Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that most notably influence dopamine (DA) release. In this thesis, I examine the role of nAChRs in mediating DA-related behaviors such as movement and drug dependence. To accomplish this, I utilized a “gain-offunction” knock-in mouse (the Leu9’Ala line) containing agonist-hypersensitive α4* nAChRs (* indicates other nAChR subunits in addition to α4 are within the receptor complex) that renders receptors 50-fold more sensitive to nicotine and acetylcholine than wild-type (WT) receptors. I found that DHβE, a selective antagonist for α4β2* nAChRs, induced reversible and robust motor dysfunction characterized by hypolocomotion, akinesia, catalepsy, tremor, and clasping in Leu9’Ala but not WT mice. Reversal of the phenotype was achieved by targeting dopamine signaling. Blockade of mutant α4* nAChRs elicited activation of brain regions in the basal ganglia including dorsal striatum and substantia nigra pars reticulata indicated by c-Fos immunoreactivity. These data indicate that blocking α4* nAChRs in Leu9’Ala mice activates the indirect motor pathway resulting in a motor deficit. We also determined that α4* nAChRs involved in motor behaviors did not contain the α6 subunit, a nAChR subunit highly expressed in DAergic neurons suggesting that different nAChR subtypes modulating striatal DA release have separate functions in motor output. Conditioned place aversion and hypolocomotion, behaviors elicited during nicotine withdrawal, were also induced by DHβE in nicotine-naïve Leu9’Ala but not WT mice. Together these data suggest that DHβE globally reduces DA release in the CNS. In a separate project, I determined that α4* and α6* nAChRs modulate drug-induced hypnosis. Activation of nAChRs increased sensitivity to ketamine-induced hypnosis; whereas antagonizing nAChRs had the opposite effect. Additionally, α4 knockout (KO) mice were less sensitive to the hypnotic effects of ketamine, but α6 KO were more sensitive. High doses of ethanol induce an anesthesia-like state characterized by immobility, analgesia, and hypnosis. Testing the effects of ethanol hypnosis in α4 KO revealed that α4* nAChR do not play a large role in the acute effects of ethanol-induced hypnosis, but are involved in tolerance to this ethanol-induced behavior. The mechanisms of anesthetic-induced hypnosis are still largely unclear, despite the wide use of anesthesia. Future work on these receptors and their involvement in the anesthetic response will help to define a mechanism for hypnosis and improve the use of anesthetic drugs.

Recently, the medial habenula-interpeduncular (MHb-IPN) axis has been hypothesized to modulate anxiety although neuronal populations and molecular mechanisms regulating affective behaviors in this circuit are unknown. Here we show that MHb cholinergic neuron activity directly regulates anxiety-like behavior. Optogenetic silencing of MHb cholinergic IPN inputs reduced anxiety-like behavior in mice. MHb cholinergic neurons are unique in that they robustly express neuronal nicotinic acetylcholine receptors (nAChRs), although their role as autoreceptors in these neurons has not been described. nAChRs are ligand-gated cation channels that are activated by the excitatory neurotransmitter, acetylcholine (ACh), as well as nicotine, the addictive component of tobacco smoke. We expressed novel nAChR subunits that render nAChRs hypersensitive to ACh, ACh detectors, selectively in MHb cholinergic neurons of adult mice. Mice expressing these ACh detectors exhibited increased baseline anxiety-like behavior that was alleviated by blocking the mutant receptors. Under stressful conditions, such as during nicotine withdrawal, nAChRs were functionally upregulated in MHb cholinergic neurons mediating an increase in anxiety-like behavior. Together, these data indicate that MHb cholinergic neurons regulate anxiety via signaling through nicotinic autoreceptors and point toward nAChRs in MHb as molecular targets for novel anxiolytic therapeutics.

Nicotine dependence is hypothesized to be due to neuroadaptations that ultimately drive compulsive nicotine use. The studies in this thesis aim to understand how the “upregulation” of nicotinic acetylcholine receptors (nAChRs) caused by chronic exposure to nicotine contributes to nicotine reward and nicotine withdrawal. Previous studies have shown that chronic nicotine induces upregulation of nAChRs containing the α4 subunit (α4* nAChR) within the Ventral Tegmental Area (VTA), a brain region critical for the rewarding properties of all illicit drugs. Curiously, α4* nAChR upregulation occurs specifically in the inhibitory GABAergic neuronal subpopulation of the VTA. To determine if increased expression and activation of α4* nAChRs in VTA GABAergic neurons contributes to nicotine dependence behaviors, I devised a viral-mediated, Creregulated gene expression system that selectively expressed α4 nAChR subunits containing a “gain-of-function” point mutation (a leucine mutated to a serine residue at the TM2 9´ position: Leu9´Ser) in VTA GABAergic neurons of adult mice. Sub-reward doses of nicotine were sufficient to activate VTA GABAergic neurons in mice expressing Leu9´Ser α4 nAChR subunits in VTA GABAergic neurons (Gad2VTA: Leu9´Ser mice) and exhibited acute hypolocomotion upon initial injection of low doses of nicotine that developed tolerance with subsequent nicotine exposures compared to control animals. In the conditioned place preference procedure, nicotine was sufficient to condition a significant place preference in Gad2VTA: Leu9´Ser mice at low nicotine doses that failed to condition control animals. I conclude from these data that upregulating α4* nAChRs on VTA GABAergic neurons increases sensitivity to nicotine reward. In a separate study testing the hypothesis that overexpression of Leu9´Ser α4* nAChRs in VTA GABAergic neurons disrupts baseline behavior and promotes anxiety-like behaviors, I found that overexpressing Leu9´Ser α4* nAChRs in VTA GABAergic neurons had a minimal effect on unconditioned anxiety-like behaviors. Drug naïve Gad2VTA: Leu9´Ser and control mice failed to exhibit any behavioral differences in the open-field, marble burying test and elevated plus maze compared to control. Together, these data indicate that overexpression of the “gain-of-function” α4* nAChRs in VTA GABAergic neurons contributes to reward sensitivity without increasing susceptibility to nicotine withdrawal symptoms. My data indicates that nAChRs expressed in VTA GABAergic neurons may be a suitable target for the development of better smoking cessation aids.

Indiana University-Purdue University Indianapolis (IUPUI) / Nicotine use among individuals with schizophrenia occurs at extremely high rates. The prevailing theory is that individuals with schizophrenia smoke as a form of self-medication to ameliorate sensory and cognitive deficits. However, these individuals also have enhanced rates of addiction to several drugs of abuse and may therefore smoke as a result of enhanced addiction liability. The experiments described herein explored these two hypotheses by assessing the effect that nicotine has on working memory, addiction vulnerability (locomotor sensitization and self-administration), and nicotinic acetylcholine receptor (nAChR) expression as well as the developmental expression of these characteristics in the neonatal ventral hippocampal (NVHL) neurodevelopmental animal model of schizophrenia. The results from these studies indicate that NVHLs had working memory impairments in both adolescence and adulthood, with nicotine having a negligible effect. Additionally, NVHLs displayed enhanced locomotor sensitization to nicotine which emerged in adulthood as well as an enhanced acquisition of nicotine self-administration, administering more nicotine overall. These behavioral differences cannot be attributed to nAChR expression as nicotine upregulated nAChR to a similar extent between NVHL and SHAM control animals. These data indicate that the enhanced rates of nicotine use among individuals with schizophrenia may occur as a result of an enhanced vulnerability to nicotine addiction.

Myasthenia gravis (MG) is an autoimmune disease whose name means "grave muscular weakness". MG is a rare disease affecting only 200-400 persons per million and the characteristic symptoms include muscle weakness, particularly in highly active voluntary muscles. MG affects the neuromuscular junction in an antibody-mediated manner, resulting in impaired nerve-muscle cell communication in affected individuals. Specifically, two main proteins are targeted: nicotinic acetylcholine receptors (ACh receptors) and a muscle-specific tyrosine kinase (MuSK). Previous studies have discovered the mechanism of MG pathogenesis but the exact mechanisms which cause the failure to maintain self-tolerance have not been discovered. Based on current knowledge of MG, this paper will explore potential causes of the disease and provide numerous hypotheses directed at future research opportunities.

The current project examined the ability of a novel series of guandine and amidine-containing nicotine analogs to interact with several native and recombinantlyexpressed mammalian neuronal nicotinic-acetylcholine receptor (nAChR) subtypes. Rational drug design methods and parallel organic synthesis was used to generate a library of guanidine-containing nicotine (NIC) analogs (AH compounds). A smaller series of amidine-containing nicotine analogs (JC compounds) were also synthesized. In total, >150 compounds were examined. Compounds were first assayed for affinity in a high-throughput [3H]epibatidine radioligand-binding screen. Lead compounds were evaluated in subtype-selective binding experiments to probe for affinity at the α4β2* and α7* neuronal nAChRs. Several compounds were identified which possess affinity and selectivity for the α4β2* subtype [AH-132 (Ki=27nm) and JC-3-9 (Ki=11nM)]. Schild analysis of binding suggests a complex one-site binding interaction at the desensitized high-affinity nAChR. Whole-cell functional fluorescence (FLIPR) assays revealed mixed subtype pharmacology. AH-compounds were identified which act as activators and inhibitors at nAChR subtypes, while lead JC-compounds were found which possess full agonist activity at α4β2* and α3β4* subtypes. Compounds were identified as partial agonists, full agonists and inhibitors of multiple nAChR subtypes. Several SAR-based, ligand-receptor pharmacophore models were developed to guide future ligand design. Second-generation lead compounds were identified.

Traumatic brain injury (TBI) is a leading cause of death and long-term disability worldwide, and survivors are often left with cognitive deficits and significant problems with day to day tasks. To date, therapeutic pharmacological treatments of TBI remain elusive despite numerous clinical trials. An improved understanding of the molecular and cellular response to injury may help guide future treatment strategies. One promising marker for brain injury is the translocator protein (TSPO), which is normally expressed at a low level, but is highly expressed following brain damage and is associated with neuroinflammation. The isoquinoline carboxamide PK11195 binds selectively to the TSPO in many species, and has therefore become the most-studied TSPO ligand. To characterize the time-course of TSPO expression in the controlled cortical injury (CCI) model of TBI we subjected Sprague-Dawley rats to CCI and euthanatized them after 30 minutes, 12 hours, 1, 2, 4, or 6 days. Autoradiography with radiolabelled PK11195 was used to assess the time-course of TSPO binding following CCI. Autoradiographs were compared to adjacent tissue slices stained with the microglia/macrophage marker ED-1, with which a moderate positive correlation was discovered. PK11195 autoradiography was used as a tool with which to assess neuroinflammation following CCI and the administration of an α7 nAChR antagonist, methyllycaconitine (MLA). We hypothesized that blocking the calcium permeable α7 nAChR after brain injury would have a neuroprotective effect by attenuating excitotoxicity in the shortterm. Our study revealed clear dose-dependent tissue sparing in rats administered MLA after trauma and a modest improvement in functional outcome. The relatively modest recovery of function with MLA, which could be due to prolonged α7 nAChR blockade or downregulation lead us to explore the potential of α7 nAChR partial agonists in treating TBI. The α7 nAChR partial agonists tropisetron, ondansetron, and DMXB-A produced a moderate attenuation of cognitive deficits, but did not have a neuroprotective effect on tissue sparing. These studies show that following TBI, α7 nAChR modulation can have neuroprotective effects and attenuate cognitive deficits. Whether this modulation is best achieved through partial agonist treatment alone or a combination antagonist/agonist treatment remains to be determined.

Upregulation of nicotinic acetylcholine receptors (nAChRs) is a well-documented response to chronic nicotine exposure. Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels consisting of alpha (α2-10) and beta (β2-4) subunits. Nicotine, an agonist of nAChRs, alters trafficking and assembly of some subtypes of nAChRs, leading to an increase in expression of high sensitivity receptors on the plasma membrane. These physiological changes in nAChRs are believed to contribute to nicotine addiction, although the mechanism of these processes has not been resolved. Recently, many studies have converged on the idea that nicotine induces upregulation by an intracellular mechanism. In this dissertation, expression levels of nAChRs were quantified upon exposure to nicotine and its primary metabolite, cotinine. A pH sensitive variant of GFP, super ecliptic pHluorin (SEP), was integrated with a nAChR subunit to study expression and trafficking of nAChRs by differentiating intracellular and plasma membrane inserted receptors. In this work, cotinine is shown to increase the number of α4β2 nAChRs within a cell. Cotinine also affects trafficking of α4β2, evident by a redistribution of intracellular receptors and an increase in single vesicle insertion events on the plasma membrane. This work shows both nicotine and cotinine alter the overall assembly of α4β2 to favor the high sensitivity (α4)2(β2)3 version. Since cotinine and nicotine induce similar physiological changes in nAChRs, the metabolite potentially plays a role in the mechanism of nicotine addiction. Although an intracellular mechanism for upregulation has been supported, a shift in assembly to the high sensitivity (α4)2(β2)3 version exclusively in the endoplasmic reticulum has not previously been detected. In order to study organelle specific changes in stoichiometry, a novel method was developed to isolate single nAChRs in nanovesicles derived from native cell membranes. Separation of nanovesicles originating from the endoplasmic reticulum and plasma membrane, encompassing isolated nAChRs, allows precise changes in stoichiometry to be monitored in subcellular regions. In this work, single molecule bleaching steps of green fluorescent protein (GFP) encoded in each alpha subunit of the pentamer are detected. The number of bleaching steps, or transitions to a nonfluorescent state upon continuous excitation, corresponds to the number of GFP-labeled alpha subunits present. Therefore, the stoichiometry can be deduced by detection of two bleaching steps, as in (α4)2(β2)3, or three bleaching steps, seen in (α4)3(β2)2. Using this method on isolated nAChRs, a shift to assembly of high sensitivity (α4)2(β2)3 receptors is detected definitively within the endoplasmic reticulum. In addition, an increase in (α4)2(β2)3 receptors located on the plasma membrane is shown when nicotine is present. This work provides convincing evidence that nicotine acts intracellularly, within the endoplasmic reticulum, to alter stoichiometry of nAChRs.

The α7 nicotinic acetylcholine receptor (nAChR) subtype is abundantly expressed in the central nervous system (CNS) and in the periphery. Positive allosteric modulators (PAMs) of the α7 increase the response to an agonist and are divided into two types depending on whether they also decrease desensitization of the receptor (type II) or not (type I). Therefore, this study aims to investigate whether the enhancement of endogenous α7 nAChR function will result in a beneficial effect in nociceptive, inflammatory and chronic neuropathic pain models. While NS1738 and PNU-120596 were not active to reduce acute thermal pain, measured by hot-plate and tail-flick tests, only PNU-120596 dose-dependently attenuated paw-licking behavior in the formalin test. Our results with selective (MEK) inhibitor U0126 argues for an important role of extracellular signal-regulated kinase (ERK1/2) pathways activation in PNU-120596’s antinociceptive effects in formalin-induced pain. The α7 antagonist MLA, via intrathecal and intraplantar administration, reversed PNU-120596’s effects, confirming PNU-120596’s action through central and peripheral α7 nAChRs. Tolerance to PNU-120596 was not developed after chronic treatment of the drug. Furthermore, mixtures of PNU-120596 and choline, an endogenous α7 nAChR agonist, synergistically reduced formalin-induced pain, while interactions of non-antinociceptive doses of PNU-120596 and PHA-543613, a selective α7 nAChR agonist, or nicotine resulted in antinociception. In contrast, PNU-120596 failed to enhance nicotine-induced convulsions, -hypomotility and –antinociception in acute pain models. Surprisingly, it enhanced nicotine-induced hypothermia via α7 nAChRs. In the carrageenan inflammatory test both NS1738 and PNU-120596 significantly reduced thermal hyperalgesia, while only PNU-120596 significantly reduced edema. Importantly, PNU-120596 reversed established thermal hyperalgesia and edema induced by carrageenan. In the chronic neuropathic pain (CCI) model, PNU-120596 had long-lasting (up to 6 hrs), dose-dependent anti-hyperalgesic and anti-allodynic effects after a single injection, while NS1738 was inactive. Subcutaneous and intrathecal administration of MLA reversed PNU-120596’s effects, suggesting the involvement of α7 nAChRs. Finally, PNU-120596 enhanced an ineffective dose of selective agonist PHA-543613 to produce anti-allodynic effects in the CCI model. Our results show a fundamental in vivo difference between type I and II α7 nAChR PAMs, and demonstrate type II’s potential for the treatment of chronic inflammatory pain.