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

Hengen KB, Nelson NR, Stang KM, Johnson SM, Smith SM, Watters JJ, Mitchell GS, Behan M - PLoS ONE (2015)

Bottom Line: The parameters governing GABAA receptor subtype expression patterns are not well understood, although significant shifts in subunit expression may support key physiological events.We hypothesized that this plasticity may be a compensatory response to a chronic increase in inhibitory tone caused by increased central neurosteroid levels.We hypothesize that increased inhibitory tone in the respiratory control network and cortex causes a compensatory increase in ε-subunit-containing GABAARs.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Training Program, University of Wisconsin, Madison, Madison, Wisconsin, United States of America.

ABSTRACT
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.

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GABAAR ε subunit protein expression in medulla and cortex of isoflurane-treated rats.A, Medullary slices used for in vitro electrophysiology were immunohistochemically labeled for the GABAAR ε subunit (red, top) and NK1-R (green, middle). Images were acquired in the location of electrode placement. The GABAAR ε and NK1-R exhibited >90% colocalization on neurons in the preBötC of the medulla (merge, bottom). Example image was acquired in the preBötC of a virgin female rat. B, Cortical slices previously used for in vitro electrophysiology were sectioned and immunohistochemically labeled for the GABAAR ε subunit. Example image was acquired in the cortex of a 30d isoflurane-treated male rat.
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pone.0119351.g003: GABAAR ε subunit protein expression in medulla and cortex of isoflurane-treated rats.A, Medullary slices used for in vitro electrophysiology were immunohistochemically labeled for the GABAAR ε subunit (red, top) and NK1-R (green, middle). Images were acquired in the location of electrode placement. The GABAAR ε and NK1-R exhibited >90% colocalization on neurons in the preBötC of the medulla (merge, bottom). Example image was acquired in the preBötC of a virgin female rat. B, Cortical slices previously used for in vitro electrophysiology were sectioned and immunohistochemically labeled for the GABAAR ε subunit. Example image was acquired in the cortex of a 30d isoflurane-treated male rat.

Mentions: To confirm the presence or absence of GABAAR ε subunit expression at the in vitro recording sites, medullary slices from recording experiments were treated with an antibody against the GABAAR ε subunit. Medullary sections were co-labeled for GABAAR ε subunit and for NK1-R (Substance P receptor) that is used as a marker for putative respiratory neurons in the VRC and preBötC [41, 42]. NK1-R and the GABAAR ε subunit exhibited >90% colocalization, both in the nucleus ambiguus (subcompact and compact; Lin et al., 2008), which was used as a landmark for electrode placement, and in the VRC (Fig. 3A; [36]). Neurons in the VRC displayed enriched somatic ε subunit staining as well as in primary neuronal processes (Fig. 3A). The pentobarbital-insensitive GABAARs identified in vitro were functionally consistent with immunohistochemically identified ε subunit-containing GABAARs in the VRC. To our surprise, the GABAAR ε subunit was detectable in CTX neurons in all groups as well. Layer 5 pyramidal cells demonstrated the most robust expression, and staining appeared to be largely restricted to the soma (Fig. 3B).


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

Hengen KB, Nelson NR, Stang KM, Johnson SM, Smith SM, Watters JJ, Mitchell GS, Behan M - PLoS ONE (2015)

GABAAR ε subunit protein expression in medulla and cortex of isoflurane-treated rats.A, Medullary slices used for in vitro electrophysiology were immunohistochemically labeled for the GABAAR ε subunit (red, top) and NK1-R (green, middle). Images were acquired in the location of electrode placement. The GABAAR ε and NK1-R exhibited >90% colocalization on neurons in the preBötC of the medulla (merge, bottom). Example image was acquired in the preBötC of a virgin female rat. B, Cortical slices previously used for in vitro electrophysiology were sectioned and immunohistochemically labeled for the GABAAR ε subunit. Example image was acquired in the cortex of a 30d isoflurane-treated male rat.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4352015&req=5

pone.0119351.g003: GABAAR ε subunit protein expression in medulla and cortex of isoflurane-treated rats.A, Medullary slices used for in vitro electrophysiology were immunohistochemically labeled for the GABAAR ε subunit (red, top) and NK1-R (green, middle). Images were acquired in the location of electrode placement. The GABAAR ε and NK1-R exhibited >90% colocalization on neurons in the preBötC of the medulla (merge, bottom). Example image was acquired in the preBötC of a virgin female rat. B, Cortical slices previously used for in vitro electrophysiology were sectioned and immunohistochemically labeled for the GABAAR ε subunit. Example image was acquired in the cortex of a 30d isoflurane-treated male rat.
Mentions: To confirm the presence or absence of GABAAR ε subunit expression at the in vitro recording sites, medullary slices from recording experiments were treated with an antibody against the GABAAR ε subunit. Medullary sections were co-labeled for GABAAR ε subunit and for NK1-R (Substance P receptor) that is used as a marker for putative respiratory neurons in the VRC and preBötC [41, 42]. NK1-R and the GABAAR ε subunit exhibited >90% colocalization, both in the nucleus ambiguus (subcompact and compact; Lin et al., 2008), which was used as a landmark for electrode placement, and in the VRC (Fig. 3A; [36]). Neurons in the VRC displayed enriched somatic ε subunit staining as well as in primary neuronal processes (Fig. 3A). The pentobarbital-insensitive GABAARs identified in vitro were functionally consistent with immunohistochemically identified ε subunit-containing GABAARs in the VRC. To our surprise, the GABAAR ε subunit was detectable in CTX neurons in all groups as well. Layer 5 pyramidal cells demonstrated the most robust expression, and staining appeared to be largely restricted to the soma (Fig. 3B).

Bottom Line: The parameters governing GABAA receptor subtype expression patterns are not well understood, although significant shifts in subunit expression may support key physiological events.We hypothesized that this plasticity may be a compensatory response to a chronic increase in inhibitory tone caused by increased central neurosteroid levels.We hypothesize that increased inhibitory tone in the respiratory control network and cortex causes a compensatory increase in ε-subunit-containing GABAARs.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Training Program, University of Wisconsin, Madison, Madison, Wisconsin, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus