The restructuring of muscarinic receptor subtype gene transcripts in c-fos knock-out mice

Brain Research Bulletin 94 (2013) 30–39

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The restructuring of muscarinic receptor subtype gene transcripts in c-fos knock-out mice Jan Benes a,∗ , Boris Mravec b,c , Richard Kvetnansky c , Jaromir Myslivecek a a b c

Institute of Physiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia

a r t i c l e

i n f o

Article history: Received 12 December 2012 Received in revised form 17 January 2013 Accepted 29 January 2013 Available online 5 February 2013 Keywords: c-fos Muscarinic receptors Muscarinic receptor mRNA Cerebral cortex Cerebellum Hypothalamus Striatum Hippocampus

a b s t r a c t Although c-Fos plays a key role in intracellular signalling, the disruption of the c-fos gene has only minor consequences on the central nervous system (CNS) function. As muscarinic receptors (MR) play important roles in many CNS functions (attention, arousal, and cognition), the c-fos knock-out might be compensated through MR changes. The aim of this study was to evaluate changes in the M1 –M5 MR mRNA in selected CNS areas: frontal, parietal, temporal and occipital cortex, striatum, hippocampus, hypothalamus and cerebellum (FC, PC, TC, OC, stria, hip, hypo, and crbl, respectively). Knocking out the c-fos gene changed the expression of MR in FC (reduced M1 R, M4 R and M5 R expression), TC (increased M4 R expression), OC (decreased M2 R and M3 R expression) and hippocampus (reduced M3 R expression). Moreover, gender differences were observed in WT mice: increased expression of all M1 –M5 R in the FC in males and M1 –M4 R in the striatum in females. A detailed analysis of MR transcripts showed pre-existing correlations in the amount of MR–mRNA between specific regions. WT mice showed three major types of cortico-cortical correlations: fronto-occipital, temporo-parietal and parieto-occipital. The cortico-subcortical correlations involved associations between the FC, PC, TC and striatum. In KO mice, a substantial rearrangement of the correlation pattern was observed: only a temporo-parietal correlation and correlations between the FC and striatum remained, and a new correlation between the hypothalamus and cerebellum appeared. Thus, in addition to the previously described dopamine receptor restructuring, the restructuring of MR mRNA correlations reveals an additional mechanism for adaptation to the c-fos gene knockout. © 2013 Published by Elsevier Inc.

1. Introduction C-Fos is a protein product derived from the so-called immediate early genes (IEGs) (Hughes and Dragunow, 1995) that plays a role as an inducible transcription factor. Together with other proteins (FosB and Fos-related antigens – Fra-1 and Fra-2), c-Fos is a part of a family of transcription factors that form heterodimeric complexes with Jun proteins to generate transcription factor activator protein1 (AP-1) (Herdegen and Leah, 1998). Subsequently, AP-1 regulates the expression of target genes by binding to the AP-recognition sequence identified in a variety of target genes (Greenberg and Ziff, 1984). Knocking out the c-fos gene is surprisingly not lethal, and therefore the functions of the c-fos gene and protein can be studied using

∗ Corresponding author at: Institute of Physiology, 1st Faculty of Medicine, Charles University, Albertov 5, 128 00 Prague, Czech Republic. Tel.: +420 732 816 242; fax: +420 224 918 816. E-mail addresses: [email protected], [email protected] (J. Benes). 0361-9230/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.brainresbull.2013.01.010

this technique (Wang et al., 1992). However, c-fos KO mice exhibit severe developmental impairments, characterised by hypotrophia and osteopetrosis, but the alteration of the CNS function is mild and not consistent with the principal role of c-fos in signalling. Although, the assessment of the central nervous system function in c-fos KO mice is biased by confounding motor disorders and other CNS dysfunctions (Paylor et al., 1994), the role of c-Fos has been studied in mice carrying a CNS-specific c-fos knock-out (c-fos CNS ). Experiments with these mice have revealed that c-fos CNS mice have selective deficits in hippocampus-dependent learning and a reduced magnitude of long-term potentiation (LTP) at hippocampal CA3 -to-CA1 synapses (Fleischmann et al., 2003). However, c-fos KO mice do not exhibit long-term memory deficits in aversive taste learning (Yasoshima et al., 2006). The expression of c-Fos can be influenced through multiple receptor systems, including dopamine (Pennypacker, 1995) and muscarinic receptors (Pennypacker et al., 1995). In contrast, changes in the receptor densities and receptor signalling potentially play a considerable role in managing c-fos knock-out. In a previous study (Benes et al., 2012a), we showed that c-fos

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Fig. 1. The changes in the expression of muscarinic receptor subtypes in the frontal cortex between males and females and wild type (WT) and knock-out (KO) animals. The yellow colour indicates WT, and the violet colour indicates KO. Refer to the figure legend below for explanations. *p < 0.05 between males and females, # p < 0.05 between WT and KO, A.U.: arbitrary units. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

KO mice experience a reorganisation of the entire dopaminergic signalling system, including receptor density changes and changes in dopamine receptor transcripts that involve multiple areas of the CNS. Muscarinic receptors (mAChRs) are metabotropic receptors that respond to acetylcholine. These receptors are coupled to G-proteins that transmit the signal further in the cell. There are five subtypes of mAChRs, which are distinct gene products (Nathanson, 2000). In general, M1 , M3 and M5 receptors activate phospholipase C (PLC), employing pertussis toxin-insensitive G-proteins of the Gq superfamily, which do not inhibit adenylyl cyclase, and M2 and M4 receptors inhibit adenylyl cyclase through pertussis toxinsensitive G-proteins of the Gi /G0 family, which do not stimulate PLC. However, this specificity is not absolute, as M2 and M4 receptors can potentially activate PLC when expressed at high levels in certain cell types due to the release of beta-gamma subunits from Gi /G0 (Katz et al., 1992). Muscarinic receptor signalling is complex. Under certain circumstances, both Gi and Gq -specific mAChRs can be potentially coupled to Gs and stimulate adenylyl cyclase activity (Migeon and Nathanson, 1994). Muscarinic receptors play important roles in many CNS functions in humans (Ellis et al., 2006) and experimental animals (Bymaster et al., 1993), and these functions have been further confirmed using knock-out studies (Wess, 2003; Wess et al., 2003, 2007). Regarding specific muscarinic receptor subtypes, M1 (Yamasaki et al., 2010), M2 and M4 (Ohno et al., 1994; Tzavara et al., 2003) have been implicated in attention, arousal and cognitive processes. M2 receptors mediate whole body tremor, hypothermia and analgesia (Gomeza et al., 2001). M3 receptors are involved in certain types of learning (Poulin et al., 2010) and are required for the proper functioning of hypothalamic GHRH neurons (Gautam et al., 2009). M4 receptors are involved in the inhibitory control of

striatal projecting neurons, where they control D1 receptormediated locomotor stimulation (Gomeza et al., 2001). Furthermore, M4 muscarinic receptors are involved in mediating parkinsonian tremor which is distinct of whole body tremor mediated by M2 subtype (Betz et al., 2007, 2009; Mayorga et al., 1999; Salamone et al., 2001). M5 receptors are likely involved in the acetylcholine-mediated vasodilatation of cerebral arteries and arterioles. Moreover, M5 receptors are expressed in the ventral tegmental area where they are involved in mesolimbic dopaminergic pathway for the regulation of morphine reward and withdrawal symptoms (Yamada et al., 2003). The purpose of this study was to evaluate the role of muscarinic receptor subtypes in c-fos KO mice with a targeted disruption of the c-fos gene (Johnson et al., 1992). Because neither immunohistochemistry nor binding studies confer the necessary specificity for discriminating between receptor subtypes (Pradidarcheep et al., 2009), we employed a transcription analysis of muscarinic receptor subtypes in selected CNS areas (frontal, parietal, temporal and occipital cortex, striatum, hippocampus, hypothalamus and cerebellum). Moreover, we analysed pre-existing correlations in the mRNA of muscarinic receptor subtypes in wild type mice compared with their c-fos KO counterparts. Hence, we propose that the restructuring of muscarinic receptor signalling maintains the relatively preserved CNS functions in this genetic mutant. 2. Methods 2.1. Animals The c-fos mutation was produced through gene targeting (Johnson et al., 1992). The mice were obtained from the Institute for Molecular Pathology, University of Vienna, Austria and maintained on a mixed genetic background derived from

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Fig. 2. The changes in the gene expression of muscarinic receptor subtypes in the striatum between males and females and wild type (WT) and knock-out (KO) animals. The yellow colour indicates WT, and the violet colour indicates KO. Refer to the figure legend below for explanations. *p < 0.05 between males and females, # p < 0.05 between WT and KO, n.s.: not significant, A.U.: arbitrary units. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Fig. 3. The changes in the gene expression of muscarinic receptor subtypes in the temporal cortex, occipital cortex and hippocampus between males and females and wild-type (WT) and knock-out (KO) animals. The yellow colour indicates WT, and the violet colour indicates KO. Refer to the legend below for explanations. *p < 0.05 between males and females, # p < 0.05 between WT and KO, A.U.: arbitrary units. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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Fig. 4. Schematic representation of the correlations between the levels of muscarinic receptor mRNA expression within the cortical regions and the cerebellum. The green line with arrows indicates a positive correlation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.) (C57BL/6Jx129/SvJ)F1 mice. The mice were maintained at constant temperature (21 ◦ C ± 1) under a 12-h light/12-h dark cycle (lights on at 6:00 AM) with food and water ad libitum. Wild type (WT, +/+) and knock-out (KO, −/−) mice were generated through crosses using heterozygous (+/−) adult mice (2 months old). Male and female control (n = 16) and KO (n = 11) mice (20–25 g of body weight) were used in this study. WT and KO genotypes were confirmed using PCR as previously described (Johnson et al., 1992). The mice were sacrificed through decapitation and exsanguination, and samples of the frontal (FC), temporal (TC), parietal (PC), and occipital (OC) cortex, cerebellum (CRBL), hypothalamus (Hypo), striatum (Stria), and hippocampus (Hip) were isolated using a punch method, isolating as much tissue as possible. Tissue was flash frozen in liquid nitrogen and stored at −80 ◦ C until further analysis. All experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996), the UK Animals (Scientific Procedures) Act 1986 and associated guidelines of the European Communities Council Directive of 24 November 1986 (86/609/EEC). The Committee for Animal Care of the Institute of Experimental Endocrinology in Bratislava, Slovakia approved all experiments.

2.2. Quantitative real-time PCR Total mRNA was isolated using chloroform–isopropanol method (RNA Bee, TelTest, TX, USA) according to the manufacturer’s instructions. The yield and integrity of the RNA were evaluated spectrophotometrically using a Tecan Infinite 200 Nanoquant (Tecan, Maennedorf, Switzerland) microplate reader at A1 = 260 nm and A1 /A2 = 260/280 nm, respectively. The samples with A1 /A2 between 1.7 and 1.95 were used in additional experiments. Total RNA was purified using recombinant DNAse (Ambion, TX, USA) to eliminate potential contaminating genomic DNA. After purification, the final concentration of purified mRNA was assessed using the Tecan Infinite 200 Nanoquant microplate reader (Tecan, Maennedorf, Switzerland). A 1000-ng sample of purified mRNA was subsequently transcribed into cDNA using Ready-To-Go You-Prime First-Strand Beads and pd(N)6 primers (GE Healthcare, Little Chalfont, UK). The qPCR reaction was performed with TaqMan probes (Applied Biosystems, Carlsbad, CA, USA) using following probes: Hs 99999901 s1 for 18SrRNA, Mm01231010 m1 for M1 , Mm01167087 m1 for M2 , Mm01338409 m1 for M3 , Mm0133561 s1 for M4 , and Mm01701883 s1 for M5 and Roche qPCR

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Fig. 5. Schematic representation of the correlation between the levels of muscarinic receptor mRNA expression within the subcortical regions. The green line with arrows indicates a positive correlation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Mastermix (Roche, Pleasanton, CA, USA) in a final volume 10 ␮L using the following protocol: 2 min at 50 ◦ C and 10 min at 95 ◦ C, followed by 50 cycles of 15 s at 95 ◦ C and 1 min at 60 ◦ C. All experiments were performed in duplicates. The gene expression levels in each sample were calculated using the following formula: normalised ratio = 2expCT1−CT2 , where CT is the cycle threshold, CT1 is the CT of the reference housekeeper transcript, and CT2 is the CT of the target transcript. No template controls (NTC) and no reverse transcription (NRT) reactions were performed as negative controls.

3.1.2. Comparison of the gene expression in wild type and knock-out animals Higher levels of M1 R, M4 R and M5 R mRNA were observed in the frontal cortex of knock-out animals (Fig. 1). Increased M4 R mRNA expression was also detected in the temporal cortex (Fig. 3). In contrast, reduced levels of M2 R and M3 R mRNA in the occipital cortex and M3 R-mRNA in the hippocampus were observed in knock-out animals (Fig. 3).

2.3. Data and statistical analyses Muscarinic receptor subtype gene expression (M1 R-mRNA-M5 R-mRNA) normalised to the housekeeper gene (18SrRNA) was evaluated using GraphPad software (San Diego, CA, USA). The statistical significance was determined using 2-way ANOVA calculating the effect of sex (males vs. females) and gene knock-out (wild type vs. knock-out). Subsequently, the relative amounts of each receptor subtype mRNA (M1 R-mRNA-M5 R-mRNA) were plotted against each other. The correlations were analysed using Pearson’s correlation coefficient (r), the coefficient of determination (r2 ), and the corresponding p-values (using Statistica software) for all correlations. In all cases, a value of p < 0.05 was considered as statistically significant. A positive correlation was described when an increase in the expression of one receptor subtype mRNA was accompanied by an increase in the expression of another receptor subtype. Similarly, a negative correlation was described when the increased expression of one receptor subtype mRNA was accompanied by the decreased expression of another receptor subtype.

3. Results

3.2. Muscarinic receptor transcript analysis 3.2.1. Intraregional correlations The correlation analysis revealed that intraregional correlations within specific cortical (FC, PC, TC, OC) and subcortical (striatum, hypothalamus, hippocampus, cerebellum) regions showed similar patterns in both WT and KO mice. There were only slight differences observed: the wild-type mice lacked M1 –M5 , M2 –M5 and M4 –M5 correlations in the PC, M1 –M5 , M3 –M5 , M4 –M5 correlations in the TC, an M1 –M5 correlation in the OC, an M1 –M5 correlation in the striatum and M1 –M2 , M2 –M3 and M2 –M4 correlations in the cerebellum, and the knock-out mice lacked an M1 –M2 correlation in the hypothalamus and M3 –M5 and M4 –M5 correlations in the cerebellum. The intraregional correlations observed in these regions were positive, and no negative correlation was detected (Figs. 4 and 5).

3.1. Gene expression 3.1.1. Comparison of gene expression in males and females Gender-specific differences in gene expression of muscarinic receptor subtypes were observed in the frontal cortex and the striatum of wild-type mice. The increased expression of all M1 –M5 receptors was detected in the frontal cortex of male mice (Fig. 1). In contrast, higher levels of M1 –M4 mRNA were detected in the striatum of female mice, and no difference in the M5 R mRNA expression was observed (Fig. 2). Moreover, there were no differences in muscarinic receptor mRNA levels in the other CNS regions examined (data not shown).

3.2.2. Interregional correlations, cortico-cortical correlations The wild-type mice showed three major types of corticocortical correlations: fronto-occipital, parieto-temporal and parieto-cerebellar correlations. A minor occipito-cerebellar correlation was also observed in these mice. All fronto-occipital correlations were negative, whereas all remaining correlations were positive. The c-fos KO mice showed a markedly reduced amount of preserved cortico-cortical correlations (positive correlations between temporal M2 receptors and all parietal receptor subtypes). The other correlations observed in WT mice were absent in the KO mice (Fig. 6A and B).

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4. Discussion The results obtained in this study showed that knocking out the c-fos gene changed the expression of muscarinic receptors in four specific brain regions, namely the frontal cortex (increased M1 R, M4 R and M5 R mRNA), temporal cortex (increased M4 R mRNA), occipital cortex (decreased M2 R and M3 R mRNA) and hippocampus (decreased M3 R-mRNA). A detailed analysis of muscarinic receptor transcripts showed that there were pre-existing correlations in amount of MR mRNA between specific regions. Knocking out the cfos gene affected interregional correlations, as both cortico-cortical and cortico-subcortical correlations were significantly altered. In contrast, the vast majority of intraregional correlations (intracortical and intrasubcortical) were preserved. Wild type mice showed three major types of cortico-cortical correlations: fronto-occipital, temporo-parietal and parieto-occipital correlations. In c-fos KO mice, only temporo-parietal correlations, including the same type of receptors, were observed; the other correlations were lost and no new correlations were generated. Correlations between the cortex and striatum were the main types of cortico-subcortical correlations detected in wild-type mice. The frontal, parietal and temporal cortex showed the tightest correlations with the striatum. In addition, minor correlations between the frontal cortex and hypothalamus and striatum and hippocampus were also observed. The c-fos knock-out completely altered the correlation patterns observed in WT mice. The only preserved correlation patterns were between the frontal cortex and striatum, but both the correlation types and participating receptors were changed, i.e., a positive correlation was observed in WT mice between the striatal M5 subtypes and a negative correlation was observed in KO mice between the striatal M1 and M4 subtypes. None of the other correlation patterns detected in WT mice were observed in KO mice. Moreover, a tight correlation between the hypothalamus and cerebellum was observed in the KO mice, which was not present in WT mice. Fig. 6. Schematic representation of the correlations between the levels of muscarinic receptor mRNA expression within the cortical regions and the cerebellum. The green line with arrows indicates a positive correlation, and the red dashed line with circles indicates a negative correlation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

3.2.3. Interregional, cortico-subcortical, and subcortical correlations Positive correlations between the FC and striatum (FC: M1 , M2 , M3 , M4 -stria: M5 ), striatum and hippocampus (stria: M4 -hip: M2 , M4 ), and hypothalamus and TC (hypo: M4 -TC:M1 ) were observed in WT mice. Negative correlations between the FC and hypothalamus (FC: M1 , M3 , M4 -hypo: M4 , FC: M4 -hypo: M1 ), PC and striatum (PC: M5 -stria: M1 , M2 , M3 , M4 ), TC and striatum (TC: M1 -stria: M2 , TC: M2 -stria: M1 , M2 , M3 ), PC and hippocampus (PC: M5 -hippo: M4 ) and hypothalamus and cerebellum (hypo: M5 -crbl: M4 ) were also observed. In contrast, c-fos KO mice showed a profoundly different correlation pattern. The only positive correlations were between the hypothalamus and cerebellum (hypo: M1 -crbl: M1 , M2 , M3 , M5 , hypo: M3 -crbl: M1 , M2 , M3 , hypo: M5 -crbl: M1 , M2 , M3 ), cerebellum and striatum (crbl: M5 -stria M1 , M4 ) and hypothalamus and hippocampus (hyp: M2 -hippo: M2). The correlations between the frontal cortex and striatum were negative and involved different receptor subtypes (stria: M1 –FC: M1 –M5 , stria: M4 -FC: M1 , M2 , M3 , M5 ); negative correlations were also observed between the occipital cortex and hippocampus (OC: M3 -hippo: M3 ). The correlations observed in WT mice were missing in the KO mice (Fig. 7). Examples of positive and negative correlations are shown in Fig. 8.

4.1. Gender differences The results obtained in this study revealed the increased expression of all M1 –M5 receptors in the frontal cortex in males and the increased expression of M1 –M4 receptors in the striatum in females, with no other differences in muscarinic receptor mRNA quantities in other regions. Many processes involving muscarinic receptors show distinct gender-specific patterns. Gender differences were shown in a variety of learning (reviewed in (Jonasson, 2005)) and pathological states, including the effects of methamphetamine (Siegel et al., 2010). However, studies on the differences in the muscarinic receptors in both the central nervous system and extracerebral tissues between genders are scarce. A previous study (Novakova et al., 2010) showed gender-specific changes in muscarinic receptors upon stress in mice. The results from additional studies in mice (Rhodes et al., 2005, 2008) and rats (Rhodes et al., 2001a,b) suggested that cholinergic receptors act on the hypothalamic–pituitary–adrenal axis in a gender-specific manner. Moreover, gender-specific changes in cholinergic function resulting from the knock-out of a specific gene involved in the development of CNS have been observed (Fragkouli et al., 2006). In humans, an increase in muscarinic receptor binding sites in the central nervous system in the female brain has been documented (Yoshida et al., 2000). In the present study, we systematically examined the expression of muscarinic receptor subtypes in all major cortical and subcortical areas and observed significant gender-specific differences in the frontal cortex where the increased expression of all M1 –M5 receptors was detected in males and in the striatum where the increased expression of M1 –M4 receptors was detected in females.

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Fig. 7. Schematic representation of the correlations between the levels of muscarinic receptor mRNA expression between the cortical and subcortical regions. The green line with arrows indicates a positive correlation, and the red dashed line with circles indicates a negative correlation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

These results are consistent with those of a previously published study (Benes et al., 2012a) that also showed gender-specific changes in dopamine receptor transcripts in the striatum of c-fos knock-out animals. Namely, the increased expression of dopamine D2 -receptor and acetylcholinesterase mRNA and the reduced expression of dopamine D3 -receptor mRNA was observed in females. The existence of a balance between dopamine and acetylcholine signalling in the striatum has been proposed (Gomeza et al., 2001), suggesting that M1 , M4 and D1 receptors are postsynaptically located, whereas D2 and M2 are presynaptically located to inhibit the release of the particular neurotransmitters in the synapse. Thus, complex changes involving the majority of muscarinic receptors, several dopamine receptor subtypes and the expression of acetylcholinesterase support the idea of a complex interplay between cholinergic and dopaminergic systems in the striatum. 4.2. Differences between wild type and knock-out animals Differences between wild type and knock-out animals were observed in the frontal (higher levels of M1 R, M4 R, and M5 R mRNA in KO mice), temporal (higher level of M4 R mRNA in KO mice), and occipital cortex (lower levels of M2 R and M3 R mRNA in KO mice) and the hippocampus (lower level of M3 R mRNA in KO mice). Moreover, a considerable restructuring of muscarinic receptor transcripts was also observed (cortical fronto-occipital and

parieto-occipital correlations disappeared, fronto-striatal correlations changed in terms of correlation type and receptor subtypes involved, parieto- and temporo-striatal correlations disappeared and hypothalamo–cerebellar correlations were generated). A previous study (Nelson et al., 2005) showed that changes in the levels of neurotransmitter in the prefrontal cortex influence the levels of other neurotransmitters in the parietal cortex (the administration of AMPA and carbachol, but not nicotine, in the prefrontal cortex increased the levels of acetylcholine in the posterior parietal cortex), documenting the existence of broader correlations between cortical structures. Moreover, the administration of carbachol and nicotine in the prefrontal cortex increased the efflux of acetylcholine from the same region. Thus, based on the evidence of this study, it is reasonable to propose that pre-existing interactions also occur between receptor systems within one cerebral region and remote regions. However, the limitation of the present study is that mRNA quantification and correlation does not exactly describe how muscarinic signalling is changed in c-fos KO. To our knowledge, this is the first study examining muscarinic receptors on the whole-brain level and we are currently not aware of any study quantifying the expression of muscarinic receptors on the protein level (performing either binding studies or immunohistochemistry). Each approach has limitations in terms of limited receptor-subtype specificity, though (Michel et al., 2009; Pradidarcheep et al., 2009). When new radioligands or antibodies with improved specificity are developed, it

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Fig. 8. Representative results from the regression analysis. Correlation between M1 and M4 mRNA (top, left) in the hippocampus of wild-type mice. Correlation between M1 mRNA in the frontal cortex and M5 mRNA in the occipital cortex (bottom, left) of wild-type mice. Correlation between M1 and M2 mRNA in the hippocampus of knock-out mice (top, right). Correlation between M2 mRNA in the frontal cortex and M2 -mRNA in the striatum of knock-out mice (bottom, right).

would be of considerable interest to perform a quantification of muscarinic receptor subtypes on the protein level. Similarly, the precise relationship between the restructuring of muscarinic receptor transcripts and the relatively preserved global function of the CNS in c-fos KO animals is also not absolutely clear, but there are several indirect hints suggesting the possible mechanisms in changed muscarinic signalling. The Fos family of proteins (c-Fos, FosB, Fra-1, Fra-2) are a part of the AP-1 transcription factor complex (Jochum et al., 2001) and the functional substitution of one transcription factor with another can occur as Fra-1 (a component of AP-1) was proved to functionally substitute for c-Fos in Fos1 knock-in mice (Fleischmann et al., 2000). The purpose of this study was not to quantify the possible functional substitution in AP-1 transcription complex; however, such quantification would certainly help to resolve this question. Second, muscarinic receptors are interrelated with other receptor systems (Gomeza et al., 2001), (i.e., dopaminergic). Thus the influence of dopaminergic receptors on muscarinic signalling is likely to occur also under the conditions of c-fos knock-out. Indeed, our previous study proved that c-fos knock-out also involves complex changes in dopaminergic system (Benes et al., 2012a). Specific receptor subtypes can activate multiple signalling pathways and fine tuning of signalling through many types of receptors can occur (Tomankova and Myslivecek, 2011; Xu et al., 2007). Moreover, signal transduction can be modified on the level of G protein by shuttling between trimeric and small G

proteins (Saini et al., 2009). Receptor activity-modifying proteins provide a further possibility to regulate signalling (Hay et al., 2006). 4.3. The impact of knock-out studies The present study have broader implications for understanding the functional impact of knock-out studies. Knock-out studies provide a valuable insight into the function of many genes. The easily interpreted results can be obtained when there is the knockout of a gene with well-defined function. Straightforward use of knock-out study is to demonstrate that a certain property persists even in the situation of the gene knock-out, i.e., unexpected maintaining of the hypoglycaemic effect of metformin in the absence of AMP-activate protein kinase (Foretz et al., 2010). Knock-out studies have been used in receptor physiology as well. Our recent study (Benes et al., 2012b) demonstrated the role of muscarinic M2 muscarinic receptors in the heart and the impact of its knock-out on other heart receptor systems (mainly adrenoceptors). Knock-out studies can also show that certain genes are surprisingly not necessary for survival, i.e., animals with whole-body (Hrabovska et al., 2010) as well as CNS specific (Farar et al., 2012) knock-out of acetylcholinesterase are viable and even (as in the case of CNS specific knock-out) can have only mild functional impairment. Similarly, c-fos knock-out animals are viable but with apparent functional consequences. However, knocking-out of genes with multiple

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functions usually triggers complex adaptation changes. A negative effect of a KO can merely mean that plastic changes have taken place. A positive effect of a KO could be due to the loss of the particular receptor or protein, but also could be due to the induction of plastic changes in the nervous system, and the resulting changes in chemical signalling. These views were discussed recently (Crusio et al., 2009; Dhein et al., 2013). We thus conclude that in wild-type mice, correlations exist between the muscarinic receptor subtypes within specific regions of the central nervous system and different brain regions. In c-fos KO mice, intraregional correlations are well preserved, but interregional correlations are significantly altered. The changes in the expression of muscarinic receptor transcripts might be a mechanism for the maintenance of the integrity and relatively preserved functions of the CNS in c-fos KO mice.

Acknowledgements This research was supported through a grant (GACR 309/090/406) from the Grant Agency of the Czech Republic. The authors would like to thank Ms. Petra Svatosova for excellent technical assistance.

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