GFP-expressing locus ceruleus neurons from Prp57 transgenic mice exhibit CO₂/H⁺ responses in primary cell culture

Abstract

Johnson SM, Haxhiu MA, Richerson GB. GFP-expressing locus ceruleus neurons from Prp57 transgenic mice exhibit CO 2/H ϩ responses in primary cell culture. J Appl Physiol 105: 1301-1311. First published July 17, 2008 doi:10.1152/japplphysiol.90414.2008.-The locus ceruleus (LC) contains neurons that increase their firing rate (FR) in vitro when exposed to elevated CO2/H ϩ and have been proposed to influence the respiratory network to make compensatory adjustments in ventilation. Prp57 transgenic mice express green fluorescent protein (GFP) in the LC and were used to isolate, culture, and target LC neurons for electrophysiological recording. We hypothesized that GFP-LC neurons would exhibit CO2/H ϩ chemosensitivity under primary culture conditions, evidenced as a change in FR. This is the first study to quantify CO2/H ϩ responses in LC neuron FR in cell culture. Neurons were continuously bathed with solutions containing antagonists of glutamate and GABA receptors, and the acidbase status was changed from control (5% CO2; pH ϳ7.4) to hypercapnic acidosis (9% CO 2; pH ϳ7.2) and hypocapnic alkalosis (3% CO 2; pH ϳ7.6). FR was quantified during perforated patch current clamp recordings. Approximately 86% of GFP-LC neurons were stimulated, and ϳ14% were insensitive to changes in CO2/H ϩ . The magnitude of the response of these neurons depended on the baseline FR, ranging from 155.9 Ϯ 6% when FR started at 2.95 Ϯ 0.49 Hz to 381 Ϯ 55.6% when FR started at 1.32 Ϯ 0.31 Hz. These results demonstrate that cultured LC neurons from Prp57 transgenic mice retain functional sensing molecules necessary for CO 2/H ϩ responses. Prp57 transgenic mice will serve as a valuable model to delineate mechanisms involved in CO2/H ϩ responsiveness in catecholaminergic neurons.CO2/H ϩ chemosensitivity; central respiratory chemoreceptors; carbon dioxide; primary cell culture; pH response CENTRAL RESPIRATORY CHEMORECEPTORS detect fluctuations in CO 2 and/or pH, alter their neuronal activity, and subsequently influence the respiratory network to appropriately adjust ventilation. Chemoreceptors were first proposed to be located in distinct cellular aggregations at or near the ventrolateral medullary surface (37, 57). However, it is now well established that there are many neurons widely distributed along the neuraxis that increase their firing rate (FR) in response to acidosis, including cells within the ventrolateral medullary surface (36,37,57), medullary raphé (6,49,51,70,71,74), pre-Bötzinger complex (34,52,61), retrotrapezoid nucleus (RTN) (20, 27, 56), hypothalamus (15, 16, 75), nucleus tractus solitarius (NTS), and locus ceruleus (LC) (12,18,22,23,28,33,40,42,53,55). It has been proposed that all of these groups of pH-sensitive neurons are central respiratory chemoreceptors, each one contributing to the increased respiratory motor output to ensure that the pH of the blood and cerebrospinal fluid is restricted to normal physiological limits (9, 10, 41). The relative importance of each one to the overall response, and the conditions under whi...

Involvement of TRP channels in the CO2 chemosensitivity of locus coeruleus neurons

“…Whole cell current clamp was performed in Ͼ200 LC neurons. As shown previously (Johnson et al 2008;Zhang et al 2010a), most LC neurons fired spontaneously at the basal condition in the presence of 5% CO 2 . In this study, we were focused on the LC neurons in which activity was stimulated by hypercapnia.…”

“…Under this condition, 56 out of 159 (35%) LC neurons were found to raise their FR by Ͼ10% when exposed to 8% CO 2 (Fig. 1) compared with 86% without Kir channel blockade as reported previously (Johnson et al 2008). Detailed data analysis of cellular responses to hypercapnia was performed in 21 LC neurons before and after the treatment with a TRPC blocker (see below).…”

“…Several groups of brain stem neurons, including those in the locus coeruleus (LC), play a role in the central CO 2 chemosensitivity (Ballantyne and Scheid 2000; Gargaglioni et al 2010;Jiang et al 2005;Putnam 2010). The LC neurons are CO 2 chemosensitive (Erlichman et al 2009;Johnson et al 2008;Oyamada et al 1998;Pineda and Aghajanian 1997;Stunden et al 2001). These norepinephrinergic (NE) neurons modulate firing activity of brain stem respiratory neurons (Biancardi et al 2008).…”

“…The NE neurons in the LC are intrinsically CO 2 chemosensitive (Johnson et al 2008;Nichols et al 2008;Oyamada et al 1998). An ionic mechanism has been shown to underlie the CO 2 chemosensitivity of these neurons.…”

Characterization of the chemosensitive response of individual solitary complex neurons from adult rats

“…However, the CI of SC neurons activated by HA recorded using sharp-tip electrodes (133 Ϯ 5%) is significantly (P Ͻ 0.001) lower than the CI found for activated SC neurons using the whole cell pipettes (177 Ϯ 8%). It is not clear why the CI is higher for activated SC neurons using the whole cell technique compared with the sharp-tip technique, but it may be due to the somewhat higher initial firing rate measured with sharp-tip vs. whole cell pipettes, which has been shown to affect the calculated values of CI (19). We note that the absolute increase in firing rate induced by HA is nearly identical when measured with whole cell (1.4 Hz) vs. sharp-tip (1.3 Hz) pipettes.…”