Hippocampal Acetylcholine Increases During Eyeblink Conditioning in the Rabbit

Hippocampal Acetylcholine Increases During Eyeblink Conditioning in the Rabbit

Physiology & Behavior, Vol. 60, No. 5, pp. 1199–1203, 1996 Copyright C) 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/9...

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Physiology & Behavior, Vol. 60, No. 5, pp. 1199–1203, 1996 Copyright C) 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/96 $15.00 + .00

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CLASSICAL conditioning of the rabbit nictitating membrane reflex (NMR) has been widely studied to elucidate the neurobiology of learning and memory. The neuronal circuitry that mediates the NMR has been fairly well established. The essential neuromd plasticity occurs in the cerebellum ( 14). The hippocampus plays a modulatory role in this form of learning. Several changes associated with NMR acquisition have been reported in the rabbit hippocarnpus. Electrical activity increased in CA1 pyramidal cells in response to conditioned stimulus (CS ) presentation after acquisition of the conditioned response (CR) (6). Protein kinase C translocation from cytosol to cell membrane was seen after classical conditioning ( 1). Additionally, a decrease in a 20-kDa conditioning-associated GTP binding protein has been reported following NMR training ( 15). Acetylcholine (ACh) appears to play a vital role in the processes of learning and memory (28). Alterations in hippocampal ACh activity are associated with changes in NMR acquisition. Lesions of the medial septum, which has the cell bodies of neurons that release ACh throughout the hippocarnpus (11), disrupts NMR acquisition (7), as demonstrated by learning curve suppression. Administration of the anticholinergic drug scopolamine, either directly into the medial septum ( 18) or systemically (17,19) also attenuated NMR acquisition. The association between hippocarnpal cholin6rgic system function and NMR ac‘C 2 w

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Hippocampalacetylcholineincreases during eyeblink conditioningin the

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quisition could be further studied by measuring ACh release in the hippocampus during this behavior. This was done in the present study. Additionally, ACh release was studied in rabbits that had received aluminum (Al) injections into their lateral ventricles, which has been shown to reduce hippocampal cholinergic function. Aluminum is a neurotoxin that has been administered to rabbits to model neuropathological and behavioral changes associated with human aging and Alzheimer’s disease (27). It disrupts hippocarnpal cholinergic function. It produces deficits in hippocampal choline acetyltransferase (CAT) activity ( 12) and high affinity choline uptake into hippocampal synaptosomes ( 13), resulting in a decreased capacity for ACh synthesis. Intracerebroventriculm injections of Al attenuated potassium-evoked hippocampal ACh release in rabbits not engaged in NMR acquisition (unpublished observations). The measurement of neurotransrnitters in brain extracellular fluid, as a measure of their neuronrd release, can be accomplished with microdialysis. This technique has been used to study neurotrrmsrnitter release in discrete brain regions of awake and freely behaving animrds (5,22). In the present study, ACh release in the ventral hippocampus was measured during NMR acquisition in control and Al-intoxicated rabbits, to test the hypothesis that its release is associated with NMR acquisition.

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MEYER, ALLEN AND YOKEL METHODS

Subjects Sixteen male New Zealand white rabbits weighing 3–4 kg were used. They were housed for the duration of the study in a climate-controlled AAALAC accredited facility, under the care of laboratory animal veterinarians. They had free access to food and water throughout the study. They were anesthetized with an intramuscular injection of ketamine/acepromazine (55/0.55 mg/ kg) to enable intracerebroventricular (ICV) injections and implantation of a microdialysis probe guide. The rabbit’s head was secured in a stereotaxic frame. Rabbits received bilateral ICV injections (P:2 and L:7 from bregma, V:5 mm from the surface of the brain) of either 5 ~moles Al (50 pl of 0.1 M Al lactate; n = 6; Al-intoxicated group) or 15 pmoles of sodium lactate (n = 6; control group). A microdialysis probe guide (Carnegie Medicin) was implanted to terminate in the right ventral hippocampus (P:13.8, L:1O.8 from bregma and 35° from vertical toward the midline) at a depth where the 4-mm exposed membrane of a microdialysis probe would extend 4–8 mm below the surface of the brain. This implantation site was selected because it enabled microdialysis of the greatest hippocamprd area. The probe guide was held in place with skull screws and dental cement. Rabbits received 1 ml of procaine penicillin (Ambi-Pen@) following surgery. NMR Classical Conditioning and Microdialysis Rabbits were prepared for classical conditioning and microdialysis 7 days following ICV injections. A rnicrodialysis probe (CMA/ 10, 4 mm exposed membrane; Carnegie Medicin) was inserted into the probe guide. The rabbits were acclimated to the restrainer and chamber used for NMR conditioning, as previously described (24). Classical conditioning began 24 h after microdialysis probe implantation and was completed within 60 h. This temporal window was considered to be optimal because it avoided the short-term effects of microdialysis probe implantation on glucose metabolism and blood flow that are absent by this time, and it preceded the cellular infiltration of the probe membrane after 2–3 days that would reduce probe function (4). Two conditioning sessions were conducted at approximately 0930 and 1630 h each day for 2 consecutive days, to assure sufficient conditioning trials to enable NMR acquisition during the period of optimal microdialysis probe functioning. Each conditioning session consisted of 100 paired presentations of a 600 ms 1 kHz tone CS and a 50 ms 3 mA shock unconditioned stimulus (US) delivered to the paraorbital region. The CS and the US coterminated, resulting in a delay conditioning paradigm. Paired stimulus presentations were delivered, on average, every 30 s (range, 20-40 s). The 400 paired CS/US presentations were sufficient for control rabbits to attain the CR at a level of greater than 80%, which has been used as a measure of CR acquisition ( 16). A CR was defined as a 0.5-mm extension of the nictitating membrane with onset 50–550 ms after CS onset. Microdialysis was initiated 30 tin prior to and continued throughout each conditioning session. The microdialysis probe was perfused with artificial cerebrospinal fluid (aCSF) containing 5 PM neostigmine. The aCSF was delivered at 3 pllrnin by a CMA/ 100 syringe pump. Dialysate samples were collected every 15 min by a CMA/ 170 refrigerated microfraction collector at 4“C. Each dialysate sample collection tube contained 5 @ of 0.5 mM HCI to minimize ACh hydrolysis. Two baseline samples were collected before and 4 samples were collected during each NMR conditioning session. To correct for changes in microdialysis probe recovery during and between microdialysis sessions, antipyrine (AP) was dosed

to steady-state during each conditioning day (10 mglkg bolus; 5.6 mglkglh infusion throughout the conditioning day). Antipyrine has been validated as a measure of microdialysis probe efficiency over time (25). A blood sample was taken at the end of the second and fourth conditioning session to determine AP blood concentration. To ascertain the effects of CS and US presentation on ACh release and to assure that NMR conditioning was not due to nonassociative processes, such as sensitization or pseudoconditioning, a separate group of rabbits was studied. Four rabbits received bilateral ICV injections of sodium lactate and presentation of the conditioning stimuli during microdialysis, as above, except that the CS and US presentations were explicitly unpaired. Each session consisted of 50 CS-alone and 50 US-alone presentations. Unpaired stimulus presentations were delivered, on average, every 30 s (range, 20–40 s). The rabbits were killed at the end of the fourth conditioning session and their brains removed and frozen. Microdialysis probe placement was verified visually after sectioning the frozen brain with a microtome. The probes were correctly placed in the rabbits reported herein. Acetylcholine Analysis Acetylcholine was measured by high-performance liquid chromatography (HPLC) with electrochemical detection using described methods (2,10). The HPLC system was composed of a Beckman 116 System Gold pump, a reverse-phase Cls analytical column and a postcohunn immobilized enzyme reactor (Chrompack). The immobilized enzyme reactor was prepared by injection of 500 ,u1of a mobile phase containing 80 U of acetylcholinesterase and 40 U of choline oxidase. The enzymes generated betaine and hydrogen peroxide from ACh. The hydrogen peroxide was detected by an ESA Coulochem 51OOAelectrochemical detector with a model 5012 platinum wall jet electrode. The voltage applied to the platinum electrode was +300 mV (vs. the solid-state reference electrode incorporated into the analytical cell). The detector signal was monitored by a Shimadzu CR601 printing integrator. Ethylhomocholine (50 pl of a 1 pM solution) was added to each sample and standard as an internal standard. The mobile phase consisted of 0.1 M phosphate with 1 mM sodium octane sulfonic acid and 1.2 mM tetrarnethyl- ammonium chloride at pH 8. The mobile phase flow rate was 0.6 mlhnin. A 4-point standard curve (ACh concentrations from 0.01 to 0.5 wM) was run each day and a standard analyzed after every 4-6 samples. The standard curve was linear over the concentration range used (mean r = 0.995). Replicate analysis of z 6 samples within 1 day yielded a relative standard deviation of < 4Y0 on each of 4 occasions. Across-day variability was 6.9?lo.The detection limit of ACh was 100 fmoles per 20 @ injection. Antipyrine Analysis Antipyrine was quantified by HPLC as described (25). Dialysate samples were injected directly into the HPLC system. Antipyrine in blood was extracted into methylene chloride ( 1 ml) along with phenacetin (25 ,u1of a 50 #g/ml solution) as an internal standard. The methylene chloride was evaporated under nitrogen and the sample reconstituted in mobile phase prior to analysis. Data Analysis and Statistics Dialysate ACh concentrations were determined by comparison of peak height ratios of ACh/intemal standard to those obtained from standards. To account for changes in microdialysis

ACETYLCHOLINE IN EYEBLINK CONDITIONING

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probe recovery, all ACh values were corrected using AP recovery. AP recovery was determined by: AP recovery = IAp]dialY.a,./[Ap]blood Each ACh dialysate concentration was corrected for changes in microdialysis probe recovery by: [ACh] = IAP recovery for the same dialysate sample. The average of the two baseline ACh concentrations in the samples collected prior to each conditioning session (baseline samples) was defined as IOO?iO ACh release. Acetylcholine concentrations in samples collected during each conditioning session were expressed as a percent of this precession baseline. Significant differences in baseline ACh release and the percentage of CRS, as well as ACh release as a percentage of baseline ACh release during the conditioning sessions among the 3 groups, were determined by 2-way repeated measures analysis of variance (ANOVA). These tests were conducted on the mean precession or session values from each subject. The least significant difference (LSD) post hoc test was used following a significant ANOVA. Acetylcholine release during each conditioning session was also compared to ACh release prior to that session by a paired t-test. Significance for all statistical tests was accepted at p <0.05.

RESULTS

Acquisition of the NMR Control rabbits acquired the NMR using this paradigm, exhibiting CRSduring 91.2%( t 0.2% SE) of the trials of the fourth conditioning session (Fig. 1). Aluminum-intoxicated rabbits did not learn the response as well, reaching a level of 37.6% ( t 2.4’%o SE) CRS during the fourth conditioning session (Fig. 1). The pseudoconditioned rabbits did not exhibit CRS, indicating that no association between the conditioning stimuli was made. There was a significant difference among the 3 groups, F’(2,3 ) = 7.99, p < 0.01; across the 4 classical conditioning sessions, F(3,36) = 8.02, p < 0.01; and a significant interaction F(6,36) = 3.75, p <0.01. The LSD test showed that the Al-intoxicated and pseudoconditioned groups were significantly different from the control group during the second, third, and fourth conditioning sessions. The Al-intoxicated group was significantly different from the pseudoconditioned group during the third and fourth conditioning sessions. ACh Release Precession ACh release was significantly different among the treatment groups (F(2,3) = 31.76, p < 0.01), but was not dif-

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ferent across the 4 sessions. The interaction term was not significant. Precession release was significantly greater in the control (0.045 pM * 0.005) than the A1-tieated rabbits (0.016 AM f 0.002). Precession ACh was also greater in the pseudoconditioned group (0.036 WM t 0.009) than in the Al-treated group. Acetylcholine release increased significantly over precession baseline release during the second and third conditioning sessions in the control group, but did not significantly increase in the other 2 groups (Fig. 2). Acetylcholine release did not increase significantly in the Al-intoxicated group during any of the conditioning sessions (Fig. 2). ANOVA revealed a significant effect among the 3 treatment groups (F(2,3) = 7.80, p < 0.01) and across the 4 conditioning sessions (F(3,36) = 8.02, p < 0.01) and a significant interaction (F(6,36) = 3.75, p < 0.01). The LSD test showed a significant difference between the Al-intoxicated and control groups during the second and third conditioning sessions. The pseudoconditioned group was also significantly different from the control group during the third session.

ditionally, Al-intoxicated rabbits exhibited a deficit in NMR acquisition and a lack of significant increase in hippocampal ACh release. These results are consistent with other studies that demonstrate a positive correlation between cholinergic activity and NMR acquisition in the rabbit (7,17,19). The hippocampus appears to be involved in the consolidation of information from immediate to long-term memory through its interconnections with the neocortex (21). Medial septal lesions retard, but do not eliminate, NMR acquisition (7), suggesting that the septohippocampal system is involved in the early stages of NMR acquisition. The results of the present study are consistent with such a position. Acetylcholine release significantly increased in the control group during the second and third NMR conditioning sessions, concomitant with the period of greatest CR acquisition, when CRSincreased from 5.2% to 85.5%. During the fourth conditioning session, ACh release was no longer significantly elevated and the increase in percentage of CRS was small. This would suggest that the increase in ACh release is associated with CR acquisition, not performance. The increase in ACh release would appear to be associated with NMR acquisition and not simply conditioning stimuli presentation, because pseudoconditioned rabbits that received the same stimuli, but ex-

DISCUSSION

This study demonstrates an increase in hippocamprd ACh release that accompanies NMR acquisition in control rabbits. Ad-

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plicitly unpaired, did not display a significant increase in hippocampal ACh release. Aluminum can reduce cholinergic function by decreasing CAT activity and high-affinity choline uptake, hindering the ability of neurons to synthesize ACh ( 12,13). The Al-intoxicated rabbits in the present study demonstrated a lack of significant increase in ACh release during NMR training. They also exhibited a deficit in NMR acquisition, which is consistent with previous results ( 16,24). This deficit in NMR acquisition is similar to the deficit found in rabbits administered the anticholinergic drug scopolamine ( 17,18,19). The ability of scopolamine to interfere with NMR acquisition suggests that cholinergic function is associated with this learned behavior. The increase in ACh release during NMR acquisition and the subsequent loss of increased ACh release with continued performance of this behavior is consistent with a role of ACh in NMR acquisition. The attenuation of increased ACh release in Al-intoxicated rabbits concurrent with the attenuation of NMR acquisition is also consistent with a role of ACh in this behavior. These results provide further evidence for the deleterious effects of Al on cholinergic function. The hippocampus is a common site of pathology in human disease states that affect cognition, such as Alzheimer’s disease (AD) (9). Similar deficits in NMR acquisition have been reported in Al-intoxicated rabbits (24) and in the analogous eye-

blink reflex in Alzheimer’s disease subjects (20,23,26). This similarity suggests a similar mechanism for this behavioral deficit. The essential neuronal circuitry for the NMR in the rabbit lies within the cerebellum. The cerebellum does not demonstrate deficits in Al toxicity in the rabbit (3), suggesting that the more likely site of Al-induced alteration of NMR acquisition is elsewhere, perhaps the hippocampus. Similarly, cerebella deficits are rarely seen in AD (8). The injection of Al into the lateral ventricles would be expected to produce a much greater effect on structures in close proximity, such as the hippocampus, than distant structures, such as the cerebellum. Hippocampal cholinergic activity is adversely affected in both Al-intoxication and AD. The negative effects of anticholinergic drugs on NMR acquisition and the positive association between ACh release and NMR acquisition suggest that a possible mechanism of deficits in eyeblink and NMR acquisition in AD subjects and Al-intoxicated rabbits may be attenuated hippocampal cholinergic function. ACKNOWLEDGEMENTS

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