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Concurrent muscarinic and β-adrenergic blockade in rats impairs place-learning in a water maze and retention of inhibitory avoidance
Brain Research, 513 (1990) 81-85 Elsevier
81
BRES 15330
Concurrent muscarinic and fl-adrenergic blockade in rats impairs place-learning in a water maze and retention of inhibitory avoidance Michael W. Decker, T. Michael Gill and James L. McGaugh Department of Psychobiology and Center for the Neurobiology of Learning and Memory, University of California at Irvine, lrvine, CA 92717 (U.S.A.)
(Accepted 29 August 1989) Key words: Muscarinic receptor; fl-Adrenergic receptor; Inhibitory avoidance; Spatial learning; Scopolamine; Propranolol; Neurotransmitter interaction; Rat
These experiments examined the effects of separate and concurrent muscarinic cholinergic and fl-adrenergic blockade on inhibitory (passive) avoidance performance and spatial learning in the Morris water maze. Pretraining systemic administration of either scopolamine (0.3 or 1.0 mg/kg) or propranolol (3.0 or 10.0 mg/kg) had no significant effect on one-day retention of step-through inhibitory avoidance training. Similarly, pretraining administration of either 0.3 mg/kg scopolamine or 10 mg/kg propranolol did not affect spatial learning in the Morris water maze. However, combined administration of scopolamine and 10.0 mg/kg of propranolol impaired performance on these tasks. These findings further support a role for interactions between norepinephrine and acetylcholine in the modulation of learning and memory and implicate the participation of fl-adrenergic mechanisms in this interaction. Because cholinergic and noradrenergic deterioration is found in aging and Alzheimer's disease, these results also have implications regarding the role of age-related noradrenergic and cholinergic dysfunction in cognitive decline. INTRODUCTION Interactions between norepinephrine (NE) and acetylcholine (ACh) in the brain appear to play an important role in neural plasticity. For example, the shift in ocular dominance columns normally observed in kittens with visual input restricted to one eye is unaffected by depletion of either NE or ACh alone but is blocked by combined depletion of these transmitters 7. An interaction between NE and ACh also appears to be important in learning and memory. Kruglikov 24 reported that neither lesions of the locus coeruleus (LC) - source of forebrain NE - nor systemic administration of 0.5 mg/kg of the muscarinic antagonist scopolamine affected retention of active avoidance training, but that administration of scopolamine to LC-lesioned rats produced a profound deficit. Similarly, while NE-depletion produced by injection of 6 - O H D A into the dorsal noradrenergic bundle does not, by itself, impair radial arm maze performance, this NE-depletion significantly potentiates the disruptive effects of scopolamine on this spatial memory task 16. Several interactions between NE and ACh have been established biochemically and electrophysiologically in brain structures thought to be important for learning and memory. NE applied to the cortex enhances the response
of cortical neurons to iontophoretically applied mCh 39. NE also decreases the release of ACh from cholinergic terminals in the cortex both in slices and in vivo 31'38, an effect apparently mediated both directly via a-adrenergic receptors on cholinergic terminals and indirectly via NE modulation of G A B A release 6. In contrast, NE release in the medial septal area appears to increase ACh turnover in the hippocampus 35, an effect that may be related to changes in hippocampal 0-activity produced by intraseptal application of NE 3°. Even in the striatum, which has sparse noradrenergic input, fl-noradrenergic regulation of muscarinic-stimulated dopamine turnover has been reported 4°. Cholinergic-mediated modification of NE function has also been reported. In hippocampal synaptosomal preparations, muscarinic stimulation inhibits NE synthesis via action at M 1 receptors and NE release via M 2 receptors9; and neurons in the locus coeruleus are influenced by ACh through an action at M 2 receptors 19. To investigate further the interaction of NE and ACh in learning and memory, the present study examined the effects of separate and concurrent administration of the muscarinic cholinergic antagonist scopolamine and the fl-adrenergic antagonist propranolol on two learning and memory tasks - inhibitory (passive) avoidance and spatial learning in the Morris water maze 32.
Correspondence.. M. Decker, Center for the Neurobiology of Learning and Memory, Bonney Center, University of California. Irvine, CA 92717, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
82 MATERIALS AND METHODS
RESULTS
Sub]eels
Inhibitor), avoidance
Male, Sprague-Dawley rats (250-350 g) were obtained from Charles River Laboratories and housed in climate-controlled vivarium on a 12:12 h light:dark cycle (lights on at 07.00 h). All training was conducted during the light portion of the cycle. Food and water were available ad lib.
T h e d a t a f r o m 5 rats w h o s e l a t e n c i e s to cross i n t o t h e d a r k c o m p a r t m e n t o n t h e t r a i n i n g trial w e r e g r e a t e r t h a n 2 rain a n d o n e r a t t h a t d i d n o t r e a c t to t h e s h o c k w e r e excluded
form
t h e analysis.
Drug
treatment
did n o t
significantly affect t h e r a t s ' r e a c t i o n to t h e s h o c k a p p l i e d
Inhibitory (passive) avoidance Step-through inhibitory avoidance training was conducted in a trough-shaped apparatus divided into two compartments by a guillotine door 26. The floors and walls of the apparatus were constructed of metal plates divided longitudinally by a 1.0 cm gap. To begin the training session, the rat was placed into the smaller compartment which was illuminated with a tensor lamp. Ten seconds later the guillotine door was opened and the rat was allowed access to the dark compartment. When the rat stepped completely into the dark compartment, the door was closed and a mild footshock (0.5 mA, 2.0 s, constant current) was administered through the metal floorplates. The rat's latency to enter the dark compartment and reaction to the shock was recorded. Reaction to the shock was judged using a 4-point scale: no reaction was assigned a score of 0, a flinch or crouch response received a score of 1, a jump or other locomotor response a score of 2, and vocalization a score of 3. Retention was tested 24 h later by placing the rat into the light (safe) compartment and measuring the latency to enter the dark compartment (maximum latency of 600 s). Longer latencies were interpreted as indicating better retention. Scopolamine (0.3 or 1.0 mg/kg) and m.-propranolol (3.0 or 10.0 mg/kg) were injected independently or in combination 20 min prior to training. Vehicle (distilled water) was administered in control injections.
d u r i n g t r a i n i n g ( P > 0.05, K r u s k a l - W a l l i s test; d a t a n o t s h o w n ) . T h e l a t e n c i e s to cross i n t o t h e d a r k c o m p a r t m e n t d u r i n g t h e r e t e n t i o n test a r e i l l u s t r a t e d in Fig. 1. R a t s receiving scopolamine alone or propranolol alone did not differ s i g n i f i c a n t l y f r o m c o n t r o l s in t h e i r r e t e n t i o n of inhibitory avoidance training. In contrast, administration of t h e h i g h e r d o s e (10 m g / k g ) o f p r o p r a n o i o l c o m b i n e d with
either
dose
of s c o p o l a m i n e
markedly
impaired
p e r f o r m a n c e ( P < 0.005 for 10 m g / k g p r o p r a n o l o l + 0.3 m g / k g s c o p o l a m i n e ; P < 0.001 f o r 10 m g / k g p r o p r a n o l o l + 1.0 m g / k g s c o p o l a m i n e ; M a n n - W h i t n e y
U).
Morr& water m a z e A s c a n b e s e e n in Fig. 2, l a t e n c i e s to find t h e e s c a p e p l a t f o r m d e c r e a s e d a c r o s s t r a i n i n g b l o c k s ( F < ~92 = 24.13, P < 0.001). M o r e i m p o r t a n t l y , a s i g n i f i c a n t d r u g effect was o b s e r v e d (F~.32 = 9.11, P < 0.001): i n d e p e n d e n t a d m i n i s t r a t i o n of e i t h e r s c o p o l a m i n e o r p r o p r a n o l o l did n o t s i g n i f i c a n t l y affect t h e e s c a p e l a t e n c y m e a s u r e , b u t
Morris water maze The water maze and the procedures used have been described previously 17. Rats were trained to find a 12 x 14 cm escape platform in a circular (1.83 m diameter) pool. The platform was located in a fixed position relative to cues available in the room and was slightly submerged such that it could not be seen by the rat. At the start of each trial, the rat was placed in the water and allowed to swim to the platform. Rats not finding the platform within 90 s were gently guided to it. Rats were allowed to remain on the platform for 20 s at the end of each trial. Four different starting positions, equally spaced around the perimeter of the pool, were used; and the platform was located in the center of one of the quadrants defined by these starting positions. The starting point used for each trial was varied in a quasirandom fashion with the constraints that the total distance between the platform and the start location be equal for each block of 2 trials and that no start location be used more than once per day. Rats were trained for 4 trials per day (2 blocks/day) for 3 days and for 2 trials on the 4th day. A free swim probe trial was conducted immediately after the 2nd trial on day 4. During this 60-s free swim trial, the platform was removed and the rat's swim pattern was recorded on video tape. The number of times the rat crossed 12 × 14 cm regions located in the center of each quadrant was recorded. Scopolamine (0.3 mg/kg) and propranolol (10.0 mg/kg) were administered separately or in combination to different groups of animals 20 min before training on each day. A vehicle (water) control group was also included.
concurrent
administration
of these
d u c t e d i m m e d i a t e l y a f t e r t h e 2 n d trial o n d a y 4, c o n f i r m e d this f i n d i n g . D u r i n g this 60-s trial, r a t s g i v e n a
T{
INHIBITORY
600
AVOIDANCE
///
[] []
CONTROL PROP (3)
MI
PROP
(10)
NN
/i/ z// 400 ,N.
?
...1 ~ 2O0
.H
~NNN
.H ~H H.
///
O
0.3 DOSE S C O P O L A M I N E
Drugs (-)Scopolamine HBr and DL-propranolol HCI (Sigma) were administered intraperitoneaily in a volume of 1.0 ml/kg. Doses were calculated using the salt weight, and drugs were dissolved in distilled water. Combined administration of scopolamine and propranolol was by a single injection of a cocktail. Drug vials were coded so that the behavioral experiments could be run 'blind'.
drugs significantly
i m p a i r e d p e r f o r m a n c e . T h e f r e e s w i m p r o b e trial, con-
1.0 (mg/kg)
Fig. 1. Median step-through latencies and interquartile ranges for the 24 h inhibitory avoidance retention test. Rats had been treated with scopolamine, propranolol, scopolamine + propranolol, or vehicle prior to inhibitory avoidance training conducted on the previous day (n = 8-13/group). (**P < 0.005, ***P < 0.001, differences from vehicle group determined by Mann-Whitney U-test).
83
MORRIS WATER MAZE 90
• CONTROL ['] SCOPOLAMINE 4- P I ~ q A N O L O L
A
~. 6 0
~ 3o
IA
1B
2A
2B
3A
3B
4A
TRAINING BLOCK
Fig. 2. Escape latencies of rats during training on the Morris water maze (means). Combined treatment with scopolamine and propranolol impaired performance relative to vehicle injections or treatment with either drug by itself. See text for statistical analysis (n = 9/group).
combined injection of scopolamine and propranolol exhibited a lower percentage (P < 0.05) of target location crossings (38.6 + 9.4%) than rats receiving injections of either scopolamine (59.6 + 4.1%) or propranolol (59.2 + 4.4%) alone. DISCUSSION The findings of these experiments indicate that doses of scopolamine and propranolol that did not affect performance when administered independently profoundly impaired acquisition of inhibitory avoidance as well as spatial learning in a water maze when administered in combination. These results are not readily interpreted in terms of drug effects on non-associative factors affecting performance. Reaction to the inhibitory avoidance training footshock was not altered by combined administration of scopolamine and propranolol, and findings based on latency data in the water maze were confirmed by free swim data insensitive to swim speed. These two observations support the interpretation that combined administration of scopolamine and propranolol primarily affects learning and memory processes. Of course, effects on non-associative performance factors can never be completely excluded when drugs are administered before training. The demonstration of an interaction between noradrenergic and muscarinic blockade in these two very different learning and memory tasks is consistent with previous reports of synergistic effects of NE-depletion and muscarinic blockade on learning and memory 16'24 and strongly suggests that NE and ACh interact in the modulation of learning and memory. Further, the results of the current study suggest
that this interaction between NE and ACh might involve fl-adrenergic mechanisms. Additional studies will be necessary to evaluate the participation of a-adrenergic mechanisms in this interaction. Some recent studies have either failed to observe an interaction between NE-depletion and muscarinic blockade 11 or found an interaction best described as additive 18. It is notable, however, that these studies utilized the systemically administered neurotoxin, DSP-4, and investigated the effects of relatively modest depletions of NE. Such findings could be interpreted as evidence of a 'threshold' effect: extensive NE-depletion interacts with muscarinic blockade to produce severe learning and memory deficits, while slightly more modest NE-depletion does not. Our findings are consistent with this interpretation: a dose of 10.0 mg/kg of oL-propanolol combined with muscarinic blockade significantly impaired inhibitory avoidance in the current study, whereas a lower dose (3.0 mg/kg) of DL-propranolol combined with muscarinic blockade did not. As the experimental parameters used in the present study were chosen to produce consistently high retention performance in control animals, the resulting ceiling effects may have obscured an effect of scopolamine alone on inhibitory avoidance. We have previously observed deficits in inhibitory avoidance at these doses of scopolamine in mice 18, and deficits on this task are commonly (but not universally) found with muscarinic antagonist treatment 3. In preliminary studies, we found that a higher dose of scopolamine alone (1.0 mg/kg) impairs spatial learning in the water maze under the same conditions used in the present experiment. This observation is consistent with previous reports of the effects of muscarinic blockade on this task 1°'42. Thus, both inhibitory avoidance and Morris water maze performance can be impaired by muscarinic blockade. However, the present findings clearly indicate that such effects are potentiated by concurrent fl-adrenergic blockade. Concurrent deterioration of the noradrenergic and cholinergic systems is found in aging and Alzheimer's disease 14'15'27. The examination of the role these systems play in impaired cognitive functioning, however, has largely ignored interactions between them. The development of animal models of age-related cognitive deficits, for example, has typically focussed on experimentally induced cholinergic deficits. Lesions that impair cholinergic function and pharmacological treatments that block cholinergic neurotransmission disrupt performance on a variety of learning and memory tasks 3-5'33,37. Although the evidence from these studies is consistent with the view that cholinergic dysfunction may, in part, underlie the cognitive deficits associated with aging and Alzheimer's disease, it has become increasingly evident that
84 cholinergic dysfunction cannot provide a complete account of these deficits. In contrast to the effects of cholinergic manipulations, experimentally induced depletion of NE does not significantly affect performance on many learning and memory tasks, including the Morris water maze 32, the radial arm maze 12'16, and inhibitory (passive) avoidance ~'l~ - all of which are sensitive to cholinergic blockade and are performed poorly by aged animals 2°'2t. Further, systemic administration of adrenergic antagonists does not impair either inhibitory avoidance 29 or radial arm maze s'23 performance. Because NE-depletion by itself has, at best, only modest effects on learning and memory in comparison to experimental disruption of cholinergic function (see ref. 41 for a direct comparison), it is usually concluded that N E plays a minor role in age-related memory deficits. There is, however, other evidence for the involvement of noradrenergic dysfunction in age-related memory decline. For example, although inhibitory avoidance learning and the acquisition of spatial information in a water maze do not appear to be affected by NE depletion, the long-term retention of these tasks is impaired in NEdepleted animals 1s'34, a deficit similar to the 'rapid forgetting' characteristic of aged animals 21"28'36'44. In addition, inhibitory avoidance performance in aged mice is highly correlated with the integrity of the noradrenergic system 25. Further, N E manipulations can ameliorate some age-related learning and memory deficits. NE agonists such as clonidine can reverse cognitive deficits REFERENCES 1 Archer, T., Jonsson, G. and Ross, S.B., Active and passive avoidance following the administration of systemic DSP4, xylamine or p-chloro-amphetamine, Behav. Neural Biol., 43 (1985) 238-249. 2 Arnsten, A.ET. and Goldman-Rakic, P.S., az-Adrenergic mechanisms in prefrontal cortex associated with cognitive decline in aged nonhuman primates, Nature (Lond.), 230 (1985) 12731276. 3 Bammer, G., Pharmacological investigations of neurotransmitter involvement in passive avoidance responding: a review and some new results, Neurosci. Biobehav. Rev., 6 (1982) 247-296. 4 Bartus, R.T., Dean, R.L., Beer, B. and Lippa, A.S. The cholinergic hypothesis of geriatric memory dysfunction, Science, 217 (1982) 408-417. 5 Bartus, R.T., Dean, R.L., Pontecorvo, M.J. and Flicker, C., The cholinergic hypothesis: a historical overview, current perspective, and future directions, Ann. N.Y.. Acad. Sci., 44 (1985) 332-358. 6 Beani, L., Tangenel!i, S., Antonelli, T. and Bianchi, C., Noradrenergic modulation of cortical acetylcholine release is both direct and 7-aminobutyric acid-mediated, J. Pharmacol. Exp. Ther., 236 (1986) 230-236. 7 Bear, M.F. and Singer, W., Modulation of visual cortical plasticity by acetylcholine and noradrenaline, Nature (Lond.), 320 (1986) 172-176. 8 Beatty, W.W. and Rush, J.R., Spatial working memory in rats:
found in aged monkeys 2. Similarly, stimulation of NE activity improves inhibitory avoidance performance in aged mice 43, and transplantation of NE-rich fetal brain tissue or intraventricular infusion of NE reverses the age-related deficit on this task in rats ~. Reversal of cognitive deficits by enhanced NE function in aged animals is perhaps surprising in view of the minor role for NE in learning and m e m o r y implied by NEdepletion studies in young animals. However, given that NE dysfunction is accompanied by cholinergic deficits in aged animals, these therapeutic effects of enhanced NE function in aged animals support a role for interactions between NE and ACh in age-related memory decline. The combined noradrenergic/cholinergic dysfunction characteristic of aged animals produces substantially greater behavioral impairment than the cholinergic dysfunction that is found in aged animals with experimentally enhanced NE function. Further, the improved performance of aged rats with NE-rich transplants is blocked by administration of propranoloi j3, suggesting the involvement of fl-adrenergic mechanisms. Thus, the effects of cholinergic/fl-adrenergic interactions on learning and memory described in the present study may also play an important role in age-related cognitive decline.
Acknowledgements. This work was supported by grant MH12526 from NIMH and NIDA and ONR Contract N00014-87-K-0518 to J.L.M.M.W.D. was supported by NIA Postdoctoral Fellowship AG055446. The authors wish to thank Nan Collett for help preparing the manuscript.
effects of monoaminergic antagonists, Pharmacol. Biochem. Behav., 18 (1983) 7-12. 9 Birch, P.J. and Fillenz, M., Muscarinic receptor activation inhibits both release and synthesis of noradrenaline in rat hippocampal synaptosomes, Neurochem. Int., 8 (1986) 171-177. 10 Buresova, O., Bolhuis, J.J. and Bures, J., Differential effects of cholinergic blockade on performance of rats in the water tank navigation task and in a radial water maze, Behav. Neurosci., 100 (1986) 476-482. t 1 Chachich, M., Spangler, E., Smith, K., Wenk, G. and Ingram, D., Effects of noradrenergic depletion and cholinergic antagonism on complex maze performance in young F-344 rats, Soc. Neurosci, Abstr., 14 (1988) 722. 12 Chrobak, J.C., DeHaven, D.L. and Walsh, T.J., Depletion of brain norepinephrine with DSP-4 fails to alter acquisition or performance of a radial arm maze task, Behav. Neural Biol., 44 (1985) 144-150. 13 Collier, T.J., Gash, D.M. and Sladek, J.R., Jr., Transplantation of norepinephrine neurons into aged rats improves performance of a learned task, Brain Research, 448 (1988) 77-87. 14 Coyle, J.T., Price, D.L. and DeLong, M.R., Alzheimer's disease: a disorder of cortical cholinergic innervation, Science, 219 (1983) 1184-1190. 15 Decker, M.W., The effects of aging on hippocampal and cortical projections of the forebrain cholinergic system, Brain Res. Rev., 12 (1987) 423-438. 16 Decker, M.W. and Gallagher, M., Scopolamine-disruption of radial arm maze performance: modification by noradrenergic depletion, Brain Research. 417 (1987) 59-69.
85 17 Decker, M.W., Introini-Coilison, I.B. and McGaugh, J.L., Effects of naloxone on Morris water maze learning in the rat: enhanced acquisition with pretraining but not posttraining administration, Psychobiology, in press. 18 Decker, M.W. and McGaugh, J.L., Effects of concurrent manipulations of cholinergic and noradrenergic function on learning and retention in mice, Brain Research, 477 (1989) 29-37. 19 Eagan, T.M. and North, R.A., Acetylcholine acts on m2musearinic receptors to excite rat locus coeruleus neurones, nr. J. Pharmacol., 85 (1985) 733-735. 20 Gallagher, M. and Pelleymounter, M.A., Spatial learning deficits in old rats: a model for memory decline in the aged, Neurobiol. Aging, 9 (1988) 549-556. 21 Gold, P.E. and McGaugh, J.L., Changes in learning and memory during aging. In J.M. Ordy and K.R. Brizzee (Eds.), Neurobiology of Aging, Plenum, New York, 1975, pp.145-158. 22 Hagan, J.J., Alpert, J.E., Morris, R.G.M. and Iversen, S.D., The effects of central eatecholamine depletions on spatial learning in rats, Behav. Brain Res., 9 (1983) 83-104. 23 Hiraga, Y. and Iwasaki, T., Effects of cholinergic and monoaminergic antagonists and tranquilizers upon spatial memory in rats, Pharmacol. Biochem. Behav., 20 (1984) 205-207. 24 Kruglikov, R.I., On the interaction of neurotransmitter systems in processes of learning and memory. In C.A. Marsan and H. Matthies (Eds.), Neuronal Plasticity and Memory Formation, Raven, New York, 1982, pp. 339-351. 25 Leslie, F.M., Loughlin, S.E., Sternberg, D.B., McGauch, J.L., Young, L.E. and Zornetzer, S.E, Noradrenergic changes and memory loss in aged mice, Brain Research, 359 (1985) 292-299. 26 Liang, K.C., McGaugh, J.L., Martinez, J.L., Jr., Jensen, R.A., Vasquez, B.J. and Messing, R.B., Post-training amygdaloid lesions impair retention of an inhibitory avoidance response, Behav. Brain Res., 4 (1982) 237-249. 27 Mann, D.M.A., The locus coeruleus and its possible role in ageing and degenerative disease of the human central nervous system, Mech. Ageing. Devel., 23 (1983) 73-94. 28 Martinez, J.L., Jr. and Rigter, H., Assessment of retention capacities in old rats, Behav. Neural Biol., 39 (1983) 181-191. 29 McGaugh, J.L., Involvement of hormonal and neuromodulatory systems in the regulation of memory storage, Annu. Rev. Neurosci., 12 (1989) 255-287. 30 Miura, Y. Ito, T. and Kadokawa, T., Effects of intraseptally injected dopamine and noradrenaline on hippocampal synchronized theta wave activity in rats, Jpn. J. Pharmacol., 44 (1987) 471-479.
31 Moroni, E, Tanganelli, S., Antonelli, T., Carla, V., Bianchi, C. and Beani, L., Modulation of cortical acetylcholine and gammaaminobutyric acid release in freely moving guinea pigs: effects of clonidine and other adrenergic drugs, J. Pharmacol. Exp. Ther., 227 (1983) 435-440. 32 Morris, R.G.M., Spatial localization does not require the presence of local cues, Learn. Motiv., 12 (1981) 239-260. 33 Olton, D.S. and Wenk, G.L., Dementia: animal models of the cognitive impairments produced by degeneration of the basal forebrain cholinergic system. In H.Y. Meltzer (Ed.), Psychopharmacology: The Third Generation of Progress, Raven, New York, 1987, pp. 941-953. 34 Prado de Carvalho, L. and Zornetzer, S.F., The involvement of the locus coeruleus in memory, Behav. Neural Biol., 31 (1981) 173-186. 35 Robinson, S.E., Cheney, D.L. and Costa, E., Effect of Nomifensine and other antidepressant drugs on acetylcholine turnover in various regions of rat brain, Naunyn-Schmiedeberg's Arch. Pharmacol., 304 (1978) 263-269. 36 Ruthrich, H.L., Wetzel, W. and Matthies, H., Acquisition and retention of different learning tasks in old rats, Behav. Neural Biol., 35 (1982) 139-146. 37 Smith, G. Animal models of Alzheimer's disease: experimental cholinergic denervation, Brain Res. Rev., 13 (1988) 103-118. 38 Vizi, E.S., Modulation of cortical release of acetylcholine by noradrenaline released from nerves arising from the rat locus coeruleus, Neuroscience, 5 (1980) 2139-2144. 39 Waterhouse, B.D., Moises, H.C. and Woodward, D.J., Alpha receptor-mediated facilitation of somatosensory cortical neuronal responses to excitatory synaptic inputs and iontophoretically applied acetylcholine, Neuropharmacology, 20 (1981) 907-920. 40 Weinstock, M., Zavadil, A.P. III and Kopin, I.J., Differential effects of r,- and L-propranolol on dopamine turnover stimulated by oxotremorine in striatal and mesolimbic areas of rat brain, Eur. J. Pharmacol., 59 (1979) 187-193. 41 Wenk, G., Hughey, D., Boundy, V. and Kim, A., Neurotransmitters and memory: role of cholinergic, serotonergic and noradrenergic systems, Behav. Neurosci., 101 (1987) 325-332. 42 Whishaw, I.Q., Cholinergic receptor blockade in the rat impairs locale but not taxon strategies for place navigation in a swimming pool, Behav. Neurosci., 99 (1985) 979-1005. 43 Zornetzer, S.F., Catecholamine system involvement in agerelated memory dysfunction, Ann. N. Y. Acad. Sci., 444 (1985) 242-254. 44 Zornetzer, S.E, Thompson, R. and Rogers, J., Rapid forgetting in aged rats, Behav. Neural Biol., 36 (1982) 49-60.
81
BRES 15330
Concurrent muscarinic and fl-adrenergic blockade in rats impairs place-learning in a water maze and retention of inhibitory avoidance Michael W. Decker, T. Michael Gill and James L. McGaugh Department of Psychobiology and Center for the Neurobiology of Learning and Memory, University of California at Irvine, lrvine, CA 92717 (U.S.A.)
(Accepted 29 August 1989) Key words: Muscarinic receptor; fl-Adrenergic receptor; Inhibitory avoidance; Spatial learning; Scopolamine; Propranolol; Neurotransmitter interaction; Rat
These experiments examined the effects of separate and concurrent muscarinic cholinergic and fl-adrenergic blockade on inhibitory (passive) avoidance performance and spatial learning in the Morris water maze. Pretraining systemic administration of either scopolamine (0.3 or 1.0 mg/kg) or propranolol (3.0 or 10.0 mg/kg) had no significant effect on one-day retention of step-through inhibitory avoidance training. Similarly, pretraining administration of either 0.3 mg/kg scopolamine or 10 mg/kg propranolol did not affect spatial learning in the Morris water maze. However, combined administration of scopolamine and 10.0 mg/kg of propranolol impaired performance on these tasks. These findings further support a role for interactions between norepinephrine and acetylcholine in the modulation of learning and memory and implicate the participation of fl-adrenergic mechanisms in this interaction. Because cholinergic and noradrenergic deterioration is found in aging and Alzheimer's disease, these results also have implications regarding the role of age-related noradrenergic and cholinergic dysfunction in cognitive decline. INTRODUCTION Interactions between norepinephrine (NE) and acetylcholine (ACh) in the brain appear to play an important role in neural plasticity. For example, the shift in ocular dominance columns normally observed in kittens with visual input restricted to one eye is unaffected by depletion of either NE or ACh alone but is blocked by combined depletion of these transmitters 7. An interaction between NE and ACh also appears to be important in learning and memory. Kruglikov 24 reported that neither lesions of the locus coeruleus (LC) - source of forebrain NE - nor systemic administration of 0.5 mg/kg of the muscarinic antagonist scopolamine affected retention of active avoidance training, but that administration of scopolamine to LC-lesioned rats produced a profound deficit. Similarly, while NE-depletion produced by injection of 6 - O H D A into the dorsal noradrenergic bundle does not, by itself, impair radial arm maze performance, this NE-depletion significantly potentiates the disruptive effects of scopolamine on this spatial memory task 16. Several interactions between NE and ACh have been established biochemically and electrophysiologically in brain structures thought to be important for learning and memory. NE applied to the cortex enhances the response
of cortical neurons to iontophoretically applied mCh 39. NE also decreases the release of ACh from cholinergic terminals in the cortex both in slices and in vivo 31'38, an effect apparently mediated both directly via a-adrenergic receptors on cholinergic terminals and indirectly via NE modulation of G A B A release 6. In contrast, NE release in the medial septal area appears to increase ACh turnover in the hippocampus 35, an effect that may be related to changes in hippocampal 0-activity produced by intraseptal application of NE 3°. Even in the striatum, which has sparse noradrenergic input, fl-noradrenergic regulation of muscarinic-stimulated dopamine turnover has been reported 4°. Cholinergic-mediated modification of NE function has also been reported. In hippocampal synaptosomal preparations, muscarinic stimulation inhibits NE synthesis via action at M 1 receptors and NE release via M 2 receptors9; and neurons in the locus coeruleus are influenced by ACh through an action at M 2 receptors 19. To investigate further the interaction of NE and ACh in learning and memory, the present study examined the effects of separate and concurrent administration of the muscarinic cholinergic antagonist scopolamine and the fl-adrenergic antagonist propranolol on two learning and memory tasks - inhibitory (passive) avoidance and spatial learning in the Morris water maze 32.
Correspondence.. M. Decker, Center for the Neurobiology of Learning and Memory, Bonney Center, University of California. Irvine, CA 92717, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
82 MATERIALS AND METHODS
RESULTS
Sub]eels
Inhibitor), avoidance
Male, Sprague-Dawley rats (250-350 g) were obtained from Charles River Laboratories and housed in climate-controlled vivarium on a 12:12 h light:dark cycle (lights on at 07.00 h). All training was conducted during the light portion of the cycle. Food and water were available ad lib.
T h e d a t a f r o m 5 rats w h o s e l a t e n c i e s to cross i n t o t h e d a r k c o m p a r t m e n t o n t h e t r a i n i n g trial w e r e g r e a t e r t h a n 2 rain a n d o n e r a t t h a t d i d n o t r e a c t to t h e s h o c k w e r e excluded
form
t h e analysis.
Drug
treatment
did n o t
significantly affect t h e r a t s ' r e a c t i o n to t h e s h o c k a p p l i e d
Inhibitory (passive) avoidance Step-through inhibitory avoidance training was conducted in a trough-shaped apparatus divided into two compartments by a guillotine door 26. The floors and walls of the apparatus were constructed of metal plates divided longitudinally by a 1.0 cm gap. To begin the training session, the rat was placed into the smaller compartment which was illuminated with a tensor lamp. Ten seconds later the guillotine door was opened and the rat was allowed access to the dark compartment. When the rat stepped completely into the dark compartment, the door was closed and a mild footshock (0.5 mA, 2.0 s, constant current) was administered through the metal floorplates. The rat's latency to enter the dark compartment and reaction to the shock was recorded. Reaction to the shock was judged using a 4-point scale: no reaction was assigned a score of 0, a flinch or crouch response received a score of 1, a jump or other locomotor response a score of 2, and vocalization a score of 3. Retention was tested 24 h later by placing the rat into the light (safe) compartment and measuring the latency to enter the dark compartment (maximum latency of 600 s). Longer latencies were interpreted as indicating better retention. Scopolamine (0.3 or 1.0 mg/kg) and m.-propranolol (3.0 or 10.0 mg/kg) were injected independently or in combination 20 min prior to training. Vehicle (distilled water) was administered in control injections.
d u r i n g t r a i n i n g ( P > 0.05, K r u s k a l - W a l l i s test; d a t a n o t s h o w n ) . T h e l a t e n c i e s to cross i n t o t h e d a r k c o m p a r t m e n t d u r i n g t h e r e t e n t i o n test a r e i l l u s t r a t e d in Fig. 1. R a t s receiving scopolamine alone or propranolol alone did not differ s i g n i f i c a n t l y f r o m c o n t r o l s in t h e i r r e t e n t i o n of inhibitory avoidance training. In contrast, administration of t h e h i g h e r d o s e (10 m g / k g ) o f p r o p r a n o i o l c o m b i n e d with
either
dose
of s c o p o l a m i n e
markedly
impaired
p e r f o r m a n c e ( P < 0.005 for 10 m g / k g p r o p r a n o l o l + 0.3 m g / k g s c o p o l a m i n e ; P < 0.001 f o r 10 m g / k g p r o p r a n o l o l + 1.0 m g / k g s c o p o l a m i n e ; M a n n - W h i t n e y
U).
Morr& water m a z e A s c a n b e s e e n in Fig. 2, l a t e n c i e s to find t h e e s c a p e p l a t f o r m d e c r e a s e d a c r o s s t r a i n i n g b l o c k s ( F < ~92 = 24.13, P < 0.001). M o r e i m p o r t a n t l y , a s i g n i f i c a n t d r u g effect was o b s e r v e d (F~.32 = 9.11, P < 0.001): i n d e p e n d e n t a d m i n i s t r a t i o n of e i t h e r s c o p o l a m i n e o r p r o p r a n o l o l did n o t s i g n i f i c a n t l y affect t h e e s c a p e l a t e n c y m e a s u r e , b u t
Morris water maze The water maze and the procedures used have been described previously 17. Rats were trained to find a 12 x 14 cm escape platform in a circular (1.83 m diameter) pool. The platform was located in a fixed position relative to cues available in the room and was slightly submerged such that it could not be seen by the rat. At the start of each trial, the rat was placed in the water and allowed to swim to the platform. Rats not finding the platform within 90 s were gently guided to it. Rats were allowed to remain on the platform for 20 s at the end of each trial. Four different starting positions, equally spaced around the perimeter of the pool, were used; and the platform was located in the center of one of the quadrants defined by these starting positions. The starting point used for each trial was varied in a quasirandom fashion with the constraints that the total distance between the platform and the start location be equal for each block of 2 trials and that no start location be used more than once per day. Rats were trained for 4 trials per day (2 blocks/day) for 3 days and for 2 trials on the 4th day. A free swim probe trial was conducted immediately after the 2nd trial on day 4. During this 60-s free swim trial, the platform was removed and the rat's swim pattern was recorded on video tape. The number of times the rat crossed 12 × 14 cm regions located in the center of each quadrant was recorded. Scopolamine (0.3 mg/kg) and propranolol (10.0 mg/kg) were administered separately or in combination to different groups of animals 20 min before training on each day. A vehicle (water) control group was also included.
concurrent
administration
of these
d u c t e d i m m e d i a t e l y a f t e r t h e 2 n d trial o n d a y 4, c o n f i r m e d this f i n d i n g . D u r i n g this 60-s trial, r a t s g i v e n a
T{
INHIBITORY
600
AVOIDANCE
///
[] []
CONTROL PROP (3)
MI
PROP
(10)
NN
/i/ z// 400 ,N.
?
...1 ~ 2O0
.H
~NNN
.H ~H H.
///
O
0.3 DOSE S C O P O L A M I N E
Drugs (-)Scopolamine HBr and DL-propranolol HCI (Sigma) were administered intraperitoneaily in a volume of 1.0 ml/kg. Doses were calculated using the salt weight, and drugs were dissolved in distilled water. Combined administration of scopolamine and propranolol was by a single injection of a cocktail. Drug vials were coded so that the behavioral experiments could be run 'blind'.
drugs significantly
i m p a i r e d p e r f o r m a n c e . T h e f r e e s w i m p r o b e trial, con-
1.0 (mg/kg)
Fig. 1. Median step-through latencies and interquartile ranges for the 24 h inhibitory avoidance retention test. Rats had been treated with scopolamine, propranolol, scopolamine + propranolol, or vehicle prior to inhibitory avoidance training conducted on the previous day (n = 8-13/group). (**P < 0.005, ***P < 0.001, differences from vehicle group determined by Mann-Whitney U-test).
83
MORRIS WATER MAZE 90
• CONTROL ['] SCOPOLAMINE 4- P I ~ q A N O L O L
A
~. 6 0
~ 3o
IA
1B
2A
2B
3A
3B
4A
TRAINING BLOCK
Fig. 2. Escape latencies of rats during training on the Morris water maze (means). Combined treatment with scopolamine and propranolol impaired performance relative to vehicle injections or treatment with either drug by itself. See text for statistical analysis (n = 9/group).
combined injection of scopolamine and propranolol exhibited a lower percentage (P < 0.05) of target location crossings (38.6 + 9.4%) than rats receiving injections of either scopolamine (59.6 + 4.1%) or propranolol (59.2 + 4.4%) alone. DISCUSSION The findings of these experiments indicate that doses of scopolamine and propranolol that did not affect performance when administered independently profoundly impaired acquisition of inhibitory avoidance as well as spatial learning in a water maze when administered in combination. These results are not readily interpreted in terms of drug effects on non-associative factors affecting performance. Reaction to the inhibitory avoidance training footshock was not altered by combined administration of scopolamine and propranolol, and findings based on latency data in the water maze were confirmed by free swim data insensitive to swim speed. These two observations support the interpretation that combined administration of scopolamine and propranolol primarily affects learning and memory processes. Of course, effects on non-associative performance factors can never be completely excluded when drugs are administered before training. The demonstration of an interaction between noradrenergic and muscarinic blockade in these two very different learning and memory tasks is consistent with previous reports of synergistic effects of NE-depletion and muscarinic blockade on learning and memory 16'24 and strongly suggests that NE and ACh interact in the modulation of learning and memory. Further, the results of the current study suggest
that this interaction between NE and ACh might involve fl-adrenergic mechanisms. Additional studies will be necessary to evaluate the participation of a-adrenergic mechanisms in this interaction. Some recent studies have either failed to observe an interaction between NE-depletion and muscarinic blockade 11 or found an interaction best described as additive 18. It is notable, however, that these studies utilized the systemically administered neurotoxin, DSP-4, and investigated the effects of relatively modest depletions of NE. Such findings could be interpreted as evidence of a 'threshold' effect: extensive NE-depletion interacts with muscarinic blockade to produce severe learning and memory deficits, while slightly more modest NE-depletion does not. Our findings are consistent with this interpretation: a dose of 10.0 mg/kg of oL-propanolol combined with muscarinic blockade significantly impaired inhibitory avoidance in the current study, whereas a lower dose (3.0 mg/kg) of DL-propranolol combined with muscarinic blockade did not. As the experimental parameters used in the present study were chosen to produce consistently high retention performance in control animals, the resulting ceiling effects may have obscured an effect of scopolamine alone on inhibitory avoidance. We have previously observed deficits in inhibitory avoidance at these doses of scopolamine in mice 18, and deficits on this task are commonly (but not universally) found with muscarinic antagonist treatment 3. In preliminary studies, we found that a higher dose of scopolamine alone (1.0 mg/kg) impairs spatial learning in the water maze under the same conditions used in the present experiment. This observation is consistent with previous reports of the effects of muscarinic blockade on this task 1°'42. Thus, both inhibitory avoidance and Morris water maze performance can be impaired by muscarinic blockade. However, the present findings clearly indicate that such effects are potentiated by concurrent fl-adrenergic blockade. Concurrent deterioration of the noradrenergic and cholinergic systems is found in aging and Alzheimer's disease 14'15'27. The examination of the role these systems play in impaired cognitive functioning, however, has largely ignored interactions between them. The development of animal models of age-related cognitive deficits, for example, has typically focussed on experimentally induced cholinergic deficits. Lesions that impair cholinergic function and pharmacological treatments that block cholinergic neurotransmission disrupt performance on a variety of learning and memory tasks 3-5'33,37. Although the evidence from these studies is consistent with the view that cholinergic dysfunction may, in part, underlie the cognitive deficits associated with aging and Alzheimer's disease, it has become increasingly evident that
84 cholinergic dysfunction cannot provide a complete account of these deficits. In contrast to the effects of cholinergic manipulations, experimentally induced depletion of NE does not significantly affect performance on many learning and memory tasks, including the Morris water maze 32, the radial arm maze 12'16, and inhibitory (passive) avoidance ~'l~ - all of which are sensitive to cholinergic blockade and are performed poorly by aged animals 2°'2t. Further, systemic administration of adrenergic antagonists does not impair either inhibitory avoidance 29 or radial arm maze s'23 performance. Because NE-depletion by itself has, at best, only modest effects on learning and memory in comparison to experimental disruption of cholinergic function (see ref. 41 for a direct comparison), it is usually concluded that N E plays a minor role in age-related memory deficits. There is, however, other evidence for the involvement of noradrenergic dysfunction in age-related memory decline. For example, although inhibitory avoidance learning and the acquisition of spatial information in a water maze do not appear to be affected by NE depletion, the long-term retention of these tasks is impaired in NEdepleted animals 1s'34, a deficit similar to the 'rapid forgetting' characteristic of aged animals 21"28'36'44. In addition, inhibitory avoidance performance in aged mice is highly correlated with the integrity of the noradrenergic system 25. Further, N E manipulations can ameliorate some age-related learning and memory deficits. NE agonists such as clonidine can reverse cognitive deficits REFERENCES 1 Archer, T., Jonsson, G. and Ross, S.B., Active and passive avoidance following the administration of systemic DSP4, xylamine or p-chloro-amphetamine, Behav. Neural Biol., 43 (1985) 238-249. 2 Arnsten, A.ET. and Goldman-Rakic, P.S., az-Adrenergic mechanisms in prefrontal cortex associated with cognitive decline in aged nonhuman primates, Nature (Lond.), 230 (1985) 12731276. 3 Bammer, G., Pharmacological investigations of neurotransmitter involvement in passive avoidance responding: a review and some new results, Neurosci. Biobehav. Rev., 6 (1982) 247-296. 4 Bartus, R.T., Dean, R.L., Beer, B. and Lippa, A.S. The cholinergic hypothesis of geriatric memory dysfunction, Science, 217 (1982) 408-417. 5 Bartus, R.T., Dean, R.L., Pontecorvo, M.J. and Flicker, C., The cholinergic hypothesis: a historical overview, current perspective, and future directions, Ann. N.Y.. Acad. Sci., 44 (1985) 332-358. 6 Beani, L., Tangenel!i, S., Antonelli, T. and Bianchi, C., Noradrenergic modulation of cortical acetylcholine release is both direct and 7-aminobutyric acid-mediated, J. Pharmacol. Exp. Ther., 236 (1986) 230-236. 7 Bear, M.F. and Singer, W., Modulation of visual cortical plasticity by acetylcholine and noradrenaline, Nature (Lond.), 320 (1986) 172-176. 8 Beatty, W.W. and Rush, J.R., Spatial working memory in rats:
found in aged monkeys 2. Similarly, stimulation of NE activity improves inhibitory avoidance performance in aged mice 43, and transplantation of NE-rich fetal brain tissue or intraventricular infusion of NE reverses the age-related deficit on this task in rats ~. Reversal of cognitive deficits by enhanced NE function in aged animals is perhaps surprising in view of the minor role for NE in learning and m e m o r y implied by NEdepletion studies in young animals. However, given that NE dysfunction is accompanied by cholinergic deficits in aged animals, these therapeutic effects of enhanced NE function in aged animals support a role for interactions between NE and ACh in age-related memory decline. The combined noradrenergic/cholinergic dysfunction characteristic of aged animals produces substantially greater behavioral impairment than the cholinergic dysfunction that is found in aged animals with experimentally enhanced NE function. Further, the improved performance of aged rats with NE-rich transplants is blocked by administration of propranoloi j3, suggesting the involvement of fl-adrenergic mechanisms. Thus, the effects of cholinergic/fl-adrenergic interactions on learning and memory described in the present study may also play an important role in age-related cognitive decline.
Acknowledgements. This work was supported by grant MH12526 from NIMH and NIDA and ONR Contract N00014-87-K-0518 to J.L.M.M.W.D. was supported by NIA Postdoctoral Fellowship AG055446. The authors wish to thank Nan Collett for help preparing the manuscript.
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