Perforated Patch Clamp in Non-Neuronal Cells, the Model of Mammalian Sperm Cells

Editor: Fatima Shad KaneezThis chapter deals with the steps required to obtain perforated patch-clamp recordings from mammalian sperm cells. (in spite of the fact that these cells) In spite of the fact that these cells are not electrically excitable, they possess a number of conductances due to their expression of ion channels and even neurotransmitter-gated ion-channels, many of which remain to be explored functionally and structurally. Detailed methods for obtaining cells suitable for electrophysiological recordings and protocols to perform patch-clamp recordings are outlined in the text

Estudios funcionales y moleculares de receptores GABAc

Tesis (Doctorado en Ciencias) UANLUANLhttp://www.uanl.mx

An Update on GABAρ Receptors

The present review discusses the functional and molecular diversity of GABAρ receptors. These receptors were originally described in the mammalian retina, and their functional role in the visual pathway has been recently elucidated; however new studies on their distribution in the brain and spinal cord have revealed that they are more spread than originally thought, and thus it will be important to determine their physiological contribution to the GABAergic transmission in other areas of the central nervous system. In addition, molecular modeling has revealed peculiar traits of these receptors that have impacted on the interpretations of the latest pharmacolgical and biophysical findings. Finally, sequencing of several vertebrate genomes has permitted a comparative analysis of the organization of the GABAρ genes

Knock-Down of Specific Thyroid Hormone Receptor Isoforms Impairs Body Plan Development in Zebrafish

The role of thyroid hormones (THs) in development has been extensively studied, however, the specific molecular mechanisms involved are far from being clear. THs act by binding to TH nuclear receptors (TR) that act as ligand-dependent transcription factors to regulate TH-dependent gene expression. Like vertebrates, zebrafish express different isoforms of functional Tr alpha and beta, some of which can bind alternative ligands like 3,5-T2. In this study, we first analyzed the effects of exogenous T3 and 3,5-T2 exposure during embryogenesis. The percentage of affected embryos was similar to those vehicle-injected, suggesting that the early exposure to low TH levels is not sufficient to elicit effects upon the phenotype of the embryo. We then generated crispants for four isoforms of thr to learn more about the role of these receptors in early development. We found that crispant larvae from thraa and a newly identified l-thrb+, but not thrab and canonical thrb1 showed profound deleterious effects upon symmetry and laterality, suggesting early novel roles for these Tr isoforms in the body plan developmental program. Since critical events that determine cell fate start in the late gastrula, we tested if some genes that are expressed during early developmental stages could indeed be TH targets. We identify early development genes, like sox10 and eve, that were specifically over-expressed in thraa and l-thrb+ crispants, suggesting that these specific thr isoforms function as transcription repressors for these genes, while transcription of zic and ets appear to be thraa and l-thrb+-mediated, respectively. Overall, present results show that TH signaling participates in early zebrafish development and identify Tr isoform-specific mediated regulation of early gene expression

Mutants of the Zebrafish K+ Channel Hcn2b Exhibit Epileptic-like Behaviors

Epilepsy is a chronic neurological disorder that affects 50 million people worldwide. The most common form of epilepsy is idiopathic, where most of the genetic defects of this type of epilepsy occur in ion channels. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization, and are mainly expressed in the heart and central and peripheral nervous systems. In humans, four HCN genes have been described, and emergent clinical data shows that dysfunctional HCN channels are involved in epilepsy. Danio rerio has become a versatile organism to model a wide variety of diseases. In this work, we used CRISPR/Cas9 to generate hcn2b mutants in zebrafish, and characterized them molecularly and behaviorally. We obtained an hcn2b mutant allele with an 89 bp deletion that produced a premature stop codon. The mutant exhibited a high mortality rate in its life span, probably due to its sudden death. We did not detect heart malformations or important heart rate alterations. Absence seizures and moderate seizures were observed in response to light. These seizures rarely caused instant death. The results show that mutations in the Hcn2b channel are involved in epilepsy and provide evidence of the advantages of zebrafish to further our understanding of the pathogenesis of epilepsy