the brain region essential for initiation of seizures, and (2) testing the anticonvulsant effect of galanin overexpression. Whole mount immunofluorescence staining and pentylenotetrazole (PTZ)-seizure model in larval zebrafish using automated analysis of motor function and qPCR were used in the study. Immunohistochemical staining of zebrafish larvae revealed numerous galanin-IR fibers innervating the subpallium, but only scarce fibers reaching the dorsal parts of telencephalon, including lateral pallium. In three-month old zebrafish, galanin-IR innervation of the telencephalon was similar; however, many more galanin-IR fibers reached the dorsal telencephalon, but in the lateral pallium only scarce galanin-IR fibers were visible. qRT-PCR revealed, as expected, a strong increase in the expression of galanin in the Tg(hsp70l:galn) line after heat shock; however, also without heat shock, the galanin expression was several-fold higher than in the control animals. Galanin overexpression resulted in downregulation of c-fos after PTZ treatment. Behavioral analysis showed that galanin overexpression inhibited locomotor activity in PTZ-treated and control larvae. The obtained results show that galanin overexpression reduced the incidence of seizure-like behavior episodes and their intensity but had no significant effect on their duration. The findings indicate that in addition to antiepileptic action, galanin modulates arousal behavior and demonstrates a sedative effect. The current study showed that galanin overexpression correlated with a potent anticonvulsant effect in the zebrafish PTZ-seizure model.

Pituitary adenylate cyclase-activating polypeptide (PACAP-38) plays an inhibitory role against inflammation induced by chemical damage to zebrafish hair cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP-38) is a common neuropeptide exerting a wide spectrum of functions in many fields, including immunology. In the present study, 5-day post-fertilization (dpf) zebrafish larvae of three diverse genetic lines [transgenic lines Tg(MPX:GFP) with GFP-labelled neutrophils and Tg(pou4f3:GAP-GFP) with GFP-labelled hair cells and the wild-type Tuebingen] were used to investigate an inhibitory role of PACAP-38 in inflammation associated with damaged hair cells of the lateral line. Individuals of each genetic line were assigned to four groups: (1) control, and those consisting of larvae exposed to (2) 10 µM CuSO4, (3) 10 µM CuSO4+100 nM PACAP-38 and (4) 100 nM PACAP-38, respectively. Forty-minute exposure to CuSO4 solution was applied to evoke necrosis of hair cells and consequent inflammation. The inhibitory role of PACAP-38 was investigated in vivo under a confocal microscope by counting neutrophils migrating towards damaged hair cells in Tg(MPX:GFP) larvae. In CuSO4-treated individuals, the number of neutrophils associated with hair cells was dramatically increased, while PACAP-38 co-treatment resulted in its over 2-fold decrease. However, co-treatment with PACAP-38 did not prevent hair cells from extensive necrosis, which was found in Tg(pou4f3:GAP-GFP) individuals. Real-Time PCR analysis performed in wild-type larvae demonstrated differential expression pattern of stress and inflammation inducible markers. The most significant findings showed that CuSO4 exposure up-regulated the expression of IL-8, IL-1ß, IL-6 and ATF3, while after PACAP-38 co-treatment expression levels of these genes were significantly decreased. The presence of transcripts for all PACAP receptors in neutrophils was also revealed. Adcyap1r1a and vipr1b appeared to be predominant forms. The present results suggest that PACAP-38 should be considered as a factor playing an important regulatory role in inflammatory response associated with pathological processes affecting zebrafish hair cells and it cannot be excluded that this interesting property has more universal significance.