The role of cortical sympathetic ingrowth in the behavioral effects of nucleus basalis magnocellularis lesions

Brain Research, 474 (1988) 353-358 Elsevier

353

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The role of cortical sympathetic ingrowth in the behavioral effects of nucleus basalis magnocellularis lesions Lindy E. Harrell and Dee S. Parsons Departments of Neurology and Psychology, VeteransAdministration Medical Center and University of Alabama Medical Center, Birmingham, Alabama 35294 ( U.S. A.) (Accepted 2 August 1988) Key words: Nucleus basalis magnocellularis lesion; Learning/memory; Sympathetic nervous system; Neuronal reorganization

Following cholinergic denervation of the neocortex by nucleus basalis magnocellularis (NBM) lesions, peripheral sympathetic fibers grow into the neocortex. Two experiments were performed to determine the behavioral effects of this neuronal rearrangement. Group I animals underwent training on a standard radial-8-arm maze task, while Group II animals learned a modified version (i.e. 4 arms baited). Following acquisition, NBM lesions were performed. Animals with lesions but without sympathetic ingrowth performed consistently better in both behavioral paradigms, than animals with NBM lesions and sympathetic ingrowth. These studies suggest that cortical sympathetic ingrowth can alter behavior and is detrimental to the learning of a spatial memory paradigm. Following cholinergic denervation of the neocortex by nucleus basalis magnocellularis (NBM) lesions 7 or the hippocampal formation by medial septal, fimbria-fornix, or anterior hippocampal 6 lesions, peripheral sympathetic fibers, originating from the superior cervical ganglia, grow into the neocortex and hippocampus. Although a great deal of information is available regarding the anatomical, physiological 6, and behavioral -~'l~-jS'js effects of hippocampal sympathetic ingrowth (HSI), little is known about cortical sympathetic ingrowth (CSI). Recent studies ]'~z1"~'25 have suggested that the NBM is important for learning/memory, as destruction of this area produces deficits in a variety of behavior paradigms employed to assess learning/memory. Since we have previously demonstrated that HSI is detrimental to the recovery of spatial learning paradigms, we were particularly interested in assessing the effect of CSI on this type of behavior. Therefore, we investigated the effect of CSI on reacquisition of both a simple and a complicated spatial memory task. Subjects and experimental groups. Male Charles

River S p r a g u e - D a w l e y rats with an initial weight of 200 g were housed in a temperature- and light- (12 h L:12 h D) controlled colony room. Animals were deprived to 85% of their ad libitum weight with food restriction designed to allow 5 g b. wt. gain per week. Water was continuously available. Prior to habituation, 33 animals were assigned to Group I while 30 animals were assigned to Group II. Those animals in Group I were tested on the standard version of the radial-8-arm maze with all arms baited, while those assigned to Group II underwent testing on a modified version of the radiai-8-arm maze task in which only 4 of the 8 arms were baited. Apparatus. Both groups of animals were tested on a standard wooden, 8-arm maze (the center platform was octagonal in shape, 30 cm in diameter) with arms (75 cm long and 7 cm wide) spaced equidistant around the center platform. The entire apparatus was elevated 100 cm from the floor and was placed in a room with numerous visual cues. Procedure. Both groups of animals received 4 days of habituation to the maze with all arms baited (1/4 of a Kellogg's Froot L o o p cereal). In Group I, acquisi-

Correspondence: L.E. Harrell. Department of Neurology, Jefferson Tower 1210, University of Alabama Medical Center, Birmingham, Alabama 35294, U.S.A. 0006-8993/88/$03.5/) © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

354 tion trials and subsequent behavioral testing were accomplished by placing 1/4 of a Fruit Loop in the recessed food cup at the end of each arm. At the beginning of a trial, the animal was placed on the center platform and allowed to freely explore the maze and collect food rewards. A trial was terminated when the animal had entered each arm and consumed all baits or after 5 minutes had elapsed. After attainment of criterion (see below), animals were randomly assigned to one of 3 surgical conditions: (1) CON (vehicle NBM injection + sham superior cervical ganglionectomy), (2) NBM + Gx (NBM lesion + ganglionectomy), or (3) NBM (NBM lesion + sham ganglionectomy). Because of the aphagia and adipsia associated with NBM lesions animals were maintained by intragastric tube feeding until this resolved (about 4 days). A further 4 days of free feeding was allowed prior to reducing the animals to 85%. of b. wt. and behavioral testing reinitiated. Trials were continued until criterion was reachieved. The learning criterion, both pre- and post-surgery, was defined as visiting and consuming the food in each of the 8 arms, in the first i0 selections over 5 consecutive days. Errors were defined as reentering previously visited baited arms. Group II animals were treated in a similar fashion except that during acquisition trials and subsequent behavioral testing, one-quarter of a Froot Loop was placed in 4 of the arms (baited), while the others remained unbaited. The location of the food was determined by the use of random number tables and remained constant throughout the experiment for a given rat. A trial continued until all 4 baited arms were visited and the food consumed, or until 10 min elapsed. Each rat received one trial a day. Criterion of learning was defined as 4 correct responses (visiting baited arms) in the first 5 choices during a block of 5 consecutive trials. Errors were defined as either visiting unbaited arms or reentering previously visited baited arms. Surgery. All surgery was carried out under ketamine (87 mg/kg) and xylazine (13 mg/kg) anesthesia. Neocortical AChE depletion was induced by infusing ibotenic acid (5 #g, Sigma), mixed with 1 ~1 of phosphate-buffered saline, over 5 rain into the region of the NBM (AP -0.7; ML +_ 2.5; DV -6.2). The needle was left in place for a further 5 rain to prevent diffusion along the needle tract. Vehicle injections (1

,ul phosphate-buffered saline) were made in a similar fashion. Ganglionectomy was performed by making a midline neck-incision, the carotid bifurcation exposed by blunt dissection, and the superior cervical ganglion visualized and removed. Sham animals were treated in a similar fashion except the ganglion was left intact.

Histology. At completion of the experiment the animals were decapitated and the brain removed and frozen in cold 2-methylbutane (-60 °C). Coronal sections (20/~m) were taken in triplet every 100/~m starting at the rostral forebrain and continuing through the temporal cortex. Sections were stained with cresyl violet for Nissl substance, acetylcholinesterase (ACHE), to determine the extent of cholinergic denervation 24, and processed for catecholamine histofluorescence 7, to assess peripheral sympathetic ingrowth. Data analysis. A repeated measures analysis of variance (ANOVA) was used to assess treatment, trial effects, and their interaction on total arm selections, total errors (Group l), and errors to baited and unbaited arms (Group II). A one-way ANOVA followed by Duncan's multiple range test was used to examine the effect of the surgical treatments on number of trials to pre- and post-surgical learning criterion. Anatomical results. Fifteen animals from Group 1 and 11 animals from Group II were excluded from data analysis due to perioperative death. This high incidence of mortality is not uncommon with lesions of the NBM area due to the aphagia and adipsia associated with these lesions. Additionally, another 4 animals from Group I and 6 animals from Group II were excluded due to lesion misplacement. These animals for the most part had lesions which were too dorsal or lateral producing damage in the globus pallidus and/ or amygdala. None of these animals had AChE depletion in the neocortex, nor was sympathetic ingrowth observed. No animals were excluded on behavioral criteria. As shown in Fig. 1 the typical NBM lesion was located slightly ventral to the ventromedial corner of the globus pallidus and in some animals extended to the nucleus preopticus lateralis and/or magnocellularis. The maximum extent of the lesion occurred at about the level of the A6360 plate from the K6nig and Klippei rat atlas 2° and extended about

355 1.5 mm in a rostral-caudal plane. AChE-staining sections revealed loss of AChE-staining magnocellular cells and the AChE fiber plexus in the area of the lesion. There was no difference in lesion Size or location between those animals assigned to the two lesioned groups in either experimental Group. Control animals demonstrated only gliosis around the injection tract. Examination of AChE staining in the neocortex revealed extensive loss of AChE-positive fibers and terminals. This loss was confined to the dorsal-lateral cortex and extended from the prefrontal region to the parietal-temporal cortex with sparing of the cingulate, perirhinal, periform, entorhinal and occipital cortex. There were no major differences in AChE loss between lesion groups. Control groups demonstrated dense AChE staining in the neocortex (Fig.

2A-D). Central and peripheral noradrenergic fibers were easily distinguished on the basis of their appearance, with central fibers demonstrating thin, small varicosities and peripheral fibers demonstrating bright, coarse, thick, knobby varicosities. As expected, animals without lesions had widespread central noradrenergic (NE) innervation of the neocortex, with peripheral NE fibers confined to pial blood vessels (which did not accompany penetrating arterioles), choroid plexus and pineal gland. Those animals within the NBMGx groups demonstrate similar central NE fluorescence as controls, however, peripheral fibers were absent. In animals with NBM lesions and without ganglionectomy two types of adrenergic fibers were observed in the neocortex. The first were small, thin fibers, consistent with center NE fibers, which were observed throughout the neocortex in a

similar distribution as in control animals. The second fiber type was a large, coarse fiber that was similar to those observed on blood vessels, choroid plexus and in the hippocampus after cholinergic denervation of that structure 6. These fibers were seen arising from the pial vessels (Fig. 2E) and penetrating into the neocortex (Fig. 2F). At times they appeared to follow the course of the small penetrating arteries (a phenomenon never seen in control animals). They eventually appeared to ramify in layers IV and V of the neocortex. It was unclear whether synaptic contact was made in these regions. These fibers were scattered diffusely throughout all regions of the neocortex (prefrontal, frontal, parietal, temporal) that were cholinergically denervated, but were never observed in any area with normal AChE staining. Behavior of Group I. Prior to surgery all animals were able to master the task with no statistical differences in number of trials to criterion (Table I). No differences among grouPs were observed in total arm selections or total errors. Analysis of number of trials to achieve criterion following surgical procedures revealed a statistically significant group effect (F = 3.8 (2, 13) P < 0.05). Post-hoc analysis revealed that animals with NBM lesions and ingrowth took significantly longer to reach postoperative criterion than either the NBM + Gx or control group, which were equivalent in behavior (Table I). Total number of arm selections was not effected by the various treatment conditions and remained fairly constant over time (ANOVA). Total number of errors, however, was effected by the various treatments (F = 7.13 (2, 44) P < 0.002) with post-hoc testing demonstrating that the NBM group made signifi-

A Fig. 1. Schematic representation of lesion location and size from an animal with a typical NBM lesion.

t~

357 TABLE I

Mean number (+ S.E.M.) of trials to criterion Presurgery

Postsurgery

10.0 _+ 2.19 11.2 _+ 3.9 11.5 __+4.5

10.2 + 2.8 13.8 + 2.5 34.2 + 12.2

Group 1 CON (n = 5) NBM + Gx (n = 5) NBM(n = 4)

Group H CON (n = 4) NBM + Gx(n = 5) NBM (n = 5)

37.5 ___6.0 41.6_.+7.3 43.4 _+ 8.3

13 + 1.65 31+7.1 59 + 7.5

cantly more number of errors than either the CON or NBM + Gx groups (Duncan P < 0.05). Errors were found to decrease over time (F = 1.97 (14, 44) P < 0.04). Behavior of Group IL All animals were able to master the task. Trials to initial criterion ranged between 19 and 70 with no statistical differences among animals assigned to the various experimental groups (Table I). No differences in total arm selection, total errors, or errors to baited and unbaited arms were observed among groups pre-operatively. Analysis of number of trials to achieve learning criterion following surgical procedures revealed a significant group effect (F = 11.9 (2, 13) P < 0.007). Post-hoc analysis revealed that animals with NBM lesions and ingrowth took significantly longer to achieve criterion, than the NBM group, which in turn took longer than the controls (Duncan P < 0.05). Total arm selections, total errors and errors to unbaited arms were not affected by the various surgical treatments or number of trials. Errors to baited arms were found to be significantly altered by the various treatments (F = 4.69 (2, 73) P < 0.01), with post-hoc testing demonstrating significantly more of these types of errors in the lesion groups than the control group (Duncan P < 0.05). The results of this study suggest that cortical peripheral sympathetic ingrowth, which occurs following lesions of the NBM, has a detrimental effect on

recovery of a spatial learning/memory task. This effect is most likely due to some interaction of ingrowth within the neocortex, as a previous study revealed no effect of isolated ganglionectomy on radial-8-arm maze performance 12. The mechanism by which this effect was mediated is totally unknown. Sympathetic neurons are known to contain both NE and polypeptides with opiate-like immunoreactivity 6'9. Since both NE 18 and opiates 2 have been implicated in normal learning/memory, perhaps release of these substances into the neocortex during ingrowth altered behavior. The factors which mediate, control, and/or induce cortical sympathetic ingrowth are unknown. However, since the cholinergic neurons which form the NBM and medial septum are basically part of the same basal forebrain cholinergic neuronal population with just different terminal projections (i.e. neocortex and hippocampus, respectively), it seems reasonable to assume that CSI is regulated by the same factors as HSI 6. Although HSI has been reported to be facilitory for behavioral recovery by other investigators 5,~s, in our studies 13'14 both CSI and HSI appear to affect recovery of a spatial learning/memory task in a detrimental fashion. Whether CSI will alter other types of learning/memory or will be similar to HSI, where no effects have been observed on passive 15 or active avoidance (unpublished observation) learning, remains to be determined. In agreement with previous investigations 1'16'1725, we found that NBM lesions disrupted retention of a spatial memory paradigm. Analysis of data from our Group II animals suggested that this arose secondary to deficits in working (trial-dependent) memory as errors were directed toward the re-entry of previously baited arms. This finding is in direct conflict with the studies of Murray and Fibigera2,23 which have suggested that NBM lesions typically produce deficits in reference (trial-independent) memory (i.e. visiting unbait arms), but in agreement with other investigators t6't739, who have found deficits only in working memory.

Fig. 2. Photomicrographs of normal cortical AChE staining (A and B), cortical AChE staining following NBM lesions (C and D) and cortical sympathetic ingrowth (E and F). The black boxes in A and C indicate where higher power photomicrographs were obtained. In E cortical sympathetic ingrowth is delineated by the central white arrow. Note that this fiber has the same characteristics as those surrounding the pial blood vessels (bv, marked by the other white arrow). Central NE fibers are delineated by the open black arrow. cc, corpus callosum; cn, caudate nucleus; bv, blood vessel. Magnification: A and C ×4; B and D ><20; E ×4; F ×120.

358 It is interesting to note that the postsurgical learning of the NBM + Gx animals in G r o u p 1 was not impaired. This was not explained by differences in lesion size or cholinergic loss (as determined by A C h E stains), as these were similar between NBM + Gx an-

In summary, our data suggest that CS1 can alter behavior in a detrimental m a n n e r . These results may have clinical relevance as this same type of neuronal reorganization occurs in Alzheimer's disease 3'4. Its role, however, in the clinical pathology is unclear.

imals assigned to Group I and Group II. Perhaps this finding relates to the complexity/difficulty of the two paradigms, as previous studies I"1~'have demonstrated

We thank Joanne Cage and Nancy Hodges for sec-

that by stressing the ' m e m o r y ' of 'recovered' NBM lesioned animals clinical deficits once again emerge.

retarial assistance. Work support by VA Merit Review (LEH).

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