Chronic modulation of the GABAA receptor ... - Semantic Scholar
Neuropharmacology 39 (2000) 227–234 www.elsevier.com/locate/neuropharm
Chronic modulation of the GABAA receptor complex regulates Y1 receptor gene expression in the medial amygdala of transgenic mice Alessandra Oberto a, Giancarlo Panzica b, Fiorella Altruda c, Carola Eva
a,*
a
b
Sezione di Farmacologia, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Universita` di Torino, Via Pietro Giuria 13, 10125 Torino, Italy Sezione di Anatomia, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Universita` di Torino, Corso Massimo d’Azeglio, 52, 10126 Torino, Italy c Dipartimento di Genetica, Biologia e Biochimica, Universita` di Torino, Via Santena, 5 bis, 10126 Torino, Italy Accepted 21 May 1999
Abstract NPY exerts anxiolytic effects, which are mediated by activation of Y1 receptors in the amygdala. It has been shown that diazepam counteracts the anxiogenic effect of Y1 receptor antagonists, suggesting that NPYergic and GABAergic systems are coupled in the regulation of anxiety. We used a transgenic mouse model, expressing a mouse Y1 receptor-β-galactosidase fusion gene (Y1R/LacZ), to study the effect of positive or negative modulators of GABAA receptors on Y1 receptor gene expression. Mice were treated for 14 days with diazepam (4 or 20 mg/kg), the anxiolytic β-carboline-derivative abecarnil (0.3 or 6 mg/kg) and the anxiogenic βcarboline FG7142 (20 mg/kg). Transgene expression was determined by quantitative analysis of β-galactosidase histochemical staining in the medial amygdala and in the medial habenula as a control region. Chronic treatment with 20 mg/kg diazepam or 6 mg/kg abecarnil significantly increased, whereas FG 7142 decreased, transgene expression in the medial amygdala. A transient decrease in transgene expression was observed in the medial amygdala six hours after the acute treatment with 20 mg/kg FG 7142 but not with diazepam or abecarnil. No significant changes were observed in the medial habenula. These data suggest that modulation of GABAA receptor function may regulate Y1 receptor gene expression in medial amygdala. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Neuropeptide Y Y1 receptor; Amygdala; Transgenic mice; Diazepam; Abecarnil; FG 7142
1. Introduction GABA-mediated neurotransmission in the CNS can be differentially modulated by diverse compounds that bind to allosteric modulatory sites named benzodiazepine/ω (BZ/ω) sites associated with GABAA receptors (Barnard et al., 1998). Benzodiazepine and non benzodiazepine ligands that are currently in clinical use, such as diazepam, act as positive allosteric modulators of GABA-gated Cl⫺ channels, enhance GABA-ergic transmission and produce both anxiolytic and anticon-
* Corresponding author. Tel.: +39-011-6707718; fax: +39-0116707788. E-mail address: [email protected] (C. Eva)
vulsant effects on behavior (Costa et al., 1975; Macdonald and Barker, 1978; Haefely, 1986). On the other hand, compounds acting as negative allosteric modulators, such as the β-carboline FG 7142, which also bind to BZ/ω sites but inhibit, rather than enhance GABAA receptor function, are referred to as inverse agonists and exhibit anxiogenic (Dorow et al., 1983) and proconvulsant activity (Little et al., 1984; Corda et al., 1985). The opposite effects of positive and negative allosteric modulators of GABAA receptors are also evident after chronic treatments. Both clinical and experimental evidence indicate that chronic treatment with BZ/ω full agonists, such as diazepam, results in the development of tolerance, dependence and potential of drug abuse (File, 1985; Haigh and Feely, 1988; Woods et al., 1991). Conversely, depending on the schedule of drug delivery,
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chronic exposure of rats to FG 7142 may result either in an increased sensitivity to its proconvulsant activity (chemical kindling) or in the up-regulation of GABAA receptor function and anticonvulsant activity (Little et al., 1984; Marley et al., 1991). The discovery of the existence of multiple GABAA receptors has suggested that subtype-selective agonists, such as the β-carboline derivative abecarnil, display some but not all the pharmacological effects of classical benzodiazepines and display a reduced tolerance and dependence potential in different animal species including humans (Stephens et al., 1990; Steppuhn et al., 1993; Serra et al., 1994). Recent studies suggest that compensatory changes in GABAA receptor subunit gene expression may underlie development of tolerance to these drugs following chronic administration (Zhao et al., 1994; O’Donovan et al., 1992; Impagnatiello et al., 1996; Holt et al., 1996). While evidence is accumulating on the molecular mechanisms underlying alteration in sensitivity to GABA following chronic administration of modulators of GABAA receptors, little is known, as yet, on the potential neuropharmacological interaction of the GABA-ergic system with other receptor systems known to modulate behavioral manifestation of anxiety. Several lines of evidence suggest that neuropeptide Y (NPY), the most prominent and abundant neuropeptide in the mammalian CNS, elicits sedative/anxiolytic effects which may be mediated by activation of the Y1 receptor subtype in the amygdala. NPY-Y1 receptor agonists administered intracerebroventricularly, or directly into the central nucleus of the amygdala, elicit behavioral responses indistinguishable from clinically effective benzodiazepines. This has been tested in animal models of anxiety, including the conflict test (Heilig et al., 1993; Britton et al., 1997), the elevated plus maze (Heilig et al., 1993; Broqua et al., 1995; Kask et al., 1996), and the fear-potentiated startle paradigm (Wettstein et al., 1994; Broqua et al., 1995). In contrast, behavioral signs of anxiety are elicited by centrally administered Y1 receptor antagonists or antisense oligonucleotides complementary to the Y1 receptor mRNA (Wahlestedt, 1994; Heilig, 1995; Kask et al., 1997). It has been demonstrated recently that the anxiolytic benzodiazepine diazepam counteracts the anxiogenic effect of the non-peptide Y1 receptor antagonist BIBP3226 suggesting that the equilibrium between GABA-ergic and NPY-ergic neurotransmission may be important for the regulation of an animal’s emotional state (Kask et al., 1996). We have recently generated transgenic mouse lines carrying the 1.3 Kb 5⬘ flanking region of the mouse Y1 receptor promoter fused to the coding region of the Escherichia coli LacZ gene (Eva et al., 1992). In a previous study we have shown that this construct contains sufficient information to replicate the expression pattern of the endogenous Y1 receptor gene in a CNS-restricted
and developmental stage-specific manner, although differences in the relative levels of the expression of the endogenous gene and the native protein could be seen (Oberto et al., 1998). In the present work we have investigated the effect of treatment with anxiolytic and/or anxiogenic drugs, acting either as positive or negative allosteric modulators of the GABAA receptor complex, on the Y1 receptor function in the amygdala, using Y1R/LacZ transgene expression as a marker of altered signal transduction. In particular, changes in transgene expression were analyzed in the medial amygdala since, in this nucleus, β-galactosidase positive cells are numerous, intensely stained and suitable for automated measurements. Transgene expression was evaluated in parallel in the medial habenula, since this brain region also expresses high levels of β-galactosidase-positive cells but is not involved in the regulation of anxiety behavior.
2. Methods 2.1. Animals Adult male Y1R/LacZ transgenic mice from transgenic line 27 (25–30 gm) were used in these experiments (Oberto et al., 1998). Mice were housed in cages with free access to food and water and maintained on a 12h light/12-h dark cycle and a constant temperature of 22°±2°C. Experiments were performed at the same time on each day to avoid any circadian effects. At the end of each experiment the brains were quickly excised following cervical dislocation. In all cases, at least three animals per experimental group were used. Animal care and handling throughout the experimental procedure were in accordance with the European Community Council Directive of 24 November 1986 (86/609/EEC). 2.2. Treatments FG 7142 was obtained from Tocris (Bristol, United Kingdom) and diazepam from Sigma (Milan, Italy). Abecarnil was a gift of Schering AG (Berlin, Germany). For acute treatments, animals were injected with diazepam (20 mg/kg, i.p.), abecarnil (6 mg/kg, s.c.) or FG 7142 (20 mg/kg, i.p.) and killed 6, 9 and 12 hours after the treatment. Diazepam and FG 7142 were suspended in distilled water with a drop of Tween–80 per 5 ml and injected in a volume of 10 ml per kilogram of body mass. Abecarnil was dissolved in sesame oil and administered in a volume of 4 ml/kg. Habituating the mice to the injection procedure for four consecutive days minimized the stress associated with the pharmacological treatment. For chronic treatments, diazepam (4 mg/kg or 20 mg/kg), abecarnil (0.3 mg/kg), and FG 7142 (20 mg/kg)
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were suspended in distilled water with a drop of Tween– 80 and administered i.p. once a day for 15 days. A parallel group of mice were treated subcutaneously once a day for 15 days with abecarnil (6 mg/kg) dissolved in sesame oil and administered in a volume of 4 ml/kg. Control mice received an equivalent volume of vehicle. Mice did not show convulsions when observed for ⱖ4 hours after the treatment with FG 7142. 2.3. X–gal staining LacZ expression was determined by β-galactosidase staining of coronal sections from male transgenic mice as previously described (Oberto et al., 1998). Mice were killed by cervical dislocation, brains were quickly excised and immediately frozen in 10% embedding medium (Bio–optica, Milano, IT) in phosphate buffer saline (PBS) (v/v) on the surface of liquid nitrogen. Twenty five µm-thick coronal sections of brain were cut on a cryostat at ⫺20°C to ⫺25°C, dehydrated for 5 min on ice with acetone–chloroform (1:1), air dried and fixed for ten seconds in 2.5% glutaraldehyde in PBS (v/v) in a microwave oven at the maximum power for ten seconds. Sections were washed twice in 1X PBS at 25°C and incubated in a solution containing 1 mg/ml X–gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% Triton X–100 in 1X PBS, for 48 hours at 30°C. Slides were washed twice in water for five minutes, then counterstained with nuclear fast red and coverslipped with DPX mounting medium (Fluka, Buchs, Switzerland).
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images were recorded from the sections while increasing their contrast and the visibility of blue dots by acting on the look-up-table registers of the board which are controlled via the software. If the boundaries of the nuclei were not visible, the section was observed with a builtin green filter in order to better define the area of interest (AOI, see below). The AOI was defined by drawing a line following the boundaries of the medial amygdaloid nucleus or the medial habenula. Using a manual thresholding method, dots were selected in red colour, avoiding as far as possible, the fusion of the closest ones. The image was hence binarized and the number of dots and the extension of the AOI were automatically recorded. For each animal the cumulative number of dots and the cumulative areas of the analyzed sections (three for the medial amygdala, four for the medial habenula) were considered to obtain the density of expression of the transgene, represented as dots per µm2. This method provides semiquantitative analysis of changes in β-galactosidase expression, reflecting changes in promoter activity. 2.5. Data analysis Data are expressed as means±standard errors of the means (±S.E.M.). They were statistically examined using one way analysis of variance (ANOVA) and the appropriate contrasts analyzed by the Newman–Keuls test for multiple comparisons.
3. Results 2.4. Quantitation of transgene expression as determined by b-galactosidase histochemistry and data analysis For computer-assisted quantitation of the Y1R/LacZ transgene expression we have examined three standardized sections of comparable levels of the left medial amygdaloid complex and four standardized sections of the left medial habenular nucleus, defined by neuroanatomical criteria (extension and shape of the amygdaloid complex and the presence of other neuroanatomical landmarks at the same level). To facilitate the neuroanatomical identification of the regions, sections were counterstained with neutral fast red. The expression of the transgene appears as medium-sized blue dots. Selected sections were placed on a Zeiss Axioplan I microscope, observed by means of a ×10 objective, and the corresponding image was transferred via a black and white CCD camera (PCO, VC44, Keilheim, Germany) to a digitizing board (Scion LG-3, Scion Co, Frederick, MD, USA) placed in a PowerPC 8200 Macintosh computer. The software was NIH-Image version 1.62, a public domain program written by W. Rasband at U.S. National Institutes of Health (Bethesda, USA). The
3.1. The effect of the acute treatment with diazepam, abecarnil or FG 7142 on Y1R/LacZ transgene expression The effects of three pharmacologically and structurally different allosteric modulators of GABAA receptors were examined. Mice were treated with diazepam, a clinically effective BZ/ω full agonist, with abecarnil, an anxiolytic/anticonvulsant β-carboline acting as a subtype selective agonist and with the β-carboline FG 7142, an anxiogenic and proconvulsant BZ/ω inverse agonist. Transgene expression was determined 6, 9 and 12 hours following the drug administration by histochemical βgalactosidase staining with chromogenic substrate X–gal of brain coronal sections of the medial amygdaloid nucleus and of the medial habenula as the control region. Fig. 1 shows typical images used for computer-assisted quantitation of β-galactosidase expression in the medial amygdaloid nucleus six hours after a single dose administration of vehicle (A), 20 mg/kg diazepam (B), 6 mg/kg abecarnil (C) or 20 mg/kg FG 7142 (D). Compared with controls, acute administration of diazepam (20 mg/kg, i.p.) or abecarnil (6 mg/kg, s.c.) did not affect β-galacto-
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Fig. 1. The effects of acute treatments with positive and negative modulators of GABAA receptors on Y1R/LacZ expression detected by means of β-galactosidase histochemistry in coronal sections of mouse medial amygdala. Transgene expression is shown six hours after acute treatment with vehicle (A), diazepam (20 mg/kg, i.p.; B), abecarnil (6 mg/kg., s.c.; C) and FG 7142 (20 mg/kg, i.p; D). The white lines were drawn on the digitized images to trace the boundaries of the medial amygdaloid nucleus (based on the cellular density differences with the surrounding structures) and to define the area of interest used for quantitation of transgene expression. Bar=100 µm.
sidase expression in the medial amygdaloid nucleus (Fig. 2a). In contrast FG 7142 (20 mg/kg) induced a transient decrease in transgene expression in the medial amygdaloid nucleus six hours after a single dose administration, whereas no changes were measured 9 and 12 hours after the treatment (Fig. 2a). No significant changes of βgalactosidase expression were observed in the medial habenula after each of the treatments (Fig. 2b). 3.2. The effect of the chronic treatment with diazepam, abecarnil or FG 7142 on Y1R/LacZ transgene expression In order to study the effect of chronic administration of benzodiazepine receptor ligands to Y1R/LacZ trans-
Fig. 2. Quantitation of Y1R/LacZ gene expression in the medial amygdala (a) and in the medial habenula (b) 6, 9 and 12 hours following acute treatment with vehicle, 20 mg/kg, i.p. diazepam, 6 mg/kg, s.c abecarnil or 20 mg/kg, i.p. FG 7142. Data are expressed as the density of blue dots and are the mean±SEM from 5–8 mice. (a) Oneway ANOVA: F(1,11)=4.165. P⬍0.001. *P⬍0.001 versus vehicle treated mice, by Newman–Keuls test. (b) One-way ANOVA: F(1,11)=0.861. P=0.581.
gene expression, mice were injected for 15 days with diazepam (4 or 20 mg/kg, i.p) abecarnil (0.3 mg/kg., i.p. or 6 mg/kg, s.c.) or FG7142 (20 mg/kg, i.p.) and βgalactosidase expression was determined 24 hours after the final injection. The treatment with 20 mg/kg diazepam or with 6 mg/kg abecarnil produced a statistically significant increase in β-galactosidase expression in the medial amygdaloid nucleus of Y1R/LacZ transgenic mice (Figs. 3 and 4a). Lower doses of both drugs did not produce significant changes in Y1R/LacZ transgene expression (Fig. 4a). Conversely FG 7142 (20 mg/kg) induced a significant decrease of transgene expression in the medial amygdala (Figs. 3 and 4a). Chronic treatment with diazepam, abecarnil or FG 7142 failed to affect βgalactosidase expression in the medial habenula (Fig. 4b).
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Fig. 3. Changes of Y1R/LacZ transgene expression in the medial amygdala after chronic treatment (15 days) with vehicle (A), 20 mg/kg, i.p diazepam (B), 6 mg/kg., s.c. abecarnil (C) or 20 mg/kg, i.p FG 7142 (D). The white lines surrounding the medial amygdaloid nucleus define the areas used for quantitation of transgene expression. Bar=100 µm.
4. Discussion In rodents, a marked plasticity in the expression of the Y1 receptor and its mRNA in the CNS can be induced by different circumstances. For instance, electrical kindling and kainic acid-induced epilepsy decrease Y1 receptor and Y1 receptor mRNA expression in rat hippocampus (Kofler et al., 1997; Gobbi et al., 1998). In the arcuate nucleus food deprivation decreases, and hyperfagia increases, Y1 receptor gene expression (Cheng et al., 1998; Kalra et al., 1998). Thus compensatory changes in Y1 receptor gene expression may reflect parallel changes in functional activity of NPY-Y1 receptor mediated neurotransmission. In this study we used a Y1R/LacZ transgenic model to examine the effects of acute and chronic treatment with anxiolytic and anxiogenic drugs, acting as positive or negative modulators of GABAA receptors, on Y1 receptor gene expression. The advantages of the transgenic
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Fig. 4. Quantitation of Y1R/LacZ gene expression in the medial amygdala (a) and in the medial habenula (b) after chronic treatment with vehicle (V), diazepam, 4 mg/kg (D4) and 20 mg/kg (D20) i.p, abecarnil, 0.3 mg/kg i.p. (A=0.3) and 6 mg/kg s.c (A6) and FG 7142 20 mg/kg, i.p (F20). (a) Data are the mean±SEM from 7–22 mice. One-way ANOVA: F(1,5)=14.713. P⬍0.05. *P⬍0.005; **P⬍0.001 versus vehicle treated mice, by Newman–Keuls test. (b) Data are the mean±SEM from 3–16 mice. One-way ANOVA: F(1,5)=0.424. P=0.8286.
model are that it is more sensitive and more readily quantifiable than in situ hybridization and that genomic sequences responsible for the observed regulation are known to be contained within the transgene, making subsequent analysis of the transcriptional mechanisms possible. In our model, chronic treatment with BZ/ω agonists significantly increases, and inverse agonists decrease, Y1R/LacZ gene expression in the medial amygdala but not in the medial habenula, another region in which the transgene is highly expressed. Our results are consistent with the hypothesis that, in the amygdala, the NPY-Y1 receptor-mediated transmission and GABA-ergic system are closely coupled and that NPY and GABA may functionally interact in the regulation of anxiety behavior
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(Kask et al., 1996). Previous studies have suggested that the anxiolytic effect of NPY is mediated by the activation of Y1 receptors in the central nucleus of the amydgala (Heilig, 1995). We have now demonstrated that chronic treatment with positive or negative regulators of GABAA receptors might also modulate Y1 receptor-mediated transmission in the medial amygdala, another amygaloid nucleus that has been shown to play a role in anxiety (Adamec and McKay, 1993; Adamec and Morgan, 1994; Duncan et al., 1996; Duxon et al., 1997). Changes in Y1R/LacZ transgene expression were observed only after a chronic treatment with positive modulators of the GABAA receptor complex and could not be induced by a single dose administration. In fact acute treatment with diazepam and abecarnil failed to affect transgene expression in the medial amygdala 6, 9 and 12 hours after the treatment. Moreover a single dose administration of the proconvulsant β-carboline FG 7142 decreases β-galactosidase expression six hours after the injection, however, transgene expression returns to control values nine hours after the treatment. It has been recently demonstrated that acute treatment with FG 7142 transiently elevates neuroactive steroids and corticosterone in brain and plasma, respectively (Barbaccia et al., 1996). It is possible that the transient decrease of Y1R/LacZ transgene observed six hours after the treatment with FG 7142 is due to an increase in steroid concentrations that in turn might regulate, by a genomic action or a transynaptic mechanism, the transcriptional activity of the gene. To answer this question we are currently analyzing the modulation of Y1R/LacZ transgene expression by neuroactive steroids in primary cultures of neuronal cells. The molecular mechanisms that underlie changes in Y1R/LacZ transgene expression induced by prolonged administration of BZ/ω ligands remain to be determined. On chronic treatment, positive or negative modulators of GABAA receptors were shown to affect the GABAA receptor complex in a different manner, depending on the treatment schedule and on the drug used in the different investigations. Chronic administration of diazepam and other BZ/ω site agonists results in a decrease of GABAA receptor function that has been demonstrated biochemically, physiologically and behaviorally (File, 1985; Haigh and Feely, 1988; Woods et al., 1991). In contrast, repeated administration of FG 7142 produces an increase in sensitivity to its proconvulsant activity (Little et al., 1984). Furthermore, the steady state levels of different GABAA receptor subunit mRNA are influenced by chronic exposure to diazepam and FG 7142 in a different manner (Primus and Gallager, 1992). We suggest that altered GABAA receptor function may also induce changes in Y1 receptor gene expression. In this work we have also compared the effects of the classical benzodiazepine full agonist diazepam with
those induced in the same brain areas of mice by a similar protracted treatment with equipotent anxiolytic doses of abecarnil, a subtype selective agonist with a restricted pharmacological profile. This drug is an anxiolytic and anticonvulsant β-carboline derivative that lacks the significant sedative and muscle-relaxant effects exhibited by diazepam and, on chronic treatment, fails to induce behavioral tolerance and physical dependence (Serra et al., 1994; Stephens et al., 1990; Steppuhn et al., 1993; Turski et al., 1990). Furthermore, on chronic treatment, abecarnil and diazepam were shown to differentially affect the expression of GABAA receptor subunit mRNA in the rat cortex (Holt et al., 1996). The atypical pharmacological profile of abecarnil is likely to be due to the fact that this drug binds with different affinity, and displays varying efficacy towards, GABAA receptors that are composed of different subunit isoforms. Our experiments have shown that diazepam and abecarnil produce similar effects on the expression of the Y1R/LacZ transgene in the medial amygdala. This suggests that compensatory changes in NPY-Y1 neurotransmission are unlikely to account for diazepam dependence and tolerance and that they may be triggered by the activation of those GABAA receptor subtypes that are bound with high affinity by abecarnil. In conclusion, our results are consistent with the hypothesis that NPY-ergic and GABA-ergic systems are functionally coupled in the amygdaloid complex. The neuroanatomical basis of this interaction still remains to be determined. It has been proposed that NPY and GABA may be co-existent within the same neurons in cortical areas (Hendry et al., 1984). Preliminary results obtained in our laboratory by double histochemical and immunohistochemical staining of brain sections from the Y1R/LacZ transgenic mice indicate that GABAimmunoreactive neurons are scattered within the medial amygdala and in several cases they also bear the histochemical staining for Y1/LacZ. As a working hypothesis we suggest that chronic treatment with modulators of GABAA receptor complex may induce compensatory changes in the firing rate of NPY-containing neurons which, in turn, might be responsible of changes in Y1 receptor gene transcriptional activity.
Acknowledgements This work has been carried out under a research contract with NE.FA.C Pomezia, Italy, within the Neurobiological Systems Research Plan of the Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica to C.E. and was supported by Telethon, project N. 29 to F.A. We thank Prof. M.L. Barbaccia, Dipartimento di Neuroscienze, Universita` di Tor Vergata, Roma, for the critical reading of this manuscript.
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Chronic modulation of the GABAA receptor complex regulates Y1 receptor gene expression in the medial amygdala of transgenic mice Alessandra Oberto a, Giancarlo Panzica b, Fiorella Altruda c, Carola Eva
a,*
a
b
Sezione di Farmacologia, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Universita` di Torino, Via Pietro Giuria 13, 10125 Torino, Italy Sezione di Anatomia, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Universita` di Torino, Corso Massimo d’Azeglio, 52, 10126 Torino, Italy c Dipartimento di Genetica, Biologia e Biochimica, Universita` di Torino, Via Santena, 5 bis, 10126 Torino, Italy Accepted 21 May 1999
Abstract NPY exerts anxiolytic effects, which are mediated by activation of Y1 receptors in the amygdala. It has been shown that diazepam counteracts the anxiogenic effect of Y1 receptor antagonists, suggesting that NPYergic and GABAergic systems are coupled in the regulation of anxiety. We used a transgenic mouse model, expressing a mouse Y1 receptor-β-galactosidase fusion gene (Y1R/LacZ), to study the effect of positive or negative modulators of GABAA receptors on Y1 receptor gene expression. Mice were treated for 14 days with diazepam (4 or 20 mg/kg), the anxiolytic β-carboline-derivative abecarnil (0.3 or 6 mg/kg) and the anxiogenic βcarboline FG7142 (20 mg/kg). Transgene expression was determined by quantitative analysis of β-galactosidase histochemical staining in the medial amygdala and in the medial habenula as a control region. Chronic treatment with 20 mg/kg diazepam or 6 mg/kg abecarnil significantly increased, whereas FG 7142 decreased, transgene expression in the medial amygdala. A transient decrease in transgene expression was observed in the medial amygdala six hours after the acute treatment with 20 mg/kg FG 7142 but not with diazepam or abecarnil. No significant changes were observed in the medial habenula. These data suggest that modulation of GABAA receptor function may regulate Y1 receptor gene expression in medial amygdala. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Neuropeptide Y Y1 receptor; Amygdala; Transgenic mice; Diazepam; Abecarnil; FG 7142
1. Introduction GABA-mediated neurotransmission in the CNS can be differentially modulated by diverse compounds that bind to allosteric modulatory sites named benzodiazepine/ω (BZ/ω) sites associated with GABAA receptors (Barnard et al., 1998). Benzodiazepine and non benzodiazepine ligands that are currently in clinical use, such as diazepam, act as positive allosteric modulators of GABA-gated Cl⫺ channels, enhance GABA-ergic transmission and produce both anxiolytic and anticon-
* Corresponding author. Tel.: +39-011-6707718; fax: +39-0116707788. E-mail address: [email protected] (C. Eva)
vulsant effects on behavior (Costa et al., 1975; Macdonald and Barker, 1978; Haefely, 1986). On the other hand, compounds acting as negative allosteric modulators, such as the β-carboline FG 7142, which also bind to BZ/ω sites but inhibit, rather than enhance GABAA receptor function, are referred to as inverse agonists and exhibit anxiogenic (Dorow et al., 1983) and proconvulsant activity (Little et al., 1984; Corda et al., 1985). The opposite effects of positive and negative allosteric modulators of GABAA receptors are also evident after chronic treatments. Both clinical and experimental evidence indicate that chronic treatment with BZ/ω full agonists, such as diazepam, results in the development of tolerance, dependence and potential of drug abuse (File, 1985; Haigh and Feely, 1988; Woods et al., 1991). Conversely, depending on the schedule of drug delivery,
0028-3908/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 1 2 9 - X
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chronic exposure of rats to FG 7142 may result either in an increased sensitivity to its proconvulsant activity (chemical kindling) or in the up-regulation of GABAA receptor function and anticonvulsant activity (Little et al., 1984; Marley et al., 1991). The discovery of the existence of multiple GABAA receptors has suggested that subtype-selective agonists, such as the β-carboline derivative abecarnil, display some but not all the pharmacological effects of classical benzodiazepines and display a reduced tolerance and dependence potential in different animal species including humans (Stephens et al., 1990; Steppuhn et al., 1993; Serra et al., 1994). Recent studies suggest that compensatory changes in GABAA receptor subunit gene expression may underlie development of tolerance to these drugs following chronic administration (Zhao et al., 1994; O’Donovan et al., 1992; Impagnatiello et al., 1996; Holt et al., 1996). While evidence is accumulating on the molecular mechanisms underlying alteration in sensitivity to GABA following chronic administration of modulators of GABAA receptors, little is known, as yet, on the potential neuropharmacological interaction of the GABA-ergic system with other receptor systems known to modulate behavioral manifestation of anxiety. Several lines of evidence suggest that neuropeptide Y (NPY), the most prominent and abundant neuropeptide in the mammalian CNS, elicits sedative/anxiolytic effects which may be mediated by activation of the Y1 receptor subtype in the amygdala. NPY-Y1 receptor agonists administered intracerebroventricularly, or directly into the central nucleus of the amygdala, elicit behavioral responses indistinguishable from clinically effective benzodiazepines. This has been tested in animal models of anxiety, including the conflict test (Heilig et al., 1993; Britton et al., 1997), the elevated plus maze (Heilig et al., 1993; Broqua et al., 1995; Kask et al., 1996), and the fear-potentiated startle paradigm (Wettstein et al., 1994; Broqua et al., 1995). In contrast, behavioral signs of anxiety are elicited by centrally administered Y1 receptor antagonists or antisense oligonucleotides complementary to the Y1 receptor mRNA (Wahlestedt, 1994; Heilig, 1995; Kask et al., 1997). It has been demonstrated recently that the anxiolytic benzodiazepine diazepam counteracts the anxiogenic effect of the non-peptide Y1 receptor antagonist BIBP3226 suggesting that the equilibrium between GABA-ergic and NPY-ergic neurotransmission may be important for the regulation of an animal’s emotional state (Kask et al., 1996). We have recently generated transgenic mouse lines carrying the 1.3 Kb 5⬘ flanking region of the mouse Y1 receptor promoter fused to the coding region of the Escherichia coli LacZ gene (Eva et al., 1992). In a previous study we have shown that this construct contains sufficient information to replicate the expression pattern of the endogenous Y1 receptor gene in a CNS-restricted
and developmental stage-specific manner, although differences in the relative levels of the expression of the endogenous gene and the native protein could be seen (Oberto et al., 1998). In the present work we have investigated the effect of treatment with anxiolytic and/or anxiogenic drugs, acting either as positive or negative allosteric modulators of the GABAA receptor complex, on the Y1 receptor function in the amygdala, using Y1R/LacZ transgene expression as a marker of altered signal transduction. In particular, changes in transgene expression were analyzed in the medial amygdala since, in this nucleus, β-galactosidase positive cells are numerous, intensely stained and suitable for automated measurements. Transgene expression was evaluated in parallel in the medial habenula, since this brain region also expresses high levels of β-galactosidase-positive cells but is not involved in the regulation of anxiety behavior.
2. Methods 2.1. Animals Adult male Y1R/LacZ transgenic mice from transgenic line 27 (25–30 gm) were used in these experiments (Oberto et al., 1998). Mice were housed in cages with free access to food and water and maintained on a 12h light/12-h dark cycle and a constant temperature of 22°±2°C. Experiments were performed at the same time on each day to avoid any circadian effects. At the end of each experiment the brains were quickly excised following cervical dislocation. In all cases, at least three animals per experimental group were used. Animal care and handling throughout the experimental procedure were in accordance with the European Community Council Directive of 24 November 1986 (86/609/EEC). 2.2. Treatments FG 7142 was obtained from Tocris (Bristol, United Kingdom) and diazepam from Sigma (Milan, Italy). Abecarnil was a gift of Schering AG (Berlin, Germany). For acute treatments, animals were injected with diazepam (20 mg/kg, i.p.), abecarnil (6 mg/kg, s.c.) or FG 7142 (20 mg/kg, i.p.) and killed 6, 9 and 12 hours after the treatment. Diazepam and FG 7142 were suspended in distilled water with a drop of Tween–80 per 5 ml and injected in a volume of 10 ml per kilogram of body mass. Abecarnil was dissolved in sesame oil and administered in a volume of 4 ml/kg. Habituating the mice to the injection procedure for four consecutive days minimized the stress associated with the pharmacological treatment. For chronic treatments, diazepam (4 mg/kg or 20 mg/kg), abecarnil (0.3 mg/kg), and FG 7142 (20 mg/kg)
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were suspended in distilled water with a drop of Tween– 80 and administered i.p. once a day for 15 days. A parallel group of mice were treated subcutaneously once a day for 15 days with abecarnil (6 mg/kg) dissolved in sesame oil and administered in a volume of 4 ml/kg. Control mice received an equivalent volume of vehicle. Mice did not show convulsions when observed for ⱖ4 hours after the treatment with FG 7142. 2.3. X–gal staining LacZ expression was determined by β-galactosidase staining of coronal sections from male transgenic mice as previously described (Oberto et al., 1998). Mice were killed by cervical dislocation, brains were quickly excised and immediately frozen in 10% embedding medium (Bio–optica, Milano, IT) in phosphate buffer saline (PBS) (v/v) on the surface of liquid nitrogen. Twenty five µm-thick coronal sections of brain were cut on a cryostat at ⫺20°C to ⫺25°C, dehydrated for 5 min on ice with acetone–chloroform (1:1), air dried and fixed for ten seconds in 2.5% glutaraldehyde in PBS (v/v) in a microwave oven at the maximum power for ten seconds. Sections were washed twice in 1X PBS at 25°C and incubated in a solution containing 1 mg/ml X–gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% Triton X–100 in 1X PBS, for 48 hours at 30°C. Slides were washed twice in water for five minutes, then counterstained with nuclear fast red and coverslipped with DPX mounting medium (Fluka, Buchs, Switzerland).
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images were recorded from the sections while increasing their contrast and the visibility of blue dots by acting on the look-up-table registers of the board which are controlled via the software. If the boundaries of the nuclei were not visible, the section was observed with a builtin green filter in order to better define the area of interest (AOI, see below). The AOI was defined by drawing a line following the boundaries of the medial amygdaloid nucleus or the medial habenula. Using a manual thresholding method, dots were selected in red colour, avoiding as far as possible, the fusion of the closest ones. The image was hence binarized and the number of dots and the extension of the AOI were automatically recorded. For each animal the cumulative number of dots and the cumulative areas of the analyzed sections (three for the medial amygdala, four for the medial habenula) were considered to obtain the density of expression of the transgene, represented as dots per µm2. This method provides semiquantitative analysis of changes in β-galactosidase expression, reflecting changes in promoter activity. 2.5. Data analysis Data are expressed as means±standard errors of the means (±S.E.M.). They were statistically examined using one way analysis of variance (ANOVA) and the appropriate contrasts analyzed by the Newman–Keuls test for multiple comparisons.
3. Results 2.4. Quantitation of transgene expression as determined by b-galactosidase histochemistry and data analysis For computer-assisted quantitation of the Y1R/LacZ transgene expression we have examined three standardized sections of comparable levels of the left medial amygdaloid complex and four standardized sections of the left medial habenular nucleus, defined by neuroanatomical criteria (extension and shape of the amygdaloid complex and the presence of other neuroanatomical landmarks at the same level). To facilitate the neuroanatomical identification of the regions, sections were counterstained with neutral fast red. The expression of the transgene appears as medium-sized blue dots. Selected sections were placed on a Zeiss Axioplan I microscope, observed by means of a ×10 objective, and the corresponding image was transferred via a black and white CCD camera (PCO, VC44, Keilheim, Germany) to a digitizing board (Scion LG-3, Scion Co, Frederick, MD, USA) placed in a PowerPC 8200 Macintosh computer. The software was NIH-Image version 1.62, a public domain program written by W. Rasband at U.S. National Institutes of Health (Bethesda, USA). The
3.1. The effect of the acute treatment with diazepam, abecarnil or FG 7142 on Y1R/LacZ transgene expression The effects of three pharmacologically and structurally different allosteric modulators of GABAA receptors were examined. Mice were treated with diazepam, a clinically effective BZ/ω full agonist, with abecarnil, an anxiolytic/anticonvulsant β-carboline acting as a subtype selective agonist and with the β-carboline FG 7142, an anxiogenic and proconvulsant BZ/ω inverse agonist. Transgene expression was determined 6, 9 and 12 hours following the drug administration by histochemical βgalactosidase staining with chromogenic substrate X–gal of brain coronal sections of the medial amygdaloid nucleus and of the medial habenula as the control region. Fig. 1 shows typical images used for computer-assisted quantitation of β-galactosidase expression in the medial amygdaloid nucleus six hours after a single dose administration of vehicle (A), 20 mg/kg diazepam (B), 6 mg/kg abecarnil (C) or 20 mg/kg FG 7142 (D). Compared with controls, acute administration of diazepam (20 mg/kg, i.p.) or abecarnil (6 mg/kg, s.c.) did not affect β-galacto-
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Fig. 1. The effects of acute treatments with positive and negative modulators of GABAA receptors on Y1R/LacZ expression detected by means of β-galactosidase histochemistry in coronal sections of mouse medial amygdala. Transgene expression is shown six hours after acute treatment with vehicle (A), diazepam (20 mg/kg, i.p.; B), abecarnil (6 mg/kg., s.c.; C) and FG 7142 (20 mg/kg, i.p; D). The white lines were drawn on the digitized images to trace the boundaries of the medial amygdaloid nucleus (based on the cellular density differences with the surrounding structures) and to define the area of interest used for quantitation of transgene expression. Bar=100 µm.
sidase expression in the medial amygdaloid nucleus (Fig. 2a). In contrast FG 7142 (20 mg/kg) induced a transient decrease in transgene expression in the medial amygdaloid nucleus six hours after a single dose administration, whereas no changes were measured 9 and 12 hours after the treatment (Fig. 2a). No significant changes of βgalactosidase expression were observed in the medial habenula after each of the treatments (Fig. 2b). 3.2. The effect of the chronic treatment with diazepam, abecarnil or FG 7142 on Y1R/LacZ transgene expression In order to study the effect of chronic administration of benzodiazepine receptor ligands to Y1R/LacZ trans-
Fig. 2. Quantitation of Y1R/LacZ gene expression in the medial amygdala (a) and in the medial habenula (b) 6, 9 and 12 hours following acute treatment with vehicle, 20 mg/kg, i.p. diazepam, 6 mg/kg, s.c abecarnil or 20 mg/kg, i.p. FG 7142. Data are expressed as the density of blue dots and are the mean±SEM from 5–8 mice. (a) Oneway ANOVA: F(1,11)=4.165. P⬍0.001. *P⬍0.001 versus vehicle treated mice, by Newman–Keuls test. (b) One-way ANOVA: F(1,11)=0.861. P=0.581.
gene expression, mice were injected for 15 days with diazepam (4 or 20 mg/kg, i.p) abecarnil (0.3 mg/kg., i.p. or 6 mg/kg, s.c.) or FG7142 (20 mg/kg, i.p.) and βgalactosidase expression was determined 24 hours after the final injection. The treatment with 20 mg/kg diazepam or with 6 mg/kg abecarnil produced a statistically significant increase in β-galactosidase expression in the medial amygdaloid nucleus of Y1R/LacZ transgenic mice (Figs. 3 and 4a). Lower doses of both drugs did not produce significant changes in Y1R/LacZ transgene expression (Fig. 4a). Conversely FG 7142 (20 mg/kg) induced a significant decrease of transgene expression in the medial amygdala (Figs. 3 and 4a). Chronic treatment with diazepam, abecarnil or FG 7142 failed to affect βgalactosidase expression in the medial habenula (Fig. 4b).
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Fig. 3. Changes of Y1R/LacZ transgene expression in the medial amygdala after chronic treatment (15 days) with vehicle (A), 20 mg/kg, i.p diazepam (B), 6 mg/kg., s.c. abecarnil (C) or 20 mg/kg, i.p FG 7142 (D). The white lines surrounding the medial amygdaloid nucleus define the areas used for quantitation of transgene expression. Bar=100 µm.
4. Discussion In rodents, a marked plasticity in the expression of the Y1 receptor and its mRNA in the CNS can be induced by different circumstances. For instance, electrical kindling and kainic acid-induced epilepsy decrease Y1 receptor and Y1 receptor mRNA expression in rat hippocampus (Kofler et al., 1997; Gobbi et al., 1998). In the arcuate nucleus food deprivation decreases, and hyperfagia increases, Y1 receptor gene expression (Cheng et al., 1998; Kalra et al., 1998). Thus compensatory changes in Y1 receptor gene expression may reflect parallel changes in functional activity of NPY-Y1 receptor mediated neurotransmission. In this study we used a Y1R/LacZ transgenic model to examine the effects of acute and chronic treatment with anxiolytic and anxiogenic drugs, acting as positive or negative modulators of GABAA receptors, on Y1 receptor gene expression. The advantages of the transgenic
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Fig. 4. Quantitation of Y1R/LacZ gene expression in the medial amygdala (a) and in the medial habenula (b) after chronic treatment with vehicle (V), diazepam, 4 mg/kg (D4) and 20 mg/kg (D20) i.p, abecarnil, 0.3 mg/kg i.p. (A=0.3) and 6 mg/kg s.c (A6) and FG 7142 20 mg/kg, i.p (F20). (a) Data are the mean±SEM from 7–22 mice. One-way ANOVA: F(1,5)=14.713. P⬍0.05. *P⬍0.005; **P⬍0.001 versus vehicle treated mice, by Newman–Keuls test. (b) Data are the mean±SEM from 3–16 mice. One-way ANOVA: F(1,5)=0.424. P=0.8286.
model are that it is more sensitive and more readily quantifiable than in situ hybridization and that genomic sequences responsible for the observed regulation are known to be contained within the transgene, making subsequent analysis of the transcriptional mechanisms possible. In our model, chronic treatment with BZ/ω agonists significantly increases, and inverse agonists decrease, Y1R/LacZ gene expression in the medial amygdala but not in the medial habenula, another region in which the transgene is highly expressed. Our results are consistent with the hypothesis that, in the amygdala, the NPY-Y1 receptor-mediated transmission and GABA-ergic system are closely coupled and that NPY and GABA may functionally interact in the regulation of anxiety behavior
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(Kask et al., 1996). Previous studies have suggested that the anxiolytic effect of NPY is mediated by the activation of Y1 receptors in the central nucleus of the amydgala (Heilig, 1995). We have now demonstrated that chronic treatment with positive or negative regulators of GABAA receptors might also modulate Y1 receptor-mediated transmission in the medial amygdala, another amygaloid nucleus that has been shown to play a role in anxiety (Adamec and McKay, 1993; Adamec and Morgan, 1994; Duncan et al., 1996; Duxon et al., 1997). Changes in Y1R/LacZ transgene expression were observed only after a chronic treatment with positive modulators of the GABAA receptor complex and could not be induced by a single dose administration. In fact acute treatment with diazepam and abecarnil failed to affect transgene expression in the medial amygdala 6, 9 and 12 hours after the treatment. Moreover a single dose administration of the proconvulsant β-carboline FG 7142 decreases β-galactosidase expression six hours after the injection, however, transgene expression returns to control values nine hours after the treatment. It has been recently demonstrated that acute treatment with FG 7142 transiently elevates neuroactive steroids and corticosterone in brain and plasma, respectively (Barbaccia et al., 1996). It is possible that the transient decrease of Y1R/LacZ transgene observed six hours after the treatment with FG 7142 is due to an increase in steroid concentrations that in turn might regulate, by a genomic action or a transynaptic mechanism, the transcriptional activity of the gene. To answer this question we are currently analyzing the modulation of Y1R/LacZ transgene expression by neuroactive steroids in primary cultures of neuronal cells. The molecular mechanisms that underlie changes in Y1R/LacZ transgene expression induced by prolonged administration of BZ/ω ligands remain to be determined. On chronic treatment, positive or negative modulators of GABAA receptors were shown to affect the GABAA receptor complex in a different manner, depending on the treatment schedule and on the drug used in the different investigations. Chronic administration of diazepam and other BZ/ω site agonists results in a decrease of GABAA receptor function that has been demonstrated biochemically, physiologically and behaviorally (File, 1985; Haigh and Feely, 1988; Woods et al., 1991). In contrast, repeated administration of FG 7142 produces an increase in sensitivity to its proconvulsant activity (Little et al., 1984). Furthermore, the steady state levels of different GABAA receptor subunit mRNA are influenced by chronic exposure to diazepam and FG 7142 in a different manner (Primus and Gallager, 1992). We suggest that altered GABAA receptor function may also induce changes in Y1 receptor gene expression. In this work we have also compared the effects of the classical benzodiazepine full agonist diazepam with
those induced in the same brain areas of mice by a similar protracted treatment with equipotent anxiolytic doses of abecarnil, a subtype selective agonist with a restricted pharmacological profile. This drug is an anxiolytic and anticonvulsant β-carboline derivative that lacks the significant sedative and muscle-relaxant effects exhibited by diazepam and, on chronic treatment, fails to induce behavioral tolerance and physical dependence (Serra et al., 1994; Stephens et al., 1990; Steppuhn et al., 1993; Turski et al., 1990). Furthermore, on chronic treatment, abecarnil and diazepam were shown to differentially affect the expression of GABAA receptor subunit mRNA in the rat cortex (Holt et al., 1996). The atypical pharmacological profile of abecarnil is likely to be due to the fact that this drug binds with different affinity, and displays varying efficacy towards, GABAA receptors that are composed of different subunit isoforms. Our experiments have shown that diazepam and abecarnil produce similar effects on the expression of the Y1R/LacZ transgene in the medial amygdala. This suggests that compensatory changes in NPY-Y1 neurotransmission are unlikely to account for diazepam dependence and tolerance and that they may be triggered by the activation of those GABAA receptor subtypes that are bound with high affinity by abecarnil. In conclusion, our results are consistent with the hypothesis that NPY-ergic and GABA-ergic systems are functionally coupled in the amygdaloid complex. The neuroanatomical basis of this interaction still remains to be determined. It has been proposed that NPY and GABA may be co-existent within the same neurons in cortical areas (Hendry et al., 1984). Preliminary results obtained in our laboratory by double histochemical and immunohistochemical staining of brain sections from the Y1R/LacZ transgenic mice indicate that GABAimmunoreactive neurons are scattered within the medial amygdala and in several cases they also bear the histochemical staining for Y1/LacZ. As a working hypothesis we suggest that chronic treatment with modulators of GABAA receptor complex may induce compensatory changes in the firing rate of NPY-containing neurons which, in turn, might be responsible of changes in Y1 receptor gene transcriptional activity.
Acknowledgements This work has been carried out under a research contract with NE.FA.C Pomezia, Italy, within the Neurobiological Systems Research Plan of the Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica to C.E. and was supported by Telethon, project N. 29 to F.A. We thank Prof. M.L. Barbaccia, Dipartimento di Neuroscienze, Universita` di Tor Vergata, Roma, for the critical reading of this manuscript.
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