the selective 7 nicotinic acetylcholine receptor agonist ... · this deficit leads to disrupted...
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The Selective α7 Nicotinic Acetylcholine Receptor Agonist PNU-282987
Enhances GABAergic Synaptic Activity in Brain Slices and
Restores Auditory Gating Deficits in Anesthetized Rats
M. Hajós, R. S. Hurst, W. E. Hoffmann, M. Krause1, T. M. Wall2, N. R. Higdon2, and
V. E. Groppi
Department of Neuroscience (MH, RSH, MK, TMW, NRH) and
CNS Molecular Sciences (VEG),
Pfizer Global Research & Development, Pfizer Inc. Groton, CT and Ann Arbor, MI, USA
JPET Fast Forward. Published on November 2, 2004 as DOI:10.1124/jpet.104.076968
Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title: PNU-282987, a novel α7 nAChR agonist
Corresponding author: Mihály Hajós, PharmD, Ph.D.
Department of Neuroscience
Pfizer Global Research and Development
Eastern Point Road
Groton, CT 06340, USA
Telephone: (860) 686-6967
Fax: (860) 715-2349
E-mail: [email protected]
Number of text pages: 34
Number of tables: 0
Number of figures: 8
Number of words in the abstract: 219
Number of words in the introduction: 593
Number of words in the discussion: 1098
Number of References: 39
Recommended Section: Neuropharmacology
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Abbreviations:
BPS, Phosphate Buffer Saline; C, conditioning; CHRNA7, α7 nicotinic acetylcholine receptor
subunit gene; CNQX, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate 6-
cyano-7-nitroquinoxaline-2,3-dione; EEG, electroencephalograph; GABA, gamma-aminobutyric
acid; GTS-21, 3-[(2,4-Dimethoxy)benzylidene]-anabaseine dihydrochloride (DMXBA); MLA,
methyllycaconitine; nAChR, nicotinic acetylcholine receptor; N40, auditory evoked potential,
negative deflection at 40 ms latency; nRT, reticular thalamic nucleus; P20, auditory evoked
potential, positive deflection at 20 ms latency; P50, auditory evoked potential, positive deflection
at 50 ms latency; PNU-282987, (N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide
hydrochloride; PSTH, peristimulus time histograms; TTX, tetrodotoxin
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ABSTRACT Schizophrenic patients are thought to have an impaired ability to process sensory information.
This deficit leads to disrupted auditory gating measured electrophysiologically as a reduced
suppression of the second of paired auditory-evoked responses (P50), and is proposed to be
associated with decreased function and/or expression of the homomeric α7 nicotinic
acetylcholine receptor (nAChR). Here we provide evidence that N-[(3R)-1-azabicyclo[2.2.2]oct-
3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), a novel selective agonist of the α7
nAChR, evoked whole-cell currents from cultured rat hippocampal neurons that were sensitive
to the selective α7 nAChR antagonist methyllycaconitine (MLA), and enhanced GABAergic
synaptic activity when applied to hippocampal slices. Amphetamine-induced sensory gating
deficit, determined by auditory evoked potentials in hippocampal CA3 region, was restored by
systemic administration of PNU-282987 in chloral hydrate anaesthetized rats. Auditory gating
of rat reticular thalamic neurons was also disrupted by amphetamine, however PNU-282987
normalized gating deficit only in a subset of tested neurons (6 out of 11). Furthermore, PNU-
282987 improved the inherent hippocampal gating deficit occurring in a subpopulation of
anaesthetized rats, and enhanced amphetamine-induced hippocampal theta oscillation. We
propose, that the α7 nAChR agonist PNU-282987, via modulating/enhancing hippocampal
GABAergic neurotransmission, improves auditory gating and enhances hippocampal oscillatory
activity. These results provide further support for the concept that drugs that selectively activate
α7 nAChRs may offer a novel, potential pharmacotherapy in treatment of schizophrenia.
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INTRODUCTION
It is recognized that development of schizophrenia is genetically influenced, and a subset of
genes are predisposing to the illness. Among a number of genetic linkage sites, the homomeric,
α7 nicotinic acetylcholine receptor (α7 nAChR) subunit gene, CHRNA7 has been implicated in
schizophrenia (Stassen et al., 2000; Gault et al., 2003). Thus, a genetic linkage of the 15q13-15
region of chromosome 15 containing CHRNA7 has been established to impaired auditory gating,
a presumed indicator of dysfunctional sensory processing in schizophrenia (Freedman et al.,
1997; Leonard et al., 2002). Deficiency in auditory (P50) gating has been regarded as a
manifestation of an impaired sensory filtering mechanism leading to inefficient sensory
processing and disturbed perception in schizophrenic patients (Light and Braff, 1999; Freedman
et al., 2003; Thoma et al., 2003). Based on the previous clinical observation that nicotine
transiently improves auditory gating in schizophrenics (Adler et al., 1993) and the association
between α7 nAChRs and auditory gating in preclinical models (e.g. Stevens et al., 1998), it has
been proposed that activation of α7 nAChRs would improve sensory processing and thus provide
benefit for positive and/or negative symptoms, or impaired cognitive function in schizophrenic
patients (Stevens et al., 1998; Bodnar et al., 2004; Hajos et al., 2003b; Martin et al., 2004).
We have recently described PNU-282987 (N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-
chlorobenzamide hydrochloride) as a potent and selective α7 nAChR agonist (Bodnar et al.,
2004). This compound showed high affinity for the rat α7 nAChR (Ki = 26 nM) and activity at
the α7-5-HT3 chimera (EC50 = 128 nM) and showed a negligible block of α1β1γδ and α3β4
nAChRs (> 60 µM). In addition, PNU-282987 was found to be inactive at all tested monoamine,
muscarine, glutamate and GABA receptors at 1 µM concentration, except 5-HT3 receptors (Ki =
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930 nM; Bodnar et al., 2004). In the present study, we further evaluated its in vitro
pharmacological characteristics and its action on auditory gating processes. PNU-282987 was
compared to reference α7 nAChR agonists for functional activity using cultured rat hippocampal
neurons and for the ability to modulate GABAergic synaptic activity in isolated rat hippocampal
slices. In order to evaluate the in vivo activity of PNU-282987, auditory gating experiments
were carried out in anaesthetized rats. Physiological gating in the hippocampal CA3 region or
reticular thalamic nucleus (nRT) was disrupted by systemic administration of amphetamine
(Stevens et al., 1996; Krause et al., 2003), and the efficacy of PNU-282987 to reverse the
amphetamine-induced gating deficit was determined. The efficacy of the partial α7 nAChR
agonist GTS-21 (Briggs et al., 1997) was also evaluated in our hippocampal gating model since
GTS-21 has been shown previously to improve the inherent auditory gating deficits in DBA mice
(Stevens et al., 1998) or in isolation reared rats (O’Neill et al., 2003).
It is known that enhanced catecholamine neurotransmission in the hippocampal formation leads
to synchronized activity, i.e. theta oscillation in the hippocampus (Berridge and Foote, 1991;
Hajos et al., 2003a), and pronounced hippocampal theta activity has been demonstrated after
systemic administration of amphetamine (Krause et al., 2003). Since hippocampal theta activity
is thought to be associated with synaptic plasticity and hippocampal-dependent cognitive
processes (Buzsaki 2002; Seager et al., 2002), and cognitive-enhancing compounds have been
shown to augment evoked theta activity (Kinney et al., 1999), possible modulations of
amphetamine-induced hippocampal theta activity by PNU-282987 were also analyzed.
Interestingly, a subset of rats used in the present study (approximately 5%) showed consistent
impairment in hippocampal auditory gating at control measurements. These animals were not
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treated with amphetamine, instead the ability of PNU-282987 and GTS-21 was tested to
normalize their inherent gating deficit.
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METHODS
Cell Isolation and Culture Conditions: Sprague-Dawley rats (postnatal day 3) were killed by
decapitation and their brains were removed and placed in ice cold Hibernate-A medium.
Hippocampal regions were gently removed, cut into small pieces and placed in Hibernate-A
medium with 1 mg/ml papain for 60 min at 35°C. After digestion, the tissues were washed
several times in Hibernate-A media and transferred to a 50 ml conical tube containing 6 ml
Hibernate-A medium with B27 supplement (2%). Neurons were dissociated by gentle trituration
through a series of three 9 inch Pasteur pipettes with decreasing tip diameters. Cells were
purified over a Nycoprep gradient according to the methods of Brewer (1997). Cells were plated
onto poly-D-lysine/laminin coated coverslips at a density of 300 – 700 cells/mm2, allowed to
adhere for 1 hour at room temperature and then transferred to 24-well tissue culture plates
containing warmed culture medium composed of Neurobasal-A medium, B27 supplement (2%),
L-glutamine (0.5 mM), 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml
Fungizone. Cells were maintained in a humidified incubator at 37°C and 6% CO2 for 1 – 2
weeks. The medium was changed after 24 hours and then approximately every three days
thereafter.
Brain slice isolation: Spague-Dawley rats ranging from postnatal day 16 to 21 were anesthetized
with Halothane, decapitated, and the brains were removed and blocked. The region containing
the hippocampus was sectioned into 350 micron slices (Microslicer, DSK model 1500E) under
ice-cold slicing buffer composed of (in mM): NaCl (130), NaHCO3 (26), NaH2PO4 (1.25), KCl
(3), CaCl2 (0.5), MgCl2 (10), glucose (10), ascorbic acid (0.4), lidocaine (0.2) continuously
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bubbled with a mixture of O2/CO2 (95:5). Slices were warmed slowly to room temperature in the
same bath solution as above but with 1 mM Ca2+ and no lidocaine; the slices were allowed to
recover for at least 1 hour before recording.
Patch-Clamp Electrophysiology: Whole cell currents were recorded using an Axopatch 200B
amplifier (Axon Instruments, Union City, CA). Analog signals were filtered at 1/5 the sampling
frequency, digitized, stored, and measured using pCLAMP software (Axon Instruments). Patch
pipettes were pulled from borosilicate capillary glass using a Flaming/Brown micropipette puller
(P97, Sutter Instrument, Novato, CA) and filled with an internal pipette solution composed of (in
mM): CsCH3SO3 (126), CsCl (10), NaCl (4), MgCl2 (1), CaCl2 (0.5), EGTA (5), HEPES (10),
ATP-Mg (3), GTP-Na (0.3), phosphocreatin (4), pH 7.2. QX314 (4 mM) was included in the
pipette solution for experiments measuring synaptic activity in brain slices. The resistances of
the patch pipettes when filled with internal solution ranged between 3 – 6 MΩ. All experiments
were conducted at room temperature. Cultured cells were continuously superfused with an
external bath solution containing (in mM): NaCl (140), KCl (5), CaCl2 (2), MgCl2 (1), HEPES
(10), glucose (10), pH 7.4. Bicuculline (10 µM), CNQX (5 µM) and tetrodotoxin (TTX, 0.5 µM)
were included in the bath solution to diminish spontaneous synaptic activity. Compounds were
delivered via a multibarrel fast perfusion exchange system (Warner Instrument, Hamden, CT).
For experiments with brain slices, tissue was transferred to a recording chamber superfused with
a recording buffer composed of (in mM): NaCl (130), NaHCO3 (26), NaH2PO4 (1.25), KCl (3),
CaCl2 (2), MgCl2 (1), glucose (10), ascorbic acid (0.4), AP-5 (0.01), CNQX (0.005), saturated
with O2/CO2 (95:5). The recording chamber was mounted on the stage of a Zeiss Axioscope
microscope with IR-DIC optics and water immersion objectives. Slices were continuously
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superfused with the recording buffer at 3 to 4 ml per minute. Either PNU-282987 or DMSO was
applied by bath application; solution exchange was achieved in < 2 min. All data are reported as
mean ± SEM. Statistical analysis was performed with a two-tailed Students t-test for populations
of unequal variance.
Animals and Surgical Procedures: Experiments were performed on male Sprague-Dawley rats
(weighing 250–300 g) in chloral hydrate anesthesia (400 mg/kg, IP), under an approved animal
use protocol and were in compliance with the Animal Welfare Act Regulations (9 CFR parts 1,
2, and 3) and with the Guide for the Care and Use of Laboratory Animals, National Institutes of
Health guidelines. The femoral artery and vein were cannulated for monitoring arterial blood
pressure and administration of test compounds or additional doses of anesthetic, respectively.
The anesthetized rat was placed in a Kopf stereotaxic frame, and unilateral craniotomy was
performed above the regions of the reticular thalamus or CA3 region of the hippocampus. Body
temperature of the rat was maintained at 37o C by means of an isothermal (37o C) heating pad
(Braintree Scientific, Brain-tree, MA). After conclusion of experiments, animals were
euthanized with an IV bolus of chloral hydrate; brains were removed, blocked and frozen for
histological verification of electrode placement.
Hippocampal EEG recordings: Field potentials (electroencephalogram, EEG) were recorded
from the CA3 region of the left hippocampus, 3.8 mm ventral, 3.5 mm posterior, and 3.0 mm
lateral from bregma (Paxinos and Watson 1986), using a monopolar, stainless steel
macroelectrode (Rhodes Medical Instruments, Woodland Hills, CA). Data were digitized and
stored using the Spike2 software package (Cambridge Electronic Design, Cambridge, UK).
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Rhythmic synchronized (theta) and large-amplitude irregular hippocampal activities were
distinguished in the EEG; quantitative EEG analysis was performed by means of Fast Fourier
transformation (Hajos et al., 2003a). Power spectrum density of EEG was calculated between 0
and 12 Hz, and determined in periods of 10 minutes prior or after drug treatment. Theta peak
was defined as the highest power between 3 and 6 Hz. Auditory evoked potentials were
determined by measuring the potential difference between the positive and the negative
deflection 20 and 40 ms after stimulation (P20 and N40), respectively. For quantification, 50
sweeps were averaged, and the amplitude was determined and the ratio of the response after the
second stimulus (test, T) and the first stimulus (conditioning, C) was calculated. This T/C ratio
is used as a measure of sensory (auditory) gating. Statistical significance was determined by
means of two-tailed paired Student’s t-test.
Single unit recordings from reticular thalamic nucleus: Glass microelectrodes filled with 2
mol/L NaCl and 2% pontamine sky blue (impedance 4-10 MΩ) were lowered 5.2–5.6 mm into
the left nRT (3.0 mm posterior and 3.6 mm lateral with respect to bregma), using a hydraulic
microdrive (Kopf Instruments, Tujunga, CA). In order to identify neurons in the auditory sector
of the reticular thalamus, continuous auditory stimulation was presented during electrode
descent. Spontaneously active nRT neurons were recorded extracellularly, and only those
neurons that responded with activation to auditory stimuli were selected for our studies (Krause
et al., 2003). Extracellular signals were amplified, low-pass filtered, and action potentials
discriminated on-line (Neurolog System, Hertfordshire, UK). At the end of each experiment, dye
was deposited iontophoretically from the recording electrode, and location of the electrode tip
was verified by microscopic inspection of slide-mounted and cresyl violet–stained sections.
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Data were digitized, stored, and analyzed using the Spike3 software package. Firing rates and
interspike time interval histograms were determined at baseline and after drug administration.
Raster displays and peristimulus time histograms (PSTH) were constructed from the evoked
responses to auditory stimulation on-line. The number of events (i.e. extracellularly recorded
action potentials) before auditory stimulation and after the conditioning and test stimuli were
determined using PSTHs. The number of events after the test stimulus divided by the number of
events after the conditioning stimulus was called the T/C ratio. Statistical significance was
determined by means of two-tailed paired Student’s t-test.
Auditory Stimulation: Auditory stimulation consisted of two consecutive tone bursts 10 ms
duration at a frequency of 5 kHz. The sound pressure level was 95 dB between the ear bars as
determined with a sound level meter (RadioShack, Fort Worth, TX). Tones were delivered
through hollow earbars. Recording hippocampal auditory gating, delay between the first
“conditioning” stimulus and second “test” stimulus was 0.5 s. Due to the long-lasting activation
of nRT neurons to auditory stimulus, gating of nRT neurons was tested by paired tones with 1 s
interval between conditioning and test stimuli. The time interval between tone-pairs was 10 s for
both hippocampal and nRT recordings.
Experimental design and Drug treatment: Baseline auditory gating was determined by an
average of 50 sequential evoked potentials (hippocampal CA3 region) or PSTH (nRT neurons) in
response to conditioning and test stimuli. Amphetamine (D-amphetamine sulfate, 1 mg/kg, IV)
was administered in order to disrupt sensory gating. Recordings of evoke potentials or PSTHs
commenced 5 min after amphetamine administration, and 4 blocks of 25 evoked potentials were
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computed. Disruption in auditory gating was affirmed if the mean of the last 50 evoked
potentials showed gating deficit equal or exceeding a 0.2 increase in T/C ratio. Auditory gating
measurements started 5 min after IV administration of the drug or vehicle. Levels of auditory
gating (T/C rations) have been determined from means of 50 subsequent evoked potentials at
time intervals between 5 and 15 minutes, as well as between 15 and 30 minutes after drug or
vehicle administration. In addition, auditory gating was calculated from all 100 evoked
potentials after drug or vehicle treatment.
Materials: Cell culture reagents were purchased from Life Technologies. (-)-Nicotine tartrate
salt, papain, bicuculline methiodide, CNQX, D-amphetamine sulfate and terodotoxin (TTX) with
citrate buffer were purchased from Sigma. MLA was purchased from Research Biochemicals.
PNU 282987 (N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride) and
GTS-21 (DMXBA; 3-[(2,4-Dimethoxy)benzylidene]-anabaseine dihydrochloride) were obtained
from the Medicinal Chemistry Department, Pfizer, Inc. (Kalamazoo, MI). For auditory gating
experiments, compounds were dissolved in Phosphate Buffer Saline (BPS) based upon their salt
weights and the concentrations were adjusted so that injection volumes equaled 1ml/kg body
weight. Control animals received PBS.
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RESULTS
Activation of α7 nAChRs on cultured rat hippocampal neurons by PNU-282987 and
reference agonists.
Examples of whole-cell currents evoked by the α7 nAChR agonists nicotine, GTS-21 and PNU-
282987 are shown in Fig. 1A. Agonists were applied for 1 s once every 30 s at a series of
concentrations. Because hippocampal neurons express varying levels of functional α7 nAChRs,
the nonselective agonist (-)-nicotine (100 µM) was applied to each cell to normalize the data for
comparisons between cells. In addition, because multiple nicotinic receptor subtypes are
expressed by these neurons (e.g., Alkondon and Albuquerque, 1993), nicotine-evoked currents
were recorded in the absence and presence of the selective α7 nAChR antagonist
methyllycaconitine (MLA). To minimize the influence of other receptor subtypes, cells were
included in this study only if the current evoked by nicotine was inhibited >90% by 10 nM
MLA. As illustrated in Fig. 1B, some cells did express a small but measurable amount of
nicotine-evoked current that was resistant to MLA, reflecting the fraction of current mediated by
non-α7 nAChRs (traces shown in Fig. 1B were recorded from the same cell as those shown in
the third row in Fig. 1A; peak nicotine-evoked currents were –289 pA and -19 pA in the absence
and presence of MLA, respectively). In contrast, the current evoked by PNU-282987 was
completely inhibited by 10 nM MLA in every cell tested, even those that had a MLA-resistant
component to the nicotine response (e.g., Fig. 1B). These results suggest that PNU-282987
activated only MLA-sensitive or α7-containing nAChRs on the cell soma and/or proximal
dendrites. The concentration-response of the three compounds are shown in Fig. 1C normalized
to the peak current evoked by 100 µM nicotine.
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PNU-282987 elevates spontaneous GABAergic synaptic activity in hippocampal slices.
Previous work has demonstrated that within the rat hippocampus, nAChRs are predominantly
expressed on GABAergic interneurons and that activation of those receptors modulates
GABAergic synaptic activity (Alkondon et al., 1997; Jones and Yakel, 1997; Köfalvi et al.,
2000; Ji and Dani, 2000). We therefore evaluated the ability of PNU-282987 to modulate
GABAergic synaptic activity in acutely isolated rat hippocampal slices. Spontaneous
GABAergic synaptic events were recorded from CA1 pyramidal neurons for 3 to 10 min. under
baseline conditions and then for an additional 10 min. in the presence of either vehicle (0.1%
DMSO) or PNU-282987. Bath application of 30 nM and 300 nM PNU-282987 more than
doubled frequency of synaptic activity in about half the cells tested (3 of 6 cells and 5 of 11 cells
for 30 nM and 300 nM, respectively), but the average change in frequency was significantly
different from the vehicle control only for cells treated with 300 nM PNU-282987 (p = 0.002,
Fig. 2). No clear effect was observed with 1 µM PNU-282987, possibly reflecting the
desensitization of α7 nAChRs during the 10 min. treatment.
Effects of nAChR agonists on auditory gating in anaesthetized rats
Auditory Gating in the Hippocampus
Hippocampal field potential recordings revealed evoked responses to auditory stimulation in
anesthetized rats. Auditory gating, expressed as the ratio between evoked potentials to paired
conditioning (C) and testing (T) stimuli was determined at baseline by an average of 50
subsequently evoked potentials. Systemic administration of amphetamine (1 mg/kg, IV)
disrupted auditory gating in the majority of the treated rats, as indicated by a significant increase
of the T/C ratio (Figs. 3 & 4A, B). The increase in T/C ratio was due both to an increase in
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amplitude in response to the test stimuli and a decrease in amplitude in response to the
conditioning stimuli (Fig. 3 & 4C). Because the absolute level of disruption induced by
amphetamine was somewhat variable, only rats showing ≥ 0.2 increase in the T/C ratio
(approximately 70% of tested animals) were used for subsequent evaluation of α7 nAChR
agonists or vehicle. In addition, rats with appropriate auditory gating displayed average
amplitudes of conditioning evoked potential over 100 µV, providing excellent signal/noise ratio
for evaluating parallel changes in amplitudes of evoked potentials to conditioning and test
stimuli induced by drug treatments.
Administration of vehicle (PBS, 1 ml/kg, IV, n=6) did not alter amphetamine-induced gating
deficit as determined from the average of 50 evoked potentials measured between 5 to 15
minutes after vehicle application (Fig 4A). Disrupted auditory gating prevailed for at least 30
minutes following amphetamine administration, as indicated by a significant increase in T/C
ratio calculated over this time period from 100 evoked potentials (T/C; 0.59 + 0.11; p < 0.02 vs.
control). In contrast, administration of PNU-282987 (1 mg/kg, IV, n=6) significantly restored
auditory gating (Fig. 4B; determined from the average of 50 evoked potentials), by reversing the
action of amphetamine on the amplitude of evoked potentials, particularly on test stimuli (Fig.
4C). Significant drug action was also established when the degree of auditory gating was
calculated from 100 evoked potentials (T/C; 0.37 + 0.07; p < 0.03 vs. amphetamine). The partial
α7 nAChR agonist GTS-21 also reversed amphetamine-induced gating deficit: T/C values were
0.14 + 0.04 at baseline, 0.48 + 0.03 after amphetamine (1 mg/kg, IV; p < 0.005) and 0.09 + 0.05
after subsequent administration of GTS-21 (1 mg/kg, IV; p < 0.005, vs. amphetamine, n=4).
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In agreement with our previous observations (Krause et al., 2003), administration of
amphetamine resulted in synchronization of hippocampal EEG (Fig. 5). Quantitative EEG
analysis showed a significant increase in EEG power resulting in a peak frequency of 4.4 + 0.1
Hz (baseline value: 1.6 + 0.2 Hz; p < 0.0001; n=12), thereby indicating an increased synchrony
in the theta frequency range (Figs. 5 & 6). Interestingly, amphetamine elicited pronounced
hippocampal theta activity irrespective of its effect on auditory gating, indicating different
mechanisms involved in these two pharmacological responses. Subsequent administration of
vehicle (PBS, 1 ml/kg, IV) or PNU-282987 (1 mg/k, IV) did not alter peak frequency of
hippocampal EEG (data not shown) however PNU-282987 significantly enhanced theta power
(Fig. 6).
Although most of chloral hydrate anaesthetized rats showed normal auditory gating
(characterized by a T/C ration lower than 0.2), approximately 5% of rats displayed persistent
auditory gating deficits (monitored by blocks of subsequent averages of 25 evoked potentials)
with a T/C ratio > 0.5 under baseline conditions. Administration of the α7 nAChR partial
agonist GTS 21 (1 mg/kg, IV, n=4) or the α7 nAChR agonist PNU-282987 (1 mg/kg, IV, n=4)
significantly improved auditory gating in these rats (Fig. 7).
Auditory Gating in the Thalamic Reticular Nucleus
Reticular thalamic neurons responded to auditory stimuli with a typical discharge of bursts of
action potentials (n =11 neurons from 11 rats; Fig. 8). Auditory evoked activity of reticular
thalamic neurons showed oscillations at 7–12 Hz, with each burst comprising ~6 action
potentials, as it has been described previously (Krause et al., 2003). The number of evoked
potentials was determined in 800 ms post-stimulus interval after conditioning and test stimuli,
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and the ratio between the number of spikes after test stimuli and the number of spikes after the
conditioning stimuli represented auditory gating (Krause et al., 2003). Administration of
amphetamine (1 mg/kg, IV, n=11) disrupted auditory gating in each tested neuron (Fig. 8).
Subsequent administration of the α7 nAChR agonist PNU-282987 (1 mg/kg, IV) restored
auditory gating in about half of reticular thalamic neurons (n=6 out of 11; Fig 8B,C). As has
been reported previously (Krause et al., 2003), amphetamine changed the firing pattern of
reticular thalamic neurons from burst firing to single-spike firing mode (Fig. 8A).
Administration of PNU-282987 did not reverse amphetamine-induced firing pattern change in
reticular thalamic neurons (n=11).
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DISCUSSION
Previous studies have shown that within the rat hippocampus α7 nAChRs are expressed
predominantly on GABAergic interneurons where they function to modulate inhibitory synaptic
transmission (Alkondon et al., 1997; Jones and Yakel, 1997; Köfalvi et al., 2000; Ji and Dani,
2000). Impaired function of these interneurons, due in part to decreased expression of α7
nAChRs, has been proposed to contribute to the neuropathology of schizophrenia (Freedman et
al., 2000). Thus, activation of α7 nAChRs by selective agonists could provide an effective
therapy for treating the cognitive deficits of schizophrenia (e.g. Levin and Rezvani, 2002). We
recently reported that PNU-282987 is a potent and selective agonist of human and rat α7
nAChRs (Bodnar et al., 2004). When applied to cultured rat hippocampal neurons, PNU-282987
evoked MLA-sensitive currents that were readily detectable when briefly applied at
concentrations ≥ 300 nM or approximately 30-fold lower than that required for either nicotine or
GTS-21 (Fig. 1). It should be noted however that while these results provide good evidence that
PNU-282987 selectively activated α7-containing nAChRs on the cell body and/or proximal
dendrites, they do no exclude the possibility that PNU-282987 activated MLA-resistant currents
in the axon terminals. The effects of prolonged application of PNU-282987 on GABAergic
synaptic transmission was evaluated in acutely isolated rat hippocampal slices. In agreement
with the reported role of the α7 nAChR in the hippocampus, bath application of 30 nM and 300
nM PNU-282987 increased the frequency of synaptic events by >2-fold in about half the cells
tested although the average effect was significant only for the 300 nM group, and no clear effect
was observed at the highest tested concentration of 1 µM. These results suggest that 300 nM
PNU-282987 activated a sufficient number of receptors to produce a measurable change in
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synaptic activity and that a balance was achieved between receptor activation and receptor
desensitization that allowed for a relatively long lasting response. The high cell to cell
variability observed with 30 nM and 300 nM PNU-282987 likely reflects both inhibitory and
disinhibitory actions produced by excitation of multiple hippocampal interneurons within the
circuit influencing the recorded pyramidal cell (Ji and Dani, 2000).
In order to analyze in vivo activity of α7 nAChR agonists, auditory gating experiments were
carried out in anaesthetized rats. Physiological auditory gating was disrupted by amphetamine
since impaired hippocampal gating is well demonstrated following systemic administration of
amphetamine (Stevens et al., 1996; Krause et al., 2003). Impairment of gating was apparent as a
result of a significant decrease in amplitude of evoked potentials to conditioning stimuli, and a
significant increase in amplitude of evoked potentials to test stimuli, leading to an increased T/C
ratio. Since dopamine D2 receptor antagonists reverse the amphetamine-induced gating deficit,
it has been proposed that enhanced dopamine neurotransmission results in disrupted gating
(Stevens et al., 1996; Krause et al., 2003). Furthermore, enhanced catecholamine
neurotransmission by amphetamine (Light et al., 1999) or cocaine (Adler et al., 2001) leads to
impaired gating in humans. Interestingly, it has been demonstrated that amphetamine- or
cocaine-induced gating deficit can be reversed not only with D2 antagonists, but with nicotine or
nicotinic agonists as well (Stevens et al., 1995; Stevens et al., 1999; Adler et al., 2001),
presumably interacting with inhibitory neuronal circuitry involved in gating, i.e. GABAergic
interneurons in the hippocampus (Stevens et al., 1999; Freedman et al., 2000). Subsequent
experiments indicated a key role for the α7 nAChR in nicotine-induced improvement in auditory
gating and in gating mechanisms in general. It was shown that α7 nAChR stimulation
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normalizes chronic cocaine-induced loss of hippocampal sensory inhibition in C3H mice
(Stevens et al., 1999). Furthermore, inherently impaired auditory gating in DBA/2 mice was
normalized by GTS-21 (1 to 10 mg/kg, SC.; Stevens et al., 1998). Our current findings provide
further evidence that α7 nAChR agonists can normalize abnormal auditory sensory gating, since
we confirmed the efficacy of the partial agonist GTS-21, and demonstrated that the structurally
distinct, highly selective and potent PNU-282987 reversed amphetamine-induced gating deficit.
Interestingly, a subset of rats in our experiments showed a gating deficit at baseline
measurements. Although it is unclear what mechanisms contributed to this pathological gating,
it was normalized by both GTS-21 and PNU-282987. Recently it has been reported that social
isolation (O’Neill et al., 2003) or early maternal deprivation (Ellenbroek et al., 2004) can also
impair sensory gating in adult rats suggesting that early life events such as stress could contribute
to gating abnormality. Similar to our current finings, GTS-21 can normalize auditory gating
deficits in isolation-reared rats (O’Neill et al., 2003).
Systemic administration of amphetamine disrupted auditory gating in nRT neurons as we
reported previously (Krause et al., 2003). Although PNU-282987 reversed hippocampal gating
deficit in all amphetamine-treated rats, it reversed gating deficit only in half of the tested nRT
neurons. The reason for the heterogeneous response of the nRT neurons is presently unknown,
but could reflect heterogeneity in expression of α7 nAChRs by nRT neurons, or a disparity in the
synaptic input/circuit connectivity of nRT neurons. Although within the human thalamus the
highest alpha-bungarotoxin binding, reflecting α7 nAChR expression has been localized in nRT
(Spurden et al., 1997), recent publication on rat brain nAChRs indicates a predominant presence
of heteromeric nAChRs (labeled with epibatidine) in the thalamus, including nRT (Tribollet et
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al., 2004). In addition, PNU-282987 did not modify amphetamine-induced changes in firing
pattern characteristics, in contrast to the D2 antagonists, haloperidol (Krause et al, 2003).
In line with our previous findings, amphetamine not only disrupted auditory gating in
anaesthetized rats, but also induced a slow rhythmic activity in the hippocampal EEG, with a
significant increase in theta power and frequency (Krause et al, 2003). Subsequent
administration of the selective α7 nAChR agonist PNU-282987 further enhanced hippocampal
rhythmic activity as revealed by a significant increase in theta power. In contrast, administration
of vehicle, (or the D2 antagonist haloperidol, Krause et al., 2003) did not heighten theta activity,
although haloperidol normalized amphetamine-induced gating deficit. The fact that PNU-
282987 further synchronized hippocampal activity, and significantly augmented theta power
could be a contributing mechanism to pro-cognitive actions of α7 nAChR agonists described
recently both in animal models (Van Kampen et al., 2004; Young et al., 2004) and humans
(Kitagawa et al., 2003).
In conclusion, the highly selective and potent α7 nAChR agonist PNU-282987 enhances
GABAergic synaptic activity in the hippocampus in vitro, and reverses amphetamine-induced
auditory gating deficit in anaesthetized rats. In addition, PNU-282987 improves the inherent
gating deficit observed in a subset of rats, and enhances amphetamine-induced hippocampal
theta activity. These results support the concept that α7 nAChR agonists represent a novel,
potential pharmacotherapy in treatment of schizophrenia.
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FOOTNOTES
Address correspondence to: Mihály Hajós, PharmD, PhD, Neuroscience Department, Pfizer
Global Research and Development, Eastern Point Road, MS 8220-4083, Groton, CT 06340,
USA. E-mail: [email protected]
1 Current address: Baylor College of Medicine, Division of Neuroscience, Houston, TX 77030 2 Current address: MPI-CardIon Laboratories, Kalamazoo, MI 49008
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LEGENDS FOR FIGURES
Figure 1
Agonist-activation of nAChRs on cultured rat hippocampal neurons. A. Whole-cell currents
evoked by 1 s applications of nicotine (100 µM) and three concentrations of either nicotine
(upper row), GTS-21 (middle row) and PNU-282987 (lower row). Sequential agonist challenges
were separated by a 30 s wash-out period. Traces shown on any row were all recorded from the
same cell. B. Example of currents evoked by nicotine (100 µM) and PNU-282987 (30 µM) in
the presence of 10 nM MLA. Both traces were recorded from the same cell as the traces shown
in the third row of panel A. C. Concentration-response relationships for nicotine, GTS-21 and
PNU-282987. Data points represent the peak current evoked by the indicated concentration of
the test compound normalized to the peak current evoked by 100 µM nicotine from the same cell.
Figure 2 PNU-282987 produces a long-lasting enhancement of GABAergic synaptic activity in
hippocampal slices. A. Example of synaptic events recorded under baseline conditions (left)
and in the presence of 300 nM PNU-282987 (right). B. Summary of change in frequency of
spontaneous synaptic activity relative to baseline for 0.1% DMSO (vehicle) and PNU-282987
(30, 300, 1000 nM). The mean change in synaptic activity was evaluated by comparing the
activity measured during a 3 – 10 min. baseline period to the activity measured during 10 min.
treatement with the vehile or PNU-282987 from the same cell. The mean change in frequency
was as follows: -8% ± 9% (n=10) for 0.1% DMSO, 143 % ± 65% (n=6) for 30 nM PNU-282987,
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103% ± 28% (n=11) for 300 nM PNU-282987, and –13% ± 30% (n=6) for 1000 nM PNU-
282987.
Figure 3
Typical hippocampal auditory evoked potentials (summation of 50 subsequent evoked potentials)
in response to conditioning and test stimuli (intertone interval 0.5 s) in control conditions, after
administration of Amphetamine (AMP, 1.0 mg/kg, IV) and following a subsequent
administration of (A) phosphate buffered saline (PBS) or (B) PNU-282987 (1 mg/kg, IV).
Figure 4
Hippocampal auditory gating expressed as a ratio between evoke potential amplitudes (n=50) to
test and conditioning stimuli (T/C ratio). Administration of amphetamine (1.0 mg/kg, IV)
disrupted auditory gating indicated by an increase in T/C ration. Following a subsequent
administration of vehicle (PBS, 1 ml/kg, IV, n=6) auditory gating remained disrupted (A).
Administration of PNU-282987 (1 mg/kg, IV) restored auditory gating (n = 6; p < 0.01; B).
Amplitudes of hippocampal evoked potentials: Amphetamine-induced decrease in the amplitude
of the conditioning response and an increase in the amplitude of the test response were reversed
by PNU-282987 (C).
Figure 5
Effects of amphetamine and PNU-282987 on rhythmic activity in the hippocampal
electroencephalogram (EEG). Hippocampal EEG (left) and power spectra (right) under control
conditions, after administration of amphetamine (1.0 mg/kg, IV), and after subsequent
administration of PNU-282987 (1 mg/kg, IV). Amphetamine induced a slow rhythmic activity
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in the hippocampal EEG in the theta frequency range, indicated by an increase in power between
3 and 6 Hz. The power of the rhythmic theta activity was enhanced after administration of PNU-
282987.
Figure 6
Summary graph showing changes in EEG power at peak theta frequency after amphetamine and
a subsequent administration of either vehicle (PBS, 1 ml/kg, IV, n=7) or PNU-282987 (1 mg/kg,
IV, n=5).
Figure 7
Effects of the α7 nAChR partial agonist GTS-21 and full agonist PNU-282987 on auditory
gating in rats showing inherent auditory gating deficit. Both compounds improved gating as
indicated by a significant reduction in T/C ratio.
Figure 8
A: Typical recordings from a single unit in the reticular nucleus of the thalamus showing
auditory gating.
Control: Distribution of spikes over a period of 24 stimulations (upper panel) and post stimulus
time histogram (lower panel, bin size 2 ms) after conditioning I and test pulse (T) of a single unit
recorded in the reticular nucleus of the thalamus.
Amphetamine: Administration of amphetamine (1 mg/kg, IV) reduces the number of spikes
after the conditioning stimulus and increases the number of spikes after the test stimulus. At the
same time burst firing is abolished and the unit fires in a phasic fashion
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34
Amphetamine + PNU-282987: Subsequent administration of the α7 nAChR agonist PNU-
282987 (1.0 mg/kg, IV) restores amphetamine-induced gating deficit. Note that amphetamine-
induced tonic-firing mode is still prevailing.
B: Summary graphs for auditory gating in single units in the reticular thalamic nucleus when
PNU-282987 restored amphetamine-induced gating deficit (n = 6 animals). Gating is expressed
as a ratio of number of spikes after test pulse and conditioning pulse. Averaged T/C ratios after
administration of amphetamine (AMP, 1.0 mg/kg, IV) were significantly higher from control
condition; PNU-282987 (PNU282, 1.0 mg/kg, IV) significantly reversed this effect.
C: Summary graphs for auditory gating in single units in the reticular thalamic nucleus when
PNU-282987 failed to restore amphetamine-induced gating deficit (n = 5 animals). Gating is
expressed as a ratio of number of spikes after test pulse and conditioning pulse. Averaged T/C
ratios after administration of amphetamine (AMP, 1.0 mg/kg, IV) or amphetamine + PNU-
282987 (1 mg/kg, IV) were significantly different from control condition.
0
30
60
90
120
150
180
0.1 1 10 100Concentration (µM)
Res
po
nse
(%
Nic
oti
ne) PNU-282987
Nicotine
GTS-21
PNU-282987 (0.3, 3, 30 µM)
Nicotine (10, 30, 100 µM)
GTS-21 (1, 10, 100 µM)
A.
B.
NIC (100 µM)
NIC (100 µM)
NIC (100 µM)
C.
Nicotine (100 µM) PNU-282987 (30 µM)
+ MLA (10 nM)
20 pA
1 s
1 s
100 pA
1 s
100 pA
1 s
100 pA
Fig.1.
Baseline PNU-282987 (300 nM)
2 s
50 pA
b0725
Syn
aptic
Act
ivity
(% c
hang
e fr
om b
asel
ine)
DM
SO
(0
.1%
)
PNU-282987 (nM)
A.
B.
-100
0
100
200
300
400
30 300 1000
Fig.2.
100 ms
100 µ
V
Control AMP (1mg/kg) AMP & 282987 (1mg/kg)
Conditioning Response
100 ms
100 µ
V10
0 µV
Conditioning Response
B
100 ms
100 µ
VControl AMP (1mg/kg) AMP & PBS (1ml/kg)
Conditioning Response
100 ms
100 µ
V
Conditioning Response
A
Fig. 3
Test Response
Test Response
0.00
0.20
0.40
0.60
0.80T
/C R
atio
ControlAMPAMP & PBS
**
* P < 0.02 vs. Control
A
0.00
0.20
0.40
0.60
0.80
T/C
Rat
io
ControlAMPAMP & 282*
#
* p < 0.01 vs. Control
# p < 0.01 vs. AMP
B
0
50
100
150
200
250
Control AMP AMP & 282
* p < 0.05 vs. Control
# p < 0.05 vs. AMP
*
*
#
Res
pons
e A
mpl
itud
e (µ
V)
C
Fig. 4
ConditioningTest
Control
Amphetamine
Amphetamine + PNU-282987
12
8
4
12
8
4
Pow
er, µ
V2 x
103
1 s
0.5 mV
12
8
4
2 6 10Frequency, Hz
Fig. 5
0
5
10
15
20
25
PBS (1ml/kg, n=7) 282987 (1 mg/kg, n=5)
The
ta P
ower
(
µ
V2
x 10
3 )
Control AMP AMP + Drug
* p < 0.05 vs. Control
# p < 0.05 vs. AMP
*
*
*
* #
Fig. 6
0
0.2
0.4
0.6
0.8
T/C
Rat
io
*
*
Control
Drug (1.0 mg/kg, IV)
* p < 0.05 vs. Control
GTS-21 PNU-282987
Fig. 7
20100
5
00 1 2 3
# S
pike
s#
Stim
ulat
ions
C T#
Spi
kes
20100
5
00 1 2 3
# S
timul
atio
ns
AControl
20100
5
00 1 2 3
# S
pike
s#
Stim
ulat
ions
Amphetamine
Amphetamine + PNU-282987
Time [s]
C
0.3
0.6
0.9
1.2
1.5
T/C
0.3
0.6
0.9
1.2
1.5
Control AMP PNU282
p < 0.05
B
T/C
Control PNU282AMP
p < 0.050.05 p 0.01p 0.01
0.3
0.6
0.9
1.2
1.5
0
<
Fig. 8