ABSTRACT
To obtain insights into the emergence of rhythmogenic circuits supporting respiration, we monitored spontaneous activities in isolated brainstem and medullary transverse slice preparations of mouse embryos, combining electrophysiological and calcium imaging techniques. At embryonic day 15 (E15), in a restricted region ventral to the nucleus ambiguus, we observed the onset of a sustained high-frequency (HF) respiratory-like activity in addition to a preexisting low-frequency activity having a distinct initiation site, spatial extension, and susceptibility to gap junction blockers. At the time of its onset, the HF generator starts to express the neurokinin 1 receptor, is connected bilaterally, requires active AMPA/kainate glutamatergic synapses, and is modulated by substance P and the mu-opioid agonist D-Ala2-N-Me-Phe4-Glycol5-enkephalin. We conclude that a rhythm generator sharing the properties of the neonatal pre-Bötzinger complex becomes active during E15 in mice.
Subject(s)
Brain Stem/cytology , Motor Neurons/physiology , Periodicity , Respiration , Respiratory Center/physiology , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Age Factors , Analgesics, Opioid/pharmacology , Animals , Calcium/metabolism , Cholera Toxin/pharmacology , Diagnostic Imaging/methods , Dose-Response Relationship, Radiation , Electric Stimulation/methods , Embryo, Mammalian , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Female , Fluorescent Antibody Technique/methods , In Vitro Techniques , Male , Mice , Patch-Clamp Techniques/methods , Pregnancy , Receptors, Neurokinin-1/metabolism , Respiratory Center/drug effects , Respiratory Center/embryology , Substance P/pharmacology , gamma-Aminobutyric Acid/pharmacologyABSTRACT
Establishment of neuronal identity requires co-ordinated expression of specific batteries of genes. These programs of gene expression are executed by activation of neuron-specific genes in neuronal cells and their repression in non-neuronal cells. Such co-ordinate regulation requires that individual activators and repressors regulate transcription from specific subsets of their potential target genes, yet we know little of the mechanisms that underlie this selective process. The RE-1 silencing transcription factor (REST) is a repressor that is proposed to silence transcription of numerous neuron-specific genes in non-neuronal cells via recruitment of two independent histone deacetylase (HDAC)-containing co-repressor complexes. However, in vivo, REST appears to be an obligate silencer for only a minority of RE-1-bearing genes. Here we examine the interaction of REST, Co-REST, Sin3A, HDAC1, and HDAC2 with two archetypical endogenous target genes, the M4 muscarinic receptor and the sodium type II channel (NaV1.2) genes. We find that these genes are present in distinct chromosomal domains. The NaV1.2 gene is actively transcribed but repressed by REST independently of histone deacetylation or DNA methylation and does not co-localize with epigenetic markers of silence, including dimethylation of H3K9 and HP1. In contrast, the M4 gene is maintained in a silent state independently of REST and co-localizes with dimethylated H3K9 and HP1alpha and HP1gamma, characteristic of silenced or senescent euchromatic DNA. This contrasts with the coordinate REST-dependent regulation of this locus reported previously. Taken together, we infer that distinct repressor complexes and mechanisms are operative at particular loci even in cell lines derived from a common embryological origin.
