Brain Research. 507 (1990) 261-266
261
Elsevier BRES 15139
Restoration of cholinomimetic activity by clonidine in cholinergic plus noradrenergic lesioned rats Vahram Haroutunian, Philip D. Kanof, Gabriel Tsuboyama and Kenneth L. Davis Department of Psychiatry, The Mount Sinai School of Medicine, New York, N Y 10029 (U.S.A, ~ and The Bronx Veterans Administration Medical Center, Bronx, N Y 10468 fU.S.A.I
(Accepted 20 June 1989) Key words: Nucleus basalis; Noradrenergic lesion: Mcmory; Physostigmine; Oxotremorine" Clonidinc
The effects of combined lesions of forebrain cholinergic and noradrenergic systems on memory and responsivity to the memory enhancing effects of cholinomimetics were investigated in rats. Forebrain noradrenergic deficits produced by the injection of 6-hydroxydopaminc into the ascending noradrenergic bundle (ANB) blocked the ability of cholinomimetics such as physostigmine and oxotrcmorinc to enhance retention test performance in nucleus basalis of Meynert lesioned rats. Low doses of the noradrcnergic receptor agonist clonidine, when administcrcd in conjunction with cholinomimetics reversed this blockade. These results suggest that combined cholinergic/noradrenergic therapy may be of value in the treatment of some Alzheimer's disease patients. INTRODUCTION Results of studies on autopsy and biopsy materials obtained from Alzheimer's disease ( A D ) patients have overwhelmingly established the involvement of forebrain cholinergic systems in A D ~'34"~7"4". A m o n g the major cholinergic pathways of interest to the study of A D are the cholinergic cells of the medial septum which project to the hippocampus and the cholinergic cells of the nucleus basalis of Meynert (nbM) which project to the neocortex 14'2s'4~. It is the degeneration of these cells in A D which is responsible for the loss of cholinergic marker activity in the cortex and hippocampus. Animal studies indicate that lesions of the forebrain cholinergic system result in significant learning and memory impairmerits ~s's~ which are often reversed by cholinomimetic drugs lv'~s'31. Clinical trials with cholinomimetic therapy, however, have led to only partial or marginal improvements in mnemonic function ~. This discrepancy between animal model studies and clinical trials has given impetus to the view that non-cholinergic neurotransmitter and neuroanatomical deficits must also play a role in the cognitive dysfunctions associated with this disease. In addition to the cholinergic deficits, noradrenergic 2~. serotonergi¢', somatostatinergi¢ ~'t"36, and corticotropin releasing factor ~ systems are among the most severely affected. The often replicated finding of considerable cortical noradrenergic depletion and decreased norepinephrinc
(NE) turnover 2"~' in A D provides a strong basis for the belief that noradrenergic deficits contribute to the pathophysiology of A D in at least a significant subpopulation of A D patients. The importance of the noradrenergic deficit in A D is underscored by the significant correlations reported between brain NE markers and performance on tests of cognition ~'2~. The involvement of the noradrenergic system in cognitive processes has been confirmed in animal studies which have established that experimental lesions of forebrain catecholaminergic systems and age-related forebrain NE deficits result in cognitive impairments in monkeys 3 ~. Furthermore, noradrenergic receptor stimulation by high doses of systemically administered ckmidine, or another ~2-agonist, guanfacine, has been found to attenuate these age-related cognitive deficits in monkeys ~, to prevent the amnesia which results from cycloheximide administration to rats 2~, and to enhance memory in Korsakoff's psychosis -~" 77. In addition to direct effects of noradrenergic deficits on cognitive performance, locus coeruleus cell loss and forebrain noradrenergic deficits could affect cognitive performance indirectly by (a) depriving forebrain cholinergic neurons of their noradrenergic influences or (b) altering the action of acetylcholine at a postsynaptic site where NE and ACh systems converge and interact. The functional effects of NE and acetylcholine tfltimately depend upon the interaction of these transmitters with receptors on postsynaptic target cells. Hectrophysiological, pharmacological and behavioral data indicate
Correspondence: \!. Haroutunian, Psychiatry Service, Bronx VA Medical Center, 130 West Kingsbridgc Road, Bronx, N'~ 1()46g, U.S.A.
262
rat.
avoidance protocol and drug administration, each rat wa~ returned to its home cage and was left undisturbed until testing 72 h later. The passive avoidance retention test procedure was identical to that used during training except that upon completion of the crossthrough response into the dark compartment, the rat was not shocked. The latency to enter the dark, shock-associated compartment was taken as the dependent measure of memory. Within one week of behavioral testing each rat was sacrificed by decapitation, the brain was removed and cortices were dissected and assayed for choline acetyltransferase ~, acetvlcholinesterase 2. ancl norcpinephfine 2~' In one study, a second cognitive test, fear potentiated acoustic startle ~, was used to assess the generality of the passive avoidance findings. Fourteen to 21 days after the lesion procedure shamoperated and nbM+ANB-lesioned rats were randomly assigned to conditioned vs pseudoconditioned groups (n - 8). Each rat assigned to the conditioned group was placed in a light- and sound-attenuated chamber and received 30 trials in which a 15 s h m g light stimulus (7.5 W house light) was paired and co-terminated with a 1 s hmg 1 mA scrambled footshock during a 30 min conditioning session. Rats assigned to the pseudoconditioning group received an identical number of light and shock stimuli, but these stimuli were randomly and independently distributed over the 30 min session. These same procedures were repeated 24 h later. Two days after the last conditioning session each rat was placed in an acoustic startle apparatus (22 x 10 x ll) cm). Thirty, l l/) dB (SPL), 45 ms long white noise stimuli were presented to each rat and its startle response, transduced by an accelerometer attached to the startle chamber, was measured. On 50~ of the trials the acoustic startle stimulus was preceded by the light stimulus which had been presented during the two day 'fear' conditioning session. Fear potentiated startle was defined as the percent change in mean startle amplitude on light vs no light trials.
MATERIALS AND METHODS
RESULTS
that t h e s e t r a n s m i t t e r s m a y h a v e synergistic effects. For e x a m p l e , n o r e p i n e p h r i n e and c~-adrenergic agonists applied at d o s e s which by t h e m s e l v e s h a v e no e l e c t r o p h y sioiogical effects g r e a t l y p o t e n t i a t e the r e s p o n s i v e n e s s of somatosensory
cortex
cells to
a c e t y l c h o l i n e 37"-~. T h e
ability of c h o l i n e r g i c agonists to p r o d u c e the b e h a v i o r a l s y n d r o m e of c a t a l e p s y is d e p e n d e n t u p o n the integrity of the a s c e n d i n g a d r e n e r g i c p r o j e c t i o n to the c o r t e x and h i p p o c a m p u s 25. N o r a d r e n e r g i c lesions e n h a n c e the amnesic effects o f low d o s e s c o p o l a m i n e in radial m a z e l e a r n i n g p a r a d i g m s 13. C a t e c h o l a m i n e synthesis inhibition by a - m e t h y l - p - t y r o s i n e effects
of the
blocks the m e m o r y e n h a n c i n g
cholinomimeffc
o x o t r e m o r i n e 2°. T h e s e
studies a n d t h o s e d e s c r i b e d in this r e p o r t suggest that the noradrenergic
lesions in A D
m a y not only influence
c o g n i t i v e f u n c t i o n , but m a y also c o n t r i b u t e to the d e g r e e to which c h o l i n o m i m e t i c s can e n h a n c e c o g n i t i v e performance. In an effort to m o r e closely a p p r o x i m a t e the n e u r o c h e m i c a l deficits of A D and to study the i n t e r a c t i o n of c e n t r a l c h o l i n e r g i c and n o r a d r e n e r g i c systems, we h a v e e x a m i n e d the c o n s e q u e n c e s o f c o m b i n e d f o r e b r a i n cholinergic a n d n o r a d r e n e r g i c deficits on c o g n i t i v e perform a n c e and r e s p o n s i v i t y to c h o l i n o m i m e t i c agents in the
Male Sprague-Dawley rats (225-250 g) were anesthetized using a combination of ketamine (60 mg/kg i.m.) and pentobarbital (21 mg/kg i.p.). Following the induction of anesthesia, the rats were positioned in a stereotaxic apparatus with the upper incisor bar set level with the intra-aural line. After a midline incision, burr holes were drilled at coordinates bregma -0.3, +2.8 mm lateral to midline. A 33-gauge stainless steel hypodermic cannula was slowly lowered 7.5-8.0 mm ventral to the surface of the skull. One microliter of ibotenic acid (5/~g/ktl of phosphate buffer) was infused into the nbM area. The ascending noradrenergic bundle was lesioned by the injection of two microliters of a 4 ~g/~l solution of 6-hydroxydopamine (6-OHDA) dissolved in a 0.1% solution of ascorbic acid at stereotaxic coordinates bregma -6.0, 0.8 mm lateral to midline and 6.5 mm ventral to skull. Sham lesioned rats received identical treatments except for the infusion of neurotoxins, Behavioral testing began two weeks after the surgical procedures. In the majority of the experiments, a 72 h retention of one trial passive avoidance test procedure was used to assess mnemonic function and the effects of cholinornimetic drugs. After a 15 min adaptation period to the behavioral testing rooms, rats were placed singly inside the bright, white painted compartment of a two compartment, black/white, shuttle box (28 cm × 14 cm × 15 cm). Sixty seconds later, a guillotine door separating the two compartments was raised and the rat's latency to cross from the bright compartment into the dark, black painted compartment was measured. Upon entry into the dark compartment, the guillotine door was lowered and the rat received a two second long, 1).6 mA scrambled foot shock delivered through the grid floor of the apparatus. The rat was confined to the dark chamber for an additional 60 s after which it was removed and injected with the pharmacological agent under study. The drugs administered to the rats were always prepared on the day of the experiment, and were pH adjusted to 7.4. Following the acquisition phase of the passive
S e v e r a l initial studies s h o w e d that c o g n i t i v e f u n c t i o n , as assessed by tests of l e a r n i n g and m e m o r y in a passive a v o i d a n c e p a r a d i g m , was not i m p a i r e d by n o r a d r e n e r g i c lesions alone, In the first study, 12 rats r e c e i v e d bilateral i n j e c t i o n s of 6 - O H D A bundle
(ANB).
into the a s c e n d i n g n o r a d r e n e r g i c
A second group
ibotenic acid-induced
of 12 rats r e c e i v e d
lesions o f the n b M .
An
equal
n u m b e r of rats r e c e i v e d s i m u l t a n e o u s bilateral lesions of the n b M and A N B . A f o u r t h g r o u p of 12 rats r e c e i v e d e i t h e r s h a m A N B o r s h a m n b M lesions. All rats w e r e trained on the o n e trial passive a v o i d a n c e task two w e e k s after the lesion. T h e results i n d i c a t e d
that rats with
lesions of the n b M o r lesions o f the n b M + A N B w e r e s e v e r e l y i m p a i r e d with r e s p e c t to the 72 h r e t e n t i o n of passive a v o i d a n c e , w h e r e a s A N B lesions by t h e m s e l v e s did not i m p a i r test p e r f o r m a n c e . M e a n c r o s s - t h r o u g h latencies w e r e 306.4 _+ 68, 343.2 __+ 71, 113.4 _ 56 and 106.7
+
48 for s h a m - ,
ANB-,
nbM-
and
ANB
+
n b M - l e s i o n e d rats, r e s p e c t i v e l y (F~,44 = 7.2, P < 0.001). N e u r o c h e m i c a l l y , n b M - and n b M + A N B - l e s i o n e d rats displayed 2 6 . 4 % and 2 8 . 1 % r e d u c t i o n s in cortical c h o l i n e acetyitransferase (CHAT) and acetylcholinesterase ( A C H E ) activity, r e s p e c t i v e l y ( P < 0.01 vs s h a m - o p e r a t e d c o n t r o l s ) , w h e r e a s A N B - and n b M + A N B - l e s i o n e d rats d i s p l a y e d a 9 5 . 4 % r e d u c t i o n ( P < 0.01 vs sham-
263 TABLE I
Effect of sham, nbM, A N B and nbM + A N B lesions on cortical neurochemical markers Results are expressed as m e a n s +_ S.E,M. (n).
Sham nbM % dep ANB % dep nbM + ANB % dep
ChAT (nmol A CH/rnin prot)
AChE (nmol/ rain~rag prot)
NE (ng/lO0 mg tissue)
DA (ng/lO0 mg/mg tissue)
1.66 _+ I).06 (12) 1.17 + I).(17 (14) 29.5* 1,71) +/I.05 (12) 2.4 1.19 ± 0.06 (13) 27.7*
2509 _+ 41 1804 __ 83 28.0* 2461 +_ 77 1.9 1847 ± 65 26.4*
30.1 +__0.85 29.2 ± (I.92 3.1 1.9 -_~ 1.22 93.7* 3.5 _+ 2.21 88.4*
6.7 ± 0.24 (12) 6.3 ± 0.28 (14) 5.2 6.4 ± 11.34 (12) 3.8 5.9 ___0.36 (13) 11.2
(12) (14) (12) (13)
(12) (14) (12) (13)
* vs Sham, P < 0.111.
operated controls) in cortical NE activity (Table I). Lesions of the nbM did not affect cortical NE activity and lesions of the ANB did not alter cortical CHAT, AChE or dopaminergic markers (P > 0.1). These results suggest that lesions which deplete cortical cholinergic markers can severely impair performance on a learning and memory task, while noradrenergic lesions alone do not have a similar effect on passive avoidance retention test performance. This pattern of results is in agreement with earlier studies of cholinergic and adrenergic lesion effects on relatively simple tests of learning and memory in r o d e n t s 26,29.
Although cognitive deficits independent of performance variables have been repeatedly demonstrated in nbM-lesioned rats, the failure of the nbM + ANBlesioned rats to perform appropriately in the passive avoidance test could have resulted from performance artifacts or response inhibition deficits rather than learning and memory deficits per se. To test this possibility, an ancillary study was conducted in which groups of shamlesioned and nbM + ANB-lesioned rats were compared
I000 o9 v >(...) 8 0 0 Z W d I
using a fear potentiation of acoustic startle paradigm. The results of this study revealed a significant conditioning vs pseudoconditioning effect (Fl.m, = 8.8, P < 0.008) as well as a significant effect of the nbM + ANB lesion (F1,2o : 10.5, P < 0.005). In sham-operated rats, the amplitude of the startle response was significantly raised 1167%) when the acoustic startle stimulus was preceded by the shock-associated light stimulus (P < 0.02). In contrast, no significant effect of the light stimulus (121%) could be demonstrated in the nbM + ANB-lesioned rats (P > 0.05). These data taken together with the results of the passive avoidance study discussed above suggest that nbM + ANB lesions lead to profound learning and memory deficits which are independent of the measures used to assess learning and memory. The most striking effects of ANB lesions became evident when physostigmine was used in an attempt to augment the passive avoidance retention test performance of nbM + ANB-lesioned rats. The hypothesis tested by this experiment was that noradrenergic lesions may hamper the ability of cholinomimetics to potentiate performance on cognitive tasks. Different groups (n = 7) of sham-operated, nbM-lesioned, ANB-lesioned or nbM + ANB-lesioned rats received a s.c. injection of either saline or 0.06 mg/kg physostigmine immediately follow-
~700
600
600~500-
~. 400(/3 (,9
~400-
n-c.)
~ 300-
o z
200
200"
w
,~ '00O-
SAL
PHYS
SHAM
SAL
PHYS
ANB
SAL
PHYS
nbM
SAL
PHYS
ANB + nbM
Fig. 1. Effects of a 0.06 mg/kg dose of physostigmine on the 72 h retention of passive avoidance in sham-, nbM-, A N B - and nbM + ANB-lesioned rats.
0 -
0,015
006 SHAM
0,03
0.12
0,24
mq/kq
0.015 0.05 0.06 nbM + ANB
0.12
0.24
Fig. 2. Effects of different doses of physostigmine on the 72 h retention of one trial passive avoidance in s h a m - o p e r a t e d and nbM + ANB-lesioned rats.
2~4 ing the acquisition phase of a one trial passive avoidance response. When retention for passive avoidance was assessed 72 h later, the nbM-lesioned and nbM + ANB-lesioned rats receiving saline showed poor memory of the passive avoidance response relative to shamoperated controls (E~.4~ = 9.7, P 0.4). These results are depicted in Fig. 1. It is noteworthy that the effects of physostigmine in shamoperated and nbM-lesioned rats directly replicate earlier reports of physostigmine enhancement of retention test performance in nbM-lesioned rats 17. The failure of physostigmine to affect the retention test performance of the nbM + ANB-lesioned rats suggests that the combination of cholinergic and noradrenergic deficits blocks the ability of physostigmine to enhance mnemonic processes. It can be argued that the failure of physostigmine to affect retention test performance in nbM + ANBlesioned rats was due to an ANB lesion-induced shift in physostigmine dose-response relationships. To test this possibility different groups of 11-12 rats each with either sham or nbM + ANB lesions were prepared. Two weeks following the lesion procedure the rats were trained in the one trial passive avoidance paradigm. Immediately following passive avoidance training, 6 doses of physostigmine (0.0, 0.015, 0.03, 0.06, 0.12 and 0.24 mg/kg) were administered to the rats in the two lesion groups. Upon retention testing 72 h later (Fig. 2), the nbM + ANB lesions led to profound memory impairments (ANOVA, Fl,10 4 = 12.4, P < 0.001). In addition, there were significant effects of physostigmine (F5:04 = 4.5, P < 0.002) and lesion condition by physostigmine interactions (F5.1o 4 = 3.5, P < 0.01). The 0.03 mg/kg dose of physostigmine enhanced the retention test performance of sham-operated rats (Newman-Keuls, P < 0.0l) replicating earlier findings ~~, but the retention test performance of the nbM + ANB-lesioned rats was not improved by any of the doses of physostigmine investigated (P > 0.10). Examination of cholinergic and noradrenergic neurochemical changes in the lesioned rats revealed an 88.4% depletion of cortical NE in nbM + ANB-lesioned rats and 27.7% and 26.4% depletion of frontal cortical ChAT and AChE activity (Ps < 0.01). Since a rather wide dose range of physostigmine was used in this experiment, it is unlikely that the failure of physostigmine to potentiate retention test performance in nbM + ANB-lesioned rats was attributable to lesioninduced shifts in dose-response relationships, The results of this experiment suggest that one reason for the
apparent impotency of cholinomimetics in it significant proportion of AD victims may' bc the presence of noradrenergic as well as cholinergic deficits. The final experiments assessed the possibility that the memory impairments which result from forebrain cholinergic and noradrenergic deficits can be reversed by pharmacological treatments which augment cholinergic and noradrenergic function simultaneously. To this end, 68 nbM + ANB-lesioned rats and 68 sham-operated controls were trained in the one trial passive avoidance paradigm. Different groups of lesioned and control rats (n = 11-12) received one trial passive avoidance training followed immediately by s.c. injections of either 0.06 mg/kg physostigmine alone, or 0.06 mg/kg physostigmine plus one of several doses (0, 0.01 or 0.5 mg/kg) of the a2-adrenergic receptor agonist clonidine. Retention of passive avoidance was assessed 72 h later. Clonidine was selected as the noradrenergic drug of choice as some experiments 4's'22'27 have shown that clonidine effectively reverses the cognitive deficits which are evident in aged rhesus monkeys suffering forebrain noradrenergic deficits and Korsakoff's psychosis patients with compromised central noradrenergic systems. The results of this experiment are depicted in Fig. 3. Analyses of variance revealed significant lesion (F1.,j 3 = 5.2, P < 0.03), drug (F5.103 -- 5.4, P < 0.001) and lesion by drug condition interactions Es,lo3 = 5.9, P < 0.001). Independent group comparisons showed that nbM + ANB lesion-induced memory deficits were reversed by either high doses (0,5 mg/kg) of clonidine alone or much smaller doses of cionidine (0.01 mg/kg) administered in conjunction with 0.06 mg/kg physostigmine (Ps < 0.01). This same combination of physostigmine and ctonidine had an amnestic effect on the performance of the sham-operated rats (P < 0.03). In an additional experiment using the same passive avoidance training and testing and drug administration procedures, the dose of physostigmine
0.0
.ol
.50 0.0 SHAM
.01
.so
0.0
.ol
.so
0.0
.oI
.~eLo~
N B M + .~I~IB Lesioned
* • V S S H A M P
261
Elsevier BRES 15139
Restoration of cholinomimetic activity by clonidine in cholinergic plus noradrenergic lesioned rats Vahram Haroutunian, Philip D. Kanof, Gabriel Tsuboyama and Kenneth L. Davis Department of Psychiatry, The Mount Sinai School of Medicine, New York, N Y 10029 (U.S.A, ~ and The Bronx Veterans Administration Medical Center, Bronx, N Y 10468 fU.S.A.I
(Accepted 20 June 1989) Key words: Nucleus basalis; Noradrenergic lesion: Mcmory; Physostigmine; Oxotremorine" Clonidinc
The effects of combined lesions of forebrain cholinergic and noradrenergic systems on memory and responsivity to the memory enhancing effects of cholinomimetics were investigated in rats. Forebrain noradrenergic deficits produced by the injection of 6-hydroxydopaminc into the ascending noradrenergic bundle (ANB) blocked the ability of cholinomimetics such as physostigmine and oxotrcmorinc to enhance retention test performance in nucleus basalis of Meynert lesioned rats. Low doses of the noradrcnergic receptor agonist clonidine, when administcrcd in conjunction with cholinomimetics reversed this blockade. These results suggest that combined cholinergic/noradrenergic therapy may be of value in the treatment of some Alzheimer's disease patients. INTRODUCTION Results of studies on autopsy and biopsy materials obtained from Alzheimer's disease ( A D ) patients have overwhelmingly established the involvement of forebrain cholinergic systems in A D ~'34"~7"4". A m o n g the major cholinergic pathways of interest to the study of A D are the cholinergic cells of the medial septum which project to the hippocampus and the cholinergic cells of the nucleus basalis of Meynert (nbM) which project to the neocortex 14'2s'4~. It is the degeneration of these cells in A D which is responsible for the loss of cholinergic marker activity in the cortex and hippocampus. Animal studies indicate that lesions of the forebrain cholinergic system result in significant learning and memory impairmerits ~s's~ which are often reversed by cholinomimetic drugs lv'~s'31. Clinical trials with cholinomimetic therapy, however, have led to only partial or marginal improvements in mnemonic function ~. This discrepancy between animal model studies and clinical trials has given impetus to the view that non-cholinergic neurotransmitter and neuroanatomical deficits must also play a role in the cognitive dysfunctions associated with this disease. In addition to the cholinergic deficits, noradrenergic 2~. serotonergi¢', somatostatinergi¢ ~'t"36, and corticotropin releasing factor ~ systems are among the most severely affected. The often replicated finding of considerable cortical noradrenergic depletion and decreased norepinephrinc
(NE) turnover 2"~' in A D provides a strong basis for the belief that noradrenergic deficits contribute to the pathophysiology of A D in at least a significant subpopulation of A D patients. The importance of the noradrenergic deficit in A D is underscored by the significant correlations reported between brain NE markers and performance on tests of cognition ~'2~. The involvement of the noradrenergic system in cognitive processes has been confirmed in animal studies which have established that experimental lesions of forebrain catecholaminergic systems and age-related forebrain NE deficits result in cognitive impairments in monkeys 3 ~. Furthermore, noradrenergic receptor stimulation by high doses of systemically administered ckmidine, or another ~2-agonist, guanfacine, has been found to attenuate these age-related cognitive deficits in monkeys ~, to prevent the amnesia which results from cycloheximide administration to rats 2~, and to enhance memory in Korsakoff's psychosis -~" 77. In addition to direct effects of noradrenergic deficits on cognitive performance, locus coeruleus cell loss and forebrain noradrenergic deficits could affect cognitive performance indirectly by (a) depriving forebrain cholinergic neurons of their noradrenergic influences or (b) altering the action of acetylcholine at a postsynaptic site where NE and ACh systems converge and interact. The functional effects of NE and acetylcholine tfltimately depend upon the interaction of these transmitters with receptors on postsynaptic target cells. Hectrophysiological, pharmacological and behavioral data indicate
Correspondence: \!. Haroutunian, Psychiatry Service, Bronx VA Medical Center, 130 West Kingsbridgc Road, Bronx, N'~ 1()46g, U.S.A.
262
rat.
avoidance protocol and drug administration, each rat wa~ returned to its home cage and was left undisturbed until testing 72 h later. The passive avoidance retention test procedure was identical to that used during training except that upon completion of the crossthrough response into the dark compartment, the rat was not shocked. The latency to enter the dark, shock-associated compartment was taken as the dependent measure of memory. Within one week of behavioral testing each rat was sacrificed by decapitation, the brain was removed and cortices were dissected and assayed for choline acetyltransferase ~, acetvlcholinesterase 2. ancl norcpinephfine 2~' In one study, a second cognitive test, fear potentiated acoustic startle ~, was used to assess the generality of the passive avoidance findings. Fourteen to 21 days after the lesion procedure shamoperated and nbM+ANB-lesioned rats were randomly assigned to conditioned vs pseudoconditioned groups (n - 8). Each rat assigned to the conditioned group was placed in a light- and sound-attenuated chamber and received 30 trials in which a 15 s h m g light stimulus (7.5 W house light) was paired and co-terminated with a 1 s hmg 1 mA scrambled footshock during a 30 min conditioning session. Rats assigned to the pseudoconditioning group received an identical number of light and shock stimuli, but these stimuli were randomly and independently distributed over the 30 min session. These same procedures were repeated 24 h later. Two days after the last conditioning session each rat was placed in an acoustic startle apparatus (22 x 10 x ll) cm). Thirty, l l/) dB (SPL), 45 ms long white noise stimuli were presented to each rat and its startle response, transduced by an accelerometer attached to the startle chamber, was measured. On 50~ of the trials the acoustic startle stimulus was preceded by the light stimulus which had been presented during the two day 'fear' conditioning session. Fear potentiated startle was defined as the percent change in mean startle amplitude on light vs no light trials.
MATERIALS AND METHODS
RESULTS
that t h e s e t r a n s m i t t e r s m a y h a v e synergistic effects. For e x a m p l e , n o r e p i n e p h r i n e and c~-adrenergic agonists applied at d o s e s which by t h e m s e l v e s h a v e no e l e c t r o p h y sioiogical effects g r e a t l y p o t e n t i a t e the r e s p o n s i v e n e s s of somatosensory
cortex
cells to
a c e t y l c h o l i n e 37"-~. T h e
ability of c h o l i n e r g i c agonists to p r o d u c e the b e h a v i o r a l s y n d r o m e of c a t a l e p s y is d e p e n d e n t u p o n the integrity of the a s c e n d i n g a d r e n e r g i c p r o j e c t i o n to the c o r t e x and h i p p o c a m p u s 25. N o r a d r e n e r g i c lesions e n h a n c e the amnesic effects o f low d o s e s c o p o l a m i n e in radial m a z e l e a r n i n g p a r a d i g m s 13. C a t e c h o l a m i n e synthesis inhibition by a - m e t h y l - p - t y r o s i n e effects
of the
blocks the m e m o r y e n h a n c i n g
cholinomimeffc
o x o t r e m o r i n e 2°. T h e s e
studies a n d t h o s e d e s c r i b e d in this r e p o r t suggest that the noradrenergic
lesions in A D
m a y not only influence
c o g n i t i v e f u n c t i o n , but m a y also c o n t r i b u t e to the d e g r e e to which c h o l i n o m i m e t i c s can e n h a n c e c o g n i t i v e performance. In an effort to m o r e closely a p p r o x i m a t e the n e u r o c h e m i c a l deficits of A D and to study the i n t e r a c t i o n of c e n t r a l c h o l i n e r g i c and n o r a d r e n e r g i c systems, we h a v e e x a m i n e d the c o n s e q u e n c e s o f c o m b i n e d f o r e b r a i n cholinergic a n d n o r a d r e n e r g i c deficits on c o g n i t i v e perform a n c e and r e s p o n s i v i t y to c h o l i n o m i m e t i c agents in the
Male Sprague-Dawley rats (225-250 g) were anesthetized using a combination of ketamine (60 mg/kg i.m.) and pentobarbital (21 mg/kg i.p.). Following the induction of anesthesia, the rats were positioned in a stereotaxic apparatus with the upper incisor bar set level with the intra-aural line. After a midline incision, burr holes were drilled at coordinates bregma -0.3, +2.8 mm lateral to midline. A 33-gauge stainless steel hypodermic cannula was slowly lowered 7.5-8.0 mm ventral to the surface of the skull. One microliter of ibotenic acid (5/~g/ktl of phosphate buffer) was infused into the nbM area. The ascending noradrenergic bundle was lesioned by the injection of two microliters of a 4 ~g/~l solution of 6-hydroxydopamine (6-OHDA) dissolved in a 0.1% solution of ascorbic acid at stereotaxic coordinates bregma -6.0, 0.8 mm lateral to midline and 6.5 mm ventral to skull. Sham lesioned rats received identical treatments except for the infusion of neurotoxins, Behavioral testing began two weeks after the surgical procedures. In the majority of the experiments, a 72 h retention of one trial passive avoidance test procedure was used to assess mnemonic function and the effects of cholinornimetic drugs. After a 15 min adaptation period to the behavioral testing rooms, rats were placed singly inside the bright, white painted compartment of a two compartment, black/white, shuttle box (28 cm × 14 cm × 15 cm). Sixty seconds later, a guillotine door separating the two compartments was raised and the rat's latency to cross from the bright compartment into the dark, black painted compartment was measured. Upon entry into the dark compartment, the guillotine door was lowered and the rat received a two second long, 1).6 mA scrambled foot shock delivered through the grid floor of the apparatus. The rat was confined to the dark chamber for an additional 60 s after which it was removed and injected with the pharmacological agent under study. The drugs administered to the rats were always prepared on the day of the experiment, and were pH adjusted to 7.4. Following the acquisition phase of the passive
S e v e r a l initial studies s h o w e d that c o g n i t i v e f u n c t i o n , as assessed by tests of l e a r n i n g and m e m o r y in a passive a v o i d a n c e p a r a d i g m , was not i m p a i r e d by n o r a d r e n e r g i c lesions alone, In the first study, 12 rats r e c e i v e d bilateral i n j e c t i o n s of 6 - O H D A bundle
(ANB).
into the a s c e n d i n g n o r a d r e n e r g i c
A second group
ibotenic acid-induced
of 12 rats r e c e i v e d
lesions o f the n b M .
An
equal
n u m b e r of rats r e c e i v e d s i m u l t a n e o u s bilateral lesions of the n b M and A N B . A f o u r t h g r o u p of 12 rats r e c e i v e d e i t h e r s h a m A N B o r s h a m n b M lesions. All rats w e r e trained on the o n e trial passive a v o i d a n c e task two w e e k s after the lesion. T h e results i n d i c a t e d
that rats with
lesions of the n b M o r lesions o f the n b M + A N B w e r e s e v e r e l y i m p a i r e d with r e s p e c t to the 72 h r e t e n t i o n of passive a v o i d a n c e , w h e r e a s A N B lesions by t h e m s e l v e s did not i m p a i r test p e r f o r m a n c e . M e a n c r o s s - t h r o u g h latencies w e r e 306.4 _+ 68, 343.2 __+ 71, 113.4 _ 56 and 106.7
+
48 for s h a m - ,
ANB-,
nbM-
and
ANB
+
n b M - l e s i o n e d rats, r e s p e c t i v e l y (F~,44 = 7.2, P < 0.001). N e u r o c h e m i c a l l y , n b M - and n b M + A N B - l e s i o n e d rats displayed 2 6 . 4 % and 2 8 . 1 % r e d u c t i o n s in cortical c h o l i n e acetyitransferase (CHAT) and acetylcholinesterase ( A C H E ) activity, r e s p e c t i v e l y ( P < 0.01 vs s h a m - o p e r a t e d c o n t r o l s ) , w h e r e a s A N B - and n b M + A N B - l e s i o n e d rats d i s p l a y e d a 9 5 . 4 % r e d u c t i o n ( P < 0.01 vs sham-
263 TABLE I
Effect of sham, nbM, A N B and nbM + A N B lesions on cortical neurochemical markers Results are expressed as m e a n s +_ S.E,M. (n).
Sham nbM % dep ANB % dep nbM + ANB % dep
ChAT (nmol A CH/rnin prot)
AChE (nmol/ rain~rag prot)
NE (ng/lO0 mg tissue)
DA (ng/lO0 mg/mg tissue)
1.66 _+ I).06 (12) 1.17 + I).(17 (14) 29.5* 1,71) +/I.05 (12) 2.4 1.19 ± 0.06 (13) 27.7*
2509 _+ 41 1804 __ 83 28.0* 2461 +_ 77 1.9 1847 ± 65 26.4*
30.1 +__0.85 29.2 ± (I.92 3.1 1.9 -_~ 1.22 93.7* 3.5 _+ 2.21 88.4*
6.7 ± 0.24 (12) 6.3 ± 0.28 (14) 5.2 6.4 ± 11.34 (12) 3.8 5.9 ___0.36 (13) 11.2
(12) (14) (12) (13)
(12) (14) (12) (13)
* vs Sham, P < 0.111.
operated controls) in cortical NE activity (Table I). Lesions of the nbM did not affect cortical NE activity and lesions of the ANB did not alter cortical CHAT, AChE or dopaminergic markers (P > 0.1). These results suggest that lesions which deplete cortical cholinergic markers can severely impair performance on a learning and memory task, while noradrenergic lesions alone do not have a similar effect on passive avoidance retention test performance. This pattern of results is in agreement with earlier studies of cholinergic and adrenergic lesion effects on relatively simple tests of learning and memory in r o d e n t s 26,29.
Although cognitive deficits independent of performance variables have been repeatedly demonstrated in nbM-lesioned rats, the failure of the nbM + ANBlesioned rats to perform appropriately in the passive avoidance test could have resulted from performance artifacts or response inhibition deficits rather than learning and memory deficits per se. To test this possibility, an ancillary study was conducted in which groups of shamlesioned and nbM + ANB-lesioned rats were compared
I000 o9 v >(...) 8 0 0 Z W d I
using a fear potentiation of acoustic startle paradigm. The results of this study revealed a significant conditioning vs pseudoconditioning effect (Fl.m, = 8.8, P < 0.008) as well as a significant effect of the nbM + ANB lesion (F1,2o : 10.5, P < 0.005). In sham-operated rats, the amplitude of the startle response was significantly raised 1167%) when the acoustic startle stimulus was preceded by the shock-associated light stimulus (P < 0.02). In contrast, no significant effect of the light stimulus (121%) could be demonstrated in the nbM + ANB-lesioned rats (P > 0.05). These data taken together with the results of the passive avoidance study discussed above suggest that nbM + ANB lesions lead to profound learning and memory deficits which are independent of the measures used to assess learning and memory. The most striking effects of ANB lesions became evident when physostigmine was used in an attempt to augment the passive avoidance retention test performance of nbM + ANB-lesioned rats. The hypothesis tested by this experiment was that noradrenergic lesions may hamper the ability of cholinomimetics to potentiate performance on cognitive tasks. Different groups (n = 7) of sham-operated, nbM-lesioned, ANB-lesioned or nbM + ANB-lesioned rats received a s.c. injection of either saline or 0.06 mg/kg physostigmine immediately follow-
~700
600
600~500-
~. 400(/3 (,9
~400-
n-c.)
~ 300-
o z
200
200"
w
,~ '00O-
SAL
PHYS
SHAM
SAL
PHYS
ANB
SAL
PHYS
nbM
SAL
PHYS
ANB + nbM
Fig. 1. Effects of a 0.06 mg/kg dose of physostigmine on the 72 h retention of passive avoidance in sham-, nbM-, A N B - and nbM + ANB-lesioned rats.
0 -
0,015
006 SHAM
0,03
0.12
0,24
mq/kq
0.015 0.05 0.06 nbM + ANB
0.12
0.24
Fig. 2. Effects of different doses of physostigmine on the 72 h retention of one trial passive avoidance in s h a m - o p e r a t e d and nbM + ANB-lesioned rats.
2~4 ing the acquisition phase of a one trial passive avoidance response. When retention for passive avoidance was assessed 72 h later, the nbM-lesioned and nbM + ANB-lesioned rats receiving saline showed poor memory of the passive avoidance response relative to shamoperated controls (E~.4~ = 9.7, P 0.4). These results are depicted in Fig. 1. It is noteworthy that the effects of physostigmine in shamoperated and nbM-lesioned rats directly replicate earlier reports of physostigmine enhancement of retention test performance in nbM-lesioned rats 17. The failure of physostigmine to affect the retention test performance of the nbM + ANB-lesioned rats suggests that the combination of cholinergic and noradrenergic deficits blocks the ability of physostigmine to enhance mnemonic processes. It can be argued that the failure of physostigmine to affect retention test performance in nbM + ANBlesioned rats was due to an ANB lesion-induced shift in physostigmine dose-response relationships. To test this possibility different groups of 11-12 rats each with either sham or nbM + ANB lesions were prepared. Two weeks following the lesion procedure the rats were trained in the one trial passive avoidance paradigm. Immediately following passive avoidance training, 6 doses of physostigmine (0.0, 0.015, 0.03, 0.06, 0.12 and 0.24 mg/kg) were administered to the rats in the two lesion groups. Upon retention testing 72 h later (Fig. 2), the nbM + ANB lesions led to profound memory impairments (ANOVA, Fl,10 4 = 12.4, P < 0.001). In addition, there were significant effects of physostigmine (F5:04 = 4.5, P < 0.002) and lesion condition by physostigmine interactions (F5.1o 4 = 3.5, P < 0.01). The 0.03 mg/kg dose of physostigmine enhanced the retention test performance of sham-operated rats (Newman-Keuls, P < 0.0l) replicating earlier findings ~~, but the retention test performance of the nbM + ANB-lesioned rats was not improved by any of the doses of physostigmine investigated (P > 0.10). Examination of cholinergic and noradrenergic neurochemical changes in the lesioned rats revealed an 88.4% depletion of cortical NE in nbM + ANB-lesioned rats and 27.7% and 26.4% depletion of frontal cortical ChAT and AChE activity (Ps < 0.01). Since a rather wide dose range of physostigmine was used in this experiment, it is unlikely that the failure of physostigmine to potentiate retention test performance in nbM + ANB-lesioned rats was attributable to lesioninduced shifts in dose-response relationships, The results of this experiment suggest that one reason for the
apparent impotency of cholinomimetics in it significant proportion of AD victims may' bc the presence of noradrenergic as well as cholinergic deficits. The final experiments assessed the possibility that the memory impairments which result from forebrain cholinergic and noradrenergic deficits can be reversed by pharmacological treatments which augment cholinergic and noradrenergic function simultaneously. To this end, 68 nbM + ANB-lesioned rats and 68 sham-operated controls were trained in the one trial passive avoidance paradigm. Different groups of lesioned and control rats (n = 11-12) received one trial passive avoidance training followed immediately by s.c. injections of either 0.06 mg/kg physostigmine alone, or 0.06 mg/kg physostigmine plus one of several doses (0, 0.01 or 0.5 mg/kg) of the a2-adrenergic receptor agonist clonidine. Retention of passive avoidance was assessed 72 h later. Clonidine was selected as the noradrenergic drug of choice as some experiments 4's'22'27 have shown that clonidine effectively reverses the cognitive deficits which are evident in aged rhesus monkeys suffering forebrain noradrenergic deficits and Korsakoff's psychosis patients with compromised central noradrenergic systems. The results of this experiment are depicted in Fig. 3. Analyses of variance revealed significant lesion (F1.,j 3 = 5.2, P < 0.03), drug (F5.103 -- 5.4, P < 0.001) and lesion by drug condition interactions Es,lo3 = 5.9, P < 0.001). Independent group comparisons showed that nbM + ANB lesion-induced memory deficits were reversed by either high doses (0,5 mg/kg) of clonidine alone or much smaller doses of cionidine (0.01 mg/kg) administered in conjunction with 0.06 mg/kg physostigmine (Ps < 0.01). This same combination of physostigmine and ctonidine had an amnestic effect on the performance of the sham-operated rats (P < 0.03). In an additional experiment using the same passive avoidance training and testing and drug administration procedures, the dose of physostigmine
0.0
.ol
.50 0.0 SHAM
.01
.so
0.0
.ol
.so
0.0
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.~eLo~
N B M + .~I~IB Lesioned
* • V S S H A M P
