Effects of different culture media on optimization of primary neuronal cell culture for in vitro models assay

BACKGROUND: In vitro model studies are becoming increasingly popular for experimental research designs. They include isolation and expansion of cells of a particular tissue, such as the nervous tissue which contributes to understanding the underlying mechanisms in many pathologies. It enables the scrutinization of intracellular signaling pathways responsible for cell death.

OBJECTIVES: In the literature, there are different methods for the isolation and culture of rat embryonic cortical neurons. However, this study developed a feasible, rapid and easily performable method.

METHODS: Isolation of neurons was performed without using enzymatic digestion. Primary cortical cultures neurite outgrowth and neuron numbers per field of common mediums were compared for neuronal cells isolation and expansion. In this study, three different culture mediums were considered: Medium I: Neurobasal medium, B-27 and L-glutamine; Medium II: DMEM, FBS and L-glutamine; and Medium III: DMEM/F-12, FBS and L-glutamine.

RESULTS: High survival rate and number of neurons was obtained with the current method. The best neuronal growth was achieved by Medium I, while Medium II and III had moderate effect on the neurite outgrowth.

CONCLUSIONS: Enzyme-free treatment was introduced and Medium I was used as an alternative method for optimal neuron isolation and expansion. The neuronal cultures are similar to nervous tissue in physiological aspects. Hence, Medium I is more similar to the in vivo condition compared to Mediums II and III.

[ role of glycine and NMDA glutamate receptor on central regulation of feed intake of broiler cockerels]

Glycine is an inhibitory neurotransmitter in central nervous system and plays a certain role in food intake in mammalian. The purpose of the present study was to investigate the role of glycine in central regulation of feed intake of broiler cockerels [Ross 308] during six sequential phases. At 1, 2 and 3 phases, glycine [50, 100 and 200 nmol], NFPS [inhibitor of glycine transporter at 25, 50 and 100 nmol] and hydrochloride strychnine [competitive antagonist of presynaptic of glycine at 10, 50 and 250 nmol] were injected intracerebroventriculary [ICV]. At 4, 5 and 6 phases, the effect of pretreatment of NFPS [100 nmol], strychnine [250 nmol] and DL-AP5 [antagonist of glutamate NMDA receptors, 5 nmol] on cumulative feed intake induced by glycine was evaluated. During this study, the control group was injected ICV by sterile physiological serum. Thereafter, Cumulative feed intake was measured at 15, 30, 60, 120 and 180 min after injection. According to the results, ICV injection of 200 nmol glycine significantly reduced the feed intake [p<0.05]. Moreover, the injection of NFPS at 50 and 100 nmol, significantly increased the feed intake [p<0.05], while strychnine had no effect. Additionally, pretreatment with NFPS and DL-AP5 significantly attenuated the feed intake induced by glycine [p<0.05], whereas strychnine had no effect [p>0.05]. These results showed that the inhibitory effect of glycine on feed intake is not associated with neurotransmitory function of glycine, but is due to its neuromodulatory effect which is probably mediated via NMDA glutamate receptors in birds

[ effect of intracerebroventricular injection of CB[1] antagonist on feed consumption in broiler cockerels]

Endocannabinoid system play a critical role in the regulation of appetite in mammals. In the present study, the effect of avian brain CB[1] receptor on food intake of broilers was studied. ACEA, a potent CB[1] agonist, and AM28 1, a potent CB[1] antagonist, were injected into the chicken right lateral cerebral ventricle and food intake was measured 15, 30, 60, 120, and 180 minutes post injection. The results indicate that CB[1] agonist and antagonist increase and decrease food intake, respectively. Also, pre treatment with CB1 antagonistfully inhibits the CB[1]-agonist-induced food intake. The results of the study is consistent with the experiments carried out in mammals

RNA extraction and leptin receptor mRNA detection in bull ejaculated spermatozoa

Leptin, known as a potential satiety factor, plays an important role in both metabolism and reproduction. The presence of leptin in human seminal plasma and human spermatozoa has been shown; recently, leptin receptors [Ob-R] have been localized in human spermatozoa, thus suggesting a possible action of this hormone even on these cells. Our aim was to detect leptin receptor mRNA in bull ejaculated spermatozoa by reverse transcriptase-polymerase chain reaction [RT-PCR]. Total RNA was isolated from bull ejaculated spermatozoa and purified by different methods. Our results have revealed that sodium dodecylsulphate [SDS] and SDS/citric acid extraction methods are superior to guanidinium isothiocyanate in terms of yield and reproducibility of RNA recovery. The mRNA for Ob-Rb was detected in all samples examined. We conclude that Ob-R mRNA is present in bull spermatozoa where seminal plasma leptin can exert its effects

[Detection of leptin and leptin receptor mRNA expression in the bovine testis]

Leptin, hormonal product of the ob gene, is known to regulate food intake, energy metabolism and reproductive functions in mammals. The mechanism by which leptin affect male reproductive system, in contrast to its well proven effects in female fertility, has been a matter of debate. Expression of leptin and its receptor in some reproductive organs suggest that leptin has both endocrine and paracrine/autocrine effects on reproduction. Various evidences have pointed to a direct role of leptin in the control of rodent testicular function such as steroidogenesis and spermatogenesis. So, detection of leptin and leptin receptor mRNA in bovine testis will be the first crucial step to an understanding of its paracrine/autocrine effect on testes in cattle. In the present study, we showed the expression of leptin mRNA as well as its functional receptor [Ob-Rb] mRNA in whole testis of Holstein cattle using reverse transcription and polymerase chain reaction [RT-PCR] analysis. To confirm the first results, RT-PCR products were amplified with Nested PCR using inner leptin primer pairs designed on different exons. Based on our results, although we could not determine the exact cell source of leptin in / testis, it suggests that besides its primary actions at the hypothalamic-pituitary level, leptin can also involved in autocrine and/or paracrine mechanisms in testicular physiology in cattle

Glutamate ionotropic and metabotropic receptors affect feed intake in broiler cockerels

In this study the role of the glutamatergic system on feed intake in 24-hour-feed-deprived broiler cockerels was investigated. ICV injection of 0, 0.675, 1.25, and 2.5 nmol of glutamate reduced feed intake dose-dependently, and increased the latency time to start feeding. Pretreatment with 2.5 nmol HQCA, an ionotropic glutamate antagonist resulted in both an increase in feed intake and a decrease in latency of birds to start feeding. Pretreatment with 2nmol of MSPG, a metabotropic glutamate receptor antagonist, severely reduced feed intake and increased the latency to start feeding. These findings suggest, for the first time, that glutamate, acting as a neurotransmitter, is involved in feed intake regulation in broiler cockerels. This effect is probably mediated by both ionotropic and metabotropic receptors. It appears that both postsynaptic and presynaptic glutamate receptors are involved