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Genetic reduction of GABAA receptor γ2 subunit expression potentiates the immobilizing action of isoflurane
Neuroscience Letters 472 (2010) 1–4
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Genetic reduction of GABAA receptor ␥2 subunit expression potentiates the immobilizing action of isoflurane Kenji Seo a,∗ , Hiroyuki Seino a , Hiroyuki Yoshikawa a , Andrey B. Petrenko b , Hiroshi Baba b , Naoshi Fujiwara c , Genji Someya a , Yoshiro Kawano d , Takeyasu Maeda d , Masato Matsuda e , Takashi Kanematsu f , Masato Hirata g a
Division of Dental Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan Division of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan c Department of Medical Technology, Niigata University School of Health Sciences, Niigata 951-9518, Japan d Division of Oral Anatomy, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8514, Japan e Niigata University Center for Integrated Human Brain Science, Brain Research Institute, Niigata 951-8585, Japan f Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8553, Japan g Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science and Station for Collaborative Research, Kyushu University, Fukuoka 812-8582, Japan b
a r t i c l e
i n f o
Article history: Received 12 November 2009 Received in revised form 15 January 2010 Accepted 15 January 2010 Keywords: GABAA receptors PRIP-1 PRIP-2 Double knockout Isoflurane Minimum alveolar concentration
a b s t r a c t Potentiation of inhibitory ␥-aminobutyric acid subtype A (GABAA ) receptor function is involved in the mechanisms of anesthetic action. The present study examined the immobilizing action of the volatile anesthetic isoflurane in mice with double knockout (DKO) of phospholipase C-related inactive protein (PRIP)-1 and -2. Both of these proteins play important roles in the expression of GABAA receptors containing the ␥2 subunit on the neuronal cell surface. Immunohistochemistry for GABAA receptor subunits demonstrated reduced expression of ␥2 subunits in the spinal cord of the DKO mice. Immunohistochemistry also revealed up-regulation of the ␣1 and 3 subunits even though there were no apparent differences in the immunoreactivities for the 2 subunits between wild-type and DKO mice. The tailclamp method was used to evaluate the anesthetic/immobilizing effect of isoflurane and the minimum alveolar concentration (MAC) was significantly lower in DKO mice compared with wild-type controls (1.07 ± 0.01% versus 1.36 ± 0.04% atm), indicating an increased sensitivity to isoflurane in DKO mice. These immunohistochemical and pharmacological findings suggest that reduced expression of the GABAA receptor ␥2 subunit affects the composition and function of spinal GABAA receptors and potentiates the immobilizing action of isoflurane. © 2010 Elsevier Ireland Ltd. All rights reserved.
Volatile anesthetics such as isoflurane are commonly used for the anesthetic management of surgical patients. They are considered to induce anesthesia by potentiating the function of inhibitory ␥aminobutyric acid subtype A (GABAA ) receptors. Native GABAA receptors are composed of multiple subunits, including ␥2 subunits that are ubiquitously present in the central nervous system. Although the GABAA ␥2 subunits have been reported to represent a target for mediating the anxiolytic and sedative effects of benzodiazepines [2], their roles in the mechanism of anesthesia remain unclear. Phospholipase C-related but catalytically inactive protein (PRIP) was first identified as an inositol-1,4,5-trisphosphate binding protein [5], and was subsequently revealed to play important roles in the intracellular trafficking and phospho-regulation of GABAA
∗ Corresponding author. Tel.: +81 25 227 2971; fax: +81 25 227 0812. E-mail address: [email protected] (K. Seo). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.01.031
receptors [4,13]. PRIP-1 and -2 genetically deficient mice show some alterations in GABAA receptors that are related to the ␥2 subunit. PRIP-1/-2 double knockout (DKO) mice were reported to show reduced cell surface expression of the GABAA receptors containing the ␥2 subunit, as shown by decreased binding of the GABAA receptor ␥2 subunit-specific agonist flumazenil to hippocampal neurons [10]. Moreover, the saturation binding of GABAA receptor-specific agonist muscimol was elevated in mutant mice [10]. In the present study, we first immunohistochemically evaluated the effect of DKO on GABAA receptor subunit expression in the spinal cord and then used the tail-clamp assay to pharmacologically examine the effect of disrupted ␥2 subunit function on the anesthetic properties of isoflurane. This study was approved and performed based on the guidelines of the Niigata University Intramural Animal Use and Care Committee. All procedures were performed on male adult C57BL/6 wildtype (WT) and PRIP-1/-2 DKO mice. The animal genotypes were determined by polymerase chain reaction (PCR) using DNA
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K. Seo et al. / Neuroscience Letters 472 (2010) 1–4
Fig. 1. Genotyping of wild-type (WT) and PRIP-1/-2 double knockout (DKO) mice using PCR and agarose gel electrophoresis of DNA specimens. The PCR primers and products were as follows: PRIP-1 forward (F2 primer), 5 -ACACAGTCACATGGATTCTGTCTGC-3 ; PRIP-1 reverse (R1 primer; KO), 5 -GTGGGGGTGGGGTGGGATTA-3 ; PRIP-1 reverse (R2 primer; PRIP-1 product sizes, WT), 5 -CAAGCGGTTGCTTACTACGAGAGACC-3 ; 830 bp for WT and 540 bp for PRIP-1 KO; PRIP-2 forward (F3 primer), 5 -AAGAATAGATGCTCCCCGAAGC-3 ; PRIP-2 reverse (Neo primer; KO), 5 -CCTGTGCTCTAGTAGCTTTACG-3 ; PRIP-2 reverse (R2 primer; WT), 5 GAAGGAATCTGTACTCGGCTAG-3 ; PRIP-2 product sizes, 420 bp for WT and 120 bp for PRIP-2 KO.
extracted from ear specimens taken from the mice (Fig. 1). Although the gross appearances did not differ between the two genotypes, the DKO mice tended to exhibit slower movements and lower overall activity. The DKO mice did not show any spontaneous seizures, as previously described [10]. The animals were housed under a standard 12 h/12 h light/dark cycle, and water and food were available ad libitum. For immunohistochemical examination of GABAA receptor ␥2, ␣1, 2 and 3 subunit expression in the spinal cord dorsal horn, the animals were transcardially perfused with a fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.4) under deep anesthesia by an intraperitoneal injection of 8% chloral hydrate (400 mg/kg). Tissue blocks, including the cervical spinal cord, were cut horizontally at a thickness of 10 m using a cryostat (Cryostat M2; Ames, Elkhart, IN). The sections were primarily incubated with a polyclonal antiserum against GABAA receptor ␥2 subunit (1:1000, Biomol International, Plymouth Meeting, PA), and subsequently incubated with an FITCconjugated donkey anti-rabbit IgG (1:400, Santa Cruz Biochemistry, Inc., Santa Cruz, CA). Other sections were primarily incubated with antiserum against GABAA receptor ␣1, 2 and 3 subunits (1:200, Santa Cruz biotechnology, Inc, CA), and subsequently incubated with FITC donkey anti-goat IgG (1:200, Santa Cruz biotechnology, Inc, CA). All sections were cover-slipped with a Vectashield (Vector Laboratories, Burlingame, CA) and examined under a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). The immunoreactivity of ␣1, 2, 3 and ␥2 subunits in the spinal cord dorsal horn was quantitatively analyzed using ImageJ® (http://rsb.info.nih.gov/ij/), and the total immunoreactive area for each subunit was calculated and compared between the wild-type and DKO mice. For anesthetic sensitivity measurements, a classic tail-clamp paradigm was applied for the genotyped mice to determine the minimum alveolar anesthetic concentration (MAC) values, which reflect the immobilizing properties of isoflurane. Animals were individually placed in small plastic chambers connected to a vaporizer and an oxygen source, and their body temperatures were actively maintained between 36 and 38 ◦ C by warming with a plastic plate filled with circulating hot water placed below the chambers. Isoflurane (Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan) was vaporized in 2 l/min oxygen. The concentration of the anesthetic, which was maintained for a minimum equilibration period of 20 min, was continually monitored using an infrared gas
analyzer (Capnomac Ultima; Instrumentarium Corp., Helsinki, Finland). The rectal temperatures were also measured with a digital thermometer (TD-300; Shibaura Electronics, Saitama, Japan) before and after each measurement. Isoflurane was administered to each chamber at an initial concentration of 0.8% atm. After the equilibration period, the movement in response to a tail clamp was tested by placing an alligator clip on the proximal one-third of the tail. The concentration of isoflurane was then increased by 0.1% atm and, after another equilibration period, the tail clamp response was tested again. If no movement was observed, the clamp was left in place for a 1-min period. The concentration at which the mouse lost its tail-clamp reflex was noted. The MAC was calculated by averaging the two concentrations at which the mouse either retained or lost the movement response to the tail clamp. MAC values and total areas immunoreactive to each of the stained GABAA receptor subunits were compared between the two experimental groups using Student’s t-test (SigmaStat ver3.11; Systat Software Inc., Richmond, CA). Values of p < 0.05 were considered to indicate a significant difference. First, we performed immunohistochemical staining to examine the effect of DKO of PRIP-1 and -2 on GABAA receptor ␥2 subunit expression in the spinal cord, a primary site for mediating the anesthetic/immobilizing action of inhaled anesthetics [11]. In the spinal cord of WT mice, the immunoreactivity for GABAA receptor ␥2 subunit was exclusively localized to the cell surfaces in laminae I and II and deeper layers, the latter showing very high levels of ␥2 subunit expression (Fig. 2). In contrast, DKO mice showed reduced ␥2 subunit immunoreactivity (Fig. 2). The immunoreactivities for ␣1, 3 and 2 subunits were scattered throughout the superficial and deeper layers, and there were no apparent differences in 2 subunit expression patterns between the two genotypes (Fig. 2). Statistical analysis of the average total immunoreactive areas indicated significant differences between WT and DKO mice for the expression levels of ␣1, 3 and ␥2 subunits (t-test, p < 0.05), except for 2 subunits (Student’s t-test, p > 0.05) (Table 1). Next, we evaluated the anesthetic properties of isoflurane in DKO and WT mice. An anesthetic state was induced in both genotypes, with subsequent complete and uneventful recovery after the cessation of anesthetic inhalation. The MAC values for isoflurane were 1.36 ± 0.04% atm (n = 10) and 1.07 ± 0.01% atm (n = 10) in the WT and DKO mice, respectively (Fig. 3), indicating the DKO mice were more susceptible to the immobilizing effect of isoflurane anesthesia (Student’s t-test, p < 0.001). Anesthesia is a multicomponent state and anesthetic-induced immobility is one of its major determinants. Several studies, including those performed in decerebrated animals, led to the conclusion that the spinal cord is the primary site that mediates the ability of volatile anesthetics such as isoflurane to produce immobility [11]. Volatile anesthetics show prominent potentiation of GABAA receptor function [14]. Therefore, determining the subunit composition Table 1 Comparison of the total areas of immunoreactivity to ␣1, 2, 3 and ␥2 subunits in WT and DKO mice. The average immunoreactive areas for ␣1 and 3 subunits were significantly larger in DKO than in WT mice (t-test, p < 0.05). The average immunoreactive areas for the ␥2 subunit were significantly smaller in DKO mice (t-test, p < 0.05). There was no significant difference between two groups of mice in the expression of the 2 subunit (t-test, p > 0.05). Values are means (standard deviation). * The absolute values of ␥2 subunit immunoreactivity are different from those for other subunits. This is speculated to be due to differences in antibody sensitivity. Subunit
Wild
DKO
␣1 2 3 ␥2
380.21 (148.47) 403.72 (200.32) 189.69 (98.74) 0.011 (0.044)
790.85 (125.16)* 681.58 (391.65) 430.86 (91.14)* 0.003 (0.002)*
Mean (SD) m2 . * p < 0.05.
K. Seo et al. / Neuroscience Letters 472 (2010) 1–4
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The present immunohistochemical observations indicate that PRIP-1/-2 depletion not only drastically reduced the expression of the ␥2 subunit in the spinal cord dorsal horn neurons, but also increased the expression of ␣1 and 3 subunits in the spinal cord. This finding is consistent with earlier ligand-binding assays performed in PRIP-1/-2 DKO mice, which revealed greater expression of GABAA receptors concomitant with lower ␥2 subunit expression, and an increase in the cell surface GABA binding sites in hippocampal neurons [10]. Therefore, the action of isoflurane, which potentiates GABAA receptor function, is likely to affect these GABAA receptors by increasing the expression of the ␣1 and 3 subunits, thus enhancing GABA activity in DKO mice. Several lines of evidence indicate that, of the various GABAA receptor subunits, the 3 subunits may have a particular role in isoflurane-induced immobility. It has been shown that the MAC for isoflurane was unaltered after knock-in of the ␣1 subunit [6,12], while ␣2 subunit knockout did not change the GABA currents after exposure to isoflurane [7]. On the other hand, the MAC values for isoflurane were larger in 3 subunit (N265M) knock-in mice than in wild-type controls [9]. Furthermore, 3(N265M) knock-in mice displayed reduced sensitivity to the immobilizing action of isoflurane [8], even though there were no detectable differences in motor activity or sensitivity to noxious thermal stimuli versus wild-type control mice [3]. Therefore, it is reasonable to consider that a variation in the 3 subunit composition is critical for the ability of inhaled anesthetics to enhance the GABAA receptor function, as evidenced by the ability of volatile anesthetics to produce immobility in response to noxious stimulation. It is possible that changes in the expression and/or function of other GABAA receptor subunits, or even other neurotransmitter systems relevant to anesthetic mechanisms, may be responsible for our observations. However, we suggest that genetic reduction of ␥2 subunit expression in DKO animals affected the composition and function of other GABAA receptor subunits and increased the inhibitory tone and facilitated the effect of GABAergic compounds, such as isoflurane. Further studies are needed to fully understand the relationship between anesthetic action and GABAA receptor function.
Fig. 2. Immunohistochemical staining of the spinal cord dorsal horn. (A) A picture of a horizontal section of the cervical spinal cord. Immunoreactivity for GABAA receptor subunits in the outlined area is shown at a higher magnification in (B). Tr: spinal cord tract. (B) Immunoreactivity for ␣1, 2, 3 and ␥2 subunits. WT: wild type mouse; DKO: PRIP-1/-2 double knockout mouse; Tr: spinal cord tract; I: lamina I; II: lamina II. ␥2, ␣1, 2 and 3 indicate the respective GABAA receptor subunits. Scale bar: 50 m.
and expression patterns of different GABAA receptor subunits in the spinal cord is particularly important. As demonstrated in a previous immunohistochemical study, most neurons in the spinal cord express GABAA receptors subunit triplets ␣1–6/2,3/␥2. However, the 2,3 subunits were not detected in motor neurons, which may possess “atypical” receptors subtypes (i.e. ␣2/␥2 and ␣2/␣5/␥2) [1]. The effect of anesthetic agents on motor neurons is considered to be more important for achieving immobility than their effect on other neuronal subtypes. However, PRIP requires the presence of  subunits to execute the facilitation of surface expression of GABAA receptors containing ␥2 subunits [10]. Therefore, the absence of  subunits in motor neurons suggests that depletion of PRIP-1 and -2 is unlikely to affect these neurons. This suggests that the enhanced immobilizing action of isoflurane observed in the DKO mice was probably mediated through the perceptive neurons present in the dorsal horn, rather than in the motor neurons of the ventral horn.
Fig. 3. Distribution of the MAC values for isoflurane in wild-type (WT) and PRIP-1/2 double knockout (DKO) mice. There is a significant difference between the mean isoflurane MAC values (p < 0.001, Student’s t-test).
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Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (No. 20390511). References [1] S. Bohlhalter, O. Weinmann, H. Mohler, J.M. Fritschy, Laminar compartmentalization of GABAA-receptor subtypes in the spinal cord: an immunohistochemical study, J. Neurosci. 16 (1996) 283–297. [2] U. Gunther, J. Benson, D. Benke, J.M. Fritschy, G. Reyes, F. Knoflach, F. Crestani, A. Aguzzi, M. Arigoni, Y. Lang, H. Bluethmann, H. Mohler, B. Lüscher, Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 7749–7753. [3] R. Jurd, M. Arras, S. Lambert, B. Drexler, R. Siegwart, F. Crestani, M. Zaugg, K.E. Vogt, B. Ledermann, B. Antkowiak, U. Rudolph, General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit, FASEB J. 17 (2003) 250–252. [4] T. Kanematsu, I.S. Jang, T. Yamaguchi, H. Nagahama, K. Yoshimura, K. Hidaka, M. Matsuda, H. Takeuchi, Y. Misumi, K. Nakayama, T. Yamamoto, N. Akaike, M. Hirata, Role of the PLC-related, catalytically inactive protein p130 in GABA(A) receptor function, EMBO J. 21 (2002) 1004–1011. [5] T. Kanematsu, H. Takeya, Y. Watanabe, S. Ozaki, M. Yoshida, T. Koga, S. Iwanaga, M. Hirata, Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol, J. Biol. Chem. 267 (1992) 6518–6525. [6] J. Kim, R. Atherley, D.F. Werner, G.E. Homanics, E. Carstens, J.F. Antognini, Isoflurane depression of spinal nociceptive processing and minimum alveolar anesthetic concentration are not attenuated in mice expressing isoflurane resistant gamma-aminobutyric acid type-A receptors, Neurosci. Lett. 420 (2007) 209–212.
[7] M.D. Krasowski, N.L. Harrison, General anaesthetic actions on ligand-gated ion channels, Cell. Mol. Life Sci. 55 (1999) 1278–1303. [8] S. Lambert, M. Arras, K.E. Vogt, U. Rudolph, Isoflurane-induced surgical tolerance mediated only in part by beta3-containing GABA(A) receptors, Eur. J. Pharmacol. 516 (2005) 23–27. [9] M. Liao, J.M. Sonner, R. Jurd, U. Rudolph, C.M. Borghese, R.A. Harris, M.J. Laster, E.I. Eger 2nd, Beta3-containing gamma-aminobutyric acidA receptors are not major targets for the amnesic and immobilizing actions of isoflurane, Anesth. Analg. 101 (2005) 412–418, table of contents. [10] A. Mizokami, T. Kanematsu, H. Ishibashi, T. Yamaguchi, I. Tanida, K. Takenaka, K.I. Nakayama, K. Fukami, T. Takenawa, E. Kominami, S.J. Moss, T. Yamamoto, J. Nabekura, M. Hirata, Phospholipase C-related inactive protein is involved in trafficking of gamma2 subunit-containing GABA(A) receptors to the cell surface, J. Neurosci. 27 (2007) 1692–1701. [11] J.M. Sonner, J.F. Antognini, R.C. Dutton, P. Flood, A.T. Gray, R.A. Harris, G.E. Homanics, J. Kendig, B. Orser, D.E. Raines, I.J. Rampil, J. Trudell, B. Vissel, E.I. Eger 2nd, Inhaled anesthetics and immobility: mechanisms, mysteries, and minimum alveolar anesthetic concentration, Anesth. Analg. 97 (2003) 718–740. [12] J.M. Sonner, D.F. Werner, F.P. Elsen, Y. Xing, M. Liao, R.A. Harris, N.L. Harrison, M.S. Fanselow, E.I. Eger 2nd, G.E. Homanics, Effect of isoflurane and other potent inhaled anesthetics on minimum alveolar concentration, learning, and the righting reflex in mice engineered to express alpha1 gamma-aminobutyric acid type A receptors unresponsive to isoflurane, Anesthesiology 106 (2007) 107–113. [13] M. Terunuma, I.S. Jang, S.H. Ha, J.T. Kittler, T. Kanematsu, J.N. Jovanovic, K.I. Nakayama, N. Akaike, S.H. Ryu, S.J. Moss, M. Hirata, GABAA receptor phospho-dependent modulation is regulated by phospholipase C-related inactive protein type 1, a novel protein phosphatase 1 anchoring protein, J. Neurosci. 24 (2004) 7074–7084. [14] T. Yamakura, E. Bertaccini, J.R. Trudell, R.A. Harris, Anesthetics and ion channels: molecular models and sites of action, Annu. Rev. Pharmacol. Toxicol. 41 (2001) 23–51.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Genetic reduction of GABAA receptor ␥2 subunit expression potentiates the immobilizing action of isoflurane Kenji Seo a,∗ , Hiroyuki Seino a , Hiroyuki Yoshikawa a , Andrey B. Petrenko b , Hiroshi Baba b , Naoshi Fujiwara c , Genji Someya a , Yoshiro Kawano d , Takeyasu Maeda d , Masato Matsuda e , Takashi Kanematsu f , Masato Hirata g a
Division of Dental Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan Division of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan c Department of Medical Technology, Niigata University School of Health Sciences, Niigata 951-9518, Japan d Division of Oral Anatomy, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8514, Japan e Niigata University Center for Integrated Human Brain Science, Brain Research Institute, Niigata 951-8585, Japan f Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8553, Japan g Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science and Station for Collaborative Research, Kyushu University, Fukuoka 812-8582, Japan b
a r t i c l e
i n f o
Article history: Received 12 November 2009 Received in revised form 15 January 2010 Accepted 15 January 2010 Keywords: GABAA receptors PRIP-1 PRIP-2 Double knockout Isoflurane Minimum alveolar concentration
a b s t r a c t Potentiation of inhibitory ␥-aminobutyric acid subtype A (GABAA ) receptor function is involved in the mechanisms of anesthetic action. The present study examined the immobilizing action of the volatile anesthetic isoflurane in mice with double knockout (DKO) of phospholipase C-related inactive protein (PRIP)-1 and -2. Both of these proteins play important roles in the expression of GABAA receptors containing the ␥2 subunit on the neuronal cell surface. Immunohistochemistry for GABAA receptor subunits demonstrated reduced expression of ␥2 subunits in the spinal cord of the DKO mice. Immunohistochemistry also revealed up-regulation of the ␣1 and 3 subunits even though there were no apparent differences in the immunoreactivities for the 2 subunits between wild-type and DKO mice. The tailclamp method was used to evaluate the anesthetic/immobilizing effect of isoflurane and the minimum alveolar concentration (MAC) was significantly lower in DKO mice compared with wild-type controls (1.07 ± 0.01% versus 1.36 ± 0.04% atm), indicating an increased sensitivity to isoflurane in DKO mice. These immunohistochemical and pharmacological findings suggest that reduced expression of the GABAA receptor ␥2 subunit affects the composition and function of spinal GABAA receptors and potentiates the immobilizing action of isoflurane. © 2010 Elsevier Ireland Ltd. All rights reserved.
Volatile anesthetics such as isoflurane are commonly used for the anesthetic management of surgical patients. They are considered to induce anesthesia by potentiating the function of inhibitory ␥aminobutyric acid subtype A (GABAA ) receptors. Native GABAA receptors are composed of multiple subunits, including ␥2 subunits that are ubiquitously present in the central nervous system. Although the GABAA ␥2 subunits have been reported to represent a target for mediating the anxiolytic and sedative effects of benzodiazepines [2], their roles in the mechanism of anesthesia remain unclear. Phospholipase C-related but catalytically inactive protein (PRIP) was first identified as an inositol-1,4,5-trisphosphate binding protein [5], and was subsequently revealed to play important roles in the intracellular trafficking and phospho-regulation of GABAA
∗ Corresponding author. Tel.: +81 25 227 2971; fax: +81 25 227 0812. E-mail address: [email protected] (K. Seo). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.01.031
receptors [4,13]. PRIP-1 and -2 genetically deficient mice show some alterations in GABAA receptors that are related to the ␥2 subunit. PRIP-1/-2 double knockout (DKO) mice were reported to show reduced cell surface expression of the GABAA receptors containing the ␥2 subunit, as shown by decreased binding of the GABAA receptor ␥2 subunit-specific agonist flumazenil to hippocampal neurons [10]. Moreover, the saturation binding of GABAA receptor-specific agonist muscimol was elevated in mutant mice [10]. In the present study, we first immunohistochemically evaluated the effect of DKO on GABAA receptor subunit expression in the spinal cord and then used the tail-clamp assay to pharmacologically examine the effect of disrupted ␥2 subunit function on the anesthetic properties of isoflurane. This study was approved and performed based on the guidelines of the Niigata University Intramural Animal Use and Care Committee. All procedures were performed on male adult C57BL/6 wildtype (WT) and PRIP-1/-2 DKO mice. The animal genotypes were determined by polymerase chain reaction (PCR) using DNA
2
K. Seo et al. / Neuroscience Letters 472 (2010) 1–4
Fig. 1. Genotyping of wild-type (WT) and PRIP-1/-2 double knockout (DKO) mice using PCR and agarose gel electrophoresis of DNA specimens. The PCR primers and products were as follows: PRIP-1 forward (F2 primer), 5 -ACACAGTCACATGGATTCTGTCTGC-3 ; PRIP-1 reverse (R1 primer; KO), 5 -GTGGGGGTGGGGTGGGATTA-3 ; PRIP-1 reverse (R2 primer; PRIP-1 product sizes, WT), 5 -CAAGCGGTTGCTTACTACGAGAGACC-3 ; 830 bp for WT and 540 bp for PRIP-1 KO; PRIP-2 forward (F3 primer), 5 -AAGAATAGATGCTCCCCGAAGC-3 ; PRIP-2 reverse (Neo primer; KO), 5 -CCTGTGCTCTAGTAGCTTTACG-3 ; PRIP-2 reverse (R2 primer; WT), 5 GAAGGAATCTGTACTCGGCTAG-3 ; PRIP-2 product sizes, 420 bp for WT and 120 bp for PRIP-2 KO.
extracted from ear specimens taken from the mice (Fig. 1). Although the gross appearances did not differ between the two genotypes, the DKO mice tended to exhibit slower movements and lower overall activity. The DKO mice did not show any spontaneous seizures, as previously described [10]. The animals were housed under a standard 12 h/12 h light/dark cycle, and water and food were available ad libitum. For immunohistochemical examination of GABAA receptor ␥2, ␣1, 2 and 3 subunit expression in the spinal cord dorsal horn, the animals were transcardially perfused with a fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.4) under deep anesthesia by an intraperitoneal injection of 8% chloral hydrate (400 mg/kg). Tissue blocks, including the cervical spinal cord, were cut horizontally at a thickness of 10 m using a cryostat (Cryostat M2; Ames, Elkhart, IN). The sections were primarily incubated with a polyclonal antiserum against GABAA receptor ␥2 subunit (1:1000, Biomol International, Plymouth Meeting, PA), and subsequently incubated with an FITCconjugated donkey anti-rabbit IgG (1:400, Santa Cruz Biochemistry, Inc., Santa Cruz, CA). Other sections were primarily incubated with antiserum against GABAA receptor ␣1, 2 and 3 subunits (1:200, Santa Cruz biotechnology, Inc, CA), and subsequently incubated with FITC donkey anti-goat IgG (1:200, Santa Cruz biotechnology, Inc, CA). All sections were cover-slipped with a Vectashield (Vector Laboratories, Burlingame, CA) and examined under a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). The immunoreactivity of ␣1, 2, 3 and ␥2 subunits in the spinal cord dorsal horn was quantitatively analyzed using ImageJ® (http://rsb.info.nih.gov/ij/), and the total immunoreactive area for each subunit was calculated and compared between the wild-type and DKO mice. For anesthetic sensitivity measurements, a classic tail-clamp paradigm was applied for the genotyped mice to determine the minimum alveolar anesthetic concentration (MAC) values, which reflect the immobilizing properties of isoflurane. Animals were individually placed in small plastic chambers connected to a vaporizer and an oxygen source, and their body temperatures were actively maintained between 36 and 38 ◦ C by warming with a plastic plate filled with circulating hot water placed below the chambers. Isoflurane (Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan) was vaporized in 2 l/min oxygen. The concentration of the anesthetic, which was maintained for a minimum equilibration period of 20 min, was continually monitored using an infrared gas
analyzer (Capnomac Ultima; Instrumentarium Corp., Helsinki, Finland). The rectal temperatures were also measured with a digital thermometer (TD-300; Shibaura Electronics, Saitama, Japan) before and after each measurement. Isoflurane was administered to each chamber at an initial concentration of 0.8% atm. After the equilibration period, the movement in response to a tail clamp was tested by placing an alligator clip on the proximal one-third of the tail. The concentration of isoflurane was then increased by 0.1% atm and, after another equilibration period, the tail clamp response was tested again. If no movement was observed, the clamp was left in place for a 1-min period. The concentration at which the mouse lost its tail-clamp reflex was noted. The MAC was calculated by averaging the two concentrations at which the mouse either retained or lost the movement response to the tail clamp. MAC values and total areas immunoreactive to each of the stained GABAA receptor subunits were compared between the two experimental groups using Student’s t-test (SigmaStat ver3.11; Systat Software Inc., Richmond, CA). Values of p < 0.05 were considered to indicate a significant difference. First, we performed immunohistochemical staining to examine the effect of DKO of PRIP-1 and -2 on GABAA receptor ␥2 subunit expression in the spinal cord, a primary site for mediating the anesthetic/immobilizing action of inhaled anesthetics [11]. In the spinal cord of WT mice, the immunoreactivity for GABAA receptor ␥2 subunit was exclusively localized to the cell surfaces in laminae I and II and deeper layers, the latter showing very high levels of ␥2 subunit expression (Fig. 2). In contrast, DKO mice showed reduced ␥2 subunit immunoreactivity (Fig. 2). The immunoreactivities for ␣1, 3 and 2 subunits were scattered throughout the superficial and deeper layers, and there were no apparent differences in 2 subunit expression patterns between the two genotypes (Fig. 2). Statistical analysis of the average total immunoreactive areas indicated significant differences between WT and DKO mice for the expression levels of ␣1, 3 and ␥2 subunits (t-test, p < 0.05), except for 2 subunits (Student’s t-test, p > 0.05) (Table 1). Next, we evaluated the anesthetic properties of isoflurane in DKO and WT mice. An anesthetic state was induced in both genotypes, with subsequent complete and uneventful recovery after the cessation of anesthetic inhalation. The MAC values for isoflurane were 1.36 ± 0.04% atm (n = 10) and 1.07 ± 0.01% atm (n = 10) in the WT and DKO mice, respectively (Fig. 3), indicating the DKO mice were more susceptible to the immobilizing effect of isoflurane anesthesia (Student’s t-test, p < 0.001). Anesthesia is a multicomponent state and anesthetic-induced immobility is one of its major determinants. Several studies, including those performed in decerebrated animals, led to the conclusion that the spinal cord is the primary site that mediates the ability of volatile anesthetics such as isoflurane to produce immobility [11]. Volatile anesthetics show prominent potentiation of GABAA receptor function [14]. Therefore, determining the subunit composition Table 1 Comparison of the total areas of immunoreactivity to ␣1, 2, 3 and ␥2 subunits in WT and DKO mice. The average immunoreactive areas for ␣1 and 3 subunits were significantly larger in DKO than in WT mice (t-test, p < 0.05). The average immunoreactive areas for the ␥2 subunit were significantly smaller in DKO mice (t-test, p < 0.05). There was no significant difference between two groups of mice in the expression of the 2 subunit (t-test, p > 0.05). Values are means (standard deviation). * The absolute values of ␥2 subunit immunoreactivity are different from those for other subunits. This is speculated to be due to differences in antibody sensitivity. Subunit
Wild
DKO
␣1 2 3 ␥2
380.21 (148.47) 403.72 (200.32) 189.69 (98.74) 0.011 (0.044)
790.85 (125.16)* 681.58 (391.65) 430.86 (91.14)* 0.003 (0.002)*
Mean (SD) m2 . * p < 0.05.
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The present immunohistochemical observations indicate that PRIP-1/-2 depletion not only drastically reduced the expression of the ␥2 subunit in the spinal cord dorsal horn neurons, but also increased the expression of ␣1 and 3 subunits in the spinal cord. This finding is consistent with earlier ligand-binding assays performed in PRIP-1/-2 DKO mice, which revealed greater expression of GABAA receptors concomitant with lower ␥2 subunit expression, and an increase in the cell surface GABA binding sites in hippocampal neurons [10]. Therefore, the action of isoflurane, which potentiates GABAA receptor function, is likely to affect these GABAA receptors by increasing the expression of the ␣1 and 3 subunits, thus enhancing GABA activity in DKO mice. Several lines of evidence indicate that, of the various GABAA receptor subunits, the 3 subunits may have a particular role in isoflurane-induced immobility. It has been shown that the MAC for isoflurane was unaltered after knock-in of the ␣1 subunit [6,12], while ␣2 subunit knockout did not change the GABA currents after exposure to isoflurane [7]. On the other hand, the MAC values for isoflurane were larger in 3 subunit (N265M) knock-in mice than in wild-type controls [9]. Furthermore, 3(N265M) knock-in mice displayed reduced sensitivity to the immobilizing action of isoflurane [8], even though there were no detectable differences in motor activity or sensitivity to noxious thermal stimuli versus wild-type control mice [3]. Therefore, it is reasonable to consider that a variation in the 3 subunit composition is critical for the ability of inhaled anesthetics to enhance the GABAA receptor function, as evidenced by the ability of volatile anesthetics to produce immobility in response to noxious stimulation. It is possible that changes in the expression and/or function of other GABAA receptor subunits, or even other neurotransmitter systems relevant to anesthetic mechanisms, may be responsible for our observations. However, we suggest that genetic reduction of ␥2 subunit expression in DKO animals affected the composition and function of other GABAA receptor subunits and increased the inhibitory tone and facilitated the effect of GABAergic compounds, such as isoflurane. Further studies are needed to fully understand the relationship between anesthetic action and GABAA receptor function.
Fig. 2. Immunohistochemical staining of the spinal cord dorsal horn. (A) A picture of a horizontal section of the cervical spinal cord. Immunoreactivity for GABAA receptor subunits in the outlined area is shown at a higher magnification in (B). Tr: spinal cord tract. (B) Immunoreactivity for ␣1, 2, 3 and ␥2 subunits. WT: wild type mouse; DKO: PRIP-1/-2 double knockout mouse; Tr: spinal cord tract; I: lamina I; II: lamina II. ␥2, ␣1, 2 and 3 indicate the respective GABAA receptor subunits. Scale bar: 50 m.
and expression patterns of different GABAA receptor subunits in the spinal cord is particularly important. As demonstrated in a previous immunohistochemical study, most neurons in the spinal cord express GABAA receptors subunit triplets ␣1–6/2,3/␥2. However, the 2,3 subunits were not detected in motor neurons, which may possess “atypical” receptors subtypes (i.e. ␣2/␥2 and ␣2/␣5/␥2) [1]. The effect of anesthetic agents on motor neurons is considered to be more important for achieving immobility than their effect on other neuronal subtypes. However, PRIP requires the presence of  subunits to execute the facilitation of surface expression of GABAA receptors containing ␥2 subunits [10]. Therefore, the absence of  subunits in motor neurons suggests that depletion of PRIP-1 and -2 is unlikely to affect these neurons. This suggests that the enhanced immobilizing action of isoflurane observed in the DKO mice was probably mediated through the perceptive neurons present in the dorsal horn, rather than in the motor neurons of the ventral horn.
Fig. 3. Distribution of the MAC values for isoflurane in wild-type (WT) and PRIP-1/2 double knockout (DKO) mice. There is a significant difference between the mean isoflurane MAC values (p < 0.001, Student’s t-test).
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