Nicotinic Acetylcholine Receptor Antagonists / Blockers for Use in Increasing Dopamine

Abstract

The present invention relates to methods of increasing dopamine levels in a subject. The present invention also relates to the treatment of conditions or disorders involving reduced dopamine levels, and to medicaments for use in such treatment.

Description

[Technical field]

[0001] The present invention relates to treatments and methods for increasing dopamine levels in a subject, particularly in the central nervous system (CNS).The present invention also relates to the treatment of conditions or disorders involving reduced dopamine levels, and to drugs for use in such treatments. [Background technology]

[0002] Striatal cholinergic interneurons (ChIs), acting at nicotinic acetylcholine receptors (nAChRs) on dopamine axons, are thought to facilitate dopamine output. Synchronized activity in ChIs can enhance dopamine release (Cachope, R. et al. Selective activation of cholinergic interneurons enhances accumbal phasic dopamine release: setting the tone for reward processing. Cell Rep 2, 33-41, doi:10.1016 / j.celrep.2012.05.011 (2012) and Threlfell, S. et al. Striatal dopamine release is triggered by synchronized activity in cholinergic interneurons. Neuron 75, 58-64, doi:10.1016 / j.neuron.2012.04.038 (2012)), whereas pauses in ChIs enhance dopamine release during bursts of high-frequency dopamine neuron activity (Cragg, SJ Meaningful silences: how dopamine listens to the ACh pause. Trends Neurosci 29, 125-131, doi:10.1016 / j.tins.2006.01.003 (2006), and Rice, ME & Cragg, SJ Nicotine amplifies reward-related dopamine signals in striatum. Nat Neurosci 7, 583-584, doi:10.1038 / nn1244 (2004). However, an important unexplained observation is that when nAChRs are activated, dopamine release can be significantly reduced compared to when they are inactivated (Rice & Cragg, 2004), raising the question of whether ChIs can enhance or attenuate dopamine output.

[0003] Decreased dopamine levels have been associated with several conditions, including addiction, Alzheimer's disease, attention deficit hyperactivity disorder (ADHD), bipolar disorder, dopa-responsive dystonia and DRD plus, Huntington's disease, multiple sclerosis, obsessive-compulsive disorder (OCD), Parkinson's disease (including Parkinson's rest tremor), schizophrenia, seizures (i.e. repetitive involuntary movements or sounds) and Tourette's syndrome.

[0004] Conventional treatments such as levodopa for diseases such as Parkinson's disease are believed to result in a general increase in dopamine levels in the striatum, such as by release from inappropriate non-dopaminergic inputs. This can lead to serious side effects years later. Therefore, alternative therapeutic approaches are needed to reduce the risk of such side effects.

[0005] Parkinson's disease is the second most common neurodegenerative disorder. Core motor symptoms are caused by loss of midbrain dopamine (DA) neurons. Levodopa, a precursor of DA, provides the most effective symptomatic treatment by enhancing DA release from remaining neurons. However, chronic administration of levodopa at concentrations sufficient to achieve symptomatic relief produces a side effect that causes motor dysfunction, called levodopa-induced dyskinesia (LID), especially in late-stage PD patients or those receiving long-term levodopa treatment. Summary of the Invention

[0006] The present inventors have found that striatal cholinergic interneurons can potently inhibit dopamine release in the striatum after activating nicotinic acetylcholine receptors (nAChRs) on dopamine axons.It is expected that administration of nAChR antagonists / blockers can relieve the inhibition of dopamine release caused by striatal cholinergic interneurons, thereby providing effective treatment for conditions or disorders involving reduced dopamine levels.Therefore, the present invention targets the reduced dopamine levels seen in some conditions.

[0007] The present invention recognizes that treatments involving administration of nicotinic acetylcholine receptor (nAChR) antagonists / blockers, or pharma- ceutically acceptable salts thereof, result in increased dopamine levels in the CNS. Administration of nAChR antagonists / blockers itself targets inhibition of dopamine release caused by striatal cholinergic interneurons acting at nAChR.

[0008] As used herein, the term "nicotinic acetylcholine receptor antagonist / blocker" (also known as nicotine antagonist / blocker) refers to a compound that inhibits the action of acetylcholine (ACh) at the nicotinic acetylcholine receptor. The term "antagonist / blocker" includes competitive antagonists and non-competitive antagonists.

[0009] Therapeutic Uses and Applications In a first aspect of the present invention, there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in the treatment of a condition or disorder involving reduced dopamine levels.

[0010] In another aspect of the present invention, there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in the treatment of a condition or disorder involving reduced dopamine levels in the central nervous system (CNS).

[0011] In another aspect of the present invention, there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in the treatment of a condition or disorder involving reduced dopamine levels in the brain.

[0012] In another aspect of the invention, there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in the treatment of a condition or disorder involving reduced dopamine levels in the striatum.

[0013] In another aspect of the invention, there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in the treatment of a condition or disorder involving decreased dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0014] In another aspect of the present invention, there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in the treatment of a condition or disorder involving reduced dopamine levels in the dorsal striatum (including the caudate and putamen).

[0015] In a further aspect of the invention there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in increasing dopamine levels in the CNS.

[0016] In another aspect of the invention there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in increasing dopamine levels in the brain.

[0017] In another aspect of the invention there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in increasing dopamine levels in the striatum.

[0018] In another aspect of the invention, there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in increasing dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0019] In another aspect of the present invention, there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in increasing dopamine levels in the dorsal striatum (including the caudate and putamen).

[0020] In another aspect of the invention, there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in increasing dopamine levels in the CNS of a subject in the treatment of a condition or disorder involving decreased dopamine levels in the CNS.

[0021] In another aspect of the invention, there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in increasing dopamine levels in the brain of a subject in the treatment of a condition or disorder involving decreased dopamine levels in the brain.

[0022] In another aspect of the invention, there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in increasing dopamine levels in the striatum of a subject in the treatment of a condition or disorder involving decreased dopamine levels in the striatum.

[0023] In another aspect of the invention, there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in increasing dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra of a subject in the treatment of a condition or disorder involving decreased dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0024] In another aspect of the invention, there is provided a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for use in increasing dopamine levels in the dorsal striatum (including the caudate nucleus and putamen) of a subject in the treatment of a condition or disorder involving decreased dopamine levels in the dorsal striatum (including the caudate nucleus and putamen).

[0025] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in treating a subject suffering from a disorder associated with reduced dopamine levels in the CNS.

[0026] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in treating a subject suffering from a disorder associated with reduced dopamine levels in the brain.

[0027] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in treating a subject suffering from a disorder associated with reduced dopamine levels in the striatum.

[0028] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in treating a subject suffering from a disorder associated with decreased dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0029] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in treating a subject suffering from a disorder associated with reduced dopamine levels in the dorsal striatum (including the caudate and putamen).

[0030] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in increasing dopamine levels in the CNS.

[0031] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in increasing dopamine levels in the brain.

[0032] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in increasing dopamine levels in the striatum.

[0033] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in increasing dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0034] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in increasing dopamine levels in the dorsal striatum (including the caudate and putamen).

[0035] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in increasing dopamine levels in the CNS in the treatment of a subject suffering from a disorder associated with decreased dopamine levels in the CNS.

[0036] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in increasing dopamine levels in the brain in the treatment of a subject suffering from a disorder associated with decreased dopamine levels in the brain.

[0037] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in increasing dopamine levels in the CNS in the treatment of a subject suffering from a disorder associated with decreased dopamine levels in the CNS.

[0038] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in increasing dopamine levels in the striatum in the treatment of a subject suffering from a disorder associated with decreased dopamine levels in the striatum.

[0039] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in increasing dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra in the treatment of a subject suffering from a disorder associated with decreased dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0040] In another aspect of the invention, there is provided the use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in increasing dopamine levels in the dorsal striatum (including the caudate nucleus and putamen) in the treatment of a subject suffering from a disorder associated with decreased dopamine levels in the dorsal striatum (including the caudate nucleus and putamen).

[0041] In another aspect of the invention, there is provided the use of a nAChR antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for the manufacture of a medicament for increasing dopamine levels in the CNS in the treatment of a condition or disorder involving decreased dopamine levels in the CNS.

[0042] In another aspect of the invention, there is provided the use of a nAChR antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for the manufacture of a medicament for increasing dopamine levels in the brain in the treatment of a condition or disorder involving decreased dopamine levels in the brain.

[0043] In another aspect of the invention, there is provided the use of a nAChR antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for the manufacture of a medicament for increasing dopamine levels in the striatum in the treatment of a condition or disorder involving decreased dopamine levels in the striatum.

[0044] In another aspect of the invention, there is provided the use of a nAChR antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for the manufacture of a medicament for increasing dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra in the treatment of a condition or disorder involving decreased dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0045] In another aspect of the present invention, there is provided the use of an nAChR antagonist / blocker, or a pharma- ceutically acceptable salt thereof, for the manufacture of a medicament for increasing dopamine levels in the dorsal striatum (including the caudate nucleus and putamen) in the treatment of a condition or disorder involving decreased dopamine levels in the dorsal striatum (including the caudate nucleus and putamen).

[0046] Treatment In another embodiment of the present invention, there is provided a method of treating a condition or disorder involving reduced dopamine levels in the CNS, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof.

[0047] In another aspect of the present invention, there is provided a method of treating a condition or disorder involving reduced dopamine levels in the brain, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof.

[0048] In another aspect of the present invention, there is provided a method of treating a condition or disorder involving reduced dopamine levels in the striatum, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof.

[0049] In another embodiment of the present invention, there is provided a method of treating a condition or disorder involving decreased dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof.

[0050] In another embodiment of the present invention, there is provided a method of treating a condition or disorder involving reduced dopamine levels in the dorsal striatum (including the caudate and putamen), the method comprising administering to a subject in need of treatment a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof.

[0051] In another aspect of the present invention, there is provided a method of increasing dopamine levels in the CNS of a subject, the method comprising administering to the subject a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof.

[0052] In another aspect of the present invention, there is provided a method for increasing dopamine levels in the brain of a subject, the method comprising administering to the subject a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof.

[0053] In another aspect of the present invention, there is provided a method for increasing dopamine levels in the striatum of a subject, the method comprising administering to the subject a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof.

[0054] In another embodiment of the present invention, there is provided a method for increasing dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra of a subject, the method comprising administering to the subject a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof.

[0055] In another aspect of the present invention, there is provided a method for increasing dopamine levels in the dorsal striatum (including the caudate and putamen) of a subject, the method comprising administering to the subject a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof.

[0056] In another embodiment of the present invention, a method is provided for treating a condition or disorder involving decreased dopamine levels in the CNS, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, wherein administration of the nicotinic acetylcholine receptor antagonist / blocker increases dopamine levels in the CNS.

[0057] In another aspect of the present invention, there is provided a method for treating a condition or disorder involving decreased dopamine levels in the brain, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, wherein administration of the nicotinic acetylcholine receptor antagonist increases dopamine levels in the brain.

[0058] In another aspect of the present invention, a method is provided for treating a condition or disorder involving decreased dopamine levels in the striatum, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, wherein administration of the nicotinic acetylcholine receptor antagonist / blocker increases dopamine levels in the striatum.

[0059] In another embodiment of the present invention, there is provided a method of treating a condition or disorder involving decreased dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, wherein administration of the nicotinic acetylcholine receptor antagonist / blocker increases dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0060] In another aspect of the present invention, there is provided a method for treating a condition or disorder involving decreased dopamine levels in the dorsal striatum (including the caudate nucleus and putamen), the method comprising administering to a subject in need of treatment a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker, or a pharma- ceutically acceptable salt thereof, wherein administration of the nicotinic acetylcholine receptor antagonist / blocker increases dopamine levels in the dorsal striatum (including the caudate nucleus and putamen).

[0061] Condition being treated Some conditions are associated with decreased dopamine levels.The present inventors show that administering nAChR antagonist / blocker acts to increase dopamine levels in CNS (including, for example, brain, striatum) to treat such conditions.Suitably, administering nAChR antagonist / blocker acts to increase dopamine levels in dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.Most suitably, administering nAChR antagonist / blocker acts to increase dopamine levels in dorsal striatum (including caudate and putamen).

[0062] Suitably, the condition or disorder is selected from addiction (including overeating, nicotine addiction and alcohol addiction), Alzheimer's disease, attention deficit hyperactivity disorder (ADHD), bipolar disorder, dopa-responsive dystonia and DRD plus, Huntington's disease, multiple sclerosis, obsessive-compulsive disorder (OCD), Parkinson's disease (including Parkinson's resting tremor), schizophrenia, convulsions (i.e. repetitive involuntary movements or vocalizations), Tourette's syndrome, depression and reward deficiency syndrome.More suitably, the condition or disorder is selected from Parkinson's disease or ADHD.Most suitably, the condition or disorder is Parkinson's disease.

[0063] In some situations, a subject may experience side effects as a result of taking a dopamine precursor (e.g., levodopa) or a dopamine agonist to treat a disorder involving reduced dopamine levels in the CNS. Suitably, the subject suffers from levodopa-induced dyskinesia.

[0064] Suitably, the dopamine levels in the CNS, brain and / or striatum of the subject are decreased. More suitably, the dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra of the subject are decreased. Most suitably, the dopamine levels in the dorsal striatum (including the caudate nucleus and putamen) of the subject are decreased.

[0065] Suitably, administration of nicotinic acetylcholine receptor antagonist / blocker promotes dopamine release in CNS, brain and / or striatum.More suitably, administration of nicotinic acetylcholine receptor antagonist / blocker promotes dopamine release in dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.Most suitably, administration of nicotinic acetylcholine receptor antagonist / blocker promotes dopamine release in dorsal striatum (including caudate and putamen).

[0066] nAChR antagonists / blockers Suitably, the nAChR antagonist / blocker is selected from the group consisting of dihydro-β-erythroidine (DHβE), mecamylamine, bPiDDP (1,1'-(1,12-dodecanediyl)bis[3-methylpyridinium] dibromide), methyllycaconitine, MG 624 (N,N,N-triethyl-2-[4-(2-phenylethenyl)phenoxy]ethanaminium iodide), SR 16584 (1,3-dihydro-1-(3-exo)-9-methyl-9-azabicyclo[3.3.1]non-3-yl]-2H-indol-2-one), catestatin, chlorisondamine diiodide, 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), chlorisondamine, α-conotoxin (e.g. ACV 1, AuIB, EI, ImI, MII, PIA, PnIA), A 85380 (3-[(2S)-2-azetidinylmethoxy]-pyridine), ABT 089 (2-methyl-3-[(2S)-pyrrolidinylmethoxy]pyridine), ABT 594 ((R)-5-(azetidin-2-ylmethoxy)-2-chloropyridine), Dianiline ((5aS,8S,10aR)-5a,6,9,10-tetrahydro-7H,11H-8,10a-methanopyrido[2',3':5,6]pyrano[2,3-d]azepine), RJR 2403 ((E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine), TC 2559 (4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine), varenicline (7,8,9,10-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine), hexamethonium bromide, tubocurarine chloride, alpha-bungarotoxin, COG 133, D-amphetamine nitrate, PAMP-20, or a pharma- ceutically acceptable salt or derivative thereof.

[0067] Suitably, the nAChR antagonist / blocker is selected from the group consisting of dihydro-β-erythroidine (DHβE), mecamylamine, bPiDDP (1,1'-(1,12-dodecanediyl)bis[3-methylpyridinium] dibromide), methyllycaconitine, MG 624 (N,N,N-triethyl-2-[4-(2-phenylethenyl)phenoxy]ethanaminium iodide), SR 16584 (1,3-dihydro-1-(3-exo)-9-methyl-9-azabicyclo[3.3.1]non-3-yl]-2H-indol-2-one), catestatin, chlorisondamine diiodide, 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), chlorisondamine, α-conotoxin (e.g. ACV 1, AuIB, EI, ImI, MII, PIA, PnIA), A 85380 (3-[(2S)-2-azetidinylmethoxy]-pyridine), ABT 089 (2-methyl-3-[(2S)-pyrrolidinylmethoxy]pyridine), ABT 594 ((R)-5-(azetidin-2-ylmethoxy)-2-chloropyridine), Dianiline ((5aS,8S,10aR)-5a,6,9,10-tetrahydro-7H,11H-8,10a-methanopyrido[2',3':5,6]pyrano[2,3-d]azepine), RJR 2403 ((E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine), TC 2559 (4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine), varenicline (7,8,9,10-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine), or a pharma- ceutically acceptable salt or derivative thereof.

[0068] More suitably, the nicotinic acetylcholine receptor antagonist / blocker is selected from dihydro-β-erythroidine (DHβE), mecamylamine, bPiDDP, methyllycaconitine, MG 624, SR 16584, catestatin, chlorisondamine diiodide, 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), chlorisondamine and α-conotoxins (e.g. ACV 1, AuIB, EI, ImI, MII, PIA, PnIA), or a pharma- ceutically acceptable salt or derivative thereof.

[0069] Most suitably, the nicotinic acetylcholine receptor antagonist / blocker is selected from dihydro-β-erythroidine (DHβE), mecamylamine, chlorisondamine diiodide, 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), chlorisondamine, or a pharma- ceutically acceptable salt or derivative thereof.

[0070] The nAChR antagonist / blocker may be a derivative of mecamylamine, such as one or more of the compounds described in WO2013 / 026852, which is incorporated by reference in its entirety. The nAChR antagonist / blocker may be a derivative of dihydro-β-erythroidine.

[0071] Suitably, nAChR antagonist / blocker is an antagonist for α4β2 nAChR receptor.More suitably, nAChR antagonist / blocker is selective for α4β2 receptor.Antagonist / blocker selective for α4β2 receptor is believed to reduce the risk of side effects in treated subjects.

[0072] Certain α4β2 ligands with antagonistic activity are described in US Patent No. 9,029,557 and US Patent No. 5,691,365 (the entirety of which is incorporated by reference).Other known antagonists / blockers include lophotoxin, neosurugatoxin and erysodine (J. Med. Chem. (1997) 40: 4169-4194).Those skilled in the art are aware of such nAChR antagonists / blockers, for example, those described in Nicotinic ACh Receptors Scientific Review, Tocris Scientific Review Series (Wonnacott, Tocris Scientific Review Series, 2014, https: / / www.tocris.com / literature / scientific-reviews).

[0073] Further examples of nAChR antagonists / blockers selective for the α4β2 receptor include A 85380 dihydrochloride (3-[(2S)-2-azetidinylmethoxy]-pyridine dihydrochloride), ABT 089 (2-methyl-3-[(2S)-pyrrolidinylmethoxy]pyridine), ABT 594 ((R)-5-(azetidin-2-ylmethoxy)-2-chloropyridine), dianiline ((5aS,8S,10aR)-5a,6,9,10-tetrahydro-7H,11H-8,10a-methanopyrido[2',3':5,6]pyrano[2,3-d]azepine), RJR 2403 ((E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine), TC 2559 (4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine) and varenicline (7,8,9,10-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine tartrate), or a pharma- ceutical acceptable salt or derivative thereof.

[0074] Pharmaceutical Compositions and Routes of Administration Effective amounts of nAChR antagonists / blockers may be administered by a variety of modes of administration including, for example, rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenous, intrathecal, intracranial, intraperitoneal, intraventricular, parenteral, intramuscular, subcutaneous, oral, topical, intranasal, as an inhalant, or by an impregnated or coated device such as a stent.

[0075] Suitably, the nAChR antagonists / blockers are administered orally, intranasally, intraperitoneally, intracerebroventricularly, intracranially, intrathecally, subcutaneously or intravenously.

[0076] Suitably, the nAChR antagonist / blocker is capable of crossing the blood-brain barrier. The nAChR antagonist / blocker that is capable of crossing the blood-brain barrier may be administered by a number of routes as described herein.

[0077] Suitably, the nAChR antagonist / blocker is administered directly to the CNS, which is believed to reduce the risk of peripheral side effects associated with administration of the nAChR antagonist / blocker.

[0078] Suitably, the nAChR antagonist / blocker is administered by local injection into the CNS or by direct injection into the CNS. Administration into the CNS can be achieved by intracranial injection, such as intrastriatal injection, intraputamen injection, intracaudate injection, intracerebroventricular injection, intraparenchymal injection and intracortical injection.

[0079] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a nAChR antagonist / blocker as defined above, or a pharma- ceutically acceptable salt thereof, and one or more pharma- ceutically acceptable excipients.

[0080] Suitably, the pharmaceutical composition further comprises one or more further pharma- ceutical active agents, as described herein.

[0081] Suitably, the pharmaceutical compositions described herein are for use in the treatment of a condition or disorder as described herein.

[0082] The compositions of the invention may be in a form suitable for oral administration (e.g., as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), topical administration (e.g., as creams, ointments, gels, aqueous or oily solutions or suspensions), inhalation administration (e.g., as finely divided powders or liquid aerosols), insufflation administration (e.g., as finely divided powders) or parenteral administration (e.g., as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular administration, or as a suppository for rectal administration).The compositions of the invention may be in a form suitable for direct administration to the CNS (e.g., as a sterile aqueous or oily solution for local infusion or direct injection into the CNS).

[0083] The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical additives well known in the art. Thus, compositions intended for oral use may contain, for example, one or more coloring agents, sweeteners, flavoring agents and / or preservatives.

[0084] An effective amount of a nAChR antagonist / blocker for use in treatment in an amount sufficient to treat or prevent a condition involving reduced dopamine levels in the CNS as referred to herein slows its progression and / or alleviates the symptoms associated with the condition.

[0085] The amount of active ingredient that is combined with one or more additives to produce a single dosage form will necessarily vary depending on the individual being treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, 0.5 mg to 0.5 g of active material (more appropriately, 0.5 to 100 mg, e.g., 1 to 30 mg), combined with an appropriate and convenient amount of an additive, which may vary from about 5 to about 98% by weight of the total composition.

[0086] The dose of nAChR antagonists / blockers for therapeutic or prophylactic purposes will of course vary according to the nature and severity of the condition, the age and sex of the animal or patient, and the route of administration, in accordance with well-known principles of medicine.

[0087] When using nAChR antagonists / blockers for therapeutic or prophylactic purposes, a daily dose ranging from, for example, 0.1 to 75 mg / kg body weight is generally administered, in divided doses as necessary. Generally, when using parenteral routes, lower doses are administered. Thus, for example, for intravenous or intraperitoneal administration, doses ranging from, for example, 0.1 to 30 mg / kg body weight are generally used. Similarly, for inhalation administration, doses ranging from, for example, 0.05 to 25 mg / kg body weight are used. Oral administration may also be appropriate, particularly in tablet form. Typically, a unit dosage form contains about 0.5 mg to 0.5 g of nAChR antagonists / blockers.

[0088] combination The nicotinic acetylcholine receptor antagonist / blocker treatment defined above can be applied as a single treatment, or can include, in addition to the nicotinic acetylcholine receptor antagonist / blocker, other drugs that are conventionally used to treat related conditions.Thus, the nicotinic acetylcholine receptor antagonist / blocker or pharmaceutical composition can be administered in combination with one or more additional medicaments.

[0089] Such combination treatment may be achieved by the simultaneous, sequential or separate administration of the individual components of the treatment. Such combination products utilize the nicotinic acetylcholine receptor antagonist / blocker within its approved dosage range as described above and the other pharmacologic active agent within its approved dosage range.

[0090] In a further aspect of the present invention, there is provided a nicotinic acetylcholine receptor antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in combination with another pharmaceutical agent for use in the treatment of a condition or disorder involving reduced dopamine levels in the CNS. The condition or disorder may be any of those defined herein.

[0091] Suitably, the further medicament active substance is a substance that increases the dopamine level in CNS by a mechanism of action different from that of nicotinic acetylcholine receptor antagonist / blocker.Examples of such substances include dopamine precursors (e.g. levodopa, cocalerdopa, coveneldopa), dopamine agonists (e.g. pramipexole, ropinirole, apomorphine, rotigine), MAO-B inhibitors (e.g. rasagiline, selegiline, safinamide), COMT inhibitors (e.g. entacapone, opicapone), amantadine, anticholinergics (e.g. procyclidine, trihexyphenidyl) or Ritalin.

[0092] In a further embodiment of the present invention, there is provided a nicotinic acetylcholine receptor antagonist / blocker, or its pharmaceutically acceptable salt, for use in the treatment of Parkinson's disease, in combination with a dopamine precursor (e.g. levodopa, cocalerdopa, coveneldopa), a dopamine agonist (e.g. pramipexole, ropinirole, apomorphine, rotigine), an MAO-B inhibitor (e.g. rasagiline, selegiline, safinamide), a COMT inhibitor (e.g. entacapone, opicapone), amantadine or an anticholinergic drug (e.g. procyclidine, trihexyphenidyl).Suitably, the nicotinic acetylcholine receptor antagonist / blocker, or its pharmaceutically acceptable salt, for use in the treatment of Parkinson's disease, in combination with a dopamine precursor (e.g. levodopa, cocalerdopa, coveneldopa).

[0093] In this specification, when the term "combination" is used, it is understood to refer to simultaneous, separate or sequential administration. In one embodiment of the present invention, "combination" refers to simultaneous administration. In another embodiment of the present invention, "combination" refers to separate administration. In a further embodiment of the present invention, "combination" refers to sequential administration. When administration is sequential or separate, the delay in administration of the second component should not be such that the beneficial effect of the combination is lost.

[0094] According to a further aspect of the present invention, there is provided a pharmaceutical composition comprising a nicotinic acetylcholine receptor antagonist / blocker, or a pharma- ceutically acceptable salt thereof, in combination with a substance that increases dopamine levels in the CNS by a mechanism of action different from that of the nicotinic acetylcholine receptor antagonist / blocker, and a pharma- ceutically acceptable diluent or carrier.

[0095] In another embodiment, the present invention relates to a therapeutic combination comprising a nicotinic acetylcholine receptor antagonist / blocker and an additional agent that increases dopamine levels in the CNS by a mechanism of action different from the nicotinic acetylcholine receptor antagonist / blocker. [Brief description of the drawings]

[0096] Embodiments of the present invention will now be further described with reference to the accompanying drawings, in which: [Figure 1] We show that the activity of ChIs inhibits the subsequent DA release evoked by electrical stimulation. [Figure 1a] Schematic of the stimulus configuration for panels c–j. Blue light stimulation of ChR2-eYFP-expressing ChIs (Lstim), focal electrical stimulation in striatal slices (Estim) of ChAT-Cre:Ai32 mice. [Figure 1b] ChR2-eYP expression in choline acetyltransferase (ChAT)-immunoreactive striatal neurons. Scale bar, 40 μm. [Figure 1c.1i]Top shows average transients (± SEM) from a representative experiment of [DA]o evoked by single pulses of Lstim (light grey line, left), or Estim (light grey line, left), or Estim (black line, right), or combined responses to Lstim and Estim pulse pairs (purple) at ISIs of 8–200 milliseconds (ms) in the DLS (c) and NAcc (i). Dark dashed lines indicate [DA]o resulting from paired Estim after subtraction of [DA]o due to Lstim. Bottom shows average transients (± SEM) of [DA]o evoked by single or paired electrical pulses (dark grey lines) in the presence of DHβE (1 μM) in the DLS (c) and NAcc (i). Light dashed lines indicate [DA]o resulting from paired Estim after subtraction of [DA]o due to single Estim. [Figure 1d.1j] Mean peak [DA]o evoked by paired Estims normalized to [DA]o evoked by single Estims over ISIs in the DLS (d, N = 5 animals) and NAcc (j, N = 5 animals) (± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Fisher's LSD post-hoc test. [Figure 1e.1k] Mean transients (±SEM) from a representative experiment of [DA]o evoked by a subthreshold light pulse (Lstim), a single Estim, or an Lstim and Estim pair at ISIs of 25–100 ms in the DLS (e) or NAcc (k). [Fig. 1f.1l] Mean peak [DA]o evoked by dual stimulation normalized to [DA]o evoked by single Estim versus ISI in DLS (f, N = 5 animals) and NAcc (l, N = 5 animals) (± SEM). *P<0.05, **P<0.01, ***P<0.001, one-sample t-test versus single electrical stimulation. [Diagram 2] We show that the activity of ChIs inhibits subsequent DA release induced by optogenetic stimulation. [Figure 2a]Schematic of the stimulus configuration for panels m-t. Local electrical stimulation in striatal slices (Estim) and blue light stimulation of ChR2-eYFP-expressing DA axons (Lstim). [Figure 2b] Exemplary ChR2-eYFP expression in midbrain DA neurons co-immunoreactivity to DAT in DAT-Cre mice after VTA injection. Scale bar, 400 μm. [Fig. 2c.2g] Average transients from a representative experiment of [DA]o evoked by single pulses of Estim (gray lines, left) or Lstim (light gray lines, right), or Estim and Lstim pulse pairs (dark gray), at ISIs between 8 and 200 ms in the DLS (c) and NAcc (g) when nAChRs are allowed to activate (no DhβE) (top) or when nAChRs are inhibited (DhβE present) (bottom) in DAT-Cre mice. The dark dashed lines show [DA]o due to paired Estim after subtraction of [DA]o due to Estim without DhβE, and the light dashed lines show [DA]o due to paired Lstim in the presence of DhβE. [Figure 2d.2h] Mean peak [DA]o evoked by paired Lstim normalized to [DA]o evoked by single Lstim over ISI in DLS (d, N = 5 animals) and NAcc (h, N = 5 animals) (± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Fisher's LSD post-hoc test. [Figure 2e.2i] Average transients (±SEM) from a representative experiment of [DA]o evoked by a single maximal intensity Estim (gray line, left, top), paired Estim (light gray line, top), or single low intensity Estim (Estim50, light gray line, bottom), paired maximal intensity Estim (dark gray, bottom) at ISIs of 25–100 ms in the DLS (e) and NAcc (i) in wild-type animals. Thin (bottom) and dark (top) dashed lines indicate [DA]o resulting from paired Estim after subtraction of [DA]o due to Estim 1p. [Figure 2f.2j]Mean peak [DA]o (±SEM) evoked by paired stimuli versus ISI in the DLS (F, N=5 animals) and NAcc (J, N=5 animals). DhβE was present in e, f, i, and j. **P<0.01, ***P<0.001, two-way ANOVA with Fisher's LSD post-hoc test. [Diagram 3] We show that ChI and nAChR activation inhibits repetitive axonal depolarization and calcium accumulation in DA axons, resulting in apparent nAChR desensitization over longer intervals. [Figure 3a] Images of the VTA and SNc from DAT-Cre:Ai95D mice showing GCaMP6f-eGFP expression (top) in TH-immunopositive neurons (middle). [Figure 3b] Diagram of stimulation placement in biological tissue. [Figure 3c] Examples of Ca2+ imaging responses (change in GCaMP6f fluorescence, ΔF / F) (mean ± SEM from two replicates) in populations of DA axons imaged with DLS in response to single or trains of electrical pulses (4p) at 100 Hz in control conditions (left) and in the presence of DHβE (1 μM) (right). [Figure 3d] Mean peak values ​​(±SEM) for GCaMP6fΔF / F versus pulse number. Data are normalized to values ​​for one pulse (N=5 animals). ***P<0.0001, paired t-test for [DA]o from 4 pulses of stimulation. [Figure 3e] Images of the VTA and SNc from DAT-Cre mice with ASAP3 expression (top) and TH-positive neurons (middle). [Figure 3f] Diagram of stimulation placement in biological tissue. [Figure 3g]Average voltage sensor transients (change in ASAP3 fluorescence, -ΔF / F, mean ± SEM) in striatal dopamine axons to single or four electrical pulses at 50 Hz before and after blocking nAChRs with DHβE. Scale bar ordinate applies to 1p data only. For four-pulse data, the peak value seen after the first pulse of stimulation is scaled to match the peak of the transient seen for a single pulse (n=7 recordings in N=4 animals). [Figure 3h] Average response over time after successive pulses in pre- and post-DHβE pulse trains (gray in G). [Fig.3i.3k] Mean transients (±SEM) from a representative experiment of [DA]o evoked by single pulses of Lstim (left, top) or Estim (left, bottom), or Lstim plus either 1 or 3 Estim pulse pairs (labeled), at ISIs of 25-400 ms spanning activation, desensitization, and resensitization in the DLS (i) and NAcc (k) in the striatum of ChAT-Cre:Ai32 mice. Light and dark dashed lines indicate [DA]o resulting from paired stimuli after Lstim subtraction. [Fig.3j.3l] Top panel shows mean peak [DA]o (±SEM) evoked by paired Estims for 1p (circles) and 3p (squares) normalized to single Estim-evoked [DA]o versus ISI for DLS (j, N=5 animals) and NAcc (l, N=5 animals). Bottom panel shows difference between Estim-evoked [DA]o for 3p and 1p. *P<0.05, **P<0.01, one-way ANOVA with post-hoc Tukey test. [Figure 4] We show that nAChR antagonism in the DLS in vivo promotes DA release, conditioned place preference, and ameliorates motor deficits in a mouse model of PD. [Figure 4a] Schematic of the placement of recording and stimulating electrodes in DLS. [Figure 4b]Color plots and representative line plots of voltammetric DA current vs. time oxidation current (from the dashed series at approximately +0.7 V) and corresponding DA cyclic voltammograms (from the black dotted time point) before and after mecamylamine (ip). [Figure 4c] DA signals (mean + SEM) evoked by 1 or 10 pulses at 100 Hz before (black) and after (red) mecamylamine (2 mg / kg ip). [Figure 4d] Mean peak DA currents (±SEM). **P<0.01, Sidak's post hoc multiple comparison test (N=4 animals). [Figure 4h] Schematic diagram of the bilateral cannula system for localized injection into the dorsal striatum, and an exemplary semi-thin section with DAPI staining. [Figure 4i] Conditioning paradigm. Mice were conditioned four times (20 min per session) over two days following local injection of mecamylamine (10 μg / side, right chamber, grey) or saline (0.5 μl over 1 min (min), left chamber, black), whereas a control group received saline for both chambers. [Figure 4j] Representative chase traces on the day after conditioning. [Figure 4k] Preference for the right chamber after conditioning. *P<0.05, paired t-test, N=5 animals per test. [Figure 4l] Total distance traveled and movement speed in the open field test (N=5 animals). [Figure 4m] Schematic diagram of TH staining after unilateral 6-OHDA injury. [Figure 4n] Cylinder test paradigm. Mice were tested before and after 6-OHDA injection on the day of injury, and before and after mecamylamine infusion through the same cannula on the test day. [Figure 4o] Striatal mecamylamine infusion reduces the deficit in contralateral forelimb contact seen after injury. *P<0.05, **P<0.01, paired t-test, N=6 animals per test. [Diagram 5]We show that ChI-induced inhibition governs striatal DA release in a computational model. [Figure 5a] Values ​​used in the model for the strength of ChI-induced inhibition of DA release (dark grey) and normalized levels of apparent nAChR desensitization (light grey) versus time after ChI activation in the DLS (top) and NAcc (bottom). [Figure 5b] When the firing rate of DA neurons (labeled) is constant, a brief decrease (solid line) or increase (dotted line) in ChI activity (labeled) can increase or decrease [DA]o in the DLS (labeled) and NAcc (labeled), respectively. [Figure 5c] The top row shows inputs of polyphasic ChI responses with (left) or without (right) an initial excitation phase (labeled, light grey) and DA neuron burst activity (labeled, black)16 to predict striatal DA release in the DLS and NAcc (bottom line) when tonic levels of ChI-induced inhibition of DA release were set to 0% (labeled, upper middle line), 50% (labeled, lower middle line), and 100% (labeled, bottom line). [DA]o is normalized to that seen with zero tonic ChI inhibition (labeled, no ChI effect). [Figure 5d] Summary of peak [DA]o (open circles) and area under the peak [DA]o curve (closed circles) in the DLS (gray) and NAcc (black) with (left) and without (right) early excitation in ChI polyphasic activity when ChI-evoked inhibition was set to 0%, 50%, and 100% in ChI tonic activity. [Figure 5e] Peak [DA]o (left) and ratio of area under the [DA]o curve (right) release for ChIs without and with early excitation. [Figure 5f] Schematic showing that activation of ChIs and nAChRs on DA axons limits action potential propagation. [Figure 6] Light stimulation (Lstim0) intensity is shown to be subthreshold for eliciting detectable DA release. [Figure 6a]The intensity of Lstim0 used to stimulate ChIs in ChAT-Cre:Ai32 mice in Figure 1 was significantly lower than Lstim in both the DLS (left side, dark grey) and NAcc (right side, grey). N = 5 animals. ***P < 0.001, one-sample t test. [Figure 6b] Schematic of the stimulus configuration for the data in c. Blue light stimulation (Lstim) of ChR2-eYFP-expressing ChIs in striatal slices from ChAT-Cre:Ai32 mice, followed by local electrical stimulation (Estim). [Figure 6c] Mean transients (±SEM) from a representative experiment in DLS showing [DA]o evoked by either a subthreshold light pulse (Lstim0), a single Estim, or a pair of Lstim and Estim at an ISI of 25 ms when GABAA and GABAB receptors are inhibited with bicuculline (10 μM) and CGP 55845 hydrochloride (2 μM), respectively. Mean peak [DA]o evoked by dual stimulation normalized to [DA]o evoked by a single Estim when GABA receptors are inhibited (n=5 recordings, N=3 animals). ***P<0.001, one-sample t-test against Estim. [Figure 7] We show that expression of ASAP3 in DA neurons and dynamic DA release responses following ChI light activation (100–400 ms interval) are sustained in DLS in the presence of antagonists for either muscarinic or D2 receptors. [Figure 7a] Expression of ASAP3-GFP after virus injection in DA neurons in the midbrain co-labeled for TH immunoreactivity. [Figure 7b] Co-expression of ASAP3 and TH in dopamine axons in the DLS. CC: corpus callosum (scale bar 400 μm). [Figure 7c] Schematic of the stimulation configuration used in d and e. Blue light stimulation (Lstim) of ChR2-eYFP-expressing ChIs in striatal slices from ChAT-Cre:Ai32 mice, followed by local electrical stimulation (Estim). [Figure 7d.7e]Left: Mean peak [DA]o (±SEM) evoked in DLS followed by either 1p (grey) or 3p 100Hz (black) Estim after Lstim over a range of ISIs, normalized to [DA]o evoked by a single Estim after antagonizing either muscarinic receptors with atropine (2 μM) (d, n=5 recordings, N=3 animals) or D2 receptors with L-741,626 (1 μM) (e, n=5 recordings, N=4 animals). Right: Difference between [DA]o evoked by 3p and 1p Estim. Significant effect of IPI seen in bell-shaped curves for both. P<0.001, one-way ANOVA. [Figure 8] We show that the computational model predicts the empirical ex vivo data. The computational model built from the experimental data in Fig. 1n,r predicted the effect of prior activation of ChI on subsequent DA release by electrical stimulation similar to Fig. 1d,h. Because electrical stimulation (second stimulation in Fig. 1d,h) induced DADA and DAChI, but not only DADA induced by light stimulation (second stimulation in Fig. 1n,r), the predicted data (triangle plots) were scaled as a whole curve to match the overall average of the experimental data (circle plots) in the DLS (left) and NAcc (right) to overcome the unknown ChI-induced inhibition of DAChI in this experimental paradigm. [Figure 9] We show that nAChR 'desensitization' plays a limiting role in altering phasic DA release in a computational model during polyphasic ChI activity. [Figure 9a] Concurrent phasic DA neuron firing (labeled, dark grey) and polyphasic ChI activity (labeled, light grey) with (left) or without (right) initial excitation in the ChI. Inset shows firing rates normalized to baseline rates. [Figure 9b]Phasic DA release was altered by ChI activity both with (solid lines) and without (dashed lines) a “desensitization” component in models (derived from Fig. 2b) for the DLS (top) and NAcc (bottom) (compare with no ChI effect, labeled, upper lines). Background levels of tonic inhibition of DA output by ChI included in the models were 0, 50, and 100%. Inclusion of a dynamic desensitization component in the model affected phasic DA release only when there was an initial ChI excitation (left) and only in the DLS, where it acted to slightly increase phasic DA release by preventing nAChRs from inhibiting DA release during the rebound activity phase at the ChI. This effect was more pronounced in the DLS (top) than in the NAcc (bottom), consistent with a stronger ChI-induced inhibition of DA release in the DLS than in the NAcc. As expected, excluding the dynamic desensitization component from the model did not affect DA release in the absence of initial excitation, as there was no initial increase in nAChR activation. [Figure 10] We show that the tonic level of ChI-induced inhibition of DA release alters the relative influence of ChI polyphasic activity on phasic DA release in a computational model. [Figure 10a] The top panel shows a model of simultaneous phasic DA neuron firing (labeled, dark grey) and polyphasic ChI activity (labeled, light grey) with (left) or without (right) initial excitation in the ChI. The bottom panel shows a comparison of phasic DA signals at three different background levels (0, 50, 100%) of tonic inhibition of DA output before (dashed lines) versus after (solid lines) the onset of polyphasic activity. At low-to-moderate levels (≦50%) of tonic ChI inhibition of DA release, polyphasic ChI activity reduced phasic DA release, whereas at very high levels (50-100%) of tonic ChI inhibition of DA release, ChI polyphasic activity enhanced phasic DA release. [Figure 10b] Summary of the effects of polyphasic ChI activity on phasic DA release at different background levels of tonic inhibition of DA release by tonic ChI activity. Changes in phasic DA signal amplitude and area under the curve (AUC) with (left) or without (right) an initial ChI excitation phase. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] Mesentriatal dopamine (DA) neurons play a key role in action selection and learning 1-4 Striatal DA release has long been thought to be determined primarily by action potentials generated in midbrain DA cell bodies, but the kinetics of striatal DA release is not locally controlled by DA axons. 5-7 , but there is growing evidence that in behaving animals, it can be decoupled from the firing of midbrain DA neurons. 8、9 Striatal ChIs acting on nAChRs on DA axons may play an important role. 5-7、10、11 In vivo ChIs exhibit dynamic tonic activity during spontaneous behavior. 12、13 During learning, the brain also produces polyphasic excitation-pause-rebound activity, or the so-called "pause response," which lasts for 200-400 ms. 14、15 , consistent with phasic activity of DA neurons. 16、17 Somewhat paradoxically, both excitation and quiescence of ChIs have been suggested to promote DA release. 5、6、18 Activation of a small number of ChIs promotes DA release via activation of nAChRs 5、6 Furthermore, loss or cessation of striatal nAChR activity by antagonism, nicotine desensitization, or forebrain ACh depletion enhances frequency-dependent DA release. 7、11、18、19 .

[0098] However, we noted that the highest levels of DA production were elicited when nAChRs were inactivated, not activated. 6、7 This long-overlooked result may suggest that there are significant and unexplored adverse effects of DA activating nAChRs. Here, by activating ChIs and DA axons ex vivo, in vivo and in silico, we show that ChI excitation at levels above or below the threshold that promotes DA release acts on nAChRs to induce action potential-dependent depolarization and Ca upregulation in DA axons. 2+We show that preventing accumulation inhibits subsequent DA release with short latency. Relieving this ChI-induced inhibition of DA release can ameliorate motor deficits in a mouse model of PD. These data provide a new framework for understanding how ACh controls DA release, which is governed by the dynamic inhibition of DA after nAChR activation.

[0099] We show that striatal cholinergic interneurons exert strong inhibition on dopamine axons, limiting their release after activating nicotinic acetylcholine receptors (nAChRs) on the axons, and this inhibition is particularly strong in the dorsal striatum, the human equivalent of the caudate / putamen (CPu), the region most affected by loss of dopamine neurons in the substantia nigra pars compacta (SNpc).

[0100] The inventors have shown that blockade of nAChRs can enhance dopamine signals ex vivo and in vivo and induce conditioned place preference in mice. Administration of nAChR antagonists / blockers relieves the inhibition of dopamine release caused by striatal cholinergic interneurons. Compared with conventional treatments for disorders such as Parkinson's disease, blockade of nicotinic acetylcholine receptors amplifies the physiological pattern of endogenous dopamine release that follows the firing pattern of dopamine neurons. Administration of nAChR antagonists / blockers is not expected to distort dopamine release as does the dopamine precursor levodopa, which may result in a general increase in dopamine levels in the striatum, including release from inappropriate non-dopaminergic inputs, which may lead to the development of serious side effects after several years. This new treatment may extend the treatment period of levodopa and reduce the movement disorders of Parkinson's disease.

[0101] In the present invention, we show that in the mouse striatum, brief activation of ChIs and nAChRs markedly inhibits dopamine release for 50-100 ms afterward with short latency (<7 ms), and the inhibition is more pronounced in the dorsolateral striatum than in the ventral striatum. The inhibition of dopamine output is not due to depletion but is reflected by intracellular calcium dynamics in dopamine axons. Correspondingly, antagonism of nAChRs in vivo promotes dopamine release in the dorsal striatum and induces conditioned place preference. Computational models show that ChI-induced inhibition of dopamine release predominates during both polyphasic burst-pause and sustained activity in ChIs, especially in the dorsal striatum. Thus, the present invention demonstrates that, contrary to prevailing view, ChIs exert region-specific effects on dopamine output that are primarily inhibitory, uncoupling dopamine release from somatic activity and with the power to critically shape dopamine function. EXAMPLES

[0102] (material and method) animal Male adult mice used in ex vivo experiments and in vivo DA recordings were C57Bl6 / J mice (Charles River, UK) (21–40 days old), ChAT-cre:Ai32 (6–16 weeks old), DAT-IRES-Cre (8–16 weeks old), or DAT-Cre:Ai95D (4–7 weeks old). + / + Mouse (B6;129S6-Chat tm2(cre)Lowl / J, JAX stock number 006410) Ai32 + / + Mouse (B6; 129S-Gt(ROSA) 26Sor tm32(CAG-COP4*H134R / EYFP)Hze / J, JAX stock number 012569) to obtain heterozygous ChAT-Cre:Ai32 mice. + / - Mouse (B6.SJL-Slc6 a3tm1.1(cre)BkmnpAAV-double floxed-hChR2(H134R)-EYFP-WPRE-pA was injected into the midbrain of mice (8–16 weeks of age) (J, JAX stock number 006660) to express ChR2 in DA axons. + / - Mouse Ai95D + / + Mouse (B6:129S-Gt(ROSA)26Sor tm95.1(CAG-GCaMP6f)Hze / J) to obtain heterozygous DAT-Cre:Ai95D mice.

[0103] Male C57BL / 6N mice (Charles River, Beijing, China) (42–50 days old) were used for behavioral experiments. Animals were group-housed, maintained on a 12-h light / dark cycle, and had free access to food and water. Procedures for ex vivo recordings and in vivo DA recordings under anesthesia were performed in accordance with the Animals (Scientific Procedures) Act 1986 (Amended 2012) with ethical approval from the University of Oxford and under the authority of a Project License granted by the UK Home Office. Behavioral experiments were performed using protocols approved by the Animal Care & Use Committees of the Chinese Institute for Brain Research (#CIBR-IACUC-007) and in accordance with the guidelines set out by the US National Institutes of Health.

[0104] Virus injection Heterobred DAT-IRES-Cre mice were injected intracerebrally with a Cre-inducible recombinant AAV serotype 5 vector (pAAV-double floxed-hChR2(H134R)-EYFP-WPRE-pA) (titer = 1E+12 vg / ml, University of North Carolina Vector Core) containing an inverted gene for channelrhodopsin 2 fused in-frame with a gene encoding enhanced yellow fluorescent protein, or a Cre-inducible recombinant AAV serotype 5 vector (titer = 2.4E+12 vg / ml) (Stanford Gene Vector and Virus Core) containing an inverted gene for ASAP3 with the EF1A promoter without a somatic targeting signal and WPRE RNA stabilizing element (Stanford Gene Vector and Virus Core). Mice were positioned in a stereotaxic frame under isoflurane anesthesia, and a craniotomy was performed over the injection site. Virus was injected 1 μL unilaterally or bilaterally into either the VTA (coordinates (mm) from bregma: AP -3.1, ML ±0.5, DV -4.4) or SNc (mm from bregma: AP -3.5, ML ±1.2, DV -4.0) using a 2.5 μL 33-gauge Hamilton syringe at 0.2 μL / min with a microinjector. The syringe was left in place for 5 min after each injection and then slowly retracted. Animals were maintained for at least 3 weeks after surgery to allow virus expression in the striatum.

[0105] Ex vivo thin-section voltammetry For fast-scan cyclic voltammetry (FCV) in acute coronal slices, animals were anesthetized with isoflurane in ice-cold high MgCl2 buffer containing 85 mM NaCl, 25 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 0.5 mM CaCl2, 7 mM MgCl2, 10 mM glucose, and 65 mM sucrose. 2+Brains were quickly transferred to the cutting solution. Brains were then blocked and 300 μm coronal slices were cut with a vibratome (Leica VT1200S) between +1.5 and +0.5 mm from bregma, including the caudate-putamen and nucleus accumbens. After sectioning, slices were allowed to recover for 30–40 min at 32°C and then kept at room temperature. Slices were maintained and recorded in artificial cerebrospinal fluid (aCSF) containing 130 mM NaCl, 25 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 2.5 mM CaCl2, 2 mM MgCl2, and 10 mM glucose. aCSF was saturated with 95% O2 / 5% CO2, and recordings were performed at 32–33°C. Extracellular DA concentration ([DA] o ) was measured as previously described using an FCV equipped with a 7 μm diameter carbon fiber microelectrode (CFM; tip length 50–100 μm) and a Miller voltammeter (Julian Millar, Barts and the London School of Medicine and Dentistry). 55 The voltage was applied as a triangular waveform (-0.7 to +1.3 V vs. Ag / AgCl) at a scan rate of 800 V / s, and data were sampled at 8 Hz.

[0106] For optogenetic stimulation, ChI or DA axons expressing ChR2 were activated using a 473 nm diode laser (DL-473, Rapp Optoelectronic) connected to the microscope with a fiber optic cable (200 μm multimode, NA 0.22). The spot illumination was 30 μm in diameter under a ×40 immersion objective. The laser pulse was 2 ms in duration and 23 mW / mm in the specimen. 2 Lstim0 was achieved by lowering the laser intensity to the point where the induced FCV signal was no longer detectable above noise in either online or offline analysis (Zoom in Figure 1).

[0107] For electrical stimulation, a 0.65 mA current (200 μs duration) was delivered through a superficial bipolar concentric Pt / Ir electrode (125 μm outer diameter, 25 μm inner diameter, FHC, USA) placed approximately 100 μm from the recording electrode.50 is the induced [DA] o The stimulation current was approximately 50% of that seen with normal stimulation (0.65 mA) during online analysis. Stimulation was timed to avoid FCV scanning.

[0108] Calcium imaging Previous research 21 As in, an Olympus BX51WL microscope equipped with an OptoLED light system (CAIRN Research), a Prime Scientific CMOS (sCMOS) camera (Teledyne Photometrics) and a ×40 / 0.8 NA underwater objective (Olympus UK) was used for wide-field fluorescence imaging of GCaMP6f at dopaminergic axons in DLS of ex vivo slices in response to single and continuous (4 pulses, 100 Hz) electrical stimulation pulses. Images were acquired at a frame rate of 16.6 Hz every 2.5 min using Micro-Manager 1.4, and stimulation and recording were synchronized using a custom-written procedure in Igor Pro 6 (WaveMetrics) and an ITC-18 A / D board (Instrutech). Image files were analyzed using Matlab R2019b and Fiji 1.5. We extracted the fluorescence intensity from regions of interest (ROIs) and equivalent background regions (over the stimulating electrodes) that had no expression of GCaMP6f. After background subtraction, the Ca 2+ Transients were bleach-corrected by fitting an exponential curve function through both the baseline (2 seconds (s) before stimulation) and the last second of the 7.2 s recording period. The sequence of single and train stimuli was evenly distributed in alternation, and data were collected in duplicate before and after changes in extracellular experimental conditions. Data are expressed as ΔF / F, where F is the fitted curve.

[0109] Voltage Sensor Imaging An Olympus BX51Wl microscope equipped with an OptoLED light system (CAIRN Research), an iXon EMCCD camera (ANDOR) and a ×40 / 0.8 NA medium objective (Olympus UK) was used for wide-field fluorescence imaging of ASAP3 in dopaminergic axons in DLS of ex vivo slices in response to single and train (4 pulses, 500 Hz) electrical stimulation pulses. Images were acquired at a frame rate of 660 Hz every 2.5 min using Micro-Manager 1.4, and stimulation and recording were synchronized using PClamp. Image files were analyzed using Matlab R2019b and Fiji 1.5. We extracted the fluorescence intensity from regions of interest (ROIs). ASPA3 transients were bleach-corrected by fitting an exponential curve function. The order of single and train stimulations was evenly distributed in alternation, and data were collected in duplicate before and after changes in extracellular experimental conditions. Data are expressed as ΔF / F, where F is the fitted curve.

[0110] In vivo voltammetry under anesthesia Wild-type mice were anesthetized with urethane (1.4–1.9 g / kg, i.p.; Biolab), with additional urethane (0.2 g / kg) every 1–2 hours (hr) as needed. All wounds and pressure points were infiltrated with bupivacaine (0.5%). Once surgical anesthesia was reached, the head was fixed in a stereotaxic frame (Kopf, USA). Core body temperature was maintained at 35–36°C using a homeothermic blanket and monitored by a rectal probe (TR-100, Fine Science Tools). A circular section of the skull overlying the left hemisphere was removed to target the DLS (AP +1.0 mm to bregma, ML 1.6 mm, DV 2.2 mm). A stimulation and recording array (MS303 / 3-A / SPC, P1 Technology), consisting of a carbon fiber microelectrode and a bipolar stimulating electrode, was placed in the DLS. An Ag / AgCl reference electrode was implanted in another part of the forebrain. [DA] owas measured using an FCV equipped with a 7 μm diameter carbon fiber microelectrode (CFM; tip length 50–100 μm) and a Tarheel system (University of Washington, Seattle, US). Voltage was applied as a triangular waveform (−0.4 to +1.3 V vs. Ag / AgCl) at a scan rate of 400 V / s, and data were sampled at 10 Hz. The position of the FCV electrode tip was confirmed histologically.

[0111] For striatal electrical stimulation, 0.65 mA current (200 μs) was delivered through a bipolar stimulating electrode (0.005 in., MS303 / 3-A / SPC, P1 Technologies). The tips of the stimulating electrodes were approximately 500 μm apart and glued to the FCV recording electrode, with the tip of the FCV electrode fixed between the two stimulating electrodes.

[0112] Behavioral Record Cannula placement: Male C57BL / 6N mice (42-50 days old) were anesthetized with isoflurane (5% induction, 1.5-2% maintenance) and placed in a stereotaxic frame for surgery. Bilateral injection needles (outer diameter 0.21 mm, inner diameter 0.11 mm, RWD, China) and guide cannulas (outer diameter 0.41 mm, inner diameter 0.25 mm, RWD, China) were implanted into the dorsal striatum either vertically or slightly tilted from vertical, with the tip of each cannula aimed at the following coordinates: AP +1.0 mm to bregma; ML ±1.6 mm; DV -2.4 mm (from the dura). Mice were allowed to recover for 3 days after surgery.

[0113] Conditioned place preference test: In the CPP experiments, mice were placed in a 40 cm × 40 cm transparent Plexiglas arena, which was divided into two equal chambers by a doorway. The chambers were decorated with horizontal or vertical stripes. Animal movements were recorded and analyzed using Smart V3.0 tracking software (Panlab, Spain).

[0114] On day 1, mice were allowed to move freely between the two chambers and baseline place preference was assessed, expressed as the percentage of time spent in the right chamber. Mice were conditioned on days 2 and 3, and either mecamylamine (10 μg / side) or saline vehicle (0.9%) was injected into the striatum in alternating bilateral increments of 0.5 μl over 1 min in the morning and afternoon. Animals were then restrained for 20 min in the right or left chamber, respectively. Treatments were counterbalanced by time of day. On day 4, post-conditioning chamber preference was calculated as the percentage of time spent in the right mecamylamine-associated chamber compared to day 1 before conditioning. On the next 2 days (days 5–6), animals were injected with saline bilaterally and explored both chambers for 20 min, after which the place preference disappeared. The conditioning procedure was then repeated for both chambers with saline bilaterally, with pre-conditioning testing on day 7, 2 days of conditioning on days 8–9, and post-conditioning testing on day 10. To minimize place preference bias at baseline, in each test, the five animals with the lowest place preference on the day before conditioning (mecamylamine 42%-58%; control 45%-55%) were selected for subsequent conditioning. For the open field experiment, mice were injected with either saline vehicle or mecamylamine (10 µg / side) in the striatum on both sides and then placed in the open field chamber to assess their total running distance and average speed within 20 min.

[0115] 6-OHDA Lesion and Reaching Task: On the day of lesion, 6-hydroxydopamine was injected into the right dorsolateral striatum over 5 min via a pre-implanted cannula (4 μg / μL 6-OHDA (Sigma-Aldrich) and 1 μl of 0.9% saline with 0.02% acetic acid). On the day of testing, mecamylamine (10 μg / side) was injected into the lesioned (right) striatum over 1 min via a cannula in a volume of 0.5 μl. Animals were sacrificed immediately after behavioral testing. 6-OHDA-induced loss of dopamine axons was confirmed by a ∼50% decrease in fluorescence intensity of TH staining in thin sections compared to the contralateral DLS measured with ImageJ (1.52p). The anterior section was used as 0% background fluorescence. During injection of 6-OHDA and mecamylamine, animals were briefly sedated with isoflurane.

[0116] To confirm the acute effects of 6-OHDA, the cylinder test was performed before and 10 min after 6-OHDA injection. 56 The test was performed before and again 10 min after mecamylamine injection. Mice were not allowed to habituate to the cylinder. During the test, mice were placed individually in a glass cylinder (19 cm diameter, 25 cm height). A mirror was placed behind the cylinder to allow complete observation and recording of contacts with the wall. Only contacts with one paw (ipsilateral or contralateral forelimb) with the wall were counted in the analysis. Data are expressed as the percentage of contralateral contacts over the total number of contacts with one paw.

[0117] immunocytochemistry After voltammetric recordings in acute slices, slices were fixed with 4% paraformaldehyde dissolved in PBS containing 0.2% picric acid. Slices were fixed overnight at 4°C and then stored in PBS. Free-floating sections were then washed five times for 5 min in PBS and incubated in 0.5% Triton X-100 and 10% normal donkey serum.

[0118] ChIs expressing ChR2-eYFP were identified as ChAT immunoreactive as previously (Zhang et al., 2018). Fixed and washed slices were incubated overnight with goat anti-ChAT 1:100 antibody (Millipore) dissolved in PBS containing 0.5% Triton X-100 and 3% normal donkey serum. Sections were then washed 5 times for 5 min with PBS and incubated for 2 h at room temperature with 1:1000 Alexa Fluor 568 donkey anti-goat antibody (Invitrogen) dissolved in PBS containing 0.5% Triton X-100 and 3% normal donkey serum.

[0119] DA neurons co-expressing ChR2-eYFP, GCaMP6f-eYFP or ASAP3, and striatal DA axons were analyzed using 21The sections were identified by immunoreactivity against tyrosine hydroxylase (TH) as in Example 1. Fixed and washed sections were incubated overnight with 1:2000 rabbit anti-TH antibody (Sigma) dissolved in PBS containing 0.5% Triton X-100, 1% normal goat serum, and 1% fetal bovine serum. Sections were washed five times for 5 min in PBS and incubated for 2 h at room temperature with 1:1000 (DyLight 594 goat anti-rabbit antibody (Jackson) dissolved in PBS containing 0.5% Triton X-100, 1% normal goat serum, and 1% fetal bovine serum.

[0120] Sections processed as above were washed in PBS, mounted on gelled slides with Vectashield mounting medium (Vector Labs) and imaged at 20×, NA 0.8 with an LSM880 confocal microscope system (Zeiss) running ZEN black version 2.3 or with a confocal microscope system (FV1000 IX81; Olympus) using a 20× / 0.75 NA objective and Fluoview software (Olympus). Maximum intensity projections from 30 μm high z-stacks were captured individually and image stacks were compressed. Red fluorescence (TH and ChAT) was captured from 638–759 nm with 633 nm excitation. Green fluorescence (GCaMP, ChR2 and ASAP3) was captured from 493–630 nm with 488 nm excitation.

[0121] To identify the location of the carbon fiber in the dorsal striatum for in vivo FCV recording, anesthetized mice were sacrificed, and the brains were quickly removed and fixed overnight in 4% paraformaldehyde (PFA). The fixed brains were then cut into 50 μm slices using a vibratome (Leica). The slices were washed three times with PBS, mounted on glass slides, and then photographed under a microscope to identify the location of the recording sites.

[0122] To confirm the placement of intrastriatal injection cannulas and 6-OHDA lesions in behavioral experiments, mice were anesthetized by intraperitoneal injection of avertin (250 mg / kg body weight) and transcardially perfused with saline and 4% PFA. Brains were dissected and post-fixed overnight in 4% PFA, followed by dehydration in 30% sucrose for 24 h. Fixed brains were then cryosectioned into 50 μm slices using a vibratome (Leica). Slices to confirm cannula placement were washed three times with PBST (PBS containing 0.1% Tween 20) and mounted on glass slides containing 5 μg / ml DAPI. Slices to confirm 6-OHDA were stained with immunoreactivity for TH in the same way as slices from ex vivo experiments. Slices were imaged with an inverted confocal microscope (Zeiss) using a 405 nm laser for excitation.

[0123] Drugs DHβE and mecamylamine hydrochloride for ex vivo and anesthetized in vivo experiments were purchased from Tocris Bioscience (UK). Mecamylamine hydrochloride for behavioral experiments was purchased from Sigma Aldrich (China). All other chemicals were purchased from Sigma Aldrich (UK). Pharmacological drugs for ex vivo experiments were prepared as stock aliquots at 1000x final concentration in distilled deionized water and stored at -20°C. Drug stocks were then diluted to final concentrations with carbogenated aCSF immediately prior to use and bathed. Drugs for in vivo experiments were dissolved in sterile saline to final concentrations. Computational model A computational model was developed in MATLAB. The model included: (1) polynomial curve fitting of [DA] evoked by a second stimulus before and after stimulating nAChRs with DHβE; o (1) the dynamic strength of ChI-induced inhibition, determined from the ratio of [DA] induced by 3 pulses and 1 pulse in the second stimulus (Fig. 3f,h), normalized to the maximum and fitted to a polynomial curve. o (3) the profile of apparent nAChR “desensitization” estimated from the change in the difference between [DA] in vivo (Fig. 3e, g); oTo model [DA] as a scalar product of DA neuron activity, we used the [DA] observed after a single electrical stimulation ex vivo. o Dynamic release and uptake profiles of DA. We also included in the model various potential levels of background tonic ChI-induced inhibition of DA release (0, 50, 100%) resulting from tonic activity at the ChI. We found that the threshold for nAChR-mediated inhibition of DA release was lower than that required to promote release ex vivo (see FIG. 1) and was met in vivo after discrete striatal stimulation (see FIG. 4), thus allowing for some experimental scenarios (FIG. 1). 5、6 In this study, we excluded the potential component of DA release that could be facilitated by synchronous activation of ChIs. The strength of ChI-induced inhibition in (1) was measured using the [DA] concentration before and after nAChR inhibition in Fig. 1n, r. o The best fit polynomial curve to the ratio of DLS was y=1.97e+5x 2 -0.00833x+0.872, R 2 =0.99;NAcc:y=8.71e+5x 2 -0.0149x+0.611, R 2 = 0.96). The nAChR desensitization levels of (2) were obtained from Fig. 4b, d and normalized to their own maximum and minimum values. The normalized data were then best fitted to a polynomial curve (DLS: y = 2e-05x 2 -0.0087x+0.88NAcc:y=-9e-10x 4 +9e-07x 3 -0.0003x 2 +0.039x-0.5654).

[0124] Quantitative and statistical analysis Statistical analysis was performed using GraphPad Prism 6.0. Data are expressed as mean ± standard error of the mean (SEM). N value is the number of animals, n value is the number of individual records. One-sample t-test, t-test and two-way ANOVA were used.

[0125] (result) Activation of ChI inhibits subsequent DA release We tested the effect of ChI activation on subsequent DA release. In ex vivo striatal slices from ChAT-Cre:Ai32 mice (Fig. 1a, b), we used targeted optogenetic activation of ChR2-expressing ChIs to activate nAChRs and promote DA release (ChI-activated DA release, DA ChI ) 6 Then, 8–200 ms later, an electrical stimulation pulse was applied to directly stimulate the DA DA ) and indirectly through activation of ChIs (DA ChI , followed by a short latency of about 10 ms 10、20 In both the dorsolateral striatum (DLS) (Fig. 1c, d) and nucleus accumbens (NAcc) (Fig. 1i, j), targeted activation of ChIs induced DA release, followed by subsequent electrically evoked DA release 8–200 ms later, that was greater than the peak extracellular DA concentration ([DA]) evoked by electrical stimulation alone. o When interstimulus intervals were ≤50 ms in the DLS and ≤25 ms in the NAcc, this inhibition was significantly greater than that produced by the first electrical stimulus in the presence of an nAChR antagonist (DHβE, 1 μM) (Fig. 1c, d, i, j), as seen previously. 7、21 , interstimulus interval and [DA] o These data suggest that nAChRs inhibit subsequent DA release for a short period of time. D2 autoreceptors on DA axons inhibit subsequent DA release only after an interval of >200 ms. 21 , which can be excluded as a contributing mechanism. We then demonstrated that the apparent inhibition of subsequent DA release seen after ChI activation is due to depletion of the DA vesicle pool. 10 We tested whether this suggests that DA release is related to the minimal intensity required to induce detectable DA release (normal Lstim-induced DA release). ChI The ChI was activated at a low light intensity (Lstim0) (Fig. 6) below 0.5% of normal (Fig. 1e, k). Activation of the ChI with Lstim0 was induced by a subsequent electrical pulse [DA]. o induced by electrical pulses alone [DA] oDA release was maintained at 20–60% (DLS) or 60–80% (NAcc) of the normal range (Fig. 1e, f, k, l). Thus, activation of ChI / nAChRs at levels lower than those required to promote DA release inhibits subsequent DA release, independent of the DA vesicle pool or prior DA release levels. Inhibition of DA release persists in the presence of GABA receptor antagonists, and thus the inhibition of ChI 22 or DA axons 23 , from GABA neurons, and GABA tones 24 The effect was not mediated by striatal GAVA, which could result from simultaneous release from GABA (Fig. 6).

[0126] We also investigated the DA axons (DA DA We investigated whether prior ChI activation limits subsequent DA release when it is promoted by targeted optogenetic activation of DA. We first used electrical stimulation to promote DA release (DA DA +DA ChI ), which was then promoted by subsequent optical activation of ChR2-expressing DA axons in ex vivo striatal slices from DAT-Cre mice. DA In both the DLS (Fig. 2c, d) and NAcc (Fig. 2g, h), electrical stimulation induced DA release, whereas a subsequent light pulse induced [DA] o is induced by a single light pulse alone [DA] o When the interstimulus interval was less than 100 ms (DLS) or 50 ms (NAcc), this inhibition was relieved in the presence of a nAChR antagonist (DHβE, 1 μM). The reduced inhibition was not due to lower initial levels of DA release (approximately half). In wild-type mice, we found that the electrical stimulation intensity (Estim 50 ) reduced the initial DA release by half, but the [DA] induced by the subsequent maximal electrical stimulation was o We found that levels of were similar after maximal intensity stimulation in the DLS (Fig. 2e, f) and only slightly higher in the NAcc (Fig. 2i, j).

[0127] Thus, activation of nAChRs by ChIs can inhibit DA release during subsequent activation of DA axons by a mechanism independent of DA pool availability or subsequent ACh release that lasts for a time comparable to that of the ChI polyphasic pause and that is more pronounced and longer-lasting in the DLS than in the NAcc.

[0128] Activation of nAChRs induces axonal Ca 2+ Limit loading and subsequent de-electrodes DA release is Ca 2+ Upstream axon activation mechanisms and Ca influx 2+ Tightly regulated by mechanisms controlling influx, intracellular buffering and signal transduction 21、25、26 We demonstrated that striatal nAChRs induced axonal Ca 2+ In brain slices from DAT-Cre:Ai95D mice (Fig. 3a, b), we tested whether the effect of single pulses versus four-pulse trains (100 Hz) on the neuronal responses, both with and without nAChR antagonism, was previously reported. 21 Ca in DA axons during evoked DLS 2+ The reporter GCaMP6f was imaged. This stimulation protocol shows activation of nAChRs. [DA] induced by pulse train o is only slightly higher than that of a single pulse when nAChRs can be activated, but is significantly higher when nAChRs are antagonized. 7、27 In parallel, axonal GCaMP6f fluorescence was slightly higher with trains compared to single pulses when we activated nAChRs (Fig. 3c, d), but significantly higher when we antagonized nAChRs (DHβE, 1 μM) (Fig. 3c, d). These data suggest that activation of nAChRs significantly reduces axonal [Ca] responses to subsequent stimuli. 2+ ] i This indicates that the weighting of

[0129] To test whether nAChRs directly limit axonal depolarization during repetitive stimulation of dopamine axons, we used the ASAP3 voltage sensor expressed in DA axons in the DLS of brain slices during single-pulse or four-pulse (50 Hz) electrical stimulation. 28 In the absence of nAChR antagonist, dopamine axons were depolarized by the first pulse but not obviously by subsequent pulses in the train (Fig. 3g, d). In contrast, when nAChRs were antagonized, DA axons could be depolarized by every successive stimulation pulse in the train (Fig. 3g, h). These results suggest that activation of nAChRs is essential for the subsequent spike-evoked depolarization and Ca in dopamine axons. 2+ These results suggest that blocking dopamine influx limits subsequent dopamine release at short latencies.

[0130] nAChRs activated by initial excitation are switched off during ChI rebound In a polyphasic response, ChIs show a "rebound" increase in activity approximately 100-300 ms after the initial excitation. 16、29 During these intervals, the short-latency ChI-induced inhibition of DA release is lost (see Figure 1). However, for approximately 1 s thereafter, further release of DA is promoted only by direct optogenetic activation of DA axons, but not by ChIs. 6、10 This apparent refractoriness of DA release to be stimulated by ChI but not by DA axon activity suggests that nAChRs on DA axons are not responsive to ACh released during rebound ChI activity. 30-32 is suspected to play a role in DA release kinetics during stimulation trains 33 We tested whether DA release kinetics at the time of ChI rebound activity was consistent with nAChR desensitization. We took advantage of the observation that activation of nAChRs limits DA release in subsequent pulses (see Figure 1), whereas switching off or desensitizing nAChRs promotes subsequent release during trains. 7As a potential readout of the time course of nAChR desensitization, we measured the time course of [DA] elicited by triple versus single pulses (100 Hz). o We investigated how the difference in [DA] changes over time. In striatal slices from ChAT-Cre:Ai32 mice, we applied a light-activated ChI followed by a single or triple electrical pulse (100 Hz) 25–400 ms later (Fig. 3i, j; see also Fig. 7c). [DA] induced by triple versus single pulses o The differences varied with interval, peaking at approximately 200 ms in the DLS and 100 ms in the NAcc, and declining by 400 ms (Fig. 3i,j,k,l). Neither muscarinic nor D2 receptor activation was involved in these kinetics (Fig. 7d,e). These kinetics reflected the time course of nAChR desensitization and resensitization, and regional differences were consistent with the differential nAChR stoichiometry. 34、35 This may reflect a shift in the nAChRs' response to ChI stimulation, suggesting that nAChRs are at least partially desensitized during ChI rebound activity approximately 100–300 ms after initial excitation.

[0131] In vivo nAChR antagonism enhances DA release and induces conditioned place preference We investigated the effects of striatal nAChRs in vivo, where the balance of DA inhibition or desensitization to nAChR-mediated excitation may differ from ex vivo. In urethane-anesthetized wild-type mice, injection of nAChR antagonists in the DLS was elicited by local striatal stimulation with short electric pulse trains [DA]. o was significantly increased (Figure 4a-d), indicating that in vivo nAChRs can be activated to inhibit DA release, a property that is not dominated by desensitization. We also tested whether inhibition of nAChRs by bilateral injection of mecamylamine into the dorsal striatum promotes reward-associated learning in freely moving mice. 36After 2 days of daily conditioning, mice developed a conditioned place preference for the chamber conditioned with intrastriatal mecamylamine, but not saline control (Figure 4h-l). This finding is consistent with previous in vivo studies in the adjacent NAc, which showed that nAChR antagonists increased reward-induced DA levels and promoted reward-associated learning. 37、38 These data indicate that nAChR-induced inhibition of DA function predominates over directly causing axonal DA release and nAChR desensitization.

[0132] nAChR antagonism reverses motor deficits in PD models These data raise the prediction that nAChR antagonists may counteract motor deficits in PD caused by DA deficiency by preventing ChI-induced inhibition of DA release. We tested whether striatal antagonism of nAChRs could ameliorate motor deficits induced by DA lesions in mice. Striatal DA inputs in wild-type mice were partially lesioned unilaterally by local injection of 6-OHDA in the right dorsolateral striatum, which resulted in a reduction in striatal tyrosine hydroxylase immunoreactivity. We demonstrated that unilateral reaching movements of the contralateral limb were improved by 6-OHDA injections. 39 We tested the effects of nAChR antagonists on reaching (Fig. 4m-o). Striatal injection of mecamylamine ameliorated these motor deficits (Fig. 4m-o), restoring reaching to normal levels, indicating that nAChR antagonists may be a potential treatment to counteract the effects of DA impairment in PD.

[0133] Tonic and polyphasic activity in ChIs inhibits DA release in a computational model nAChR dynamics during dynamic activity patterns of ChIs [DA] in vivo o To understand how changes in sustained ChI firing rate affect sustained [DA], we developed a computational model based on the data in Figs. 2c and d and 2g and h (Fig. 5a). We confirmed that the model can simulate the ex vivo observations in Fig. 1d and h (Fig. 8). We first demonstrated that changes in sustained ChI firing rate affect sustained [DA]. oWe set an arbitrary tonic level of ChI-induced inhibition of DA release (50% of maximum) and tested how [DA] o was normalized to the level in the absence of ChI effects. The model assumes that transient (~100 ms) changes in ChI population activity can be predicted by [DA] in the absence of underlying changes in DA firing activity. o (Fig. 5b). This prediction is supported by the o may explain the recent in vivo observation that DA neuron firing rates can be altered without fundamental changes. 8 .

[0134] We then demonstrated that polyphasic ChI activity induces [DA] during the simultaneous bursting activity of DA neurons. o How to change 16 , both with and without early excitation occurring in half of the ChIs. 29 In , we tested incorporating various levels of tonic nAChR-induced inhibition of DA release (0, 50, 100%) prior to polyphasic activity. Tonic ChI activity reduced baseline DA release levels (Fig. 5c). Furthermore, during burst activity of DA neurons, DA release was reduced by polyphasic activity at the ChI, was maximal when accompanied by initial ChI excitation and lower tonic ChI activity, and was reduced more in the DLS than in the NAc (Fig. 5c–e). The reduction in burst DA release seen during polyphasic ChI activity without initial excitation results from inhibition of DA release by concurrent ChI recoil activity. Desensitization-like elements played only a minor role in these results, primarily in the DLS rather than the NAc, and were present even when initial excitation was present (Fig. 9). Overall, this model suggests that polyphasic activity at the ChI inhibits DA release during phasic activity, particularly in the DLS.

[0135] The model also trains the scenario 29、40Reflecting this, we show that tonic ChI activity in the absence of polyphasic ChI activity reduced baseline and burst-evoked levels of DA release (dashed line, Fig. 10a). At low to moderate levels of tonic ChI inhibition of DA release (<50%), polyphasic ChI activity further reduced phasic DA release. Only at very high levels of tonic inhibition (50–100%) did ChI polyphasic activity enhance phasic DA release (Fig. 10a and b), presumably due to reduced ChI inhibition during ChI rest. Thus, the level of phasic striatal DA release is a function of both tonic and polyphasic ChI activity.

[0136] (Consideration) Reconciling the diverse actions of nAChRs on DA release and revising current thinking, we show that nAChR activation leads to a predominant inhibition of DA release. The results described herein show that DA output is gated by the kinetics of nAChR activation via a rapid-onset but long-lasting inhibition of DA release (<100 ms) followed by a clear loss of nAChR control (after approximately 100-300 ms). nAChR-induced inhibition does not require depletion of the DA pool but rather requires axonal Ca2+ release. 2+ It limits summation, is detected in vivo, and affects DA-dependent learning.

[0137] Inhibition vs. enhancement of DA release We show that activation of ChIs mediates DA axon depolarization events and axonal Ca after nAChR activation. 2+ We show that ChIs limit continuous DA release by preventing summation, and computational modeling predicts that tonically active ChIs will inhibit DA release continuously in vivo. ChIs have previously been suggested to enhance DA release in response to both synchronous excitation and pauses during polyphasic activity. 5-7、11、18 Striatal ChIs can directly promote DA release after optogenetic or electrical activation of ChIs or inputs to the cortex or thalamus. 5、6、10、20、41, leading to the inference that initial excitation or rebound activity during polyphasic ChI activity in vivo may facilitate DA release. However, ex vivo and in vivo observations suggest that the levels of excitation and rebound during polyphasic ChI activity in vivo are insufficient to facilitate DA axonal release, but rather are sufficient to result in inhibition of phasic DA release, because lower levels of nAChR activation are required to inhibit rather than facilitate DA release. Pausing of ChI polyphasic responses has previously been speculated to facilitate simultaneous burst-evoked DA, because high-frequency train stimulation of both ChIs and DA axons can elicit more DA release when nAChRs are turned off. 7、18 This new finding indicates that increased levels of DA release result from disinhibition, the removal of ChI-induced inhibition. The current finding that ChIs primarily inhibit DA release also supports the notion that ChI activity is driven by excitatory inputs, e.g. from the cortex or thalamus, and the resulting activation of nAChRs on DA axons. 41、42 may inhibit striatal DA release in vivo, and conversely, may act as an inhibitory input to ChIs. 43、44 may boost DA release.

[0138] Over longer time courses (100–300 ms after the initial activity), corresponding to the rebound activity seen in ChI polyphasic activity, DA release was found to better reflect the received stimulus, consistent with nAChR unavailability. 7、11 This most likely corresponds to nAChR desensitization, suggesting that nAChRs activated during the initial excitation do not control DA release during the rebound phase.

[0139] The rapid inhibition of DA release after nAChR activation correlated with subsequent limited depolarization in response to activity and, consequently, limited axonal calcium loading. Prior DA release was not required, and suggested a rapid ionic mechanism that may occur at levels of axonal activity that are subthreshold for DA release and action potentials. The ionic mechanism is that DA output is mediated by DAT activity, K + Dependent Excitation and GABA AShunt inhibition and Na after receptor activation + This is consistent with emerging data showing that axonal excitability is tightly gated by mechanisms that include channel inactivation 21、45 Differences in the strength and duration of ChI-induced inhibition of DA release in the DLS versus the NAc were related to nAChR subtype and consequently channel properties (α4α5β2* and α4α6β2β3*, respectively). 34、35 with heterogeneity.

[0140] Consequences of ChI activity on DA release and function Our understanding of dopamine function to date has been dominated by insights into action potential activity recorded at DA cell bodies. However, by limiting subsequent depolarization events at DA axons, ChIs may prevent the propagation of action potentials and play a key role in governing DA function. The extremely rapid onset (<7 ms) of ChI-induced inhibition of DA release and the decay of this mechanism over approximately 50–100 ms allowed rapid alterations of DA release in response to dynamic changes in ChI activity without altering DA neuron activity, and conversely. This local change could result in increased DA release in the absence of any obvious changes in DA neuron activity. 8 This may be the cause of the recently reported extreme case.

[0141] Regarding burst activity in DA neurons, this model shows that the combined effects of simultaneous polyphasic ChI activity primarily inhibit DA output as a result of nAChR activation, especially in the early excitation. These effects are consistent with striatal learning. 46 , Changes in DA signals after learning 29 Moreover, the variation seen across the striatum in the strength and duration of ChI-induced inhibition, which is stronger in the DLS than in the NAc, may in turn be due to diverse DA signals and regulatory mechanisms between regions. 47-49 may contribute to.

[0142] Local antagonism of nAChRs can alter the animals' reward-related behavior and ameliorate motor deficits in 6-OHDA-lesioned mice, indicating that manipulating nAChRs on dopamine axons may be a promising treatment for PD and other dopamine-related diseases. ChI-induced inhibition of DA release was consistent with established inhibitory DA-D2 receptor inhibition of ChIs. 50-52 Together, these findings provide a mechanistic explanation for the long-suspected balance of reciprocal inhibition between striatal ACh and DA. This feed-forward loop amplifies any deviation from balance in the ACh or DA systems. For example, DA deficiency in PD exaggerates ChI-induced inhibition of DA release by disinhibiting ChIs, resulting in a maladaptive further reduction in DA output. nAChR antagonists are thought to alleviate this maladaptive inhibition of DA release. Furthermore, nicotine-induced desensitization of striatal nAChRs in smokers 7、53 also removes nAChR inhibition of striatal DA release, thereby inhibiting DA-dependent behaviors (in parallel with mechanisms operating at the level of DA neurons). 54 ) is expected to be promoted.

[0143] In summary, we have discovered that striatal nAChRs add rapid inhibition to the portfolio of mechanisms by which they gate DA release and that this mechanism predominates as a means by which nAChRs control DA output.

[0144] Throughout the description and claims of this specification, the terms "comprise" and "comprising" and variations thereof mean "including but not limited to" and are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context dictates otherwise. In particular, where the indefinite article is used, it is understood that the specification contemplates the singular and the plural unless the context dictates otherwise.

[0145] It is understood that features, integers, properties, compounds, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are also applicable to other side aspects, embodiments or examples described herein, unless inconsistent therewith. All features described herein (including the accompanying claims, abstract and drawings), and / or all steps of the methods or processes described therein, can be combined in any combination, except combinations in which at least some features and / or steps are mutually exclusive. The invention is not limited to the details of the above embodiments. The invention extends to novel or novel combinations of features described herein (including the accompanying claims, abstract and drawings), or novel or novel combinations of steps of the methods or processes described therein.

[0146] The reader is directed to all articles and documents related to this application that have been filed contemporaneously or prior to and published herewith, and the contents of all such articles and documents are incorporated herein by reference.

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Claims

1. A pharmaceutical product comprising a nicotinic acetylcholine receptor antagonist / blocker or a pharmaceutically acceptable salt thereof for the treatment of a condition or disorder involving reduced dopamine levels in the CNS.

2. The pharmacopoeia according to claim 1, wherein administration of a nicotinic acetylcholine receptor antagonist / blocker promotes dopamine release in the CNS.

3. The pharmacopoeia according to claim 1, wherein administration of a nicotinic acetylcholine receptor antagonist / blocker promotes dopamine release in the brain.

4. The pharmacopoeia according to claim 1, wherein administration of a nicotinic acetylcholine receptor antagonist / blocker promotes dopamine release in the striatum.

5. The pharmacopoeia according to claim 1, wherein administration of a nicotinic acetylcholine receptor antagonist / blocker promotes dopamine release in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

6. A pharmaceutical product according to any one of claims 1 to 5, wherein the condition or disorder involves a decrease in dopamine levels in the brain.

7. A pharmaceutical product according to any one of claims 1 to 5, wherein the condition or disorder involves a decrease in dopamine levels in the striatum.

8. The pharmacopoeia according to any one of claims 1 to 5, wherein the condition or disorder involves a decrease in dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

9. A pharmacopoeia according to any one of claims 1 to 5, wherein the condition or disorder is selected from dopamine deficiency, addiction (including binge eating, nicotine addiction and alcohol addiction), Alzheimer's disease, attention deficit hyperactivity disorder (ADHD), bipolar disorder, dopa-responsive dystonia and DRD plus, Huntington's disease, multiple sclerosis, obsessive-compulsive disorder (OCD), Parkinson's disease (including resting tremor of Parkinson's disease), schizophrenia, convulsions (i.e., recurrent involuntary movements or vocalizations), Tourette syndrome, depression and reward deficiency syndrome.

10. A pharmaceutical product according to any one of claims 1 to 5, wherein the condition or disorder is selected from Parkinson's disease or ADHD.

11. A pharmaceutical product according to any one of claims 1 to 5, wherein the condition or disorder is Parkinson's disease.

12. Nicotinic acetylcholine receptor antagonists / blockers include dihydro-β-erythroidine (DHβE), mecamylamine, bPiDDP (1,1'-(1,12-dodecanediyl)bis[3-methylpyridinium]dibromide), methyllicaconitine, MG 624 (N,N,N-triethyl-2-[4-(2-phenylethenyl)phenoxy]ethaneaminium iodide), SR 16584 (1,3-dihydro-1-(3-exo)-9-methyl-9-azabicyclo[3.3.1]nona-3-yl]-2H-indole-2-one), catestatin, chlorisondamine diiodide, 2,2,6,6-tetramethylpiperidine-4-yl heptanoate (TMPH), chlorisondamine, and α-conotoxin (e.g., ACV). 1, AuIB, EI, ImI, MII, PIA, PnIA), A 85380 (3-[(2S)-2-azetidinyl methoxy]-pyridine), ABT 089 (2-methyl-3-[(2S)-pyrrolidinyl methoxy]pyridine), ABT 594 ((R)-5-(azetidine-2-ylmethoxy)-2-chloropyridine), Dianiclean ((5aS,8S,10aR)-5a,6,9,10-tetrahydro-7H,11H-8,10a-methanopyrido[2',3':5,6]pyrano[2,3-d]azepine), RJR 2403 ((E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine), TC Selected from 2559 (4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-butene-1-amine), varenicline (7,8,9,10-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine), hexamethonium bromide, tubocurarine chloride, α-bungarotoxin, COG 133, D-amphetamine nitrate, PAMP-20, or pharmaceutically acceptable salts or derivatives thereof; Optionally, nicotinic acetylcholine receptor antagonists / blockers include dihydro-β-erythroidine (DhβE), mecamylamine, bPiDDP, methyl licaconitine, MG 624, SR 16584, catestatin, chlorisondamine diiodide, 2,2,6,6-tetramethylpiperidine-4-yl heptanoate (TMPH), chlorisondamine, α-conotoxin (e.g., ACV 1, AuIB, EI, ImI, MII, PIA, PnIA), A 85380 dihydrochloride (3-[(2S)-2-azetidinyl methoxy]pyridine dihydrochloride), ABT 089 (2-methyl-3-[(2S)-pyrrolidinyl methoxy]pyridine), ABT 594 ((R)-5-(azetidine-2-ylmethoxy)-2-chloropyridine), Dianiclin ((5aS,8S,10aR)-5a,6,9,10-tetrahydro-7H,11H-8,10a-methanopyrido[2',3':5,6]pyrano[2,3-d]azepine), RJR 2403 ((E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine), TC A pharmaceutical product according to any one of claims 1 to 5, selected from 2559 (4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-butene-1-amine), varenicline (7,8,9,10-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine tartrate), or a pharmaceutically acceptable salt or derivative thereof.

13. A pharmacopoeia according to any one of claims 1 to 5, wherein the nicotinic acetylcholine receptor antagonist / blocker is selected from dihydro-β-erythroidine (DhβE), mecamylamine, bPiDDP, methyl licaconitine, MG 624, SR 16584, catestatin, chlorisondamine diiodide, 2,2,6,6-tetramethylpiperidine-4-yl heptanoate (TMPH), chlorisondamine and α-conotoxin (e.g., ACV 1, AuIB, EI, ImI, MII, PIA, PnIA), or a pharmaceutically acceptable salt or derivative thereof.

14. The pharmaceutically acceptable pharmacopoeia according to any one of claims 1 to 5, wherein the nicotinic acetylcholine receptor antagonist / blocker is selected from dihydro-β-erythroidine (DhβE), mecamylamine, chlorisondamine diiodide, 2,2,6,6-tetramethylpiperidine-4-yl heptanoate (TMPH), chlorisondamine, or a pharmaceutically acceptable salt or derivative thereof.

15. The pharmaceutical product according to any one of claims 1 to 5, wherein the nicotinic acetylcholine receptor is selected from DHβE and mecamylamine.

16. The pharmaceutical agent according to any one of claims 1 to 5, wherein the nicotinic acetylcholine receptor antagonist / blocker is selective for the α4β2 receptor.

17. α4β2 receptor-selective nicotinic acetylcholine receptor antagonists / blockers include A 85380 (3-[(2S)-2-azetidinyl methoxy]-pyridine dihydrochloride), ABT 089 (2-methyl-3-[(2S)-pyrrolidinyl methoxy]pyridine), ABT 594 ((R)-5-(azetidine-2-ylmethoxy)-2-chloropyridine), Dianiclin ((5aS,8S,10aR)-5a,6,9,10-tetrahydro-7H,11H-8,10a-methanopyrido[2',3':5,6]pyrano[2,3-d]azepine), RJR 2403 ((E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine), TC The pharmaceutically acceptable pharmacopoeia according to claim 16, selected from 2559 (4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine) and varenicline (7,8,9,10-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine tartrate), or a pharmaceutically acceptable salt or derivative thereof.

18. A pharmacopoeia according to any one of claims 1 to 5, wherein a nicotinic acetylcholine receptor (nAChR) antagonist / blocker is administered orally, nasally, intraperitoneally, intraventricularly, intrathecally, intracranially, subcutaneously, or intravenously.

19. A pharmacopoeia according to any one of claims 1 to 5, wherein a nicotinic acetylcholine receptor (nAChR) antagonist / blocker is administered directly to the CNS.

20. A pharmacopoeia according to any one of claims 1 to 5, wherein a nicotinic acetylcholine receptor (nAChR) antagonist / blocker is administered by intraperitoneal injection, intravenous injection, local infusion, or direct injection into the CNS.

21. A pharmaceutical product containing a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for increasing dopamine levels in the CNS.

22. A pharmaceutical composition comprising a nicotinic acetylcholine receptor antagonist / blocker or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable additives, for the treatment of a condition or disorder involving a decrease in dopamine levels in the CNS.

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