Global ischaemia: Hippocampal pathology and spatial deficits in the water maze

ELSEVIER

Behavioural Brain Research 62 (1994) 41-54

BEHAVIOURAL BRAIN RESEARCH

Research Report

Global ischaemia: hippocampal pathology and spatial deficits in the water maze J . A . N u n n ~''*, E. L e P e i l l e t b, C . A . N e t t o ~''~, H . H o d g e s ~', J . A . G r a y ~', B . S . M e l d r u m b lhTmrlments O[ ~P~Tcholo,
Rcceivcd 18 October 1993: accepted 9 February 1994

Abstract Spatial deficits were assessed in male Wistar rats which had undergone 4 vessel occlusion for 5, 10, 15 or 30 min. Relationships between the extent of brain damage, the duration of 4-vessel occlusion, and the behavioural impairment consequent upon ischaemia were investigated. Starting 13-18 days after occlusion, rats were trained to find a hidden platform in a Morris water maze. All ischacmic groups were impaired on some performance indices relative to controls, in both acquisition and retention of the platform location. Increasing the duration of ischaemia increased behavioural deficits on some measures, but there was no clear-cut evidence that longer durations of ischaemia resulted in increased behavioural impairments. Histological assessment, at two coronal levels in hippocampus and four coronal levels in cortex and striatum, revealed CA1 cell loss in all ischaemic groups, which varied between 10-100'!,, across the range of durations employed. CA1 cell loss increased as both a linear and quadratic function of increasing the duration of ischaemia. In rats subjected to 5-15 rain ischaemia, cell loss was almost exclusively confined to the CA1 area. In rats subjected to 30 rain ischaemia there was additional, variable damage in hippocampal areas CA2, 3 and 4, substantial cell loss in the striatum (50-70(',,) and some neuronal damage in the cortex (largely in layer III). However correlations between CA1 cell loss in ischaemic rats and indices of spatial ability were non-significant, despite avoiding bias in the analysis by' ensuring that only' those rats with submaximal CA 1 cell loss estimates and behavioural impairments were included. Given the lack of correlation between damage to the CA I region and behaviour, it is suggested that CA I cell loss may not be the only determinant of the water maze deficits displayed by 4-vessel occlusion ischaemic rats. Key words:

Spatial learning; Water maze; Hippocampus: CA l: Forebrain ischemia: lschemic duration

1. Introduction

Clinical evidence [46] for a circumscribed anterograde amnesia and primary loss of hippocampal CA1 neurons following transient global cerebral ischaemia induced by cardiac arrest or coronary artery occlusion has provided the rationale for development of a 4-vessel occlusion (4VO) rat model [32] to study memory dysfunction after ischaemic episodes that resemble the effects of heart attack in man. This method of inducing ischaemia involves cauterisation of both vertebral arteries plus transient occlusion of the carotids; cerebral blood flow (CBF) in the forcbrain is reduced by 900o or more [32]. Although many factors, such as brain temperature [4,6,21] and plasma glucose levels [34], have been shown to influence the extent of brain damage during ischaemia, if these are equated * Corresponding author, Fax: (44)(71) 703-5796. (/160-4328~94 $7.00 O 1994 Elsevier Scicnce B.V. Air rights reserved 5-8

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across subjects the extent of4VO ischaemic brain damage is primarily related to the duration of the ischacmic episode [34,36]. With a short duration ( < 15 rain)of4VO the CA 1 region of the hippocampus appears to bc selectively vulnerable, whereas with longer periods ( >_30 min) damage also occurs in the CA2, CA3 and CA4 cell fields of the hippocampus, dorsolateral striatum and cortex [34]. Although the number of brain regions affected by 4VO is clearly related to the duration of ischaemia, the function that describes the relationship between ischaemic duration and degree of neuronal damage is not known. One aim of the present study was to determine whether CA I cell loss increases as a definable function of the duration of ischaemia. Previous studies have shown that destruction of hippocampal regions is associated with the occurrence of deficits in spatial learning when animals must use allocentric spatial cues for efficient solution of the task, i.e. 'place

42

I.A. .'\'tm~; et al

Behavioural Brain Research 6 2 : 1 9 9 4 4 1 - 5 4

learning' [24,25,45]. It is therefore not surprising that rats subjected to 15-20 rain of 4VO ischaemia are impaired in acquisition of a water maze place task [3,13,16] and rats given 30 rain of 4VO show deficits in a T-maze [31,43] and radial-maze task [10.12.41.42]. Kiyota etal. [18] have also shown that rats given shorter durations of 4VO (5 or 20 min) demonstrate impatrment of radial-maze performance, but not of acquisition of the water maze place task - - c o n t r a s t i n g with the afore-mentioned results of J aspers ct al. [16] and Block et al. [3], who used the same duration ofischaemia (20 min}. Thus the behavioural profile of rats subjected to 4VO ischaemia in the water maze place task is not yet firmly established. Another aim of the present experiment, therefore, was to clarify' whether 4VO ischaemia does impair performance in the water maze place task [26], and to determine what duration of 4VO is required for deficits to appear, by using a range ofischaemic durations: 5. 10. 15 and 30 rain. Evidence for impairment in learning and memory functions following CA1 cell loss induced by ischaemia [ 1,2,40,46] raises the possibility that the extent of deficit correlates with the extent of damage to the CA I region. Some studies suggest that this is indeed the case. Kiyota et al. [ 18] found a significant correlation between CA 1 cell loss and radial-maze reference and working memory errors following 4VO. Volpe et al. [43] showed that the extent of CA1 loss following 4VO or neurotoxic lesions correlated with goal-arm performance in a T-maze. Other findings relating the extent of CA 1 damage and behavioural impairment are conflicting. On one hand Jaspers et al. [ 16] found impalrn3ent in water maze place learning after transitorx occlusion of the carotids {2VOI, in the absence of any observable hippocampal cell loss. Similarly Lyeth et al. [221 reported prolonged spatial memory deficits in the radial-maze, without overt damage m the brain regions examined, and specifically no quantitative neuronal loss in the CA1 area. following traumatic brain injury. On the other hand Auer et al. [ 1] found onlx marginal impairment in the water maze. after 2VO. when 50",, of the CA1 cell field was necrotic, and Volt et al. [401 reported only a slight impairment on the water maze place task (i.e. ischaemic rats learnt more slowly than shamoperated rats) following 2VO ischaemia which resulted in approximately 60°0 loss of CA1 cells. Rod et al. [35! found a significant correlation between 2VO-induced CA1 cell loss and performance on Whishaw's water maze learning-set task [44]. However these researchers included sham-operated animals with uniformly absent brain damage in the correlational analysis. Such a complete absence of variation in one variable makes inclusion of this group in the analysis questionable, and in turn renders their statement that CA 1 cell loss is significantly correlated with behavioural performance less than convincing. A true cor-

relation between CA1 cell loss and performance should reveal itself without inclusion of a group lacking any CA I cell loss. In fact. reanalysis of the data set o f R o d et al. [35] without the sham-operated group reveals a re)nsignificant correlation between C*\I cell loss and tatcncy (r : 0.52. d r = 10. P = 0.08~ and between C A I cell toss and errors ( r = 0.41. d f = 10, P = 0.171. These results suggest that (1') although deficits in spatial tasks occur after damage to the CA I region. /he cell loss required may be substantia! (:> 50".,): (2) no clear correlation between extent of C kt cell loss and behaxiouralimpairment has been established; and 131 other types of neuronal dysfunction ma) produce similar impairments in the absence of CA1 cell loss. It is therefore of interest to determine whether a correlation exists between extent of C A t cell loss induced by 4VO and m3pairment m water maze place |earning. N o such correlational study has been conducted using this task. despite the fact that it is one of the few tests of spatial memory that appears to involve true allocentric Iocalisanon [37]. A range of durations of ischaemia, giving rise to a range of CA 1 cell loss. enables correlations between cell loss and behaviour in a large number of animals. In summary, the aims of the present stud,, were to determine: (at whether CA I cell loss increases as a definable function of the duration of ischaenna: (b) whether 4VO ischaemia produces impairments ilq the water maze place task and w h a t duration o f 4VO is required for deficits to appear: (c) whether a correlation exists between extent of cell loss in CA I and impairment m place learning.

2. Materials and methods 2. I. Subjects Sixty-seven naive male Wistar r a t s (Bantin and Kingman. UK), initial weight 250-280 g, were subjected to 5. 10. 15 or 30 rain 4VO ischaemia {ISC), sham-operation or no operation Animals were housed &cage in a temperature controlled environment (22 ":C), under a 14/10 Might dark cycle (lights on 07.00 to 2/.00 h) and gtven ad libitum access to food and water, t5 and 30 rain ischaemic groups were operated on and tested at a different time to that of 5 and 10 min ischaemic groups; therefore separate sham-operated control groups were created and tested in both parts of the experiment. An unoperated group, tested alongside 15 and 30 min ischaemic rats and their shamoperated controls, was included in case sham-operation resulted in cell loss.

J.A. Nunn et al. ,' Bekavioural Brain Research 62 (1994j 4 1 - 5 4

2.2. Surgery Global cerebral ischaemia was produced by the 4-vessel occlusion technique [32]. Briefly, rats were anaesthetised with halothane (2 °o) in a 3:1, N , O : O 2 gas mixture and the vertebral arteries electrocauterized through the alar foramen on the dorsolateral cervical vertebra. Silastic ligatures were also inserted around the common carotids and brought to the surface. The next day ischaemia was induced by tightening and clamping the carotid ligatures for 5, 10. 15 or 30 rain, in counterbalanced order. Rats that lose their righting reflex within 2 rain of carotid clamping, and maintain such loss throughout the ischaemic period, have been shown to have their CBF reduced to less than 10 ml/100 g/min, approximately. 103b of that of control rats [33]. Loss of righting reflex was therefore used as a selection criterion, In addition, animals which convulsed during the ischaemia or the reperfusion period were excluded, to avoid the complicating feature of generalised seizures which can cause remote, diffuse cell loss [39]. Body temperature was maintained at 37 _+0.5 °C during ischaemia and reperfusion by means of a rectal probe and heating blanket. Head temperature was monitored by means of a microprobe thermistor (Harvard) and maintained at 36.5 C using an electric lamp. Sham-operated controls underwent the same surgical procedure as ischaemic rats except for carotid occlusion. Unoperated controls were not subjected to any' surgery'.

2.3. Neuro/ogica] a.vses.s'menl Following ischaemia, animals were assessed daily for evidence of neurological deficits, which often occur following ischaemia, but which gradually subside over the first post-ischaemic week [7]. Animals were rated for the presence or absence of grasping, righting reflexes, placing reactions, equilibrium and spontaneous motility. A global neurological score was calculated using the Bures et al. [5] scoring index. Only those animals which recovered normal neurological function within 10-13 days were included in subsequent behavioural testing; however no exclusions on neurological grounds were necessary.

2.4. Behavioural testing Thirteen to 18 days were allowed for recovery between surgery and the start of place navigation testing. Spatial learning and memory were tested in Morris' water maze place task [26]. Briefly, rats were trained to find the location of a platform (10 cm diameter) submerged 3 cm below the water surface of a circular pool (2 m diameter, 0.75 m high) filled to a depth of 25 cm with water. Tem-

43

perature was maintained at approx. 26 °C [24,26]. The water was made opaque by the addition of half a pint of milk. This, plus the fact that the platform was submerged below the surface, rendered the platform invisible to the rats. Four points on the pool rim were designated as north, south, east and west such that the pool surface was divided into 4 quadrants of equal area. The platform was placed approximately at the centre of such a designated quadrant. Four trials/day/rat were given for 6 days, with an inter-trial interval of 5 min. A trial began when the rat was placed in the water facing the pool wall and ended when the rat escaped from the water by climbing onto the submerged platform. However, if a rat failed to locate the platform within 60 s it was placed on or guided to it, where it remained for 15 s before being removed and returned to a holding cage to await the next trial. The location of the hidden platform remained unchanged throughout experimentation. Since the start locations were semi-randomly varied across subsequent trials and there were no intramaze cues signalling the location of the platform, effective navigation required the use of extra-maze spatial information. Swim path, latency to reach the platform, 7o time spent in each area of the pool and heading angle (a measure of divergence from the direct path to the platform) were recorded by an HVS image analysing system (VP112, HVS Image Ltd., Hampton, UK). On Day' 7 spatial memory was assessed in a 1 min probe trial, during which the platform was removed.

2.5. HistoloKv At the end of behavioural testing (6 weeks post-surgery), rats were deeply anaesthetised with pentobarbitone sodium (40 mg/kg, i.p.). The ascending aorta was cannulated and the rat was briefly' perfused with heparinized physiologic saline followed by. 300 ml FAM fixative (formaldehyde, glacial acetic acid and methanol in the ratio 1:1:8). After fixation the brains were removed and stored in FAM fixative until they were embedded in paraffin for sectioning. Coronal sections (7/~m) were stained by the method of Klttver and Barrera [19] and examined under a light microscope. Previous studies have reported a preponderance of damage to the dorsolateral striatum, the CA1 region and the CA2, CA3 and CA4 cell fields of the hippocampus. These areas plus the cortex were assessed bilaterally by two independent observers unaware of the experimental condition. Measures were taken at four levels in cortex--10.7, 9.7, 5.7 and 3.7 mm from the interaural line ( I A L ) - - a n d at two levels in striatum and the CA 1, CA2, CA3 and CA4 cell fields of the hippocampus (5.7 and 3.7 inm from the IAL). Quantitative histology.' of ischaemic neuronal damage was based on a six point rank order scale [20], where 0 = 0-10 <, cell loss, 1 = 10-_0 ,>

44

.I.A. Nunn et al. /Behavioural Brain Research 62 1994 4 1 - 5 4

loss, 2 = 30-50°/~, loss, 3 = 50-70"~ loss, 4 = 70-90!I~a loss and 5 = 90-1003~o loss.

2.6. Statistical analysis Water maze measures were analysed by repeated measures analyses of variance (ANOVA: G E N S T A T , Rothhamstead, UK) with Groups as the between-subjects (S) factor, and Trials and Days of testing as within-S factors, followed where appropriate by Student's t-tests, using the between-S pooled ANOVA error term and degrees of freedom. Group trends were compared by the method of orthogonal polynomials. Planned comparisons of behavioural performance were made between each ischaemic group and their sham-operated control group only, and were therefore not subjected to any per comparison error rate correction. For clarity figures show means of shamoperated and unoperated groups' performance. The three control groups did not differ from each other on any water maze measure ( P > 0.05 for each comparison), and showed near-identical performance to each other. Differences between ischaemic groups in terms of cell loss were assessed by non-parametric analysis, i.e. a Kruskall-Wallis test for overall differences, since cell loss data consisted of grades. Subsequent comparisons between groups protected ~ at 0.05, using the formula :~= ~/k, where k = n u m b e r of comparisons made (the Bonferroni correction). Spearman's rank correlation coefficient was the test of choice for correlational analyses, due to the ordinal nature of cell loss indices.

Nevertheless, ISC rats reacted adequately as soon as the~ tactually located the platform. All ISC groups took longer to find the platform over the 6 days of training (see Fig. t than non-ISC controls. Analysis of variance gave significant main effects of Group (F = 5.88, df = 6.53. P < 0,001 ), Da3s(Lin) (F=29.94, df= 1.265~ P<0.001) and Trials (F=20.60, df=3.950. P<0.001} for the measure of latency. The Days(Lin) x Group interaction was significant ~F= 3.33. dr= 6.265. P < 0.0t ), and comparisons betwcen the linear trends of ISC rats and controls showed that this was dueto 10, 15 and 30min ISC, but not 5 rain ISC, rat,~ differing significantly from their sham-operated controls (I0 rain ISC: t:~,~ = 2.27. P < 0 . 0 5 : I 5 rain ISC: L,~,~= 2.50, P < 0 . 0 5 : 3 0 rain ISC: t2~,5=2.96, P < 0 . 0 5 : 5 rain ISC t2~,5 = 1.06}, in terms of linear decline in latency over da~s As can be seen in Fig, 1. all ISC groups showed a similar impairment in latency, with the exception of the 5 nun ISC group on da>s 4-6. In order to assess which of the ISC groups showed a significant Hnprovement over the 6 training days, a one-sample t-test tested whether the mean linear coefficient was significantly different from zero. Ontv 5 and 10 rain ISC rats linear coefficients differed from zero. showing that for these two groups, latencies decreased progressively over training (5 rain 1SC: t, = 4.{)(l. P < 0 . 0 1 : [ 0 rain ISC: t~=2.89. P < 0 . 0 5 : 1 5 rain ISC: t~ = 1.67:30 rain ISC: r~ = 1.961. With regard to percentage of ~ime spent in the trainin~

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In the 15 min ischaemia group, one animal failed to lose its righting reflex during carotid occlusion and was sacrificed. Three animals subjected to 30 min ischaemia died the day after occlusion. During surgery on 5 and 10 min ischaemic rats, 3 rats did not lose their righting reflex during carotid occlusion and were also sacrificed. The total number of animals that underwent behavioural testing in each group was as follows: unoperated (n = 10), sham-operated for 15 and 30 min ischaemic rats (n = 9), 15 min ischaemia (n = 9), 30 rain ischaemia (n = 8), sham-operated for 5 and 10 rain ischaemic rats (n = 7), 5 rain ischaemia (n = 9) and 10 rain ischaemia (n = 8).

3.2. Water maze place task~acquisition All groups of ISC rats were able to navigate, although poorly, searching in an apparently random fashion,

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J.A. Nunn et al. ,; Behavioural Brain Research 62 (1994) 41-54

quadrant, where the platform was located (Qua& 4), all ISC rats again showed impairment (see Fig. 2). Analysis of variance gave significant main effects of Group (F=5.74, df=6,53, P<0.001),I Days(Lin) (F=109.22, df= 1,265, P<0.001) and Trials (F=59.73, df=3,950, P<0.001). Investigation of a significant Days(Lin) x Group interaction (F= 5.01, df=6,265, P<0.001), showed that all ISC groups differed significantly from sham-operated animals (5 min ISC: t265 = 1.99, P<0.05; I0 rain ISC: t>5 = 2.97, P<0.05; 15 min ISC: t:65 = 2.01, P<0.05; 30 min 1SC: t265= 3.34, P<0.01), in terms of linear increase over days. Inspection of Fig. 2 shows that all ISC groups spent a broadly' similar reduced percentage of time in the training quadrant. In order to assess which of the ISC groups showed a significant improvement over the 6 training days in terms of percentage time spent in the training quadrant, a one-sample t-test tested whether the mean linear coefficient was zero. Only 5 and 10 min ISC rats linear coefficients differed from zero, showing that these two groups progressively increased the time they spent in the training quadrant (5 min ISC: t~=4.06, P<0.01; 10 min ISC: h = 2 . 6 6 , P<0.05; 15 min ISC: ls = 2.33; 30 min ISC: t 7 = 0.21). Heading angle, an index of divergence from a direct path to the platform, improved linearly over training for all rats

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(F=2.65, df=6,53, P<0.05), but the interaction of Days(Lin) x Group was only marginal (F= 2.12, df= 6, 265, P<0.06), reflecting only a slight impairment by ISC rats in learning the general direction in which the platform lay. Time spent in Annulus B, in which the platform was located, was similar for both ISC and non-ISC rats (main effect of G r o u p - - F < 1), as shown in Fig. 3. However ISC rats did spend more time in the outer ring of the pool (Annulus C) than sham-operated controls. Analysis of variance gave a significant main effect of Group (F= 2.80, dr= 6,53, P < 0.05). Main effects of Days(Lin) (F= 261.49, df=1,265, P<0.001) and Trials (F=51,99, df=3,950, P<0.001) were also significant, reflecting a decrease in time spent in this portion of the pool as training progressed, and as animals learnt to swim away from the side walls. A significant Days(Lin)× Group interaction was present (F= 5.25, dr= 6,265, P<0.001) and comparisons of linear trends showed that 5, 10 and 30 min, but not 15 rain ISC rats showed a slower linear decrease than controls (5 rain ISC: t265=2.95, P<0.01; 10 rain ISC: t~65=2.19, P<0.05; 30 min ISC: t2~5=4.83, P<0.001; 15 min ISC: t265 = 1.27). 30 min ISC rats spent most time in thigmotaxis, as can be seen in Fig. 4.

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Fig. 2. Effects of 5, 10, 15 and 30 rain 4VO ischaemia on percentage of time spent in the training quadrant during water maze place task acquisition. Scores are mean percentages for each day of training (4 trials/ day). Bar represents 2 S.E. of the combined groups' mean.

Fig. 3. Effects of 5, 10, 15 and 30 rain 4VO ischaemia on percentage of time spent in Annulus B (containing the platform) during water maze place task acquisition. Scores are mean percentages liar each day of training (4 trials/day). Bar represents 2 S.E. of the combined groups' mean.

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3.3. Water maze place task--probe trial

With the platform removed, all ISC groups spent less time in the previously correct quadrant than controls (main effect of G r o u p - - F = 2.74, df=6,53, P<0.05), but this difference was significant only for t0, 15 and 30 rain ISC rats (5 min ISC: t53 = 1.26; 10 min ISC: t53 = 2.35, P < 0.05; 15 rain ISC: t53 = 2.49, P < 0 . 0 5 ; 30 min ISC: t53 = 2.66, P<0.05). The 10 min ISC group spent less time in the training quadrant than other ISC groups (see Fig. 5), indicating worse recall of the platform's location. 10 rain ISC rats also tended to distribute their time amongst the other quadrants differently to that of other ISC and nonISC rats, spending relatively less time in Quad. 3 (adjacent to the training quadrant) and more time in Quads. 1 and 2. However Quad. 1 was the only quadrant in which differences between groups reached statistical significance (main effect of G r o u p - - F = 3.72, d f = 6,53, P<0.01). A t-test confirmed that the 10 rain ISC group spent significantly more time in this area than Shams (ts3 =4.27, P < 0.01). Quadrant 1, which like Quad. 3 was adjacent to the training quadrant, had fewer extra-maze cues surrounding it, which would appear to be reflected in the lesser time that all groups spent in this quadrant compared to other quadrants (see Fig. 5). In terms of the heading angle of rats, the probe trial measure distinguished between ISC and non-ISC groups

Fig. 5, Effects of 5. IlL 15 and 30 mm 4V() i s c n a e m m o n pcrcentagcnT time spent m each quadranl during the a atcr maze probe trial. Quadrant 4 represents the previousl} correct quadrant, Scores are mean perccntages. Bars represent 2SE ol" thc c o m b i n e d groups' mean tier each quadrant. Stars represent a significant difference between an experimental group and its s h a m - o p e r a t e d control group. * P < 0.05. *** P < 0.001.

better than acquisition measures. I'he main effect of Group was significant (F= 3.94. df=~6,53. P<0.01), which reflected the fact that all ISC groups were impaired in recalling the direction in which the platform lay relative to controls, as shown m Fig. 6 (5 rain ISC: z~ =2.25. P < 0 . 0 5 : 1 0 min ISC: ts~=2.(~9. P < 0 . 0 1 : 1 5 min ISC: t~ = 2.30. P < l l . 0 5 : 3 0 min IS(;: t~3 = 2.59. P < 0 . 0 5 ] Ischaemic rats showed evidence of thigmotaxls during the probe trial, as they had during acquisition, spending a greater percentage of time in Annulus C (the outer annulus) than controls 1main effecl of G r o u p ~ F = 3 . 3 6 , d f = 6.53. P<0.051. However this difference was significant only for 30 min ISC rats u,? = 3.27. P < 0 , 0 1 ) and 10 rain ISC rats (ts3 = 2.41. P < 0,t)51, All groups of rats spent a similar amount of time in Annulus B, in which the platform was located, as they had done during acquisition (main effect of G r o u p ~ F - 1.46, d f = 6.53). Throughout the 6 training days and the probe trial. ISC groups did not differ from controls in terms of swim speeds (Fs < 1), so differences in latencies were unlikely to be due t~) motor or motivational effect~ 3.4. Histoh)gic al analys'is

Sham-operated rats showed no cell loss in any brain region examined, i.e. all were given a cell loss grade of 0. representing 0 - 1 0 ° ; loss. 4VO caused bilateral cell loss m dorsal CA 1, both anterior and posterior, in all ISC groups (see Fig. 7). The amount of CA 1 cell loss increased as the

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J.A. 3,'unn et al. : Behavioural Brain Research 62 (1994) 41-54

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ISC

Fig. 6. Effects of 5, 10, 15 and 30 rain 4VO ischaemia on heading angle (an index of divergence from a direct path to the platliwm)in the water maze probe trial. Scores are mean angles in degrees. Bar represents 2 S.E. of the combined groups' mean. Stars represent a significantdifference between an experimentalgroup and its sham-operated control group. * P< 0.05, ** P< 0.01.

duration of ischaemia was increased. CA1 loss was great-

in posterior CA 1. No damage other than to CA 1 was seen

est at the anterior level studied (5.7 m m from the IAL), for all durations of ischaemia. More specifically, 5 min ischaemia produced very little CA1 loss: a graded estimate of l (10-300d, l o s s ) w a s given to 5/9 rats at the 5.7 mm level and a score of i or less was

to occur in the 5 and l0 min ISC groups. Ischaemia of 15 and 30 rain duration resulted in > 90°0 (mean graded estimate) cell loss in anterior CA 1. Specifically, 7/9 15 min ISC animals and 7/7 30 min ISC animals

given to 5/9 rats at the 3.7 m m (posterior) level. Ten rain

testing was completed) showed 90-100"~, loss at the

ischaemia produced greater CA1 cell loss, averaging around 50", cell loss in anterior CA1, and 2 0 - 3 0 ° 0 loss

5.7 mm level. At the 3.7 mm level, estimates of CA1 cell loss in both 15 and 30 min ISC rats were more variable,

00 U)

ranging from 5 0 - 1 0 0 " 0 , although rats in the 30 rain ISC group showed greater CA1 cell loss. However in the 15 min ISC group damage was almost exclusively con-

5 min ISC

O I

fined to the CA1 area; only 3/9 rats in this group exhibited loss in areas CA2, 3 and 4 of hippocampus (ranging from 10-70°o), and striatum (10-70,,,), 2 of which also showed slight cortical damage (ca. 10",,). All rats sub-

10 min ISC

.J

Ill

O b. O >nO uJ
L~

1

2 CA 1

(one animal from this group died shortly after behavioural

REGION

Fig. 7. Mean graded estimates (+ S.E.) of CAI cell loss and duration of"4VO ischaemia. Left ( 1): anlerior CA 1 loss grades (5.7ram from the IAL) for 5, 10, 15 and 30 rain of 4VO. Right (2): posterior CA1 cell loss grades (3.7 mm from the IAL) for 5, 10, 15 and 3(I rain of 4VO. Grades of cell loss: 0 I)-10~'., 1 = 10-30",,, 2 = 30-50",, 3 = 50-70°., 4 = 7090°,,, 5 = 90-100"i, loss.

jected to 30 min ischaemia, however, showed additional, variable loss in areas CA2, 3 and 4 (30-100"o), together with substantial damage in striatum ( 5 0 - 7 0 " ; ) and some loss of cortical cells (ca. 20°o), mainly in layer Ill. There was very little left/right hemisphere variability in terms of cell necrosis in any of the brain regions studied. The extent of cell loss outside the CA1 region is shown in graph form in Fig. 8. A K r u s k a l l - W a l l i s test showed a significant difference between groups in terms of median CA I cell loss estimates at both the 5.7 m m (anterior) level ( - = 2 0 . 1 8 , d r = 3 , P < 0 . 0 0 1 ) and the 3.7 mm (posterior) level (_-=22.44, d f = 3 , P < 0 . 0 0 1 ) . Subsequent multiple comparisons showed that the 5 and 10 min ISC groups did not differ

.I.,4. \:win et al.

48

if)

Behavioural Brain Research 62 1994 4 1 - 5 4

5

O "J

4

~'~

3

~

~

2

o°

15 mln ISC

3o min

ISC

,

3o 1

2

3

CORTEX

4

1

2

1

8TRIATUM

BRAIN

2

CA2

1

2 CA3

1

2 CA4

REGION

Fig. 8. M e a n g r a d e d e s t i m a t e s ~ ~ S . E , ) o f e x t r a - C A t cetl l o s s a n d d u r a t i o n o f 4 V O i s c h a e m i a at 4 levels in c o r t e x t I .. i 0 9 . 2 - 9.7. 3 -- 5, 7 . 4 -- 3.r a m f r o m t h e I A L ] , 2 levels in s t r i a t u m [ I = 10.7. 2 = 9.7 m m f r o m t h e I A L ] , a n d 2 levels in a r e a s C A 2 . 3 a n d 4 [1 ~ 5,7, 2 = 3.7 m m f r o m t h e I A t . ] . F o r 15 a n d 30 rain o f 4 V O . G r a d e s

o f cell l o s s : 0 = 0 - 1 0 ' :

. 1 - 10-30",.

significantly from each other at either level 15.7 mm: z = 1.69; 3.7 ram: z = 1.271. Similarly, 15 and 30 min ISC groups did not differ from each other at either level (5.7 mm: z = 0.48; 3.7 mm: z = 0,87). At the 5.7 m m level, where most CA1 loss occurred. 5 min ISC rats showed significantly less CA1 loss than both 15 min ISC rats (z = 3.88, P < 0.05) and 30 min ISC rats (z = 4.12. P < 0.05) whereas 10 rain I S C rats did not ( P > 0.05 for each comparison). At the 3.7 m m level. 5 min I S C rats again showed significantly less CA1 loss than both 15 min ISC rats (z = 3.56, P < 0.05) and 30 rain I S C rats {z = 4.20, P < 0.05). At this level, 10 min I S C rats also showed significantly less C A I cell loss than both the 15 min I S C group ( z = 2.81. P < 0 . 0 5 ) and the 30 rain I S C group ( z = 2 . 8 9 . P < 0 . 0 5 ) . Another A N O V A determined whether CA1 cell loss increased as a definable function of the duration of ischaemia. Both linear and quadratic effects were fitted (taking into account the experimental spacing 5.10. 15 and 30 rain), and both effects were highly significant at both levels at which CA1 loss was assessed (5.7 mm: F ( L i n ) = 52.73, d f = 1.29. P < 0 . 0 0 0 1 : F ( Q u a d ) = 19.58. d f = 1,29, P = 0 . 0 0 0 1 ; 3.7 mm: F(Lin)= 63.63. d f = 1.29. P < 0.0001; F(Quad) = 11.58. df = 1.29. P = 0.002 ),

3.5. Correlational analysis Following histological assessment, a correlational study was carried out to identify any relationship between performance in the water maze and cell loss in various brain regions. The behavioural indices chosen were: (1) mean latency to find the platform during acquisition clays 5 and

2 = 30-50' ,, 3 = 50-7(P'

. 4 = 70-90".,

5 = 9 0 - 1 0 0 ° ' loss,

6. (2) mean 7,, time spent in the training quadrant during acquisition days 5 and 6. and (3) °:; time spent in the previously correct quadrant during the 1 rain probe trial. The late phase of acquisition used (i.e. days 5 and 6) was to prevent the considerable non-specific instrumental learning that occurs early in training (e.g, learning to swim away from the side wall) from masking impairments in true place learning. In CA1. the maximal cell loss show1~ by the t5 and 30 min I S C groups atlevel 5,7 m m from the IAL precluded the inclusion o f these groups in the analysis. Therefore. at this level, only 5 and 10 rain ISC rats" cell loss estimates were correlated with behaviourai indices In = 17). At the 3.7 mm level, however, all 4 I S C groups showed variable C A 1 cell loss a n d all 4 groups were therefore included (n = 33). For other brain regions, means of cell loss grades at the 2 levels assessed (for hippocampus and striatum) or 4 levels assessed (for cortex) were used as cell loss indices. Only those rats which showed cell necrosis outside C A I were included in these analyses i.e. 3/9 of the 15 rain ISC rats and 7/7 of the 30 min I S C rats In = 10). The results are shown in T a b l e 1, along withdefinitions of behavioural indices. N o correlation was found between CA1 cell loss grade and water maze latency (e.g. L A T vs. anterior CA1 cell l o s s m r = -0.081: nor were there any significant correlations between latency and cell loss estimates in cortex, striatum or the CA2. CA3 and C A 4 hippocampal celt fields (e.g. LAT vs. CA4 celt l o s s - - r = 0 . 1 6 ; EAT vs. striatal loss r = 0.05). The correlations between CA1 cell loss grade and mean °::~ time spent in the training quadrant during acquisition were also non-significant (e.g.

49

J.A. Nunn et al. .; Behavioural Brain Research 62 (l 994) 41-54

Table 1 Correlations between cell loss grades and behavioural indices for 5. ill, 15 and 30 rain ISC groups

C A 1 cell l o s s g r a d e (e.g. 2 o P T r vs. a n t e r i o r C A 1

cell

l o s s - - r = 0.04), n o r w i t h cell l o s s e s t i m a t e s in o t h e r p a r t s o f t h e b r a i n (e.g. ° o P T r vs. C A 2 l o s s - - r = - 0.33). S c a t t e r

Variables

Spearman's rank correlation coefficient, r

n

plots showing the relationship between anterior and posterior CA1

cell l o s s a n d

water

maze

performance

are

s h o w n in F i g s . 9 a n d 10. CA1 loss ~rS• (at Level 1)

LAT +oACQ ",>PTr

--0.08 0.38 - 0.04

17 17 17

Correlations performance

between

different behavioural

in t h e w a t e r m a z e

indices of

were also analysed,

assess how different water maze measures

to

relate to each

CA1 loss ~s. (at Level 2)

LAT °oACQ ";, PTr

0.12 -0.08 0.18

33 33 33

o t h e r . T h e r e s u l t s a r e s h o w n in T a b l e 2.

CA2 loss ~s. (mean of Levels 1 and 2)

LAT +,ACQ ",, PTr

0.42 - 0.11 - 0.33

10

of acquisition (LAT) was highly negatively correlated with

10

t h e ° o o f t i m e s p e n t in t h e t r a i n i n g q u a d r a n t

LAT "~,ACQ "o PTr

0.56 - 0.19 - 0.42

10

CA3 loss vs. (mean of Levels 1 and 2) CA4 loss ~s. (mean of Levels 1 and 2) Striatal loss vs. (mean of Levels 1 and 2) Cortical loss xs. (mean of Levels 1-4)

10 10 10

LAT ",,ACQ ",, PTr

0.16 - 0.59 - 0.15

10 10 10

I.AT ",,ACQ "~,PTr

0.05 -0.48 0.19

10

LAT ~'.,ACQ ",,PTr

0.47 - 0.42 0.54

10 10 10

10

10

L a t e n c y t o f i n d t h e h i d d e n p l a t f o r m d u r i n g t h e late p h a s e

s a m e late p h a s e & a c q u i s i t i o n

during the

(°0 A C Q ) (r = - 0.84). T h i s

s h o w s t h a t r a t s w h i c h h a d l o n g l a t e n c i c s t e n d e d to s p e n d m o r e t i m e in q u a d r a n t s

not c o n t a i n i n g the p l a t f o r m t h a n

r a t s w i t h s h o r t l a t e n c i e s i.e. m i s p l a c e d s e a r c h i n g d i d n o t predominantly

take place close to the platform, but some

w a y f r o m it. ' L A T ' w a s a l s o n e g a t i v e l y c o r r e l a t e d w i t h "0 t i m e s p e n t in t h e p r e v i o u s l y c o r r e c t q u a d r a n t

in t h e p r o b e trial (!},,

P T r ) (r = - 0.37), as w a s "~, A C Q '

(r = 0.39).

These

significant correlations

failure of Table 1 correlations

s e r v e to s h o w t h a t t h e w a s n o t d u e to n o i s e in

behavioural indices. Cell loss was assessed at 2 levels in areas CA 1, 2,3 and 4 of hippocampus (1 = 5.7, 2 = 3.7 mm anterior to the Inter Aural Line (IAL)); 2 levels in striatmn (1 = 10.7, 2 - 9.7 mm anterior to the IAL) and 4 levels in cortex (I = 10.7, 2 = 9.7, 3 5.7.4 = 3.7 mm anterior to the IAL). I N D I C E S OF P E R F O R M A N C E : LAT = mean latency (s), water maze acquisition, day's 5 and 6; ", ACQ = ",, time spent in training quadrant, water maze acquisition, days 5 and 6: ",, PTr = ",, time spent in training quadrant, water maze probe trial.

'!o A C Q vs. p o s t e r i o r C A 1 cell l o s s - - r = - 0 . 0 8 ) , correlations

between

this behavioural

b r a i n r e g i o n s (e,g. ?0 A C Q

vs, C A 3

9

4o

I,

ii

as w e r e

index and lossIr=

io

other

-0.19).

F u r t h e r , t h e '~o t i m e s p e n t in t h e p r e v i o u s l y c o r r e c t q u a d -

2oo

r a n t in t h e p r o b e trial d i d n o t c o r r e l a t e s i g n i f i c a n t l y w i t h

1

Anterior

1

CA1

I

oeM 1o141

io

Table 2 Correlations between indiccs of perforlnance in the water maze for 5, 10, 15 and 30 rain ISC groups Variables

i I

gg

i

++

t5

Spearman's rank correlation coefficienL r

For dr= 3 l, r > 0.356 = P<0.05

i:

- 0.84 - 0.37

** *

#

io

°

•

40

t LAT

vs. vs.

".ACO ",, Ptr

Ii ,i

<'oACQ vs.

°oPTr

0.39

*

1NDICI£S OF+ P E R F O R M A N C E LAT = Mean latency (s)+ water maze

acquisition, da~s 5 and 6; <'o ACQ - % time spent in training quadrant, wa~.er maze acquisition, days 5 and 6: o,, PTr = '!0 time spent in training quadrant, water maze probe trial. *P<0.05: **P<0.01.

Anterior

2

I

CA1 oeH loire

Fig. 9. Scatter plots of anterior CA1 cell loss (5.7 I+11111from the IAL) vs. behavioural indices in the water maze place task fi)r 5 and 10 min 4VO. Behavioural indices: (top) = latency to find platform, mean for days 5 and 6; (bottom)= "0 time spent in the training quadrant, mean days 5 and 6+

51t

.I.A. ,\"unn et al. , Behavl'Olo'al Brain Re.~earch 0 2 , 1 9 9 4

lOO

- -

{'o ° •

:

•

! •

o

1o 1

t

PoQterlor 10

I

•

$ CA1

4 cell

losQ

.

1

I

so

o t 0

i '°

I poeterlor

i

i CAt

4 oell l o s s

Fig. 10. Scatter plots of posterior CAI cell loss (3.7 mm from the [ALl vs. behavioural indices in the water maze place task for 5, 10, 15 and 30 rain 4VO. Behavioural indices: (top) = <'~,time spent in the training quadrant during the 1 rain probe trial: (bottom)= latency to find platform. mean days 5 and 6.

4. Discussion The present results confirm that 4VO ischaemia produces a CA1 lesion and impairments in the water maze. Prolonging the duration of ischaemia from 5 - 3 0 min increased the amount of cell loss in the CA1 region of the hippocampus and, for 30 rain I S C rats, also resulted in cell loss in other brain regions susceptible to ischaemia, Increases in CA1 loss could be defined as both a linear and quadratic function of increasing the duration of ischaemia. Although increasing the duration of 4VO increased behavioural deficits on some measures, there was no clear-cut evidence that prolonging the duration of ischaemia (and thereby increasing the amount of CA1 cell loss) reveals itself behaviourally in the water maze. N o correlation was found between extent of C A I cell loss (in 5 and 10 rain ISC rats) and three indices of performance in the water maze. The finding that 4VO produces impairments in water maze place task learning is consistent with the results of Block et al. [3]; Hagan and Beaughard [13] and Jaspers et al. [ 16] (see Introduction, above), who also found water maze deficits in rats subjected to 4VO. However the present results are at odds with the findings of Kiyota et al. [ 18 ], who found no impairments in the same task, despite

41-~,4

the fact that their 4VO-ischaemLc rats sustained 71)°,, or more CA I cell loss. It is therefore unlikely that lesser C A I loss can explain these discrepant results: in the present experiment 5 and 10 min ISC rats. with considerably less than 7 0 " CA1 loss. did show water maze impatrments. Extra-CAl cell loss has no apparent explanatory power either, since 5 and 10 min ISC rats s h o ~ e d no evidence of cell necrosis outside the CA i Mternauve possibilities include subtle differences m testing procedure i Ior example. Kiyota et al. used floating styrene foam pieces to conceal the platform, rather lhan milk. which might engender the use of intra-maze cues lot locating the platforint, or neuronal dysfunction but not celt death in C A I or other regions which is not h~stologically detectable. The present results would appear to confirnt previous reports suggesting that CA 1 cell loss alone following 4VO is sufficient to cause a memory unpairment [S.10I h~ agreement with these data is the clinical case of patient 'R.B." [46]. Extensive neuropsychological tests demonstrated anterograde anmesia m this patient, who developed selective memory impairment following an ischaemic episode. During 5 years of testing prior to death, 'R B." showed little, if any, retrograde amnesia, and also showed no signs of cogmtive impairments other than memory. Thorough histological analyses revealed a circumscribed bilateral lesion almost exclusivetx confined to the CA1 fields of the hippocampus. The present results extend previous knowledge by demonstrating that C A I loss of approximately only 10-30",. (5 rain ISC groupl induced by 4VO can produce impairment m the water maze place task. The finding that such a small degree of 4VO-induced CA 1 cell loss is sufficient to disrupt water maze place task performance indicates that the functional consequences of 2VO and 4VO ischaemia may be different: Auer et al. l 1 ] and Voll et al. [40], using the 2VO ischaemia model, reported that approximately 50 and 60<~,, CA1 cell loss, respectively, resulted in only mlmw or transient deficits in water maze place task learning. Some aspects of the behavioural profile of the 4VO ischaemic rat in the water maze were revealed. All ischaemic rats searched in the appropriate annulus of the pool (Annulus BI. where the platform was located, as much of the time as non-ischaemic controls, but took much longer to find the platform during acquisition. The high negative correlation between latency and percentage of time spent in the training quadrant showed that rata with long latencies l ischaemic) tended to spend time in the wrong quadrant rather than ch~se to the platform, However. whichever quadrant ischaemlc rats were in, they spent more ume circling close to the pool rim (thigmotaxisi than controls At other times, ischaemic rats did not appear to adopt taxon-based strategies. >uch as swimming a fixed distance Dora the side walls of the pool, but rather searched

J.A. Nunn et al. / Behaviouml Bra#t Research 62 ~199& 4 1 - 5 4

all over the pool. In the probe trial, all ischaemic rats demonstrated poor recollection of the direction the platform lay in, as indexed by an increased heading angle relative to controls, lschaemic groups spent less time in the quadrant with the least number of extra-maze cues (Quad. 1) than other quadrants, indicating that, like normal rats, the presence of extra-maze cues aided navigation. The near-identical swim speeds of ischaemic and non-ischaemic rats showed that the deficits outlined above were not likely to be due to deficits in motor function, but were instead due to cognitive impairment. The present experiment also confirmed that the 4VO sham-operation (permanent cauterization of the vertebral arteries) does not result in cell loss, and showed that sham-operated rats were indistinguishable from unoperated rats in terms of behaviour. The effect of prolonging the duration of ischaemia was to increase the amount of CA1 loss, and for 30 min duration, to produce extensive, additional intra- and extrahippocampal damage, confirming previous reports [ 18,34]. These previous reports are now extended by the finding that the increase in CA1 cell loss can be defined as both a linear and quadratic function of increasing the duration of 4VO ischaemia. As can be seen in Fig. 7, additional CA1 cell loss decelerates as duration exceeds 15 min, in a fashion well described by a quadratic function. Across the range of durations employed, CA1 cell loss from ca. 10 to 100"{, was achieved. For some water maze measures, it would appear that prolonging the duration of 4VO (and therefore also increasing CA1 loss) increased behavioural impairments. For example, in terms of latency during acquisition and percentage time spent in the previously correct quadrant during the probe trial, 5 min ISC rats were not significantly impaired relative to controls, whereas other ischaemic groups which had undergone longer periods of carotid occlusion were. Moreover, only 5 and 10 min ISC rats demonstrated a significant improvement over the 6 training days in terms of latency and percentage time spent in the training quadrant, so 15 and 30 min ISC rats appeared to be more severely impaired. However, 10 rain ISC rats were worse than 15 min rats in terms of thigmotaxis, during both acquisition and the probe trial, and spent more time in quadrants not previously containing the platform during the probe trial than other ISC groups. In addition, on the measures on which 5 min ISC rats were not impaired relative to controls (e.g. latency during acquisition and percentage time spent in the training quadrant during the probe trial), all other ISC groups were impaired to a very similar degree. Thus, prolonging the duration of 4VO beyond 10 min (and augmenting CA1 cell loss) did not substantially affect performance on these measures. So it is not clear that increasing the amount of CA1 cell loss

51

increases water maze impairments in any systematic fashion. The 30 min ISC group was reliably impaired on all measures of water maze performance. This group was also the one group to exhibit cell loss outside the CA1 region of the h i p p o c a m p u s - - s o perhaps extra-CA1 loss contributed to this group's deficits. However the 10 rain ISC group was also reliably impaired throughout testing--on some measures more so than 30 min ISC rats (e.g. distribution of time amongst quadrants in the probe trial), indicating that loss outside the CA 1 contributed little additive effect to the behavioural profile ofischaemic rats in the water maze. This finding may be taken as support for Cook and Kesner's [9] suggestion that the caudate nucleus (a region of damage second only to the CA1 area in rats with 30 rain occlusion) is not required for allocentric tasks. However more extensive behavioural testing would be required to rule out the possibility that ischaemic damage in regions other than the CA1 contributes to the spatial deficits shown by 30 min ISC rats. In particular, there was some evidence that both 15 and 30 min ISC groups were exhibiting a 'floor effect' in terms of impairment during acquisition (i.e. the linear component of the functions for latency and percentage time spent in the training quadrant did not deviate from a horizontal line), thereby possibly inhibiting the display of any contribution that intra- and extra-hippocampal damage might make to ischaemic spatial deficits. Prolonging the period of behavioural testing might enable 15 and 30 min ISC rats to show significant learning and, as a result, reveal any additional deficits that 30 min ISC rats might have. It has been suggested that 4VO ischaemia of 30 min duration produces a CA I lesion so extensive that it is functionally equivalent to total ablation of the hippocampus [10]. If one considers the unidirectional flow of information through the hippocampus via either the trisynaptic circuit (dentate g y r u s - C A 3 - C A 1-subiculum) or the direct entorhinal-CA1 pathway (by-passing the dentate and CA3), it can be seen that this should indeed be true. CA1 lesions should prevent information flow along either pathway. The finding that monkeys given 15 min of ischaemia (with cell toss restricted to the CA1 and a few cells of the dentate gyrus) perform similarly to monkeys given selective lesions of the hippocampus on delayed nonmatching to sample, object discrimination and eightpair discrimination tasks [47] supports the notion that the hippocampus is equally disabled by ischaemia as by ablation of the entire hippocampus. Lesions produced in other laboratories, of the dentate gyrus and other subfields of the hippocampus, provide some support for the idea that damage to hippocampal subfields impairs performance on the same tasks that hippocampectomy also disrupts. Sutherland et al. [38] and Whishaw [45] showed

52

J.A, Nunn et al. r Behaviourat Brain Research 62 H994 4 1 - 5 4

that performance in the water maze was disrupted by colchicine-induced depletion of dentate granule cells. Handlemann and Olton [14] found that kainic-acidinduced CA3 lesions produced impairment (albeit transient) of spatial working memory in a radial-maze. However Jarrard [ 15] found that kainic-acid-induced CA3 loss did not disrupt spatial memory (working or reference) in a similar radial-maze task. In the present study, the effects of ischaemia on water maze performance were not quite the same as those reported following hippocampal removal by a neurotoxin. Morris et al. [25] found that selective hippocampal removal by ibotenic acid resulted in stereotyped swimming at a fixed distance from the side walls of the water maze. No such effect was seen in ischaemic rats: although impaired at searching for the platform, they searched in a random fashion, like controls who did not know the location of the platform. Therefore, although there is some evidence that lesions of hippocampal subfields may be equivalent, subtle differences in water maze performance may exist between CA1 lesions and total hippocampal ablation. Direct comparisons of ischaemic and hippocampectomised rats in the water maze should clarify this issue. Correlations between CA 1 cell toss at both anterior and posterior levels of CA1 (and of other brain regions affected) and 3 behavioural indices of water maze performance were non-significant, echoing a previous finding from this laboratory [27], in which a lack of correlation between degree of CA1 cell toss and a number of indices of water maze performance was also noted. Non-significant correlations are consistent with the finding that, in the main, water maze deficits were not amplified by increasing levels of CA1 loss. Variability in CA1 loss was wide and submaximal, even at the anterior level (5.7 mm from the IAL) where, to ensure that the criterion of sub-maximal CA1 loss was met, only 5 and 10 min ISC rats were included in the correlational analysis. When only 5 and 10 min ISC groups were included, the number of animals used (17) was still sufficiently large to detect an association. It is unlikely that the appropriate level of CA1 for linking to behaviour was missed since both the region where most CA1 loss is seen following ischaemia (anterior) and the region where less is seen (posterior) were sampled. Rod et al. [35] have shown by sampling CA1 loss along the entire anterior-posterior range that CA1 cell loss following ischaemia only varies in that it is reduced in the posterior part of the hippocampus. Presumably, therefore, additional correlations with other levels of CA 1 loss would have produced the same non-significant result. These points, plus the fact that the lack of correlation between CA1 loss and behaviour was absolute, makes it unlikely that use of a restricted ordinal grading biased these results.

In addition, the use of Spearman s rank correlation coetficient rules out the possibility that only a linear association is excluded by these results, because the 'rank" test detects a monotonic association of any kind. unlike, tbr example, Pearson's product moment correlation, which only detects linear relationships. These results conflict with those of Kiyota et al. [ 18] and Volpe et al. [43] (see Introduction, above) whose studies produced significant correlations between CA I loss follmving 4VO ischaemla and behavioural impairment in a radiaMnaze and a -l--maze. respectively. Several factors could account for the observed lack of correlation between behavioural deficit and CA 1 cell loss. Deficits could be manifest above a certain threshold rather than relate systematically to degree of damage. However scatter plots {see Figs. 9 and 101 indicated that deficits are not only manifest above a certain threshold of CA I loss but instead are manifest at all levels of CA1 loss. Behaw ioural deficits could reflect neuronal dysfunction, rather than actual destruction of cells, and thus involve factors that would not be detectable b~ histological methods. Onodera et al. [28-30], for instance, have shown that after 4VO isehaemia changes in neurotransmitter levels. receptor binding and second messenger systems were not confined to the lesioned CA1 field, but pervaded the hippocampus. Such findings coukt account fbr reports of spatial deficits following ischaemia in the absence of CA 1 cell loss [ 16]. However. one might expect these dysfunctions to be more pronounced with 30 than 15 or 10 rain ischaemi a. yet there were no add itive effects on behaviour. Thus, although the present experiment does not address the issue of neuronal dysfunction directly, our results tend to discount this as a sole explanation. Moreover. other studies in this laboratory [27] have recently established that grafts of foetal hippocampal tissue dissected from regio superior, so as to contain CA 1 cells~ and sited in the alveus, just above the damaged CAt field, alleviate deficits in water maze navigauon displayed by rats subjected to 15 rain of 4VO. Grafts from regio inferior, containing dentate granule cells, and chotinergic-rich grafts of tbetal basal forebrain tissue were ineffective. This evidence suggests that replacement of appropriate cells, rather than delivery of a requisite neurotransmltter (both CA I and dentate granule cells are glutamatergic), plays an important part m graft-induced functional recovery from ischaemic damage. If so. these results would also indicate that the absence of viable CA1 cells is critically involved in spatial navigational deficits in the water maze. rather than generalised neuronal dysfunction throughout the hippocampus, although neuronal dysfunction but not death remains a potential additional mediator of spatml deficits. Another possibility is that somatostatin (SS)-immunoposiuve dentate hilar neurons were depleted during

J.A. Nunn et al. / Behavioural Brain Research 62 (1994) 41-54

ischaemia, but their depletion was not apparent with the histological methods employed. Johansen etal. [17] showed that loss of SS cells in the hilus preceded the delayed loss of CA 1 neurons in the rat. Matsuyama et al. [23], using immunocytochemical analyses, demonstrated that S S hilar cells are more vulnerable than CA 1 neurons to the effects of ischaemia in the gerbil. SS neurons in the hilus decreased in number after brief (2 rain) ischaemic insults that did not destroy CA1 pyramidal neurons. If the findings of Matsuyama et al. are generalisable to the rat, the lack of correlation between CA1 cell loss and behaviour in the present study may be explicable: both hilar SS cells and CA1 pyramidal cells may have mediated the spatial deficits shown by ischaemic rats in the water maze. The discrepancy between the present results and those of Kiyota et al. [ 18] and Volpe et al. [43] (see above) may be best explained in terms of task specificity. No study has undertaken correlation analysis of 4VO-induced CA 1 cell loss and behaviour in a water maze place task. Rod et al. [35], however, found a significant correlation between CA1 loss following 2VO ischaemia and performance in a water maze learning-set task (see Introduction, above). Their correlation became non-significant, however, when sham-operated controls were removed from the analysis. Although the learning-set task predominantly tests working memory whereas the place task is a reference memory task, both make the same kind of navigational demands in the same motivational state, and may be more similar in some respects to each other, therefore, than the water maze place task is to the T-maze or the radial-maze. One of the determining factors of whether or not correlations between CA 1 loss and behaviour are significant may well be the behavioural task. Proprioceptive or kinaesthetic cues contribute to successful solution of the T-maze [13], distinguishing the T-maze from both the water maze and the radial-maze. The water maze place task, whilst extremely sensitive to the loss of merely 10-30°o of CA1 cells following 4VO, may not be the most sensitive task to detect correlations, due to the large within-animal variability in performance measures during acquisition. However the probe trial, more representative of 'steady-state' performance than acquisition trials, eliminates much of this variability, and correlations between an index of performance in the probe trial and CA1 loss were still nonsignificant. In conclusion of this point, it would appear that discussion of correlations of this nature should remain specific to the task employed. In summary, the present experiment demonstrates that across a range of durations (5-30 rain), 4VO ischaemia impairs water maze place task performance. Although the duration of 4V0 and subsequent degree of CA1 cell loss were found to be clearly related, no significant relationships between 4VO duration and behaviour, or 4VO du-

53

ration and CA1 cell loss, were detectable. CA1 cell loss as a predictor of behavioural performance may be applicable only in certain test procedures, such as the radial-maze and the T-maze, on which performance has been found to correlate with CA1 cell loss [18,43].

Acknowledgements This work was supported by the British Heart Foundation, the Bethlem Maudsley Research Funds and the Wellcome Trust.

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