WO2013013338A1 - Use of substances targeting gc-c signaling pathway in diagnosis and treatment for midbrain dopamine neurons diseases - Google Patents

Abstract

The present invention discloses the use of substances targeting GC-C signaling pathway in the diagnosis and treatment for midbrain dopamine neurons diseases. One of the uses disclosed by the present invention is the method for constructing the animal model of attention deficit hyperactivity disorder using GC-C gene as the target. The method comprises the following steps: the GC-C gene of target mammal is knocked out, and the GC-C gene knockout animal obtained is the animal model of attention deficit hyperactivity disorder. The animal model of attention deficit hyperactivity disorder can be used to screen products for preventing and/or treating diseases related with midbrain dopamine system.

Description

 Use of substances targeting the GC-C signaling pathway for the diagnosis and treatment of midbrain dopamine neuron diseases

 The present invention relates to the use of a substance targeting guanylate cyclase-C (GC-C) and protein kinase G (PKG) signaling pathways, in particular to the use of GC-C and protein kinase G (PKG) signaling pathways. Use of target substances in the diagnosis and treatment of midbrain dopamine neuronal diseases.

Background technique

 The midbrain dopamine system is a very important neurotransmitter system. Dopamine neurons distributed in the ventral tegmental area (VTA) and basal substantia nigra (SNc) of the midbrain project their axons to the forebrain and release dopamine to regulate many important behavioral processes such as exercise, cognition and learning. [References: Nestler, EJ, Is there a common molecular pathway for addiction? Nat Neurosci, 2005. 8(1 1): 1445-1449. and Bjorklund, A. and SB Dunnett, Dopamine neuron systems in the brain: an Update. Trends Neurosci, 2007. 30(5): 194-202.] The dysfunction of this system is associated with many serious mental illnesses. Specifically, projection from the VTA to the prefrontal cortex is thought to be associated with schizophrenia [References: Callicott, JH, et al., Physiological dysfunction of the dorsolateral prefrontal cortex in schizophrenia revisited. Cereb Cortex, 2000. 10(11) : 1078-1092. and Mimics, K., et al., Molecular characterization of schizophrenia viewed by microarray analysis of gene expression in prefrontal cortex. Neuron, 2000. 28(1): 53-67.], and from SNc to grain The projection of degenerative lesions is considered to be the main cause of Parkinson's disease [References: Innis, RB, et al., Single photon emission computed tomographic imaging demonstrates loss of striatal dopamine transporters in Parkinson disease. Proc Natl Acad Sci USA, 1993. 90(24): 1 1965-1 1969. and ish, SJ, K. Shannak, and O. Hornykiewicz, Uneven pattern of dopamine loss in the striatum of patients with idiopathic Parkinson's disease. Pathophysiologic and clinical implications. N Engl J Med, 1988. 318(14): 876-880.]. Drug addiction is thought to be primarily related to activities from VTA to Nucleus Accumbens [References: Cornish, JL and PW alivas, Glutamate transmission in the nucleus accumbens mediates relapse in cocaine addiction. J Neurosci, 2000. 20(15): RC89. and Di Chiara, G., Nucleus accumbens shell and core dopamine: differential role in behavior and addiction. Behav Brain Res, 2002. 137(1-2): 75-1 14.]. Receptor agonists and blockers corresponding to this variety of dopamine are used to treat diseases such as Parkinson's disease, schizophrenia, and attention deficit hyperactivity disorder [Emilien, G., et al" Dopamine receptors— Pharmacol Ther, 1999. 84(2): 133-156.] Studying how dopaminergic neurons are selectively regulated, not only helps us understand the neurobiological mechanisms of behavioral control And can provide ideas for finding more effective ways to treat mental illness.

Guanylate cyclase (GC) is a class of enzymes that convert GTP into an important intracellular second messenger cGMP. According to their different positions, the GC family can be divided into two categories: soluble guanylate cyclase and guanylate cyclase on the membrane. Soluble guanylate cyclases are distributed in the cytosol and they can be activated by the small gas molecules nitric oxide and carbon monoxide. These small molecules can freely cross the cell membrane. The guanylate cyclases on the membrane are transmembrane proteins, most of which are activated by extracellular signals. Guanylate cyclase C (GC-C) belongs to guanylate cyclization on the membrane The enzyme family is widely believed to be abundantly expressed in the small intestine of mammals, including humans [Miwatani, T. Amino-acid sequence of a heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli. Eur. J. Biochem. 129, 257-263.] . The gene sequence and amino acid sequence of GC-C were first determined by Schulz et al. [Schula S, et al. Guanylyl cyclase is a heat-stable enterotoxin receptor. Cell, 1990. 63(5): 941-948.]. Some receptors for guanylate cyclase on membranes have been discovered, but some are still unknown [Emilien, G., et al., Dopamine receptors - physiological understanding to therapeutic intervention potential. Pharmacol Ther, 1999. 84 (2): 133-156.].

 Activation of guanylate cyclase C (GC-C) increases intracellular cGMP levels. GC-C is also a key receptor for the intestinal hormones guanylin (G) and uroguanylin (UG) [Reference: Currie, MG, et al., Guanylin: an endogenous activator of intestinal guanylate cyclase. Proc Natl Acad Sci USA, 1992. 89(3): 947-951. and Hamra, FK, et al., Uroguanylin: structure and activity of a second endogenous peptide that stimulates intestinal guanylate cyclase. Proc Natl Acad Sci USA, 1993. 90(22) : 10464-10468.], and the major receptor for Escherichia coli heat-stable endotoxin STa. STa is a major cause of acute secretory diarrhea [Schulz, S., et al., Guanylyl cyclase is a heat-stable enterotoxin receptor. Cell, 1990. 63(5): 941-948.]. GC-C has been shown to be involved in the mediation of water and salt in the gut. However, GC-C knockout mice can survive, fertile or even be physiologically healthy, and they exhibit significantly normal intestinal water regulation and body weight under different food and salt intake conditions [Reference: Mann, EA, et al., Mice lacking the guanylyl cyclase C receptor are resistant to STa-induced intestinal secretion. Biochem Biophys Res Commun, 1997. 239(2): 463-466. and Schulz, S., et al., Disruption of The guanylyl cyclase-C gene leads to a paradoxical phenotype of viable but heat-stable enterotoxin-resistant mice. J Clin Invest, 1997. 100(6): 1590-1595.]. Although the guanylate cyclase on the membrane is involved in behavioral regulation in the nematode [Tsunozaki, M., SH Chalasani, and CI Bargmann, A behavioral switch: cGMP and PKC signaling in olfactory neurons reverses odor preference in C. elegans. Neuron, 2008. 59(6): 959-971.], and the cGMP signaling pathway is also thought to be involved in important behavioral and physiological regulation in multiple species [References: Lucas, KA, et al., Guanylyl cyclases and signaling by cyclic GMP. Pharmacol Rev, 2000. 52(3): 375-414. and Reaume, CJ and MB Sokolowski, cGMP-dependent protein kinase as a modifier of behaviour. Handb Exp Pharmacol, 2009(191): 423-443.] , The function of GC-C in the nervous system is currently poorly understood.

 Attention deficit hyperactivity disorder (ADHD) is one of the most common types of childhood mental disorders, probably affecting

2-12% of children, these symptoms will remain in adulthood in 60% of children, and eventually 3-5% of adults are also plagued by this mental disorder [Dopheide, JA and SR Pliszka, Attention-deficit- Hyperactivity disorder: an update. Pharmacotherapy, 2009. 29(6): 656-679.] Both genetic and pathological studies of ADHD and environmental pathology studies suggest that the dopamine system may be associated with this disease. Genetic studies have found that some genes are polymorphic in ADHD patients, with the best repeatability being two genes associated with the dopamine system, type 4 dopamine receptors and type 1 dopamine transporter genes [Swanson, JM, et Al., Etiologic subtypes of attention-deficit/hyperactivity disorder: brain imaging, molecular genetic and environmental factors and the dopamine hypothesis. Neuropsychol Rev, 2007. 17(1): 39-59.]. Environmental pathology studies have identified some important non-hereditary pathogenic factors, such as smoking in pregnant women will increase the risk of their offspring, long-term exposure to lead pollution, etc. These factors will also affect the dopamine system in the brain. Normal function [Braun, JM, et al., Exposures to environmental toxicants and attention deficit hyperactivity disorder in US children. Environ Health Perspect, 2006. 114(12): 1904-1909.]. The most direct evidence comes from recent brain imaging data collected on ADHD patients. Brain imaging data suggest that dopamine neurotransmission is blocked in ADHD patients [Reference: Ernst, Μ·, et al., High midbrain [18FJDOPA accumulation in children with attention deficit hyperactivity disorder. Am J Psychiatry, 1999. 156 (8 ): 1209-1215. Wokou Volkow, ND, et al., Brain dopamine transporter levels in treatment and drug naive adults with ADHD. Neuroimage, 2007. 34(3): 1 182-1 190. and Lou, HC, et Al., ADHD: increased dopamine receptor availability linked to attention deficit and low neonatal cerebral blood flow. Dev Med Child Neurol, 2004. 46(3): 179-183.], and this deficiency is associated with core symptoms of ADHD such as lack of attention Deficiencies in impulsiveness, impulsiveness, and rewards and driving forces are all related [References: Volkow, ND, et al., Depressed dopamine activity in caudate and preliminary evidence of limbic involvement in adults with .attention-deficit/hyperactivity disorder. Arch Gen Psychiatry, 2007. 64(8): 932-940. and Rosa Neto, P., et al., Methylphenidate-evoked potentiation of extracellular dopamine In the brain of adolescents with premature birth: correlation with attentional deficit. Ann NY Acad Sci, 2002. 965: 434-439.] Further studies more systematically demonstrate the activity of dopamine receptors and dopamine transporters in some important dopaminergic energies There is a significant decrease in brain regions such as striatum and midbrain [Volkow, ND, et al" Evaluating dopamine reward pathway in ADHD: clinical implications. JAMA, 2009. 302(10): 1084-1091.]. These evidences tell us that the activities of the dopamine system are closely related to the causes of ADHD.

Invention disclosure

 The object of the present invention is to provide a substance targeting a guanylate cyclase-C (GC-C) and protein kinase G (PKG) signaling pathway, in particular to GC-C and protein kinase G (PKG). The use of a signal pathway-targeted substance for the diagnosis and treatment of midbrain dopamine neuron disease. .

 Application of a substance that activates guanylate cyclase C to the preparation of an agonistic reaction product that enhances midbrain dopamine neurons mediated by a first type of metabotropic glutamate receptor and/or a metabotropic acetylcholine receptor The scope of protection of the invention.

 In a specific embodiment of the invention, the substance that activates guanylate cyclase C is guanylin or uroguanylin.

 The use of guanylate cyclase C knockout mice for screening for products for the prevention and/or treatment of diseases associated with the midbrain dopamine system is also within the scope of the present invention.

 The disease associated with the midbrain dopamine system may be human attention deficit hyperactivity disorder, schizophrenia, Parkinson's disease or drug addiction. In a specific embodiment of the present invention, a GC-C knockout mouse exhibiting a behavioral defect as attention deficit hyperactivity disorder is used as an animal model of attention deficit hyperactivity disorder.

Another object of the present invention is to provide an animal model of attention deficit hyperactivity disorder, the animal model being a bird A mammal in which the nucleotide cyclase C gene is knocked out.

 The method for constructing the animal model of attention deficit hyperactivity disorder is also within the scope of the present invention, and the method comprises the steps of: knocking out the guanylate cyclase C gene in the mammal of interest, and obtaining the guanylate cyclase The animal in which the C gene is knocked out is an animal model of attention deficit hyperactivity disorder.

 The method of knocking out the guanylate cyclase C gene in a mammal of interest is to exclude or replace the gene encoding the guanylate cyclase C in the mammal of interest. The animal in which the guanylate cyclase C gene is knocked out can be prepared according to the following method: knocking out the guanylate cyclase c gene in ex vivo embryonic stem cells, and then guanylate cyclase C gene The knockout embryonic stem cells are implanted into mouse blastocyst stage embryos, and finally transferred into the same mammalian uterus, and the born F1 mice are self-crossed to produce guanylate cyclase C gene knockout animals.

 The mammal can be a mouse, rat, rabbit, monkey, pig or chicken. In a specific embodiment of the invention, the mammal is a mouse.

 The use of an agonist or inhibitor of the GC-C/PKG signaling pathway in the preparation of a medicament for the prevention and/or treatment of a disease associated with the midbrain dopamine system is also within the scope of the present invention. When GC-C is activated, it produces a large number of intracellular second messengers cGMP, which can effectively activate protein kinase G (PKG) to produce a series of intracellular effects. Inhibition of PKG regulatory domain or catalytic domain by inhibitors allows group I metabotropic glutamate receptor-mediated excitatory responses to no longer change after GC-C activation; activation of PKG by activator activation The domain significantly enhances the excitatory response mediated by the Group I metabotropic glutamate receptor even when GC-C is not activated.

 In an embodiment of the present invention, the agonist of the GC-C/PKG signaling pathway is specifically an activator 8-Br-cGMP which is a protein kinase G; the inhibitor of the GC-C/PKG signaling pathway acts on Protein kinase G regulatory subunit inhibitor Rp-8-pCPT-cGMPS or an inhibitor of the functional subunit of protein kinase G KT5823; the disease associated with the midbrain dopamine system is attention deficit hyperactivity disorder, schizophrenia Symptoms, Parkinson's disease or drug addiction. In a specific embodiment of the present invention, a GC-C knockout mouse exhibiting a behavioral defect as an attention deficit hyperactivity disorder is used as an animal model of attention deficit hyperactivity disorder.

 Still another object of the present invention is to protect the use of a substance which detects the integrity of the guanylate cyclase C gene and the expression level of this protein in the preparation of a diagnostic or auxiliary diagnostic attention deficit hyperactivity disorder agent.

 Detection of the integrity of the guanylate cyclase C gene can be accomplished by sequencing the genome or GC-C gene of ADHD patients or potential patients. The substance for detecting the expression level of guanylate cyclase C may be an antibody against guanylate cyclase C (such as a monoclonal antibody or a polyclonal antibody) or an RNA hybridization encoding a ubiquitin cyclase C RNA. A needle, when detecting the expression level of guanylate cyclase C in a sample to be tested with an antibody against guanylate cyclase C or an RNA hybridization probe encoding guanylate cyclase C RNA, if The expression level of guanylate cyclase C in the sample is lower than the normal expression level of the organism or does not express guanylate cyclase C at all, suggesting that the sample to be tested may be derived from an organism with attention deficit hyperactivity disorder. If necessary, combined with other clinical diagnostic criteria for attention deficit hyperactivity disorder can be diagnosed.

DRAWINGS

Figure 1 shows GC-C expression in dopamine neurons in VTA/SNc. (AC) In the midbrain of mice, the mRNA signal of GC-C is similar to that of TH (n=4). (A) GC-C mRNA signal obtained by in situ hybridization. (B) The signal of TH on the same brain slice. (C) is the coincidence of A and B. (DF) Double immunostaining showed GC-C (red) expression in midbrain dopamine cells containing the TH signal (green). (n=4). (GI) High magnification shows that GC-C is expressed on the cell bodies and dendrites in midbrain dopamine neurons.

 Figure 2 shows GC-C in situ hybridization. (A) GC-C in situ hybridization signal in the midbrain of wild-type mice. (B) GC-C in situ hybridization signal in the midbrain of GC-C knockout mice.

 Figure 3 shows that in TH-GFP transgenic mice, GC-C was expressed on VTA/SNc cells containing green fluorescent protein (GFP) fluorescence. (A-F) At the midbrain site, cells immunostained by GC-C and cells containing GFP in TH-GFP transgenic mice exhibited similar patterns. (A) GC-C immunostaining (red). (B) GFP cells are in the same position as shown in A. (C) The coincidence of A and B. (D-F) The area inside the dotted line box in (A-C) shown at high magnification.

 Figure 4 shows that in TH-GFP transgenic mice, GC-C was not expressed in dopamine neurons located in the hypothalamus (A) GC-C immunostaining (red). (B) GFP cells are in the same position as shown in A.

 Figure 5 shows that electrophysiological properties of midbrain dopamine neurons cannot be affected by GC-C activation. (A) The data showed that the clamp current of the cells did not change significantly after the addition of G/UG [no significant difference; p = 0.35; paired t-test; n = 20]. (B) The data showed that the resistance of the cells did not change significantly after the addition of G/UG [no significant difference; p = 0.32; paired t-test; n = 20]. The comparison indicates a system without G and UG. Where G/UG means G or UG.

 Figure 6 shows that the reaction mediated by the ionotropic glutamate cyclase is not changed after GC-C activation. (A-D) G/UG does not significantly alter the fast excitatory postsynaptic current caused by electrical stimulation. (A-C) exemplified the absence of significant changes in post-synaptic excitatory currents in a midbrain dopaminergic nerve cell. (D) Group data [no significant difference; p = 0.51; paired t-test; n = 7]. (E-G) G/UG does not significantly alter the inward current caused by AMPA stimulation. (E-F) An exemplary demonstration of a midbrain dopamine neuron was added to G without significant changes in AMPA current. (G) group data [no significant difference; p = 0.14; paired t-test; n = 7]. Where G/UG means G or UG.

 Figure 7 shows that GC-C activation does not affect the inhibitory current response mediated by the GABAa receptor. (A-D) There is no significant change in the inhibitory response current mediated by GABAa receptors in a midbrain dopaminergic nerve cell recorded by the example. (E) Group data [no significant difference; p=0.56; n=7]. Where G/UG indicates G or UG.

Figure 8 shows that in the midbrain dopaminergic neurons, GC-C activation can significantly increase mediated by the first type of metabotropic glutamate receptor (group I metabotropic glutamate receptor) or metabotropic acetylcholine receptor. Excitatory response. (AD) G increases the excitatory response caused by DHPG. (A) In a midbrain dopaminergic nerve cell, DHPG of ΙΟμηι produces a more intense response in G of Ι μιη. The left panel shows the response of cells to DHPG before G is added. The right panel shows the response of cells to DHPG after G addition. (B) The inward current caused by DHPG can be significantly increased by G. The left panel shows the response of cells to DHPG before G is added. The reaction of cells to DHPG after G is added to the middle panel. The right panel shows the response of cells to DHPG after washing G. (C) A continuous reaction plot of cells to DHPG as shown in Figure B. (D) Group data [**, p<0.01 _; paired t-test; paired t-test; n = 1 1] The left columnar graph shows the change of DHPG response before and after the addition of G/UG, the right columnar chart Indicates the addition of a reagent ODQ that inhibits the intersynaptic current reagent TTX and inhibits soluble guanylate cyclase. Changes in DHPG response of post-cells before and after G/UG addition. (EH) GC-C activation increases the response caused by the ligand muscarine of the metabotropic acetylcholine receptor. (E) UG can increase the cellular action potential response caused by muscarine. The action potential recorded by the patch clamp on the left shows the excitatory response mediated by the metabotropic acetylcholine receptor in the cells recorded before the addition of UG. The right panel shows the excitability mediated by the metabotropic acetylcholine receptor in the cells after the addition of UG. The sexual response increased. (F, G) In a midbrain dopaminergic neuron, G is able to reversibly augment the response mediated by the metabotropic acetylcholine receptor [*, p<0.05; paired t-test; n = 5]. (F) The current recorded by the patch clamp on the left shows that the excitatory response mediated by the metabotropic acetylcholine receptor is recorded in the cells before G is added. The middle panel indicates that the cells are mediated by the metabotropic acetylcholine receptor after G addition. The excitatory response increased, and the right panel indicates that the excitatory response mediated by the metabotropic acetylcholine receptor was recovered in the cells after G wash-off. (G) (F) A graph showing the response of a metabotropic acetylcholine receptor in cells shown in the figure. (H) Group data [*, p<0.05; paired t-test _; n = 5]. The columnar graph indicates the change of cells in response to muscarine before and after the addition of G/UG.

Figure 9 shows that midbrain dopamine neurons of GC-C knockout mice exhibit normal intrinsic properties. (A, B) A midbrain dopamine neuron recorded in GC-C knockout mice. The data of group (C) showed that the dopaminergic neurons of GC-C knockout mice had no significant difference in H current compared with the dopamine neurons of wild type mice [no significant difference; p = 0.94 _; pair t Tested (paired t-test); n = 9]. (D) Group data showed that dopaminergic neurons in GC-C knockout mice had no significant difference in membrane resistance compared with dopamine neurons in wild-type mice [no significant difference; p = 0.56; paired t Tested (paired t-test); n = 9].

 Figure 10 shows that the potentiation of G/UG depends on the activity of GC-C and protein kinase G. (A-C) G/UG Increased DHPG response is dependent on GC-C. (A) An example demonstrates that a midbrain dopamine cell recorded in a GC-C knockout mouse is unable to increase its response to DHPG by UG. (B) A graph of the response of the same cells to DHPG shown in A. (C) Group data [no significant difference; p = 0.93; paired t-test; n = 11]. (D-F) G/UG increase reaction was eliminated by Rp-8-pCPT-cGMPS. (D, E) An example of Rp-8-pCPT-cGMPS (ΙΟμηι) incubation can eliminate the DHPG-induced increase caused by G. (F) Group data [no significant difference; p = 0.51; paired t-test; n = 9]. The (G-I) G/UG increase reaction was eliminated by KT5823. (G, H) Example KT5823 (2μπι) incubation can eliminate the DHPG-induced increase caused by G. (I) Group data [no significant difference; p = 0.95; paired t-test; paired t-test; n = 8 (J-L) 8-Br-cGMP significantly increased the excitatory response induced by DHPG. (J, K) Example 8-Br-cGMP (200μπι) incubation can increase the excitatory response of DHPG. (L) Group data [**, p<0.01; paired t-test; n = 9]. Where G/UG means G or UG.

 Figure 1 is a schematic diagram of the device for maintaining and monitoring the regular activities of animals R for a long time. The figure shows the device that is suitable for single animal activity.

 Figure 12 is a schematic diagram of the odor adaptation test device and the experimental flow arrangement. The first four odors used were Amyl Acetate, and the fifth test smell was Acetophenone.

 Figure 13 is a picture of the actual animal being collected during the experiment. In the middle is the exposed centrifuge tube cover, and three small holes are dug in the cover to facilitate the evaporation of the odor.

Figure 14 is a schematic diagram of the device used for the go/no test and the experimental flow arrangement. A. Schematic diagram of the device. B. Experiment Process arrangement. The red bar indicates the "go" training unit, the corresponding sound stimulus is S+, the 3kHzo blue bar indicates the "not going" training unit, the corresponding sound stimulus is S -, the 15kHzo water droplet represents the reward, and the lightning represents the electric shock penalty.

 Figure 15 is a timing diagram of the components in a single training unit in the first stage. The arrow shows the mouse probe time, and the horn shows the sound start time. The previous line indicates the start and end of the sound signal, and the next line indicates the start and end of the action time window.

 Figure 16 is a timing diagram of the components in a single training unit in the second phase and the test phase. Upper column: Timing in a single unit in the second phase. Lower bar: Timing within a single unit during the test phase.

 Figure 17 shows that the GC-C knockout mice exhibit a hyperactivity behavior defect similar to ADHD. (A) Long-term monitoring revealed that the level of autonomic exercise in GC-C knockout mice was significantly higher than in wild mice when they were in the dark phase of the light/dark cycle. (*, p<0.05; t-test, 5 in GC-C knockout mice and 5 in wild type and 5 in wild type) (B) GC-C knockout mice in an adapted environment Got a lot. The horizontal autonomous movement of the animals in the new market is recorded by an infrared camera for 4 hours (one point every ten minutes). In the first 100 minutes, the level of autonomic activity of GC-C knockout mice was slightly higher than that of wild-type mice [121% higher; genotypic difference p<0.001; analysis of variance (ANOVA); GC-C 6 knockout mice, 12 wild type mice]. After 100 minutes, GC-C knockout mice showed much higher levels of autonomic activity than wild-type mice [220% higher; p < 0.001; analysis of variance (ANOVA)]. Among them, the black and white bar chart on the abscissa, black means "dark" environment, white means "light" environment.

 Figure 18 shows that GC-C knockout mice exhibit impaired adaptability similar to ADHD. The odor-adaptability of GC-C knockout mice was impaired. Amyl acetate was used as the odor in the first four units, and acetophenone was used as the test odor in the fifth unit. Right column: The time to explore odor in GC-C knockout mice was significantly longer than that in wild type (*, p<0.05; **, p<0.01; t-test; 8 GC-C knockout mice, wild type 6). Left column: GC-C knockout mice also showed significant loss of adaptability.

 Figure 19 shows a similar learning curve for wild-type mice and GC-C knockout mice. The red curve is the learning curve for GC-C knockout mice. The black curve is the learning curve for wild type mice. The number of X axes is multiplied by 100 for the number of training units, and the y axis is the correct rate. Both mice achieved a 90% correct rate (no significant difference) after approximately 1500 training units.

 Figure 20 shows the impulsiveness and maintenance of attention in GC-C knockout mice (1). (A) Schematic diagram of the training method of the first stage and the hydrophobic activity corresponding to the sound stimulation after the GC-C knockout mouse and the wild type were learned in the first stage. The drowning activity is represented by a logic circuit signal, where a high level signal indicates that water is being drained. (B) GC-C knockout mice stopped significantly longer than wild-type, indicating impaired behavioral inhibition and impulsivity (***, p<0.001; t-test; GC-C KO 6 Only, wild type 10).

 Figure 21 shows the impulsive and maintenance deficits of GC-C knockout mice (2). The mice were asked to wait for a random delay of up to 2 seconds before the start of the stimulation and reaction time window. GC-C knockout mice showed significantly higher rates of abandoning the training unit before the start of stimulation (A: **, p<0.01; t-test) and a lower correct response rate than wild type (B : **, p < 0.01; t-test; 6 GC-C KO, 7 wild-type), indicating that they have poor ability to maintain attention.

Figure 22 shows that the background extracellular dopamine levels of GC-C knockout mice are significantly lower than in wild type mice. [**, p = 0.01; t-test; 15 GC-C knockout mice (black), 12 wild-type mice (grey)]. Data were normalized to the average concentration of wild type mice.

 Figure 23 is a dose-dependent effect of amphetamine on autonomic movement in mice. Low doses of amphetamine (1 mg/kg body weight) reduced the level of autonomic exercise in GC-C knockout mice (analysis of variance in the first 70 minutes p < 0.001, 6 animals).

 Figure 24 is a dose-dependent effect of amphetamine on spontaneous motor movement in mice. In contrast, the same dose of amphetamine had no effect on the level of autonomic exercise in the wild type (12 animals).

 Figure 25 shows that high doses of amphetamine enhance autonomic movement in GC-C knockout mice and wild-type. The peak of exercise caused by different doses was averaged relative to the exercise level after each animal was injected with physiological saline. The black curve is the dose-effect curve for GC-C knockout mice, and the gray curve is the dose-effect curve for wild type mice.

 Figure 26 shows that PKG agonists can reduce the autonomic activity of GC-C knockout mice. The autonomic activity of GC-C knockout mice in the new environment was reduced after injection of the PKG agonist (p<0.001 for the first 70 minutes of variance analysis, 5 animals each). The black curve is the autonomous activity curve of GC-C knockout mice after injection of 3mM 8-Br-cGMP, and the gray curve is the autonomous activity curve of GC-C knockout mice after injection of artificial cerebrospinal fluid.

The best way to implement the invention

 The experimental methods used in the following examples are all conventional methods unless otherwise specified.

 The materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The GC-C knockout mice used in this study were provided by Elizabeth Mann and Mitchell B. Cohen of the Children's Hospital of Cincinnati. The mouse terminates the original sequence of the GC-C gene by replacing it with a sequence encoding the minigene hypoxanthine phosphoribosyltransferase (HPRT) by homologous recombination in mouse embryonic stem cells. The expression of GC-C at this position [Mann, EA, et al., Mice lacking the guanylyl cyclase C receptor are resistant to STa-induced intestinal secretion. Biochem Biophys Res Commun, 1997. 239(2): 463-466. ]. The TH-GFP transgenic mouse was constructed by Kazuto Kobayashi of Fukushima Medical University, which is a C57BL/6 background. Construction of the mouse First, a plasmid containing a green fluorescent protein coding sequence directed by the mouse tyrosine hydroxylase (TM) promoter promoter was constructed, and the plasmid was linearized and injected into the fertilized egg by pronuclear injection. In the pronucleus, the treated fertilized egg is finally implanted into the surrogate mother, and the progeny are screened to obtain a transgenic mouse that specifically expresses GFP in the cell expressing the TH protein. In this strain of mice, eGFP is specifically expressed under the guidance of the TH promoter, thereby labeling only dopamine neurons. All wild type mice used in this study were C57BL/6 mice (provided by Beijing Vitalriver Laboratory Animal Co., Ltd.) except for the special instructions. The above-mentioned TH-GFP transgenic mice and GC-C knockout mice were all C57BL/6 backgrounds, raised and propagated in the SPF animal room of the Beijing Institute of Life Sciences, and were taken out until the experiment.

 Artificial cerebrospinal fluid for 5χ section: acetylcholine 1 10 mM, potassium chloride 2.5 mM, calcium chloride dihydrate 0.5 mM, magnesium chloride hexahydrate 7 mM, sodium dihydrogen phosphate 1.3 mM, sodium ascorbate 1.3 mM, sodium pyruvate 0.6 mM. Before use, take 100 mL of the stock solution to prepare 500 mL of solution, add sodium bicarbonate, and add anhydrous glucose to a final concentration of 25 mM and 20 mM, respectively, and adjust the osmotic pressure to 300 psi.

10x recording of artificial cerebrospinal fluid: sodium chloride 125 mM, potassium chloride 2.5 mM, calcium chloride dihydrate 2 mM, Magnesium chloride hexahydrate 1.3 mM, sodium dihydrogen phosphate 1.3 mM, sodium ascorbate L3 mM, sodium pyruvate 0.6 mM. On the day of use, 100 mL of the stock solution was prepared to prepare a 100 mL solution, and sodium hydrogencarbonate and anhydrous glucose were added to bring the final concentrations to 25 mM and 10 mM, respectively, and the osmotic pressure was adjusted to be consistent with the former.

 Perfusion solution: sucrose, sodium chloride 1 19 mM, potassium chloride 2.5 mM, calcium chloride dihydrate O.lmM, magnesium chloride hexahydrate 4.9 mM, sodium dihydrogen phosphate lmM, sodium bicarbonate 26.2 mM, anhydrous glucose 1.25 mM, Sodium ascorbate lmM, kynuric acid 3 mM. The prepared perfusion solution is dispensed in a volume of 50 ml and stored at -20 ° C, and can be repeatedly frozen and thawed.

 Drugs guanylin (1 μΜ), ODQ (10 μΜ), 8-Br-cGMP (200 μΜ), Rp-8pCPT-cGMPS (10 μΜ), and ΚΤ 5823 (2 μΜ) were purchased from BIOMOL; uroguanylin (Ι μΜ) was purchased from Peptide; ΤΤΧ (Ι μΜ) and picrotoxin (10 (^M) were purchased from Sigma; the original stock solution was diluted with artificial cerebrospinal fluid and added by circulation. Other drugs DHPG (5 or 10 μΜ), muscarine (50 μΜ) ΑΜΡΑ (17.5μΜ) and muscimol (50μΜ) were purchased from Sigma, and were diluted with artificial cerebrospinal fluid in the experiment and given by the eight-channel rapid micro-dosing system (eight-channel rapid micro-dosing system was purchased from Wuhan 100). Shikang Biotechnology Co., Ltd.) The micro-portion is about 500μηι from the cells. All the above drug storage solutions are dissolved in pure water or high-concentration DMSO according to the solubility characteristics of the drug to 1000 times or 2000 times the concentration of -20. Store at °C or -80 °C.

 The electrode used in the patch clamp recording technique was a borosilicate glass electrode with a filament produced by Sutter Instruments, which had an outer diameter of 1.2 mm and an inner diameter of 0.69 mm. The electrode was drawn using a P97 microelectrode puller (sutter instrument) to ensure a tip outer diameter of 2.6 μm. The specific formula of the electrode liquid is as follows: Electrolyte internal liquid component, hydrochloric acid potassium 1 15 mM, hydroxyethyl piperazine ethanesulfonic acid 40 mM, magnesium chloride 5 mM, ethylene glycol tetraacetic acid 10 mM, potassium chloride 6 mM (pH 7.2- 7.4), store at -20 ° C. The nystatin stock solution should be prepared on the day of the experiment, and the concentration is 25mg/ml. When using, add the electrode solution to the final concentration of 0.05 mg/ml-lmg/ml, taking care to avoid light.

 All operations in this study were in accordance with the relevant regulations of the Beijing Institute of Life Sciences and the National Institutes of Health (NIH) on the protection and use of laboratory animals.

 The invention will be further elucidated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.

 Example 1. Detection of GC-C expression in VTA/SNc dopamine neurons by immunohistochemistry and in situ hybridization assay

The immunohistochemistry was as follows: The mice were deeply anesthetized with an excess of pentobarbital, then placed in a tray, and the entire blood circulation system was perfused with physiological saline. Specifically, a small needle injected with physiological saline is inserted from the right ventricle, and the pulmonary vein connected to the left atrium is cut. The pre-cooled physiological saline was perfused at a rate of about 2 ml per minute to allow systemic blood to flow out from the pulmonary veins. After the replacement was cleaned, it was perfused with pre-cooled phosphate buffer containing 4% paraformaldehyde until the mice were all stiff. Air bubbles should be avoided throughout the process. The mouse brain was carefully dissected with small scissors and forceps, immersed in approximately 6 ml of 4% paraformaldehyde-containing phosphate buffer, fixed for 4 hours, and then dehydrated overnight with phosphate buffer containing 30% sucrose. After the brain was completely dehydrated, it was sectioned using a cryostat (Leica CM1900). The specific method is as follows: First, the forebrain part of the brain is roughly removed, the midbrain is retained, and the midbrain is embedded with an embedding agent (OCT) and frozen in a frozen section of the -20 ° C. After being completely frozen and fixed, the brain slices were cut into 20 μπ using a microtome, and the brain slices were immersed in citrate buffer (PBS). The prepared brain slices were washed three times with phosphate buffer, and the OCT residue was completely removed at intervals of 5 minutes. The non-specific site was then blocked with a phosphate buffer containing 10% calf serum and 0.1% triton-X for 1 hour. The sheep-derived GC-C antibody (purchased from Santa Cruz Biotechnology, inc.) was diluted with a blocking solution at a ratio of 1:1000, and placed on a brain slice and incubated for 4 hours at 4 。. After the primary antibody was incubated, it was washed three or four times with phosphate buffer for 5 minutes each time. The brain was incubated with the brain slices for two hours at room temperature with Cy3-donkey anti-goat (1:500, Jackson Immunoresearch). It was then washed three times with phosphate buffer again, 5 minutes apart. The stained brain slices were plated on glass slides, and dried and visualized with a 50% glycerol seal containing DAPI. The slides were stored at 4 °C.

 The pre-fixation, dehydration, and sectioning methods for in situ hybridization are the same as immunohistochemistry. The treated brain slices were pretreated with a transcribed GC-C probe, a 740-nucleotide RNA single strand containing a sorghum marker, and hybridized at 64 °C for more than 16 hours. After elution, the treated brain slices were incubated with an anti-digoxigenin antibody ligated with alkaline phosphatase, and then developed with the substrate NBT/BCIP.

 The midbrain immunohistochemistry results of normal mice showed that a large amount of GC-C was expressed on neurons in the VTA/SNc nucleus of the mouse midbrain (as shown in Fig. 1A, D, G), which is related to GC. The results of RNA in situ hybridization by -C were consistent (as shown in Figure 2). Careful observation of the staining signal mainly exists in the cell body and dendrites, but the Striatum nucleus mainly projected by the cells in the VTA/SNc nucleus is not. The midbrain VTA/SNc nucleus contains a large number of dopamine projection neurons and inhibitory local neurons. In order to further confirm the neuronal characteristics of GC-C, the brain slices of GC-C immunohistochemistry were used for TH immunization. Group detection. Tyrosine hydroxylase (TH) is an essential enzyme for the synthesis of dopamine neurotransmitters [Bjorklund, A. and SB Dunnett, Dopamine neuron systems in the brain: an update. Trends Neurosci, 2007. 30(5): 194-202 .], it can specifically label dopamine neurons in the expression position of the midbrain. The results of double staining showed that the expression of GC-C and TH were completely coincident at the cell level in the midbrain (as shown in Figure 1 C, F, I). Using TH-GFP transgenic mice for GC-C immunohistochemistry, it was also shown that the fluorescence signal of the VTA/SNc-labeled GC-C completely coincides with the signal of GFP (as shown in Figure 3). However, no expression signal of GC-C was observed in other dopamine neurons other than the midbrain (as shown in Figure 4).

 Example 2. Study on the physiological function of GC-C signaling pathway by using perforated patch clamp technique

The perforated patch clamp technique was used to clean the circulation tube of artificial cerebrospinal fluid with pure water before the start of the experiment. It was then replaced with artificial cerebrospinal fluid recorded with 95% oxygen and 5% carbon dioxide, and the circulation rate was adjusted to approximately 2 ml/min. Turn on the resistance heating fin wound around the inlet loop and adjust the temperature of the circulating solution to 32 °C. The well-incubated brain slices were inhaled into a small tank filled with artificial cerebrospinal fluid and pressed with a platinum ring wrapped with nylon filaments. The digital signal is monitored by Clampex 9 during recording, and the current or voltage stimulation of the cells can also be performed by editing the relevant program in Clampex 9. When recording the synaptic activity, a 0.5 M/0.5 M bipolar stainless steel electrode (manufactured by Micro Probe Inc.) was placed on the leading edge of the recorded cells at 100 to 300 μm. Using the Clampex9 editing program, an electrical stimulus with an intensity of 50-300 μΑ and a time course of 200 μ _δ is input through the digital output of the digital-to-analog converter every 15 s, usually recorded under the action of Ι Μ μΜ GABAa receptor antagonist bitter taste. Cells can be observed for excitatory postsynaptic currents above 100 pA. If it is not successful, change the direction of the stimulus current or adjust its intensity. Among them, the brain slices were prepared as follows: Prepare the ice cubes before preparation for slicing, and pre-cool the surgical instruments on ice. The perfusion solution and section were ice bathed with artificial cerebrospinal fluid and filled with 95% oxygen and 5% carbon dioxide. Approximately 200 ml of artificial cerebrospinal fluid was taken for recording, and a strainer was placed in the middle and incubated at 34 。. Anesthetize two-month adult mice with an appropriate amount of pentobarbital, fix in a tray, cut the chest, inject about 5 mL of perfusion solution into the blood circulation system with a syringe at a rate of ~ 2 ml/min, and quickly break with a large scissors. head. After simply rinsing the blood, use a small pair of scissors to quickly cut the brain and peel the brain into artificial cerebrospinal fluid for pre-cooled, aerated, saturated sections. After twenty seconds, the brain was transferred, and the midbrain portion was roughly separated by a blade, and it was quickly fixed to the stage of the vibrating slicer with a quick-drying glue. In this process, a 4% agar block can be attached to the stage in advance to keep the brain close to the edge, which prevents the brain tissue from being displaced during slicing. A horizontal slice having a thickness of 300 μm was cut with a vibrating slicer (microtome model Leica VTlOOOS). The vibration frequency of the instrument is usually set at 8-9 when slicing, and the feed speed is generally set at 2-3. The cut brain slices were transferred to the artificial cerebrospinal fluid at 34 ° C and filled with 95% oxygen and 5% carbon dioxide for at least 1 hour. .

 1. Does GC-C activation affect the electrophysiological properties of midbrain dopamine neurons?

 The perforated patch clamp was used to record the midbrain dopamine neurons in normal adult mice. The healthy cells were selected and stably recorded for more than 20 minutes. The GC-C ligand guanylin (G) was added to the circulating solution at a final concentration of ΙμΜ. Or uroguanylin (UG) for more than fifteen minutes, observe changes in cell current and resistance. Twenty dopamine cells recorded in the statistics showed no significant changes in cell current or resistance (as shown in Figure 5), indicating that GC-C activation could not open the ion channels on the cells and could not affect the dopamine neurons themselves. Basic characteristics of electrophysiology.

 2. Whether the change of ion-type glutamate cyclase mediated reaction after GC-C activation

 Detection of protein kinase G (PKG) activity by GC-C activation in mid-brain dopamine neurons of normal adult mice using a perforated patch clamp technique, which may modulate AMPA receptor mediated by dopamine neurons Reaction. At the time of recording, γ-aminobutyric acid a (GABAa) receptor blocker picrotoxin was added to the artificial cerebrospinal fluid, and the cells were clamped at -60 mv to ensure that the recorded postsynaptic current was mainly caused by α. - A-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor mediated. In all of the 7 cells recorded, the addition of G or UG had no significant effect on excitatory postsynaptic currents (EPSC) (Figure 6 B-D). In addition, cells were activated with an agonist of AMPA receptor and the effect of GC-C activation on this response was observed. In the presence of tetrodotoxin ("TTX") and 1H-[1,2,4] oxacillin and [4,3-a] quinoxalin-1-one (1H-[1, 2, 4] oxadiazolo In the case of [4, 3 -a] quinoxalin -1-one, ODQ), each time the cells were stimulated with 17.5 μΜ AMPA for 2 seconds, each time separated by two minutes, and G or UG was added after the recording was stable. The experimental results show that a small amount of AMPA can cause an inward current of about 200pA on dopamine cells, but this excitatory response is not affected by GC-C activation (as shown in Figure 6 E-G).

3. Whether GC-C activation affects the inhibitory current response mediated by GABAa receptors. The same method was used to record midbrain dopamine neurons in normal adult mice, and the active agent muscimol of GABAa receptor was applied to cells. After stabilization, add G or UG to observe changes in the cell response. The results confirmed that continuous administration of 50 μM muscimol in the 2S time course at two-minute intervals was effective in inhibiting neuronal cells, but the addition of G/UG had no significant effect on this response (as shown in Figure 7). 4. Is it possible to increase the excitatory response mediated by the first type of metabotropic glutamate receptor (group I metabotropic glutamate receptor) or metabotropic acetylcholine receptor after GC-C activation?

 Detection of midbrain dopamine neurons in normal adult mice by perforated patch clamp technique, incubation of G or UG in recorded brain slices, and found to significantly increase DHPG (ligand of metabotropic glutamate receptors) The frequency of cell excitability is shown in Figure 8A. The specific experimental methods are as follows: Select healthy midbrain dopamine neurons, record the neurons using puncturing with a perforated patch clamp, clamp the cells to -60nw, record stably for about 10 minutes, and then switch to current clamp. Mode, giving the cells about -50pA of current, to ensure that the cells have no spontaneous discharge response. The DHPG of Ι Ομπι was sprayed to the cells by a pressure system at a position of about 500 μm from the neurons, and the administration time of the pressure system was controlled by a computer program to be 2 s, thereby recording the excitatory response in the left image of A. The administration was repeated three times, and each time interval was 5 minutes, the stability reaction as shown in the left figure was obtained. Then, Ι μιη G was added to the artificial cerebrospinal fluid in which the brain slices were incubated, and the response of the cells to DHPG was continuously recorded during the process. After about 10 minutes of G addition, the response of the neuron to DHPG became a pattern as shown on the right. .

 In the voltage clamp recording mode, G or UG can significantly increase the inward current caused by DHPG (increased by nearly 70%, as shown in Figure 8 B-D), and this effect can be reversed without G or UG. The specific experimental methods were as follows: The healthy midbrain dopamine neurons were selected, and the neurons were recorded with a puncturing plaque using a perforated patch clamp, and the cells were clamped to -60 mv and stably recorded for more than 10 minutes. In the voltage clamp mode, the DHPG of Ιμμη is injected into the cell by the pressure system at a position of about 800 μm from the neuron, and the administration time of the pressure system is controlled by a computer program to be 12 s, thereby recording the excitability of about ΙΟΟρΑ. Inward current. Repeated administration 4 times, each time interval of 5 minutes, can obtain a more stable inward current. Three such inward currents are averaged, and the reaction shown in the left diagram of B is obtained. Then, G of Ιμηι was added to the artificial cerebrospinal fluid of the recorded brain slices, and the response of the cells to DHPG was continuously recorded during the process. After about 10 minutes of G addition, the response of the neuron to DHPG became the pattern shown in the figure in Figure B (average of the three reactions). Finally, the artificial cerebrospinal fluid containing G was replaced with ordinary artificial cerebrospinal fluid. After 20 minutes, the reaction of the cells was found to return to the previous level, as shown in the right figure of B (average of three reactions). Statistics 1 The intrinsic current of the recorded cells before the addition of G or UG and the inward current after 10 minutes of G or UG were added to obtain the left image of D.

Since GABA neurons in VTA/SNc express sGC, they can produce cGMP, which affects the electrical activity of peripheral dopamine neurons [Nugent, FS, EC Penick, and JA Kauer, Opioids block long-term potentiation of inhibitory synapses. Nature, 2007 446 (7139): 1086-1090·]. In order to exclude the interference of other cells on this reaction, in the experiment, sodium channel blockers Ι μΜ ΤΤΧ and sGC blocker 10μΜ ODQ were added to the artificial cerebrospinal fluid for recording [Schrammel, A., et al., Characterization Of lH-[l ,2,4]oxadiazolo[4,3-a]quinoxalin-l -one as a heme-site inhibitor of nitric oxide-sensitive guanylyl cyclase. Mol Pharmacol, 1996. 50(l ): l-5 .]. The response of the nine dopamine cells recorded in this condition to DHPG still increased with the activation of GC-C (as shown in the right panel of Figure 8D). This indicates that increasing the response of cells to DHPG after GC-C activation is achieved by changes in the cells themselves, independent of other peripheral neurons. Type 5 acetylcholine receptors are expressed in midbrain dopamine cells. In the experiment, the mAChR active agent muscarine also activated dopamine cells [Lacey, MG, P. Calabresi, and RA North, Muscarine depolarizes rat substantia nigra zona compacta and ventral tegmental neurons in vitro through Ml -like receptors. J Pharmacol Exp Ther, 1990. 253(1): 395-400.], observe GC-C activation The impact of this reaction. In the same way, 50 μΜ muscarine is given 2-10 s each time, once every six minutes. It can be observed that the reaction becomes larger after the addition of G or UG (as shown in Fig. 8 EH). The specific methods are as follows: Select healthy midbrain dopamine neurons, record the neurons with puncturing with a perforated patch clamp, clamp the cells to -60mv, record stably for about 10 minutes, and then switch to current clamp mode. Give the cells about -50pA of current to ensure that the cells do not have a spontaneous discharge response. A 30 μm muscarine was sprayed onto the cells by a pressure system at a position of about 500 μm from the neurons, and the administration time of the pressure system was controlled by a computer program to be 6 s, thereby recording the excitatory response in the left panel of the E map. The administration was repeated three times, and each time interval was 5 minutes, the stability reaction as shown in the left figure was obtained. Then, UG of Ιμηι was added to the artificial cerebrospinal fluid in which the brain slices were incubated, and the response of the cells to muscarine was continuously recorded during this process. After about 10 minutes of UG addition, the response of the neuron to muscarine became the pattern shown in the right panel of E. In the voltage clamp mode, 60 μm of muscarine was sprayed onto the cells by a pressure system at a position of about 500 μm from the neurons, and the administration time of the pressure system was controlled by a computer program to be 5 s, thereby recording an excitatory introversion of about 100 pA. Current. Repeated administration 4 times, each time interval of 5 minutes, can obtain a relatively stable inward current. Three such inward currents are averaged to obtain the reaction shown in the left diagram of F. Then, Ι μπι G was added to the artificial cerebrospinal fluid in which the brain slices were incubated, and the response of the cells to muscarine was continuously recorded during the process. After about 10 minutes of G addition, the response of the neuron to muscarine became a pattern as shown in the graph of F (average of three reactions). Finally, the artificial cerebrospinal fluid containing G was replaced with ordinary artificial cerebrospinal fluid. After 20 minutes, the reaction of the cells was restored to the previous level, as shown in the right graph of F (average of three reactions). The magnitude of the intrinsic current of the 5 recorded cells before G or UG addition and the magnitude of the inward current after 10 minutes of G or UG addition were counted to obtain an H map.

 The above experimental results demonstrate that G or UG can significantly enhance the Gq-GPCR-mediated response of dopamine cells. Example 3. Using a perforated patch clamp technique to detect whether the enhancement of G/UG depends on the activities of GC-C and PKG

 1. Whether G/UG enhances the excitatory response mediated by the first type of metabotropic glutamate receptor (I. metabotropic glutamate receptor) or metabotropic acetylcholine receptor depends on GC- Activation of C

GC-C knockout mice were used as experimental animals, and the midbrain dopamine neurons were detected by perforated patch clamp technique. The specific method is to rapidly separate the mouse brain in a low temperature environment, and use a vibrating slicer to cut a horizontal brain slice with a thickness of 300 μm, incubate in artificial cerebrospinal fluid, and select midbrain dopamine neurons to use nystatin which forms micropores on the cell membrane. Patch clamp recording. It was found that the dopaminergic neurons of GC-C knockout mice were not significantly different in morphology from wild-type mice, and the observed discharge frequency was also consistent with that of common dopamine cells, and in voltage patch clamp mode. Under the condition that the cells are hyperpolarized, the Η current of 30-200ρΑ can also be seen. The statistical results also showed that there was no significant difference in membrane resistance and enthalpy current between the dopaminergic neurons of the GC-C knockout mice and the dopamine neurons of the wild type mice (as shown in Fig. 9). The response of dopamine neurons in GC-C knockout mice to DHPG was recorded. After addition of G or UG, unlike wild type, the response of cells to DHPG was not significantly enhanced. The data of the group showed that after adding G or UG, the reaction intensity of dopamine neurons was basically the same as before the addition (as shown in Figure 10 AC). This is full The G/UG-enhanced dopaminergic cell-dependent DHPG-mediated response is dependent on GC-C activation.

 2. Detection of whether G/UG enhances the excitatory response mediated by the first type of metabotropic glutamate receptor (group I metabotropic glutamate receptor) or the acetylcholine receptor, depending on PKG

 Normal adult mice were used as experimental animals, and the midbrain dopamine neurons were detected by perforated patch clamp technique. (Specific method as described in Example 3), it was found that Rp-8-pCPT-cGMPS (an inhibitor of the protein kinase G regulatory subunit) was added [Butt, E., M. Eigenthaler, and HG Genieser, (Rp)-8-pCPT-cGMPS, a novel cGMP-dependent protein kinase inhibitor. Eur J Pharmacol, 1994. 269(2): 265-268.] Can eliminate the increasing effect of G/UG on DHPG response (as shown in Figure 10 DF). The same effect can also be modeled using KT5823, an inhibitor of selective functional subunits of protein kinase G. [References: Yu, YC, LH Cao, and XL Yang, Modulation by brain natriuretic peptide of GABA receptors on Rat retinal ON-type bipolar cells. J Neurosci, 2006. 26(2): 696-707. and Kwan, HY, Y. Huang, and X. Yao, Regulation of canonical transient receptor potential isoform 3 (TRPC3) channel by protein Kinase G. Proc Natl Acad Sci USA, 2004. 101(8): 2625-2630.] (shown in Figure 10 GI). In turn, we also used the widely used activator of protein kinase G 8-Br-cGMP [Mann, EA, et al., Mice lacking the guanylyl cyclase C receptor are resistant to STa-induced intestinal secretion. Biochem Biophys Res Commun , 1997. 239(2): 463-466.] » It was found that the nine cells recorded were observed to have an enhanced response to DHPG ten to twenty minutes after the addition of 8-Br-cGMP, which is related to G/UG. The effect is consistent (as shown in Figure 10 JL). The results of the assay demonstrate that protein kinase G mediates the potentiating effect of G/UG on DHPG responses.

 Example 4. Comparative study of behavioral defects of ADHD in GC-C knockout mice and humans

Animal behavior research methods include the following: 1. Long-term continuous maintenance and monitoring of animal daily activities: Devices that can continuously maintain and monitor animal daily activities (as shown in Figure 11) can provide fully enclosed, non-interfering for experimental animals. The living space can automatically control the supply of drinking water and food, conveniently set the light and dark rhythm in the enclosed space, and record its daily movement with an infrared camera. After the processing of the distance recording and analysis software, the observed animal can be calculated. Behavioral data such as the level of autonomous exercise, time and number of sleeps, and changes in cycle rhythm. 2. Market test: The site used for the open field test is a rectangular wooden box of 50*30 cm, 70 cm high, which contains litter and feed, and a water bottle is fixed on the wall. An infrared camera was placed at a height of about 1.5 m above the wooden box to record the mouse's autonomous movement. The camera was connected to a computer, and the data was collected and analyzed by software similar to that used for long-term monitoring. The room under test is always under an inverted 12-hour alternating light, maintaining good ventilation and shielding. The animals to be tested were pre-conditioned in the room for 4-7 days and then adapted to more than 4 hours in the wooden box. The daily dosing test was started one hour after the light was turned from dark to dark. 3. Odor adaptation test: The test site is an opaque cylinder with a height of 30 cm and a diameter of 30 cm. There is a litter and feed in the cylinder, and a water bottle on the wall. In the center of the bottom of the cylinder is a small hole with a diameter of about 2 cm. The cylinder frame is about 1 meter above the ground and has a bracket below it that just supports a 15 ml centrifuge tube head exposed from a small hole in the bottom of the tube. Above the cylinder is an infrared camera that records the mouse's odor exploration during the experiment. The odour diluted with mineral oil is contained in a 15 ml centrifuge tube and can be volatilized through a small hole drilled in the tube cover. The mice were placed in a cylinder to acclimate to the environment one day before the test. The test room has been in reverse for 12 hours Maintain good ventilation and shielding for the light. The test takes place in the first half of the dark period. The small tube containing amyl acetate was first taken out from the hole in the bottom of the tube and held for 2 minutes. The exploration of the mouse was taken and the odor was removed. Thereafter, the action was repeated every ten minutes for a total of four times, and the fifth time the test odor was changed to acetophenone (as shown in Figure 12). Note that proper dilution ensures that the vapor pressure of the latter odor is equal to the previous scent. After the video of the animal's odor is photographed, the time of the animal's exploration time, the number of explorations, and the interval of each exploration in the process of stimuli' stimuli are counted by manual timing. Exploring the odor is defined as the tip of the mouse's nose close to 1 cm of the orifice (as shown in Figure 13). 4, go / no behavior paradigm: There is a hole on the wall of the training box that only allows the mouse mouth to explore. There are infrared transceivers on both sides of the hole to determine whether the mouse is in the past. A metal faucet is connected to the sensing circuit directly below the outside of the hole. The circuit records the signal when the mouse fills the nozzle. The faucet is also connected to a water-filled container and is controlled by a solenoid valve. The bottom of the training box is a shock plate that is connected to a maximum of 60 volts DC power. There is a speaker outside the training box to provide signals of 3K Hz and 15K Hz. The speaker, shock plate and solenoid valve are controlled by software developed by our laboratory (as shown in Figure 14A). Adult mice were deprived of drinking water one day prior to training and then placed in a training box. Throughout this training they must learn to get drinking by distinguishing the sound signals they hear. The whole training process is formed by connecting the de-units and the non-de-units arranged in a pseudo-random manner. The two units appear equally many times (as shown in Figure 14B). Each unit begins with the mouse heading through the hole in the wall. After that, the sound is stimulated for 50-100 milliseconds and lasts for 1.2 seconds. 0.2 seconds after the start of the sound ^; start the action time window, and finally the window is closed with the sound (as shown in Figure 15). It is only possible to get water or electric shock when drowning in the action time window. In the de-unit, the mouse hears a 3K Hz sound, and then the water can be used as a reward. When the mouse is not heard, the sound of 15K Hz is heard, and then the water spout is punished by electric shock. After training about 1500 units in this way, most animals learn this paradigm. After learning, we can calculate the stopping time of the animal. This time is the time between the start of the sound in the unit and the stop of the drowning of the animal. After that, we increased the difficulty of training, so that the sound signal and the action time window started together, and continued to train the animals (as shown in phase 2 of Figure 16). After they learned again, we inserted a random delay of 0.2-2 seconds after the animal probe and then tested the animal (as shown in test in Figure 16). After the test is completed, we can calculate various indicators including correct response rate, abandon reaction rate, reaction time and so on.

 I. Detection of the level of autonomic activity in GC-C knockout mice

 Long-term monitoring of GC-C knockout mice and wild-type mice was carried out according to the above animal behavior study method of "long-term continuous maintenance and monitoring of animal daily activities". The results showed that after two days of adaptation in the test area, GC in the dark period The level of autonomic activity of -C knockout mice was more than twice that of wild mice, but there was no difference during the light period (as shown in Figure 17A). In the open field trials in which mice were new to the environment, GC-C knockout mice showed high activity levels only after they had been acclimated to the environment for more than 100 minutes (Figure 17B).

 2. Detection of odor adaptability of GC-C knockout mice

GC-C knockout mice and wild-type mice were tested for odor adaptation according to the animal behavior study method described above for "odor adaptation test". The results showed that the GC-C gene was compared with wild type mice. The time to knock out mice to explore odor was significantly longer than that of wild-type mice; GC-C knockout mice also showed significant loss of adaptability. This result indicates that the odor adaptability of GC-C knockout mice is impaired (as shown in the figure). 18)).

 Third, the detection of attention deficit in GC-C knockout mice

 In the experiment, GC-C knockout mice and wild-type mice were subjected to attention test experiments according to the above animal behavior research method of "go/no-behavior" to detect GC-C knockout mice. Attention deficit. We first deprive them of drinking water and then train them to lick water based on the sound signals they get. If you hear a 3KHz sound stimulus, then you can get a little water as a reward, and if you hear a 15KHz sound stimulus, then the drowning will get a mild electric shock and a period of refractory period as a punishment. (As shown in Figure 14). At the beginning of the training, after the sound stimulation started, the mice had a period of 200 milliseconds to judge the type of stimulation (as shown in Figure 15).

 Both wild-type and GC-C knockout mice were trained to respond correctly to this task, and their learning curves were similar (see Figure 19). Wild-type mice often start to drown after hearing the sound signal that can drink water, while GC-C knockout mice show a behavior that is always drowning, and they only get the signal that cannot be drenched. Stop drowning. Corresponding to this difference, their cessation response time is three times that of wild-type mice, indicating that their ability to suppress behavior corresponding to the stop signal is impaired, that is, they are more impulsive (as shown in Figure 20). After the 200 ms judgment time was removed, both wild-type mice and GC-C knockout mice were trained to switch their response to this change. At this point, they only start drowning after hearing a signal that can drown. However, the difficulty of the test was further improved by adding a random delay of up to two seconds before the start of the test and then retesting the animals (as shown in Figure 16). GC-C knockout mice showed significantly higher chances of abandoning the test before the sound signal, resulting in a significant decrease in their correct response rate, indicating that their ability to maintain attention was compromised (see Figure 21). ). The above experimental results fully demonstrate that GC-C knockout mice exhibit high activity levels, impulsivity, and poor attention to maintain attention. These behavioral phenotypes are very similar to the core features of attention deficit hyperactivity disorder [Sagvolden, T., et al., A dynamic developmental theory of attention-deficit/hyperactivity disorder (ADHD) predominantly hyperactive/impulsive and combined subtypes. Behav Brain Sci, 2005. 28(3): 397-419; discussion 419-68.].

 IV. Measurement of extracellular dopamine levels in GC-C knockout mice by in vivo microdialysis

 In order to measure extracellular dopamine levels in vivo microdialysis, the catheter was previously implanted into the brain of male GC-C knockout mice (15) or wild-type mice (n=12), with a specific position of pre-halogen forward. 0.9 mm, lateral 1.5 mm, 3 mm down from the brain plane, allowing the probe position to be finalized to the dorsal striatum. After the operation, the mice were kept alone for one week to recover. The probe used for intracranial microdialysis was CMA-7MD from CMA Microdialysis, with a membrane length of 1 mm and a cut-off size of 6 kilodaltons. After the probe was implanted into the brain, the artificial cerebrospinal fluid was continuously perfused at a rate of 0.5 microliters per minute, and its composition was 145 millimoles of sodium chloride, 2.7 millimoles of potassium chloride, 1.2 millimoles of calcium chloride, 1.0 millimoles of magnesium chloride, 0.45 mmol of sodium dihydrogen phosphate and 2.33 mmol of disodium hydrogen phosphate, pH = 7.4. After two hours of equilibration, the perfusate was collected every 30 minutes, and 2 μl of 0.2 mol of perchloric acid was added to the collection tube in advance. Four consecutive samples were classified as one tube and frozen at -80 degrees. At the end of microdialysis, the animals were injected with a lethal dose of pantobarbital, and the position of the probe was examined by coronal sectioning.

The determination of dopamine levels in microdialysis samples was performed using an electrochemical detector coupled to high performance liquid chromatography. The BASi LC-4C type detector was used in the experiment. The mobile phase composition is: 85 mmol citric acid, 100 Millimol of sodium acetate, 0.2 mmol of sodium EDTA, 2.5% methanol and 0.0025% di-n-butylamine. pH = 3.68. Melted samples and standards (50 microliters) were injected into the column (inner diameter 0.5 mm, length 10 cm) using a Shimadzu LC-10AT VP+ pump at 1.2 microliters per minute. The dopamine elution time in this system is approximately 6 minutes.

 In the experiment, extracellular dopamine levels in GC-C knockout mice were measured by in vivo microdialysis. The results showed that the background extracellular dopamine levels of GC-C knockout mice were significantly lower than those of wild-type mice (Figure 22).

 5. Can the treatment of human ADHD reduce the behavioral deficit of GC-C knockout mice? The symptoms of human ADHD can be low-dose rather than high-dose neurostimulators amphetamine and its derivatives. Treatment, these drugs can increase the extracellular dopamine concentration in the brain by increasing dopamine release and inhibiting its recovery [References: Giros, B., et al., Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature , 1996. 379 (6566): 606-612. and Spencer, T., et al., Efficacy of a mixed amphetamine salts compound in adults with attention-deficit/hyperactivity disorder. Arch Gen Psychiatry, 2001. 58(8): 775-782.]. This dose-dependent treatment of excessive activity levels is often used to determine the reliability of animal models of ADHD [References: Sagvolden, T., et al., A dynamic developmental theory of attention-deficit/hyperactivity disorder (ADHD) predominantly hyperactive/ Impulsive and combined subtypes. Behav Brain Sci, 2005. 28(3): 397-419; discussion 419-68. Hess Hess, EJ, KA Collins, and MC Wilson, Mouse model of hyperkinesis implicates SNAP-25 in behavioral regulation. J Neurosci, 1996. 16(9): 3104-31 1 1.].

 The effect of the neurostimulant amphetamine on the behavioral deficits of GC-C knockout mice was examined in the experiment. It was found that low doses of amphetamine (1 mg/kg body weight) can significantly reduce the level of autonomic activity of GC-C knockout mice in pre-adapted open field (as shown in Figure 23), while Wild type mice under conditions had no effect (as shown in Figure 24). In contrast, high doses of amphetamine (above 4 mg/kg body weight) significantly increased their activity levels in both wild-type and GC-C knockout mice (Figure 25). The efficacy of low-dose amphetamines in the treatment of excessive activity levels strongly suggests that GC-C knockout mice exhibit a behavioral phenotype similar to ADHD and support GC-C in regulating midbrain dopaminergic neuronal activity. effect.

 Since protein kinase G (PKG) mediates GC-C signaling, a PKG agonist was tested to see if it could correct behavioral defects in GC-C knockout mice. Consistent with previous data, injection of 8-Br-cGMP into the VTA/SNC region of GC-C knockout mice by bilateral intracranial administration did reduce the autonomy of GC-C knockout mice. Activity (as shown in Figure 26).

The intracranial administration method is as follows: The animal is anesthetized with discobarbital, and a bilateral long-term indwelling drug delivery catheter is implanted on the head, and the inner tube is positioned in the VTA/SNc region (the front side is 3 mm rearward, side) To 1.0 mm, 4.2 mm from the brain plane down). After a week of rest, a single GC-C knockout mouse was placed in a fresh test site, 8-Br-cGMP (0.5 μl per side, administered at a concentration of 3 mmol per liter, dissolved in ACSF) Or ACSF (as a control) was injected directly into VTA/SNc through a bilateral inner tube at a rate of 0.1 microliters per minute. Autonomous movement of the animals was started five minutes after the injection. At the end of each experiment, the animals were injected with a lethal dose of discobarbital, and the position of the probe was examined by coronal sectioning.

 The above experimental results show that the behavioral defects of GC-C knockout mice are very similar to those of human ADHD, and can be improved by the treatment of human ADHD.

Industrial application

 The present invention discloses for the first time that GC-C is specifically expressed on midbrain dopamine neurons; GC-C receptor activation can enhance the first type of metabotropic glutamate receptor (group I metabotropic glutamate receptor) And metabolic acetylcholine receptor-mediated excitatory responses. The inventors of the present invention found that the behavioral deficit of GC-C knockout mice is very similar to that of human ADHD and can be improved by the treatment of human ADHD. GC-C knockout mice were used as animal models of ADHD, and these mice were healthier than the previous typical SDHD animal model of spontaneously hypertensive rats (SHR). The GC-C knockout mouse model can further be used to screen for the prevention and/or treatment of diseases associated with midbrain dopamine neurons, particularly for the prevention and/or treatment of human ADHD. GC-C knockout mice provide a good opportunity for further study of the relationship between GC-C and midbrain dopamine neuron-associated diseases. More importantly, the experiments of the present invention demonstrate that the activity of the midbrain dopamine neuron can be selectively modulated by controlling the GC-C/PKG signaling pathway. Efforts to develop agonists or inhibitors acting on the GC-C/PKG signaling pathway will likely lead to the development of new therapeutic approaches to treat those mental disorders associated with the midbrain dopamine system, such as schizophrenia, attention deficit hyperactivity disorder , Parkinson's disease and drug addiction.

Claims

Rights request I. Application of a substance that activates guanylate cyclase C in the preparation of an agonistic reaction product that enhances midbrain dopamine neurons mediated by a first type of metabotropic glutamate receptor and/or a metabotropic acetylcholine receptor .  The use according to claim 1, characterized in that the substance which activates uridine cyclase C is guanosine or uridine.  3. Use of guanylate cyclase C knockout mice in screening for products for the prevention and/or treatment of diseases associated with the midbrain dopamine system.  4. The use according to claim 3, characterized in that the disease associated with the midbrain dopamine system is human attention deficit hyperactivity disorder, schizophrenia, Parkinson's disease or drug addiction.  5. An animal model of attention deficit hyperactivity disorder characterized in that: the animal model is a mammal in which the guanylate cyclase C gene has been knocked out.  6. A method of constructing an animal model of attention deficit hyperactivity disorder according to claim 5, comprising the steps of: knocking out the guanylate cyclase C gene in the mammal of interest, and the obtained guanylate cyclase C gene is knocked The animal to be removed is an animal model of attention deficit hyperactivity disorder.  7. The method according to claim 6, wherein: the method of knocking out a guanylate cyclase C gene in a mammal of interest is to encode the guanylate cyclase in the mammal of interest. C gene knockout or replacement.  8. A model according to claim 5 or a method according to claim 6 or 7, characterized in that the mammal is a mouse, a rat, a rabbit, a monkey, a pig or a chicken.  9. Use of an agonist or inhibitor of the GC-C/PKG signaling pathway for the preparation of a medicament for the prevention and/or treatment of a disease associated with a midbrain dopamine system.  10. The use according to claim 9, wherein: the agonist of the GC-C/PKG signaling pathway is an activator 8-Br-cGMP of protein kinase G; the GC-C/PKG signaling pathway The inhibitor is an inhibitor Rp-8-pCPT-cGMPS acting on a protein kinase G regulatory subunit or an inhibitor KT5823 acting on a functional subunit of protein kinase G; the disease associated with the midbrain dopamine system is more attention deficit Dyskine, schizophrenia, Parkinson's disease or drug addiction.  I I. The use of a substance for detecting the integrity of the guanylate cyclase C gene and the expression level of this protein for the preparation of a diagnostic or auxiliary diagnostic agent for attention deficit hyperactivity disorder.