Lungfish aestivating activities are locked in distinct encephalic γ-aminobutyric acid type A receptor α subunits

Abstract

Ammonia in dipnoans plays a crucial role on neuronal homeostasis, especially for those brain areas that maintain torpor and awakening states in equilibrium. In the present study, specific α subunits of the major neuroreceptor inhibitory complex (GABA(A) R), which predominated during some phases of aestivation of the lungfish Protopterus annectens, turned out to be key adaptive factors of this species. From the isolation, for the first time, of the encoding sequence for GABA(A) R α₁, α₄ , and α₅ subunits in Protopterus annectens, qPCR and in situ hybridization levels of α₄ transcript in thalamic (P < 0.001) and mesencephalic (P < 0.01) areas proved to be significantly higher during long aestivating maintenance states. Very evident α₅ mRNA levels were detected in diencephalon during short inductive aestivating states, whereas an α₄ /α₁ turnover characterized the arousal state. Contextually, the recovery of physiological activities appeared to be tightly related to an evident up-regulation of α₁ transcripts in telencephalic and cerebellar sites. Surprisingly, TUNEL and amino cupric silver methods corroborated apoptotic and neurodegenerative cellular events, respectively, above all in telencephalon and cerebellum of lungfish exposed to long maintenance aestivating conditions. Overall, these results tend to underlie a novel GABAergic-related ON/OFF molecular switch operating during aestivation of the lungfish, which might have a bearing on sleeping disorders.

Differential Gene Expression in the Brain of the African Lungfish, Protopterus annectens, after Six Days or Six Months of Aestivation in Air

“…Only until very recently, did Loong et al [11], [12] report on the differential gene expression, and the up-regulation of mRNA expression of carbamoyl phosphate synthetase III and ornithine-urea cycle capacity, in the liver of P. annectens during the induction phase (the first 6 days) of aestivation in air. More importantly, there are very few recent studies [13], [14] on the brain of aestivating African lungfish in spite of its possible role in coordinating a whole-body aestivation-specific response during the induction phase of aestivation. Therefore, this study was undertaken to examine, using suppression subtractive hybridization (SSH) polymerase chain reaction (PCR), the up- and down-regulation of gene expression in the brain of P. annectens during the induction phase (6 days) or the prolonged maintenance phase (6 months) of aestivation in air with reference to the freshwater control.…”

Aestivation and hypoxia-related events share common silent neuron trafficking processes

“…Afterwards all animals were sacrificed and their dorsal skull was opened in order to extract their brain, which was stored at −80°C and −20°C for future analyses. For the different experimental phases described in this study, the number of animals for each conditions (n = 4) was assessed according to Mead’s resource equation, which is in line with our previous published works [4]. …”

“…For quantitative analysis, sections of all brain levels were analyzed with a Leitz optic microscope (Dialux 20 EB; Leica, Italy; 16X magnification; 25°C) and captured via Leica Imaging software in order to identify neuronal cell groups according to previous works [63]. Cell counting of telencephalon (TEL), hypothalamus (HTH), optic tectum (Te) and cerebellum (Cb) was performed on five sections/area by using the following formula: Ns = [Σ(N/Vsection)/n] x Vref, as previously reported for neuronal quantification in lungfish [4], where Ns = stained neurons per area; N = stained neurons/single section; Vsection = section volume; n = sections per area; Vref = volume of brain area.…”

“…It has been established that some vertebrates may tolerate prolonged hypoxia and high temperatures without damaging vital organs such as the brain [2]. In this category, aestivating turtles and fish are considered valuable models for clinical studies [3,4]. Aestivation is characterized by a consistent metabolic reduction accompanied by a decrease of temperature and a down-regulation of gas exchange and heart rate [5].…”

The Relationship between Branchiosomatic and Pulmosomatic Indices in the African Lungfish, Protopterus Annectens of Anambra River, Nigeria

“…(Okafor,2012) It inhabits shallow and swampy parts of some Rivers and Lakes of some African countries during the wet season (Giusi et al, 2011;Icardo et al, 2011Icardo et al, ,2012Laberge and Walsh, 2011;) However, during the dry season, when the ambient water has totally dried up, Protopterus excavates a burrow in the soil where it resides in a dormant state until the end of the season. (Okafor et al 2011;Loong et al, 2012 a,b).…”