A nicotinic agonist (GTS-21), eyeblink classical conditioning and nicotinic receptor binding in rabbit brain

BRAIN RESEARCH ELSEVIER

Brain Research 645 (1994) 309-317

Research

Report

A nicotinic agonist (GTS-21), eyeblink classical conditioning, and nicotinic receptor binding in rabbit brain Diana S. Woodruff-Pak a,., Yong-Tong Li a, William R. Kem b a Department of Psychology, Temple University, Philadelphia, PA 19122, USA b Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, FL 32610, USA

(Accepted 1 February 1994)

Abstract

The septo-hippocampal cholinergic system is of demonstrated involvement in eyeblink classical conditioning (EBCC). To determine if a nicotinic cholinergic agonist, GTS-21, would facilitate acquisition of EBCC in older rabbits, three doses (0.1, 0.5, 1.0 mg/kg) in sterile saline vehicle and vehicle alone were administered to older rabbits (n = 48; mean age = 29.8 months). A control group of vehicle-treated young rabbits (n = 12; mean age = 3.5 months) was included. Rabbits were conditioned for fifteen 90-trial sessions in the 750 ms delay paradigm with a tone conditioned stimulus and corneal airpuff unconditioned stimulus. Dependent measures of trials to learning criterion, percentage of conditioned responses (CRs) and CR amplitude consistently showed significant improvement in older rabbits treated with 0.5 and 1.0 mg/kg of GTS-21. Acquisition was similar in vehicle-treated young and GTS-treated older rabbits. Vehicle-treated older rabbits conditioned more poorly than vehicletreated young rabbits. No non-associative learning effects were observed in GTS-21 treated animals. Nicotinic receptor binding was similar in all groups of older rabbits, indicating that GTS-21 administration over a 15-day period did not affect nicotinic receptors. Alzheimer's disease (AD) has been associated with significant loss of nicotinic cholinergic receptors, and patients diagnosed with probable AD are seriously impaired on EBCC. These results demonstrating that the nicotinic agonist, GTS-21, facilitated EBCC in older rabbits suggest that the compound should receive additional investigation for its potential to affect cognition in AD. Key words: Acetylcholine; Nicotine; Aging; Alzheimer's disease; Nicotinic receptor; Nictitating membrane; Learning; Cognition

enhancing

I. Introduction

Nicotine receptors are significantly reduced in cerebral cortex and hippocampal regions of the brain in Alzheimer's disease (AD) [1,26,39,48,54]. It has been suggested that stimulation of remaining nicotinic receptors might alleviate some cognitive deficits due to cholinergic neuron dysfunction [21,54]. Intravenous administration of nicotine to patients diagnosed as probable A D produced some improvements in cognitive performance, but anxiety and other side effects were observed [30,31,36]. There is much interest in developing new brain-selective nicotinic agonists to inhibit the progressive loss of cognitive function in A D [33].

* Corresponding author. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00173-A

Within the mammalian brain there are a number of nicotinic receptors subtypes and at least some of these possess different pharmacological properties [27]. Although nicotine and acetylcholine do not display large differences in affinity for most nicotinic receptor subtypes, a recently synthesized analog of the marine natural product anabaseine [22] called GTS-21 (chemical name: 3-(2,4-dimethoxybenzylidene)-anabaseine) has been found to preferentially interact with neuronal nicotinic receptors [63] (Kem, unpublished results). This compound is much less toxic to mice than nicotine. Its behavioral actions are currently being investigated using several rodent cognitive performance tests. Eyeblink classical conditioning (EBCC) displays natural age-related deficits in rabbits, cats and rats [8,15,16,34,52,59]. The similarities in age differences in EBCC in normal aging in rabbits and humans are

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striking [44,55,62]. Moreover, EBCC is a task on which patients diagnosed with probable AD are profoundly impaired [7,43,58]. Neurobiological age-related changes which are likely to be involved with the EBCC changes occur in humans as well as in rabbits (e.g. cerebellar Purkinje cell loss [57]; hippocampal cholinergic dysfunction and pyramidal cell loss [3,24]). Artificial induction of behavioral deficits comparable to those observed in normal aging have been reported in the EBCC literature. An example involving the nicotinic cholinergic system entails mecamylamine's antagonism of nicotinic cholinergic receptors. When mecamylamine was injected into young rabbits, disrupting hippocampal cholinergic function, acquisition was prolonged and resembled acquisition in older rabbits [61]. In addition to its parallels with human behavior, a major advantage of using the model system of EBCC in the rabbit over the behavioral models commonly used in preclinical trials is that the neural circuitry is almost completely understood [49,50]. Neuropsychological studies with human patient populations suffering from various types brain damage suggest that the neural circuitry for EBCC in humans is similar to the circuitry demonstrated in rabbits [10,11,28,46,56]. 1.1. Eyeblink classical conditioning paradigm

The standard format for the presentation of stimuli in EBCC is called the delay paradigm. A neutral stimulus such as a tone or light is called the conditioned stimulus (CS). It is presented for a duration of around half a second. While it is still on, the unconditioned stimulus (US) is presented and the CS and US coterminate 50-100 ms later. In rabbits, the US which elicits an eyeblink unconditioned response (UR) is either a shock to the infraorbital region of the eye or a corneal airpuff. The organism learns to blink to the tone before the onset of the US. This is called a conditioned response (CR). The interval between CS and US onset plays a significant role in the rate of acquisition. CS-US intervals of less than 100 ms result in little or no conditioning and CS-US intervals exceeding 500 ms make acquisition more difficult for rabbits. Much of our data on aging rabbits was collected using the 750 ms delay paradigm, a CS-US interval in which large age-related deficits are apparent [38]. This paradigm provides the opportunity to demonstrate facilitation of learning in older rabbits. Older rabbits attain a learning criterion of eight CRs in nine consecutive trials on average on the 9th training day, after 720 trials. Attainment of learning criterion by rabbits treated with GTS-21 on any training day earlier than the 9th or in significantly fewer than 720 trials would indicate facilitation of learning by the compound.

1.2. Acetylcholine and eyeblink classical conditioning

The septo-hippocampal acetylcholinergic system is of demonstrated involvement in EBCC. Pyramidal cells in the CA1 layer of the hippocampus fire in a pattern contiguous with the conditioned and unconditioned NM response [4,5]. Disruption of muscarinic receptors in the septo-hippocampal cholinergic system with scopolamine injections impairs acquisition of the conditioned NM response [17,45] and eliminates pyramidal cell activity in conjunction with the NM response [37]. Subcutaneous scopolamine injections in humans also impair acquisition of the conditioned eyeblink response [42]. Cholinergic afferents to the hippocampal formation are generated from a single source in the medial septal area [29] and both muscarinic cholinergic receptors [47] and nicotinic cholinergic receptors [40] are present in the same target regions of these afferents. Thus, it is likely that both receptor types are activated in parallel. Cholinergic modulation of hippocampal function probably reflects a complex, dynamic combination of muscarinic receptor and nicotinic receptor activation, rather than an exclusive action of either type of cholinergic receptor. Recently, we demonstrated that blockade of nicotinic cholinergic receptors with mecamylamine injections impairs acquisition of the conditioned NM response [61]. Although the acquisition of EBCC is now known to be inhibited by both muscarinic and nicotinic cholinergic antagonists, the effects of cholinergic agonists have not yet been reported for this associative learning paradigm. A major impetus for this experiment was to test the efficacy of a nicotinic agonist in older rabbits in the EBCC paradigm. 1.3. Nicotine administration and nicotinic receptors

In rats and in smoking humans, chronic nicotine administration causes a significant increase (up-regulation) in brain nicotinic receptor concentration. This nicotinic receptor up-regulation is unusual, since prolonged stimulation of a receptor by its neurotransmitter (or similar agonist) generally leads to a decrease in receptor concentration. Both Kellar's [23] laboratory and ours [61] observed no nicotinic receptor up-regulation by the specific antagonist mecamylamine. However, as previously mentioned, mecamylamine had a profound effect on NM conditioning in young rabbits. It delayed their learning so that they performed like old rabbits [61]. Because a nicotinic antagonist impaired EBCC, it is possible that a nicotinic agonist might facilitate acquisition of CRs in older rabbits. Deterioration of central cholinergic pathways has been consistently observed during aging [12,23]. Administration of nicotine has been shown to improve the performance of aged rats

D.S. Woodruff-Pak et al. /Brain Research 645 (1994) 309-317 [2]. I t is l i k e l y t h a t a d m i n i s t r a t i o n o f n i c o t i n e o r c o m p o u n d s s u c h as G T S - 2 1 t h a t a c t i v a t e n i c o t i n i c c h o l i n e r gic r e c e p t o r s w o u l d f a c i l i t a t e a c q u i s i t i o n o f C R s i n older rabbits. Whereas we predicted that the nicotinic agonist, GTS-21 would facilitate acquisition of CRs in older r a b b i t s , it w a s less c l e a r w h e t h e r n i c o t i n i c r e c e p t o r b i n d i n g d e n s i t y i n t h e r a b b i t s ' b r a i n s w o u l d b e aff e c t e d . S i n g l e d a i l y i n j e c t i o n s o f G T S - 2 1 15 m i n b e f o r e t r a i n i n g c o u l d a f f e c t l e a r n i n g s i g n i f i c a n t l y w i t h o u t alt e r i n g t h e d e n s i t y o f n i c o t i n i c r e c e p t o r s . P r e v i o u s l y it was shown that rodents receiving daily or twice-daily subcutaneous injections of nicotine displayed an upregulation in the concentration of nicotinic receptors in c o r t i c a l as w e l l as s e v e r a l o t h e r r e g i o n s o f t h e b r a i n [9,13,41]. A l t h o u g h it is n o t y e t c l e a r if t h e i n c r e a s e i n nicotinic receptors affects cognitive function, evidence for increased nicotine sensitivity has been reported for s t r i a t a l d o p a m i n e r e l e a s e [35,51]. S i n c e o u r e x p e r i m e n tal protocol for investigating GTS-21 action upon associative learning involved daily injections of this compound over a period of time sufficient for nicotine up-regulation of nicotinic receptors, we measured the concentration of cerebral cortex receptors in the brains o f t h e r a b b i t s u s e d f o r E B C C , i n o r d e r t o d e t e r m i n e if GTS-21 also influences receptor concentration.

2. Materials and methods

311

dependent variable measures of conditioning. A minitorque potentiometer on the headmount converted NM movements into electrical signals that were subjected to an analog-to-digital analysis. The digitized values were analyzed from RAM immediately after each training session and also routinely logged on to a floppy disk for permanent storage. 2.3. Procedure Rabbits were placed in restrainers for two adaptation sessions before training or drug injections began. For training, rabbits were injected subcutaneously (s.c.) with drug or vehicle 15 rain before training. Next they were placed into the restrainer, fitted with the headmount and then positioned in a ventilated and sound-attenuated experimental chamber. An 850-ms, 85 dB, 1-kHz tone CS was followed 750 ms after its onset by a 100-ms, 3-psi corneal airpuff US. The CS and US coterminated. The intertrial interval was random, ranging from 10-20 s, The entire EBCC session lasted 35-45 min. Training was completed within 1 h after drug administration while the compound was likely attaining peak blood levels. For the explicitly unpaired conditioning group, all procedures were the same as for the paired conditioning sessions, except that the tone and airpuff were not presented together. Each session consisted of 90 explicitly unpaired trials of tone or airpuff presentations. A CR was automatically scored by the computer system if the NM movement exceeded 0.5 mm and occurred in the period between 25-750 ms after onset of the tone CS. The learning criterion was the production of eight CRs in a sequence of nine consecutive trials. The unconditioned response (UR) was the reflexive blink to the corneal airpuff and was scored as any response occurring in the interval between 751-1041 ms after CS onset. Amplitude of the CR and UR were measured at the point of peak amplitude of the response. 2.4. Drugs and research design

The experiment employed a total of 60 female New Zealand white rabbits. Forty-eight were retired breeder rabbits and 12 were 3-4 months old at the time of testing. Retired breeder rabbits were a minimum of 24 months old and ranged up to 55 months old. Mean age of the older rabbits was 29.8 months. Young rabbits weighed 2.0-2.8 kg and retired breeders' weight ranged between 3.5-5 kg. All rabbits were purchased from Hazleton Research Products (Denver, PA). Birthdates of Hazleton animals were available to the investigators so that the exact age of each animal was documented. Rabbits were individually housed in stainless steel cages and fed 17 g of Purina high fiber rabbit chow per day and tap water. They were on a 12/12 h light/dark cycle.

The nicotinic cholinergic receptor agonist GTS-21 was supplied as the dihydrochloride salt. Three doses (0.1, 0.5, 1.0 mg/kg) of the compound were dissolved in a vehicle of sterile saline. Each experimental group consisted of eight rabbits. A group size of eight rabbits provides adequate statistical power to observe facilitatory effects. Six groups of older rabbits were tested as follows: groups 1, 2 and 3 received paired presentations of the tone CS and corneal airpuff US and doses of 0.1, 0.5 and 1.0 mg/kg GTS-21 respectively; group 4 received paired CS-US presentations and sterile saline vehicle; group 5 received explicitly unpaired presentation of the CS and US and a dose of 0.5 mg/kg GTS-21; group 6 received explicitly unpaired presentation of the CS and US and sterile saline vehicle. Group 7 was comprised of young rabbits injected with vehicle and tested for 10 sessions in the paired condition. Experimenters training the rabbits were blind to the dose tested in each group.

2.2. Apparatus

2.5. Nicotinic receptor analysis

Plexiglas Gormezano restrainers were used to restrain each rabbit during EBCC. Customized Gormezano restrainers built for the larger size of older rabbits were used for the retired breeders. Elastic eyelid retractors held the left eye open. A removable platform to hold the headstage was secured behind the ears and under the animal's muzzle. The headstage attached to this platform held a nozzle positioned 1 cm from the cornea through which the airpuff US was presented. Also on the headstage was a minitorque potentiometer (for NM movement measurement). The potentiometer was attached by a lever and a thread to a nylon suture loop in the NM. A computer programmed with Forth and assembly language and interfaced with the stimulus equipment controlled the presentation and duration of stimulus events, collected analog data and extracted

Three days after the last testing session, each older rabbit was deeply anesthetized with Nembutal. When it showed no reflexes to strong stimulation, it was decapitated and the brain was removed without delay and rapidly frozen prior to overnight shipment in dry ice to the University of Florida. The brains were stored at - 85°C for further analyses. High affinity nicotinic receptor concentrations were then determined by vacuum filtration using [3H]cytisine as radioligand [34]. The protocol for preparing the synaptosomal membranes has been previously described [6,61]. Radioligand binding by each cerebral cortex sample was determined in quadruplicate under three separate conditions: (1) total binding at 0.4 nM [3H]cytisine; (2) total binding at 2.0 nM [3H]cytisine; and (3) non-specific binding at 2.0 nM

2.1. Animals

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radioligand in the presence of 10 IzM nicotine (assuming that nonspecific binding is linearly proportional to radioligand concentration, specific cytisine binding at 0.4 and 2.0 nM concentrations can be calculated). After a 1 h incubation at 0°C, each synaptosome sample was quickly washed free of unbound ligand with two successive 4.0 ml aliquots of ice-cold saline by vacuum filtration. The Whatman G F / C filters were soaked for 1 h in 0.5% polyethleneimine (Sigma) dissolved in binding saline and then drained under vacuum prior to use in order to minimize non-specific binding. Synaptosomal membrane protein was measured using a Coomassie brilliant blue G250 (Pierce) dye-binding assay; bovine serum albumin was used as the protein standard. Measurement of radiolabelled cytisine binding at two concentrations allowed us to determine whether the affinity for cytisine was affected by GTS-21 administration and to determine the total receptor concentration or Bmax, assuming a linear Scatchard plot. The linearity of this binding relation was verified in two separate experiments over a wider range (0.1-10 nM) of cytisine concentrations. The cerebral cortex synaptosomes of each rabbit were analyzed for radiolabelled cytisine binding in two separate experiments. During the intervening time the homogenate was frozen at -85°C. Before the second binding experiment the synaptosomes were thawed and resuspended by gentle homogenization prior to addition of the radiolabelled cytisine.

3. Results

3.1. Behavior Trials to learning criterion were significantly different for the saline vehicle and various GTS-21 doses (Fig. 1). A one-way analysis of variance (ANOVA) comparing the effects of GTS-21 dose (1.0, 0.5, 0.1, 0.0 m g / k g [saline vehicle]) was statistically significant (F3,zs = 3.32, P < 0.05). Rabbits administered GTS-21 took fewer trials to attain learning criterion than rabbits administered saline. Post hoc Scheffe tests indicated that the 0.5 and 1.0 m g / k g doses resulted in significantly more rapid learning than saline ( F = 8.43, P < 0.01) or the 0.1 m g / k g dose of GTS-21 ( F = 4.31, P < 0.05). A second measure of EBCC, percentage of CRs over the fifteen 90-trial acquisition sessions also demonstrated significantly better learning in rabbits treated with doses of 0.5 and 1.0 m g / k g GTS-21 (Fig. 2A). A 4 by 15 repeated measures A N O V A (with planned comparisons between each dose level and vehicle) comparing the effect of four doses of GTS-21 in the paired condition over 15 testing sessions yielded significant effects (Fig. 2A). The effect of dose was significant (F3,28 = 4.36, P < 0.01) as well as the effect of repeated testing s e s s i o n s (F14,392 = 5 8 . 8 8 , P < 0.001). The dose interaction with testing sessions did not attain statistical significance. Planned comparisons indicated that the doses of 0.5 and 1.0 m g / k g resulted in statistically significantly more rapid learning than vehicle ( F 1 = 10.11, P < 0.01 and F 1 = 5.77, P < 0.05, respectively).

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To determine if GTS-21 had non-associative effects on EBCC causing rabbits to blink more or startle to the tone CS, comparisons were made of rabbits in the unpaired drug (0.5 m g / k g GTS-21) and vehicle (sterile saline, 0 m g / k g GTS-21) conditions (Fig. 2B). A 2 by 15 repeated measures A N O V A comparing the effects of dose and testing sessions yielded no statistically significant effects. GTS-21 at a dose of 0.5 m g / k g did not affect the blinking response of the rabbits. The effect of GTS-21 on EBCC in older rabbits was so dramatic, we wanted to determine if learning in these groups of treated older rabbits was equivalent to learning in untreated young rabbits. We compared percentage of CRs in the four groups of rabbits tested in the paired condition with data from the young group (Fig. 2A). The young rabbits were tested for 10 sessions, so the first 10 training sessions were analyzed for all rabbits. Thus, a 5 by 10 repeated measures A N O V A was carried out comparing five groups of rabbits over ten testing sessions. The effect of group was significant (F4,39 = 2.71, P < 0.05), along with the effect of training sessions ( F 9 , 3 5 1 = 45.71, P < 0.001) and the group by training sessions interaction (F36,351 = 2.52, P < 0.001). Post hoc Scheffe analyses indicated that there was no significant difference in percentage of CRs in older rabbits given GTS-21 dose levels of 1.0, 0.5 and 0.1 m g / k g and young rabbits given vehicle. The difference between the young rabbits' percentage CRs and the vehicle-treated older rabbits' was statistically significant (F~ = 4.64, P < 0.05). GTS-21 facilitated acquisition in older rabbits so that their performance was not significantly different from young rabbits, but vehicletreated older rabbits conditioned more poorly than young vehicle-treated rabbits. Yet another means to assess acquisition is amplitude of the CR. This measure should be near zero in

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Fig. 4. A: amplitude (in mm) of the unconditioned response (UR) in four treatment groups of older rabbits: paired presentations of tone and airpuff and doses of 0.0 (vehicle), 0.1, 0.5, 1.0 m g / k g GTS-21. Each training session was comprised of 90 trials of paired tone and airpuff presentations. B: amplitude (in mm) of the UR in two treatment groups of older rabbits: unpaired presentations of 90 tones or airpuffs and doses of 0.0 and 0.5 m g / k g GTS-21.

D.S. Woodruff-Pak et al. / Brain Research 645 (1994) 309-317

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the initial training sessions when the rabbits are producing few CRs and should increase over training sessions as more and larger CRs are produced (Fig. 3). A 4 by 15 repeated measures A N O V A (with planned comparisons between each dose and vehicle) comparing dose in the four paired groups of older rabbits over training sessions revealed a significant effect of dose (F3.28 = 4.75, P < 0.01), a significant effect of training s e s s i o n s (F14,392 = 12.37, P < 0.001) and a significant dose by training sessions interaction (/742,392 = 1.62, P < 0.01; Fig. 3A). Planned comparisons revealed significant differences between vehicle and the dose of 0.5 m g / k g ( F 1 = 5.99, P < 0.05) and 1.0 m g / k g ( F 1 = 6.63, P < 0.05). C R amplitude in the unpaired condition was not affected by GTS-21 (Fig. 3B). A 2 by 15 repeated measures A N O V A comparing the effects of dose and testing session in older rabbits in the unpaired condition yielded no statistically significant effects. Amplitude of the U R is an assessment of the motor component of classical conditioning. It is the magnitude of the N M response to the airpuff. If GTS-21 has an effect on the motor component of the task, it should be apparent in analyses of U R amplitude. A 4 by 15 repeated measures A N O V A comparing the effect of dose and training sessions in the paired condition on U R amplitude indicated that there were no significant effects of dose or training session. However, the dose by training session interaction did attain statistical significance (F42,392 = 1.80, P < 0.01; Fig. 4A). For the unpaired groups (Fig. 4B), a 2 by 15 repeated measures A N O V A comparing the effects of dose and testing sessions revealed no significant effects.

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Fig. 5. Specific [3H]cystinebinding to rabbit cerebral cortex nicotinic receptors. Each group of eight rabbits was injected with 0.0, 0.1, 0.5 or 1.0 mg/kg GTS-21 once daily for 15 days. Four groups received paired presentations of tone and airpuff, and one group received unpaired tone and airpuff presentations. The Bmax estimates were calculated using the mean cytisine K D of 1.05 nM. Error bars represent standard error of the mean.

3.2. N i c o t i n i c receptors

The nicotinic receptor estimates for the rabbits administered 0.1, 0.5 or 1.0 m g / k g of GTS-21 are compared with the receptor estimates for the vehicletreated (control) group (Fig. 5). Since the estimated KDS for the four different groups did not display statistically significant differences, we chose to estimate the Bmax for each rabbit cortex using the m e a n K D estimate of 1.05 nM. Differences between the Bmax estimate differences between each GTS-21 treated group and the saline control rabbit brains also were not statistically significant. Thus, our data did not reveal evidence for receptor up- or down-regulation by the dose regime of GTS-21 administered.

4. D i s c u s s i o n

The nicotinic agonist, GTS-21, facilitated acquisition in older rabbits in the 750 ms CS-US delay E B C C paradigm. The doses that were consistently effective in facilitating E B C C in older rabbits as assessed by trials to learning criterion, percentage CRs and C R amplitude were 0.5 and 1.0 m g / k g . These doses did not affect the motor aspects of the response ( U R amplitude). A 0.5 m g / k g dose of GTS-21 did not sensitize the older rabbits to the tone CS as the blink rate to the CS was low in the unpaired condition. The E B C C levels achieved in 15 sessions by older rabbits treated with GTS-21 are not significantly different from E B C C levels performed by young rabbits. However, there was a significant difference between the vehicle-treated older and younger rabbits. Thus, GTS-21 facilitates acquisition of the conditioned eyeblink response to the degree that older rabbits perform as well as young rabbits. The data are consistent across several measures of E B C C in identifying 0.5 and 1.0 m g / k g as effective doses for enhancing acquisition in older rabbits. None of the doses used in this experiment were overdoses. The 0.1 m g / k g dose appeared to be an underdose, as acquisition with this dose was not significantly different from vehicle using the measures of trials to criterion, percentage CRs and C R amplitude. Given that the 0.1 m g / k g dose was not significantly different from vehicle in older rabbits, it is surprising that post hoc contrasts did not attain significance when comparing young vehicle-treated rabbits' performance and older rabbits treated with 0.1 m g / k g GTS-21. This result occurred due to the conservative nature of post hoc tests and somewhat greater variability in the performance of the 0.1 m g / k g GTS-21 group. The F value of 2.44 ( P = 0.1263) for the post hoc comparison reflecting a 10.9 difference in total percentage CRs between the young vehicle (% CRs = 44.8; S.D. = 13.2)

D.S. Woodruff-Pak et al. / Brain Research 645 (1994) 309-317

and older GTS-21 (% CRs = 33.9; S.D. = 16.3) groups approached statistical significance. Total percentage of CRs for the older vehicle group was 29.8 (S.D. = 14.7) reflecting a difference of 15 between the young and older vehicle groups. Although there was only a difference of 4.1 between total percentage of CRs in the older vehicle and 0.1 m g / k g GTS-21 groups, that additional effect coupled with the smaller variability in the older vehicle group yielded a statistically significant F value of 4.64 ( P = 0.0374) for the post hoc comparison of the young and older vehicle groups. U R amplitude was similar in the four groups of older rabbits in the paired condition and in the two groups of older rabbits in the unpaired condition. There was stability in U R amplitude over the 15 acquisition trials, although for the paired groups, CR amplitude increased significantly with acquisition trials. The one anomalous result was the statistically significant interaction between dose and training trials in the older rabbits receiving paired trials. Fig. 4A illustrates these data. Visual examination of the data suggests that the cause of the significant interaction was the change over sessions in U R amplitude in the vehicletreated group. Initially, U R amplitude was lowest in the vehicle-treated group, but by Session 8, U R amplitude in the vehicle-treated group was highest of the four groups. Whereas the three groups of older rabbits treated with GTS-21 maintained relatively stable mean U R amplitude across all sessions, vehicle-treated older rabbits showed stability in U R amplitude beginning in Session 8. We have no explanation for this result apart from individual variability in rabbits. In two other sterile saline vehicle groups of comparable retired breeder rabbits receiving the identical paired tone and airpuff paradigm with a 750 ms CS-US interval, U R amplitude was stable across sessions at a mean close to 6 mm throughout 10 or 15 90-trial sessions [60]. The fact that there were no main effects of dose or training sessions coupled with the fact that animals treated with GTS-21 showed stability of U R amplitude over trials support our conclusion that the motor aspects of EBCC were not affected by GTS-21. The significance as well as the mechanism underlying nicotinic receptor up-regulation by nicotinic agonists is far from clear at the present time [9,18,25,41]. Mecamylamine, a non-competitive antagonist, also appears to increase nicotinic receptor concentration [9], in addition to nicotine. Since relatively large doses of these drugs were used to demonstrate the up-regulation of nicotinic receptors it is also possible that receptor desensitization occurred concomitantly [19]. While up-regulation of nicotinic receptors with repeated daily administration of nicotine has been consistently observed, only a few investigations have been made to determine if there are functional changes associated with this phenomenon and their conclusions

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are not so consistent. At this time the consequences of nicotinic receptor up-regulation are not clear. Some investigators have reported increased neurotransmitter release [14,35,51], while others have not observed increased sensitivity to nicotine [18,19]. In our study rabbits were exposed to relatively high single, daily doses of GTS-21 similar to the doses of nicotine administered in the rodent studies [9,41]. Although the rate of elimination of GTS-21 is similar to that of nicotine in the rat (Mahnir, unpublished resuits), the affinity of GTS-21 for high affinity brain nicotinic receptors is about 1 / 1 0 that for nicotine (Kern et al., unpublished results). Consequently the lack of observed effect of GTS-21 upon nicotinic receptor concentration in the present investigation may be due to administration of a dose regime which resulted in a much smaller degree of receptor occupation. Alternatively, GTS-21 may differ from nicotine in such a manner that it does not cause receptor up-regulation. After this investigation was completed it was found that GTS-21 possesses a mixed agonist/antagonist activity profile: it acts as a strong partial agonist upon rat o~-7 nicotinic receptors expressed in the Xenopus oocyte, but essentially as an antagonist upon oz-4-/3-2 receptors [20]. Because aging has similar effects on EBCC in rabbits and humans [55], it appears that the conditioned eyeblink response in the rabbit provides a model where basic memory storage processes can be analyzed at the neuronal level in terms of the effects of aging on the formation of memories. The knowledge generated from this approach has significance both at the level of basic understanding of some kinds of learning and memory processes in aging and at the practical level of providing the potential to alter some aspects of impaired capacity of learning and memory in the aged. GTS-21 functions as a cognition-enhancing agent in older rabbits on a task (EBCC) that is seriously impaired in AD. There is potential for nicotine and nicotinic cholinergic agonists such as GTS-21 to serve as cognition-enhancing agents for memory loss in normal aging and in AD. Many previous studies have provided evidence that nicotine may enhance cognitive processes, at least under acute administration conditions [36,53]. There is also evidence that nicotine may enhance cognitive processes in older animals [2]. Almost all previous studies administered nicotine in acute doses. If nicotinic compounds are to serve as cognition-enhancing compounds in AD, their actions must be demonstrated under conditions of chronic use for much longer periods of time than have so far been investigated. In the near future we plan to carry out additional experiments concerning the effect of chronic GTS-21 and nicotine administration upon rabbit EBCC, brain nicotinic receptor levels and alterations in responsiveness to nicotinic drugs.

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Acknowledgements We thank Dr. Katalin Prokai-Tatrai (UF) for synthesis and analysis of the GTS-21 and Mr. Abbas Kazmi (UF) for carrying out the nicotinic receptor binding experiments. We are grateful for the assistance of Mr. Di Luo and Mr. Kevin Conway (TU) who trained the rabbits in this investigation. Mr. Harry Riley (TU) of the Biological Sciences Department was most helpful in preparing the solutions and coding them so that the investigators were blind to the dose. This research was supported by Taiho Pharmaceutical Co., Ltd.

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