NeurobiologyofAging,Vol. 13, pp. 687-695, 1992
0197-4580/92 $5.00 + .00 Copyright© 1992 PergamonPressLtd.
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Age-Dependent Cerebral Metabolic Effects of Unilateral Nucleus Basalis Magnocellularis Ablation in Rats E N R I C O D E M I C H E L I A N D T I M O T H Y T. S O N C R A N T 1
Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892 R e c e i v e d 5 F e b r u a r y 1992; A c c e p t e d 15 M a y 1992 DE MICHELI E. AND T. T. SONCRANT. Age-dependent cerebralmetaboliceffects of unilateralnucleus basalis magnocellularis ablation in rats. NEUROBIOL AGING 13(6) 687-695, 1992.--To investigate the age-dependent functional importance of cholinergic neocortical inputs, and to explore whether cortical cholinergic denervation in aged animals might better model the cerebral metabolic changes of Alzheimer's disease, the effects of unilateral ablation of the nucleus basalis magnocellularis (NBM) on cerebral glucose metabolism were studied in young and aged rats. Regional cerebral metabolic rates for glucose (rCMRglc) were determined, using the [~4C]deoxyglucosemethod, in 48 brain regions of 3- and 24-month old Fischer344 rats at 3, 7, 14 and 28 days after stereotaxic injection ofibotenate into the right NBM, and in sham-operated animals at 3 and 14 days later. For both ages the peak effect of unilateral NBM ablation occurred 3 days later: in young rats, rCMRglc was significantly reduced (compared to the contralateral side) in all 24 anterior cortical areas examined (mean decline 20%), whereas in aged animals, only 9 of 24 areas showed a significant decline in glucose utilization, and the magnitude ofrCMRglc reduction (9%) was smaller. Near complete recovery of rCMRglc occurred by 7 days in young and old rats. We conclude that the basalocortical cholinergic projection plays a smaller role in neocortical function of aged rats, possibly because its tonic activity is reduced. Both young and aged rats undergo cortical metabolic normalization after unilateral NBM ablation; hence the NBMlesioned aged rat is not a better model of the progressive decline in rCMRglc that occurs in Alzheimer's disease. Aging Alzheimer's disease Nucleus basalis magnoeellularis Forebrain cholinergic neurons Excitotoxic lesions [~4C]2-deoxy-D-glucose cerebral glucose utilization Rat
D U R I N G aging and to a greater extent in Alzheimer's disease (AD), markers of neocortical presynaptic cholinergic structure and function are reduced. Choline acetyltransferase (CAT) and acetylcholinesterase (ACHE) activity, and acetylcholine synthesis and release decline during normal aging (9,27,44,59,62,69) and in AD (9,10,13,14,73). These changes reflect the loss or atrophy of neurons of the nucleus basalis magnocellularis of Meynert (NBM), a basal forebrain nucleus which supplies the major cholinergic innervation of the neocortex (62,67). Cortical cholinergic dysfunction has been implicated in cognitive deficits that occur with age and in AD. In animals, cortical cholinergic deafferentation produced by lesions of the NBM results in severe impairment of memory and learning. However, such lesions do not lead to the occurrence of Alzheimer-type pathology. Moreover, brain glucose consumption, which is progressively reduced in AD, normalizes shortly after NBM ablation in animals (32,33,35,38,47,52). The present study was conducted to determine the impact of age-related changes in cholinergic innervation on cortical function. The contribution of cholinergic inputs to cortical functional activity was assessed in young and old rats by mea-
suring the degree of reduction in cortical glucose utilization (a measure of functional neuronal activity) that occurs shortly after NBM ablation. We also wished to explore whether NBM ablation in aged animals might better model the cerebral metabolic alteration found in A D (i.e., progressive glucose metabolic deficit). Hence, the time course of regional brain glucose consumption during the 30 days after NBM ablation was measured in young and aged rats. An abstract of this work has been published (15). METHOD
Nucleus Basalis Magnocellularis Lesions Male Fischer-344 rats, 3 and 24 months old, were purchased from Charles River Breeding Laboratories (Wilmington, MA). Rats were acclimated for at least 4 days to a facility in which temperature, humidity, and light cycle (on 6.0018.00 h) were maintained and they had free access to food and water. Under ketamine (80 mg/kg, IP) anesthesia, an animal was placed in a stereotaxic apparatus (David K o p f Instruments,
~Requests for reprints should be addressed to T. T. Soncrant, NIH 10-6C103, Bethesda, MD 20892. 687
688 Tujunga, CA) and the scalp was incised. The skin was infiltrated with 1% lidocaine. Unilateral lesion of the NBM was performed by injection of 0.75 ul of a solution ofibotenic acid (Sigma, St. Louis, MO, 1 mg in 75 #I of phosphate buffer in saline, pH 7.4). A 1 ul Hamilton syringe was positioned at the NBM stereotaxic coordinates in the right side of the brain, according to Wenk et al. (72) (0.9 mm posterior to bregma, 2.6 mm lateral to bregma, 6.8 mm below the dura). The infusion was made over 5 min and the syringe was slowly removed after remaining in position for an additional l0 min. The scalp was apposed with wound clips and the rats were returned to the vivarium until the time of measurement of glucose utilization. Sham operated animals were subjected to the same surgical procedure, but the tip of the syringe was positioned in the striatum, above the NBM, and no solution was injected. This procedure was chosen in order to avoid mechanical damage to NBM neurons.
Histological Examinations Two histological methods were used to assess the effectiveness of the NBM lesions. Twenty um thick coronal brain sections were cut in the area of the injection and in the rostral part of the brain. The first series of sections were stained with cresyl violet to locate the position oftbe needle track and the presence of gliosis and neuronal loss at the site of injection. The second series were stained for ACHE, according to the technique of Karnovsky and Roots (31). Optical densities of stain product in homologous regions in the left and right sides of each brain then were measured, using an image analyzer (MCID, Imaging Research, St. Catharines, Ontario, Canada), Optical density was measured in three different sections of frontal and parietal cortical areas. Any animal in which AChE staining on the side of the lesion was reduced by less than 15% compared to the contralateral side was discarded.
Preparationfor Measurement of Brain Glucose Utilization Following 16 h of food deprivation, anesthesia was induced in a glass container with vaporized halothane and was maintained for 10-15 rain with 1% halothane in oxygen with an anesthesia machine (Fluotec MK III and Quantiflex, Fraser Harlake, Orchard Park, NY) and nose cone. Femoral venous and arterial polyethylene catheters were inserted as described previously (63). The operative site was infiltrated with lidocaine 1% and closed with wound clips. Following surgery, animals were placed in a loose-fitting plaster body cast in which the hindlimbs were restrained. This arrangement permitted free movement of head and forelimbs. Body temperature was monitored and controlled via a rectal thermoprobe connected to a thermostatic device (Indicating Controller Model 73A, Yellow Springs Instruments, Yellow Springs, OH) which activated an external warming element when body temperature fell below 35.5"C.
Regional Cerebral Metabolic Rates for Glucose (rCMRglc) rCMRglc was measured by the quantitative [~4C]2-deoxyD-glucose technique (63) in 3- and 24-month old animals at 3 and 14 days after NBM lesion or sham operation, and at 7 and 28-30 days after NBM ablation. ['4C]DG (50-55 Ci/mol) in ethanol was purchased from New England Nuclear (Boston, MA). The solution was evaporated and ['4C]DG was resuspended in isotonic saline. To ascertain the purity of the ['4C]DG, thin layer chromatography was performed. After a 4 h recovery period from surgery, ['4C]DG 125 uCi/kg was ad-
I)E MICHEL1 AND SONCRAN I ministered, by rapid IV bolus infusion, and 100 ul timed arterial blood samples were removed at 6, 18, 30, 60 s and 3, 5, 7.5, 15, 25, 35, and 45 min later. Samples were centrifuged immediately and plasma radioactivity (Model LS9000 Liquid Scintillation Counter, Beckman Instruments, Irvine, CA) and glucose concentrations (Glucose Analyzer 11, Beckman) were measured. Animals were killed 45 rain after ['4C]DG by an IV overdose of pentobarbital (60 mg in 1 ml). Brains were removed rapidly and frozen in methylbutane cooled to 40°C, then were stored at - 70°C. Coronal brain sections, 20 um thick, were prepared on a cryostat (Bright Model 5030, Hacker Instruments, Fairfield, N J) at - 200C. Sections were mounted on cover slips and dried immediately at 55°C on a hot plate. Autoradiographs then were prepared by exposing Kodak SB-5 X-ray film (Eastman Kodak, Rochester, NY) to the mounted sections and to ['4C]methyl-methacrylate standards (Amersham, Arlington Heights, IL), for 7 days. Brain radioactivity was determined by quantitative autoradiography in 48 regions on each side of the brain. Using a microdensitometer (Model 700-10-90, Gamma Scientific, San Diego, CA) a single experimenter obtained 6 separate optical density readings at each region in both left and right sides of the brain (64). rCMRglc was calculated from brain and plasma radioactivities and from plasma glucose concentrations, using equations and constants for transport and phosphorylation of [~4C]DG given by Sokoloff et al. (63), and is expressed in umol/100 g/min, mean +_ SEM.
Physiological Variables Mean arterial blood pressure and heart rate were assessed before and at fixed times during the [ ~4C]DGprocedure by connecting the arterial catheter to a pressure transducer (Statham Instruments, Hato Rey, PR) which was attached to a paper chart recorder (Model 2400 S, Gould, Cleveland, OH). Baseline and subsequent measurements of plasma glucose were made throughout the experiment. Animals were excluded from the study if the plasma glucose concentration exceeded 300 mg/dl at any time, or if baseline values were outside the following limits: mean arterial pressure 90-130 mm Hg, heart rate 350-500 per min, temperature 35-380C, plasma glucose 100-200 mg/dl. Arterial blood gases and pH were measured (Blood Gas Analyzer 1301, Instrumentation Laboratories, Lexington, MA) in 120 ul arterial blood samples taken 10 min before [~4C]DG injection.
Statistics Optical density values of AChE-stained sections taken at various times after NBM ablation were compared by age, using the Student t test. rCMRglc values for each group were tested for interhemispheric asymmetry by the paired t test: rCMRglc values measured at 3 days after NBM ablation were expressed as hemispheric ratios ([Right-Left]/Left) and were compared by age using the Student t test. RESULTS
Placement of Lesions The location and extent of excitotoxic lesions wereverified on coronal brain sections stained with cre~yl:violet. Light microscopic examination of sections revealed the infusion site as an elliptical area of neuronal loss and gliosis similar in location and extent in young and aged animals. The maximal extent of the ibotenic acid lesions is shown schematically in Fig. I.
NBM ABLATION A N D A G I N G
689
(4).92)
FIG. I. The extent and the location of damage caused by infusion of ibotenic acid into the right nucleus basalis mangocellularis,as assessed by cresyl violet histology, are indicated diagrammatically. Numbers at the top of each section refer to the distance (mm) posterior to bregma, according to Paxinos and Watson (53).
Acetylcholinesterase Staining At all times (3-30 days) after NBM lesion, AChE staining in the ipsilateral anterior cortex was markedly reduced as compared to the unlesioned hemipshere in both 3- and 24-month old rats (Fig. 2; Table 1). No significant difference in AChE staining was found between 3- and 24-month old rats at any time point.
Physiological Parameters Physiological parameters in sham-operated and NBM-iesioned rats were similar to those reported for unlesioned rats (65). Unilateral NBM lesion did not significantly affect mean arterial blood pressure, heart rate, body temperature, plasma glucose concentration, arterial blood gases, or pH in either 3or 24-month old animals at any time (compared to sham operated rats).
Regional Cerebral Metabolic Rates jbr Glucose rCMRglc values in sham-operated rats (not shown) were similar to previously reported values for awake, unoperated rats (63) (Table 2). At 3 and 14 days after sham operation, no significant right-left asymmetry between homologous brain regions was observed in either age group, except in the frontal cortex (area 10), where a consistent reduction in glucose utilization was observed ipsilateral to needle placement, probably corresponding to tissue disruption near the needle track. Unilateral NBM ablation did not significantly alter rCMRglc (compared to sham rats) in any contralateral brain area at 3 or 14 days later, in either 3 or 24 month old animals. Three-month-oldanimals. The peak effect of right NBM ablation on cortical rCMRglc occurred 3 days later. In all 24 anterior cortical areas examined, rCMRglc was significantly lower on the lesioned vs the unlesioned side of the brain. The mean asymmetry was 20%. Metabolic asymmetries (p < 0.05) were observed also in most (9 out of 12) of the posterior cortical structures examined, but they were smaller (mean 16%) and no significant asymmetry in rCMRglc was found in subcortical and brainstem areas. At later time points, rCMRglc was lower ipsilateral to NBM ablation in fewer anterior cortical regions
(7 days: 1 region, 14 days: 8, 28 days: 4). A trend toward lower rCMRglc values ipsilateral to NBM ablation was found at all later time points, but asymmetries were smaller than at 3 days. Twenty-four-month old animals. As in 3 month old rats, the peak effect of NBM ablation on rCMRglc in ipsilateral anterior cortical areas of 24-month-old rats was a 3 days, when 10 of 24 areas of the anterior neocortex had a significant asymmetry of rCMRglc. However, the magnitude of the asymmetry was smaller in 24 than in 3-month-old rats (9% vs. 20%, p < 0.05, Figs. 3, 4). Three of 12 posterior cortical regions, and no subcortical structure, showed significant metabolic asymmetry. At 7, 14, and 28 days after NBM ablation, rCMRglc in some ipsilateral anterior cortical areas was still lower in aged rats, with mean asymmetries of 6%, 5%, and 5%, respectively. DISCUSSION
Unilateral NBM ablation reduced rCMRglc 3 days later in the ipsilateral anterior cortex of both young and aged rats, but the metabolic reduction was more than two-fold larger in young than in old animals. The disparity is not attributable to differences in lesion technique, as NBM lesions in young and old rats were similar in location and extent and caused similar reductions in AChE staining in the ipsilateral frontoparietal neocortex. The age difference also was unrelated to resting glucose metabolic rates which were similar in both ages. We interpret these results to indicate that cholinergic inputs play a smaller role in the neocortical function of aged rats, possibly because their tonic activity is reduced. Ibotenic acid lesions of the NBM resulted in a large and age-independent reduction in AChE activity in the ipsilateral frontoparietal cortex that was qualitatively and quantitatively similar to previous findings (2,37). The AChE reduction reportedly is accompanied by reduced cortical choline acetyltransferase activity and high affinity choline uptake (2), implying substantial impairment of cortical cholinergic function. No spontaneous recovery of AChE activity was found after 30 days, as reported previously (5,7,12,19,49,55). The metabolic effects of unilateral excitotoxic NBM ablation have been previously investigated in young adult rats. Using a qualitative method, Lamarca and Fibiger (35) found
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I)E MICHELI AND SONCRAN !
B FIG. 2. Coronal brain sections stained tor acetylcholinesterase, taken from rats that underwent right nucleus basalis magnocellularis ablation 14 days earlier. Sections were cut in the rostral part of the brain at the levels of the pre-frontal (A) and parietal (B) cortex. Note the marked loss of acetylcholinesterase staining in the neocortex ipsilateral to the lesion (arrows).
no left-right asymmetry in brain deoxyglucose accumulation at 7 days after a unilateral ibotenate lesion. London et al. (38), using the quantitative [~4C]DG autoradiographic method, reported 20%-30% reductions in rCMRglc at 3 days after an
TABLE 1 AChE STAINING INTENSITY IN FRONTAL CORTEX AFTER NBM ABLATION 3-Month-Old Rats Days After NBM Lesion
N
3 7 14 28
10 9 10 10
Mean _+ SEM
Right-Left % Left
--21 --23 --18 --14
__+2 +__2 __+2 __+2
24-Month-Old Rats N
Right-Left % Left
7 10 10 2
--22__+ 2 --24_____ 2 --22 __+3 --15 __+5
NBM lesion in the ipsilateral neocortex and caudate nucleus, but found no asymmetry 28-32 days later. Orzi et al. (52) found 37%-38% rCMRglc reductions in ipsitateral frontal a n d parietal cortex at 4 days after NBM destruction with kainate, and a persistent 18% reduction in the ipsilateral frontal cortex 14 days later. One week after bilateral ibotenic acid injection into the NBM, a moderate (20%--25%) reduction in rCMRglc was reported by Mihara (47) throughout the neocortex, although only the frontal cortex demonstrated a statistically significant difference as compared to the sham-operated group (25%). In adult baboons. 4 days after stereotaxic unilateral electrocoagulation of the NBM (32,33) significant metabolic depression was present in the entire ipsilateral neocortex, most marked in the frontotemporal region; r C ~ c normalized within 6-13 weeks. Our findings confirm that, in rats, the peak effect of unilateral NBM albation on brain glucose metabolism occurs within a few days, when rCMRgle is reduc.~ in the anterior neocortex. They extend previous findings to demonstrate that near complete rCMRglc recovery occurs by 7 days. A trend toward asymmetry was present at 28-30 days, possibly
NBM ABLATION
AND AGING
691
TABLE 2 rCMRglc ASYMMETRY IN CORTICAL AREAS AFTER RIGHT NBM ABLATION Time After NBM Ablation Co~ical A~as
Layer
Pre-ffontal IV VB Frontal(10) IV VB Frontal(8) IV VB Pre-motor(6) IV VB Parietal(14) IV VB Motor IV VB Somatosensory IV VB
3 Days 3 Months (10)
7 Days
14 Days
28-30 Days
24 Months (7)
3 Months (9)
24 Months (10)
3 Months (10)
24 Months (10)
-16±5" --11±3" -21 ±5t - 9 ± 1~ -16±4" - 9±2* -22±4 t - 9±2* -26±4 t -14± It -22±4t-10± It -24±5t - 9±4 -23±6* - 9±3 -19±4 t - 6±3 -20±5t-12±5 -25±5~ --13±5 --19±5" - 6±4 -21±5" -- 4 ± 2 -21±6" -7±3 -16±4" - 7±2* -23±5t--16±4t --31 ± 7 t --17±6 --19±4 t -13±6 -17±5" - 2±5 -22±5t - 8±7 -16±6t - 6±2
- 8±5 -10±4 - 9±3 - 5 ±6 - 9±5 - 9±4 - 4±4 - 6±4 - 4±5 -18±7 --14±7 - 6± 7 -- 6 ± 5 -7±5 -- 2 + 6 -10±6 -11±6 - 8±5 - 6±5 -- 8 ± 5 - 4+5
7±4 - 8±2* - 5±3 - 4±2 - 8±2" -11 ±2t - 7±2* - 222 - 5 ± 1" -15±5 --10±4 - 8 +4 - 2+3 4±5 - I ±3 --13±5 --10±5 -11±3" - 2±4 - 6±2 0±2
-- 7 + 3 7+2* - 3+2 - 9+2t -11 + It - 8±2t - 3±3 - 6+3 - 7+3 -12±3" - 5±2 - 5 ± 2 - 4±2 -5+3 - 2+3 9±4 --11 ± 3t -- 7 ± 2 * - 624 - 4±3 5±3
- 323 - 7± 3 - 5 ± 1" -10±2t -10±2t - 9±2t - 3±3 - 4±3 - 3±2 - 2+ 3 - 5±3 - 5 + 3 2±4 3+2 9+~ - 6±2 --11 + 2 t 1+2 - 2+2 - 6±2* - 2±2
-
9±3* 823
-11±3" - 3±4 -2±4
-
-
8±4
-
Pre-ffontal medial 10±7t - 8±4 Insular(13) IV - 1 5 ± 4 t 4±5 Cingulate IV - 1 6 ± 3 t - 9 ± 2 * - 6 ± 3 Meanofaboveregions -20±4t9±2t
6±2*
1±5 0±2 -7±2*-4±2 6±
1# -
8+2* 4+3
5±
3 Months (10) - 4± - 6± - 4± - 6± -11 ± -10± - 4± - 5± - 4± 7± 9± 7± 52 5± 3± - 3± - 6± 7± 5± 32 - 4±
3 - 6+3 lt-6±l 2 - 2+0t 3 - 2±1 2t - 5 + 9 2* 2 + 12 2 - 0+8 3 - 4+4 2 - 2±7 4 - 14 ± 10 4 -- 7 + 9 3 5± 1 2 0+10 2 4+1 2 4±2 2 -- 4 + 2 3 -- 7 + 2 3 -10 + 0t 3 --17 + 0 t 3 --11 + 1" 2 3+0t
9±2* - I ±2 -6±2
It -
24 Months (2)
6±2"
-22+6 - 8±5 -6±1 -
5 ± I
Values are (rCMRglcngrrCMRglcLen/rCMrglc~ef~ ± SEM for n u m b e r of animals in parentheses. Difference from zero assessed by paired t test. *p < 0.05; t P < 0.01.
c o r r e s p o n d i n g to t h e p e r s i s t e n t effect o f N B M a b l a t i o n o n c o r tical r C M R g l c r e p o r t e d b y O r z i et al. (52). B o t h y o u n g a d u l t a n d a g e d r a t s d e m o n s t r a t e d a c a p a c i t y to normalize rCMRglc after cortical cholinergic deafferentation, and the recoveries followed similar time-courses. The mechan i s m o f m e t a b o l i c r e c o v e r y is u n k n o w n , b u t it c l e a r l y d o e s n o t p a r a l l e l t h o s e o f n e u r o c h e m i c a l a n d b e h a v i o r a l deficits, w h i c h d o n o t n o r m a l i z e in t h e first m o n t h ( 5 , 7 , 1 2 , 1 9 , 4 9 , 5 5 ) a n d m a y or may not do so later (5,7,12,19,22,49,56,70). How metabolic r e c o v e r y f r o m u n i l a t e r a l N B M l e s i o n o c c u r s is u n k n o w n ; it m i g h t b e d u e to o n e o r m o r e o f t h e f o l l o w i n g m e c h a n i s m s : Cholinergic reinnervation. B e c a u s e N B M n e u r o n s a r e t o p o g r a p h i c a l l y d i f f u s e , it is likely t h a t s o m e e h o l i n e r g i c n e u r o n s survived the lesion, which might assume the function of des t r o y e d cells. A l s o , t h e c o n t r a l a t e r a l N B M h a s b e e n i m p l i c a t e d in r e c o v e r y f r o m u n i l a t e r a l l e s i o n s , b e c a u s e b i l a t e r a l l y l e s i o n e d rats do not undergo neurochemical and behavioral recovery o b s e r v e d b y s o m e i n v e s t i g a t o r s in u n i l a t e r a l l y l e s i o n e d r a t s (12). C h o l i n e r g i c cells o f t h e c o n t r a l a t e r a l N B M u n d e r g o h y p e r t r o p h y a f t e r e x c i t o t o x i c u n i l a t e r a l N B M a b l a t i o n (54) a n d unilateral NBM ablation reportedly produces bilateral changes in c h o l i n e r g i c r e c e p t o r l i g a n d b i n d i n g a n d in o t h e r c h o l i n e r g i c m a r k e r s ( 5 5 , 5 7 ) a n d in n e o c o r t i c a l s l o w p o t e n t i a l s (57). F u r t h e r m o r e , c o r r e l a t i o n a n a l y s i s o f r C M R g l c v a l u e s at 2 w e e k s a f t e r u n i l a t e r a l N B M a b l a t i o n in y o u n g r a t s s u g g e s t s t h a t f u n c -
tional interactions are established between the remaining cholinergic forebrain nuclei (both ispilateral and contralateral) a n d t h e c h o l i n e r g i c a l l y d e n e r v a t e d n e o c o r t e x (66). H o w e v e r , c h o l i n e r g i c r e i n n e r v a t i o n , i f it o c c u r s , m u s t b e f a r f r o m c o m plete, b e c a u s e c h o l i n e r g i c m a r k e r s r e m a i n r e d u c e d ( v i d e supra), contralateral cholinergic projections of the NBM have n o t b e e n d e s c r i b e d in t h e r a t a n d s p r o u t i n g r e p o r t e d l y d o e s n o t o c c u r in t h e i p s i l a t e r a l c o r t e x f o r u p t o 5 m o n t h s a f t e r N B M a b l a t i o n (51). Cholinergic receptor regulation. U p r e g u l a t i o n o f p o s t s y n a p t i c c h o l i n e r g i c r e c e p t o r s c o u l d c o m p e n s a t e for r e d u c e d p r e s y n a p t i c i n p u t . A f t e r u n i l a t e r a l N B M a b l a t i o n , c o r t i c a l M 2 rec e p t o r n u m b e r r e p o r t e d l y is r e d u c e d at 3 d a y s (41) b u t i n c r e a s e d at 1 w e e k (4). L a m o u r et al. (36) s h o w e d a t r a n s i e n t b u t large i n c r e a s e in c o r t i c a l electrical r e s p o n s e s t o i o n t o p h o retic a p p l i c a t i o n o f a c e t y l c h o l i n e in r a t s 2 w e e k s a f t e r b i l a t e r a l N B M l e s i o n s . I n c r e a s e d s e n s i t i v i t y to a c e t y l c h o l i n e , m e a s u r e d a s t h e a b i l i t y o f i n t r a v e n t r i c u l a r a c e t y l c h o l i n e to p r o d u c e m o t o r s e i z u r e s , w a s f o u n d in N B M l e s i o n e d r a t s 4 w e e k s a f t e r b i l a t e r a l N B M a b l a t i o n (42). F u r t h e r m o r e , 2 w e e k s a f t e r u n i l a t e r a l N B M a b l a t i o n , t h e a d m i n i s t r a t i o n o f a r e c o l i n e (5 r a g / kg) r e s u l t e d in g r e a t e r [ I J 4 C ] a r a c h i d o n i c a c i d i n c o r p o r a t i o n into ipsilateral versus contralateral pre-frontal and frontal cort i c e s (50). O n t h e o t h e r h a n d , A i r m a n et al. (2) a t 2 w e e k s a n d M c K i n n e y a n d C o y l e (46) at 5 w e e k s a f t e r b i l a t e r a l N B M le-
692
DE MICHELI AND SONCRANI malize rCMRglc without producing recovery of cholinergic biochemical markers or of behavior. Other neurotransmitter systems are altered by NBM ablation and have been implicated in functional recovery (71). However, changes in markers for those systems do not occur early after NBM ablation (28,73). Furthermore, at 2 weeks after unilateral NBM lesion, we have been unable to demonstrate altered rCMRglc responsivity of the ipsilateral cortex to dopaminergic, serotonergic, or adrenergic drugs ( 16,17).
3O
3rno ----
24 rno
>I-I,U
I
20
=Z X >-
Resetting oJ"coupling between resting glucose metabolism and fimction. As in the present study, rapid recovery of
(n
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Age-Dependent Cerebral Metabolic Effects of Unilateral Nucleus Basalis Magnocellularis Ablation in Rats E N R I C O D E M I C H E L I A N D T I M O T H Y T. S O N C R A N T 1
Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892 R e c e i v e d 5 F e b r u a r y 1992; A c c e p t e d 15 M a y 1992 DE MICHELI E. AND T. T. SONCRANT. Age-dependent cerebralmetaboliceffects of unilateralnucleus basalis magnocellularis ablation in rats. NEUROBIOL AGING 13(6) 687-695, 1992.--To investigate the age-dependent functional importance of cholinergic neocortical inputs, and to explore whether cortical cholinergic denervation in aged animals might better model the cerebral metabolic changes of Alzheimer's disease, the effects of unilateral ablation of the nucleus basalis magnocellularis (NBM) on cerebral glucose metabolism were studied in young and aged rats. Regional cerebral metabolic rates for glucose (rCMRglc) were determined, using the [~4C]deoxyglucosemethod, in 48 brain regions of 3- and 24-month old Fischer344 rats at 3, 7, 14 and 28 days after stereotaxic injection ofibotenate into the right NBM, and in sham-operated animals at 3 and 14 days later. For both ages the peak effect of unilateral NBM ablation occurred 3 days later: in young rats, rCMRglc was significantly reduced (compared to the contralateral side) in all 24 anterior cortical areas examined (mean decline 20%), whereas in aged animals, only 9 of 24 areas showed a significant decline in glucose utilization, and the magnitude ofrCMRglc reduction (9%) was smaller. Near complete recovery of rCMRglc occurred by 7 days in young and old rats. We conclude that the basalocortical cholinergic projection plays a smaller role in neocortical function of aged rats, possibly because its tonic activity is reduced. Both young and aged rats undergo cortical metabolic normalization after unilateral NBM ablation; hence the NBMlesioned aged rat is not a better model of the progressive decline in rCMRglc that occurs in Alzheimer's disease. Aging Alzheimer's disease Nucleus basalis magnoeellularis Forebrain cholinergic neurons Excitotoxic lesions [~4C]2-deoxy-D-glucose cerebral glucose utilization Rat
D U R I N G aging and to a greater extent in Alzheimer's disease (AD), markers of neocortical presynaptic cholinergic structure and function are reduced. Choline acetyltransferase (CAT) and acetylcholinesterase (ACHE) activity, and acetylcholine synthesis and release decline during normal aging (9,27,44,59,62,69) and in AD (9,10,13,14,73). These changes reflect the loss or atrophy of neurons of the nucleus basalis magnocellularis of Meynert (NBM), a basal forebrain nucleus which supplies the major cholinergic innervation of the neocortex (62,67). Cortical cholinergic dysfunction has been implicated in cognitive deficits that occur with age and in AD. In animals, cortical cholinergic deafferentation produced by lesions of the NBM results in severe impairment of memory and learning. However, such lesions do not lead to the occurrence of Alzheimer-type pathology. Moreover, brain glucose consumption, which is progressively reduced in AD, normalizes shortly after NBM ablation in animals (32,33,35,38,47,52). The present study was conducted to determine the impact of age-related changes in cholinergic innervation on cortical function. The contribution of cholinergic inputs to cortical functional activity was assessed in young and old rats by mea-
suring the degree of reduction in cortical glucose utilization (a measure of functional neuronal activity) that occurs shortly after NBM ablation. We also wished to explore whether NBM ablation in aged animals might better model the cerebral metabolic alteration found in A D (i.e., progressive glucose metabolic deficit). Hence, the time course of regional brain glucose consumption during the 30 days after NBM ablation was measured in young and aged rats. An abstract of this work has been published (15). METHOD
Nucleus Basalis Magnocellularis Lesions Male Fischer-344 rats, 3 and 24 months old, were purchased from Charles River Breeding Laboratories (Wilmington, MA). Rats were acclimated for at least 4 days to a facility in which temperature, humidity, and light cycle (on 6.0018.00 h) were maintained and they had free access to food and water. Under ketamine (80 mg/kg, IP) anesthesia, an animal was placed in a stereotaxic apparatus (David K o p f Instruments,
~Requests for reprints should be addressed to T. T. Soncrant, NIH 10-6C103, Bethesda, MD 20892. 687
688 Tujunga, CA) and the scalp was incised. The skin was infiltrated with 1% lidocaine. Unilateral lesion of the NBM was performed by injection of 0.75 ul of a solution ofibotenic acid (Sigma, St. Louis, MO, 1 mg in 75 #I of phosphate buffer in saline, pH 7.4). A 1 ul Hamilton syringe was positioned at the NBM stereotaxic coordinates in the right side of the brain, according to Wenk et al. (72) (0.9 mm posterior to bregma, 2.6 mm lateral to bregma, 6.8 mm below the dura). The infusion was made over 5 min and the syringe was slowly removed after remaining in position for an additional l0 min. The scalp was apposed with wound clips and the rats were returned to the vivarium until the time of measurement of glucose utilization. Sham operated animals were subjected to the same surgical procedure, but the tip of the syringe was positioned in the striatum, above the NBM, and no solution was injected. This procedure was chosen in order to avoid mechanical damage to NBM neurons.
Histological Examinations Two histological methods were used to assess the effectiveness of the NBM lesions. Twenty um thick coronal brain sections were cut in the area of the injection and in the rostral part of the brain. The first series of sections were stained with cresyl violet to locate the position oftbe needle track and the presence of gliosis and neuronal loss at the site of injection. The second series were stained for ACHE, according to the technique of Karnovsky and Roots (31). Optical densities of stain product in homologous regions in the left and right sides of each brain then were measured, using an image analyzer (MCID, Imaging Research, St. Catharines, Ontario, Canada), Optical density was measured in three different sections of frontal and parietal cortical areas. Any animal in which AChE staining on the side of the lesion was reduced by less than 15% compared to the contralateral side was discarded.
Preparationfor Measurement of Brain Glucose Utilization Following 16 h of food deprivation, anesthesia was induced in a glass container with vaporized halothane and was maintained for 10-15 rain with 1% halothane in oxygen with an anesthesia machine (Fluotec MK III and Quantiflex, Fraser Harlake, Orchard Park, NY) and nose cone. Femoral venous and arterial polyethylene catheters were inserted as described previously (63). The operative site was infiltrated with lidocaine 1% and closed with wound clips. Following surgery, animals were placed in a loose-fitting plaster body cast in which the hindlimbs were restrained. This arrangement permitted free movement of head and forelimbs. Body temperature was monitored and controlled via a rectal thermoprobe connected to a thermostatic device (Indicating Controller Model 73A, Yellow Springs Instruments, Yellow Springs, OH) which activated an external warming element when body temperature fell below 35.5"C.
Regional Cerebral Metabolic Rates for Glucose (rCMRglc) rCMRglc was measured by the quantitative [~4C]2-deoxyD-glucose technique (63) in 3- and 24-month old animals at 3 and 14 days after NBM lesion or sham operation, and at 7 and 28-30 days after NBM ablation. ['4C]DG (50-55 Ci/mol) in ethanol was purchased from New England Nuclear (Boston, MA). The solution was evaporated and ['4C]DG was resuspended in isotonic saline. To ascertain the purity of the ['4C]DG, thin layer chromatography was performed. After a 4 h recovery period from surgery, ['4C]DG 125 uCi/kg was ad-
I)E MICHEL1 AND SONCRAN I ministered, by rapid IV bolus infusion, and 100 ul timed arterial blood samples were removed at 6, 18, 30, 60 s and 3, 5, 7.5, 15, 25, 35, and 45 min later. Samples were centrifuged immediately and plasma radioactivity (Model LS9000 Liquid Scintillation Counter, Beckman Instruments, Irvine, CA) and glucose concentrations (Glucose Analyzer 11, Beckman) were measured. Animals were killed 45 rain after ['4C]DG by an IV overdose of pentobarbital (60 mg in 1 ml). Brains were removed rapidly and frozen in methylbutane cooled to 40°C, then were stored at - 70°C. Coronal brain sections, 20 um thick, were prepared on a cryostat (Bright Model 5030, Hacker Instruments, Fairfield, N J) at - 200C. Sections were mounted on cover slips and dried immediately at 55°C on a hot plate. Autoradiographs then were prepared by exposing Kodak SB-5 X-ray film (Eastman Kodak, Rochester, NY) to the mounted sections and to ['4C]methyl-methacrylate standards (Amersham, Arlington Heights, IL), for 7 days. Brain radioactivity was determined by quantitative autoradiography in 48 regions on each side of the brain. Using a microdensitometer (Model 700-10-90, Gamma Scientific, San Diego, CA) a single experimenter obtained 6 separate optical density readings at each region in both left and right sides of the brain (64). rCMRglc was calculated from brain and plasma radioactivities and from plasma glucose concentrations, using equations and constants for transport and phosphorylation of [~4C]DG given by Sokoloff et al. (63), and is expressed in umol/100 g/min, mean +_ SEM.
Physiological Variables Mean arterial blood pressure and heart rate were assessed before and at fixed times during the [ ~4C]DGprocedure by connecting the arterial catheter to a pressure transducer (Statham Instruments, Hato Rey, PR) which was attached to a paper chart recorder (Model 2400 S, Gould, Cleveland, OH). Baseline and subsequent measurements of plasma glucose were made throughout the experiment. Animals were excluded from the study if the plasma glucose concentration exceeded 300 mg/dl at any time, or if baseline values were outside the following limits: mean arterial pressure 90-130 mm Hg, heart rate 350-500 per min, temperature 35-380C, plasma glucose 100-200 mg/dl. Arterial blood gases and pH were measured (Blood Gas Analyzer 1301, Instrumentation Laboratories, Lexington, MA) in 120 ul arterial blood samples taken 10 min before [~4C]DG injection.
Statistics Optical density values of AChE-stained sections taken at various times after NBM ablation were compared by age, using the Student t test. rCMRglc values for each group were tested for interhemispheric asymmetry by the paired t test: rCMRglc values measured at 3 days after NBM ablation were expressed as hemispheric ratios ([Right-Left]/Left) and were compared by age using the Student t test. RESULTS
Placement of Lesions The location and extent of excitotoxic lesions wereverified on coronal brain sections stained with cre~yl:violet. Light microscopic examination of sections revealed the infusion site as an elliptical area of neuronal loss and gliosis similar in location and extent in young and aged animals. The maximal extent of the ibotenic acid lesions is shown schematically in Fig. I.
NBM ABLATION A N D A G I N G
689
(4).92)
FIG. I. The extent and the location of damage caused by infusion of ibotenic acid into the right nucleus basalis mangocellularis,as assessed by cresyl violet histology, are indicated diagrammatically. Numbers at the top of each section refer to the distance (mm) posterior to bregma, according to Paxinos and Watson (53).
Acetylcholinesterase Staining At all times (3-30 days) after NBM lesion, AChE staining in the ipsilateral anterior cortex was markedly reduced as compared to the unlesioned hemipshere in both 3- and 24-month old rats (Fig. 2; Table 1). No significant difference in AChE staining was found between 3- and 24-month old rats at any time point.
Physiological Parameters Physiological parameters in sham-operated and NBM-iesioned rats were similar to those reported for unlesioned rats (65). Unilateral NBM lesion did not significantly affect mean arterial blood pressure, heart rate, body temperature, plasma glucose concentration, arterial blood gases, or pH in either 3or 24-month old animals at any time (compared to sham operated rats).
Regional Cerebral Metabolic Rates jbr Glucose rCMRglc values in sham-operated rats (not shown) were similar to previously reported values for awake, unoperated rats (63) (Table 2). At 3 and 14 days after sham operation, no significant right-left asymmetry between homologous brain regions was observed in either age group, except in the frontal cortex (area 10), where a consistent reduction in glucose utilization was observed ipsilateral to needle placement, probably corresponding to tissue disruption near the needle track. Unilateral NBM ablation did not significantly alter rCMRglc (compared to sham rats) in any contralateral brain area at 3 or 14 days later, in either 3 or 24 month old animals. Three-month-oldanimals. The peak effect of right NBM ablation on cortical rCMRglc occurred 3 days later. In all 24 anterior cortical areas examined, rCMRglc was significantly lower on the lesioned vs the unlesioned side of the brain. The mean asymmetry was 20%. Metabolic asymmetries (p < 0.05) were observed also in most (9 out of 12) of the posterior cortical structures examined, but they were smaller (mean 16%) and no significant asymmetry in rCMRglc was found in subcortical and brainstem areas. At later time points, rCMRglc was lower ipsilateral to NBM ablation in fewer anterior cortical regions
(7 days: 1 region, 14 days: 8, 28 days: 4). A trend toward lower rCMRglc values ipsilateral to NBM ablation was found at all later time points, but asymmetries were smaller than at 3 days. Twenty-four-month old animals. As in 3 month old rats, the peak effect of NBM ablation on rCMRglc in ipsilateral anterior cortical areas of 24-month-old rats was a 3 days, when 10 of 24 areas of the anterior neocortex had a significant asymmetry of rCMRglc. However, the magnitude of the asymmetry was smaller in 24 than in 3-month-old rats (9% vs. 20%, p < 0.05, Figs. 3, 4). Three of 12 posterior cortical regions, and no subcortical structure, showed significant metabolic asymmetry. At 7, 14, and 28 days after NBM ablation, rCMRglc in some ipsilateral anterior cortical areas was still lower in aged rats, with mean asymmetries of 6%, 5%, and 5%, respectively. DISCUSSION
Unilateral NBM ablation reduced rCMRglc 3 days later in the ipsilateral anterior cortex of both young and aged rats, but the metabolic reduction was more than two-fold larger in young than in old animals. The disparity is not attributable to differences in lesion technique, as NBM lesions in young and old rats were similar in location and extent and caused similar reductions in AChE staining in the ipsilateral frontoparietal neocortex. The age difference also was unrelated to resting glucose metabolic rates which were similar in both ages. We interpret these results to indicate that cholinergic inputs play a smaller role in the neocortical function of aged rats, possibly because their tonic activity is reduced. Ibotenic acid lesions of the NBM resulted in a large and age-independent reduction in AChE activity in the ipsilateral frontoparietal cortex that was qualitatively and quantitatively similar to previous findings (2,37). The AChE reduction reportedly is accompanied by reduced cortical choline acetyltransferase activity and high affinity choline uptake (2), implying substantial impairment of cortical cholinergic function. No spontaneous recovery of AChE activity was found after 30 days, as reported previously (5,7,12,19,49,55). The metabolic effects of unilateral excitotoxic NBM ablation have been previously investigated in young adult rats. Using a qualitative method, Lamarca and Fibiger (35) found
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I)E MICHELI AND SONCRAN !
B FIG. 2. Coronal brain sections stained tor acetylcholinesterase, taken from rats that underwent right nucleus basalis magnocellularis ablation 14 days earlier. Sections were cut in the rostral part of the brain at the levels of the pre-frontal (A) and parietal (B) cortex. Note the marked loss of acetylcholinesterase staining in the neocortex ipsilateral to the lesion (arrows).
no left-right asymmetry in brain deoxyglucose accumulation at 7 days after a unilateral ibotenate lesion. London et al. (38), using the quantitative [~4C]DG autoradiographic method, reported 20%-30% reductions in rCMRglc at 3 days after an
TABLE 1 AChE STAINING INTENSITY IN FRONTAL CORTEX AFTER NBM ABLATION 3-Month-Old Rats Days After NBM Lesion
N
3 7 14 28
10 9 10 10
Mean _+ SEM
Right-Left % Left
--21 --23 --18 --14
__+2 +__2 __+2 __+2
24-Month-Old Rats N
Right-Left % Left
7 10 10 2
--22__+ 2 --24_____ 2 --22 __+3 --15 __+5
NBM lesion in the ipsilateral neocortex and caudate nucleus, but found no asymmetry 28-32 days later. Orzi et al. (52) found 37%-38% rCMRglc reductions in ipsitateral frontal a n d parietal cortex at 4 days after NBM destruction with kainate, and a persistent 18% reduction in the ipsilateral frontal cortex 14 days later. One week after bilateral ibotenic acid injection into the NBM, a moderate (20%--25%) reduction in rCMRglc was reported by Mihara (47) throughout the neocortex, although only the frontal cortex demonstrated a statistically significant difference as compared to the sham-operated group (25%). In adult baboons. 4 days after stereotaxic unilateral electrocoagulation of the NBM (32,33) significant metabolic depression was present in the entire ipsilateral neocortex, most marked in the frontotemporal region; r C ~ c normalized within 6-13 weeks. Our findings confirm that, in rats, the peak effect of unilateral NBM albation on brain glucose metabolism occurs within a few days, when rCMRgle is reduc.~ in the anterior neocortex. They extend previous findings to demonstrate that near complete rCMRglc recovery occurs by 7 days. A trend toward asymmetry was present at 28-30 days, possibly
NBM ABLATION
AND AGING
691
TABLE 2 rCMRglc ASYMMETRY IN CORTICAL AREAS AFTER RIGHT NBM ABLATION Time After NBM Ablation Co~ical A~as
Layer
Pre-ffontal IV VB Frontal(10) IV VB Frontal(8) IV VB Pre-motor(6) IV VB Parietal(14) IV VB Motor IV VB Somatosensory IV VB
3 Days 3 Months (10)
7 Days
14 Days
28-30 Days
24 Months (7)
3 Months (9)
24 Months (10)
3 Months (10)
24 Months (10)
-16±5" --11±3" -21 ±5t - 9 ± 1~ -16±4" - 9±2* -22±4 t - 9±2* -26±4 t -14± It -22±4t-10± It -24±5t - 9±4 -23±6* - 9±3 -19±4 t - 6±3 -20±5t-12±5 -25±5~ --13±5 --19±5" - 6±4 -21±5" -- 4 ± 2 -21±6" -7±3 -16±4" - 7±2* -23±5t--16±4t --31 ± 7 t --17±6 --19±4 t -13±6 -17±5" - 2±5 -22±5t - 8±7 -16±6t - 6±2
- 8±5 -10±4 - 9±3 - 5 ±6 - 9±5 - 9±4 - 4±4 - 6±4 - 4±5 -18±7 --14±7 - 6± 7 -- 6 ± 5 -7±5 -- 2 + 6 -10±6 -11±6 - 8±5 - 6±5 -- 8 ± 5 - 4+5
7±4 - 8±2* - 5±3 - 4±2 - 8±2" -11 ±2t - 7±2* - 222 - 5 ± 1" -15±5 --10±4 - 8 +4 - 2+3 4±5 - I ±3 --13±5 --10±5 -11±3" - 2±4 - 6±2 0±2
-- 7 + 3 7+2* - 3+2 - 9+2t -11 + It - 8±2t - 3±3 - 6+3 - 7+3 -12±3" - 5±2 - 5 ± 2 - 4±2 -5+3 - 2+3 9±4 --11 ± 3t -- 7 ± 2 * - 624 - 4±3 5±3
- 323 - 7± 3 - 5 ± 1" -10±2t -10±2t - 9±2t - 3±3 - 4±3 - 3±2 - 2+ 3 - 5±3 - 5 + 3 2±4 3+2 9+~ - 6±2 --11 + 2 t 1+2 - 2+2 - 6±2* - 2±2
-
9±3* 823
-11±3" - 3±4 -2±4
-
-
8±4
-
Pre-ffontal medial 10±7t - 8±4 Insular(13) IV - 1 5 ± 4 t 4±5 Cingulate IV - 1 6 ± 3 t - 9 ± 2 * - 6 ± 3 Meanofaboveregions -20±4t9±2t
6±2*
1±5 0±2 -7±2*-4±2 6±
1# -
8+2* 4+3
5±
3 Months (10) - 4± - 6± - 4± - 6± -11 ± -10± - 4± - 5± - 4± 7± 9± 7± 52 5± 3± - 3± - 6± 7± 5± 32 - 4±
3 - 6+3 lt-6±l 2 - 2+0t 3 - 2±1 2t - 5 + 9 2* 2 + 12 2 - 0+8 3 - 4+4 2 - 2±7 4 - 14 ± 10 4 -- 7 + 9 3 5± 1 2 0+10 2 4+1 2 4±2 2 -- 4 + 2 3 -- 7 + 2 3 -10 + 0t 3 --17 + 0 t 3 --11 + 1" 2 3+0t
9±2* - I ±2 -6±2
It -
24 Months (2)
6±2"
-22+6 - 8±5 -6±1 -
5 ± I
Values are (rCMRglcngrrCMRglcLen/rCMrglc~ef~ ± SEM for n u m b e r of animals in parentheses. Difference from zero assessed by paired t test. *p < 0.05; t P < 0.01.
c o r r e s p o n d i n g to t h e p e r s i s t e n t effect o f N B M a b l a t i o n o n c o r tical r C M R g l c r e p o r t e d b y O r z i et al. (52). B o t h y o u n g a d u l t a n d a g e d r a t s d e m o n s t r a t e d a c a p a c i t y to normalize rCMRglc after cortical cholinergic deafferentation, and the recoveries followed similar time-courses. The mechan i s m o f m e t a b o l i c r e c o v e r y is u n k n o w n , b u t it c l e a r l y d o e s n o t p a r a l l e l t h o s e o f n e u r o c h e m i c a l a n d b e h a v i o r a l deficits, w h i c h d o n o t n o r m a l i z e in t h e first m o n t h ( 5 , 7 , 1 2 , 1 9 , 4 9 , 5 5 ) a n d m a y or may not do so later (5,7,12,19,22,49,56,70). How metabolic r e c o v e r y f r o m u n i l a t e r a l N B M l e s i o n o c c u r s is u n k n o w n ; it m i g h t b e d u e to o n e o r m o r e o f t h e f o l l o w i n g m e c h a n i s m s : Cholinergic reinnervation. B e c a u s e N B M n e u r o n s a r e t o p o g r a p h i c a l l y d i f f u s e , it is likely t h a t s o m e e h o l i n e r g i c n e u r o n s survived the lesion, which might assume the function of des t r o y e d cells. A l s o , t h e c o n t r a l a t e r a l N B M h a s b e e n i m p l i c a t e d in r e c o v e r y f r o m u n i l a t e r a l l e s i o n s , b e c a u s e b i l a t e r a l l y l e s i o n e d rats do not undergo neurochemical and behavioral recovery o b s e r v e d b y s o m e i n v e s t i g a t o r s in u n i l a t e r a l l y l e s i o n e d r a t s (12). C h o l i n e r g i c cells o f t h e c o n t r a l a t e r a l N B M u n d e r g o h y p e r t r o p h y a f t e r e x c i t o t o x i c u n i l a t e r a l N B M a b l a t i o n (54) a n d unilateral NBM ablation reportedly produces bilateral changes in c h o l i n e r g i c r e c e p t o r l i g a n d b i n d i n g a n d in o t h e r c h o l i n e r g i c m a r k e r s ( 5 5 , 5 7 ) a n d in n e o c o r t i c a l s l o w p o t e n t i a l s (57). F u r t h e r m o r e , c o r r e l a t i o n a n a l y s i s o f r C M R g l c v a l u e s at 2 w e e k s a f t e r u n i l a t e r a l N B M a b l a t i o n in y o u n g r a t s s u g g e s t s t h a t f u n c -
tional interactions are established between the remaining cholinergic forebrain nuclei (both ispilateral and contralateral) a n d t h e c h o l i n e r g i c a l l y d e n e r v a t e d n e o c o r t e x (66). H o w e v e r , c h o l i n e r g i c r e i n n e r v a t i o n , i f it o c c u r s , m u s t b e f a r f r o m c o m plete, b e c a u s e c h o l i n e r g i c m a r k e r s r e m a i n r e d u c e d ( v i d e supra), contralateral cholinergic projections of the NBM have n o t b e e n d e s c r i b e d in t h e r a t a n d s p r o u t i n g r e p o r t e d l y d o e s n o t o c c u r in t h e i p s i l a t e r a l c o r t e x f o r u p t o 5 m o n t h s a f t e r N B M a b l a t i o n (51). Cholinergic receptor regulation. U p r e g u l a t i o n o f p o s t s y n a p t i c c h o l i n e r g i c r e c e p t o r s c o u l d c o m p e n s a t e for r e d u c e d p r e s y n a p t i c i n p u t . A f t e r u n i l a t e r a l N B M a b l a t i o n , c o r t i c a l M 2 rec e p t o r n u m b e r r e p o r t e d l y is r e d u c e d at 3 d a y s (41) b u t i n c r e a s e d at 1 w e e k (4). L a m o u r et al. (36) s h o w e d a t r a n s i e n t b u t large i n c r e a s e in c o r t i c a l electrical r e s p o n s e s t o i o n t o p h o retic a p p l i c a t i o n o f a c e t y l c h o l i n e in r a t s 2 w e e k s a f t e r b i l a t e r a l N B M l e s i o n s . I n c r e a s e d s e n s i t i v i t y to a c e t y l c h o l i n e , m e a s u r e d a s t h e a b i l i t y o f i n t r a v e n t r i c u l a r a c e t y l c h o l i n e to p r o d u c e m o t o r s e i z u r e s , w a s f o u n d in N B M l e s i o n e d r a t s 4 w e e k s a f t e r b i l a t e r a l N B M a b l a t i o n (42). F u r t h e r m o r e , 2 w e e k s a f t e r u n i l a t e r a l N B M a b l a t i o n , t h e a d m i n i s t r a t i o n o f a r e c o l i n e (5 r a g / kg) r e s u l t e d in g r e a t e r [ I J 4 C ] a r a c h i d o n i c a c i d i n c o r p o r a t i o n into ipsilateral versus contralateral pre-frontal and frontal cort i c e s (50). O n t h e o t h e r h a n d , A i r m a n et al. (2) a t 2 w e e k s a n d M c K i n n e y a n d C o y l e (46) at 5 w e e k s a f t e r b i l a t e r a l N B M le-
692
DE MICHELI AND SONCRANI malize rCMRglc without producing recovery of cholinergic biochemical markers or of behavior. Other neurotransmitter systems are altered by NBM ablation and have been implicated in functional recovery (71). However, changes in markers for those systems do not occur early after NBM ablation (28,73). Furthermore, at 2 weeks after unilateral NBM lesion, we have been unable to demonstrate altered rCMRglc responsivity of the ipsilateral cortex to dopaminergic, serotonergic, or adrenergic drugs ( 16,17).
3O
3rno ----
24 rno
>I-I,U
I
20
=Z X >-
Resetting oJ"coupling between resting glucose metabolism and fimction. As in the present study, rapid recovery of
(n
