Overexpression of the immediate-early genes Egr1, Egr2, and Egr3 in two strains of rodents susceptible to audiogenic seizures.

Genetic animal models of epilepsy are an important tool for further understanding the basic cellular mechanisms underlying epileptogenesis and for developing novel antiepileptic drugs. We conducted a comparative study of gene expression in the inferior colliculus, a nucleus that triggers audiogenic seizures, using two animal models, the Wistar audiogenic rat (WAR) and the genetic audiogenic seizure hamster (GASH:Sal). For this purpose, both models were exposed to high intensity auditory stimulation, and 60min later, the inferior colliculi were collected. As controls, intact Wistar rats and Syrian hamsters were subjected to stimulation and tissue preparation protocols identical to those performed on the experimental animals. Ribonucleic acid was isolated, and microarray analysis comparing the stimulated Wistar and WAR rats showed that the genomic profile of these animals displayed significant (fold change, |FC|≥2.0 and p<0.05) upregulation of 38 genes and downregulation of 47 genes. Comparison of gene expression profiles between stimulated control hamsters and stimulated GASH:Sal revealed the upregulation of 10 genes and the downregulation of 5 genes. Among the common genes that were altered in both models, we identified the zinc finger immediate-early growth response gene Egr3. The Egr3 protein is a transcription factor that is induced by distinct stress-elicited factors. Based on immunohistochemistry, this protein was expressed in the cochlear nucleus complex, the inferior colliculus, and the hippocampus of both animal models as well as in lymphoma tumors of the GASH:Sal. Our results support that the overexpression of the Egr3 gene in both models might contribute to neuronal viability and development of lymphoma in response to stress associated with audiogenic seizures. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic".

Neurochemistry of the afferents to the rat cochlear root nucleus: possible synaptic modulation of the acoustic startle.

Afferents to the primary startle circuit are essential for the elicitation and modulation of the acoustic startle reflex (ASR). In the rat, cochlear root neurons (CRNs) comprise the first component of the acoustic startle circuit and play a crucial role in mediating the ASR. Nevertheless, the neurochemical pattern of their afferents remains unclear. To determine the distribution of excitatory and inhibitory inputs, we used confocal microscopy to analyze the immunostaining for vesicular glutamate and GABA transporter proteins (VGLUT1 and VGAT) on retrogradely labeled CRNs. We also used reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry to detect and localize specific neurotransmitter receptor subunits in the cochlear root. Our results show differential distributions of VGLUT1- and VGAT-immunoreactive endings around cell bodies and dendrites. The RT-PCR data showed a positive band for several ionotropic glutamate receptor subunits, M1-M5 muscarinic receptor subtypes, the glycine receptor alpha1 subunit (GlyRalpha1), GABAA, GABAB, and subunits of alpha2 and beta-noradrenergic receptors. By immunohistochemistry, we confirmed that CRN cell bodies exhibit positive immunoreaction for the glutamate receptor (GluR) 3 and NR1 GluR subunits. Cell bodies and dendrites were also positive for M2 and M4, and GlyRalpha1. Other subunits, such as GluR1 and GluR4 of the AMPA GluRs, were observed in glial cells neighboring unlabeled CRN cell bodies. We further confirmed the existence of noradrenergic afferents onto CRNs from the locus coeruleus by combining tyrosine hydroxylase immunohistochemistry and tract-tracing experiments. Our results provide valuable information toward understanding how CRNs might integrate excitatory and inhibitory inputs, and hence how they could elicit and modulate the ASR.

Characterization of two duplicate zebrafish Cb2-like cannabinoid receptors.

Several cannabinoid receptors have been detected in many organisms. The best known are CB1, mainly expressed in the central nervous system and CB2 which is almost exclusively expressed in the periphery. Here we report the molecular characterization of two duplicate CB2-like cannabinoid receptors from zebrafish (Danio rerio) (zebrafish Cb2a and zebrafish Cb2b). The amino acid sequences of these receptors present 56% identity with Takifugu rubripes CB2 sequence and 39% with human CB2 sequence and conserve some specific key residues for cannabinoid receptor function. Both duplicate receptors are expressed in peripheral tissues (gills, heart, intestine and muscle), immune tissue (spleen) and also in the central nervous system. Using in situ hybridization techniques zebrafish Cb2 mRNA expression was observed for the first time in the adenohypophysial cells of the rostral pars distalis and proximal pars distalis of the pituitary gland. Given the importance of the existence of duplication of genes in teleosts, the combined analysis of these two new cannabinoid receptors opens a new exciting door to investigate and understand cannabinoid function throughout evolution.

Transient expression pattern of prolactin in Sparus aurata.

Using reverse transcription-polymerase chain reaction and in situ hybridization, the expression of the prolactin (PRL) gene was determined during development in gilthead sea bream (Sparus aurata) for the first time. The mRNA for PRL was detected from the second day of the larval stage onwards. This transcript was also located in the adenohypophysial cells, starting at four days post-hatching and was found to be pituitary-specific. Moreover, the possible involvement of PRL in asynchronous growth in the cultivation of gilthead sea bream was also examined. No differences in the distribution of PRL cells were observed in the three sizes of juvenile gilthead sea bream studied. These results suggest that the transcription of PRL is involved in the early development stages of sea bream and that the asynchronous growth-related changes are not due to distinct distribution of PRL cells.

Pax2+ astrocytes in the fish optic nerve head after optic nerve crush.

The transcription factor Pax2 actively participates in the development of the vertebrate visual system. In adults, Pax2 expression persists in a subpopulation of Müller cells and/or astrocytes in the retina and optic nerve head (ONH), although its function remains elusive. In a previous work we showed that the pax2 gene expression is modified and the Pax2(+) astrocyte population in the ONH strongly reacted during the regeneration of the retina after a lesion in goldfish. In the present work we have analyzed Pax2 expression in the goldfish ONH after optic nerve (ON) crush. At one week post-injury, when the regenerating axons arrive at the ONH, the pax2 gene expression level increases as well as the number of Pax2(+) astrocytes in this region. These Pax2(+) astrocytes show a higher number of Cytokeratin (Ck)(+)/GFAP(+) processes compared with control animals. In contrast, a different S100(+) astrocyte population is not modified and persists similar to that of controls. Furthermore, we find a ring that surrounds the posterior ONH that is formed by highly reactive astrocytes, positive to Pax2, GFAP, Ck, S100, GS and ZO1. In this region we also find a source of new astrocytes Pax2(+)/PCNA(+) that is activated after the injury. We conclude that Pax2(+) astrocytes constitute a subpopulation of ONH astrocytes that strongly reacts after ON crush and supports our previous results obtained after retina regeneration. Altogether, this suggests that pax2 gene expression and Pax2(+) astrocytes are probably directly involved in the process of axonal regeneration.

Chronic administration of risperidone in a rat model of schizophrenia: a behavioural, morphological and molecular study.

In the present work we analyzed the effect of the chronic administration of risperidone (2mg/kg over 65 days) on behavioural, morphological and molecular aspects in an experimental model of schizophrenia obtained by bilateral injection of ibotenic acid into the ventral hippocampus of new-born rats. Our results show that during their adult lives the animals with hippocampal lesions exhibit different alterations, mainly at behavioural level and in the gene expression of dopamine D(2) and 5-HT(2A) receptors. However, at morphological level the study performed on the prefrontal cortex did not reveal any alterations in either the thickness or the number of cells immunoreactive for c-Fos, GFAP, CBP or PV. Overall, risperidone administration elicited a trend towards the recovery of the values previously altered by the hippocampal lesion, approaching the values seen in the animals without lesions. It may be concluded that the administration of risperidone in the schizophrenia model employed helps to improve the altered functions, with no significant negative effects.

Growth hormone expression in ontogenic development in gilthead sea bream.

The pattern of expression of the growth hormone (GH) gene was studied during the early development of gilthead sea bream ( Sparus aurata). The GH transcript was detected from the 2nd day of the larval stage onwards. In the next stages the expression level fluctuated, possibly due to different regulatory factors. The distribution of GH mRNA studied by in situ hybridization (ISH) was found to be pituitary specific. Hybridization signals for GH mRNA were detected for the first time in 4-day-old larvae. Throughout development the cells that express GH mRNA were mainly located in the proximal pars distalis. Mammosomatotroph cells coexpressing GH and PRL were not detected in juveniles or adults. Moreover, the possible involvement of GH in asynchronic growth in cultivation of gilthead sea bream was also examined by ISH. No differences in the distribution of GH cells were observed in the three sizes of juveniles of gilthead sea bream studied. These results suggest that the transcription of GH is involved in the early developmental stages of sea bream and the asynchronous growth-related changes are not due to distinct distribution of GH cells.