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

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

Hibernation induces pentobarbital insensitivity in medulla but not cortex

The 13-lined ground squirrel (Ictidomys tridecemlineatus), a hibernating species, is a natural model of physiological adaption to an extreme environment. During torpor, body temperature drops to 0–4°C, and the cortex is electrically silent, yet the brain stem continues to regulate cardiorespiratory function. The mechanisms underlying selective inhibition in the brain during torpor are not known. To test whether altered GABAergic function is involved in regional and seasonal differences in neuronal activity, cortical and medullary slices from summer-active (SA) and interbout aroused (IBA) squirrels were placed in a standard in vitro recording chamber. Silicon multichannel electrodes were placed in cortex, ventral respiratory column (VRC), and nucleus tractus solitarius (NTS) to record spontaneous neuronal activity. In slices from IBA squirrels, bath-applied pentobarbital sodium (300 μM) nearly abolished cortical neuronal activity, but VRC and NTS neuronal activity was unaltered. In contrast, pentobarbital sodium (300 μM) nearly abolished all spontaneous cortical, VRC, and NTS neuronal activity in slices from SA squirrels. Muscimol (20 μM; GABAA receptor agonist) abolished all neuronal activity in cortical and medullary slices from both IBA and SA squirrels, thereby demonstrating the presence of functional GABAA receptors. Pretreatment of cortical slices from IBA squirrels with bicuculline (100 μM; GABAA receptor antagonist) blocked pentobarbital-dependent inhibition of spontaneous neuronal activity. We hypothesize that GABAA receptors undergo a seasonal modification in subunit composition, such that cardiorespiratory neurons are uniquely unaffected by surges of an endogenous positive allosteric modulator

Transcriptomic cell type structures in vivo neuronal activity across multiple timescales

Summary: Cell type is hypothesized to be a key determinant of a neuron’s role within a circuit. Here, we examine whether a neuron’s transcriptomic type influences the timing of its activity. We develop a deep-learning architecture that learns features of interevent intervals across timescales (ms to >30 min). We show that transcriptomic cell-class information is embedded in the timing of single neuron activity in the intact brain of behaving animals (calcium imaging and extracellular electrophysiology) as well as in a bio-realistic model of the visual cortex. Further, a subset of excitatory cell types are distinguishable but can be classified with higher accuracy when considering cortical layer and projection class. Finally, we show that computational fingerprints of cell types may be universalizable across structured stimuli and naturalistic movies. Our results indicate that transcriptomic class and type may be imprinted in the timing of single neuron activity across diverse stimuli

Visual deprivation during mouse critical period reorganizes network-level functional connectivity

A classic example of experience-dependent plasticity is ocular dominance (OD) shift, in which the responsiveness of neurons in the visual cortex is profoundly altered following monocular deprivation (MD). It has been postulated that OD shifts also modify global neural networks, but such effects have never been demonstrated. Here, we use wide-field fluorescence optical imaging (WFOI) to characterize calcium-based resting-state functional connectivity during acute (3 d) MD in female and male mice with genetically encoded calcium indicators

Daily isoflurane exposure increases barbiturate insensitivity in medullary respiratory and cortical neurons via expression of ε-subunit containing GABA ARs.

The parameters governing GABAA receptor subtype expression patterns are not well understood, although significant shifts in subunit expression may support key physiological events. For example, the respiratory control network in pregnant rats becomes relatively insensitive to barbiturates due to increased expression of ε-subunit-containing GABAARs in the ventral respiratory column. We hypothesized that this plasticity may be a compensatory response to a chronic increase in inhibitory tone caused by increased central neurosteroid levels. Thus, we tested whether increased inhibitory tone was sufficient to induce ε-subunit upregulation on respiratory and cortical neurons in adult rats. Chronic intermittent increases in inhibitory tone in male and female rats was induced via daily 5-min exposures to 3% isoflurane. After 7d of treatment, phrenic burst frequency was less sensitive to barbiturate in isoflurane-treated male and female rats in vivo. Neurons in the ventral respiratory group and cortex were less sensitive to pentobarbital in vitro following 7d and 30d of intermittent isoflurane-exposure in both male and female rats. The pentobarbital insensitivity in 7d isoflurane-treated rats was reversible after another 7d. We hypothesize that increased inhibitory tone in the respiratory control network and cortex causes a compensatory increase in ε-subunit-containing GABAARs

Increased GABA_AR ε subunit mRNA levels in medulla and cortex in 30d isoflurane-treated rats.

<p>Medullary GABA_AR ε mRNA levels were more than 3-fold greater in 30d isoflurane-treated rats compared to untreated control rats. Likewise, cortical GABA_AR ε mRNA levels were more than 4-fold increased in 30d isoflurane-treated rats. Statistics were conducted on delta CT values. Asterisk denotes p<0.05. Error bars indicate SEM.</p

Spontaneous neuronal activity resistance to bath-applied pentobarbital in medullary and cortical slices from 7d isoflurane-treated rats.

<p>Spontaneous activity of neurons in the region anatomically consistent with the VRC and PreBötC (all labeled as VRC neurons) are insensitive to bath-applied pentobarbital (300 μM) following 7d isoflurane treatment. <b><i>A</i></b>, Mean normalized firing rate of VRC (n = 62, open circles) and CTX (n = 37, filled circles) neurons from male rats in response to 300 μM pentobarbital (applied at vertical bar, t = 30 min). <b><i>B</i></b>, Pentobarbital had equivalent depressive effects on VRC (n = 37) and CTX (n = 47) neurons from virgin female rats. <b><i>C</i></b>, Following pentobarbital treatment, spontaneous activity of VRC neurons (n = 62) from male rats treated with intermittent isoflurane for 7d were significantly more active than VRC neurons from untreated control rats. The response of cortical neurons (n = 49) to pentobarbital was non-significantly shifted in the same direction. <b><i>D</i></b>, Likewise, in female rats treated with isoflurane for 7d, pentobarbital failed to inhibit spontaneous neuronal activity in the VRC neurons (n = 40). CTX neurons (n = 51) trended non-significantly towards an increase in activity compared to untreated control female rats. <b><i>E</i></b>, This effect was reversible, as 7d of recovery following 7d of intermittent isoflurane shifted spontaneous activity of VRC (n = 27) and CTX neurons (n = 38) in rats toward baseline values. <b><i>F</i></b>., 30d of intermittent isoflurane significantly attenuated the pentobarbital response in VRC (n = 40) and CTX neurons (n = 32) in male rats. To confirm the presence of pentobarbital-insensitive GABA_ARs, muscimol (20 μM) was applied at 90 min (dashed vertical line). Nearly all neurons that were resistant to pentobarbital were inhibited by muscimol. <b><i>G</i></b>, In female rats exposed to 30d isoflurane treatment, the response of VRC (n = 123) and CTX (n = 83) neurons to pentobarbital was significantly attenuated. These neurons were also silenced by muscimol (dashed vertical line). (<b><i>H</i>,<i>I</i>)</b> The average steady-state responses to bath-applied pentobarbital are quantified for the different isoflurane treatments and recovery for male rats (<b><i>C</i></b>) and female rats (<b><i>D</i></b>). The asterisk indicates p<0.05 for comparison to control while the pound sign indicates p<0.05 for comparison to 7d treatment.</p