Increased GABAA Receptor ε-Subunit Expression on Ventral Respiratory Column Neurons Protects Breathing during Pregnancy

Abstract

GABAergic signaling is essential for proper respiratory function. Potentiation of this signaling with allosteric modulators such as anesthetics, barbiturates, and neurosteroids can lead to respiratory arrest. Paradoxically, pregnant animals continue to breathe normally despite nearly 100-fold increases in circulating neurosteroids. ε subunit-containing GABAARs are insensitive to positive allosteric modulation, thus we hypothesized that pregnant rats increase ε subunit-containing GABAAR expression on brainstem neurons of the ventral respiratory column (VRC). In vivo, pregnancy rendered respiratory motor output insensitive to otherwise lethal doses of pentobarbital, a barbiturate previously used to categorize the ε subunit. Using electrode array recordings in vitro, we demonstrated that putative respiratory neurons of the preBötzinger Complex (preBötC) were also rendered insensitive to the effects of pentobarbital during pregnancy, but unit activity in the VRC was rapidly inhibited by the GABAAR agonist, muscimol. VRC unit activity from virgin and post-partum females was potently inhibited by both pentobarbital and muscimol. Brainstem ε subunit mRNA and protein levels were increased in pregnant rats, and GABAAR ε subunit expression co-localized with a marker of rhythm generating neurons (neurokinin 1 receptors) in the preBötC. These data support the hypothesis that pregnancy renders respiratory motor output and respiratory neuron activity insensitive to barbiturates, most likely via increased ε subunit-containing GABAAR expression on respiratory rhythm-generating neurons. Increased ε subunit expression may be critical to preserve respiratory function (and life) despite increased neurosteroid levels during pregnancy.

The role of ovarian hormone-derived neurosteroids on the regulation of GABAA receptors in affective disorders

“…Contrary to the findings in mice, an increase in δ-subunit expression in the dentate gyrus has been observed in rats (Sanna et al 2009), suggesting that GABA A R plasticity maybe more complicated than a simple homeostatic mechanism or that species differences exist. However, further supporting compensatory regulation of GABA A Rs during pregnancy, increased expression of the neurosteroid resistant ε - subunit has been proposed to play an important role in preventing neurosteroid mediated inhibition of respiratory neurons during pregnancy when neurosteroids levels are particularly high (Hengen et al 2012). …”

Abrupt changes in pentobarbital sensitivity in preBötzinger complex region, hypoglossal motor nucleus, nucleus tractus solitarius, and cortex during rat transitional period (P10–P15)

“…In contrast, cortical neurons are electrically silent during torpor, remain sensitive to pentobarbital, and do not increase epsilon subunit immunoreactivity (Hengen et al, 2009; 2011). Likewise, during late pregnancy in rats, when central allopregnanolone concentrations increase 3–4 fold (Concas et al, 1998), respiratory motor output on the phrenic nerve is maintained longer during sequential pentobarbital injections compared to non-pregnant female rats (Hengen et al, 2012). Respiratory frequency is preserved rather than phrenic burst amplitude, suggesting the preBötC neurons increase epsilon subunit expression.…”

“…Afterwards, 200 and 300 μM sodium pentobarbital (Fort Dodge Animal Health, Fort Dodge, Iowa, USA) were sequentially bath-applied to the slices for 45 min each to determine neuronal sensitivity to pentobarbital. The rationale for these concentrations is based on extensive previous work (Hengen et al, 2009; 2012). The pentobarbital concentration sufficient to decrease neuronal firing rates in vitro is dependent on overall neuronal excitability in the slice, which is modulated by bath [K + ].…”

“…Raw extracellular recordings of spontaneous action potentials were processed as described previously (Hengen et al, 2009, 2012). Individual neurons were identified and separated using Principal Component Analysis (Adamos et al, 2008).…”

“…Specifically, GABA A Rs become insensitive to pentobarbital during hibernation (Hengen et al, 2009, 2011) and pregnancy (Hengen et al, 2012). This response appears to be selective for respiratory-related medullary regions, such as ventral respiratory group and NTS, since cortical neurons remain highly sensitive to pentobarbital during both conditions (Hengen et al, 2009, 2011, 2012). Since there is potentially widespread decreased excitability in the rat medulla during P10–P15, we hypothesized that respiratory-related medullary regions would increase resistance to positive allosteric modulation to maintain appropriate neuronal excitability (i.e., decrease impact of inhibition).…”