WO2002033103A2 - Non-human mammal with modified pacap type i receptor - Google Patents

Abstract

A non-human mammal is disclosed with a modified, preferably inactivated PACAP type I receptor (PAC1). The invention further relates to production of such a mammal and the use thereof for the characterisation of genes and/or testing of substances, medicaments, and therapeutic formulations with regard to disease states such as, for example, visceral pain conditions, learning difficulties, anxiety states or dilatative cardiomyopathy.

Description

Non-human mammal with an altered PACAP type I receptor

The present invention relates to a non-human mammal which has an altered, preferably inactivated, PACAP type I receptor (PACl). The present invention further relates to methods for producing such a mammal and its use for characterizing genes and / or testing substances, drugs and therapeutic approaches with regard to various diseases, such as e.g. visceral pain, learning disabilities, anxiety or dilated cardiomyopathy.

The PACAP type I receptor is a G protein-coupled receptor that binds the highly conserved neuropeptide PACAP ("pituitary adenylate cyclase activating polypeptide") with a thousand times higher affinity than the related peptide VIP. Signal transmission mediated by the PACAP Type I receptor is believed to be involved in a wide range of physiological processes including neurotransmitters / neurotrophic actions, neuronal differentiation, synaptic plasticity and fertility.

The treatment of diseases that are associated with impaired learning behavior, such as poor learning, or with pain, excessive anxiety behavior or dilated cardiomyopathy has so far been unsatisfactory, which is largely due to the fact that - if at all - only medication is available, which are relatively unspecific or have undesirable side effects. To get a new group of therapeutics too who do not have these disadvantages, it would therefore be desirable to be able to characterize target genes and their products which are associated with these diseases, the brain being the primary target organ. The identification of target genes will then enable new starting points for therapies. In this way, drugs could be developed that have a higher specificity and therefore fewer side effects compared to the drugs previously used. However, the prerequisites for the development of such drugs have so far been lacking, above all due to the lack of suitable fashion systems, that is to say, for example of genetically modified animals, which allow the effects of the change or inactivation of genes associated with these diseases to be investigated, above all because such genes were previously unknown or only insufficiently known.

The present invention is therefore based on the technical problem of providing means which allow the identification of genes which are involved in the development of diseases which are associated with impaired learning behavior, e.g. Learning deficiency, associated with pain, excessive fear behavior or dilated cardiomyopathy, and are therefore of therapeutic benefit, since the desired therapeutic effect can be achieved by influencing the expression of these genes.

This technical problem is solved by the embodiments characterized in the patent claims.

It turned out that the gene coding for the PACAP type I receptor is a gene which is very well suited for the above purposes. Therefore, the problems discussed above can be addressed by providing a model system by creating animals, eg mice, in which the gene coding for the PACAP type I receptor is changed or inactivated. In the investigations leading to the present invention two different mouse mutants were generated, which have ubiquitous inactivation and tissue-specific inactivation of the PACAP type I receptor. In both cases the Cre loxP system was used and exon 11 of the PACl gene was flanked with loxP sites. Mice with ubiquitous inactivation of the PACAP type I receptor showed reduced anxiety and increased locomotion. In addition, these animals showed a specific deficit in the hippocampus-dependent association learning. Other memory tests (declarative memory) remained unaffected. Furthermore, the visceral pain sensation of the mice was drastically reduced with ubiquitous inactivation of the receptor. In addition, these mice had dilated cardiomyopathy. Mice with a forebrain-specific inactivation of the receptor gene showed no abnormalities in anxiety or locomotion, but - like the mice with ubiquitous inactivation of the receptor gene - showed a deficit in the hippocampus-dependent association learning. Thus, it was shown that the destruction of this receptor gene in vivo far-reaching consequences for the ^"visceral pain, the association learning and anxiety has as well as causing dilated cardiomyopathy, whereby the PACAP type I receptor and the gene coding for this receptor a valuable Starting point for the development of new drugs. The knockout mice according to the invention allow the search for genes which are regulated via the PACAP type I receptor and whose misregulation in the PACAP type I receptor-deficient mice is the reason for the deficits described above. Thus, these knockout mice enable the identification of new genes that are involved in processes such as learning, pain, anxiety and dilated cardiomyopathy, and which in turn can be the starting point for the development of new drugs, e.g. anxiolytics or memory-enhancing therapeutic agents. Since the PACAP type I receptor is particularly important for the transmission of visceral pain, but not of somatic pain, very selective therapeutic agents, preferably for the treatment of tumor pain, the side effects of which may well be below those of broader-acting anaesthetics.

The present invention thus relates to a non-human mammal in which the PACAP type I receptor (PACl) is ubiquitously or tissue-specifically altered, preferably inactivated. Characteristic of this mammal with an altered PACAP type I receptor is a combination of two genetic manipulations: (a) insertion of two recognition sites for a recombinase into the gene coding for PACl in such a way that its function is not impaired, but the Excision of the information between the recognition parts leads to expression of an inactive PACl and (b) insertion of a gene which codes for a recombinase which recognizes the above recognition sites and is under the control of a tissue-specific promoter. Limiting the change, preferably inactivating the PACl in defined target types or target organs, thus allows a direct characterization of the receptor function in these cells without impairment by further influences, which would be due to an additional inactivation of the PACl in other cell types. In addition to other procedures, the procedure described above can also be used for the production of animals with a ubiquitously modified gene, the gene coding for the recombinase being under the control of a non-tissue-specific promoter.

The term "non-human mammal" as used herein encompasses any mammal whose PACl can be ubiquitously or tissue-specifically modified, preferably inactivated, for example mouse, rat, rabbit, horse, cattle, sheep, goat, monkey, pig, dog and cat, mouse is preferred.

The term "ubiquitous or tissue-specific altered PACAP type I receptor" as used herein encompasses any PACAP type I receptor. Receptor of a non-human mammal that is different from the wild type in biological function, preferably inactive. A non-human mammal according to the invention can be produced, for example, by the methods described in the examples below. The inactivation of the PACAP type I receptor can be achieved, for example, by changing the coding gene in such a way that a PACAP type I receptor is expressed in which the ligand-binding domain is inactivated or one or more transmembranes Domains are deleted. Preferably, a transmembrane domain or a portion thereof is deleted, most preferably a deletion of exon 11 which results in the deletion of most of the transmembrane domain IV of the receptor. The tissue-specific change / inactivation of the PACAP type I receptor is not restricted to specific tissues and can ultimately affect all tissues for which corresponding tissue-specific promoters are available. The function of the PACAP type I receptor is preferably inactivated in the brain, for example by using a recombinase under the control of the Nestin promoter, even more preferred is the forebrain-specific inactivation, for example by using a under the control of the CaMKIiα promoter (Kellendonk et al., J. Mol. Biol. 285 (1999), 175-182) standing recombinase can be achieved.

A non-human mammal according to the invention can preferably be obtained by a method, the following

Steps include:

(a) Transfection of embryonic stem cells of a non-human mammal with a vector which, after recombination with the genome of the non-human mammal, leads to the insertion of two recognition sites for a recombinase into the DNA coding for the PACAP type I receptor, on the one hand the insertion of the two recognition parts does not adversely affect the expression of a biologically active receptor and on the other hand, the two recognition sites flank a gene region, the loss of which leads to the expression of a receptor which is modified or inactivated in its biological activity;

(b) isolation of cells stably transfected in step (a) and introduction into a first non-human mammal;

(c) injecting oocytes from a second non-human mammal with a vector containing the gene for a recombinase under the control of a tissue-specific promoter;

(d) introducing the injected oocytes from step (c) into a second non-human mammal;

(e) crossing the non-human mammals from steps (b) and (d); and

(f) Selection of the progeny obtained after step (e) for those with a tissue-specifically modified, in particular inactivated PACAP type I receptor.

The above statements apply accordingly to the terms “non-human mammal” and “ubiquitously or tissue-specifically modified PACAP type I receptor”.

The term "embryonic stem cells" used here includes all embryonic stem cells of a non-human mammal that are suitable for mutating the DNA coding for the PACAP type I receptor. The embryonic stem cells are preferably from the mouse, in particular E14 / 1 cells.

The term "vector" as used herein refers to any vector which, after recombination with the genome of the non-human mammal, results in the insertion of two recognition sites for a recombinase into the DNA coding for the PACAP type I receptor, with regard to the function / Localization of the recognition sites apply the information given above, or (b) the integration of a coding for a recombinase, under the control of a (tissue-specific) promoter standing gene allowed into the genome, the recognition sites of the vector from (a) must be compatible with the recombinase of the vector from (b), ie recognized by this. Tissue-specific elimination of the PACAP type I promoter can be achieved, for example, by inserting the two recognition sites for the recombinase into the genome in such a way that the corresponding recombinase under the control of a tissue-specific promoter inactivates the areas of the receptor discussed above or deleted. It is also advantageous if the vector has a marker with which selection can be made for stem cells in which the desired recombination has taken place. One such marker is the loxP / tkneo cassette, which can be removed from the genome again using the Cre / loxP system.

Furthermore, the person skilled in the art knows conditions and materials in order to carry out steps (a) to (f) of the above method. For example, he will inject the cells stably transfected in step (a) into blastocysts of a mammalian donor female. The blastocysts are then introduced into sham-pregnant mammals. Some of the transferred blastocysts develop into mouse chimeras, some of which carry a modified PACAP type I receptor allele in the germ cells. Appropriate crossings then give rise to hetero- and homozygous mammals. By mating these mammals and crossing with mammals encoding a recombinase under the control of a tissue-specific promoter, mammals are obtained that are homozygous for the modified PACAP type I receptor allele and heterozygous for the recombinase gene. With regard to the selection step (f), the person skilled in the art will apply methods in which it can be demonstrated in the desired tissues that the receptor is modified or inactivated with regard to its biological activity, for example by determining to what extent the induction of PACAP type I -Receptor-dependent genes are weakened or prevented. The choice of the promoter controlling the recombinase depends on the tissue in which the biological function of the PACAP type I receptor in the non-human mammal according to the invention is to be changed, preferably inactivated. Suitable promoters are known to the person skilled in the art. Such are for example for the nervous system (neurons and glial cells) as Nestin-Cre in Zimmermann et al. , Neuron 12.

(1994), 11-24, for postnatal neurons as Thy-1/2 Cre in Vidal et al. , EMBO J. 9 (1990), 833-840, for postnatal forebrain as Camkli-Cre e.g. in Mayford et al. , Cell 81

(1995), 891-904, for hepatocytes as albumin / α-fetoprotein Cre in Pinkert et al., Genes and Dev. 1 (1987), 268-276, for T cells as Lck-Cre in Orban et al. , Proc. Natl. Acad. Be. 89 (1992), 6861-6865, and for macrophages as Lysozy-Cre.

For the non-human mammal or method according to the invention, any recognition site / recombinase system can be used which allows the effective excision of the gene or gene segment lying between the recognition sites for the recombinase. Suitable systems are known to the person skilled in the art. These include e.g. the LoxP / Cre recombinase system or the FRT / FLP recombinase system (Sauer and Hendersen, Proc. Natl. Acad. Sci. 85 (1988), 5166-5170), the LoxP / Cre system being preferred. It is particularly preferred if the LoxP sites flank exon 11 of the gene coding for the PACAP type I receptor.

The procedure described above is also suitable for the production of a non-human mammal according to the invention in which the PACAP type I receptor is ubiquitously changed / inactivated. This can be achieved by the recombinase being under the control of a non-tissue-specific promoter; see also the explanations in Example 1 below. Alternatively, such a non-human mammal according to the invention with a ubiquitously modified PACAP type I receptor can also be obtained by a process which comprises the following steps: (a) transfecting embryonic stem cells of a non-human mammal with a vector which, after homologous recombination with the genome of the non-human mammal, leads to the generation of a gene coding for an altered or inactivated PACAP type I receptor;

(b) isolating the cells obtained in step (a) and introducing them into blastocysts;

(c) introducing the injected blastocysts from step (b) into a non-human mammal; and

(d) Examination of the non-human mammal obtained after step (c) for a ubiquitously modified, in particular inactivated, PACAP type I receptor.

With the non-human mammal according to the invention, it is possible to investigate the effects of, for example tissue-specific, change or inactivation of the PACAP type I receptor, for example with regard to the change in the expression of specific genes and / or the associated phenotypic peculiarities. For example, ^'has the ubiquitous inactivation of the PACAP type I receptor considerable anxiolytic effects, ie, the PACAP type I receptor is involved in the development of anxiety. Such inactivation of the PACAP-type receptor also leads to dilated cardiomyopathy. Like the ubiquitous inactivation, the forebrain-specific inactivation leads to a deficit in the hippocampus-dependent association learning. A comparison of gene expression in animals in which the PACAP type I receptor was ubiquitously inactivated or only in the forebrain with control animals thus allows the identification of genes that are involved in processes such as learning, pain, anxiety or dilated cardiomyopathy. are involved and can in turn be the starting point for the development of new medicines, e.g. anxiolytics or memory-enhancing therapeutic agents. The knowledge of these genes and their function then allows the development of new approaches for pharmacological Influencing the processes described above, for example by providing compounds which specifically influence the expression of these genes or the activity of the gene products. Thus, with the non-human mammal according to the invention, not only can genes be identified which are under the control of the PACAP type I receptor, but also their function or the possibly pathogenic conditions triggered by a malfunction of the function can be determined. Since the PACAP type I receptor is particularly important for the transmission of visceral pain, but not of somatic pain, very selective therapeutic agents, preferably for the treatment of tumor pain, can be developed, the side effects of which are likely to be significantly lower than those of broader-acting analgesics ,

Thus, the present invention preferably also relates to the use of the non-human mammal to characterize genes and / or test substances, drugs and / or therapeutic approaches that have an impact on processes related to learning ability, pain, anxiety behavior and dilated cardiomyopathy these processes are diseases or related symptoms such as visceral pain, learning or memory impairment, anxiety or dilated cardiomyopathy.

Brief description of the figures:

Figure 1: Generation of mice with a PACl deficiency -

(a) Organization of the PACl gene with exons 7 to 13: Exon 11 was flanked with LoxP sites in two steps. The modified allele was first generated by homologous recombination in ES cells. Then the Cr e was transient expression - Rekomb inas s the Selektionskasette removed, resulting alleles for the production of PACL ^loxP and PAC1 ^~. Schemes of the wild-type gene locus, the Target vectors and the resulting alleles are shown (black triangles: LoxP; K: Kpnl; X: Xbal; A and B represent

Probes outside the homology arms that are for Southern

Blot analyzes of the ES cells treated with electroporation were used. ) (b, c) RNase protection analysis of whole brain RNA from

Wild type ^" (wt) and PACl ^{_ /"} mice

(b) The 400 bp wild-type transcript is missing in PACl ^{"/ _} brains, but a weak 340 bp fragment is detectable, which is an alternatively spliced transcript that leads to a truncated receptor protein.

(c) PACAP type II receptors (VPACl and VPAC2) are not upregulated in PACl ^"7" brains (M: lkb ladder; GAPDH: glyceraldehyde-3-phosphate dehydrogenase).

(d, e, f) In situ hybridization of control, PACl ^"_ and PACl ^CaMKCre2 brains: Compared to control (d), PAC1 mRNA is almost completely absent in the hippocampus region of PACl ^CaMKCre2 brains (f) and is also undetectable in PACl ^{_ / "} brains (e).

Figure 2: PACl ^{- / "} mice show increased locomotor activity and reduced anxiety behavior

(a) During the three day observation period, PACl ^{- "} mice showed increased horizontal (p <0.01) and vertical locomotor activity (p <0.01). The results of the first day of observation are shown.

(b, c, d, e, f) "Open field" analyzes. Two daily observation periods are shown over a period of five minutes on two consecutive days. PAC1 ^"- mice showed increased locomotor activity, which results from increased general activity (b) (p <0.0001} - and reduced rest time (c) (p <0.0001). In addition, PAC1 ^{" / -} mice less anxious compared to the wild type offspring, they spent more time in the central field (d) (p <0.005) and made more stops in the central field (e) (p <0.001), and they also had fewer faeces in the open field -Boli ab (f) (p <0.0005). (G, h) PACl ^{~ _} mice showed decreased hesitation regarding entering the open sections of the "elevated zero-maze" (g) (p <0.01; four paws outside) and spent more time (p <0.01) in the open sections (h) (four paws outside) , Wild-type controls (n = 28; open bars), PAC1 ^{_ "} (n = 28; black bars)

Figure 3: PACl ^{"/ _} - and PACl ^CaMKCrβ2 mice show a selective deficit in the hippocampus- ^dependent associative learning behavior

(a) PACl ^{_ / ~} mice (n = 14 mutants, 14 wild-type animals; p <0.005) and PACl ^CaMKCre2 mice (n = 12 mutants, 20 wild-type animals; p <0.01) showed a greatly reduced "freezing" - answer for contextual fear conditioning, but not for "cued fear" conditioning [please give German names if possible] (24 hour test).

(b) In contrast to PACl ^CaMKCre2 mice (n = 9 mutants, 12 wild-type animals), PACl ^{"/ _} mice (n = 14 mutants, 13 wild-type animals) also showed a deficit in the short-term test of contextual anxiety Conditioning (p <0.005). Wild-type controls (empty bars), PACl ^CaMKCre2 mice (gray bars), PACl ^"~ mice (black bars)

Figure 4: PACl ^{" "} mice have dilated cardiomyopathy

(a) About 90% of the PACl ^"_ mice crossed back into the C57bl6 background died on day 8-14. Furthermore, these mice showed progressive weakness and no weight gain from day 6.

(b) Furthermore, these mice had dilated cardiomyopathy, which affected all 4 ventricles. These mice also showed centroacinous fatty liver.

(c) In addition, these mice showed altered gene expression. For example, ANP (atrial natriuret. Factor) was up-regulated and SERCA (sarcoplasmatic calcium ATPase) under-regulated (about 30-40%). The invention is illustrated by the examples below.

Example 1: General procedures

(A) Preparation of transcrene mice: The PACl locus in ES cells was as described by Gu et al. , (Science 265 (1994), 103-106). The "targeting" vector was constructed from isogenic DNA (Kaestner et al., Genomics 20 (1994), 377-385). The upstream loxP site was inserted into the intron prior to exon 11 by overlapping PCR along with an additional Xbal site. The primers used were as follows:

External primer 1: 5'- G G A T C C C C A G G A A C C C C - 3 '

Inner pri er 1: 5 '- T C T A G A A T A A C T T C G T A T A

A T G T A T G C T A T A C G AAG T T AT T T T G C C C AG GA TA C T C T T A G - 3 '

External primer 2: 5 '- A G G G C C T G A G G G C A A G C -

3 '

Interior primer 2: 5 '- AT AAC T T G C TA TAG CATAC

AT TA TA C GAAG T TA TTC TAGA TGGGGC T TC CTTTTCT TGC - 3 ¹

The 5 'homology arm, a 3.5 kbp Spel / BamHI fragment, which carries exons 7 to 10 of the PACl gene (all DNA fragments used come from a genomic λ phage 18, which is from a genomic library was isolated (Kastner et al., Genomics 20 (1994), 377-385)), was subcloned into the "Bluescript" vector Bluescript IIKS ^{+ "} (Stratagene) and fused with the 0.35 kbp BamHI / HindIII fragment , the ^"includes the upstream loxP site and exon. 11 The selection cassette flanked by two loxP sites was inserted downstream of the BamHI / Hindlll fragment using a Hindlll / Xhol cleavage. Finally, the 3 'homology arm, a 4.5 kbp HindIII fragment, was first subcloned into the "Bluescript" vector Bluescript IIKKS ^{+ "} (supra), which within a destroyed Sacl site a second Xhol- Harbored site, then cut via Xhol cleavage and subcloned into the Xhol site of the "targeting" construct. It was used to select ES cells and clones in the presence of G418 (350 μg / ml). G418-resistant clones were analyzed by Southern blot using probes outside the homology arms. The 5 'probe represents a 500 bp Pstl-Spel fragment which connects directly to the 5' homology arm. The 3 'probe is a 350 bg HindIII fragment which connects directly to the 3' homology arm. The frequency of homologous recombination was 12%. Homologously recombined clones were transiently transfected with a Cre expression plasmid pHD2 (provided by F. Weih, DKFZ, Heidelberg, Germany) (20 μg) and subclones were selected in the presence of gancyclovir (1 μM). Which the PAC 1 ^_ - or PACL ^CaMKCre2 allele bearing mice were analyzed by blastocyst injection (Hogan et al, Manipulating the Mouse Embryo; Cold Spring Harbor Laboratory Press., 1994) were obtained.

To generate CaMKCre2 mice, nlsCre was cloned into a CaMKIl vector as described recently {.Kellendonk et al., J. Mol. Biol. 285 (1999), 175-182). Linearized pMM403-Cre insertion DNA was injected into the pronuclei of C57BI / 6 oocytes (Kellendonk et al., J. Mol. Biol. 285 (1999), 175-182) and various transgenic lines were obtained. In the CaMKCre2 line, the expression pattern of the Cre recombinase was defined using an anti-Cre recombinase antibody (Kellendonk et al., J. Mol. Biol. 285 (1999), 175-182). Mosaic inactivation of the Cre recombinase transgene was observed in 30% of the PAC ^CaMKCre2 mice. These mice were identified post mortem by staining with anti-Cre recombinase antibodies and excluded from further experiments.

(B) RNase protection analyzes and in situ hybridization: RNase protection analyzes and in situ hybridization were carried out as described recently (Otto et al., Mol. Brain Res. .66

(1999), 163-174). The following probes were used for the RNase protection analyzes: PACl (nt 1308-1644 of the murine PACl- cDNA; Hashimoto et al. , Biochim. Biophys. Acta 1281 (1996), 129-133), VPAC1 (nt 1--232 of the murine VPACl cDNA; Johnson et al., J. Neuroimmunol. 68 (1996), 109-119), and VPAC2 (nt 106-446 of murine VPAC2-CDNA; Inagaki et al., Proc. Natl. Acad. Sci. USA 91 (1994), 2679-2683).

(C) Behavioral studies: Mutant mice and control mice ^" were matched in terms of sex and age and the animals of a litter were housed together. The following behavioral studies were carried out: Locomotor activity test (Maldonado et al., Proc. Natl. Acad. Sci. USA 96 (1999), 14094-14099); "elevated zero-maze" (Tronche et al., Nature Genet. .96 (1999), 99-103); "elevated radial maze" (Maldonado et al., Proc. Natl. Acad. Sci. USA 96. (1999), 14094-14099); "light / dark box" (Tronche 'et al., Nature Genet. 96. (1999), 99-103); "open field" test (Leittinger et al., Behav. Genet. 24 (1994), 273-284); "morris water maze", "social transmission of food preference" and "fear conditioning" (Gass et al., Learn Mem. 5 (1998 ), 274- 288). to assess the general locomotor activity and ^'standing with anxiety related behavior different groups of mice in three different laboratories / countries were unt he seeks. The results were highly reproducible and were analyzed using the Student T-Test and ANOVA. The results are presented as mean values ± standard deviation. To directly compare the two mutant mouse strains, all experiments in this study were carried out with the same genetic background (75% C75BI / 6/25% 129 Ola).

Example 2: Studies on the lack of expression of PACl in the mutant mice obtained in Example 1

The mutant mice were produced according to Example 1 above. Exon 11, which codes for most of the transmembrane domain IV of the receptor protein, was used to inactivate all previously known splice variants of PACl inactivated. After homologous recombination in embryonic stem cells (ES), two different PACl alleles were generated (Figure la). The PAC1 ^" allele with missing exon 11 was injected into blastocysts to produce PACl ^{" / _} mice with ubiquitous inactivation of PACl. In the PACl ^loxP allele, exon 11 was flanked by two loxP recognition sites for the recombinase-controlled excision of the DNA sequence in between (FIG. 1 a). PACl ^loxP is thus inactivated in every cell that expresses the recombinase. PACl ^loxP homozygous mice appear normal and the expression of the PACl mRNA is identical to that of Wildty mice. To generate the mutant mice with a forebrain-specific inactivation of PACl (PACl ^loxp1 ° ^xp CaMKCre2, abbreviated to PACl ^CaMKCre2 ), PACl ^loxP mice were crossed with transgenic mice (CaMKCre mice), the Cre recombinase expressing under the control of the CaMotKI promoter ,

PACl ^CaMKCre mice show, according to the expression pattern for Cre recombinase, inactivation of PACl mainly in three brain areas, the olfactory bulb, the cortex areas of the forebrain and the toothed gyrus (Figure If). In contrast, PAC1 ^"- mice showed ubiquitous inactivation of PACl. Wild-type transcripts of PACl are completely missing (FIG. 1b, e). Instead, an alternatively fed transcript is detectable in PAC1 ^{" / -} brains, which 8% of the Wild-type RNA level reached (Figure lb). The sequencing of this transcript revealed an alternative splicing from exon 11 to exon 12, which leads to a reading frame shift with the formation of a subsequent stop codon and thereby to a shortened receptor protein which, due to the lack of the third intracellular loop, can no longer couple G proteins. Interestingly, the other PACAP receptors VPAC1 and VPAC2, which belong to the class of PACAP type II receptors, were not upregulated (FIG. 1c). At the time of weaning, PACl ^CaMKCre2 mice were found to have an expected Mendelian ratio (n = 381), while PAC1 ^{"/ -} mice were only found with a frequency of 19% (instead of 25%) (n = 589). Both types of mutants were fertile, appeared healthy, and were at first sight of indistinguishable from their wild-type siblings. Histological examination of the organs from both mutant mouse lines revealed no pathological abnormalities. In particular, neither neuronal proliferation or differentiation defects nor "mossy fiber" (moss fiber) anomalies were found within the hippocampus formation. The neurological examinations, which included tests on a hot plate and tests of reflexes, motor strength and coordination ("rotarod"), revealed no deficits in terms of sensory or motor skills.

Example 3: Behavioral studies (I): With regard to locomotor activity and anxiety behavior

Because of the main expression of PACl in the central nervous system, behavioral studies were of particular interest. PAC ^{_ / "} mice but not PACl ^CaMKCre2 mice showed both an increased locomotor activity and an increased exploratory behavior as well as a strongly reduced fear behavior (FIG. 2). The spontaneous locomotor activity was in an almost completely dark, unrelated environment of PAC ^{_ "} -Mäύsen clearly reinforced in both horizontal and vertical directions (Figure 2a). Even under stress-related conditions in the brightly lit open field, PAC ^{- ~} mice were more active (Figure 2b). Compared to the control mice, they walked longer distances, moved faster, examined the open area in more detail and took fewer breaks (Figure 2c). In addition, the PAC ^{- / "} mice were less anxious compared to their wild-type siblings, based on the total time spent in the center

(Figure 2d) or the amount of stops in the center of the open field (Figure 2e) is occupied. In contrast, the control animals preferred to remain in close contact with the wall and tried to avoid staying in the central field

(Figure 2e). Since defecation under stress conditions is one of the safest signs of anxiety, the greatly reduced amount of faecal boluses that were placed in the open field by the PAC ^{"/ _} mice (Figure 2f) is in accordance with the decreased anxiety behavior in these mice. The reduced anxiety behavior of the PAC ^{"/ -} mice was further confirmed by three behavioral paradigms based on the natural avoidance behavior of mice:" elevated zero-maze "," light / dark box "and" elevated zero-maze "(FIG. 2g, h,). In comparison to the control animals, PAC ^-" mice used to enter the open areas of the "elevated zero-maze" earlier with all four paws (FIG. 2g), and they also spent more time there (FIG 2h). While hippocampal damage has been reported to result in hyperactivity, the increased locomotor activity and decreased anxiety behavior seen in the PAC ^{- / "} mice are most likely not due to hippocampal inactivation of PACl, since PAC ^CaMKCre2 mice with a almost complete inactivation of PACl in the hippocampus (Figure 1f) did not show a comparable phenotype, and other structures involved in anxiety-related behavior include the amygdala, the "periaqueductal gray" and the lateral septu all strong expression of PACl, the receptor was however inactivated in these areas only in the PAC_ ^" mice and not in the PAC ^CaMKCre2 mice, which could explain the observed differences in the phenotype.

Example 4: Behavioral studies (II): With regard to learning behavior

No deficits were observed in two hippocampus-dependent tasks that simulate declarative learning and memory, the "Morris water maze" and the "social transmission of food preference". Since the hippocampus expression of PACl is almost exclusively restricted to the "ossy fiber" synapses, the lack of spatial learning deficits in the mutant mice is by no means surprising. In contrast to the synapse between CA3 and CA1, the "mossy fiber" synapses seem to be less important for spatial learning. Most of the spatial information is believed to be transmitted directly from the entorhinal cortex to pyramidal cells from CA3 and CAl, the "mossy f ibre" synapse is avoided and the traditional trisynaptic cycle is not followed. Both mouse mutants showed a selective deficit in the hippocampus-dependent association learning. Both PAC ^{"/ -} mice and PAC ^CaMKCre2 mice showed a drastic reduction in the" freezing "response in the long-term test of contextual conditioning, but not in the" cued fear "conditioning (Figure 3a). Based on these results, ie the fear conditioning in terms of sound, and the previously observed role of the hippocampus in terms of conditioning, it is assumed that the impaired "freezing" response in the long-term test of contextual fear conditioning, a hippocampus-dependent deficit reflects in terms of learning behavior brain areas with a complete inactivation of PACL both PAC ^{_. / "} Mice as well as PAC ^CaMKCre2 mice are the serrated gyrus and neocortex areas of the forebrain. Since lesions of the neocortex did not affect the contextual anxiety conditioning, the presynaptic PAC1-mediated signal transmission at the "mossy fiber" synapse seems to play a critical role for the consolidation of the contextual anxiety in long-term memory. In contrast to pvc ^CaMKCre2 mice, PAC ^{"/ _} mice were deficient in the hippocampus-dependent short-term ^test regarding the conditioning of contextual anxiety (Figure 3b). This finding is consistent with the previous and ubiquitous inactivation of PACl in these mutants.

Example 5: Investigations regarding dilated cardiomyopathy

PACl " ^7- mice were crossed back into the C57bl6 background and phenotypic and genotypic analysis of the mice was performed. It was found that 90% of the mice died on day 8-14. Furthermore, the mice showed progressive weakness and absence the weight gain from day 6 after birth (Figure 4 (a)) and the mice also showed dilated cardiomyopathy affecting all 4 ventricles. The mice also had a suitable centroacinar fatty liver, as would be expected in hypoxemia, which is due to the reduced pumping power of the heart (FIG. 4 (b)). At the molecular level, a change in gene expression with respect to ANP (up-regulation) and SERCA (under-regulation) observed.

Claims

claims 1. Non-human mammal in which the PACAP type I receptor is ubiquitous or tissue-specific. 2. The non-human mammal of claim 1, wherein the PACAP type I receptor is inactivated. 3. The non-human mammal of claim 2, wherein the PACAP type I receptor is tissue-specifically inactivated. 4. A non-human mammal according to claim 3, wherein the PACAP type I receptor is inactivated specifically to the forebrain. 5. A non-human mammal according to any one of claims 1 to 4, wherein exon 11 of the gene coding for the PACAP type I receptor is deleted. 6. A method for producing the non-human mammal according to any one of claims 1 to 5 with a tissue-specific modified PACAP type I receptor, comprising the following steps: (a) Transfection of embryonic stem cells of a non-human mammal with a vector which, after recombination with the genome of the non-human mammal, leads to the insertion of two recognition sites for a recombinase into the DNA coding for the PACAP type I receptor, on the one hand the insertio of the two recognition parts does not adversely affect the expression of a biologically active receptor and, on the other hand, the two recognition sites flank a gene region, the loss of which leads to the expression of a receptor which has changed in its biological activity, in particular inactivated; (b) isolating cells stably transfected in step (a) and introducing them into a first non-human mammal; (c) injecting oocytes of a second non-human mammal with a vector containing the gene for a recombinase under the control of a tissue-specific promoter; (d) introducing the injected oocytes from step (c) into a second non-human mammal; (e) crossing the non-human mammals from steps (b) and (d); and (f) Selection of the progeny obtained after step (e) for those with a tissue-specifically modified, in particular inactivated PACAP type I receptor. 7. The method of claim 6, wherein the recognition sites for recombinase are LoxP sites and the recombinase is Cre recombinase. 8. The method of claim 7, wherein the region of the gene coding for the PACAP type I receptor, flanked by the LoxP sites, is exon 11. 9. A method for producing the non-human mammal according to any one of claims 1 to 5 with a ubiquitously modified PACAP type I receptor, comprising the following steps: (a) transfection of embryonic stem cells of a non-human mammal with a vector which, after homologous recombination with the genome of the non-human mammal, leads to the generation of a gene coding for a modified, in particular inactivated PACAP type I receptor; (b) Isolation of the cells obtained in step (a) and insertion of these into blastocysts; (c) introducing the injected blastocysts from step (b) into a non-human mammal; and (d) Examination of the non-human mammal obtained after step (c) for a ubiquitously modified, in particular inactivated, PACAP type I receptor. 10. Use of the non-human mammal according to one of claims 1 to 5 or of the non-human mammal obtainable by the method according to one of claims 6 to 9 for the characterization of genes and / or testing of substances, drugs and / or therapeutic approaches have an impact on processes related to learning ability, pain, anxiety behavior and / or dilated cardiomyopathy. 11. The non-human mammal of any one of claims 1 to 5, the method of any one of claims 6 to 9, or the use of claim 10, wherein the non-human mammal is a mouse.