In the absence of clinically-efficacious therapies for ischemic stroke there is a critical need for development of new therapeutic concepts and approaches for prevention of brain injury secondary to cerebral ischemia. This study tests the hypothesis that administration of PNU-120596, a type-II positive allosteric modulator (PAM-II) of α7 nicotinic acetylcholine receptors (nAChRs), as long as 6 hours after the onset of focal cerebral ischemia significantly reduces brain injury and neurological deficits in an animal model of ischemic stroke. Focal cerebral ischemia was induced by a transient (90 min) middle cerebral artery occlusion (MCAO). Animals were then subdivided into two groups and injected intravenously (i.v.) 6 hours post-MCAO with either 1 mg/kg PNU-120596 (treated group) or vehicle only (untreated group). Measurements of cerebral infarct volumes and neurological behavioral tests were performed 24 hrs post-MCAO. PNU-120596 significantly reduced cerebral infarct volume and improved neurological function as evidenced by the results of Bederson, rolling cylinder and ladder rung walking tests. These results forecast a high therapeutic potential for PAMs-II as effective recruiters and activators of endogenous α7 nAChR-dependent cholinergic pathways to reduce brain injury and improve neurological function after cerebral ischemic stroke.
Citation: Sun F, Jin K, Uteshev VV (2013) A Type-II Positive Allosteric Modulator of α7 nAChRs Reduces Brain Injury and Improves Neurological Function after Focal Cerebral Ischemia in Rats. PLoS ONE 8(8): e73581. doi:10.1371/journal.pone.0073581
Editor: Alexander A. Mongin, Albany Medical College, United States of America
Received: June 18, 2013; Accepted: July 26, 2013; Published: August 9, 2013
Copyright: © 2013 Sun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the National Institutes of Health Grant DK-082625 and the Start-Up funds from UNTHSC to V.V. Uteshev. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Clinical management of neuronal damage resulting from ischemic stroke generally involves only palliative treatments. Currently, the only FDA-approved drug therapy for ischemic stroke involves the intravenous use of tissue plasminogen activator (tPA) to dissolve clots . This strategy appears to be effective in ischemic stroke, but only within the first 3 hours after the onset of ischemic stroke [2,3]. This strict limitation reduces the percent of stroke patients eligible for tPA to as low as ~2% . Although in the last two decades substantial efforts have been invested in developing anti-ischemic medicine, these efforts have not resulted in clinically-efficacious therapies for ischemic stroke . These failures highlight the need for development of new therapeutic concepts and approaches for prevention of brain injury secondary to ischemia. Among possible strategies, effective post-stroke treatments with broad therapeutic windows are likely to be the most valuable because of the unexpected nature of stroke. In this search, treatments that are based on recruiting and activating endogenous pathways receive special attention as these approaches are expected to be highly efficacious and cause fewer adverse effects than approaches that utilize exogenous agents [6–8]. To complement these needs, this study evaluates neurological benefits of enhanced activation of α7 nicotinic acetylcholine receptors (nAChRs) by endogenous nicotinic agonists 6 hours after ischemic insult induced by middle cerebral artery occlusion (MCAO) in young adult rats.
There is a substantial body of supportive evidence linking age-, disease- and trauma-related reduction in the expression and function of α7 nAChRs to neurodegenerative, sensorimotor and psychiatric disorders associated with cognitive decline and attention deficits [9–24]. By contrast, activation of α7 nAChRs has been demonstrated to enhance neuronal resistance to ischemia and other insults in in vivo, ex vivo and in vitro experimental models [6,25–39], as well as improved cognitive performance in patients and animal models of neurodegenerative conditions including dementia, schizophrenia, brain trauma and aging [14,26,31,39–61]. An important rationale for the therapeutic use of α7 nAChR agents arises from the fact that α7 nAChRs are ubiquitously expressed throughout the brain  including brain regions that are highly vulnerable to ischemia, such as cortex, striatum and hippocampus [63–66]. However, endogenous α7 nAChR agonists (i.e., choline and ACh) have not been regarded as potent therapeutic agents because physiological levels of choline/ACh do not appear to produce therapeutic levels of α7 activation . This limitation has been recently resolved by the use of Type-II positive allosteric modulators (PAMs-II) of α7 nAChRs [6,8,48,67–73]. PAMs-II do not activate α7 nAChRs, but they inhibit desensitization and enhance α7 activation by nicotinic agonists, including endogenous choline and ACh [48,67,68]. Thus, PAMs-II only amplify activation of α7 nAChRs by endogenous nicotinic agonists released naturally as needed . Accordingly, we have recently introduced a novel therapeutic paradigm  that converts endogenous choline/ACh into potent therapeutic agents for cerebral ischemia by enhancing activation of α7 nAChRs using PNU-120596, a PAM-II. In our previous proof-of-concept study , we have reported that a 3 hour pre-treatment with choline+PNU-120596 significantly delayed anoxic depolarization/injury of hippocampal CA1 pyramidal neurons in the complete oxygen/glucose deprivation model of ischemic stroke in acute hippocampal slices and activation of α7 nAChRs was required; while intravenous administration of PNU-120596 30 min post-ischemia in the MCAO model of ischemic stroke significantly reduced cerebral infarct volume . The present study extends our previous findings and the therapeutic promise of PAMs-II by revealing that PNU-120596 reduces both the focal ischemia-induced cerebral infarct volume and neurological deficits even when administered as long as 6 hours after the ischemic onset. The results of this study further support the potential therapeutic utility of PAMs-II as effective recruiters and activators of endogenous α7-dependent cholinergic pathways to reduce brain injury and improve neurological function secondary to focal cerebral ischemia.
Materials and Methods
Young adult male Sprague-Dawley (S.-D.) rats (~280 g) were used in experiments. The animal use was in accordance with the Guide for the Care and Use of Laboratory Animals (NIH 865-23, Bethesda, MD), and all experimental protocols were approved by the Institutional Animal Care and Use Committee of University of North Texas Health Science Center at Fort Worth, TX.
In total 22 animals were used in this study. Animals were housed 2 per tub in a Tecniplast Green Line IVC Sealsafe PLUS Rat rack on 1/8” corn cob bedding, with Envirodri shredded paper for enrichment. Animals were fed Purina Lab Diet 5LL2, and received filtered water via water bottles. Room lighting was kept below 50 Foot Candles (range of 30-40), and with a timer controlled 12:12 light dark cycle. Room temperature was maintained between 68–72 degrees, with humidity range of 30-70%. Cages were cleaned or changed at least once per week. The housing room contained only rats. The UNTHSC animal facility is AAALAC accredited and follows or exceeds all of the requirements of the Guide for the Care and Use of Laboratory Animals.
Middle cerebral artery occlusion (MCAO)
Transient (90 min) focal cerebral ischemia was induced using the suture occlusion technique as previously described . Animals (n=22; Charles River, Wilmington, MA) were anesthetized with 4% isoflurane mixed with 67% N2O and 29% O2 and delivered by a mask. After a midline incision in the neck, the left external carotid artery (ECA) was carefully exposed and dissected. A 19-mm, 4-0 monofilament nylon suture was inserted from the ECA into the left internal carotid artery to occlude the origin of left middle cerebral artery. After 90 min of occlusion, the thread was removed to allow reperfusion. The ECA was ligated, and the wound was closed. Rectal temperature was maintained at ~37° C using a heating pad.
A total of 22 animals were used in this study of which 1 animal from the control group died during the first hours of post-MCAO recovery prior to vehicle injections and another animal from the same control group died after vehicle injection, but prior to behavioral tests. Thus, the mortality rate was ~16.7% in the control group and 0% in the treatment group.
PNU-120596 was obtained from the National Institute of Drug Addiction through the Research Resources Drug Supply Program as well as purchased from Selleck Chemicals (Houston, TX). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
In all experiments of this study, 1 mg/kg PNU-120596 was administered via intravenous (i.v.) injections. Similar doses have been used in other studies [6,48,71,73]. To make a 50 mM stock solution (maximal achievable concentration is ~200 mM), PNU-120596 was dissolved in dimethyl sulfoxide (DMSO). The appropriate amounts of the stock solution (i.e., PNU+DMSO) or DMSO alone (i.e., vehicle) were injected as a single bolus. The amount of DMSO injected in each animal did not exceed 0.5 ml/kg.
Infarct Volume Measurements
Rats (n=10 per group) were anesthetized and euthanized by decapitation 24 hrs after MCAO. Brains were removed and coronal sections (2 mm thickness) immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) in saline for 20 min at 37° C, then fixed for 2 hrs in 4% paraformaldehyde . Infarct area, left hemisphere area, and total brain area were measured by a blinded observer using the ImageJ software, and areas were multiplied by the distance between sections to obtain the respective volumes. Infarct volume was calculated as a percentage of the volume of the contralateral hemisphere, as described previously .
Rats (n=10 per group) underwent neurobehavioral tests to evaluate functional outcome of treatments with PNU-120596. Animals were trained prior to MCAO (training period: 3 days, 3 trials per day) and deficits were assessed 24 hrs thereafter. The order of testing (Bederson➔cylinder➔ladder rung walking) was always the same to keep the testing conditions identical for all animals. Although it is unlikely that subjecting animals to early tests in the sequence facilitated or inhibited the animal performance in the later tests, we cannot completely rule out a possibility of inter-test interactions.
Bederson score was used to assess the neurological deficit using a four-level scale : 0, normal; 1, forelimb flexion; 2, decreased resistance to lateral push; 3, circling.
Forelimb use bias was analyzed by observing the rat’s movements over 3-minute intervals in a transparent, 18-cm-wide, 30-cm-high poly(methyl methacrylate) cylinder. A mirror behind the cylinder made it possible to observe and record forelimb movements when the rat was facing away from the examiner. After an episode of rearing and wall exploration, a landing was scored for the first limb to contact the wall or for both limbs if they made simultaneous contact. Percentage use of the impaired limb was calculated.
Ladder rung walking test
The ladder rung walking test is sensitive for quantifying skilled locomotion. The degree of motor dysfunction after MCAO was measured by counting the number of foot-faults of the impaired limbs per round, as described previously . Baseline and post-operative testing sessions consisted of three traverses across the ladder. An error was scored for any foot slip or misstep. The number of errors of the affected forelimb and hindlimb in each trial was counted. The mean number of errors in three traverses was calculated.
Statistical significance of differences among groups was defined by the p-value (i.e., * p<0.05; *** p<0.001) using the two-tailed Mann–Whitney U-test. A non-parametric Mann–Whitney U-test was used because this study did not assume any specific underlying distribution (i.e., Gaussian) of data and had a relatively small sample size (n=5-10). We recognize that non-parametric statistics are often less powerful than parametric statistics and thus, more prone to Type-II error (i.e., missing significance when it is present) . However, in this particular study, differences among groups have been found significant in all experiments further supporting our conclusions. The results are presented as mean+S.E.M.
PNU-120596 significantly reduces cerebral infarct volume
In the group of animals defined as treated, PNU-120596 (1 mg/kg) was administered intravenously (i.v.) 6 hrs post-MCAO and the effects of PNU-120596 on cerebral infarct volume were evaluated 24 hrs post-MCAO using the TTC staining (see Methods). In the matching control group of animals only vehicle (i.e., DMSO) was administered via i.v. injections. Only the left MCA was occluded in each experiment. The results of these experiments demonstrated significant reduction in the infarct volume of treated vs. untreated animals (two-tailed, Mann–Whitney U-test): p=0.0147 (n=10; Figure 1).
Figure 1. PNU-120596 significantly reduces the size of brain injury induced by focal cerebral ischemia.
Focal cerebral ischemia was induced by a transient (90 min) middle cerebral artery occlusion (MCAO). Then, 6 hrs post-MCAO, animals were given i.v. injections of either 1 mg/kg PNU-120596 dissolved in DMSO at 50 mM (i.e., treated group; n=10) or the matched amount of DMSO only (i.e., untreated group; n=10). Typical examples of injured whole-brain coronal sections (2 mm thick) obtained from untreated (i.e., DMSO only) (A) and treated (1 mg/kg PNU-120596) (B) animals. Treated and untreated animals were anesthetized and euthanized 24 hrs after MCAO (i.e., 18 hrs after PNU-120596 or DMSO injections) and brain sections were prepared for histological analysis. C) A summary: MCAO-induced infarct volumes were significantly smaller in treated vs. untreated animals: p=0.0147 (n=10; two-tailed, the Mann–Whitney U-test). The results are presented as mean+S.E.M.doi:10.1371/journal.pone.0073581.g001
PNU-120596 significantly improves neurological performance post-MCAO
The same treated and untreated animals that were used for histological measurements (Figure 1) were used in behavioral experiments 15 min prior to the animal anesthesia/euthanasia and brain tissue collection for histology (i.e., ~24 hrs post-MCAO). PNU-120596 significantly improved neurological function of treated (n=10) vs. untreated (n=10) animals as evidenced by the results of the following behavioral tests (two-tailed, Mann–Whitney U-test): Bederson (p=0.0385; Figure 2A), rolling cylinder (p=0.0124; Figure 2B), ladder rung walking (forelimb) (p=0.0486; Figure 2C) and ladder rung walking (hindlimb) (p=0.0007; Figure 2D). Therefore, the results of these experiments convincingly demonstrate that PNU-120596 produces significant neurological benefits even when it is administered as long as 6 hrs post-MCAO.
Figure 2. PNU-120596 significantly improves neurological function after focal cerebral ischemia.
The same treated (n=10) and untreated (n=10) animals that were used for histological analysis (Figure 1) were subjected to neurological tests 15 min prior to anesthesia/euthanasia and collection of brain sections for histological analysis. PNU-120596 significantly improved neurological function post-MCAO in treated (n=10) vs. untreated (n=10) groups of animals as evidenced by the results of the following tests (two-tailed, the Mann–Whitney U-test): A) Bederson, (p=0.0385); B) Rolling cylinder, (p=0.0124); C) Ladder rung walk, (forelimb), (p=0.0486); and D) Ladder rung walk, (hindlimb), (p=0.0007). The results are presented as mean+S.E.M.doi:10.1371/journal.pone.0073581.g002
The key finding of this study is that PNU-120596, a previously reported highly selective PAM-II of α7 nAChRs, significantly reduces cerebral infarct volume and neurological deficits in the MCAO model of ischemic stroke in rats when the drug is administered as long as 6 hrs post-MCAO. Such a remarkable persistent post-MCAO effectiveness of PNU-120596 invites more comprehensive pre-clinical studies of the PAM-II class of compounds aiming at giving health care providers an effective tool to reducing brain injury and improving neurological function secondary to cerebral ischemic stroke hours after the initial ischemic event. The therapeutic benefits produced by PNU-120596 originate from its ability to convert endogenous agonists of α7 nAChRs (i.e., choline and ACh) into highly potent therapeutic agents [6,48,67,68]. Thus, PAMs-II may create a conceptually novel family of treatments that are based on a novel and substantively different mechanism, i.e., recruiting and activating endogenous α7-dependent cholinergic pathways. Treatments that incorporate endogenous compounds and mechanisms are expected to be highly efficacious and cause fewer adverse effects than treatments that utilize exogenous agents.
These results extend our previous findings that demonstrated a high therapeutic efficacy of PNU-120596 administered intravenously 30 min after focal cerebral ischemia . Intriguingly, infarct volumes measured in animals treated with PNU-120596 30 min (n=5 ) and 6 hrs (n=10; this study) post-MCAO were not significantly different (p=0.2404, two-tailed Mann-Whitney test). Similarly, the corresponding infarct volumes measured in untreated animals (i.e., DMSO only) 30 min (n=5 ) and 6 hrs (n=10; this study) post-MCAO were also not significantly different (p=0.5921, two-tailed Mann-Whitney test). Therefore, it is likely that the therapeutic efficacy of PNU-120596 extends considerably beyond the 6 hrs post-ischemic delay tested in this study. By contrast, the therapeutic efficacy of donepezil, an inhibitor of ACh hydrolysis, has been reported to cease within the first 2 hrs post-MCAO . Although the reason for these differences is not known, it may be related to the ability of PNU-120596 to inhibit α7 desensitization and thus, generate persistent α7 nAChR-mediated currents in the presence of physiological/endogenous choline [67–69] even though these currents appear to be reduced at physiological temperatures . By inhibiting ACh hydrolysis, donepezil elevates the extracellular levels of ACh (a non-selective agonist of nicotinic and muscarinic AChRs), but does not appear to produce therapeutic levels of nicotinic and muscarinic AChR activation after 1-2 hrs post-ischemia .
The therapeutic utility of PAM-II-based strategies is supported by the ubiquitous expression of α7 nAChRs in the brain and especially, in the brain regions that are selectively vulnerable to ischemia, such as cortex, striatum and hippocampus [63–66]. Activation of α7 nAChRs has been shown to enhance neuronal resistance to ischemic and other types of insults [6,31,32,38,39,63,82] as well as improve cognitive performance in patients and animal models of schizophrenia [49,72,73,83], dementia [56,61,84] and traumatic brain injury . Moreover, PNU-120596 has been recently demonstrated to produce robust anti-nociceptive effects by enhancing the potency of endogenous choline for α7 nAChR activation [70,71]. Although choline is a full selective endogenous α7 nAChR agonist, near-physiological levels of choline (i.e., ~20 µM) [12,85–87] are sub-threshold for α7 activation (EC50~0.5 mM)  and tend to induce α7 desensitization (IC50~40 µM) . These limitations can be overcome by the use of PAMs-II, such as PNU-120596. PNU-120596 inhibits α7 desensitization and increases the potency of endogenous choline/ACh for α7 activation producing a weak persistent and tunable activation of α7 nAChRs [67–69] – an activation modality of α7 nAChRs that can benefit neuronal survival as discussed previously [6,27,31,32,38,39,63,82]. Moreover, energy deprivation and cell death/dysfunction can considerably elevate the concentration of choline in the extracellular space [89–91] providing a large source of this endogenous α7 nAChR agonist as has been recently demonstrated by direct measurements of choline/ACh levels in the ischemic core and penumbra in the MCAO model of ischemic stroke in rats . It is intriguing to hypothesize that these elevated levels of choline near the site of injury may robustly enhance neuronal resistance to ischemic injury, while PNU-120596 augments this endogenous therapeutic process .
Although the exact cellular and molecular mechanisms of the therapeutic effects of PNU-120596 are not known, α7 nAChR-mediated Ca2+-dependent activation of JAK2/AKT-dependent pathways are likely candidates [82,93–95]. These likely mechanisms would be expected to delay mitochondrial dysfunction and thus, PNU-120596-treated neurons may be able to better meet the energy demand of ischemic/hypoglycemic conditions and significantly delay the ultimate failure of the Na+/K+-ATPase pumps. Such a failure would cause a rapid loss of the neuronal trans-membrane electrochemical gradient leading to transient or terminal anoxic depolarization . It may seem counterintuitive that excitatory currents (i.e., α7 nAChR-mediated) could delay anoxic depolarization and reduce brain injury . However, this concept reflects a common motif in how central neurons respond to insults, i.e., the existence of optimal neuroprotective levels and spatiotemporal patterns of cytosolic Ca2+ elevations [8,14,27,32,96–101]. While sub-optimal levels of cytosolic Ca2+ are ineffective, excessive Ca2+ influx is toxic. By contrast, moderate elevations in cytosolic Ca2+ levels, for example, via a K+-induced depolarization or weak persistent activation of highly Ca2+-permeable α7 nAChRs [102–104] have been shown to protect neurons from injury in a variety of toxicity/insult models [6,27,28,32,33,38,98,105,106]. These therapeutic levels of α7 nAChR activation are consistent with the weak persistent modality of α7 nAChR activation generated by physiological concentrations of choline in the presence of PNU-120596 [67–69].
Moreover, the reported therapeutic efficacy of PNU-120596 may have resulted, at least in part, from enhanced activation of α7 nAChRs expressed in the autonomic neuronal circuitry which may have provided a neurogenic (e.g., adrenergic, nitrergic [107,108]) control over vascular tone and collateral blood circulation. In addition, functional α7 nAChRs are expressed in numerous non-neuronal tissues including glial [109–111] and immune cells [112–114]. Thus, several therapeutic components of α7 nAChR activation in multiple neuronal and non-neuronal tissues may have contributed to the significant therapeutic efficacy of PNU-120596 reported in this and previous in vivo studies [6,70–73,114,115]. These potential individual sources of brain protection and their relative contributions to the therapeutic effects of PNU-120596 are not known and present great interest.
One potential limitation of this study is that it does not include experiments with α7 nAChR antagonists (e.g., methyllycaconitine; MLA). Although PNU-120596 is highly selective for α7 nAChRs and to-date non-α7-mediated effects of PNU-120596 have not been reported, there is a slight chance that PNU-120596 activates both α7-dependent and yet unknown, α7-independent pathways. In that unlikely event, the use of highly selective α7 nAChR antagonists would be critical for distinguishing among α7-dependent and α7-independent components of the effects of PNU-120596. However, experiments using MLA in vivo may not be straightforward as evidenced from a previous report where the effects of MLA on certain behavioral functions were bell-shaped . Thus, a series of positive and negative controls will need to be conducted using selective α7 agonists (e.g., DMXBA; 3-(2,4-dimethoxybenzylidene)-anabaseine, also known as GTS-21) to determine the effective regimens of MLA as applicable to MCAO. This work has not yet been done in this laboratory.
Another possible limitation is that we have not tested the effects of PNU-120596 on neurological performance of control (sham) animals (i.e., in the absence of MCAO-induced injury). This is because control animals perform these tests nearly flawlessly leaving no room for significant improvement by PNU-120596. However, because of this limitation we cannot exclude the possibility that PNU-120596 is a performance enhancing drug which is also effective in the absence of MCAO-induced injury and thus, the therapeutic efficacy of PNU-120596 post-MCAO may not be directly related to MCAO-induced injury, but extends the performance-enhancing potential of PNU-120596 in the absence of injury.
Certain genetic, age- and trauma-related neurodegenerative, sensorimotor, and psychiatric disorders characterized by cognitive decline and attention deficits (e.g., schizophrenia, dementia and traumatic brain injury) are directly associated with decreased cholinergic tone and a decrease, but not disappearance, of functional α7 nAChRs [10,49,117]. By increasing and partially restoring α7-dependent cholinergic tone, PAMs-II would be expected to improve cognitive function and attention impairments in these patients and animal models [39,49,53,56,61,84]. In this regard, treatments with PNU-120596 or functionally-similar PAMs-II compounds may benefit individuals with ischemic stroke and certain age- and trauma-related cognitive deficits via multiple mechanisms and routes of action.
In conclusion, this study demonstrates a remarkable reduction in the size of cerebral injury and significant improvements in neurological function upon intravenous administration of PNU-120596 as long as 6 hours after the onset of transient focal cerebral ischemia. These results further support the potential therapeutic utility of PAMs-II as effective recruiters and activators of endogenous α7-dependent cholinergic pathways and extend the therapeutic promise of this novel class of compounds.
We thank the National Institute on Drug Abuse Research Resources Drug Supply Program for PNU-120596.
Conceived and designed the experiments: FS KJ VU. Performed the experiments: FS. Analyzed the data: FS KJ VU. Contributed reagents/materials/analysis tools: KJ VU. Wrote the manuscript: VU. Interpreted results: FS KJ VU. Edited and revised manuscript: KJ VU. Prepared figures: FS VU.
- 1. Furlan AJ (2002) Acute stroke therapy: beyond i.v. tPA. Cleve Clin J Med 69: 730-734. doi:10.3949/ccjm.69.9.730. PubMed: 12222978.
- 2. Adams HP Jr., del Zoppo G, Alberts MJ, Bhatt DL, Brass L et al. (2007) Guidelines for the early management of adults with ischemic stroke: a guideline from the American Heart Association/American Stroke Association Stroke Council, Clinical Cardiology Council, Cardiovascular Radiology and Intervention Council, and the Atherosclerotic Peripheral Vascular Disease and Quality of Care Outcomes in Research Interdisciplinary Working Groups: the American Academy of Neurology affirms the value of this guideline as an educational tool for neurologists. Stroke 38: 1655-1711. doi:10.1161/STROKEAHA.107.181486. PubMed: 17431204.
- 3. Brott T, Bogousslavsky J (2000) Treatment of acute ischemic stroke. N Engl J Med 343: 710-722. doi:10.1056/NEJM200009073431007. PubMed: 10974136.
- 4. Katzan IL, Furlan AJ, Lloyd LE, Frank JI, Harper DL et al. (2000) Use of tissue-type plasminogen activator for acute ischemic stroke: the Cleveland area experience. JAMA 283: 1151-1158. doi:10.1001/jama.283.9.1151. PubMed: 10703777.
- 5. Richard Green A, Odergren T, Ashwood T (2003) Animal models of stroke: do they have value for discovering neuroprotective agents? Trends Pharmacol Sci 24: 402-408. doi:10.1016/S0165-6147(03)00192-5. PubMed: 12915049.
- 6. Kalappa BI, Sun F, Johnson SR, Jin K, Uteshev VV (2013) A positive allosteric modulator of alpha7 nAChRs augments neuroprotective effects of endogenous nicotinic agonists in cerebral ischemia. Br J Pharmacol. doi: 10.1111/bph.12247
- 7. Lo EH (2008) A new penumbra: transitioning from injury into repair after stroke. Nat Med 14: 497-500. doi:10.1038/nm1735. PubMed: 18463660.
- 8. Uteshev VV (2012). Alpha 7 Nicotinic ACh Receptors as a Ligand-Gated Source of Ca(2+) Ions: The Search for a Ca (2+) Optimum. Adv Exp Med Biol 740: 603-638. doi: 10.1007/978-94-007-2888-2_27
- 9. Jenden DJ, Scremin OU, Roch M, Li G (1996) The influence of aging on whole body choline release and clearance. Life Sci 58: 2003-2009. doi:10.1016/0024-3205(96)00191-9. PubMed: 8637430.
- 10. Guan ZZ, Zhang X, Ravid R, Nordberg A (2000) Decreased protein levels of nicotinic receptor subunits in the hippocampus and temporal cortex of patients with Alzheimer’s disease. JNeurochem 74: 237-243. doi: 10.1046/j.1471-4159.2000.0740237.x
- 11. Nordberg A (2001) Nicotinic receptor abnormalities of Alzheimer’s disease: therapeutic implications. Biol Psychiatry 49: 200-210. doi:10.1016/S0006-3223(00)01125-2. PubMed: 11230871.
- 12. Sarter M, Parikh V (2005) Choline transporters, cholinergic transmission and cognition. Nat Rev Neurosci 6: 48-56. doi:10.1038/nrn1588. PubMed: 15611726.
- 13. Martin LF, Freedman R (2007) Schizophrenia and the alpha7 Nicotinic Acetylcholine Receptor. Int Rev Neurobiol 78: 225-246. doi:10.1016/S0074-7742(06)78008-4. PubMed: 17349863.
- 14. Thomsen MS, Hansen HH, Timmerman DB, Mikkelsen JD (2010) Cognitive improvement by activation of alpha7 nicotinic acetylcholine receptors: from animal models to human pathophysiology. Curr Pharm Des 16: 323-343. doi:10.2174/138161210790170094. PubMed: 20109142.
- 15. Leonard S, Breese C, Adams C, Benhammou K, Gault J et al. (2000) Smoking and schizophrenia: abnormal nicotinic receptor expression. Eur J Pharmacol 393: 237-242. PubMed: 10771019.
- 16. Freedman R, Adams CE, Leonard S (2000) The alpha7-nicotinic acetylcholine receptor and the pathology of hippocampal interneurons in schizophrenia. J Chem Neuroanat 20: 299-306. doi:10.1016/S0891-0618(00)00109-5. PubMed: 11207427.
- 17. Stevens KE, Freedman R, Collins AC, Hall M, Leonard S et al. (1996) Genetic correlation of inhibitory gating of hippocampal auditory evoked response and alpha-bungarotoxin-binding nicotinic cholinergic receptors in inbred mouse strains. Neuropsychopharmacology 15: 152-162. doi:10.1016/0893-133X(95)00178-G. PubMed: 8840351.
- 18. Felix R, Levin ED (1997) Nicotinic antagonist administration into the ventral hippocampus and spatial working memory in rats. Neuroscience 81: 1009-1017. doi:10.1016/S0306-4522(97)00224-8. PubMed: 9330363.
- 19. Wevers A, Witter B, Moser N, Burghaus L, Banerjee C et al. (2000) Classical Alzheimer features and cholinergic dysfunction: towards a unifying hypothesis? Acta Neurol Scand Suppl 176: 42-48. PubMed: 11261804.
- 20. Freedman R, Hall M, Adler LE, Leonard S (1995) Evidence in postmortem brain tissue for decreased numbers of hippocampal nicotinic receptors in schizophrenia. Biol Psychiatry 38: 22-33. doi:10.1016/0006-3223(94)00252-X. PubMed: 7548469.
- 21. Perry EK, Morris CM, Court JA, Cheng A, Fairbairn AF et al. (1995) Alteration in nicotine binding sites in Parkinson’s disease, Lewy body dementia and Alzheimer’s disease: possible index of early neuropathology. Neuroscience 64: 385-395. doi:10.1016/0306-4522(94)00410-7. PubMed: 7700528.
- 22. Nordberg A, Winblad B (1986) Reduced number of [3H]nicotine and [3H]acetylcholine binding sites in the frontal cortex of Alzheimer brains. Neurosci Lett 72: 115-119. doi:10.1016/0304-3940(86)90629-4. PubMed: 3808458.
- 23. Shimohama S, Taniguchi T, Fujiwara M, Kameyama M (1986) Changes in nicotinic and muscarinic cholinergic receptors in Alzheimer-type dementia. J Neurochem 46: 288-293. doi:10.1111/j.1471-4159.1986.tb12960.x. PubMed: 3940287.
- 24. London ED, Ball MJ, Waller SB (1989) Nicotinic binding sites in cerebral cortex and hippocampus in Alzheimer’s dementia. Neurochem Res 14: 745-750. doi:10.1007/BF00964952. PubMed: 2812250.
- 25. Ren K, King MA, Liu J, Siemann J, Altman M et al. (2007). Alpha 7 nicotinic receptor agonist 4OH-GTS-21 protects axotomized septohippocampal cholinergic neurons in wild type but not amyloid-overexpressing transgenic mice. Neuroscience 148: 230-237. doi: 10.1016/j.neuroscience.2007.05.013
- 26. Takeuchi H, Yanagida T, Inden M, Takata K, Kitamura Y, et al. (2009) Nicotinic receptor stimulation protects nigral dopaminergic neurons in rotenone-induced Parkinson's disease models. J Neurosci Res 87: 576-585. doi: 10.1002/jnr.21869
- 27. Shimohama S, Greenwald DL, Shafron DH, Akaika A, Maeda T et al. (1998) Nicotinic alpha 7 receptors protect against glutamate neurotoxicity and neuronal ischemic damage. Brain Res 779: 359-363. doi:10.1016/S0006-8993(97)00194-7. PubMed: 9473725.
- 28. Akaike A, Tamura Y, Yokota T, Shimohama S, Kimura J (1994) Nicotine-induced protection of cultured cortical neurons against N-methyl-D-aspartate receptor-mediated glutamate cytotoxicity. Brain Res 644: 181-187. doi:10.1016/0006-8993(94)91678-0. PubMed: 7519524.
- 29. Kaneko S, Maeda T, Kume T, Kochiyama H, Akaike A et al. (1997) Nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via alpha7-neuronal receptors and neuronal CNS receptors. Brain Res 765: 135-140. doi:10.1016/S0006-8993(97)00556-8. PubMed: 9310404.
- 30. Kihara T, Shimohama S, Sawada H, Kimura J, Kume T et al. (1997) Nicotinic receptor stimulation protects neurons against beta-amyloid toxicity. Ann Neurol 42: 159-163. doi:10.1002/ana.410420205. PubMed: 9266724.
- 31. Meyer EM, Tay ET, Zoltewicz JA, Papke RL, Meyers C et al. (1998) Neuroprotective and memory-related actions of novel à7 nicotinic agents with different mixed agonist/antagonist properties. J PharmacolExpTher 284: 1026-1032.
- 32. Li Y, Papke RL, He YJ, Millard WJ, Meyer EM (1999) Characterization of the neuroprotective and toxic effects of alpha7 nicotinic receptor activation in PC12 cells. Brain Res 830: 218-225. doi:10.1016/S0006-8993(99)01372-4. PubMed: 10366678.
- 33. Shimohama S, Kihara T (2001) Nicotinic receptor-mediated protection against beta-amyloid neurotoxicity. Biol Psychiatry 49: 233-239. doi:10.1016/S0006-3223(00)01100-8. PubMed: 11230874.
- 34. Verbois SL, Scheff SW, Pauly JR (2003) Chronic nicotine treatment attenuates alpha 7 nicotinic receptor deficits following traumatic brain injury. Neuropharmacology 44: 224-233. doi:10.1016/S0028-3908(02)00366-0. PubMed: 12623221.
- 35. Buccafusco JJ (2004) Neuronal Nicotinic Receptor Subtypes: DEFINING THERAPEUTIC TARGETS. Mol Interv 4: 285-295. doi:10.1124/mi.4.5.8. PubMed: 15471911.
- 36. Fucile S, Renzi M, Lauro C, Limatola C, Ciotti T et al. (2004) Nicotinic cholinergic stimulation promotes survival and reduces motility of cultured rat cerebellar granule cells. Neuroscience 127: 53-61. doi:10.1016/j.neuroscience.2004.04.017. PubMed: 15219668.
- 37. Rosa AO, Egea J, Gandía L, López MG, García AG (2006) Neuroprotection by nicotine in hippocampal slices subjected to oxygen-glucose deprivation: involvement of the alpha7 nAChR subtype. J Mol Neurosci 30: 61-62. doi:10.1385/JMN:30:1:61. PubMed: 17192628.
- 38. Egea J, Rosa AO, Sobrado M, Gandía L, López MG et al. (2007) Neuroprotection afforded by nicotine against oxygen and glucose deprivation in hippocampal slices is lost in alpha7 nicotinic receptor knockout mice. Neuroscience 145: 866-872. doi:10.1016/j.neuroscience.2006.12.036. PubMed: 17291692.
- 39. Guseva MV, Hopkins DM, Scheff SW, Pauly JR (2008) Dietary choline supplementation improves behavioral, histological, and neurochemical outcomes in a rat model of traumatic brain injury. Neurotrauma 25: 975-983. doi:10.1089/neu.2008.0516.
- 40. Buccafusco JJ, Letchworth SR, Bencherif M, Lippiello PM (2005) Long-lasting cognitive improvement with nicotinic receptor agonists: mechanisms of pharmacokinetic-pharmacodynamic discordance. Trends Pharmacol Sci 26: 352-360. doi:10.1016/j.tips.2005.05.007. PubMed: 15946748.
- 41. Buccafusco JJ, Terry AV Jr., Decker MW, Gopalakrishnan M (2007) Profile of nicotinic acetylcholine receptor agonists ABT-594 and A-582941, with differential subtype selectivity, on delayed matching accuracy by young monkeys. Biochem Pharmacol 74: 1202-1211. doi:10.1016/j.bcp.2007.07.010. PubMed: 17706609.
- 42. Kitagawa H, Takenouchi T, Azuma R, Wesnes KA, Kramer WG et al. (2003) Safety, pharmacokinetics, and effects on cognitive function of multiple doses of GTS-21 in healthy, male volunteers. Neuropsychopharmacology 28: 542-551. doi:10.1038/sj.npp.1300028. PubMed: 12629535.
- 43. Leiser SC, Bowlby MR, Comery TA, Dunlop J (2009) A cog in cognition: how the alpha 7 nicotinic acetylcholine receptor is geared towards improving cognitive deficits. Pharmacol Ther 122: 302-311. doi:10.1016/j.pharmthera.2009.03.009. PubMed: 19351547.
- 44. Olincy A, Stevens KE (2007) Treating schizophrenia symptoms with an alpha7 nicotinic agonist, from mice to men. Biochem Pharmacol 74: 1192-1201. doi:10.1016/j.bcp.2007.07.015. PubMed: 17714692.
- 45. Arendash GW, Sengstock GJ, Sanberg PR, Kem WR (1995) Improved learning and memory in aged rats with chronic administration of the nicotinic receptor agonist GTS-21. Brain Res 674: 252-259. doi:10.1016/0006-8993(94)01449-R. PubMed: 7796104.
- 46. Meyer EM, Tay ET, Papke RL, Meyers C, Huang GL et al. (1997) Effects of 3-[2,4-dimethoxybenzylidene]anabaseine (DMXB) on rat nicotinic receptors and memory-related behaviors. Brain Res 768: 49-56. doi:10.1016/S0006-8993(97)00536-2. PubMed: 9369300.
- 47. Ross RG, Stevens KE, Proctor WR, Leonard S, Kisley MA et al. (2010) Research review: Cholinergic mechanisms, early brain development, and risk for schizophrenia. J Child Psychol Psychiatry 51: 535-549. doi:10.1111/j.1469-7610.2009.02187.x. PubMed: 19925602.
- 48. Hurst RS, Hajos M, Raggenbass M, Wall TM, Higdon NR et al. (2005) A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization. JNeurosci 25: 4396-4405. doi: 10.1523/JNEUROSCI.5269-04.2005
- 49. Olincy A, Harris JG, Johnson LL, Pender V, Kongs S et al. (2006) Proof-of-concept trial of an alpha7 nicotinic agonist in schizophrenia. Arch Gen Psychiatry 63: 630-638. doi:10.1001/archpsyc.63.6.630. PubMed: 16754836.
- 50. Martin EJ, Panikar KS, King MA, Deyrup M, Hunter B et al. (1994) Cytoprotective actions of 2,4-dimethoxybenzylidene anabaseine in differentiated PC12 cells and septal cholinergic cells. Drug Dev Res 31: 134-141. doi: 10.1002/ddr.430310208
- 51. Briggs CA, Anderson DJ, Brioni JD, Buccafusco JJ, Buckley MJ et al. (1997) Functional characterization of the novel neuronal nicotinic acetylcholine receptor ligand GTS-21 in vitro and in vivo. Pharmacol Biochem Behav 57: 231-241. doi:10.1016/S0091-3057(96)00354-1. PubMed: 9164577.
- 52. Van Kampen M, Selbach K, Schneider R, Schiegel E, Boess F et al. (2004) AR-R 17779 improves social recognition in rats by activation of nicotinic alpha7 receptors. Psychopharmacology (Berl) 172: 375-383. doi:10.1007/s00213-003-1668-7. PubMed: 14727003.
- 53. Brown KL, Comalli DM, De Biasi M, Woodruff-Pak DS (2010) Trace eyeblink conditioning is impaired in α7 but not in β2 nicotinic acetylcholine receptor knockout mice. Front Behav Neurosci 4: 166. PubMed: 20976039.
- 54. Woodruff-Pak DS, Li YT, Kem WR (1994) A nicotinic agonist (GTS-21), eyeblink classical conditioning, and nicotinic receptor binding in rabbit brain. Brain Res 645: 309-317. doi:10.1016/0006-8993(94)91665-9. PubMed: 8062092.
- 55. Wishka DG, Walker DP, Yates KM, Reitz SC, Jia S et al. (2006) Discovery of N-[. (p. 3R)-1-azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide,an agonist of the alpha7 nicotinic acetylcholine receptor, for the potential treatment of cognitive deficits in schizophrenia: synthesis and structure--activity relationship. J Med Chem 49: 4425-4436. doi: 10.1021/jm0602413
- 56. Bitner RS, Bunnelle WH, Decker MW, Drescher KU, Kohlhaas KL et al. (2010) In vivo pharmacological characterization of a novel selective alpha7 neuronal nicotinic acetylcholine receptor agonist ABT-107: preclinical considerations in Alzheimer’s disease. J Pharmacol Exp Ther 334: 875-886. doi:10.1124/jpet.110.167213. PubMed: 20504913.
- 57. Bitner RS, Bunnelle WH, Anderson DJ, Briggs CA, Buccafusco J et al. (2007) Broad-spectrum efficacy across cognitive domains by alpha7 nicotinic acetylcholine receptor agonism correlates with activation of ERK1/2 and CREB phosphorylation pathways. J Neurosci 27: 10578-10587. doi:10.1523/JNEUROSCI.2444-07.2007. PubMed: 17898229.
- 58. Boess FG, De Vry J, Erb C, Flessner T, Hendrix M et al. (2007) The novel alpha7 nicotinic acetylcholine receptor agonist N-[. (p. 3R)-1-azabicyclo[2.2.2]oct-3-yl]-7-[2-(methoxy)phenyl]-1-benzofuran-2-carboxamide improves working and recognition memory in rodents. J Pharmacol Exp Ther 321: 716-725. doi: 10.1124/jpet.106.118976
- 59. Pichat P, Bergis OE, Terranova JP, Urani A, Duarte C et al. (2007) SSR180711, a novel selective alpha7 nicotinic receptor partial agonist: (II) efficacy in experimental models predictive of activity against cognitive symptoms of schizophrenia. Neuropsychopharmacology 32: 17-34. doi:10.1038/sj.npp.1301188. PubMed: 16936709.
- 60. Tatsumi R, Fujio M, Takanashi S, Numata A, Katayama J et al. (2006) (R)-3'-(3-methylbenzo[b]thiophen-5-yl)spiro[1-azabicyclo[2,2,2]octane-3,5'-oxazolidin]-2'-one,a novel and potent alpha7 nicotinic acetylcholine receptor partial agonist displays cognitive enhancing properties. J Med Chem 49: 4374-4383. doi:10.1021/jm060249c. PubMed: 16821797.
- 61. Ren K, Thinschmidt J, Liu J, Ai L, Papke RL et al. (2007). Alpha 7 Nicotinic receptor gene delivery into mouse hippocampal neurons leads to functional receptor expression, improved spatial memory-related performance, and tau hyperphosphorylation. Neuroscience 145: 314-322. doi: 10.1016/j.neuroscience.2006.11.023
- 62. Clarke PBS, Schwartz RD, Paul SM, Pert CB, Pert A (1985) Nicotinic binding in rat brain: autoradiographic comparison of [3 H] acetylcholine [3 H] nicotine and [125 I]-alpha-bungarotoxin. JNeurosci 5: 1307-1315.
- 63. Quik M, Huang LZ, Parameswaran N, Bordia T, Campos C et al. (2009) Multiple roles for nicotine in Parkinson’s disease. Biochem Pharmacol 78: 677-685. doi:10.1016/j.bcp.2009.05.003. PubMed: 19433069.
- 64. Breese CR, Adams C, Logel J, Drebing C, Rollins Y et al. (1997) Comparison of the regional expression of nicotinic acetylcholine receptor alpha7 mRNA and [125I]-alpha-bungarotoxin binding in human postmortem brain. J Comp Neurol 387: 385-398. doi:10.1002/(SICI)1096-9861(19971027)387:3. PubMed: 9335422.
- 65. Whiteaker P, Davies AR, Marks MJ, Blagbrough IS, Potter BV et al. (1999) An autoradiographic study of the distribution of binding sites for the novel alpha7-selective nicotinic radioligand [3H]-methyllycaconitine in the mouse brain. Eur J Neurosci 11: 2689-2696. doi:10.1046/j.1460-9568.1999.00685.x. PubMed: 10457165.
- 66. Woodruff TM, Thundyil J, Tang SC, Sobey CG, Taylor SM et al. (2011) Pathophysiology, treatment, and animal and cellular models of human ischemic stroke. Mol Neurodegener 6: 11. doi:10.1186/1750-1326-6-11. PubMed: 21266064.
- 67. Gusev AG, Uteshev VV (2010) Physiological concentrations of choline activate native alpha7-containing nicotinic acetylcholine receptors in the presence of PNU-120596 [1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea]. J Pharmacol Exp Ther 332: 588-598. doi:10.1124/jpet.109.162099. PubMed: 19923442.
- 68. Kalappa BI, Gusev AG, Uteshev VV (2010) Activation of functional α7-containing nAChRs in hippocampal CA1 pyramidal neurons by physiological levels of choline in the presence of PNU-120596. PLOS ONE 5: e13964. doi:10.1371/journal.pone.0013964. PubMed: 21103043.
- 69. Uteshev VV (2012) Somatic integration of single ion channel responses of α7 nicotinic acetylcholine receptors enhanced by PNU-120596. PLOS ONE 7: e32951. doi:10.1371/journal.pone.0032951. PubMed: 22479351.
- 70. Freitas K, Carroll FI, Damaj MI (2013) The antinociceptive effects of nicotinic receptors α7-positive allosteric modulators in murine acute and tonic pain models. J Pharmacol Exp Ther 344: 264-275. PubMed: 23115222.
- 71. Freitas K, Negus SS, Carroll FI, Damaj MI (2013) In vivo pharmacological interactions between a type II positive allosteric modulator of α7 nicotinic ACh receptors and nicotinic agonists in a murine tonic pain model. Br J Pharmacol 169: 567-579. doi:10.1111/j.1476-5381.2012.02226.x. PubMed: 23004024.
- 72. McLean SL, Grayson B, Idris NF, Lesage AS, Pemberton DJ et al. (2011) Activation of α7 nicotinic receptors improves phencyclidine-induced deficits in cognitive tasks in rats: Implications for therapy of cognitive dysfunction in schizophrenia. Eur Neuropsychopharmacol 21: 333-343. doi:10.1016/j.euroneuro.2010.06.003. PubMed: 20630711.
- 73. McLean SL, Idris NF, Grayson B, Gendle DF, Mackie C et al. (2012) PNU-120596, a positive allosteric modulator of α7 nicotinic acetylcholine receptors, reverses a sub-chronic phencyclidine-induced cognitive deficit in the attentional set-shifting task in female rats. J Psychopharmacol 26: 1265-1270. doi:10.1177/0269881111431747. PubMed: 22182741.
- 74. Jin K, Minami M, Lan JQ, Mao XO, Batteur S et al. (2001) Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proc Natl Acad Sci U S A 98: 4710-4715. doi:10.1073/pnas.081011098. PubMed: 11296300.
- 75. Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis RL et al. (1986) Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 17: 1304-1308. doi:10.1161/01.STR.17.6.1304. PubMed: 2433817.
- 76. Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C et al. (1990) A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 10: 290-293. doi:10.1038/jcbfm.1990.47. PubMed: 1689322.
- 77. Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL et al. (1986) Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17: 472-476. doi:10.1161/01.STR.17.3.472. PubMed: 3715945.
- 78. Sun F, Xie L, Mao X, Hill J, Greenberg DA et al. (2012) Effect of a contralateral lesion on neurological recovery from stroke in rats. Restor Neurol Neurosci 30: 491-495. PubMed: 22868223.
- 79. DeMuth JE.Basic Statistics and Pharmaceutical Statistical Applications. New York, N.Y.: Marcel Dekker Inc./CRC Press.
- 80. Fujiki M, Kobayashi H, Uchida S, Inoue R, Ishii K (2005) Neuroprotective effect of donepezil, a nicotinic acetylcholine-receptor activator, on cerebral infarction in rats. Brain Res 1043: 236-241. doi:10.1016/j.brainres.2005.02.063. PubMed: 15862539.
- 81. Sitzia F, Brown JT, Randall AD, Dunlop J (2011) Voltage- and Temperature-Dependent Allosteric Modulation of α7 Nicotinic Receptors by PNU120596. Front Pharmacol 2: 81. PubMed: 22207849.
- 82. Shimohama S (2009) Nicotinic receptor-mediated neuroprotection in neurodegenerative disease models. Biol Pharm Bull 32: 332-336. doi:10.1248/bpb.32.332. PubMed: 19252273.
- 83. Olincy A, Stevens KE (2007) Treating schizophrenia symptoms with an alpha7 nicotinic agonist, from mice to men. Biochem Pharmacol 74: 1192-1201. doi:10.1016/j.bcp.2007.07.015. PubMed: 17714692.
- 84. Kem WR (2000) The brain alpha7 nicotinic receptor may be an important therapeutic target for the treatment of Alzheimer’s disease: studies with DMXBA (GTS-21). Behav Brain Res 113: 169-181. doi:10.1016/S0166-4328(00)00211-4. PubMed: 10942043.
- 85. Klein J, Holler T, Cappel E, Köppen A, Löffelholz K (1993) Release of choline from rat brain under hypoxia: contribution from phospholipase A2 but not from phospholipase D. Brain Res 630: 337-340. doi:10.1016/0006-8993(93)90674-C. PubMed: 8118702.
- 86. Klein J, Köppen A, Löffelholz K (1998) Regulation of free choline in rat brain: dietary and pharmacological manipulations. Neurochem Int 32: 479-485. doi:10.1016/S0197-0186(97)00127-7. PubMed: 9676747.
- 87. Uteshev VV, Meyer EM, Papke RL (2003) Regulation of neuronal function by choline and 4OH-GTS-21 through alpha 7 nicotinic receptors. J Neurophysiol 89: 1797-1806. PubMed: 12611953.
- 88. Papke RL, Porter Papke JK (2002) Comparative pharmacology of rat and human alpha7 nAChR conducted with net charge analysis. Br J Pharmacol 137: 49-61. doi:10.1038/sj.bjp.0704833. PubMed: 12183330.
- 89. Rao AM, Hatcher JF, Dempsey RJ (2000) Lipid alterations in transient forebrain ischemia: possible new mechanisms of CDP-choline neuroprotection. J Neurochem 75: 2528-2535. PubMed: 11080206.
- 90. Gasull T, DeGregorio-Rocasolano N, Zapata A, Trullas R (2000) Choline release and inhibition of phosphatidylcholine synthesis precede excitotoxic neuronal death but not neurotoxicity induced by serum deprivation. J Biol Chem 275: 18350-18357. doi:10.1074/jbc.M910468199. PubMed: 10748226.
- 91. Djuricic B, Olson SR, Assaf HM, Whittingham TS, Lust WD et al. (1991) Formation of free choline in brain tissue during in vitro energy deprivation. J Cereb Blood Flow Metab 11: 308-313. doi:10.1038/jcbfm.1991.63. PubMed: 1997502.
- 92. Kiewert C, Mdzinarishvili A, Hartmann J, Bickel U, Klein J (2010) Metabolic and transmitter changes in core and penumbra after middle cerebral artery occlusion in mice. Brain Res 1312: 101-107. doi:10.1016/j.brainres.2009.11.068. PubMed: 19961839.
- 93. Kihara T, Shimohama S, Sawada H, Honda K, Nakamizo T et al. (2001) alpha 7 nicotinic receptor transduces signals to phosphatidylinositol 3-kinase to block A beta-amyloid-induced neurotoxicity. J Biol Chem 276: 13541-13546. PubMed: 11278378.
- 94. Akaike A, Takada-Takatori Y, Kume T, Izumi Y (2010) Mechanisms of neuroprotective effects of nicotine and acetylcholinesterase inhibitors: role of alpha4 and alpha7 receptors in neuroprotection. J Mol Neurosci 40: 211-216. doi:10.1007/s12031-009-9236-1. PubMed: 19714494.
- 95. Shaw S, Bencherif M, Marrero MB (2002) Janus kinase 2, an early target of alpha 7 nicotinic acetylcholine receptor-mediated neuroprotection against Abeta-(1-42) amyloid. J Biol Chem 277: 44920-44924. doi:10.1074/jbc.M204610200. PubMed: 12244045.
- 96. Hardingham GE, Bading H (2003) The Yin and Yang of NMDA receptor signalling. Trends Neurosci 26: 81-89. doi:10.1016/S0166-2236(02)00040-1. PubMed: 12536131.
- 97. Papadia S, Hardingham GE (2007) The dichotomy of NMDA receptor signaling. Neuroscientist 13: 572-579. doi:10.1177/1073858407305833. PubMed: 18000068.
- 98. Collins F, Schmidt MF, Guthrie PB, Kater SB (1991) Sustained increase in intracellular calcium promotes neuronal survival. J Neurosci 11: 2582-2587. PubMed: 1714495.
- 99. Koike T, Martin DP, Johnson EM Jr. (1989) Role of Ca2+ channels in the ability of membrane depolarization to prevent neuronal death induced by trophic-factor deprivation: evidence that levels of internal Ca2+ determine nerve growth factor dependence of sympathetic ganglion cells. Proc Natl Acad Sci U S A 86: 6421-6425. doi:10.1073/pnas.86.16.6421. PubMed: 2548215.
- 100. Blair LA, Bence-Hanulec KK, Mehta S, Franke T, Kaplan D et al. (1999) Akt-dependent potentiation of L channels by insulin-like growth factor-1 is required for neuronal survival. J Neurosci 19: 1940-1951. PubMed: 10066247.
- 101. Del Barrio L, Martín-de-Saavedra MD, Romero A, Parada E, Egea J et al. (2011) Neurotoxicity induced by okadaic acid in the human neuroblastoma SH-SY5Y line can be differentially prevented by α7 and β2* nicotinic stimulation. Toxicol Sci 123: 193-205. doi:10.1093/toxsci/kfr163. PubMed: 21715663.
- 102. Castro NG, Albuquerque EX (1995) alpha-Bungarotoxin-sensitive hippocampal nicotinic receptor channel has a high calcium permeability. Biophys J 68: 516-524. doi:10.1016/S0006-3495(95)80213-4. PubMed: 7696505.
- 103. Uteshev VV (2010) Evaluation of Ca2+ permeability of nicotinic acetylcholine receptors in hypothalamic histaminergic neurons. Acta Biochim Biophys Sin (Shanghai) 42: 8-20. doi:10.1093/abbs/gmp101. PubMed: 20043042.
- 104. Fucile S (2004) Ca2+ permeability of nicotinic acetylcholine receptors. Cell Calcium 35: 1-8. doi:10.1016/j.ceca.2003.08.006. PubMed: 14670366.
- 105. Bok J, Wang Q, Huang J, Green SH (2007) CaMKII and CaMKIV mediate distinct prosurvival signaling pathways in response to depolarization in neurons. Mol Cell Neurosci 36: 13-26. doi:10.1016/j.mcn.2007.05.008. PubMed: 17651987.
- 106. Vaillant AR, Mazzoni I, Tudan C, Boudreau M, Kaplan DR et al. (1999) Depolarization and neurotrophins converge on the phosphatidylinositol 3-kinase-Akt pathway to synergistically regulate neuronal survival. J Cell Biol 146: 955-966. doi:10.1083/jcb.146.5.955. PubMed: 10477751.
- 107. Si ML, Lee TJ (2001) Presynaptic alpha7-nicotinic acetylcholine receptors mediate nicotine-induced nitric oxidergic neurogenic vasodilation in porcine basilar arteries. J Pharmacol Exp Ther 298: 122-128. PubMed: 11408533.
- 108. Si ML, Lee TJ (2002) Alpha7-nicotinic acetylcholine receptors on cerebral perivascular sympathetic nerves mediate choline-induced nitrergic neurogenic vasodilation. Circ Res 91: 62-69. doi:10.1161/01.RES.0000024417.79275.23. PubMed: 12114323.
- 109. Parada E, Egea J, Buendia I, Negredo P, Cunha AC et al. (2013) The Microglial alpha7-Acetylcholine Nicotinic Receptor Is a Key Element in Promoting Neuroprotection by Inducing Heme Oxygenase-1 via Nuclear Factor Erythroid-2-Related Factor. p. 2. Antioxid Redox Signal.
- 110. Sharma G, Vijayaraghavan S (2001) Nicotinic cholinergic signaling in hippocampal astrocytes involves calcium-induced calcium release from intracellular stores. Proc Natl Acad Sci U S A 98: 4148-4153. doi:10.1073/pnas.071540198. PubMed: 11259680.
- 111. Shytle RD, Mori T, Townsend K, Vendrame M, Sun N et al. (2004) Cholinergic modulation of microglial activation by alpha 7 nicotinic receptors. J Neurochem 89: 337-343. doi:10.1046/j.1471-4159.2004.02347.x. PubMed: 15056277.
- 112. De Rosa MJ, Dionisio L, Agriello E, Bouzat C, Esandi Mdel C (2009) Alpha 7 nicotinic acetylcholine receptor modulates lymphocyte activation. Life Sci 85: 444-449. doi:10.1016/j.lfs.2009.07.010. PubMed: 19632243.
- 113. Wang H, Yu M, Ochani M, Amella CA, Tanovic M et al. (2003) Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 421: 384-388. doi:10.1038/nature01339. PubMed: 12508119.
- 114. Munro G, Hansen RR, Erichsen HK, Timmermann DB, Christensen JK et al. (2012). Alpha 7 nicotinic ACh receptor agonist compound B and positive allosteric modulator PNU-120596 both alleviate inflammatory hyperalgesia and cytokine release in the rat. Br J Pharmacol. doi: 10.1111/j.1476-5381.2012.02003.x
- 115. Callahan PM, Hutchings EJ, Kille NJ, Chapman JM, Terry AV Jr. (2013) Positive allosteric modulator of alpha 7 nicotinic-acetylcholine receptors, PNU-120596 augments the effects of donepezil on learning and memory in aged rodents and non-human primates. Neuropharmacology 67: 201-212. doi:10.1016/j.neuropharm.2012.10.019. PubMed: 23168113.
- 116. Chilton M, Mastropaolo J, Rosse RB, Bellack AS, Deutsch SI (2004) Behavioral consequences of methyllycaconitine in mice: a model of alpha7 nicotinic acetylcholine receptor deficiency. Life Sci 74: 3133-3139. doi:10.1016/j.lfs.2003.11.012. PubMed: 15081578.
- 117. Kelso ML, Pauly JR (2011) Therapeutic targets for neuroprotection and/or enhancement of functional recovery following traumatic brain injury. Prog Mol Biol. Transl Sci 98: 85-131. doi: 10.1111/j.1476-5381.2012.02003.x