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. Author manuscript; available in PMC: 2016 Mar 1.
Published in final edited form as: Brain Res Bull. 2015 Jan 31;112:35–41. doi: 10.1016/j.brainresbull.2015.01.008

High therapeutic potential of positive allosteric modulation of α7 nAChRs in a rat model of traumatic brain injury: Proof-of-concept

Joshua W Gatson 1, James W Simpkins 2, Victor V Uteshev 3,*
PMCID: PMC4352406  NIHMSID: NIHMS664276  PMID: 25647232

Abstract

There are currently no clinically-efficacious drug therapies to treat brain damage secondary to traumatic brain injury (TBI). In this proof-of-concept study, we used a controlled cortical impact model of TBI in young adult rats to explore a novel promising approach that utilizes PNU-120596, a previously-reported highly selective Type-II positive allosteric modulator (α7-PAM) of α7 nicotinic acetylcholine receptors (nAChRs). α7-PAMs enhance and prolong α7 nAChR activation, but do not activate α7 nAChRs when administered without an agonist. The rational basis for the use of an α7-PAM as a post-TBI treatment is tripartite and arises from: 1) the intrinsic ability of brain injury to elevate extracellular levels of choline (a ubiquitous cell membrane-building material and a selective endogenous agonist of α7 nAChRs) due to the breakdown of cell membranes near the site and time of injury; 2) the ubiquitous expression of functional α7 nAChRs in neuronal and glial/immune brain cells; and 3) the potent neuroprotective and anti-inflammatory effects of α7 nAChR activation. Therefore, both neuroprotective and anti-inflammatory effects can be achieved post-TBI by targeting only a single player (i.e., the α7 nAChR) using α7-PAMs to enhance the activation of α7 nAChRs by injury-elevated extracellular choline. Our data support this hypothesis and demonstrate that subcutaneous administration of PNU-120596 post-TBI in young adult rats significantly reduces both brain cell damage and reactive gliosis. Therefore, our results introduce post-TBI systemic administration of α7-PAMs as a promising therapeutic intervention that could significantly restrict brain injury post-TBI and facilitate recovery of TBI patients.

Keywords: nicotinic, PNU120596, PNU-120596, alpha7, choline, neuroprotection, traumatic brain injury

1. INTRODUCTION

Traumatic brain injury (TBI) commonly causes serious cognitive and motor impairments particularly by damaging the highly vulnerable hippocampus and cortex. TBI can induce two types of neuronal damage: primary (immediate) and secondary (delayed). Although it is not possible to eliminate the primary neuronal damage that occurs at the moment of TBI, it may be possible to alleviate the secondary damage that occurs hours or even days post-TBI and thereby improve the brain function and quality of life of TBI survivors. However, there are currently no clinically-efficacious drug therapies to treat neuronal damage secondary to TBI [35].

The existing literature indicates that expression of functional α7 nicotinic acetylcholine receptors (nAChRs) benefits survival and function of individual neurons and neuronal networks because activation of these receptors enhances neuronal resistance to injury as demonstrated in a number of recent in vivo and ex vivo experimental models of neurological disorders and TBI [8,11, 27, 47, 52, 55, 59]. In addition to neuronal expression, α7 nAChRs are broadly expressed in glial and immune cells where activation of α7 nAChRs results in a potent anti-inflammatory action [8, 38, 42, 47, 48, 60]. Both neuroprotective and anti-inflammatory effects of α7 nAChR activation are expected to benefit the post-TBI recovery of injured brain.

Alpha7 nAChRs are commonly expressed throughout the brain including the hippocampus and cortex [5, 62]. Although neuronal expression of α7 nAChRs is decreased following TBI [58], activation of the remaining post-TBI α7 nAChRs by nicotinic agonists can increase neuronal resistance to injury [57]. However, the effectiveness of α7 agonists appears to be compromised by α7 nAChR desensitization [43]. As a result, therapeutic effects of nicotinic agonists can develop tolerance [22, 31]. Positive allosteric modulation of α7 nAChRs has been proposed as a powerful alternative to desensitizing and somewhat indiscriminate action of nicotinic agonists as an approach to counteracting neurocognitive deficits [7, 24, 37, 50], acute and chronic nociception [14, 15, 38] and cerebral ischemia [27, 49, 55]. Type-II positive allosteric modulators (α7-PAMs), such as PNU-120596 (abbreviated hereafter as PNU), do not activate α7 nAChRs when administered alone. Instead, α7-PAMs enhance and prolong α7 nAChR activation by nicotinic agonists, including endogenous choline [26]. Choline is a full selective agonist of α7 nAChRs [2, 40], a ubiquitous cell building material, and a precursor-metabolite of ACh. However, the physiological level of extracellular choline (~5-10 αM) is sub-threshold for α7 activation [20, 30, 45, 56] due to the low potency of choline (EC50~0.5 mM) [41] and its tendency to induce α7 desensitization (IC50~40 μM) [56]. As a result, choline has not been previously regarded as a therapeutic agent. These limitations can be overcome by the use of α7-PAMs such as PNU [27, 49, 55]. By enhancing and prolonging α7 nAChR activation, α7-PAMs can boost the therapeutic efficacy of α7 activation by nicotinic agonists, including endogenous choline [9, 15, 20, 24, 26, 27, 31, 36, 38, 49, 52, 54, 55, 59].

In addition to choline, ACh is also an endogenous nicotinic agonist that can activate α7 nAChRs and produce neuroprotection in the presence of PNU. However, the extracellular levels of ACh are extremely low (<10 nM) due to ACh hydrolysis [23] and thus, it is the endogenous choline and possibly, the limited near-synaptic ACh that are likely to be the prime α7 agonists responsible for the α7-PAM-enhanced activation of α7 nAChRs near the site and time of injury.

α7-PAMs only amplify the endogenous α7-dependent cholinergic tone which is expected to be elevated in a spatiotemporally restricted manner during TBI due to the breakdown of cell membrane phosphatidylcholine to choline and diacylglycerol [4, 16, 25, 28, 30, 46] providing a large focal source of this selective α7 nAChR agonist. Thus, α7-PAM-based treatments post-TBI may convert endogenous nicotinic agonists (i.e., choline and ACh) into potent therapeutic agents near the site and time of injury. A similar α7-PAM/choline-dependent mechanism has been proposed for the injury-activated endogenous brain self-protection in experimental models of ischemic stroke [27, 49, 55]. This hypothesis is tested in the present study. PNU is found to significantly reduce both brain cell damage and reactive gliosis in a controlled cortical impact (CCI) experimental model of TBI in young adult rats.

2. METHODS

2.1 Animals

The experimental animals were housed and cared for by the Animal Resource Center (ARC) at the University of Texas Southwestern Medical Center (UTSWMC), which is certified by the Association for Assessment and Accreditation of Laboratory Animal Care. All procedures listed in this article were approved by the Institutional Animal Care and Use Committee at UTSWMC.

2.2. Rat TBI Model and Treatment

Adult Sprague Dawley male rats (weighing 300-325 grams) were anesthetized with isoflurane (3-5%) and placed in an adapted nosecone device. A midline incision was made on the head; then, the craniotomy and brain injury procedures were performed. In brief, using the dremel drill, a circular section (6-7 mm diameter) of the skull was removed (adjacent to the sagittal suture). The dura matter was left intact during this process. Using the controlled cortical impact device (Leica Microsystems), a cortical contusion was delivered to the right hemisphere (parietal-temporal cortex). The impact device is well described [61] and consists of a 4-mm flat tip impounder that delivers a velocity of 3.5 m/sec, depth of 2.5 mm, dwell time of 200 ms. Following impact, the skin was closed with surgical wound clips. The animals were treated subcutaneously with PNU (30 mg/kg) or vehicle/placebo (DMSO) either at 30 minutes prior to TBI or 5 minutes after TBI. Sham (craniotomy only) animals were included as controls. Ten animals per group were used in this study. Damage to the hippocampal structures below the injury zone was measured.

2.3. TUNEL Assay

2.3.1. Pretreatment of Paraffin-Embedded Tissues

Briefly, to detect brain cell injury, the brain was intra-cardially perfused with 4% formaldehyde in PBS and harvested at 24 hours after trauma. After fixation and paraffin embedding, the brain tissue was sectioned. The tissue sections were then deparaffinized in fresh xylene twice for 5 minutes and washed in 100% ethanol for 5 minutes at room temperature. The slides were then rehydrated by graded ethanol washes (100%, 95%, 85%, 70%, 50%) for 3 minutes each at room temperature followed by a wash in saline and then, PBS each for 5 minutes at room temperature. The tissue sections were then fixed to the slides using a 4% methanol-free formaldehyde solution in PBS. Proteinase K (100 μl of 20 μg/ml) was added for 8-10 minutes at room temperature to permeabilize the tissue. The slides were then washed in PBS for 5 minutes at room temperature.

2.3.2. Detection of Cellular Damage

The tissue sections were covered with 100 micro-liters of equilibration buffer, then the equilibration buffer was removed and 50 μl of rTdT incubation buffer was added to the tissue sections. The treated sections were then incubated at 37°C for 60 minutes. Following incubation, the sections were treated with 2× SSC for 15 minutes at room temperature. The sections were washed twice in fresh PBS for 5 minutes at room temperature and mounted on glass coverslips in Anti-Fade solution (Molecular Probes Cat.# S7461). The samples were immediately analyzed using a fluorescent microscope. Positive and negative controls were also prepared as recommended by the manufacturer. The standard fluorescein filter set was used to detect fluorescent light at ~520 nm and blue DAPI at ~460 nm. The slides were stored at −20°C in darkness.

2.3.3. Immunohistochemistry

To detect changes in astrocyte activity, the brain was intra-cardially perfused with 4% formaldehyde in PBS and harvested at 72 hours after trauma. Following the fixation steps, paraffined sections were cut at 5 αm thickness and mounted on the microscope slides. The sections were then treated with the primary antibodies (rabbit, anti-GFAP) at 4°C overnight. Secondary antibodies were conjugated with Alexa fluorophore. All antibodies were purchased from ThermoFischer Scientific (Waltham, MA). Reactive astrocytes were detected using a Zeiss Imager A.2 microscope. For detection and count of astrocytes in the prepared tissue sections, nine randomly selected fields of view were evaluated for each animal (i.e., three fields per coronal section and three sections per animal).

2.4. Drugs and Doses

PNU-120596 was purchased from Tocris (Tocris Bioscience, Bristol, UK) or received through the NIDA Research Resources Drug Supply Program. The s.c. dose of PNU-120596 was similar to that used and found effective in experiments utilizing a middle cerebral artery occlusion model of cerebral ischemic stroke in young adult rats [27].

2.5. Statistical Analysis

Data obtained from no fewer than three independent experiments were analyzed using analysis of variance (ANOVA), followed by Tukey’s post-hoc test (GraphPad; San Diego, CA). Groups were considered significantly different if p<0.05. The data are presented as a bar graph depicting the mean±SEM, using the GraphPad Software.

3. RESULTS

In this study, we analyzed brain sections near the injury zone (right parietal temporal cortex) and found that the hippocampus exhibited a considerable amount of brain cell damage at 24 hrs post-CCI (panel B vs. panel A; Figures 1-2). Pre-treatment with subcutaneous (s.c.) 30 mg/kg PNU did not have an obvious robust effect on the TBI-induced cell damage in the placebo versus 30 minute pretreatment groups in the DG (p=0.069, n=10) and CA3 (p=0.062, n=10) hippocampal brain regions and was dismissed from further investigation. Instead, the prime focus was on post-TBI s.c. administration of PNU. Very little TUNEL staining and thus, brain cell damage was observed in the group treated with PNU at 5 minutes after TBI supporting protective effects of PNU post-CCI (panel D vs. panels B-C; Figures 1-2). Specifically, while the CCI significantly (p<0.0002, n=10) increased the number of TUNEL-positive neurons in the hippocampal CA3 region, cellular injury in the CA3 was significantly reduced by s.c. treatments with PNU at 5 minutes post-injury (p<0.0005, n=10) (Figure 3A). The DG region of the hippocampus was affected in a similar manner (Figure 3B): compared to the control group, a significant increase of TUNEL+ cells was found in the placebo+TBI group (p<0.0003, n=10), while treatment with PNU 5 min post-CCI significantly (p<0.0001, n=10) lowered the number of TUNEL+ cells. No significant injury was observed on the contralateral side (data not shown).

Figure 1. Treatment with PNU results in a decrease in the number of TUNEL-positive cells in the CA3 region of the hippocampus.

Figure 1

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TUNEL staining is illustrated in panels A-D. Panels E-H are DAPI stained cells. The groups studied here are: control (A, E); placebo+TBI (B, F); PNU 30 minute pre-treatment+TBI (C, G); PNU treatment at 5 minutes after TBI (D, H).

Figure 2. PNU treatment decreases the number of TUNEL-positive cells in the DG region of the hippocampus.

Figure 2

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TUNEL staining is illustrated in panels A-D. Panels E-H are DAPI stained cells. The groups studied here are: control (A, E); placebo+TBI (B, F); PNU 30 minute pre-treatment+TBI (C, G); PNU treatment at 5 minutes after TBI (D, H).

Figure 3. Quantification of PNU effects after TBI.

Figure 3

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Administration of PNU at 5 minutes after TBI resulted in a significant decrease of TUNEL+cells in the CA3 (A) and DG (B) hippocampal brain regions. The data are presented as a bar graph depicting the mean±SEM. CA3, placebo±TBI vs. PNU+TBI (* p<0.0005); DG, placebo+TBI vs. PNU+TBI (** p<0.0001).

On day 3 after injury, a significant increase in the number of reactive astrocytes was detected in the CA3 (p<0.001, n=10) and DG (p<0.0003, n=10) hippocampal regions of rats treated with placebo after TBI group (panel II vs. panel I in Figure 4A; and Figure 4B). Within the group treated with PNU at 5 minutes after TBI, there was a significant decrease in the number of GFAP+ cells in the CA3 (p<0.01, n=10) and DG (p<0.0006, n=10) hippocampal brain regions compared to the placebo+TBI group (panel III vs. panel II in Figure 4A; and Figure 4B). No significant injury was observed on the contralateral side (data not shown). These results support our central hypothesis that post-TBI treatments with PNU can be highly effective in alleviating cellular injury secondary to TBI.

Figure 4. Quantification of activated astrocytes within the hippocampus (CA3 and DG).

Figure 4

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Staining with GFAP (panel I) showed a significant increase in activated astrocytes in the CA3 and DG (panel II), which was abolished by PNU (panel III) (A). PNU treatment after TBI led to a significant decrease in the levels of activated astrocytes in the CA3 and DG regions (B). The data are presented as a bar graph depicting the mean±SEM. CA3, placebo+TBI vs. PNU+TBI (* p<0.01); DG, placebo+TBI vs. PNU+TBI (** p<0.006).

4. DISCUSSION

This proof-of-concept study demonstrates that subcutaneous administration of an α7-PAM post-CCI in young adult rats significantly reduces both brain cell damage and reactive gliosis. Therefore, our data introduce post-TBI systemic administration of α7-PAMs as a promising therapeutic intervention that could significantly restrict brain injury secondary to TBI and facilitate recovery of TBI patients. The exact mechanism of α7-PAM-mediated protection post-TBI is not known and an important target of future investigation. However, the rational basis for the use of an α7-PAM as a post-TBI treatment is tripartite and supported by: 1) the intrinsic ability of brain injury to elevate extracellular levels of choline due to the breakdown of cell membranes near the site and time of injury [10, 16, 27, 28, 46, 49, 55, 64]; 2) the ubiquitous expression of functional α7 nAChRs in neuronal and glial/immune brain cells [5, 8, 38, 42, 44, 47, 48, 60, 62, 63]; and 3) the potent neuroprotective and anti-inflammatory effects of α7 nAChR activation [3, 11, 18, 27, 33, 38, 42, 57]. Therefore, both neuroprotective and anti-inflammatory effects can be achieved post-TBI by targeting only a single player (i.e., the α7 nAChR) using α7-PAMs to enhance α7 nAChR activation by injury-elevated extracellular choline.

A previous mass-spectrometry analysis of brain and blood samples collected 3 hrs after s.c. administration of PNU at doses similar to those used in this study (i.e., 30 mg/kg) [27], determined that the amounts of PNU in the hippocampus, frontal cortex and striatum are elevated to ~150 ng/g, a quantity somewhat greater than that detected in blood plasma (~60 nM). These measurements are comparable to those reported elsewhere [37]. Thus, sub-μM to low-μM concentrations of PNU are expected in the brain within the first 3 hrs after s.c. injection.

The plasma half-life time of PNU is ~8 hrs [37]. Thus, pretreatment with PNU would be expected to be neuroprotective. However, s.c. PNU treatment 30 min prior to CCI produced only sub-significant benefits (p~0.06) in our experiments. This apparent reduction in the therapeutic efficacy of PNU treatment before trauma as compared to post-CCI treatments may reflect the absence of elevated levels of extracellular choline before the injury.

Because α7-PAMs, like PNU, are inactive in the absence of α7 nAChR agonists [20, 26], the challenge of precise delivery of α7-PAMs to the site of TBI is naturally eliminated: α7-PAMs could be administered systemically (e.g., s.c.) to be somewhat homogenously distributed throughout the body by circulation, while their therapeutic action would take place mostly, or even exclusively, in the brain areas with elevated extracellular levels of choline [16, 28] and thus, exactly where and when it is needed: i.e., near the site and time of injury [55]. Thus, the high spatiotemporal precision of α7-PAM-based therapies is an important benefit of this novel approach.

The anticipated clinical utility of PNU is likely to extend to certain genetic and age-related neurodegenerative, sensorimotor, and psychiatric disorders characterized by cognitive decline and attention deficits (e.g., schizophrenia and dementia) since these pathologies are also associated with a decreased cholinergic tone and a decrease, but not total disappearance, of functional α7 nAChRs [12, 13, 19, 32]. Thus, in addition to protecting brain against secondary post-TBI injury, drugs that enhance α7 nAChR activation are expected to improve cognitive function and attention impairments in patients and animal models by increasing and partially restoring the endogenous cholinergic tone [6, 21, 29, 39]. In this regard, treatments with PNU are expected to benefit individuals with TBI and certain age-related cognitive deficits via multiple mechanisms and routes of action. Accordingly, subcutaneous injections of 10 mg/kg PNU has been shown to improve cognitive performance in a rat model of schizophrenia [37], while PNU remained in plasma at concentrations of ~50 nM for several hours (t1/2~8 hrs and AUC0-last ~500 nM.h). These results fall within the range of PNU activity seen ex vivo [17, 26, 27] and are consistent with our present results supporting functional relevance of s.c. injections of α7-PAMs.

Moreover, activation of α7 nAChRs post-TBI by exogenous agents has been shown to facilitate functional recovery as evidenced by a reduction of cortical and hippocampal lesion volumes, corrected levels of α7 expression in the hippocampus and improved cognitive performance in Morris Water Maze induced by dietary choline supplementation and systemic administration of nicotine in rats [21, 57]. However, although exogenous α7 nAChR ligands present valuable tools in treatment of neurological dysfunctions [15, 31, 38, 55, 57, 59], therapies that could amplify the brain’s innate ability to protect itself from various injuries, such as TBI, serve as intriguing, powerful alternatives [34, 55] and have not been fully explored.

The mechanisms of α7-dependent neuroprotection have been discussed previously for treatments that utilize α7 agonists. These mechanisms include Ca2+-dependent JAK2-PI3K/Akt and ERK1/2 dependent molecular pathways that enhance neuronal resistance to energy/ATP deprivation [1, 51]. PNU-like α7-PAMs only amplify α7 activation by agonists and thus, would be expected to amplify the same α7-/Ca2+-dependent molecular pathways. While neuroprotection against NMDA excitotoxicity requires inhibition of NMDARs; the α7-dependent neuroprotection requires enhanced α7 activation [53] and PNU-like α7-PAMs appear to augment the therapeutic action of α7 nAChR activation [55].

One limitation of this study is that it evaluates the therapeutic efficacy of PNU only in the hippocampus. The effects of PNU in other brain regions susceptible to TBI (e.g., the cortex) may differ from those determined in this study and need to be explored. Moreover, the maximal effective delay, optimal doses and routes of PNU treatment post-TBI have not been determined. While this study provides a proof-of-concept for the therapeutic effects of PNU in TBI, it is uncertain whether PNU indeed irreversibly reduces, not only delays, severe brain tissue damage after TBI. Another limitation is that we did not evaluate whether PNU alters the normal physiological state of neurons and neuronal networks. The approach that we employ in this study evaluates the number of damaged brain cells and the therapeutic efficacy of PNU after injury (i.e., cortical impact). Thus, it is not readily applicable in the absence of injury. These limitations will be addressed in our future studies where the effects of PNU in multiple brain regions and at multiple time points post-TBI will be determined to provide a more comprehensive characterization of the therapeutic efficacy of PNU in TBI.

In conclusion, this proof-of-concept study demonstrated that augmenting activation of α7 nAChRs post-TBI by systemic subcutaneous administration of a Type-II α7-PAM post-CCI in young adult rats significantly reduced both brain cell damage and reactive gliosis. Therefore, our results introduce post-TBI treatment with α7-PAMs as a highly promising therapeutic intervention that could significantly restrict brain injury secondary to TBI and facilitate recovery of TBI survivors.

Highlights.

  • PNU-120596 inhibits brain cell injury and gliosis in a CCI model of TBI

  • PNU-120596 augments the therapeutic efficacy of endogenous choline

  • PNU-120596 could restrict brain injury and facilitate recovery post-TBI

ACKNOWLEDGEMENTS

This study was supported by a grant DK082625 from the National Institute of Health to VVU. We thank the NIDA Research Resources Drug Supply Program for PNU-120596.

LIST OF ABBREVIATIONS USED

CCI

controlled cortical impact

DAPI (4′,6-diamidino-2-phenylindole)

a fluorescent dye that binds to DNA

DMSO

dimethyl sulfoxide

nAChRs

nicotinic acetylcholine receptors

PBS

phosphate buffer solution

PNU or PNU-120596 (N-(5-Chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea)

a positive allsoteric modulator of α7 nAChRs

PAM

positive allosteric modulator

s.c.

subcutaneous

TBI

traumatic brain injury

Footnotes

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REFERENCES

  • [1].Akaike A, Takada-Takatori Y, Kume T, Izumi Y. Mechanisms of neuroprotective effects of nicotine and acetylcholinesterase inhibitors: role of alpha4 and alpha7 receptors in neuroprotection. J.Mol.Neurosci. 2010;40:211–216. doi: 10.1007/s12031-009-9236-1. [DOI] [PubMed] [Google Scholar]
  • [2].Alkondon M, Pereira EF, Cortes WS, Maelicke A, Albuquerque EX. Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons. Eur.J.Neurosci. 1997;9:2734–2742. doi: 10.1111/j.1460-9568.1997.tb01702.x. [DOI] [PubMed] [Google Scholar]
  • [3].Arendash GW, Sengstock GJ, Sanberg PR, Kem WR. Improved learning and memory in aged rats with chronic administration of the nicotinic receptor agonist GTS-21. Brain Res. 1995;674:252–259. doi: 10.1016/0006-8993(94)01449-r. [DOI] [PubMed] [Google Scholar]
  • [4].Bertrand N, Ishii H, Spatz M. Cerebral ischemia in young and adult gerbils: effects on cholinergic metabolism. Neurochem.Int. 1996;28:293–297. doi: 10.1016/0197-0186(95)00086-0. [DOI] [PubMed] [Google Scholar]
  • [5].Breese CR, Adams C, Logel J, Drebing C, Rollins Y, Barnhart M, Sullivan B, Demasters BK, Freedman R, Leonard S. Comparison of the regional expression of nicotinic acetylcholine receptor alpha7 mRNA and [125I]-alpha-bungarotoxin binding in human postmortem brain. J. Comp. Neurol. 1997;387:385–398. doi: 10.1002/(sici)1096-9861(19971027)387:3<385::aid-cne5>3.0.co;2-x. [DOI] [PubMed] [Google Scholar]
  • [6].Brown KL, Comalli DM, Biasi MD, Woodruff-Pak DS. Trace Eyeblink Conditioning is Impaired in alpha7 but not in beta2 Nicotinic Acetylcholine Receptor Knockout Mice. Front Behav Neurosci. 2010;4:166. doi: 10.3389/fnbeh.2010.00166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Callahan PM, Hutchings EJ, Kille NJ, Chapman JM, Terry AV., Jr. 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. 2013;67:201–212. doi: 10.1016/j.neuropharm.2012.10.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].De Rosa MJ, Dionisio L, Agriello E, Bouzat C, Esandi Mdel C. Alpha 7 nicotinic acetylcholine receptor modulates lymphocyte activation. Life Sci. 2009;85:444–449. doi: 10.1016/j.lfs.2009.07.010. [DOI] [PubMed] [Google Scholar]
  • [9].Dinklo T, Shaban H, Thuring JW, Lavreysen H, Stevens KE, Zheng L, Mackie C, Grantham C, Vandenberk I, Meulders G, Peeters L, Verachtert H, De Prins E, Lesage AS. Characterization of 2-[[4-fluoro-3-(trifluoromethyl)phenyl]amino]-4-(4-pyridinyl)-5-thiazoleme thanol (JNJ-1930942), a novel positive allosteric modulator of the {alpha}7 nicotinic acetylcholine receptor. J. Pharmacol. Exp. Ther. 2011;336:560–574. doi: 10.1124/jpet.110.173245. [DOI] [PubMed] [Google Scholar]
  • [10].Djuricic B, Olson SR, Assaf HM, Whittingham TS, Lust WD, Drewes LR. Formation of free choline in brain tissue during in vitro energy deprivation. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 1991;11:308–313. doi: 10.1038/jcbfm.1991.63. [DOI] [PubMed] [Google Scholar]
  • [11].Egea J, Rosa AO, Sobrado M, Gandia L, Lopez MG, Garcia AG. Neuroprotection afforded by nicotine against oxygen and glucose deprivation in hippocampal slices is lost in alpha7 nicotinic receptor knockout mice. Neuroscience. 2007;145:866–872. doi: 10.1016/j.neuroscience.2006.12.036. [DOI] [PubMed] [Google Scholar]
  • [12].Felix R, Levin ED. Nicotinic antagonist administration into the ventral hippocampus and spatial working memory in rats. Neuroscience. 1997;81:1009–1017. doi: 10.1016/s0306-4522(97)00224-8. [DOI] [PubMed] [Google Scholar]
  • [13].Freedman R, Adams CE, Leonard S. The alpha7-nicotinic acetylcholine receptor and the pathology of hippocampal interneurons in schizophrenia. J. Chem. Neuroanat. 2000;20:299–306. doi: 10.1016/s0891-0618(00)00109-5. [DOI] [PubMed] [Google Scholar]
  • [14].Freitas K, Carroll FI, Damaj MI. The Antinociceptive Effects of Nicotinic Receptors alpha7-Positive Allosteric Modulators in Murine Acute and Tonic Pain Models. J. Pharmacol. Exp. Ther. 2012;344:264–275. doi: 10.1124/jpet.112.197871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Freitas K, Negus SS, Carroll FI, Damaj MI. In vivo pharmacological interactions between a type II positive allosteric modulator of alpha7 nicotinic ACh receptors and nicotinic agonists in a murine tonic pain model. Br. J. Pharmacol. 2013;169:567–579. doi: 10.1111/j.1476-5381.2012.02226.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Friedman SD, Brooks WM, Jung RE, Hart BL, Yeo RA. Proton MR spectroscopic findings correspond to neuropsychological function in traumatic brain injury. AJNR. American journal of neuroradiology. 1998;19:1879–1885. [PMC free article] [PubMed] [Google Scholar]
  • [17].Gronlien JH, Hakerud M, Ween H, Thorin-Hagene K, Briggs CA, Gopalakrishnan M, Malysz J. Distinct profiles of alpha7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes. Mol.Pharmacol. 2007;72:715–724. doi: 10.1124/mol.107.035410. [DOI] [PubMed] [Google Scholar]
  • [18].Guan YZ, Jin XD, Guan LX, Yan HC, Wang P, Gong Z, Li SJ, Cao X, Xing YL, Gao TM. Nicotine Inhibits Microglial Proliferation and Is Neuroprotective in Global Ischemia Rats. Mol. Neurobiol. 2014 doi: 10.1007/s12035-014-8825-3. [DOI] [PubMed] [Google Scholar]
  • [19].Guan ZZ, Zhang X, Ravid R, Nordberg A. Decreased protein levels of nicotinic receptor subunits in the hippocampus and temporal cortex of patients with Alzheimer’s disease. J.Neurochem. 2000;74:237–243. doi: 10.1046/j.1471-4159.2000.0740237.x. [DOI] [PubMed] [Google Scholar]
  • [20].Gusev AG, Uteshev VV. 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. 2010;332:588–598. doi: 10.1124/jpet.109.162099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Guseva MV, Hopkins DM, Scheff SW, Pauly JR. Dietary choline supplementation improves behavioral, histological, and neurochemical outcomes in a rat model of traumatic brain injury. J.Neurotrauma. 2008;25:975–983. doi: 10.1089/neu.2008.0516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Harris JG, Kongs S, Allensworth D, Martin L, Tregellas J, Sullivan B, Zerbe G, Freedman R. Effects of nicotine on cognitive deficits in schizophrenia. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 2004;29:1378–1385. doi: 10.1038/sj.npp.1300450. [DOI] [PubMed] [Google Scholar]
  • [23].Hartmann J, Kiewert C, Duysen EG, Lockridge O, Klein J. Choline availability and acetylcholine synthesis in the hippocampus of acetylcholinesterase-deficient mice. Neurochem. Int. 2008;52:972–978. doi: 10.1016/j.neuint.2007.10.008. [DOI] [PubMed] [Google Scholar]
  • [24].Hurst RS, Hajos M, Raggenbass M, Wall TM, Higdon NR, Lawson JA, Rutherford-Root KL, Berkenpas MB, Hoffmann WE, Piotrowski DW, Groppi VE, Allaman G, Ogier R, Bertrand S, Bertrand D, Arneric SP. A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization. J.Neurosci. 2005;25:4396–4405. doi: 10.1523/JNEUROSCI.5269-04.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Jope RS, Gu X. Seizures increase acetylcholine and choline concentrations in rat brain regions. Neurochem.Res. 1991;16:1219–1226. doi: 10.1007/BF00966699. [DOI] [PubMed] [Google Scholar]
  • [26].Kalappa BI, Gusev AG, Uteshev VV. Activation of functional alpha7-containing nAChRs in hippocampal CA1 pyramidal neurons by physiological levels of choline in the presence of PNU-120596. PloS one. 2010;5:e13964. doi: 10.1371/journal.pone.0013964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Kalappa BI, Sun F, Johnson SR, Jin K, Uteshev VV. A positive allosteric modulator of alpha7 nAChRs augments neuroprotective effects of endogenous nicotinic agonists in cerebral ischaemia. Br. J. Pharmacol. 2013;169:1862–1878. doi: 10.1111/bph.12247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Kiewert C, Mdzinarishvili A, Hartmann J, Bickel U, Klein J. Metabolic and transmitter changes in core and penumbra after middle cerebral artery occlusion in mice. Brain Res. 1312;2010:101–107. doi: 10.1016/j.brainres.2009.11.068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Kitagawa H, Takenouchi T, Azuma R, Wesnes KA, Kramer WG, Clody DE, Burnett AL. Safety, pharmacokinetics, and effects on cognitive function of multiple doses of GTS-21 in healthy, male volunteers. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 2003;28:542–551. doi: 10.1038/sj.npp.1300028. [DOI] [PubMed] [Google Scholar]
  • [30].Klein J, Koppen A, Loffelholz K. Regulation of free choline in rat brain: dietary and pharmacological manipulations. Neurochem.Int. 1998;32:479–485. doi: 10.1016/s0197-0186(97)00127-7. [DOI] [PubMed] [Google Scholar]
  • [31].Lendvai B, Kassai F, Szajli A, Nemethy Z. alpha7 nicotinic acetylcholine receptors and their role in cognition. Brain Res. Bull. 2013;93:86–96. doi: 10.1016/j.brainresbull.2012.11.003. [DOI] [PubMed] [Google Scholar]
  • [32].Leonard JR, Maris DO, Grady MS. Fluid percussion injury causes loss of forebrain choline acetyltransferase and nerve growth factor receptor immunoreactive cells in the rat. Journal of neurotrauma. 1994;11:379–392. doi: 10.1089/neu.1994.11.379. [DOI] [PubMed] [Google Scholar]
  • [33].Li Y, Papke RL, He YJ, Millard WJ, Meyer EM. Characterization of the neuroprotective and toxic effects of alpha7 nicotinic receptor activation in PC12 cells. Brain Res. 1999;830:218–225. doi: 10.1016/s0006-8993(99)01372-4. [DOI] [PubMed] [Google Scholar]
  • [34].Lo EH. A new penumbra: transitioning from injury into repair after stroke. Nat. Med. 2008;14:497–500. doi: 10.1038/nm1735. [DOI] [PubMed] [Google Scholar]
  • [35].Loane DJ, Faden AI. Neuroprotection for traumatic brain injury: translational challenges and emerging therapeutic strategies. Trends Pharmacol. Sci. 2010;31:596–604. doi: 10.1016/j.tips.2010.09.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [36].Malysz J, Gronlien JH, Anderson DJ, Hakerud M, Thorin-Hagene K, Ween H, Wetterstrand C, Briggs CA, Faghih R, Bunnelle WH, Gopalakrishnan M. In vitro pharmacological characterization of a novel allosteric modulator of alpha 7 neuronal acetylcholine receptor, 4-(5-(4-chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonami de (A-867744), exhibiting unique pharmacological profile. J. Pharmacol. Exp. Ther. 2009;330:257–267. doi: 10.1124/jpet.109.151886. [DOI] [PubMed] [Google Scholar]
  • [37].McLean SL, Idris NF, Grayson B, Gendle DF, Mackie C, Lesage AS, Pemberton DJ, Neill JC. PNU-120596, a positive allosteric modulator of alpha7 nicotinic acetylcholine receptors, reverses a sub-chronic phencyclidine-induced cognitive deficit in the attentional set-shifting task in female rats. J Psychopharmacol. 2012;26:1265–1270. doi: 10.1177/0269881111431747. [DOI] [PubMed] [Google Scholar]
  • [38].Munro G, Hansen R, Erichsen H, Timmermann D, Christensen J, Hansen H. The alpha7 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. 2012;167:421–435. doi: 10.1111/j.1476-5381.2012.02003.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [39].Olincy A, Harris JG, Johnson LL, Pender V, Kongs S, Allensworth D, Ellis J, Zerbe GO, Leonard S, Stevens KE, Stevens JO, Martin L, Adler LE, Soti F, Kem WR, Freedman R. Proof-of-concept trial of an alpha7 nicotinic agonist in schizophrenia. Archives of general psychiatry. 2006;63:630–638. doi: 10.1001/archpsyc.63.6.630. [DOI] [PubMed] [Google Scholar]
  • [40].Papke RL, Bencherif M, Lippiello P. An evaluation of neuronal nicotinic acetylcholine receptor activation by quaternary nitrogen compounds indicates that choline is selective for the alpha 7 subtype. Neurosci.Lett. 1996;213:201–204. doi: 10.1016/0304-3940(96)12889-5. [DOI] [PubMed] [Google Scholar]
  • [41].Papke RL, Papke JKP. Comparative pharmacology of rat and human alpha7 nAChR conducted with net charge analysis. Br J of Pharmacol. 2002;137:49–61. doi: 10.1038/sj.bjp.0704833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [42].Parada E, Egea J, Buendia I, Negredo P, Cunha AC, Cardoso S, Soares MP, Lopez MG. 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 2. Antioxid. Redox Signal. 2013 doi: 10.1089/ars.2012.4671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [43].Quick MW, Lester RA. Desensitization of neuronal nicotinic receptors. J.Neurobiol. 2002;53:457–478. doi: 10.1002/neu.10109. [DOI] [PubMed] [Google Scholar]
  • [44].Quik M, Huang LZ, Parameswaran N, Bordia T, Campos C, Perez XA. Multiple roles for nicotine in Parkinson’s disease. Biochem. Pharmacol. 2009;78:677–685. doi: 10.1016/j.bcp.2009.05.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [45].Sarter M, Parikh V. Choline transporters, cholinergic transmission and cognition. Nat.Rev.Neurosci. 2005;6:48–56. doi: 10.1038/nrn1588. [DOI] [PubMed] [Google Scholar]
  • [46].Scremin OU, Jenden DJ. Time-dependent changes in cerebral choline and acetylcholine induced by transient global ischemia in rats. Stroke. 1991;22:643–647. doi: 10.1161/01.str.22.5.643. [DOI] [PubMed] [Google Scholar]
  • [47].Sharma G, Vijayaraghavan S. Nicotinic cholinergic signaling in hippocampal astrocytes involves calcium-induced calcium release from intracellular stores. Proceedings of the National Academy of Sciences of the United States of America. 2001;98:4148–4153. doi: 10.1073/pnas.071540198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [48].Shytle RD, Mori T, Townsend K, Vendrame M, Sun N, Zeng J, Ehrhart J, Silver AA, Sanberg PR, Tan J. Cholinergic modulation of microglial activation by alpha 7 nicotinic receptors. J. Neurochem. 2004;89:337–343. doi: 10.1046/j.1471-4159.2004.02347.x. [DOI] [PubMed] [Google Scholar]
  • [49].Sun F, Jin K, Uteshev VV. A Type-II Positive Allosteric Modulator of alpha7 nAChRs Reduces Brain Injury and Improves Neurological Function after Focal Cerebral Ischemia in Rats. PloS one. 2013;8:e73581. doi: 10.1371/journal.pone.0073581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [50].Thomsen MS, El-Sayed M, Mikkelsen JD. Differential immediate and sustained memory enhancing effects of alpha7 nicotinic receptor agonists and allosteric modulators in rats. PloS one. 2011;6:e27014. doi: 10.1371/journal.pone.0027014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [51].Thomsen MS, Hansen HH, Timmerman DB, Mikkelsen JD. Cognitive improvement by activation of alpha7 nicotinic acetylcholine receptors: from animal models to human pathophysiology. Curr.Pharm.Des. 2010;16:323–343. doi: 10.2174/138161210790170094. [DOI] [PubMed] [Google Scholar]
  • [52].Timmermann DB, Gronlien JH, Kohlhaas KL, Nielsen EO, Dam E, Jorgensen TD, Ahring PK, Peters D, Holst D, Christensen JK, Malysz J, Briggs CA, Gopalakrishnan M, Olsen GM. An allosteric modulator of the alpha7 nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivo. J. Pharmacol. Exp. Ther. 2007;323:294–307. doi: 10.1124/jpet.107.120436. [DOI] [PubMed] [Google Scholar]
  • [53].Uteshev VV. alpha7 Nicotinic ACh Receptors as a Ligand-Gated Source of Ca(2+) Ions: The Search for a Ca (2+) Optimum. Adv. Exp. Med. Biol. 2012;740:603–638. doi: 10.1007/978-94-007-2888-2_27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [54].Uteshev VV. Somatic Integration of Single Ion Channel Responses of alpha7 Nicotinic Acetylcholine Receptors Enhanced by PNU-120596. PloS one. 2012;7:e32951. doi: 10.1371/journal.pone.0032951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [55].Uteshev VV. The therapeutic promise of positive allosteric modulation of nicotinic receptors. Eur. J. Pharmacol. 2014;727:181–185. doi: 10.1016/j.ejphar.2014.01.072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [56].Uteshev VV, Meyer EM, Papke RL. Regulation of neuronal function by choline and 4OH-GTS-21 through alpha 7 nicotinic receptors. J. Neurophysiol. 2003;89:1797–1806. doi: 10.1152/jn.00943.2002. [DOI] [PubMed] [Google Scholar]
  • [57].Verbois SL, Hopkins DM, Scheff SW, Pauly JR. Chronic intermittent nicotine administration attenuates traumatic brain injury-induced cognitive dysfunction. Neuroscience. 2003;119:1199–1208. doi: 10.1016/s0306-4522(03)00206-9. [DOI] [PubMed] [Google Scholar]
  • [58].Verbois SL, Sullivan PG, Scheff SW, Pauly JR. Traumatic brain injury reduces hippocampal alpha7 nicotinic cholinergic receptor binding. Journal of neurotrauma. 2000;17:1001–1011. doi: 10.1089/neu.2000.17.1001. [DOI] [PubMed] [Google Scholar]
  • [59].Wallace TL, Porter RH. Targeting the nicotinic alpha7 acetylcholine receptor to enhance cognition in disease. Biochem. Pharmacol. 2011;82:891–903. doi: 10.1016/j.bcp.2011.06.034. [DOI] [PubMed] [Google Scholar]
  • [60].Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, Li JH, Yang H, Ulloa L, Al-Abed Y, Czura CJ, Tracey KJ. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature. 2003;421:384–388. doi: 10.1038/nature01339. [DOI] [PubMed] [Google Scholar]
  • [61].Whalen MJ, Carlos TM, Dixon CE, Schiding JK, Clark RS, Baum E, Yan HQ, Marion DW, Kochanek PM. Effect of traumatic brain injury in mice deficient in intercellular adhesion molecule-1: assessment of histopathologic and functional outcome. Journal of neurotrauma. 1999;16:299–309. doi: 10.1089/neu.1999.16.299. [DOI] [PubMed] [Google Scholar]
  • [62].Whiteaker P, Davies AR, Marks MJ, Blagbrough IS, Potter BV, Wolstenholme AJ, Collins AC, Wonnacott S. 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. 1999;11:2689–2696. doi: 10.1046/j.1460-9568.1999.00685.x. [DOI] [PubMed] [Google Scholar]
  • [63].Woodruff TM, Thundyil J, Tang SC, Sobey CG, Taylor SM, Arumugam TV. Pathophysiology, treatment, and animal and cellular models of human ischemic stroke. Mol Neurodegener. 2011;6:11. doi: 10.1186/1750-1326-6-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [64].Zapata A, Capdevila JL, Trullas R. Region-specific and calcium-dependent increase in dialysate choline levels by NMDA. J. Neurosci. 1998;18:3597–3605. doi: 10.1523/JNEUROSCI.18-10-03597.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]

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