The Role of the Hyperpolarization-Activated Current in Modulating Rhythmic Activity in the Isolated Respiratory Network of Mice

Abstract

We examined the role of the hyperpolarization-activated current (I(h)) in the generation of the respiratory rhythm using a spontaneously active brainstem slice of mice. This preparation contains the hypoglossus (XII) nucleus, which is activated in-phase with inspiration and the pre-Bötzinger complex (PBC), the presumed site for respiratory rhythm generation. Voltage-clamp recordings (n = 90) indicate that cesium (Cs) (5 mM) blocked 77.2% of the I(h) current, and ZD 7288 (100 microM) blocked 85.8% of the I(h) current. This blockade increased the respiratory frequency by 161% in Cs and by 150% in ZD 7288 and increased the amplitude of integrated population activity in the XII by 97% in Cs and by 162% in ZD 7288, but not in the PBC (Cs, by 19%; ZD 7288, by -4.56%). All inspiratory PBC neurons (n = 44) recorded in current clamp within the active network revealed a significantly decreased frequency of action potentials during the interburst interval and an earlier onset of inspiratory bursts after I(h) current blockade. However, hyperpolarizing current pulses evoked only in a small proportion of inspiratory neurons (0% of type I; 29% of type II neurons) a depolarizing sag. Most of the neurons expressing an I(h) current (86%) were pacemaker neurons, which continued to generate rhythmic bursts after inactivating the respiratory network pharmacologically with CNQX alone or with CNQX, AP-5, strychnine, bicuculline, and carbenoxolone. Cs and ZD 7288 increased the frequency of pacemaker bursts and decreased the frequency of action potentials between pacemaker bursts. Our findings suggest that the I(h) current plays an important role in modulating respiratory frequency, which is presumably mediated by pacemaker neurons.

Expression of Functional Tyrosine Kinase B Receptors by Rhythmically Active Respiratory Neurons in the Pre-Bötzinger Complex of Neonatal Mice

“…Whole-cell patch-clamp recordings (n ϭ 37) were performed under visual control using differential interference contrast and infrared video microscopy, an Axoclamp2A amplifier (Axon Instruments, Foster city, CA), a digitizing interface (Digidata 1322A, Axon Instruments), and the software program pClamp8 (Axon Instruments). Neurons examined were type 2 nonpacemaker inspiratory neurons (Rekling et al, 1996;Thoby-Brisson et al, 2000) located ventral to the nucleus ambiguus and within 80 -100 m of the extracellular electrode. Patch electrodes were manufactured from borosilicate glass tubes containing a filament (Clark GC 150TF, Pangbourn, UK) and filled with a solution containing (in mM): 140 K-gluconic acid, 1 CaCl 2 , 10 EGTA, 2 MgCl 2 , 4 Na 2 -ATP, 10 HEPES, pH 7.2, and 0.05 mg/l Alexa Fluor 594 (Molecular Probes, Eugene, OR).…”

“…The PBC contains all elements sufficient for generation of the respiratory rhythm in slice preparations (Smith et al, 1991), and lesioning of the PBC in vivo abolishes breathing (Koshiya and Guyenet, 1998;Ramirez et al, 1998). The PBC includes a glutamatergic network of rhythmic neurons (Bianchi et al, 1995;Ramirez et al, 1997) that express the hyperpolarization-activated cationic current I h (Rekling et al, 1996;Thoby-Brisson et al, 2000) and the substance P receptor neurokinin 1 (NK1) (Gray et al, 1999;Wang et al, 2001).…”

Emergence of the Pre-Bötzinger Respiratory Rhythm Generator in the Mouse Embryo

“…This group is readily identifiable and amenable to analysis in in vitro preparations. The respiratory neural network in the PBC region contains rhythmic neurons connected by glutamatergic synapses, some of them exhibiting pacemaker properties (Johnson et al, 1994;Koshiya and Smith, 1999;Thoby-Brisson et al, 2000;Thoby-Brisson and Ramirez, 2001;Pena et al, 2004). The destruction of neurons expressing the NK1 receptor (NK1R) leading to an ataxic respiration (Gray et al, 2001) supports the view that the PBC plays a primary role in respiratory rhythmogenesis.…”

Endogenous Activation of Serotonin-2A Receptors Is Required for Respiratory Rhythm GenerationIn Vitro

“…After blockade of respiratory network activity with CNQX, some inspiratory neurons continue to exhibit endogenously generated pacemaker activity Thoby-Brisson et al, 2000;Thoby-Brisson and Ramirez, 2001;Del Negro et al, 2002). Because of their potential role in generating the respiratory rhythm (Rekling and Feldman, 1998;Butera et al, 1999;McCrimmon et al, 2000;Smith et al, 2000), we examined the effects of 5-HT 2A antagonists on this pacemaker activity.…”