Nicotinic acetylcholine receptor antagonists / blockers for increased dopamine

Abstract

The present invention relates to methods of increasing dopamine levels in a subject. The invention also relates to the treatment of conditions or disorders associated with reduced levels of dopamine, as well as agents useful 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 treatments for conditions or disorders associated with decreased dopamine levels, and agents for use in such treatments. Background Art

[0002] Striatal cholinergic interneurons (ChIs) acting on nicotinic acetylcholine receptors (nAChRs) on dopamine axons are thought to facilitate dopamine output. Synchronous activity in ChI can drive dopamine release (Cachope, R et al. Selective activation of cholinergic interneurons enhances accumbal phasicdopamine 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)), while ChI pauses 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 unexplained and key observation is that dopamine release when nAChR is activated may be significantly lower than when nAChR is inactivated (Rice & Cragg, 2004), which questions whether ChI promotes or attenuates dopamine output.

[0003] Reduced dopamine levels have been linked to a variety of 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 resting tremor in Parkinson's disease), schizophrenia, tics (i.e., repetitive involuntary movements or vocalizations), and Tourette syndrome.

[0004] Conventional treatments for diseases such as Parkinson's disease, such as levodopa, are thought to provide overall elevated dopamine levels in the striatum, including by releasing inappropriate non-dopaminergic input. This can lead to the development of serious side effects years later. Therefore, alternative treatments are needed to reduce the risk of such side effects.

[0005] Parkinson's disease is the second most common neurodegenerative disorder. Core motor symptoms result from the loss of midbrain dopamine (DA) neurons. Levodopa, a DA precursor, provides the most effective symptomatic treatment by increasing DA release from remaining neurons. However, chronic administration of levodopa at concentrations sufficient to achieve symptomatic relief, particularly in patients with advanced PD or those receiving long-term levodopa therapy, can lead to the development of disabling motor side effects, termed levodopa-induced dyskinesia (LID). Summary of the invention

[0006] The inventors have found that striatal cholinergic interneurons can strongly inhibit dopamine release in the striatum after activating nicotinic acetylcholine receptors (nAChR) on dopamine axons. It is expected that the administration of nAChR antagonists / blockers can alleviate the inhibition of dopamine release caused by striatal cholinergic interneurons, thereby providing effective treatment for conditions or disorders related to reduced dopamine levels. Therefore, the present invention is directed to reduced dopamine levels that occur in a variety of conditions.

[0007] The present invention is based on the recognition that therapy involving the administration of nicotinic acetylcholine receptor (nAChR) antagonists / blockers or pharmaceutically acceptable salts thereof results in an increase in dopamine levels in the CNS. Administration of nAChR antagonists / blockers per se targets the inhibition of dopamine release caused by striatal cholinergic interneurons acting on nAChRs.

[0008] As used herein, the term "nicotinic acetylcholine receptor antagonists / blockers" (also referred to as nicotinic antagonists / blockers) refers to compounds that inhibit the action of acetylcholine (ACh) at nicotinic acetylcholine receptors. The term "antagonist / blocker" encompasses both competitive antagonists and non-competitive antagonists.

[0009] Therapeutic uses and applications

[0010] A first aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for use in treating a condition or disorder associated with reduced dopamine levels.

[0011] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for use in treating a condition or disorder associated with reduced dopamine levels in the central nervous system (CNS).

[0012] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for use in treating a condition or disorder associated with reduced dopamine levels in the brain.

[0013] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for use in treating a condition or disorder associated with decreased dopamine levels in the striatum.

[0014] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for use in treating conditions or disorders associated with decreased dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0015] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for use in treating a condition or disorder associated with decreased dopamine levels in the dorsal striatum (including the caudate nucleus and putamen).

[0016] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for use in increasing dopamine levels in the CNS.

[0017] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for use in increasing dopamine levels in the brain.

[0018] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for increasing dopamine levels in the striatum.

[0019] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for increasing dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0020] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for increasing dopamine levels in the dorsal striatum (including the caudate nucleus and putamen).

[0021] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for use in increasing dopamine levels in the CNS of a subject in the treatment of a condition or disorder associated with reduced dopamine levels in the CNS.

[0022] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for use in increasing dopamine levels in the brain of a subject in the treatment of a condition or disorder associated with reduced dopamine levels in the brain.

[0023] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for use in increasing dopamine levels in the striatum of a subject in the treatment of a condition or disorder associated with decreased dopamine levels in the striatum.

[0024] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for use in increasing dopamine levels in a subject's dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra in the treatment of a condition or disorder associated with decreased dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0025] Another aspect of the present invention provides a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically 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 associated with decreased dopamine levels in the dorsal striatum (including the caudate nucleus and putamen).

[0026] Another aspect of the present invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a subject suffering from a disorder associated with reduced dopamine levels in the CNS.

[0027] Another aspect of the present invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a subject suffering from a disorder associated with reduced dopamine levels in the brain.

[0028] Another aspect of the present invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a subject suffering from a disorder associated with reduced dopamine levels in the striatum.

[0029] Another aspect of the present invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a subject suffering from a condition associated with decreased dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0030] Another aspect of the present invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a subject suffering from a condition associated with decreased dopamine levels in the dorsal striatum (including the caudate nucleus and putamen).

[0031] Another aspect of the present invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing dopamine levels in the CNS.

[0032] Another aspect of the present invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing dopamine levels in the brain.

[0033] Another aspect of the present invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing dopamine levels in the striatum.

[0034] Another aspect of the present invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0035] Another aspect of the present invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing dopamine levels in the dorsal striatum (including the caudate nucleus and putamen).

[0036] Another aspect of the invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing dopamine levels in the CNS in the treatment of a subject suffering from a disorder associated with reduced dopamine levels in the CNS.

[0037] Another aspect of the invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing dopamine levels in the brain in the treatment of a subject suffering from a disorder associated with reduced dopamine levels in the brain.

[0038] Another aspect of the invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing dopamine levels in the CNS in the treatment of a subject suffering from a disorder associated with reduced dopamine levels in the CNS.

[0039] Another aspect of the invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing dopamine levels in the striatum in the treatment of a subject suffering from a disorder associated with reduced dopamine levels in the striatum.

[0040] Another aspect of the invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in 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 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.

[0041] Another aspect of the invention provides a use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing dopamine levels in the dorsal striatum (including the caudate and putamen) in the treatment of a subject suffering from a disorder associated with decreased dopamine levels in the dorsal striatum (including the caudate and putamen).

[0042] Another aspect of the present invention provides a use of a nAChR antagonist / blocker or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for increasing dopamine levels in the CNS in the treatment of a condition or disorder associated with decreased dopamine levels in the CNS.

[0043] Another aspect of the present invention provides a use of a nAChR antagonist / blocker or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for increasing dopamine levels in the brain in the treatment of a condition or disorder associated with reduced dopamine levels in the brain.

[0044] Another aspect of the present invention provides a use of a nAChR antagonist / blocker or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for increasing dopamine levels in the striatum in the treatment of a condition or disorder associated with decreased dopamine levels in the striatum.

[0045] Another aspect of the present invention provides a use of a nAChR antagonist / blocker or a pharmaceutically 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 conditions or disorders associated with decreased dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

[0046] Another aspect of the present invention provides a use of a nAChR antagonist / blocker or a pharmaceutically 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 conditions or disorders associated with decreased dopamine levels in the dorsal striatum (including the caudate nucleus and putamen).

[0047] Treatment

[0048] Another aspect of the invention provides a method of treating a condition or disorder associated with reduced dopamine levels in the CNS, the method comprising administering to a subject in need thereof a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof.

[0049] Another aspect of the invention provides a method of treating a condition or disorder associated with reduced dopamine levels in the brain, the method comprising administering to a subject in need thereof a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof.

[0050] Another aspect of the invention provides a method of treating a condition or disorder associated with reduced dopamine levels in the striatum, the method comprising administering to a subject in need thereof a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof.

[0051] Another aspect of the present invention provides a method for treating a condition or disorder associated with 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 thereof a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof.

[0052] Another aspect of the invention provides a method for treating a condition or disorder associated with decreased dopamine levels in the dorsal striatum (including the caudate nucleus and putamen), the method comprising administering to a subject in need thereof a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof.

[0053] Another aspect of the present invention provides 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 pharmaceutically acceptable salt thereof.

[0054] Another aspect of the present invention provides a method of 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 pharmaceutically acceptable salt thereof.

[0055] Another aspect of the present invention provides a method of 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 pharmaceutically acceptable salt thereof.

[0056] Another aspect of the present invention provides a method of 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 pharmaceutically acceptable salt thereof.

[0057] Another aspect of the invention provides a method of increasing dopamine levels in the dorsal striatum (including the caudate nucleus 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 pharmaceutically acceptable salt thereof.

[0058] Another aspect of the present invention provides a method of treating a condition or disorder associated with reduced dopamine levels in the CNS, the method comprising administering to a subject in need thereof a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof, wherein administration of the nicotinic acetylcholine receptor antagonist / blocker increases dopamine levels in the CNS.

[0059] Another aspect of the present invention provides a method of treating a condition or disorder associated with reduced dopamine levels in the brain, the method comprising administering to a subject in need thereof a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof, wherein administration of the nicotinic acetylcholine receptor antagonist / blocker increases dopamine levels in the brain.

[0060] Another aspect of the present invention provides a method for treating a condition or disorder associated with reduced dopamine levels in the striatum, the method comprising administering to a subject in need thereof a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof, wherein administration of the nicotinic acetylcholine receptor antagonist / blocker increases dopamine levels in the striatum.

[0061] Another aspect of the present invention provides a method for treating a condition or disorder associated with 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 thereof a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically 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.

[0062] Another aspect of the present invention provides a method for treating a condition or disorder associated with decreased dopamine levels in the dorsal striatum (including the caudate nucleus and putamen), the method comprising administering to a subject in need thereof a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically 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).

[0063] Condition to be treated

[0064] Many conditions are associated with reduced dopamine levels. The inventors have demonstrated that the use of nAChR antagonists / blockers will play a role in increasing the dopamine levels in the CNS (e.g., brain, including striatum), thereby treating such conditions. Suitably, the use of nAChR antagonists / blockers will play a role in increasing the dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia, and / or substantia nigra. Most appropriately, the use of nAChR antagonists / blockers will play a role in increasing the dopamine levels in the dorsal striatum (including caudate nucleus and putamen).

[0065] Suitably, the condition or disorder is selected from 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 in Parkinson's disease), schizophrenia, tics (i.e., repetitive involuntary movements or vocalizations), Tourette 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.

[0066] In some cases, the subject may have experienced side effects from taking a dopamine precursor (e.g. levodopa) or a dopamine agonist to treat a condition associated with reduced dopamine levels in the CNS. Suitably, the subject suffers from levodopa-induced dyskinesia.

[0067] Suitably, the dopamine levels in the CNS, brain and / or striatum of the subject are reduced. More suitably, the dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra of the subject are reduced. Most suitably, the dopamine levels in the dorsal striatum (including the caudate nucleus and putamen) of the subject are reduced.

[0068] Suitably, use nicotinic acetylcholine receptor antagonists / blockers to promote dopamine release in CNS, brain and / or striatum. More suitably, use nicotinic acetylcholine receptor antagonists / blockers to promote dopamine release in dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra. Most suitably, use nicotinic acetylcholine receptor antagonists / blockers to promote dopamine release in dorsal striatum (including caudate nucleus and putamen).

[0069] nAChR antagonists / blockers

[0070] Suitably, the nAChR antagonist / blocker is selected from dihydro-β-erythrin (DHβE), mecamylamine, bPiDDP (1,1'-(1,12-dodecanediyl)bis[3-methylpyridinium] dibromide), methyltaurine, MG 624 (N,N,N-triethyl-2-[4-(2-phenylvinyl)phenoxy]ethanammonium iodide), SR 16584 (1,3-dihydro-1-(3-exo)-9-methyl-9-azabicyclo[3.3.1]non-3-yl]-2H-indol-2-one), Catestatin, chloroisoindolium diiodide, 2,2,6,6-tetramethylpiperidin-4-ylheptanoate (TMPH), chloroisoindolium, α-conotoxin (such as 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-methoxypyrido[2',3':5,6]pyrano[2,3-d]azepine ), RJR 2403 ((E)-N-methyl-4-(3-pyridyl)-3-butene-1-amine), TC 2559 (4-(5-ethoxy-3-pyridyl)-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, myostatin chloride, α-bungarotoxin, COG 133, D-amphetamine sulfate, PAMP-20, or a pharmaceutically acceptable salt or derivative thereof.

[0071] Suitably, the nAChR antagonist / blocker is selected from dihydro-β-erythrin (DHβE), mecamylamine, bPiDDP (1,1'-(1,12-dodecanediyl)bis[3-methylpyridinium] dibromide), methyltaurine, MG 624 (N,N,N-triethyl-2-[4-(2-phenylvinyl)phenoxy]ethanammonium iodide), SR 16584 (1,3-dihydro-1-(3-exo)-9-methyl-9-azabicyclo[3.3.1]non-3-yl]-2H-indol-2-one), casein peptide, chloroisoindolium diiodide, 2,2,6,6-tetramethylpiperidin-4-ylheptanoate (TMPH), chloroisoindolium, α-conotoxin (such as 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), Dianicline ((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-pyridyl)-3-butene-1-amine), TC 2559 (4-(5-ethoxy-3-pyridyl)-N-methyl-(3E)-3-butene-1-amine), varenicline (7,8,9,10-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine ), or a pharmaceutically acceptable salt or derivative thereof.

[0072] More suitably, the nicotinic acetylcholine receptor antagonist / blocker is selected from dihydro-β-erythrin (DHβE), mecamylamine, bPiDDP, methyltaurine, MG 624, SR 16584, casein peptide, diiodide chloroisoindole ammonium, 2,2,6,6-tetramethylpiperidin-4-ylheptanoate (TMPH), chloroisoindole ammonium and α-conotoxin (such as ACV 1, AuIB, EI, ImI, MII, PIA, PnIA), or a pharmaceutically acceptable salt or derivative thereof.

[0073] Most suitably, the nicotinic acetylcholine receptor antagonist / blocker is selected from dihydro-β-erythrin (DHβE), mecamylamine, chloroisoindolium diiodide, 2,2,6,6-tetramethylpiperidin-4-ylheptanoate (TMPH), chloroisoindolium, or a pharmaceutically acceptable salt or derivative thereof.

[0074] The nAChR antagonist / blocker may be a derivative of mecamylamine, such as one or more of the compounds disclosed in WO2013 / 026852, the entire contents of which are incorporated herein by reference. The nAChR antagonist / blocker may be a derivative of dihydro-β-erythrin.

[0075] Suitably, the nAChR antagonist / blocker is an antagonist of the α4β2 nAChR receptor. More suitably, the nAChR antagonist / blocker is selective for the α4β2 receptor. It is expected that an antagonist / blocker selective for the α4β2 receptor will reduce the risk of side effects in the subject to be treated.

[0076] Certain α4β2 ligands with antagonistic activity are described in U.S. Pat. No. 9,029,557 and U.S. Pat. No. 5,691,365, the entire contents of which are incorporated by reference. Other known antagonists / blockers include lophotoxin, neosurugatoxin, and erysodine (J. Med. Chem. (1997) 40: 4169-4194). Those skilled in the art will be aware of such nAChR antagonists / blockers, such as those disclosed in Nicotinic ACh Receptors Scientific Review, Tocris Scientific Review Series (Wonnacott, Tocris Scientific Review Series, 2014, https: / / www.tocris.com / literature / scientific-reviews).

[0077] Other examples of nAChR antagonists / blockers selective for α4β2 receptors 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), Dianicline ((5aS,8S,10aR)-5a,6,9,10-tetrahydro-7H,11H-8,10a-methoxypyrido[2',3':5,6]pyrano[2,3-d]azepine ), RJR 2403 ((E)-N-methyl-4-(3-pyridyl)-3-butene-1-amine), TC 2559 (4-(5-ethoxy-3-pyridyl)-N-methyl-(3E)-3-butene-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.

[0078] Pharmaceutical compositions and routes of administration

[0079] An effective amount of a nAChR antagonist / blocker can be administered by a variety of modes of administration, including, for example, rectal, oral, intranasal, and transdermal routes, by intraarterial injection, intravenous, intrathecal, intracranial, intraperitoneal, intracerebroventricular, parenteral, intramuscular, subcutaneous, orally, topically, nasally, as an inhalant, or by impregnation or coating devices such as stents.

[0080] Suitably, the nAChR antagonist / blocker is administered orally, intranasally, intraperitoneally, intracerebroventricularly, intracranially, intrathecally, subcutaneously or intravenously.

[0081] Suitably, the nAChR antagonist / blocker is capable of penetrating the blood-brain barrier.The nAChR antagonist / blocker capable of penetrating the blood-brain barrier may be administered by a variety of routes, such as those described herein.

[0082] Suitably, the nAChR antagonist / blocker is administered directly to the CNS. It is expected that direct administration to the CNS will reduce the risk of peripheral side effects associated with administration of nAChR antagonists / blockers.

[0083] Suitably, the nAChR antagonist / blocker is administered by local infusion into the CNS or by direct injection into the CNS. Administration to the CNS may be achieved by intracranial infusion, such as intrastriatal infusion, intraputamen infusion, intracaudate nucleus infusion, intracerebroventricular infusion, intraparenchymal infusion and intracortical infusion.

[0084] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the nAChR antagonist / blocker as defined above or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0085] Suitably, the pharmaceutical composition further comprises one or more additional pharmaceutically active agents as defined herein.

[0086] Suitably, the pharmaceutical compositions described herein are for use in treating a condition or disorder as defined herein.

[0087] The compositions of the invention may be in a form suitable for oral use (e.g., as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), in a form suitable for topical use (e.g., as creams, ointments, gels, or aqueous or oily solutions or suspensions), in a form suitable for administration by inhalation (e.g., as finely divided powders or liquid aerosols), in a form suitable for administration by insufflation (e.g., as finely divided powders), or in a form suitable for parenteral administration (e.g., as sterile aqueous or oily solutions for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular administration or suppositories for rectal administration). The compositions of the invention may be in a form suitable for direct administration to the CNS (e.g., as sterile aqueous or oily solutions for local infusion into the CNS or for direct injection into the CNS).

[0088] The compositions of the present invention can be obtained by conventional procedures using conventional pharmaceutical excipients well known in the art. Thus, compositions for oral use may contain, for example, one or more coloring agents, sweeteners, flavoring agents and / or preservatives.

[0089] An effective amount of a nAChR antagonist / blocker for use in treatment is an amount sufficient to treat or prevent a condition associated with reduced dopamine levels in the CNS (mentioned herein), slow its progression, and / or alleviate the symptoms associated with the condition.

[0090] The amount of active ingredient combined with one or more excipients 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 for oral administration to humans typically contains, for example, 0.5 mg to 0.5 g of active agent (more suitably 0.5 to 100 mg, such as 1 to 30 mg), compounded with an appropriate and convenient amount of excipients, which may comprise about 5% to about 98% by weight of the total composition.

[0091] According to well-known medical principles, the size of the dose of nAChR antagonists / blockers for therapeutic or prophylactic purposes will naturally vary depending on the nature and severity of the condition, the age and sex of the animal or patient and the route of administration.

[0092] When using nAChR antagonists / blockers for treatment or prevention purposes, it is usually applied in the following manner: the daily dose accepted ranges from, for example, 0.1 mg / kg body weight to 75 mg / kg body weight, if necessary, in divided doses. Generally, when parenteral routes are adopted, lower doses are applied. Therefore, for example, for intravenous or intraperitoneal administration, a dosage of, for example, 0.1 mg / kg body weight to 30 mg / kg body weight will generally be used. Similarly, for administration by inhalation, a dosage of, for example, 0.05 mg / kg body weight to 25 mg / kg body weight will be used. Oral administration may also be suitable, particularly in tablet form. Generally, a unit dosage form will contain about 0.5 mg to 0.5 g of nAChR antagonists / blockers.

[0093] combination

[0094] The nicotinic acetylcholine receptor antagonist / blocker treatment defined above can be applied as a monotherapy, or, in addition to the nicotinic acetylcholine receptor antagonist / blocker, can also involve other medicaments conventionally used to treat related conditions. Therefore, the nicotinic acetylcholine receptor antagonist / blocker or pharmaceutical composition can be used in combination with one or more additional pharmaceutically active agents.

[0095] Such combination therapy may be achieved by simultaneous, sequential or separate administration of the individual components of the treatment.Such combination products employ the nicotinic acetylcholine receptor antagonist / blocker within the dosage range approved hereinabove and the other pharmaceutically active agent within the approved dosage range.

[0096] Another aspect of the invention provides a nicotinic acetylcholine receptor antagonist / blocker or a pharmaceutically acceptable salt thereof for use in combination with another agent in the treatment of a condition or disorder associated with reduced dopamine levels in DNS. The condition or disorder may be any condition or disorder as defined herein.

[0097] Suitably, the additional pharmaceutically active agent is an agent that increases dopamine levels in the CNS by a mechanism of action different from that of nicotinic acetylcholine receptor antagonists / blockers. Examples of such agents include dopamine precursors (such as levodopa, co-careldopa, cobeneldopa), dopamine agonists (such as pramipexole, ropinirole, apomorphine, rotigine), MAO-B inhibitors (such as rasagiline, selegiline, safinamide), COMT inhibitors (such as entacapone, opicapone), amantadine, anticholinergics (such as procyclidine, trihexyphenidyl) or ritalin.

[0098] Another aspect of the present invention provides nicotinic acetylcholine receptor antagonist / blocker or its pharmaceutically acceptable salt, for treating Parkinson's disease, it and following combination: dopamine precursor (such as levodopa, carbendazim, cobeneldopa), dopamine agonist (such as pramipexole, ropinirole, apomorphine, rotizine), MAO-B inhibitor (such as rasagiline, selegiline, safinamide), COMT inhibitor (such as entacapone, opicapone), amantadine or anticholinergic drug (such as procyclidine, trihexyphenidyl). Suitably, nicotinic acetylcholine receptor antagonist / blocker or its pharmaceutically acceptable salt and dopamine precursor (such as levodopa, carbendazim, cobeneldopa) are combined for treating Parkinson's disease.

[0099] In this article, when the term "combination" is used, it should be understood to refer to simultaneous, separate or sequential administration. In one aspect of the invention, "combination" refers to simultaneous administration. In another aspect of the invention, "combination" refers to separate administration. In another aspect of the invention, "combination" refers to sequential administration. When administered sequentially or separately, a delay in administering the second component should not result in a loss of the beneficial effects of the combined treatment.

[0100] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising a nicotinic acetylcholine receptor antagonist / blocker or a pharmaceutically acceptable salt thereof, in combination with a medicament that increases the level of dopamine in the CNS by a different mechanism of action from the nicotinic acetylcholine receptor antagonist / blocker, and a pharmaceutically acceptable diluent or carrier.

[0101] In another embodiment, the invention relates to a therapeutic combination comprising a nicotinic acetylcholine receptor antagonist / blocker and another agent that increases dopamine levels in the CNS by a different mechanism of action than the nicotinic acetylcholine receptor antagonist / blocker. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] The embodiments of the present invention are further described below in conjunction with the accompanying drawings, which are as follows:

[0103] Figure 1 .Activation of ChI inhibits subsequent DA release evoked by electrical stimulation.

[0104] a, Schematic diagram of stimulation configurations for panels cj. Blue light stimulation (Lstim) of ChI expressing ChR2-eYFP, local electrical stimulation (Estim) in striatal slices of ChAT-Cre:Ai32 mice.

[0105] b, ChR2-eYP expression in choline acetyltransferase (ChAT)-immunoreactive striatal neurons.

[0106] Scale bar, 40 μm.

[0107] c, i, Top, [DA] evoked by single pulses of Lstim (light gray line, left) or Estim (light gray line, left) or Estim (black line, right), or the composite response of paired Lstim+Estim pulses (purple) in the DLS (c) and NAcc (i). o Mean transients (±SEM) of a representative experiment with ISIs of 8–200 ms. Dark dashed traces, minus the [DA] evoked by Lstim o The latter is attributable to the paired Estim [DA] o Lower panel, [DA] evoked by single or paired electrical pulses (dark grey lines) in the presence of DHβE (1 μM) in DLS (c) and NAcc (i). o The light dashed trace is the mean transient (± SEM) of [DA] induced by a single Estim minus the [DA] o The latter is attributable to the paired Estim [DA] o .

[0108] d, j, Mean peak [DA] evoked by paired Estim in the DLS (d, N = 5 animals) and NAcc (j, N = 5 animals). o (±SEM) (normalized to [DA] induced by a single Estim) o) Graph relative to ISI. *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA with Fisher's LSD post hoc test.

[0109] (e, k) [DA] induced by subthreshold light pulses (Lstim), single Estim, or pairs of Lstim and Estim in DLS (e) or NAcc (k) o Mean transients (±SEM) of a representative experiment with an ISI of 25–100

[0110] ms.

[0111] f, l, Mean peak [DA] evoked by double stimulation in the DLS (f, N = 5 animals) and NAcc (l, N = 5 animals). o (±SEM) (normalized to [DA] induced by a single Estim) o ) Graph relative to ISI. *P<0.05, **P<0.01, ***P<0.001, one-sample t-test vs. single electrical stimulation.

[0112] Figure 2 Activation of ChI inhibits subsequent DA release induced by optogenetic stimulation

[0113] a, Schematic diagram of the stimulation configuration of Fig. MT. Local electrical stimulation (Estim) and blue light stimulation (Lstim) of ChR2-eYFP-expressing DA axons in striatal slices.

[0114] b, ChR2-eYFP expression in midbrain DA neurons co-immunoreactive with DAT in DAT-Cre mice following e.g. VTA injection. Scale bar, 400 μm.

[0115] c, g, [DA] evoked by single pulses of Estim (gray lines, left) or Lstim (light gray lines, right) or paired Estim+Lstim pulses (dark gray) in the DLS (c) and NAcc (g) when (upper) nAChRs are active (without DHβE) or (lower) when nAChRs are inhibited (with DHβE) in DAT-Cre mice. o Mean transients (±SEM) of a representative experiment with an ISI of 7–200 ms. Dark dashed trace, subtracted [DA] evoked by Estim in the absence of DHβE o The latter is attributable to the paired Lstim [DA] o , light dashed line, [DA] attributed to paired Lstim in the presence of DHβE o .

[0116] d, h, Mean peak [DA] evoked by paired Lstim in the DLS (d, N = 5 animals) and NAcc (h, N = 5 animals). o (±SEM) (normalized to [DA] evoked by a single Lstim) o ) Graph relative to ISI. *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA with Fisher's LSD post hoc test.

[0117] e, i, In wild-type animals, the DLS (e) and NAcc (i) were stimulated by a single full-strength Estim (gray line, left, top), paired Estim (gray shading, top), or a single low-strength Estim (Estim 50 , light grey line, lower figure) and full force Estim (dark grey, lower figure) in pairs [DA] o Mean transients (±SEM) of a representative experiment with an ISI of 25–100 ms. Light (lower) and dark (upper) dashed traces, [DA] evoked by Estim 1p subtracted o The latter is attributable to the paired Estim [DA] o f, j, Mean peak [DA] evoked by paired stimulation in the DLS (F, N = 5 animals) and NAcc (J, N = 5 animals). o (±SEM) Plotted relative to ISI. In e, f, i and j, DHβE is present. **P<0.01, ***P<0.001, two-way ANOVA with Fisher's LSD post hoc test.

[0118] Figure 3 Activation of ChI and nAChRs inhibits repeated axonal depolarization and calcium accumulation in DA axons and leads to pronounced nAChR desensitization over longer time intervals

[0119] a, Images of the VTA and SNc of DAT-Cre:Ai95D mice show GCaMP6f-eGFP expression (top) in TH-immunopositive neurons (middle).

[0120] b, Schematic representation of the stimulation configuration in living tissue.

[0121] c, Ca in DA axon populations imaged in DLS in response to single electrical pulses or trains of 4 electrical pulses (4p) at 100 Hz under control conditions (left) and in the presence of DHβE (1 μM) (right). 2+Examples of imaging responses (GCaMP6f fluorescence change, ΔF / F) (mean ± SEM obtained from replicates).

[0122] d, Plot of mean peak (±SEM) of GCaMP6fΔF / F vs. number of pulses. Data were normalized to the value of 1 pulse (N=5 animals). ***P<0.0001, paired t-test, [DA] o From 4 stimulation pulses.

[0123] e, Images of the VTA and SNc of DAT-Cre mice with ASAP3 expression (top) and TH-positive neurons (middle).

[0124] f, Schematic representation of stimulation configuration in living tissue.

[0125] g, Average transients of voltage sensors in striatal dopamine axons to single or 4 electrical pulses at 50 Hz before and after nAChR blockade with DHβE (ASAP3 fluorescence change, -ΔF / F, mean ± SEM). Scale bar vertical axis applies only to 1p data. For 4-pulse data, the peak seen after the first stimulus pulse was scaled to match the peak transient seen with a single pulse (n = 7 recordings from N = 4 animals).

[0126] h, Average responses at the time following consecutive pulses in the pulse train before and after DHβE (gray in G).

[0127] i, k, [DA] evoked by single pulses of Lstim (left, upper panels) or Estim (left, lower panels), or by pairs of Lstim with 1 or 3 Estim pulses (labeled) in the DLS (i) and NAcc (k) in the striatum of ChAT-Cre:Ai32 mice. o Mean transients (±SEM) of a representative experiment with an ISI of 25–400 ms across activation, desensitization, and resensitization. Light and dark dashed traces, [DA] attributable to paired stimulation after subtraction of Lstim. o .

[0128] j, l, Top, Mean peak [DA] evoked by paired Estim at 1p (circles) and 3p (squares) for DLS (j, N = 5 animals) and NAcc (l, N = 5 animals). o (±SEM) (normalized to [DA] induced by a single Estim) o ) versus ISI. Below, [DA] caused by 3p and 1p Estim o *P < 0.05, **P < 0.01, one-way ANOVA with post hoc Tukey test.

[0129] Figure 4 In a PD mouse model, nAChR antagonism in DLS promoted DA release, conditioned place preference and improved motor deficits in vivo.

[0130] a, Schematic diagram of the recording and stimulation electrode configuration in DLS.

[0131] b, Color plot and representative line graph of voltammetric DA current versus time of oxidation current (from dashed line at approximately +0.7 V) and corresponding DA cyclic voltammograms (from time points of black dotted line) before and after mecamylamine (ip).

[0132] c, DA signals (mean + SEM) evoked by 1 pulse or 10 pulses at 100 Hz before (black) and after (red) mecamylamine (2 mg / kg, ip).

[0133] d, Mean peak DA current (±SEM). **P<0.01, Sidak post hoc multiple comparison test (N=4 animals).

[0134] h, Schematic diagram of the bilateral cannula system used for local infusion in the dorsal striatum and example of a half-section stained with DAPI.

[0135] i, Conditioning paradigm. Mice were locally infused with mecamylamine (10 μg / side, right ventricle, gray) or saline (0.5 μl, within 1 min, left ventricle, black) and then conditioned four times (20 min each) over two days, while the control group received saline infusions in both ventricles.

[0136] j, Representative follow-up traces from the date after conditioning.

[0137] k, Preference for the right ventricle after conditioning. *P < 0.05, paired t-test, N = 5 animals in each test.

[0138] 1, Total distance traveled and locomotor speed in the open field test (N=5 animals).

[0139] m, Schematic diagram of TH staining after unilateral 6-OHDA injury.

[0140] n, Cylinder test paradigm. Mice were tested before and after 6-OHDA injection on the lesion day and before and after mecamylamine infusion from the same cannula on the test day.

[0141] o, Intrastriatal mecamylamine infusion reduces contralateral forelimb reaching deficits seen after injury, *P < 0.05, **P < 0.01, paired t-test, N = 6 animals in each test.

[0142] Figure 5 In the computational model, ChI-induced inhibition dominates striatal DA release.

[0143] a, Values ​​used in the model for the potency of ChI-induced inhibition of DA release (dark gray) and normalized levels of apparent nAChR desensitization (light gray) in the DLS (upper panel) and NAcc (lower panel) are plotted versus time after ChI activation.

[0144] b, When the firing rate of DA neurons (labeled) is constant, a brief decrease (solid line) or increase (dashed line) in ChI activity (labeled) can increase or decrease [DA] in the DLS (labeled) and NAcc (labeled), respectively. o .

[0145] c, Top row, Multiphase ChI responses (labeled, light grey) with (left) or without (right) an initial excitation phase and from 16 DA neuron burst activity (markers, black) predicts striatal DA release in the DLS and NAcc (lower row) when the tonic level of ChI-induced inhibition of DA release was set to 0% (markers, upper middle line), 50% (markers, lower middle line), and 100% (markers, lower bottom line). o Normalization was to the value seen with zero tonic ChI inhibition (marker, no ChI effect).

[0146] d, Peak [DA] in the DLS (grey) and NAcc (black) with (left) and without (right) initial excitation during ChI multiphasic activity. o (open circle) and [DA] o Summary of the area under the curve (filled circles) when ChI-induced inhibition was set at 0%, 50% and 100% in ChI tonic activity.

[0147] e, Peak [DA] when ChI was not and was stimulated initially o (Left) and [DA] o The area under the curve (right panel) is the ratio of release.

[0148] f, Schematic diagram showing that activation of ChI and nAChR on DA axons limits action potential propagation.

[0149] Figure 6 .Subthreshold light stimulation (Lstim0) intensity that induces detectable DA release.

[0150] a, Figure 1 The intensity of Lstim0 used to stimulate ChI in ChAT-Cre:Ai32 mice was significantly lower than Lstim in both the DLS (left, dark gray) and NAcc (right, gray). N = 5 animals, ***P < 0.001, one-sample t-test.

[0151] b, Schematic diagram of the stimulation configuration for the data in c. Blue light stimulation (Lstim) of ChI expressing ChR2-eYFP followed by local electrical stimulation (Estim) in striatal slices of ChAT-Cre:Ai32 mice.

[0152] c, Mean transients (±SEM) from a representative experiment in DLS showing inhibition of GABA by bicuculline (10 μM) and CGP 55845 hydrochloride (2 μM), respectively. A and GABA B receptors, induced by a subthreshold light pulse (Lstim0), a single Estim, or a pair of Lstim and Estim [DA] o , ISI is 25ms. When GABA receptors are inhibited, the average peak [DA] evoked by double stimulation o (±SEM), normalized to [DA] induced by a single Estim o (n=5 recordings, N=3 animals). ***P<0.001, one-sample t-test vs. Estim.

[0153] Figure 7 ASAP3 expression and dynamic DA release responses in DA neurons following photoactivation of ChI (100-400ms interval) persisted in DLS in the presence of muscarinic receptor or D2 receptor antagonists.

[0154] a, Expression of ASAP3-GFP in DA neurons co-labeled with TH immunoreactivity in the midbrain after viral injection. b, Co-expression of ASAP3 and TH in dopamine axons in DLS. CC: corpus callosum (scale bar, 400 μm). Schematic diagram of the stimulation configuration used in c, d, and e. Blue light stimulation (Lstim) of ChI expressing ChR2-eYFP followed by local electrical stimulation (Estim) in striatal slices of ChAT-Cre:Ai32 mice. d, e, Left, Average peak [DA] evoked by subsequent Estim of 1 p (grey) or 3 p 100 Hz (black) within the ISI range after Lstim in DLS following antagonism of muscarinic receptors with atropine (2 μM) (d, n = 5 recordings, N = 3 animals) or antagonism of D2 receptors with L-741,626 (1 μM) (e, n = 5 recordings, N = 4 animals). o(±SEM), normalized to [DA] induced by a single Estim o Right: [DA] caused by 3p and 1p Estim o The bell curves of both groups showed a significant effect of IPI, P < 0.001, one-way ANOVA.

[0155] Figure 8 . Computational model predictions from empirical in vitro data.

[0156] Figure 1 The computational model based on empirical data in n and r predicts Figure 1 d, h show the effect of previous activation of ChI on subsequent DA release induced by electrical stimulation. Figure 1 The second stimulus in d and h) caused DA DA and DA ChI , rather than just light stimulation ( Figure 1 n, r) induced by the second stimulus DA , the predicted data (triangular plots) were therefore scaled to match the overall mean of the experimental data (circular plots) in DLS (left) and NAcc (right) to overcome the unknown ChI-induced DA in this experimental paradigm. ChI of inhibition.

[0157] Fig. 9 During multiphasic ChI activity, "desensitization" of nAChRs plays a limited role in modifying phasic DA release in computational models.

[0158] a, Synchronous phasic DA neuron firing (labeled, dark gray) and multiphasic ChI activity (labeled, light gray) with (left) or without (right) initial firing in ChI. Inset, firing rate normalized to baseline rate.

[0159] b, In the DLS (upper panel) and NAcc (lower panel) models (from Figure 2b) Phasic DA release is altered by ChI activity both with (solid line) and without (dashed line) a "desensitizing" component (marked, top line compared to no ChI effect). Background levels of tonic inhibition of DA output by ChI included in the model were 0%, 50%, and 100%. Adding a dynamic desensitizing component to the model affected phasic DA release only when there was an initial ChI challenge (left panel), and only in the DLS did it slightly increase phasic DA release by preventing nAChRs from inhibiting DA release during the ChI rebound activity phase. This effect was more pronounced in the DLS (top panel) than in the NAcc (bottom panel), consistent with a stronger ChI-induced inhibition of DA release in the DLS than in the NAcc. As expected, excluding the dynamic desensitizing component from the model did not affect DA release when the initial challenge was absent because there was no initial increase in nAChR activation.

[0160] Fig.10 In the computational model, the tonic level of ChI-induced inhibition of DA release altered the relative influence of ChI multiphasic activity on phasic DA release.

[0161] a, Top, Model of synchronized phasic DA neuron firing ((markers, dark gray) and multiphasic ChI activity ((markers, light gray)) with (left) or without (right) initial firing in ChI. Bottom, Comparison of phasic DA signals at three different background levels (0, 50, 100%) of tonic inhibition of DA output before (dashed lines) and after (solid lines) the development of multiphasic activity. At low-moderate levels of tonic ChI inhibition of DA release (≤50%), multiphasic ChI activity reduces phasic DA release, whereas at very high levels of tonic ChI inhibition of DA release (50-100%), ChI multiphasic activity enhances phasic DA release.

[0162] b, Summary of the effects of multi-phase ChI activity on phasic DA release under tonic inhibition of DA release by tonic ChI activity at different background levels. Changes in phasic DA signal amplitude and area under the curve (AUC) with (left) or without (right) an initial ChI excitation phase. DETAILED DESCRIPTION

[0163] Mesostriatal dopamine (DA) neurons play an important role in action selection and behavioral learning 1-4 Striatal DA release has long been thought to be primarily determined by action potentials generated by DA cell bodies in the midbrain, but increasing evidence suggests that the dynamics of striatal DA release can be controlled locally in DA axons. 5-7 , and in behaving animals can be uncoupled from DA neuron firing in the midbrain8,9 Striatal ChI acting on nAChRs on DA axons may play a key role 5-7,10,11 In vivo, ChI has dynamic tonic activity during spontaneous behavior 12,13 , and also forms a multi-stage excitation-pause-rebound activity during the learning process, that is, the so-called "pause response" lasting 200-400ms 14,15 , which is consistent with phasic activity in DA neurons 16,17 Somewhat paradoxically, both ChI excitation and pausing are thought to enhance DA release. 5,6,18 Activation of a small fraction of ChIs drives DA release via nAChR activation 5,6 In addition, loss or suspension of striatal nAChR activity due to antagonism, nicotine desensitization, or forebrain ACh depletion promotes the frequency-dependent release of DA. 7,11,18,19 .

[0164] However, the inventors noted that the highest levels of DA output were not elicited when nAChRs were activated, but rather when nAChRs were inactivated. 6,7 This long-overlooked result may indicate that activation of nAChRs has a critical, unexplored negative effect on DA. Here, by activating ChI and DA axons in vitro, in vivo, and in silico, the inventors reveal that stimulation of ChI at levels above or below the threshold for driving DA release acts on nAChRs by preventing action potential-dependent depolarization and Ca spiking in DA axons. 2+ Accumulation, with a short-latency inhibition of subsequent DA release. Relieving this ChI-induced inhibition of DA release can improve motor deficits in PD mouse models. Our data provide a new framework to understand how ACh controls DA release, primarily through dynamic inhibition of DA after nAChR activation.

[0165] The inventors have found that striatal cholinergic interneurons exert a strong inhibitory effect on dopamine axons after activating nicotinic acetylcholine receptors (nAChRs) on dopamine axons, thereby limiting the release of dopamine. This inhibitory effect is particularly strong in the human dorsal striatum (equivalent to the caudate nucleus / putamen (CPu)), which is the area most severely affected by the loss of dopamine neurons in the substantia nigra pars compacta (SNpc).

[0166] The inventors have demonstrated that blocking nAChR can enhance dopamine signals in vitro and in vivo, and can induce conditioned place preference in mice. The administration of nAChR antagonists / blockers will alleviate the inhibition of dopamine release caused by striatal cholinergic interneurons. Compared with traditional treatments for conditions such as Parkinson's disease, blocking nicotinic acetylcholine receptors will amplify the physiological pattern of endogenous dopamine release that follows the firing pattern of dopamine neurons. The administration of nAChR antagonists / blockers is not expected to distort the release of dopamine like the dopamine precursor levodopa, which may cause a general increase in dopamine levels in the striatum, including by releasing inappropriate non-dopamine inputs, and may cause serious side effects after a few years. This new treatment will have the potential to extend the therapeutic window of levodopa and reduce the motor deficits of Parkinson's disease.

[0167] In the present invention, it is shown in the mouse striatum that the brief activation of ChI and nAChR subsequently significantly inhibits dopamine release with a short delay (<7ms) lasting 50-100ms, and the inhibitory effect in the dorsolateral striatum is stronger than that in the ventral striatum. The inhibition of dopamine output is not caused by exhaustion, but is reflected by the intracellular calcium dynamics in dopamine axons. Accordingly, the antagonism of nAChR promotes dopamine release in the dorsal striatum in vivo and induces the position preference of conditioned action. Computational models show that ChI-induced dopamine release inhibition is dominant during the multi-stage burst-pause and tonic activity of ChI, especially in the dorsal striatum. Therefore, the present invention reveals that ChI exerts a regional specific effect on dopamine output, which, contrary to the general view, is mainly inhibitory, with the ability to uncouple dopamine release from somatic activity and critically shape dopamine function.

[0168] Materials and methods

[0169] animal

[0170] Male adult mice used for in vitro experiments and in vivo DA recordings were C57Bl6 / J mice (Charles River, UK) (21-40 days), ChAT-cre:Ai32 (6-16 weeks), DAT-IRES-Cre (8-16 weeks), or DAT-Cre:Ai95D (4-7 weeks). + / + Mouse (B6; 129S6-Chat tm2(cre)Lowl / J, JAX stock number 006410) and Ai32 + / + Mouse (B6; 129S-Gt(ROSA)26Sor tm32(CAG-COP4*H134R / EYFP)Hze / J, JAX stock number 012569) to generate heterozygous ChAT-Cre:Ai32 mice. pAAV-double floxed-hChR2(H134R)-EYFP-WPRE-pA was injected into DAT-IRES-Cre + / - Mouse (B6.SJL-Slc6 a3tm1.1(cre)Bkmn / J, JAX stock number 006660) to express ChR2 in DA axons (8-16 weeks). + / - Mice and Ai95D + / + Mouse (B6:129S-Gt(ROSA)26Sor tm95.1(CAG -GCaMP6f)Hze Heterozygous DAT-Cre:Ai95D mice were generated.

[0171] Male C57BL / 6N mice (Charles River, Beijing, China) (42–50 days old) were used for behavioral experiments. Animals were group-housed and maintained on a 12-h light / dark cycle with free access to food and water. Procedures for ex vivo recordings and in vivo DA recordings under anesthesia were in accordance with the Animals (Scientific Procedures) Act 1986 (as amended in 2012) and ethical approval from the University of Oxford, and were performed under project permission granted by the UK Home Office. Behavioral experiments were performed using protocols approved by the Animal Care and Use Committee of the Chinese Institute for Brain Research (#CIBR-IACUC-007) and in accordance with guidelines established by the National Institutes of Health.

[0172] Virus injection

[0173] Heterozygous DAT-IRES-Cre mice were injected intracerebrally with a Cre-inducible recombinant AAV serotype 5 vector containing an inverted gene for channelrhodopsin 2 fused in frame to a gene encoding enhanced yellow fluorescent protein (pAAV-doublefloxed-hChR2(H134R)-EYFP-WPRE-pA) (titer = 1E+12 vg / ml, University of North Carolina Vector Center) or a Cre-inducible recombinant AAV serotype 5 vector containing the EF1A promoter and the ASAP3 inverted gene (titer = 2.4E+12 vg / ml) without a somatic targeting signal and a WPRE RNA stabilizing element (Stanford Gene Vectors and Virus Core). Mice were placed in a stereotaxic frame under isoflurane anesthesia and a craniotomy was performed above the injection site. 1 μL of virus was injected unilaterally or bilaterally into the VTA (coordinates (mm) from bregma: AP-3.1, ML±0.5, DV-4.4) or SNc (coordinates (mm) from bregma: AP-3.5, ML±1.2, DV-4.0) using a 2.5 μL 33-gauge Hamilton syringe with a microinjector at 0.2 μL / min. After each injection, the syringe was left in place for 5 minutes and then slowly retracted. After surgery, the animals were maintained for at least 3 weeks to allow viral expression in the striatum.

[0174] In vitro slice voltammetry

[0175] To perform fast scan cyclic voltammetry (FCV) in acute coronal slices, animals were anesthetized with isoflurane. The brain was quickly moved into an ice-cold high Mg 2+ Cutting solution, which contains (in mM): 85NaCl, 25NaHCO3, 2.5KCl, 1.25NaH2PO4, 0.5CaCl2, 7MgCl2, 10 glucose, 65 sucrose. The brain was then blocked and 300μm coronal sections were cut using a vibrating slicer (LeicaVT1200S) between +1.5 and +0.5mm from the anterior bregma containing the caudate putamen and nucleus accumbens. After dissection, the slices were recovered at 32°C for 30-40 minutes and then stored at room temperature. The slices were maintained and recorded in artificial cerebrospinal fluid (aCSF) containing the following substances in mM: 130NaCl, 25NaHCO3, 2.5KCl, 1.25NaH2PO4, 2.5CaCl2, 2MgCl2, 10 glucose. The aCSF was saturated with 95% O2 / 5% CO2; recordings were performed at 32-33°C. FCV was used to measure the extracellular DA concentration ([DA] o), using a 7 μm diameter carbon fiber microelectrode (CFM; tip length 50-100 μm) and a Millar voltammeter (Julian Millar, Barts and the London School of Medicine and Dentistry) as previously described. 55 The voltage was applied as a triangular waveform (ranging from -0.7 to +1.3 V, relative to Ag / AgCl) with a scan rate of 800 V / s and a data sampling rate of 8 Hz.

[0176] 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 via a fiber optic cable (200 μm multimode, NA 0.22). Point illumination was 30 μm in diameter under a x40 immersion objective. The laser pulse duration at the sample was 2 ms, 23 mW / mm 2 In online or offline analysis, Lstim0 (extended Figure 1 ).

[0177] For electrical stimulation, 0.65 mA current (200 μs pulse width) was delivered through a surface bipolar concentric Pt / Ir electrode (125 μm outer diameter, 25 μm inner diameter, FHC, USA) placed approximately 100 μm away from the recording electrode. 50 Caused during online analysis [DA] o Approximately [DA] obtained with normal stimulation (0.65 mA) o The stimulation current was 50%. The timing of stimulation was designed to avoid FCV scanning.

[0178] Calcium imaging

[0179] As previously studied 21Widefield fluorescence imaging of GCaMP6f in dopaminergic axons in DLS in ex vivo slices in response to single and series (4 pulses, 100Hz) electrical stimulation pulses was performed using an Olympus BX51Wl microscope equipped with an OptoLED Lite system (CAIRN Research), a PrimeScientific CMOS (sCMOS) camera (Teledyne Photometrics), and a x40 / 0.8NA water objective (Olympus UK). Images were acquired every 2.5 minutes at a frame rate of 16.6Hz using Micro-Manager 1.4, and stimulation and recording were synchronized using a custom-written program in Igor Pro 6 (WaveMetrics) and an ITC-18A / D board (Instrutech). Image files were analyzed using Matlab R2019b and Fiji 1.5. The inventors extracted fluorescence intensities from regions of interest (ROIs) and equal background areas (on the stimulating electrodes) without GCaMP6f expression. After background subtraction, Ca was measured by fitting an exponential curve function through the baseline (2 s before stimulation) and the last 1 s of the 7.2 s recording window. 2+ Transients were corrected for photobleaching. The order of single and train stimulations was alternating and evenly spaced, and data were collected repeatedly before and after the extracellular experimental conditions were changed. Data are expressed as ΔF / F, where F is the fitted curve.

[0180] Voltage sensor imaging

[0181] Widefield fluorescence imaging of ASAP3 in dopaminergic axons in DLS in ex vivo slices in response to single and trains of (4 pulses, 500 Hz) electrical stimulation pulses was performed using an Olympus BX51Wl microscope equipped with an OptoLED Lite system (CAIRN Research), an iXon EMCCD camera (ANDOR), and a x40 / 0.8NA water objective (Olympus UK). Images were acquired at a frame rate of 660 Hz every 2.5 minutes using Micro-Manager 1.4, and stimulation and recording were synchronized using PClamp. Image files were analyzed using Matlab R2019b and Fiji 1.5. The inventors extracted fluorescence intensity from the region of interest (ROI). ASPA3 transients were bleached corrected by fitting an exponential curve function. The order of single stimulation and sequence stimulation was alternating and evenly distributed, and data were repeatedly collected before and after the extracellular experimental conditions were changed. The data are expressed as ΔF / F, where F is the fitted curve.

[0182] Anesthesia in vivo voltammetry

[0183] Wild-type mice were anesthetized with urethane (1.4–1.9 g / kg, intraperitoneal injection; Biolab) and supplemented with urethane (0.2 g / kg) every 1–2 h as needed. All wounds and pressure points were infiltrated with bupivacaine (0.5%). After surgical anesthesia was achieved, the head was fixed in a stereotaxic frame (Kopf, USA). The core temperature was maintained at 35–36 °C using a homeothermic blanket and monitored by a rectal probe (TR-100, Fine Science Tools). A circular piece of skull covering the left hemisphere was removed to target the DLS (AP +1.0 mm, ML 1.6 mm, DV 2.2 mm to bregma). A stimulation and recording array consisting of carbon fiber microelectrodes and bipolar stimulating electrodes (MS303 / 3-A / SPC, P1 Technology) was placed in the DLS. An Ag / AgCl reference electrode was implanted in another part of the forebrain. [DA] was measured using FCV with a 7-μm diameter carbon fiber microelectrode (CFM; tip length 50–100 μm) and the Tarheel system (University of Washington, Seattle, USA). o The voltage was applied as a triangular waveform (ranging from -0.4 to +1.3 V vs. Ag / AgCl) with a scan rate of 400 V / s and a data sampling rate of 10 Hz. The position of the FCV electrode tip was confirmed by histological methods.

[0184] For striatal electrical stimulation, 0.65 mA current (200 μs) was delivered through bipolar stimulating electrodes (0.005 inches, MS303 / 3-A / SPC, P1 Technologies). The stimulating electrode tips were approximately 500 μm apart and glued to the FCV recording electrode to fix the FCV electrode tip between the two stimulating electrodes.

[0185] Behavior Records

[0186] Cannula placement: Male C57BL / 6N mice (42-50 days old) were anesthetized with isoflurane (5% induction; 1.5-2% maintenance) and placed on a stereotaxic frame for surgery. Bilateral injection needles (OD 0.21 mm, ID 0.11 mm, RWD, China) were implanted vertically or at a slight angle to the vertical into the dorsal striatum with a guide cannula (OD 0.41 mm, ID 0.25 mm, RWD, China), with the tip of each cannula aligned to the following coordinates: AP +1.0 mm; ML + / -1.6 mm to bregma; DV -2.4 mm (from dura). Mice recovered for three days after surgery.

[0187] Conditioned place preference test: In the CPP experiment, mice were placed in a 40 cm × 40 cm transparent plexiglass arena divided into two equal chambers by a doorway. The chambers were decorated with horizontal or vertical stripes. The movement of the animals was recorded and analyzed using SmartV3.0 tracking software (Panlab, Spain).

[0188] On the 1st day, mice were allowed to shuttle freely between the two chambers to assess the position preference at the baseline, expressed as the percentage of time spent in the right chamber. On the 2nd and 3rd days, mice were conditioned, and mecamylamine (10 μg / side) or saline vehicle (0.9%) was injected alternately into the bilateral striatum of the animals within 1 minute in the morning and afternoon, and the injection volume was 0.5 μl. The animals were then confined to the right or left chamber for 20 minutes respectively. Treatment was balanced according to the time of the day. On the 4th day, the chamber preference after conditioning was calculated as the percentage of time spent in the chamber of mecamylamine on the right side compared to the 1st day before conditioning. In the next two days (5th-6th day), animals received bilateral saline injections and explored the two chambers for 20 minutes, after which the position preference disappeared. The conditioning procedure was then repeated, but both chambers used bilateral saline, and the 7th day was tested before conditioning, the 8th-9th day was conditioned for two days, and the 10th day was tested after conditioning. To minimize bias in place preference at baseline, five animals that showed the least place preference on the day before conditioning were selected for subsequent conditioning in each test (mecamylamine 42%-58%; control group 45%-55%). For the open field test, mice were injected with saline vehicle or mecamylamine (10 μg / side) bilaterally into the striatum and then placed in an open field chamber to assess total running distance and average speed over 20 minutes.

[0189] 6-OHDA injury and reaching task: On the injury day, 6-hydroxydopamine (1 μl 0.9% saline solution containing 4 μg / μL 6-OHDA (Sigma-Aldrich) and 0.02% acetic acid) was injected into the right dorsolateral striatum through a pre-implanted cannula within 5 minutes. On the test day, a volume of 0.5 μl of mecamylamine (10 μg / side) was injected into the injured (right) striatum through the cannula within 1 minute. Animals were killed immediately after behavioral testing. The fluorescence intensity of TH staining in the slices decreased by approximately 50% compared to the contralateral DLS measured with ImageJ (1.52p), confirming the loss of dopamine axons induced by 6-OHDA. The anterior commissure was used as 0% background fluorescence. During the injection of 6-OHDA and mecamylamine, the animals were briefly sedated with isoflurane.

[0190] The cylinder test was performed before and 10 min after 6-OHDA injection to confirm the acute effect of 6-OHDA.56 , and the test was performed before and again 10 minutes after mecamylamine injection. Mice were not habituated 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 fully observe and record the wall touches. Only touches of the wall with a single paw (ipsilateral or contralateral forelimb) were counted in the analysis. Data are contralateral touches, expressed as a percentage of the total number of single paw touches.

[0191] Immunocytochemistry

[0192] After voltammetric recordings in acute slices, slices were fixed in 4% paraformaldehyde in PBS containing 0.2% picric acid. Slices were fixed overnight at 4°C and then stored in PBS. Free-floating slices were then washed five times for 5 minutes in PBS and incubated in 0.5% Triton X-100 and 10% normal donkey serum.

[0193] ChI expressing ChR2-eYFP were identified as ChAT immunoreactive as described previously (Zhang et al., 2018). Fixed and rinsed sections 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 five times with PBS for 5 minutes each and incubated 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 for 2 hours at room temperature.

[0194] DA neurons and striatal DA axons co-expressing ChR2-eYFP, GCaMP6f-eYFP, or ASAP3 were identified by immunoreactivity for tyrosine hydroxylase (TH) as described previously. 21 The fixed and rinsed 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. The sections were then washed five times with PBS for 5 minutes each and incubated for 2 hours 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.

[0195] The sections treated as above were then washed with PBS and mounted on gel slides with Vectashield mounting medium (Vector Labs) and imaged using a Zeiss LSM880 confocal microscope system running ZEN black version 2.3 (Zeiss) at 20×, NA0.8, or using a 20× / 0.75NA objective and Fluoview software (Olympus) on a confocal microscope system (FV1000IX81; Olympus). Maximum intensity projections from z-stacks with a height of 30 μm were captured individually and the image stacks were compressed. Red fluorescence (TH and ChAT) was captured at 638–759 nm using 633 nm excitation. Green fluorescence (GCaMP, ChR2, and ASAP3) was captured at 493–630 nm using 488 nm excitation.

[0196] To verify the location of the carbon fiber in the dorsal striatum for in vivo FCV recording, the anesthetized mice were sacrificed and the brains were quickly removed and fixed in 4% paraformaldehyde (PFA) overnight. The fixed brains were then cut into 50 μm slices using a vibratome (Leica). The slices were rinsed 3 times with PBS and mounted on glass slides and then imaged under a microscope to determine the location of the recording site.

[0197] To verify the location of the intrastriatal injection cannula and 6-OHDA lesion in the behavioral experiment, mice were anesthetized by intraperitoneal injection of Avertin (250 mg / kg body weight) and cardiac perfusion with saline and 4% PFA. The brain was dissected and fixed in 4% PFA overnight, then dehydrated with 30% sucrose for 24 hours. The fixed brain was then frozen and sectioned into 50 μm slices using a vibrating microtome (Leica). The slices used to verify the placement of the cannula were rinsed 3 times with PBST (PBS containing 0.1% Tween20) and then mounted on glass slides with 5 μg / ml DAPI. Like the slices from the in vitro experiment, the slices used to verify 6-OHDA were stained with immunoreactivity to TH. The slices were imaged under an inverted confocal microscope (Zeiss) using 405nm laser excitation.

[0198] drug

[0199] DHβE and mecamylamine hydrochloride for in vitro and anesthesia 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 in vitro experiments were made into aliquots of 1000 times the final concentration in distilled deionized water and stored at -20°C. The drug stock solution was then diluted to the final concentration in carbonated aCSF immediately before use and applied by bathing. Drugs for in vivo experiments were dissolved in sterile saline to the final concentration.

[0200] Computational Model

[0201] The computational model was written in MATLAB. The model includes: (1) the dynamic strength of ChI-induced inhibition, which is caused by [DA] before and after inhibition of nAChR with DHβE under the second stimulus o Ratio determination (from Figure 1 n, r; DAT-Cre, light stimulation), which is fitted as a polynomial curve; (2) the apparent nAChR "desensitization" curve ( Figure 3 e, g), [DA] caused by 3 pulses and 1 pulse under the second stimulus o The difference between the change estimates (from Figure 3 f, h), which were normalized to the maximum value and fitted with a polynomial curve; and (3) [DA] observed after a single electrical stimulation in vitro o Dynamic release and uptake profiles to determine the in vivo [DA] o Modeled as a scalar product of DA neuron activity. The inventors also incorporated into the model a range of potential levels (0, 50, 100%) of background tonic ChI-induced inhibition of DA release caused by tonic activity in ChI. The inventors excluded potential components of DA release that could be driven by synchronous activation of ChI in certain experimental scenarios ( Figure 1 ) 5,6 , because the threshold for nAChR-mediated inhibition of DA release was found to be lower than that required to drive release in vitro (see Figure 1 ) and is satisfied in vivo after discrete striatal stimulation (see Figure 4 ). The strength of ChI-induced inhibition (1) is the best-fit polynomial curve, which is fitted as Figure 1 [DA] before and after nAChR inhibition in n and r o Ratio. (DLS: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). nAChR desensitization level (2) is from Figure 4 b, d, and normalized to its own maximum and minimum values. The normalized data were then best fitted with 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).

[0202] Quantification and statistical analysis

[0203] Statistical analysis was performed using GraphPad Prism 6.0. Data are presented as mean ± standard error of the mean (SEM). N values ​​are the number of animals and n values ​​are the number of individual records. One-sample t-test, t-test, and two-way ANOVA were used.

[0204] result

[0205] Activation of ChI inhibits subsequent DA release

[0206] The inventors tested the effect of ChI activation on subsequent DA release. In isolated striatal slices from ChAT-Cre:Ai32 mice ( Figure 1 a, b), the inventors used targeted optogenetic activation of ChI expressing ChR2 to activate nAChR and drive DA release (ChI-activated DA release, DA ChI ) 6 , and then an electrical stimulation pulse was applied 8-200ms later to directly drive DA release (DA DA ) and indirectly drive DA release by activating ChI (DA ChI , its delay time is short, about 10ms 10,20 ). In the dorsolateral striatum (DLS) ( Figure 1 c, d) and nucleus accumbens core (NAcc) ( Figure 1 In both cases (i and j), targeted activation of ChI induces DA release and then inhibits subsequent DA release induced by electrical stimulation 8–200 ms later, to the peak extracellular DA concentration ([DA] o For the cases where the stimulation interval was ≤50 ms in DLS and ≤25 ms in NAcc, this inhibition was significantly greater than the inhibition produced by the initial electrical stimulation in the presence of nAChR antagonist (DHβE, 1 μM) ( Figure 1c, d, i, j), while the stimulus interval and [DA] o There is a strong inverse relationship between 7,21 These data suggest that nAChRs inhibit subsequent DA release for a short period of time. D2 autoreceptors on DA axons can be ruled out as a contributing mechanism because D2 receptors only function after intervals exceeding 200 ms. 21 The inventors then tested whether the pronounced subsequent inhibition of DA release observed after ChI activation involved depletion of the proposed DA vesicle pool 10 The inventors used low light intensity (Lstim0) to activate ChI ( Figure 6 ), which is lower than the minimum intensity required to trigger detectable DA release (less than the DA release induced by normal Lstim ChI 0.5%)( Figure 1 e, k). Using Lstim0 to activate ChI continues to inhibit [DA] induced by subsequent electrical pulses o , until at least [DA] induced by a single electrical impulse o 20-60% (DLS) or 60-80% (NAcc) ( Figure 1 e, f, k, l). Thus, when the level of ChI / nAChR activation is below that required to drive DA release, subsequent DA release is inhibited regardless of the DA vesicle pool or the level of previous DA release. The inhibition of DA release is not mediated by striatal GABA, but rather by GABA from ChI. 22 or DA axons 23 , GABA neurons and tone from GABA 24 co-release of GABA receptors because it persists in the presence of GABA receptor antagonists ( Figure 6 ).

[0207] The inventors also explored the effects of targeted optogenetic activation of DA axons (DA DA ) is driven by whether the previous ChI activation will limit the subsequent DA release. The inventors used electrical stimulation to initially drive DA release (DA DA +DA ChI ), and then assessed DA activation driven by subsequent light activation of ChR2-expressing DA axons in isolated striatal slices from DAT-Cre mice. DA ( Figure 2 a, b). In DLS( Figure 2 c, d) and NAcc ( Figure 2 In g and h), electrical stimulation induces DA release, but the release is different from that induced by a single light pulse. o Compared to the [DA] caused by the subsequent light pulse oThis inhibition was relieved in the presence of an nAChR antagonist (DHβE, 1 μM) for interstimulus intervals <100 ms (DLS) or <50 ms (NAcc). The relief of inhibition was not due to a lower initial DA release level (approximately half): in wild-type mice, the inventors reduced the intensity of electrical stimulation (Estim 50 ) reduced the initial DA release to half, but found that subsequent full-intensity electrical stimulation induced a o The level is the same as that induced by full-intensity stimulation in DLS ( Figure 2 e, f), but only slightly higher in NAcc ( Figure 2 i, j).

[0208] Thus, ChI activation of nAChRs can inhibit DA release during subsequent DA axon activation or subsequent ACh release through a mechanism that is independent of DA pool availability, with a duration comparable to that of a multistage pause in ChI and that is more pronounced and prolonged in the DLS than in the NAcc.

[0209] Activation of nAChRs limits axonal Ca 2+ accumulation and subsequent depolarization

[0210] DA release is mainly regulated by Ca 2+ Axonal activation mechanisms upstream of Ca entry and control 2+ Mechanistic control of entry, intracellular buffering, and signaling 21,25,26 The inventors tested whether striatal nAChRs limit axonal Ca during continuous pulses. 2+ In brain slices of DAT-Cre:Ai95D mice ( Figure 3 a, b), the inventors investigated the Ca2+ in DA axons in DLS 2+ The reporter GCaMP6f was imaged as previously described. 21 , which were evoked by single pulses and 4-pulse trains (100 Hz) with or without nAChR antagonism. This stimulation protocol indicates nAChR activation: When nAChRs are activated, the pulse train-evoked [DA] o Only slightly higher than a single pulse, but when nAChR is antagonized, [DA] o Significantly higher 7,27 At the same time, when nAChRs are active, the axonal GCaMP6f fluorescence of pulse trains is only slightly stronger than that of single pulses ( Figure 3 c, d), but when the inventors antagonized nAChR (DHβE, 1 μM), the axonal GCaMP6f fluorescence was significantly stronger ( Figure 3 c, d). These data suggest that nAChR activation limits axonal [Ca2+ ] i accumulation.

[0211] To test whether nAChRs directly limit axonal depolarization during continuous stimulation of DA axons, we characterized ASAP3 voltage sensors expressed in DA axons in DLS in brain slices during single or 4-pulse (50 Hz) electrical stimulation. 28 The imaging was performed ( Figure 3 e, f, Figure 8 ). In the absence of nAChR antagonists, dopamine axons are depolarized by the first pulse but apparently not by subsequent pulses in the train ( Figure 3 g, d). In contrast, when nAChRs are antagonized, DA axons are able to depolarize with each successive stimulus pulse during the train ( Figure 3 g, h). These results suggest that nAChR activation prevents subsequent spike-induced depolarization and Ca 2+ Entering dopamine axons with a short latency limits subsequent dopamine release.

[0212] nAChRs activated by initial stimulation close during ChI rebound

[0213] In a multi-phase response, ChI shows a "rebound" increase in activity approximately 100-300ms after the initial stimulation 16,29 During these intervals, the short-latency ChI-induced inhibition of DA release has dissipated (see Figure 1 ). However, in the ensuing ~1 second, further DA release could only be driven by direct optogenetic activation of DA axons, but not by ChI. 6,10 This apparent refractory nature of DA release driven by ChI rather than DA axon activity suggests that nAChRs on DA axons are unresponsive to ACh released in response to rebound ChI activity. 30-32 Suspected to play a role in the dynamics of DA release during stimulation trains 33 The inventors tested whether the dynamics of DA release at the time of ChI rebound activity is consistent with nAChR desensitization. The inventors took advantage of the observation that activation of nAChRs limits DA release in subsequent pulses (e.g. Figure 1 ), whereas closing or desensitizing nAChRs promotes subsequent release during the pulse train. 7 As a potential readout of the time course of nAChR desensitization, the inventors explored the effects of triple- and single-pulse (100 Hz) pulses on [DA] oHow the differences change over time. In striatal slices from ChAT-Cre:Ai32 mice, the inventors activated ChI with light and then applied single or triple electrical pulses (100 Hz) 25-400 ms later ( Figure 3 i, j, see also Figure 7 c). [DA] caused by triple pulse and single pulse o The difference varied with interval, peaking at approximately 200 ms in the DLS and 100 ms in the NAcc, and decaying by 400 ms ( Figure 3 i, j, k, l). These dynamics are not caused by activation of either muscarinic receptors or D2-receptors ( Figure 7 d, e). These dynamics could reflect the time course of nAChR desensitization and resensitization, with regional differences reflecting distinct nAChR stoichiometries. 34,35 , and showed that nAChRs are at least partially desensitized during a period of ChI rebound activity approximately 100–300 ms after initial excitation.

[0214] In vivo nAChR antagonism promotes DA release and induces conditioned place preference

[0215] The inventors explored the effects of striatal nAChRs in vivo, where the balance or desensitization of nAChR-mediated DA excitation and inhibition may be different from that in vitro. In wild-type mice anesthetized with urethane, injection of nAChR antagonists into the DLS significantly increased the [DA] induced by local striatal stimulation with brief electrical pulse trains. o ( Figure 4 ad), indicating that nAChRs in vivo can be activated to inhibit DA release; their properties are not controlled by desensitization. In addition, the inventors tested whether inhibition of nAChRs by bilateral injections of mecamylamine in the dorsal striatum could promote reward-related learning in freely moving mice. 36 After two days of daily conditioning, mice developed a conditioned place preference for the chamber conditioned with intrastriatal mecamylamine but not saline controls ( Figure 4 This finding is consistent with previous in vivo studies in the adjacent NAc showing that nAChR antagonists increase reward-evoked DA levels and promote reward-related learning. 37,38 These data suggest that nAChR-induced inhibition of DA function predominates over direct triggering of axonal DA release and nAChR desensitization.

[0216] nAChR antagonists reverse motor deficits in PD models

[0217] These data raise the prospect that antagonists of nAChRs may have the potential to counteract motor deficits in PD caused by DA deficiency by preventing ChI-induced inhibition of DA release. The inventors tested whether striatal antagonism of nAChRs could rescue motor deficits caused by DA lesions in mice. Wild-type mice were subjected to unilateral partial lesions of striatal DA inputs by local injection of 6-OHDA in the right dorsolateral striatum, which resulted in decreased striatal tyrosine hydroxylase immunoreactivity. The inventors tested the effects of nAChR antagonists on single-paw reaching movements in the contralateral limb. 39 Impact Figure 4 Intrastriatal injection of mecamylamine improved these motor deficits ( Figure 4 This suggests that nAChR antagonists may be a potential therapeutic approach to counteract the effects of DA deficiency in PD.

[0218] Computational modeling reveals that tonicity and multiphasic activity in ChI inhibit DA release

[0219] To understand how nAChR dynamics during dynamic activity patterns of ChIs affect [DA] in vivo o , the inventors based on Figure 2 C&D and Figure 2 A computational model was developed using data from G&H. Figure 5 a). The inventors verified that the model can simulate Figure 1 D&H in vitro observation results ( Figure 8 The inventors first tested how changing the tonic ChI firing rate would modulate tonic [DA] o The inventors set an arbitrary tonic level (50% of maximum) for ChI-induced inhibition of DA release and set [DA] o Normalized to the level observed in the absence of ChI action. The model predicts that in the absence of underlying changes in DA firing activity, transient (~100 ms) changes in ChI population activity will be o The opposite changes occur in Figure 5 b) This prediction could explain recent in vivo observations that [DA] o Can be modified without causing potential changes in DA neuron firing rates 8 .

[0220] The inventors then tested how multi-phase ChI activity modifies [DA] in DA neurons during concurrent bursting activity. o 16 , with and without initial excitation occurring in half of the ChI 29, and combined with a range of levels of tonic nAChR-induced inhibition of DA release (0, 50, 100%) before multiphasic activity. Tonic ChI activity reduced baseline DA release levels ( Figure 5 c). In addition, for DA neuron burst activity, DA release was reduced by multiphasic activity in ChI, with the greatest reduction occurring in response to initial ChI excitation and lower tonic ChI activity, and to a greater extent in DLS than in NAc ( Figure 5 ce). The reduction in burst DA release observed during multiphasic ChI activity in the absence of an initial challenge is due to the inhibition of DA release by concurrent ChI rebound activity. Desensitization-like components play only a minor role in these results and are primarily in the DLS rather than the NAc and are not abolished when an initial challenge is present ( Fig. 9 ). Overall, this model suggests that multiphasic activity in ChI inhibits DA release during phasic activity, particularly in DLS.

[0221] The model also suggests that tonic ChI activity in the absence of multi-stage ChI activity (a reflection of the previous learning context) 29,40 ) decreased baseline and burst-induced DA release levels (dashed lines, Fig.10 a). At low to moderate levels of tonic ChI inhibition of DA release (<50%), multiphasic ChI activity further reduced phasic DA release. Only at very high levels of tonic inhibition (50-100%) did ChI multiphasic activity enhance phasic DA release ( Fig.10 a and b), presumably due to reduced ChI inhibition during the ChI pause. Therefore, the level of phasic striatal DA release will depend on both tonic and multiphasic activity in ChI.

[0222] discuss

[0223] The inventors have reconciled the diverse roles of nAChRs on DA release and revised current concepts to show that nAChR activation results in a significant inhibition of DA release. The results described herein demonstrate that DA output is gated by nAChR activation dynamics, through a rapid onset but prolonged inhibition of DA release (<100 ms) followed by a significant loss of nAChR control (after approximately 100-300 ms). The inhibition of nAChR-induced DA release does not require depletion of DA pools, but rather limits axonal Ca 2+ accumulate, are detected in vivo and influence DA-dependent learning.

[0224] Inhibition vs. enhancement of DA release

[0225] The inventors found that ChI activation can prevent DA axon depolarization events and axonal Ca 2+ ChIs accumulate to limit continuous DA release, and computational models predict that tonic-active ChIs will continuously inhibit DA release in vivo. ChIs have previously been shown to enhance DA release in response to synchronized firing and pauses during multiphasic activity. 5-7,11,18 Striatal ChIs can directly drive DA release following optogenetic or electrical activation of ChIs or their cortical or thalamic inputs 5,6,10,20,41 , which led to the speculation that initial excitation or rebound activity during multiphasic ChI activity in vivo could drive DA release. However, observations in vitro and in vivo now suggest that the levels of excitation and rebound during multiphasic activity of the ChI in vivo are not sufficient to drive DA axonal release, but rather are sufficient to cause inhibition of phasic DA release because the levels of nAChR activation required to inhibit rather than drive DA release are low. It was previously speculated that pauses in the multiphasic responses of the ChI would promote simultaneous bursts of DA release. 7,18 , because when nAChRs are turned off, high-frequency train stimulation of ChI and DA axons can induce more DA release. The new findings suggest that the increased DA release level is caused by disinhibition (i.e., the elimination of ChI-induced inhibition). The current discovery that ChI mainly inhibits DA release also suggests that ChI activity driven by excitatory inputs (such as those from the cortex and thalamus) and the resulting activation of nAChRs on DA axons 41,42 , which may inhibit striatal DA release in vivo, and vice versa, inhibitory input to ChI 43,44 May promote DA release.

[0226] During the longer time course corresponding to the rebound activity observed in the ChI multiphasic activity (100-300ms after the initial activity), DA release was found to better reflect the received stimulation, consistent with the unavailability of nAChRs. 7,11 This most likely corresponds to nAChR desensitization and suggests that nAChRs activated during the initial excitation do not gate DA release during the rebound phase.

[0227] The rapid inhibition of DA release following nAChR activation correlates with limited depolarization in the subsequent active response and, therefore, limited axonal calcium accumulation. The absence of a prior DA release together suggests a rapid ionic mechanism that can occur at subthreshold axonal activity levels for DA release and action potentials. The ionic mechanism is consistent with emerging data showing that DA output is strongly gated by mechanisms that regulate axonal excitability, including DAT activity, K + Dependent excitability and GABA A -Shunt inhibition after receptor activation and Na + Channel inactivation21,45 Differences in the magnitude and duration of ChI-induced inhibition of DA release in the DLS versus the NAc are accompanied by heterogeneity in nAChR subtypes and, therefore, channel properties (α4α5β2* and α4α6β2β3*, respectively). 34,35 .

[0228] Effects of ChI activity on DA release and function

[0229] To date, the understanding of dopamine function has been dominated by the knowledge of action potential activity recorded in DA cell bodies. However, by limiting subsequent depolarizing events in DA axons, ChIs may prevent action potential propagation and play an important role in controlling DA function. The extremely rapid onset of ChI-induced inhibition of DA release (<7ms) and the decay of this mechanism within ~50-100ms could rapidly alter DA release in response to dynamic changes in ChI activity without altering DA neuron activity. This local regulation may account for the extreme case recently reported of increased DA release without observable changes in DA neuron activity. 8 .

[0230] For burst activity in DA neurons, the model suggests that the combined effects of simultaneous multistage ChI activity primarily inhibit DA output, especially in the case of initial excitation, as a result of nAChR activation. These effects may have implications for striatal learning 46 , changes in DA signals after learning 29 Furthermore, the differences in the magnitude and duration of ChI-induced inhibition observed throughout the striatum, which were stronger in the DLS than in the NAc, may in turn result in different DA signaling and regulatory mechanisms between different regions. 47-49 .

[0231] Local antagonism of nAChRs altered the animals' reward-related behaviors and successfully rescued the motor deficits of 6-OHDA-lesioned mice, suggesting that manipulation of nAChRs on dopamine axons could be a promising approach for treating PD and other dopamine-related diseases. 50-52 , providing a mechanistic explanation for the long-suspected balance of mutual inhibition between striatal ACh and DA. This feed-forward loop will amplify any deviation from balance in the ACh or DA system. For example, DA deficiency in PD would exacerbate ChI-induced inhibition of DA release by relieving ChI inhibition and leading to a further maladaptive reduction in DA output. nAChR antagonists can alleviate this maladaptive inhibition of DA release. In addition, nicotine-induced desensitization of striatal nAChRs in smokers7,53 It is also expected to remove nAChR inhibition of striatal DA release, thereby promoting DA-dependent behaviors (with mechanisms acting at the level of DA neurons 54 Together).

[0232] In summary, the inventors added fast inhibition to the repertoire of mechanisms by which striatal nAChRs gate DA release and found that this mechanism predominates as the means by which nAChRs control DA output.

[0233] Throughout the description and claims of this specification, the words "include" and "comprises" and their variations mean "including but not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the description and claims of this specification, the singular includes the plural, unless the context requires otherwise. In particular, where the indefinite article is used, the invention should be understood to include both the plural and the singular, unless the context requires otherwise.

[0234] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All features disclosed in this specification (including any accompanying claims, abstract and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except that at least some of these features and / or steps are mutually exclusive combinations. The invention is not limited to the details of any foregoing embodiments. The invention extends to any novel one or any novel combination of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one or any novel combination of the steps of any method or process so disclosed.

[0235] The reader is advised that all papers and documents related to this application that are filed concurrently with or prior to this specification are open to public inspection with this specification, and the contents of all these papers and documents are incorporated herein by reference.

[0236] References

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Claims

1. A nicotinic acetylcholine receptor antagonist / blocker or a pharmaceutically acceptable salt thereof for use in treating a condition or disorder associated with decreased dopamine levels in the CNS.

2. The nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to claim 1, wherein administration of the nicotinic acetylcholine receptor antagonist / blocker promotes dopamine release in the CNS.

3. The nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to claim 1 or 2, wherein administration of the nicotinic acetylcholine receptor antagonist / blocker promotes dopamine release in the brain.

4. A nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein administration of the nicotinic acetylcholine receptor antagonist / blocker promotes dopamine release in the striatum.

5. A nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein administration of the 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 nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein the condition or disorder is a condition or disorder associated with reduced dopamine levels in the brain.

7. A nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein the condition or disorder is a condition or disorder associated with reduced dopamine levels in the striatum.

8. A nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein the condition or disorder is a condition or disorder associated with reduced dopamine levels in the dorsal striatum, ventral striatum, ventral tegmental area, basal ganglia and / or substantia nigra.

9. A nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, 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 in Parkinson's disease), schizophrenia, tics (i.e. repetitive involuntary movements or vocalizations), Tourette's syndrome, depression and reward deficiency syndrome.

10. A nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein the condition or disorder is selected from Parkinson's disease or ADHD.

11. A nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein the condition or disorder is Parkinson's disease.

12. A nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein the nicotinic acetylcholine receptor antagonist / blocker is selected from dihydro-β-erythrinidine (DHβE), mecamylamine, bPiDDP (1,1'-(1,12-dodecanediyl)bis[3-methylpyridinium] dibromide), methyltaurine, MG 624 (N,N,N-triethyl-2-[4-(2-phenylvinyl)phenoxy]ethanammonium iodide), SR 16584 (1,3-dihydro-1-(3-exo)-9-methyl-9-azabicyclo[3.3.1]non-3-yl]-2H-indol-2-one), casein peptide, chloroisoindole ammonium diiodide, 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), chloroisoindole ammonium, α-conotoxins (such as 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), Dianicline ((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-pyridyl)-3-butene-1-amine), TC 2559 (4-(5-ethoxy-3-pyridyl)-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, myostatin chloride, α-bungarotoxin, COG 133, D-amphetamine sulfate, PAMP-20, or a pharmaceutically acceptable salt or derivative thereof; Optionally, the nicotinic acetylcholine receptor antagonist / blocker is selected from dihydro-β-erythrin (DHβE), mecamylamine, bPiDDP, methyltaurine, MG 624, SR 16584, casein peptides, chloroisoindole ammonium diiodide, 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), chloroisoindole ammonium, α-conotoxin (such as ACV 1, AuIB, EI, ImI, MII, PIA, PnIA), 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), Dianicline ((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-pyridyl)-3-butene-1-amine)、TC2559 (4-(5-ethoxy-3-pyridyl)-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 nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein the nicotinic acetylcholine receptor antagonist / blocker is selected from dihydro-β-erythrin (DHβE), mecamylamine, bPiDDP, methyltaurine, MG 624, SR 16584, casein peptides, chloroisoindole ammonium diiodide, 2,2,6,6-tetramethylpiperidin-4-ylheptanoate (TMPH), chloroisoindole ammonium and α-conotoxin (such as ACV 1, AuIB, EI, ImI, MII, PIA, PnIA), or a pharmaceutically acceptable salt or derivative thereof.

14. A nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein the nicotinic acetylcholine receptor antagonist / blocker is selected from dihydro-β-erythrin (DHβE), mecamylamine, chloroisoindole ammonium diiodide, 2,2,6,6-tetramethylpiperidin-4-ylheptanoate (TMPH), chloroisoindole ammonium, or a pharmaceutically acceptable salt or derivative thereof.

15. A nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein the nicotinic acetylcholine receptor is selected from DHβE and mecamylamine.

16. A nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein the nicotinic acetylcholine receptor antagonist / blocker is selective for the α4β2 receptor.

17. The nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to claim 16, wherein the nicotinic acetylcholine receptor antagonist / blocker selective for α4β2 receptors is selected from: A85380 (3-[(2S)-2-azetidinylmethoxy]-pyridine dihydrochloride), ABT 089 (2-methyl-3-[(2S)-pyrrolidinylmethoxy]pyridine), ABT594 ((R)-5-(azetidin-2-ylmethoxy)-2-chloropyridine), Dianicline ((5aS,8S,10aR)-5a,6,9,10-tetrahydro-7H,11H-8,10a-methoxypyrido[2',3':5,6]pyrano[2,3-d]azepine ), RJR 2403 ((E)-N-methyl-4-(3-pyridyl)-3-butene-1-amine), TC 2559 (4-(5-ethoxy-3-pyridyl)-N-methyl-(3E)-3-butene-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. The nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein the nicotinic acetylcholine receptor (nAChR) antagonist / blocker is administered orally, nasally, intraperitoneally, intracerebroventricularly, intrathecally, intracranially, subcutaneously or intravenously.

19. A nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein the nicotinic acetylcholine receptor (nAChR) antagonist / blocker is administered directly to the CNS.

20. The nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use according to any one of the preceding claims, wherein the nicotinic acetylcholine receptor (nAChR) antagonist / blocker is administered by intraperitoneal injection, intravenous injection, local infusion into the CNS or direct injection into the CNS.

21. A nicotinic acetylcholine receptor (nAChR) antagonist / blocker for use in increasing dopamine levels in the CNS.

22. Use of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof for increasing dopamine levels in the CNS.

23. A method of treating a condition or disorder associated with reduced dopamine levels in the CNS, the method comprising administering to a subject in need thereof a therapeutically effective amount of a nicotinic acetylcholine receptor (nAChR) antagonist / blocker or a pharmaceutically acceptable salt thereof.

24. 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.

25. A pharmaceutical composition comprising a nicotinic acetylcholine receptor antagonist / blocker or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients for use in treating a condition or disorder associated with reduced dopamine levels in the CNS.

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