Dissociation of spatial navigation and visual guidance performance in Purkinje cell degeneration (pcd) mutant mice

Behavioural Brain Research, 47 (1992) 129-141 9 1992 Elsevier Science Publishers B.V. All rights reserved. 0166-4328/92[$05.00

129

BBR 01265

Dissociation of spatial navigation and visual guidance performance in Purkinje cell degeneration (pod) mutant mice Charles R. Goodlett, Kristin M. Hamre and James R. West Alcohol and Brahl Research Laboratory, Department of Anatomy, College of Medichre, University of lowa, Iowa City, IA 52242 (USA) (Received 13 February 1991) (Revised version received 9 December 1991) (Accepted 10 December 1991)

Key words: Cerebellum; Hippoeampus; Purkinje cell degeneration; pcd; Neurological mutant; Spatial navigation; Spatial learning

Spatial learning in rodents requires normal functioning ofhippocampal and cortical structures. Recent data suggest that the cerebellum may also be essential. Neurological mutant mice with dysgenesis of the cerebellum provide useful models to examine the effects of abnormal cerebellar function. Mice with one such mutation, Purkinje cell degeneration (pcd), in which Purkinje cells degenerate between the third and fourth postnatal weeks, were evaluated for performance of spatial navigation learning and visual guidance learning in the Morris maze swim-escape task. Unaffected littermates and C57BL/6J mice served as controls. Separate groups ofpcd and control mice Were tested at 30, 50 and 110 days of age. At all ages, pcd mice had severe deficits in distal-cue (spatial) navigation, failing to decrease path lengths over training and failing to express appropriate spatial biases on probe trials. On the proximal-cue (visual guidance) task, whenever performance differences between groups did occur, they were limited to the initial trials. The ability of the pcd mice to perform the proximal-cue but not the distal-cue task indicates that the massive spatial navigation deficit was not due simply to motor dysfunction. Histological evaluations confirmed that thepcd mutation resulted in Purkinje cell loss without significant depletion of cells in the hippocampal formation. These data provide further evidence that the cerebellum is vital for the expression of behavior directed by spatial cognitive processes.

INTRODUCTION

Efficient spatial performance in rodents is known to depend upon the normal function of the hippocampal formation 36"45"47"48"s4'55, septal region t4.57 and entorhinal cortex ~L53. In addition, damage to related cortical regions, including the medial frontal cortex18A9"42"54 and parietal cortex s'~9, also impairs spatial learning. While much attention has been directed toward the forebrain structures involved in spatial learning, evidence from rodent studies is accumulating which suggests that the cerebellum also serves a critical role in spatial performance, which extends beyond postural control or motor coordination. Work by Altman and his collaborators, which examined the effects of various types of cerebellar cell loss induced by early postnatal exposure to X-irradiation, indicated that maze performance based on spatial cues was severely affected 49. More recently, Goldowitz and K o c h 9 found that the staggerer neurological mutant, which has severe cerebel-

Correspondence: Charles R. Goodlett, Department of Anatomy, College of Medicine, University of Iowa, Iowa City, IA 52242, USA.

lar dysgenesis 3t without hippocampal pathology, failed to acquire a radial arm maze task. Several reports by Lalonde and colleagues have shown that different neurological mutant mice, including the staggerer, weaver, Purkinje cell degeneration, nervous and htrcher mutants, each of which has cerebellar defects resulting from different mechanisms, show impairments in a variety of tasks which incorporate spatial learning or memory, including spontaneous alternation 22"~3-~5-29 and maze navigation 23,24. The above studies with neurological mutants did not evaluate the extent to which spatial learning deficits could be dissociated from performance on other types of learning tasks. With the renewed interest in cerebellar contributions to l e a r n i n g 16"33"34"6~ including potential contributions to human cognitive processes a'3'6"32, it is important to establish whether cerebellar dysfunction disrupts specific types of learning, i.e. whether the deficits can be dissociated on the basis of the type of learning required. It is also important to determine whether performance deficits are secondary consequences of generalized motor dysfunction. One such dissociation commonly used in recent years in rodents is the water maze navigation task devised by

130 Morris 35"37. In this task, spatial navigation performance requiring the integration of multiple distal cues (considered to involve 'spatial mapping' strategies to find a hidden escape platform) can be distinguished from performance based on local guidance cues (involving 'taxis' strategies to approach a visible platform). Lesions or pharmacological disruptions of the septo-hippocampal system 14"38"39"46"55"56, entorhinal cortex 53, parietal cortex 5.19, medial frontal cortex 19"4z'54, or massive decortication 6z impair spatial mapping performance, but spare proximal-cue visual guidance performance. These selective deficits in distal-cue navigation have been characterized as revealing disruption of neural components necessary for the generation or utilization of representations of the spatial organization of the environment 36"4s. Perhaps due to the emphasis on forebrain substrates of spatial mapping, the cerebellum has not been considered as a component of the neural substrates of spatial mapping. The two versions of the Morris maze task have not been directly tested in neurological mutant mice, despite the deficits found in recent studies using other tests of spatial performance. Although the Morris maze was initially devised to test spatial navigation of rats, it has been used to identify spatial performance differences between inbred strains of mice 58. Thus, we evaluated the Purkinje cell degeneration (pcd) mutant, which loses nearly all Purkinje cells between the third and fourth postnatal weeks 3~ for acquisition of swim-escape navigation learning of both the visible and hidden platform versions in the Morris maze task. Other neural degenerative effects also occur in pcd mice over a much slower time course, including neurochemical changes in the deep cerebellar nuclei in response to terminal degeneration s~ loss of neurons in the inferior olivary complex 8, secondary degeneration of cerebellar granule cells 7 and of certain thalamic nuclei43, and retinal degeneration4~ Therefore, different groups of mice were tested at three different ages (30, 50 and 110 days) to evaluate whether behavioral profiles on the two versions of the Morris maze changed with increasing time following the primary Purkinje cell loss. In addition, the Purkinje cell loss was confirmed histologically, and cells in the hippocampus and dentate gyrus were quantified to determine whether any previously unreported secondary neuronal depletion may occur in the hippocampal formation.

MATERIALS AND METHODS

Subjects The pcd mice are homozygous for an autosomal recessive mutation (see Mullen, Eicher and Sidman 4~),

which has been backcrossed to the C57BL/6J strain. The mutants are maintained through heterozygous (+ ]pod) matings. Unaffected littermate controls have at least one wild-type allele and are designated + ]?. All mice were obtained from Jackson Laboratories (Bar Harbor, ME). The mice were group-housed with littermates with ad libitum access to food and water throughout the experiment. The vivarium was kept on a 12 h : 12 h light/dark cycle.

Behavioral testing The behavioral testing was conducted in a water tank located in a room (4.7 • 2.4 m) separate from the vivarium. Constant visual cues were provided by items placed in fixed locations at various heights around the outside ofthe tank, many within 25 cm of the tank. The tank was circular (122cm diameter, 6 1 c m deep), painted white and filled to within 15 cm of the top with 25 ~ water. Powdered milk was added to the water, rendering it opaque. A Plexiglas platform (11 x I2 cm) covered with gray fiberglass screen was used during the visible platform portion of the task, with the platform extending 2.5 cm above the water level. During the hidden platform portion, the fiberglass screen was removed and the platform was submerged 1 cm below the surface of the water. Visible platform training. A schedule was devised in which both the platform location (any point within the tank) and the starting point (any point around the perimeter) were randomized across trials. The schedules of designated platform locations and starting points across trials were the same for all mice within a given age group. On each visible platform trial, the platform was properly positioned and the subject was placed in the tank facing the wall and allowed to swim until escaping onto the platform, or until a maximum of 45 s had elapsed. Ifa mouse failed to reach the platform within 45 s, it was placed on the platform by hand. The path taken by the mouse was traced by hand onto a scaled diagram, along with the latency to escape. The path lengths were determined later using computerassisted planar morphometry (SMI, Inc.). The mice were allowed to remain on the platform for 10 s before being removed and returned to an incubator (30 ~ where they remained during the intertrial interval. If a mouse left the platform before the 10 s elapsed, it was returned by hand to the platform. The mice were tested in squads of 4-5, which resulted in an intertrial interval of approximately four minutes. At the end of testing each day, the mice were kept in the incubators until dry (about 10 rain) before being returned to the vivarium. Hidden platform training. The platform was placed in a fixed location in the center of one of the quadrants of

131 the tank, I cm beneath the surface of the water. Over blocks of four trials, the mice were started from each of four compass points around the tank (N, S, E, W), determined by a random schedule. The absence of local guidance cues in this condition required the use of distal cues to locate the hidden platform efficiently35. Otherwise, testing procedures were the same as for the visible platform condition, including a maximum of 45 s for each trial. After the last trial on the hidden platform problem, the mice were given a 30 s (30-day-olds) or a 45 s (50- and 110-day-olds) 'probe' trial, in which the platform was removed from the tank and the path taken and the time spent in the quadrant that had contained the platform were recorded by two observers. For the probe trial, the mice were started in the quadrant opposite that which had contained the escape platform. The dependent measures analyzed from the probe trials included the per cent of time spent in the target quadrant, plus the number of crossings of the platform area (target quadrant) and crossings of comparable areas of the other three quadrants (non-target quadrants).

problem (distal cues), with four, eight and eight trials per day, respectively. On the seventh test day, the first four trials were on the hidden platform problem, and the last four trials were on the visible platform problem. Between the last hidden platform trial and the first subsequent visible platform trial on the last test day, a 45 s probe trial was performed.

30-day groups

Histology

Four litters (in which 1/4 oftheoffspring are expected to be homozygous pod mice) were obtained from Jackson Laboratories when the mice were 25 days old, and testing of subjects (5-7 mice per litter) began when they were 29-31 days old. These mice were tested in the Morris maze to determine whether behavioral effects were present at a time when the Purkinje cell degeneration was nearly complete but when the secondary degeneration was only in its initial stages. They were given two days of visible platform training with 12 trials per day, followed by three days of hidden platform training (also 12 trials per day). On the sixth training day, the mice were given six trials on the hidden platform followed by the 30 s probe trial. The mice were tested in squads offour. In an attempt to limit the effects of fatigue at this age, a 5 min interval (spent in the incubator) was interposed between sets of 6 trials on the first 5 training days.

The mice in the 50-day groups were used for cell counts. All mice were perfused intracardially at 60 + 3 days of age with 0.9~o saline followed by a fixative of 1~o (w/v) paraformaldehyde and 1.25~ (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed and weights of the whole brain, forebrain, brainstem and cerebellum were obtained. The brains were then post-fixed for at least one week in additional fixative. For paraffin embedding, the brains were incubated in a series of increasing concentrations of ethanol, followed by butanol and finally into several changes of paraffin. The cerebellum was embedded to be cut in the sagittal plane, while the forebrain was cut in the horizontal plane. Sections were cut at 5/lm, and every fifth section was saved and mounted on slides. After drying, the sections were stained with Cresyl violet, dehydrated and coverslipped. Cell counts and diameters were measured using an image analysis system (SMI, Inc.) interfaced with an IBM-AT. A mid-line vermal section of the cerebellum was used to count all Purkinje cells in each of the lobules. Since the boundary between lobules I and II and that between lobules IV and V were arbitrary, these lobules were counted together. In the forebrain, hippocampal pyramidal cells were counted from the midtemporal horizontal section that was 25 itm below the last section in which the ventral hippocampal commissure could be Seen. Pyramidal cells were counted in CA4, CA2/3 and CAI (see Fig. 1C and D for demar-

50-day groups Six male pcd and six male littermate controls ( + / ? ) from four litters, and six male C57BL/6J mice from three litters were obtained from Jackson Laboratories. The mice were 4-6 weeks old upon arrival. The m i c e ranged in age from 48-55 days at the start of testing, which lasted a total of seven days. On the first three days, training was given on the visible platform problem (proximal cue), using four trials on the first day and eight trials on the next two. On test days four, five and six, the mice were given training on the hidden platform

1lO-day groups The mice were 4-5 weeks old upon arrival (n = 4 males each of pcd, +/?, and C57BL/6J), and were group housed in the vivarium until testing began when the mice were 110 days old ( + 5 days). Behavioral testing was similar to that of the 50-day groups, with the following exceptions. Three days of initial visible platform testing were given, with eight trials per day. This was followed by five days of testing using the hidden platform, also with 8 trials per day. The ninth testing day included four trials on the hidden problem, followed by a 45 s probe trial, and finishing with four trials on the visible platform problem.

132 cation). Cell diameters of each of these regions were also obtained. In addition, densities of granule cells of the dentate gyrus were obtained from the same histological section. Using a 100 x oil-immersion objective, the number of granule cells were counted in a 30.2 pm x 33.1 pm box (1000 pm2). For each subject, densities were obtained from a total of five locations along the granule cell layer of the dentate gyrus, and averaged to give a mean density for each mouse. The cerebella of mice tested at other ages were examined to confirm that the pcd and + / ? animals were properly classified. Animals were perfused using the same protocol as previously described. Brains were post-fixed in fixative plus 3 0 ~ sucrose. Sections were cut at 40 Fm using a freezing microtome, mounted and stained with Cresyl violet, and examined for the presence of Purkinje cells. Statistics

Latencies and distances in the Morris maze tasks were highly correlated within groups for all groups at all ages (r's ranged from 0.85 to 0.99), so only distances will be reported here. The data were averaged over blocks of four trials for the 50- and 110-day groups, or over blocks of six trials for the 30-day groups. For the 30-day group, data from littermate controls were averaged within litters and analyzed as a single observation per litter (5-7 individuals in each litter). In all other cases, the individual observation was the unit of analysis. The data for each platform condition (visible or hidden) were analyzed using separate repeated measures analyses of variance (ANOVAs), with group as a between-group factor and trial block as a repeated factor. Specific post-hoe comparisons between groups were performed using Newman-Keuls tests (c~ = 0.05). In order to assess statistically the potential interactions of group and training condition, repeated measures ANOVAs were conducted on matched trial blocks of the visible and hidden training condition, with condition and trial block as repeated factors. From the probe trial records, the number of crossings of the platform area in the target quadrant and the number of crosses of an equivalent area in each of the other three quadrants were determined. The number of target area crossings, non-target area crossings, and the proportion of target area crossings to total crossings were analyzed using one-way ANOVAs; specific comparisons between the pcd group and controls used least significant difference tests. The proportion oftime spent in the target quadrant was analyzed in the same way. Cell counts from the four counts from the hippocampal formation of each subject (CA4, CA2/3, CA 1, and the dentate gyrus) were analyzed with a multi-

variate ANOVA with group as the between-groups factor. The counts from each region were also evaluated with separate univariate ANOVAs. Total Purkinje cell counts for the C57BL/6J and littermate controls were analyzed with a univariate ANOVA; counts as a function of lobule were analyzed for these two groups with a MANOVA. The distributions of cell diameters in the three groups were analyzed with a 7.2. All parametric statistics were performed using the mainframe BMDP statistical package.

RESULTS Brahl weight and cell counts The pcd mice had lower body weights at each age

(Table I). The absolute weights of forebrain, cerebellum, and brainstem were also significantly lower than littermate ( + / ? ) controls at each age. Nissl-stained sections of the cerebellum confirmed that all pcd mice at each age had near total loss of Purkinje cells (Fig. 1A TABLE 1 Mean (+ S.E.M.) body and brain weights of the mice at each age Group

NI

Body (g)

Forebrain Ong)

Cerebelhml+ Brainstem O,,g)

pcd

4

+/?

4

9.4** +_0.7 15.3 +_0.4

228** +7 270 _+3

71"* +3 86 +_2

17.2'* +0.5 23.9 ## +-0.4 20.6 +-0.2

288* +-6 318'~ _+7 300 +-5

63** +2 89 +-2 84 +-2

17.9'* +-0.2 26.4## +-0.4 24.1 +0.3

253** +-4 298 _+2 292 +5

61"* +_3 91 ' ~ +-2 82 +-2

30 Day

50 Day pcd

6

+/?

6

C57BL]6J

6

110 Day ped

4

+/?

4

C57BL/6J

4

Group numbers for +/? at 30 days represent the numberof litters, since observations of littermate controls were averaged within litters (5-7 subjects in each litter). At the other two ages the group numbers indicate the number of individuals. ** pcdwas significantlydifferentfrom + / - (30 day)or both control groups (50 and 110), P < 0.01. # # + / - was significantlydifferentfrom C57BL/6J,P < 0.01. * pcd was significantlydifferentfrom + / - , P < 0.01. ~' + [- was significantlydifferentfrom C57BL/6J,P < 0.05.

133

":5

9

....~

'~ .... " " :

"

'"

Fig. 1. A and B: micrographs ofsagittal sections oflobule I of the cerebellum of a littermate control (A) and aped mouse (B) from the 50-day groups. M, molecular layer; G, granular layer. The large arrows in A denote Purkinje cells. Note the absence of Purkinje cells in B. Mag. bar = 25 lira. C and D; micrographs of horizontal sections through the hippoeampus from a littermate control (C) arid apcd mouse (D). The CA fields (where pyramidal cells were counted) are indicated. CA4 is outlined bydashed lines; the CA2]3-CAI border is labeIIed by the small arrow; the large arrowhead denotes the CAl-subicular border9 Bar = 250/zm.

134 T A B L E ll

Mean (+- S.E.M.) cell counts in the different regions of the hippocampal formation I and in different lobules of the cerebellum2

b,

Hippoeampal formation Region

C57BL/6J

+/?

CAI CA3 CA4 DG

359.1 326.6 112.5 18.7

372 310 102.8 19.8

+ 20.3 + 17.7 + 9.1 + 1.0

pcd + 20.3 + 28.1 + 8.2 +_ 0.6

306.6 363.8 136.2 19.4

_+ 31.0 _+ 19.6 _+ 10.1 + 1.1

A

Cerebellum Lobules

C57BL/6J

+/?

I & II III IV & V VI VII VIII IX X

57.0+ 64.7 + 103.8 + 71.2 + 21.8 + 43.5 + 82.0 + 41.3 +

49.2_+ 64.6_+ 104.4 + 69.4 + 18.2 + 44.4 +_ 77.0 + 38.2 +

5.9 3.7 3.0 4.2 1.9 2.8 3.3 2.0

pcd 5.8 7.6 5.6 6.2 1.2 3.7 7.7 2.8

' Counts for CAI and CA213 are the mean number of pyramidal cells in each region; CA4 counts are the mean number ofneurons (multiple cell types) in the hilus (see Fig. 1); D G counts are the mean densities of granule cells (per 1000 Fm 2) in the dentate gyrus. All counts were obtained from single, 5-Fm-thick horizontal sections through the mid-temporal hippocampal formation. There were no significant group differences on any measure. 2 Counts for each eerebellar lobule are the mean number of Purkinje cells in a single, 5-Fm-thick mid-sagittal section through the vermis. There were no significant differences between C57BL/6J and + [ - mice in any lobule.

and B). There were no significant differences in the number of Purkinje cells between the + / ? and C57BL/6J mice (Table II). In the hippocampus (Fig. 1C and D), the multivariate ANOVA revealed no significant differences in cell numbers among the three groups for the three hippocampal subfields nor in granule cell density in the dentate gyrus (Table II). Furthermore, the lower CA 1 counts of the pod mice did not approach statistical significance. The only region for which the counts approached significance (CA4, P < 0.059) was one in which the pod mice had higher numbers of cells. The distributions of cell diameters also did not differ among groups. Motor behavior Thepcd mice were readily identifiable by their ataxia. Despite their gait abnormalities, swimming ability was surprisingly competent (Fig. 2). The pcd mice could at times adopt normal swimming posture comparable to littermate controls, with their head above the water,

B .4

"

''J

.-

. G-

;,

g

C Fig. 2. Swimming posture of the mice from the 50-day groups. A: typical swimming posture of + / ? mouse with forepaws inhibited and tail underwater. B: similar posture in aped mouse. Thepcdmice were unable to maintain this posture for extended periods of time. They often changed to the 'dog-paddle' posture shown in C, in which the forepaws were disinhibited and used in swimming, and the tail thrashed above the water.

forepaws inhibited, propelling with alternating hind limb kicks and using the tail in the water. Nevertheless, unlike controls, they did not maintain this swimming pattern for long distances, and frequently broke into a 'dog-paddle' (Fig. 2C), in which they used both the forelimbs and hindlimbs for swimming. Morris maze - 30 days As shown in Fig. 3, the distances travelled to reach the visible platform decreased with training for both groups [F3.~8 = 37.3, P < 0.001]. The pod group did not differ from littermate controls in this respect, and neither the main effect of group nor the group x trial block interaction approached significance. Thus, the improvement in performance on the proximal-cue task was comparable between groups. In contrast, acquisition performance on the hidden platform task did differentiate the pod mice from littermate controls. There was a significant reduction in path lengths over trial blocks [/76.36 = 3.02, P < 0.01], pre-

135 1000'

30 DAYS OLD

900

:'

800 E

r

700

uJ o z I--

6OO

z IJJ

.....

+/?

/

500" 400

,

xxx

/

/

/

3002 I

200"-

Im

100 0

V'I

V'2

V'3

V'4

H'I

H'2

H'3

VISIBLE

H'4

H'5

H'6

H'7

HIDDEN TRIAL BLOCK

Fig. 3. The mean path length distances (in cm) averagedfor blocks of six trials from the 30-daygroups. The mice were tested on the visible platform problem(24 trials) and the hidden platformproblem (42 trials). The acquisitionof navigationperformanceofpcdmice is comparable to control levels on the visible platform problem, but severe deficits are apparent on the hidden platform problem. dominantly due to the improvement of the controls over training. The pcd group had longer path lengths on all trial blocks and did not significantly reduce their path lengths over trials, yielding a significant main effect of group [ F ] , 6 = 41.1, P < 0 . 0 0 1 ] . The group x trial block interaction did not approach statistical significance, in part because the controls at this age did not reach asymptotic performance levels. Nevertheless, the controls had significantly shorter path lengths than pears on each of the last four trial blocks of hidden platform training (P < 0.05). In keeping with the incomplete acquisition ofefficient performance on the distal-cue problem of the controls, the probe trial search strategies of the 30-day-old + / ? mice were not as localized to the target quadrant as for controls at older ages (Table III). Nevertheless, the probe trials still confirmed significant group differences at this age in the degree of spatial bias in the search patterns (Table III). Thepcd mice crossed the platform area significantly less frequently than + / ? controls, both in terms of absolute number [ F l , 6 = 10.57, P < 0 . 0 5 ] and per cent of all area crossings [ F l , 6 = 13.39, P < 0.05]. The two groups did not differ significantly in the number of non-target area crossings. The lack of an appropriate spatial bias of the pcd group was also reflected in the significant group differences in the per cent of time spent in the target quadrant [ F I , 6 = 32.08, P < 0.01].

Morris maze - 50 days For the C57BL/6J mice and littermate controls ( +/?), the distances travelled to reach the escape platform declined with training for both the visible and hidden platform conditions (Fig. 4). In contrast, for the pcdmice, the distances decreased over trials only for the visible platform training; the pcd mice failed to acquire the hidden platform problem. The ANOVA on the five blocks of visible training indicated significant effects of group [F2.15 = 21.16, P < 0.001], trial block [F4.6o = 108.66, P < 0.001] and a group x trial block interaction [F8,6o= 5.62, P < 0.001 ]. The pcd mice took significantly (P < 0.05) longer paths to the visible platform (relative to littermate controls) on the first four trial blocks. However, by the fifth visible trial block, and on the visible block following the hidden training, the pcd mice were not significantly different from littermate controls. The ANOVA on the six trial blocks of the hidden training also yielded a significant group x trial block interaction [ F l o , 7 5 = 2.33, P < 0.05] in addition to the main effects of group [F2.~5 = 12.20, P < 0.001] and trial block [F5.75 = 3.87, P < 0.01]. However, in contrast to the visible condition, the pcd mice failed to reduce their path lengths with training, and the pcd distances were significantly longer than controls from the third through the last trial block. The differential rate of improvement of the two

136 TABLE III

Probe trial performance I Group

Platform area crossings

Time

Target (TGT)

Non- TGT

% Thru TGT (TGT/Total)

% hz Target Quadrant

0.3_+0.3" 2.5-+0.6

3.8-+0.7 2.6+0.2

8+ 46+

8* 6

6 + 3* 29-+ 3

!.3 + 0.8* 3.8+0.5 4.7 + 0.6

4.3 _+ 0.8 4.7_+0.6 5.3 + 0.7

21 + 12' 46+ 6 47 -+ 5

16 + 8* 36+ 3 39 + 3

0.8 + 0.7* 3.5+0.9 4.8 + I

4.5 + 0.5 3.5+0.3 3.8 + 1.4

11 + I1" 48+ 5 58 + 14

I1 _+ 10' 40+ 4 45 + 6

30 Day

pcd +/? 50 Day

pcd +/? C57BL/6J i 10 Day

pcd +/? C57BL/6J

Data for the platform area crosses are the mean (+ S.E.M.) number of crosses of the platform area of the correct quadrant (Target) and the sum of the number of crosses through equivalent areas in the other three quandrants (Non-target); the percent of all area crosses that were through the target area is given in the third column. The per cent of the total probe trial time that was spent in the target quadrant is shown in the last column. Abbreviations: TGT, target quadrant, i.e. the one that had contained the platform; NON-TGT, the other three quadrants. * Significantly lower than the +/? group (30-day) or than both control groups (50-day and 110-day groups) (P < 0.05).

50 DAYS OLD

10001

9

:1 Eo

7001

I.U Oc,.

600i

,r162 Ir - .

r

z,,:r ILl

--,-o--.

pod

I [

+/?

..... 9&"..... C57BIJ6J

I

500: 4001 ',, ".,

300i

~

200!

100i V'I

V'2

V'3

VISIBLE

V'4

V'5

H'I

H'2

H'3

H'4

HIDDEN

H'5

H'6

V'F

VISIBLE

TRIAL BLOCK Fig. 4. The mean path length distance (in cm) averaged across blocks of four trials for the animals in the 50 day groups. The testing consisted of the visible platform problem (20 trials), the hidden platform problem (24 trials) followed by a final visible platform portion (4 trials). Note the failure of the ped group to improve on the hidden problem of the task, while approaching control levels on the visible training.

137 groups as a function of task was confirmed by the repeated measures ANOVA on the first five trial blocks of the two training conditions. There was a significant group • task • trial block interaction [F8,6o = 3.84, P < 0.001] and condition • trial block interaction [F4.6o = 4.03, P < 0.01], along with the significant main effects of group [F2.~5 = 16.87, P < 0.001], condition [F~.~5 = 80.32, P < 0.001] and trial block [F4.6o -- 30.20, P < 0.001 ]. The three-way interaction confirmed that the performance deficits of the pod mice depended upon the type of learning required. Thus, the failure of the pcd mice to improve on the hidden task was not simply a function of motor impairment. The probe trials conducted after the hidden platform task at this age (Table III) confirmed significant group differences in the acquisition of place learning. There were significant differences in the platform area crossings in the target quadrant [F2,~5 = 7.62, P < 0.01] and per cent of time spent in the target quadrant [F2,~5 -- 5.88, P < 0.05]. Thepcdmice crossed the platform area significantly less often than either the + ]? controls or the C57BL/6J controls (P < 0.05), and thepcd mice spent less time in the appropriate quadrant (P < 0.05). The groups did not differ significantly in the number of non-target area crossings. In summary, at 50 days of age, the pod mice learned to perform the visible platform task but failed to acquire the hidden platform task. The deficits in acquisition of the distal-cue task were accompanied by a failure to express spatially biased search strategies in the probe trials. This dissociation of performance on the two tasks is succinctly illustrated by examples of performance on the last hidden platform trial and the subsequent visible platform trial (Fig. 5).

Morris maze - 110 days As with the 30- and 50-day groups, the pcd mice at this age reached control levels of performance by the end of visible platform training, but failed to acquire the hidden platform problem (Fig. 6). This was the case even though more extended training was used at this age. Analysis of the visible platform distance data yielded a significant effect of group [Fz.9 = 4.46, P < 0.05] and trial block [F5.45 = 32.08, P < 0.001]. The path lengths of the pod mice differed from the + [? and the C57BL/6J mice only on the third trial block of visible training, and were not significantly different from controls on the first two or the last three training blocks. The groups also did not differ significantly on the final block of visible platform training that followed hidden platform training. For the hidden platform training, the repeated meas-

4-I?

N

N

pcd

N

N

Fig. 5. Examples of paths taken by a +/? mouse (left panel) and a

pod mouse (right panel) from the 50-day groups. The examples were from the last trial on the hidden platform problem and the first trial on the final visible platform problem. The time (in seconds) as well as the distance travelled (in era) is given.

ures ANOVA for the 11 trial blocks confirmed a significant group • trial block interaction [Fzo,9o = 2.13, P < 0.01], in addition to the main effect of group [Fz,9 = 14.34, P < 0.01] and trial block [Flo,9 o = 4.73, P < 0.001]. Between-group comparisons indicated that the pod mice had significantly longer path lengths than either control group on all but the first four blocks and the eighth trial block of hidden platform training. The repeated measures ANOVA on the first six trial blocks ofthe two training conditions (visible and hidden platform) yielded a significant group • condition interaction [F2.9 = 5.59, P < 0.05], along with the significant main effects of group [/72,9 = 7.80, P < 0.05], condition [ F I , 9 = 105.44, P < 0.001] and trial block [F5.45 = 13.10, P < 0.001]. Thus, as for the other two ages, spatial navigation performance of the pod mice was profoundly deficient, while visual guidance performance was not as severely affected. Probe trials at the end of the hidden training again revealed the absence of appropriate search strategies in the target area by the pcd mice, while controls had significant preferences for the target quadrant (Table III). This was confirmed by significant group differences in the number [Fz.9 = 5.29, P < 0.05] and proportion [F2.9 = 5.61, P < 0.05] of platform area crossings in the target quadrant, as well as for the per cent of time spent in the target quadrant [F2.9 = 6.49, P < 0.05]. The pcd group was significantly lower on each measure of target quadrant preference (P < 0.05). The groups did not differ significantly in terms of the number of non-target area crossings.

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TRIAL BLOCK Fig. 6. The mean path length distances (in cm) averaged for blocks of four trials for the mice from the 110-day groups. The mice were tested on the visible platform problem (24 trials), the hidden platform problem (44 trials) and final visible training (4 trials). Notice the improvement of the pcd mice on the visible platform task, while no significant improvement was observed on the hidden platform task.

DISCUSSION

This study demonstrated that pcd neurological mutant mice failed to acquire efficient behavior in the distal-cue version of the Morris maze navigation task. Littermate ( + / ? ) and C57BL/6J controls reliably decreased path lengths over training on the distal-cue problem and showed appropriate place preferences on the probe trials. When the platform was visible and served as a guidance cue, the pcd mice could reach performance levels comparable to controls. This dissociation between spatial navigation and visual guidance inpcdmice was present at all three ages tested, and the behavioral profiles did not change with age. The deficits at 30 days of age were particularly meaningful, since this is the age at which most of the Purkinje cells had degenerated but before the secondary neural degeneration had substantially progressed. Furthermore, the neurological mutants at 60 days had virtually complete loss of cerebellar Purkinje cells, but no pyramidal cells loss was evident in a midtemporal section of the hippocampus. Thus, the spatial navigation deficits of the pcd mice were not simply a function of motor or visual sensory deficits, nor could

they be attributed to any secondary loss ofhippocampal pyramidal or granule cells. Interpretation of the behavior ofpcd mice is complicated by the secondary degeneration found in other regions of their central nervous system. For example, mitral cells in the olfactory bulb also show a slow pattern of degeneration that begins at approximately 2 months of age 13. An assortment of thalamic nuclei, including the central portion of the medial dorsal nucleus (MD), the ventral medial geniculate, ventrolateral and posteromedial nuclei, also undergo neuronal degeneration43. The degeneration of the MD nucleus may be relevant to this task, given its connections to the prefrontal cortex 2~, though lesion studies in rats suggest MD is not a crucial component of spatial navigation 2~ Nevertheless, since evidence of degenerating cells is not observed until 50-60 days of age43, the deficits ofpcd's at 30 days could not be a result of these sources of secondary degeneration. The pcd mutation also produces slow, progressive degeneration of photoreceptor cells, beginning at approximately 3-4 weeks of age. However, the number of photoreceptors is near normal at 30 days, and is decreased by approximately 3 0 ~ and 5 0 ~ at 60 and

139 110 days, respectively4~ With respect to this retinal degeneration, the dissociation between performance on the proximal-cue versus the distal-cue condition at all ages indicates that visual capabilities were sufficient to guide behavior when the platform extended above the water. Attributing the spatial deficits to impaired visual perception would require that thepcd mice were visually compromised in such a way that the proximal but not the distal visual stimuli could be processed as cues, a circumstance that seems unlikely since several of the large distal cues were present just beyond the edge ofthe tank. Swimming places heavy demands on motor performance. Although thepcdmice were capable of the posture and organized motor pattern typical of the species, they did have difficulty maintaining it over time. This decreased limb stability during swimming may have contributed to performance differences between pcd mice and controls (i.e. more turning and less straightline swimming). However, the dissociation between spatial navigation and visual guidance performance in thepcd mice cannot be ascribed simply to a generalized deficiency in motor control or less stable swimming, since those demands are the same for both tasks. It is also unlikely that the pcd mice wereespecially subject to fatigue, since the procedure was designed to limit fatigue and hypothermia, and decrements in performance (e.g. swimming speed) ofpcd's did not emerge on the later trials of a given day. Another possible confounding variable is that thepcd mice were underweight compared to + / ? and C57BL/6J mice (Table I), indicating that developmental undernutrition likely occurs in affected offspring. However, nutritional studies in rats demonstrate that while severe undernutrition can delay development of spatial navigation, the ability is not compromised at 30 days or older4,12. Thus, it appears unlikely that the deficits at the ages tested in the mice in this study were due to undernutrition of the pcd mice. Despite the dissociation between the proximal- and distal-cue performance, the interpretation that the cerebellum serves an essential role in spatial mapping but not in visual guidance learning in rodents, as has been postulated for the hippocampus36"44"45, still must be viewed with caution. Under the training conditions used in this study, it is clear that the proximal-cue task is much easier to learn than the distal-cue task. Thus, the failure ofthepcd mice on the distal-cue task may be more an interactive function of task difficulty than a specific deficiency in spatial mapping abilities. The results do show, however, that cerebellar dysfunction can severely impair performance of spatial navigation while sparing visual guidance learning.

Studies by other investigators using other cerebellar mutants have found performance deficits in different tasks incorporating spatial cues. Lalonde and colleagues have shown deficits in spontaneous alternation in the staggerer23"28, weaver2z, htrcher29, nervotts25, and pcd26"27 mutant mice, and inability to navigate a maze in stagger23 and weaver24 mutants. Goldowitz and Koch 9 showed deficits in performance of radial arm maze tasks in staggerer mutants. Pellegrino and Altman 49 showed deficits in maze learning in rats given X-irradiation lesions of the cerebellum as neonates. Note that none of the above has the same type of secondary neuronal degeneration as that of the pcd mice. These findings, taken together with the present study, support the contention that one of the effects of developmental cerebellar dysfunction is deficient spatial performance which extends beyond generalized deficiencies in motor control. These data have important implications for ontogenetic studies of spatial learning, which is usually linked to the functional development of the hippocampal formation 5t,Sz. The cerebellum, like the hippocampal formation, undergoes extensive postnatal development in rodents ~, and the present data suggest that the development of spatial navigation may also require maturation of the cerebellum. Many manipulations during the preweanling period (e.g. undernutrition, exposure to. neurotoxic agents, or environmental enrichment or restriction) have the potential to affect both structures. Consequently, it is risky to interpret the results of studies of spatial learning involving such manipulations only in the context of effects on hippocampal development. For example, studies in our laboratory of early alcohol exposure revealed severe deficits in the development of spatial navigation. We suggested that this reflected hippocampal damage ~~ but the fact that the cerebellum is also severely affected by the alcohol treatment 6' poses difficulties in linking spatial deficits specifically to the hippocampus. Similar complications may also confound the interpretation of spatial learning deficits resulting from other developmental insults which may affect both the hippocampus and the cerebellum, including malnutrition4a2,15"t7 and perinatal glucocorticoid exposure 59. The present study supports the contention that the cerebellum serves an important role in the performance of spatial tasks. How the cerebellum exerts its influence on spatial learning has not been clearly specified. The possibilities may include regulating executive motor commands derived from spatial mapping processes (usually considered as resulting from hippocampalcortical interactions), providing critical sensorimotor feedback concerning movement to those spatial map-

140 ping processes, or even directly contributing to the formation of spatial maps. While these functions need to be better specified with respect to spatial information processing, the present data clearly demonstrate that visual guidance based on a single proximal cue remains viable in thepcd mutants, while place navigation based on multiple distal cues is severely impaired.

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