Effects of quisqualic acid nucleus basalis lesioning on cortical EEG, passive avoidance and water maze performance

Brain Research Bulletin, Vol. 24, pp. 839-842. 0 Pergamon Press plc, 1990. Printed in the U.S.A.

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Effects of Quisqualic Acid Nucleus Basalis Lesioning on Cortical EEG, Passive Avoidance and Water Maze Performance PAAVO RIEKKINEN, JR. ,*t’ JOUNI SIRVIij,* TUULA HANNILA,* RIITTA MIETTINEN*I_ AND PAAVO RIEKKINEN* Departments of *Neurology and ?Pathology, University of Kuopio, Kuopio, Finland Received 20 February 1990

RIEKKINEN, P., JR., J. SIRVIO, T. HANNILA, R. MIETTINEN AND P. RIEKKINEN. Effects ofquisquak acid nucleus basalis lesioning on corfical EEG, passive avoidance and water maze performance. BRAIN RES BULL 24(6) 839-842, 1990.-The study examines the effects of unilateral quisqualic acid nucleus basalis (NB) lesioning on cortical EEG and learning behavior. Lesions produced both gliosis in the ventral pallidum and a marked reduction in the cortical ChAT activity. Normal cortical EEG activity was abolished on the side of NB lesion, i.e., slow wave activity and the incidence of high voltage spindles was higher on the side of lesion compared with the control side. NB lesioning impaired passive avoidance retention, but not spatial learning ability. These results suggest that EEG and passive avoidance deficits induced by NB quisqualic acid lesion may result from the damage specifically to cholinergic neurons. Thus, the restoration of EEG and passive avoidance performance defects in quisqualic-lesioned rats may be used as an index of the efficacy of the cholinergic replacement therapies. Nucleus basalis

Spectral powers

High voltage spindles

Passive avoidance

to Paavo Riekkinen,

Quisqualic

acid

ing nuclei than ibotenic acid at doses that produces the same reduction in cortical choline acetyltransferase (ChAT) activity (5). Moreover, among the proliferated glial elements in the quisq&ate-infused area, some small noncholinergic neurons were spared (13). The chemical identity or connective patterns of these neurons is at present unknown. Also, recent studies have revealed that learning deficits induced by quisqualic acid NB lesion are much smaller than those induced by ibotenic acid NB lesion (5,13). Thus, it is reasonable to believe that the ibotenic acid-induced leaming deficits, although associated with cholinergic neuron loss, may partially result from nonspecific subcortical damage in the basal forebrain (5, 12, 13). The results of the previous studies which determine the role of cholinergic NB in the regulation of neocortical EEG are controversial. Electrolytic NB lesions produced a depression of EEG total amplitude whereas kainic acid NB lesioning increased EEG slow waves during waking-immobility (9,15). However, both the destruction of fibers of passage induced by electrolytic lesioning and large subcortical damage in surrounding nuclei induced by the

central nervous system cholinergic transmission has been proposed to be involved significantly in learning behavior and regulation of cortical electrical activity (3, 4, 11, 12). Secondly, the memory deficits and EEG slowing found in patients with Alzheimer’s disease may be attributable to degeneration of the nucleus basalis (NB) cholinergic neurons (2,14). Thus, animal models of AD have concentrated on the mnemonic and neurophysiological consequences of destruction of the NB, the major source of extrinsic cholinergic innervation of the neocortex and reticular nucleus of the thalamus (1,8). Several of the studies have used intracerebral ibotenic acid infusions to produce both a cholinergic cell loss and deficits of learning and memory. However, the specificity of this preparation as a model for experimental cholinergic denervation is debatable. Ibotenic acid infusions destroy magnocellular cholinergic cells as well as noncholinergic neurons located in the ventral pallidum, and also produce large nonspecific subcortical damage in adjacent structures (5). Quisqualic acid, another exicotoxin, infused into the ventral pallidum induces less nonspecific damage in surroundTHE

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Spatial learning

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P.O.B.

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RIEKKINEN

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FIG. 2. Frontal waking-immobility-related EEG amplitude values (FV) recorded ipsilaterally to the intracerebral infusions. The values are expressed as mean?S.D. Frequency bands: 1.0-4 Hz (delta), 4-8 Hz (theta), 8-12 Hz (alpha), 12-20 Hz (beta). See group abbreviations from Table 2. *p
FIG. 1. Reconstructions of maximum (on the left hemisphere) and minimum (on the right hemisphere) extent of quisqualic acid-induced gliosis.

kainate, make it difficult to estimate the importance of the cholinergic neuron loss in the EEG changes reported in these studies (9,15). Recent studies using quantitative EEG methods to study the effects of ibotenic acid NB lesioning on cortical EEG have demonstrated an increase in slow wave similar to that observed after scopolamine injections (3,ll). Moreover, NB lesioning releases high voltage spindle (HVS) generation in the reticular nucleus of thalamus (3). However, the effect of quisqualic acid-induced NB lesion on neocortical EEG activity has not been reported previously. Therefore, the effects of unilateral quisqualic acid lesioning of the NB were evaluated on the 1) cortical EEG activity, 2) on the passive avoidance (PA) and 3) Morris water maze (MWM) performance. These were done in order to elucidate the validity of this preparation for further pharmacological experiments involving the restorative effects of cholinergic treatments. METHOD Male Kuo:Wistar rats were used in this study (n = 17, 245-270 g). The rats were housed singly with food and water freely available. The light period was 14 hr (light on at 0700). The animals were anaesthetized with chloral hydrate (350 mgikg) and placed in a stereotaxic frame with the incisor bar set at - 3.3 mm.

An quisqualic acid infusion (0.12 M, 1.25 (*l, in phosphate buffered saline, pH 7.4) was used to unilaterally lesion the NB (AP: -0.8 mm, DV: -7.7 mm, ML: 2.6 mm relative to the bregma) (n = 5). Five minutes after the infusion the needle was slowly retracted. Control NB-lesioned (n = 5) rats received vehicle infusions of equal volume. Seven rats received no intracerebral infusions. The active recording electrodes (stainless steel screws 0.5 mm in diameter) were symmetrically located on both sides of the skull at the following coordinates: AP: 2.0 mm and - 7.0 mm relative to the bregma, ML: 3.0 mm. The reference electrode and ground electrodes were located in the midline above the cerebellum. The screw electrodes and connected female pins were embedded in dental acrylic. A recovery period lasting 20 days was allowed before recordings were taken. The EEG values of freely moving rats were recorded between 9.00 and 13.00 hr. Two behavioral states were selected for spectral power components: waking-immobility and movement. Five 4-second artefact-free epochs were digitized using a sampling rate of 250 Hz. For the Fast Fourier transformation, squared cosine sections were used. The absolute amplitudes in the following frequency bands were used: delta= l-4 Hz, theta=4-8 Hz, alpha= 8-12, beta= 12-20 Hz. Spike and wave HVSs (3) (6-8 Hz, bilaterally synchronous appearance) were recorded during a 25min period of cumulative waking-immobility. The total duration (mean duration x incidence) was evaluated from the polygraph charts by using a ruler. The EEG values were recorded after a recovery period of 10 days and after the behavioral testing was done (25 days recovery after operations). The water-maze system is described in detail elsewhere (12). The swim paths of the rats in the circular pool were monitored by a videocamera linked to a computer through an image analyser. The computer calculated the total distance swum as well as the path lengths in all quadrants and annuli separately. The timing of the latency was started and ended by an experimenter. Testing consisted of seven consecutive days of training (2 x 80 set trial per day, 10 set reinforcement on the platform and 30 set recovery between trials). Rats that failed to find the platform were placed on it. On the eighth day a spatial probe trial was assessed (platform withdrawn) to measure the spatial bias (the percentage of the total distance swum in the previous training quadrant).

QUISQUALIC

841

ACID NB LESIONING

TABLE 1 REGIONAL CHOLINE ACETYLTRANSFERASE (ChAT) ACTIVITIES Nucleus Basalis-Lesioned (n=5)

Vehicle Controls (n=5)

Brain Area

Frontal cortex Parietooccipital cortex Hippocampus

1.13 + 0.2 0.95 i: 0.3

0.68 2 0.2* 0.56 ? 0.2*

1.45 * 0.3

1.39 2 0.3

Mean 2 S.D. ChAT levels are expressed as nmol/mg *p
I

proteinimin.

The Plexiglas passive avoidance box was divided into a dark and lighted compartment by a metal sliding guillotine door. The dark compartment had a metal grid floor. Rats were placed on the lighted side. After 10 set a door opened into the lighted side. Five set after entry into the dark chamber, a l.O-mA shock was initiated and maintained, until the rat escaped from the dark chamber and until a training criterion of avoiding the dark chamber for a 1-min interval was attained. The latency to enter the dark chamber and the number of reentries were measured. One hour after training, rats were placed in the lighted side of the apparatus and the door was opened. The latency to enter the dark chamber was measured during the retention trial. After decapitation of the intracerebrally infused rats, a coronal slice containing NB tissue was placed into 4% formalin (0.1 M phosphate buffered saline, pH 7.4). After eight hours the tissue was immersed in 30% sucrose. Serial sections were stained with hematoxylin-eosin (H-E) and acetylcholinesterase (AChE) to locate NB lesions. After collecting the histological sample, dorsal frontal neocortex (40-60 mg), parietooccipital cortex (50-75 mg) and hippocampus (60-80 mg) were dissected on ice and these tissue pieces were stored at - 72°C. ChAT activity was measured according to the method of Fonnum (7). RESULTS

Microscopic examination of the H-E- and AChE-stained sections revealed marked gliosis and cell loss in the ventral globus pallidus. The largest (left) and smallest (right) lesions are shown in Fig. 1. On the side of lesions, the NB-lesioned rats had lowered frontal (Mann-Whitney U-test, pcO.05) and occipital (MannWhitney U-test, ~~0.05) ChAT activities than the control NBlesioned. Cortical ChAT activities contralateral to the lesioning and hippocampal ChAT activities remained unchanged (MannWhitney U-test, p>O.O5) (Table 1).

2

3

4

5

6

FIG. 3. Escape latencies (second) during the training trials. The values are expressed as mean?S.D. See group abbreviations from Table 2. The MANOVA test did not reveal any significant differences between the groups. Solid line = controls, bars = nucleus basalis-lesioned, bars + dots = nucleus basalis control-lesioned.

Due to the similar response of the frontal and occipital EEG activity induced by NB lesioning, the frontal and occipital EEG values were pooled for the statistical analysis. Irma-group comparisons revealed no changes in the EEG values during the course of experiment (recordings 10 or 25 days after surgery) (Wilcoxon signed ranks test, p>O.O5 in all comparisons). Thus, the data obtained during the first recording session was used in all statistical analysis. Figure 2 shows frontal EEG values recorded from the side of brain ipsilateral to the intracerebral infusions during wakingimmobility. Movement and waking-immobility-related EEG recordings show that NB lesioning increased EEG slow waves [delta, F(2,33) = 3.2, pcO.05; Duncan, pO.O5; Duncan, p>O.O5]. Beta activity was decreased in NB-lesioned rats, F(2,33) = 3.3, pcO.05. The total duration of HVSs was increased by NB lesions [F(2,33) = 4.7, pcO.05; Duncan, p0.05 vs. controls in all comparisons). The MANOVA test revealed no significant main group effect on the following parameters measuring water-maze performance; escape latency, F(42,196)=0.3, p>O.O5 (Fig. 3), path length, F(42,196)=0.5, p>O.O5 (data not shown) or swim speed, F(42,196) =0.8, p>O.O5 (data not shown). Furthermore, Duncan’s test revealed no significant group differences either in these

250 ~200 T 150

TABLE 2 THE TOTAL DURATION (SECOND) OF HIGH VOLTAGE SPINDLES (HVS) Q)

Group

Total Duration

NB NBC C

145 2 36* 23 ” 13 18 ? 14

Values are expressed as mean f S.D. Abbreviations: NB = nucleus basalis-lesioned, NBC = nucleus basalis control-lesioned, C = controls. *p
t

100

l

250 0t,

NEIL

NBC

PIG. 4. Passive avoidance entry latency (late) retention test. Values are expressed as mean? lesioned rats=dark bar, nucleus basalis controls controls = lightly shaded bars. *p
C (second, s) during the S.D. Nucleus basalis= heavily shaded bar, groups (Duncan’s post

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RIEKKINEN

TABLE 3 PASSIVEAVOIDANCEENTRYLATENCY(SECOND)DURING THE TRAININGTRIAL Entry Latency

Group

24 f 21 30 2 45 22 * 20

NB NBC C

Values are expressed as mean f S.D. See group abbreviations from Table 2. Duncan’s post hoc multiple group comparison did not reveal any significant group differences. parameters (pBO.05 in all comparisons) or spatial bias [F(2,16) = 0.6, p>O.O5; Duncan, pBO.05 in all comparisons] (data not shown). No significant overall group effect was seen in either the latency to enter [F(2,16) =0.6, pBO.05; Duncan, p>O.O5] or number of reentries [F(2,16)=0.4, pBO.05, Duncan, p>O.O5] during the PA training trial (Table 3). However, during the retention trial a significant main group effect on the entry latency was found, F(2,16) = 3.1, pcO.05 (Fig. 4). NB-lesioned rats were impaired (Duncan, pcO.05) compared with the other groups.

DISCUSSION The present results demonstrate that passive avoidance but not water maze acquisition is impaired following unilateral quisqualate NB lesioning. Moreover, cortical EEG slow wave activity and HVSs were increased on the side of lesion. Furthermore, no recovery of EEG activity occurred during the course of experiment (25 days). Our results further support the fact that NB cortical cholinergic system is not significantly involved in spatial navigation (5, 12, 13). It has been proposed that the learning deficits induced by ibotenic acid NB lesioning are not as a consequence of the cholinergic deafferentation of the cortex, but may result from a damage to frontostriatal system, the output of which courses

ET AL.

through the ventral and dorsal globus pallidus (13). Since previous studies have shown that the globus pallidus is damaged by ibotenate infusions, the spatial navigation impairments may result from the accidental lesioning of the frontostriatal system (5, 12, 13). Another possibility is that ibotenate-induced behavioral deficits result from damage to some other neurochemical system in the NB which is spared following quisqualate lesioning (13). We are presently investigating that possibility. Our result further supports the importance of the NB neurons in regulating the passive avoidance acquisition. Since scopolamine injections and cortical lesioning result in similar memory deficits (6). the passive avoidance impairment observed in the present study may result from the loss of cortical cholinergic fibers. Interestingly, the passive avoidance deficits were associated with neocortical electrical arousal deficits (increase of slow waves and HVSs), which further supports the importance of cholinergic deafferentation of the cortex in avoidance performance impairments. However, the contribution from noncholinergic NB neurons damaged to the passive avoidance impairments cannot be excluded. Our neurophysiological results demonstrating EEG slowing induced by restricted quisqualic acid NB lesioning further support the importance of the NB cholinergic neurons in direct cortical activation and suppression of the rhythmic activity of the nucleus reticularis thalamus. Previous studies support our conclusion. It has been shown that scopolamine, a muscarinic antagonist, produced smaller EEG change in ibotenate NB-lesioned rats than in controls (3). Moreover, the EEG slow wave activity correlated with the cortical ChAT activity in NB-lesioned rats. In our recent study we have shown that NB lesioning-induced EEG changes are partially reversed by either muscarinic agonist or cholinesterase inhibitor (10). In conclusion, the present results show for the first time that quisqualic acid NB lesioning abolishes both desynchronized EEG activity and impairs passive avoidance learning. Thus, our results suggest that these deficits may result from the loss of cholinergic neurons in the NB and furthermore that in future pharmacological studies quisqualate NB lesioning may be a suitable preparation for studying the effectiveness of cholinergic replacement therapies.

REFERENCES 1. Armstrong, D. M.; Saper, C. B.; Levey, A. I. Distribution of cholinergic neurons in rat brain: demonstrated by the immunohistochemical localisation of choline acetyltransferase. J. Comp. Neurol. 216:53-68; 1983. 2. Bartus, R. T.; Dean, R. L.; Beer, B.; Lippa, A. S. The cholinergic hypothesis of geriatric memory dysfunction. Science 217408417; 1982. 3. Buzsaki, G.; Bickford, R. G.; Ponomareff, Cl.; Thai, L. J.; Mandel, R.; Gage, F. H. Nucleus basalis and thalamic control of neocortical activity in the freely moving rat. J. Neurosci. 84007-4026; 1988. 4. Dunnett, S. B.; Low, W. C.; Iversen, S. D.; Stenevi, U.; Bjorklund, A. Septal transplants restore maze learning in rats with fomix-fimbria lesions. Brain Res. 251:335-348; 1982. 5. Dunnett, S. B.; Whishaw, I. Q.; Jones, G. H.; Bunch, S. T. Behavioural, biochemical and histochemical effects of different neurotoxic amino acids injected into nucleus basalis magnocellularis of rats. Neuroscience 20:653-669; 1987. 6. Flood, J.; Cherkin, A. Scopolamine effects on memory retention on mice: A model of dementia? Behav. Neural Biol. 45:169-184; 1986. 7. Fonnum, F. A rapid radiochemical method of the determination of choline acetyltransferase. J. Neurochem. 24:407X)9; 1975. 8. Levey, A. I.; Hallonger, A. E.; Wainer, B. H. Cholinergic nucleus basalis neurons may influence the cortex via the thalamus. Neurosci. Lea. 74:7-13; 1987. 9. Lo Conte, G.; Casamenti, F.; Bigl, V.; Milaneschi, E.; Pepeu, G. Effects of magnocellular forebrain nuclei lesions on acetylcholine output from the cerebral cortex, electmcorticogram and behavior.

Arch. Ital. Biol. 120:17&188; 1982. 10. Riekkinen, P., Jr.; JXkalB, P.; Sirvio, J.; Riekkinen, P. Effects of THA on scopolamine and nucleus basalis lesioning induced EEG slowing. Submitted. 11. Riekkinen, P., Jr.; Sin%, J.; Riekkinen, P. J. Relationship between the cortical ChAT content and EEG delta activity. Neurosci. Res.; in press. 12. Riekkinen, P., Jr.; Sirvio, J.; Riekkinen, P. Similar memory impairments found in medial septal-vertical diagonal band of Broca and nucleus basalis lesioned rats: are memory defects induced by nucleus basalis lesions related to the degree of non-specific subcortical cell loss? Behav. Brain Res. 37:81-88; 1990. 13. Robbins, T. W.; Eve&t, B. J.; Marston, H. M.; Wilkinson, J.; Jones, G. H.; Page, K. J. Comparative effects of ibotenic acid and quisqualic acid-induced lesions of the substantia innominata on attentional function in the rat: further implications for the role of the cholinergic neurons of the nucleus basalis in cognitive processes. Behav. Brain Res. 35:221-240; 1989. 14. Soininen, H.; Partanen, J.; Laulumaa, V.; HelkalaE.-L.; Laakso, M.; Riekkinen, P. J. Longitudinal EEG spectral analysis in early stage of Alzheimer’s disease. Electroencephalogr. Clin. Neurophysiol. 72: 290-297; 1989. 15. Stewart, D. J.; MacFabe, D. F.; Vanderwolf, C. H. Cholinergic activation of the electrocorticogram: Role of substantia innominata and effects of atropine and quinuclidinyl benzylate. Brain Res. 322:219-232; 1984.