The histopathological, behavioral and neurochemical effects of intraventricular injection of ethylcholine mustard aziridinium (AF64A) in the neonatal rat

Developmental Brain Research, 61 (1991) 249-257 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0165-3806/91/$03.50 ADONIS 016538069151308V

249

BRESD 51308

The histopathological, behavioral and neurochemical effects of intraventricular injection of ethylcholine mustard aziridinium (AF64A) in the neonatal rat John N. Armstrong and Bruce A. Pappas Department of Psychology, Carleton University, Ottawa, Ont. (Canada) (Accepted 30 April 1991) Key words: Cholinergic neurotoxin; Ethylcholine aziridinium; Hippocampus; Spatial memory

This study investigated the histopathological, behavioral and neurochemical effects of bilateral injection of 2.0, 0.5 and 0.1 nmol/ventricle ethylcholine aziridinium (AF64A) on postnatal day (PND) 2. The rats showed a significant, but non-dose-related reduction of choline acetyltransferase (CHAT) in the hippocampus but not the cerebral cortex or the caudate nucleus when sacrificed on PND 16. No effect on ChAT was found in any region at PND 58. The group given 2 nmol/ventricle were hyperactive and showed a deficit in spatial learning when tested on the Morris water maze at PND 38-43. No such differences were observed for the rats injected with 0.1 or 0.5 nmol/ventricle AF64A. This spatial learning impairment in the 2 nmol group was associated with non-specific tissue damage seen only in animals from this group that were sacrificed at PND 40. This tissue damage was most evident in the left medial frontal cortex, the caudate nuclei and the anterior dorsal hippocampus.

INTRODUCTION Clement and Colhoun 4 were the first to demonstrate that acetylcholine mustard aziridinium ion subjected to alkaline hydrolysis could inhibit the high affinity uptake of choline ( H A C U ) . Subsequently, other researchers have used aziridinium choline analogs, such as the ethylcholine ion (AF64A), to destroy cholinergic terminals in the central nervous system of the rat 7'15. Injection of A F 6 4 A into the lateral ventricles has been reported to result in a significant reduction of acetylcholine (ACh), choline acetyltransferase (CHAT), and H A C U in the hippocampus and/or cortex in rats l°. These effects are reported to be dose dependent, take 2-3 days to develop, asymptote at 7 days, and persist up to 12 months. Transient reductions of other neurochemicals such as norepinephrine 15'31, dopamine x5 and serotonin 31 have also been reported. It is not clear whether these reductions are the result of reduced cholinergic activity or toxicity of A F 6 4 A to noncholinergic terminals. Recently, it has been shown that pretreatment with hemicholinium, an inhibitor of H A C U , prior to intracerebroventricular (i.c.v.) infusion of A F 6 4 A reduces damage to noncholinergic neurons 31. Bilateral infusion of 3 nmol/ventricle A F 6 4 A into the

adult rat has been reported to result in 43% reduction in hippocampal CHAT. Such animals exhibited a short-term reversible impairment in reference m e m o r y and a severe and persistent impairment in working m e m o r y when assessed by a multiple component task in a T-maze 3. This is similar to that reported after electrolytic lesion of the fimbria/fornix 16, or ibotenic acid lesion of medial septurn 13. However, this behavioural impairment may not necessarily be the result of cholinergic hypofunction. This dose has also been reported to cause localized noncholinergic cell damage 16"22. For example, Jarrard et al. t6 made unilateral injections of 3.0 nmol A F 6 4 A into the substantia nigra of an adult rat and found extensive damage at the injection site. Animals displayed ipsilateral turning one day and contralateral turning two days postlesion in addition to a significant reduction in striatal dopamine. These results are similar to those reported after destruction of dopaminergic cells using 6-hydroxydopamine and clearly illustrate the need for detailed histology following A F 6 4 A infusion. Recently Speiser et al. 3z have compared the specificity of cholinergic hypofunction induced by anoxia to that produced by bilateral infusion of A F 6 4 A in the neonatal rat. They reported a modest decrease in C h A T in the

Correspondence: B.A. Pappas, Department of Psychology, Carleton University, Ottawa, Ont., Canada KIS 5B6.

250 f r o n t a l c o r t e x ( 1 7 % ) a n d c a u d a t e n u c l e u s ( 1 0 % ) 14 d a y s a f t e r i.c.v, i n j e c t i o n o f A F 6 4 A .

In addition, AF64A-

t r e a t e d a n i m a l s e x h i b i t e d a l o n g - t e r m e l e v a t i o n in locomotor

a c t i v i t y a n d t o o k less t i m e to r e - e n t e r a d a r k

chamber

where

they had experienced

f o o t s h o c k 48 h

earlier. Anoxia-treated rats displayed a similar elevation in l o c o m o t o r similar

activity that lasted until PND

impairment

in p a s s i v e

avoidance.

a heating pad to raise its core body temperature to normal. Animals recovered completely within 30 min of surgery, and no adverse effects of the glue were evident.

Behavioural tests All behavioural tests were recorded using a ceiling mounted overhead videocamera.

42, a n d a

Spatial learning

They

Spatial learning was tested in 32 animals (8 from each group), at approximately 38 days of age using the Morris water maze task, The water maze consisted of a large cylindrical tank (150 cm in diameter, 45 cm deep) filled to a depth of 30 cm with room temperature water made opaque by the addition of powdered milk. A transparent plexigiass platform was placed in the middle of one quadrant of the tank, 1 cm below the surface of the water so that it remained invisible for the rat. The pool was placed in the middle of a small storage room containing many extramaze cues including shelves, filing cabinets, bulletin boards and windows. During spatial navigation rats were given 4 trials on each day for 5 consecutive days. At the beginning of each day a random sequence of 4 starting locations (90° apart) was generated. All animals followed this sequence for that day. For each trial the rat was placed in the water facing the wail at the designated start location. The rat was given 120 s to find the platform, climb onto it and rest for 30 s. The latency to reach the platform was recorded. If the rat was unable to locate the platform in the given time the experimenter lifted the animal out of the water and placed it on the platform for the test interval. All animals were then removed from the platform and placed at the next start location. On the sixth day of testing the platform was removed from the pool and the rat was given a spatial probe trial. Each rat was given 60 s to swim about the platformless pool. The distance that the rat swam in the quadrant previously containing the platform (the training quadrant) was compared to the total distance swam. On the seventh day of testing a black platform was placed in the pool and raised to the surface of the water. Each rat was then given four cued navigation trials. Rats were given 60 s to locate the easily visible black platform. As in the spatial navigation trials a random sequence of the four starting locations was generated and followed for each of the animals. The latency to reach the platform was recorded and used to evaluate each rat's swimming ability.

also

e x h i b i t e d a n i m p a i r m e n t in a c t i v e a v o i d a n c e t h a t w a s n o t a p p a r e n t in t h e A F 6 4 A - t r e a t e d a n i m a l s . A g a i n , h o w e v e r , it is n o t c l e a r w h e t h e r t h e i m p a i r m e n t in t h e A F 6 4 A t r e a t e d r a t w a s t h e r e s u l t o f t h e c h o l i n e r g i c deficit since S p e i s e r e t al. 32 d i d n o t e x a m i n e t h e h i s t o p a t h o l o g i c a l effect of AF64A. The present study further characterized the effects of

A F 6 4 A in t h e n e o n a t a l r a t b y e x a m i n i n g t h e r e l a t i o n s h i p b e t w e e n n e o n a t a l c h o l i n e r g i c deficit, b e h a v i o u r a l i m p a i r m e n t a n d t i s s u e loss.

MATERIALS AND METHODS

Animals The litters of 20 pregnant female Long-Evans hooded rats, bred in our colony, were used. Each litter was culled to approximately eight pups, four male and four female, and placed into standard maternity cages (lights off at 08.00 h, on at 20.00 h) with their natural mother. All pups in a litter received the same treatment to avoid differential maternal treatment of the pups. On PND 25 animals were weaned and placed individually into standard wire mesh hanging cages. For the remainder of the experiments these rats were divided into 4 groups: vehicle-injected controls (n = 5 litters), and those injected with either 2.0, 0.5 or 0.1 nmoFventriele AF64A (n = 5 litters for each group).

Preparation of AF64A AF64A (Research Biochemicais Inc.) was prepared from acetylethylcholine mustard chloride s by dissolving 3 samples (4.6, 1.15 and 0.25 mg) of the starting material in 10 ml of distilled water. An additional vial was prepared in a parallel fashion without the addition of mustard hydrochioride. It served as the vehicle control. The pH of each vial was adjusted to 11.5-11.7 with dilute NaOH. This was maintained for 30 rain before being lowered to 7.0 with dilute HCI. A small amount of NaCO 3 was then added to raise the pH to 7.4. All solutions were prepared immediately prior to use and maintained at 0-4 °C.

Surgery Pups underwent neonatal stereotaxic surgery on PND 2. After being immersed in ice (5 min), each pup received an incision (1 cm) along the midline suture to expose bregma. Pups were then placed in a Kopf stereotaxic apparatus that had been modified for neonatal surgery according to the method described by Heller et ai.12. All animals received bilateral i.c.v, injections of AF64A or vehicle at an infusion rate of 1 /~l/min. Initial placement of the cannula (right versus left) was counterbalanced within each litter. The injections were made 0.5 nun anterior from bregma, 1.5 mm on either side of the midline and 3.0 mm ventral from the surface of the skull. The cannula remained in place for 1 min immediately following the injection to allow for diffusion of the drug into the cerebrospinal fluid of the lateral ventricles. After removal of the cannula from the second ventricle the animal was removed from the stereotaxic apparatus, the incision on its scalp was glued closed using Lepage's Instabound Super Glue and the pup was placed on

Elevated-plus maze Spontaneous behaviour was measured from another 8 rats per group, at approximately 38 days of age using the elevated-plus maze 3°. This maze consisted of two open arms (50 x 10 cm), and two enclosed arms (50 x 10 x 40 cm) with an open roof. The maze was designed so that the two open arms were opposite each other and the entire apparatus was elevated to a h e ~ t of 50 cm. Each rat was evaluated on the number of open/closed arm entries during a 5 rain exposure to the maze. Activity was measured by calculating the total number of arm entries for each animal and the ratio of open to total arm entries. Normally, rats show an aversion towards the open arm 3°.

Brain histology After completion of testing in the elevated-plus maze, 8 rats from each group, at 40 days of age, were perfused with 10% formalin after a lethal intraperitoneal injection of pentobarbitol (100 mg/kg). All brains were removed within 12 h, stored in formalin and later sectioned on a microtome at 50 /~m in the coronal plane. Approximately every fifth section was mounted on a slide, stained with Cresyl violet and examined microscopically. Tissue was quantified using a modification of the procedure described by Diamond et a l : "6. This procedure was simplified by the use of commercially available software (Imaging Research, St. Catherines, Ont.) that captured and digitized an image of each coronal section. The digitized image was projected onto a monitor and measurements were taken either manually or by automated

251 RESULTS

subroutine to estimate the extent of damage caused by the drug. Six sections from each of the 32 animals were used for quantitative analysis. These sections included one taken at the most rostral section showing the decussation of the corpus callosum (section CC), one taken at the decussation of the anterior commissure (section AC), and finally one taken just caudal to the appearance of the posterior commissure (section PC) so as to include the ventral hippocampus. Thickness measurements were taken from dorsal, lateral and ventral cortex for both the left and right hemispheres. These corresponded to Diamond's cortical areas B, C and D 5'6. Anatomically these represent the frontal cortex, the forelimb area of the cortex and the parietal cortex at section CC, and also at section AC. They represent medial occipital cortex, lateral occipital/parietal cortex and temporal cortex at section PC. Additional measurements taken at sections CC and AC included the area of the corpus callosum and the caudate and for section AC the area of both the lateral ventricles. Measurements taken at section PC included thickness of the entorhinal cortex, area of the granule cell layer in the dentate gyrus, area of CA3 cell field, and the area of the most lateral cell field of Ammon's Horn made up by both CA1 ad CA2. The area of the dorsal hippocampus was estimated by averaging the bilateral area of this structure for the 3 successive sections which were immediately rostral to the rostral-most appearance of the posterior commissure and which did not display ventral hippocampus.

Biochemistry C h A T activity at P N D 16. T h r e e s e p a r a t e A N O V A s r e v e a l e d that C h A T in t h e h i p p o c a m p u s ( s h o w n in T a b l e I) was significantly a f f e c t e d by A F 6 4 A t r e a t m e n t (F3,3s = 6.00, P < 0.01). T h e r e was n o e f f e c t of A F 6 4 A t r e a t m e n t of C h A T in the c a u d a t e (F3,38 = 2.86, P > 0.05) o r t h e

cortex (F3,37 =

1.80, P > 0.05). F u r t h e r

analyses using

Newman-Keuls post hoc multiple comparison procedure ( P < 0.05) r e v e a l e d that w h e n c o m p a r e d to c o n t r o l s , t h e animals i n j e c t e d w i t h 2.0, 0.5 and 0.1 n m o l / v e n t r i c l e A F 6 4 A h a d a significant ( 4 2 % for the c o m b i n e d groups) d e p l e t i o n of C h A T activity in t h e h i p p o c a m p u s . T h e s e 3 g r o u p s failed to differ f r o m e a c h o t h e r statistically.

C h A T activity at P N D 58 No differences were observed in hippocampal (F3,31 = 0.82, P > 0.05), caudate (F3,31 -~- 1.92, P > 0.05) or cortical (F3,3~ = 0.73, P > 0.05) tissue samples on PND 58 (Table I).

Biochemical analyses Seventy-two animals were sacrificed by decapitation. The brains from all animals were rapidly removed and dissected over a saline-rinsed ice cold plate into cortical, hippocampal and caudate brain parts. Forty of these animals were approximately 16 (range 15-17) days of age. Both hemispheres were assayed for CHAT. The second group of 32 animals were approximately 58 (range 57-60) days of age, and had completed testing in the Morris water maze ten days earlier. In this latter group brain parts from the right hemisphere were immersed in liquid nitrogen and stored at -70 °C before undergoing monoamine assay using reverse-phase ionpairing high performance liquid chromatography TM.Brain parts from the left hemisphere were homogenized and assayed for ChAT according to the procedure of Fonnum 9. All protein analyses were performed using the method described by Lowry et al. 2°.

Monoamine assay at P N D 58 As shown in Table II, no statistically significant between-group differences were apparent after separate ANOVAs were performed on norepinephrine (NE) in the c o r t e x (F3,27 = 0.04, P > 0.05), dopamine (DA) in the cortex (F3,27 = 2.22, P > 0.05), D A in the caudate nucleus (F3,24 = 2.56, P > 0.05) or NE in the hippocampus (F3,26 = 0.47, P > 0.05). Despite the absence of significance, it may be worthy to note that there was a dose-dependent depletion of cortical D A in the drug

TABLE I ChATactivity at PND 16 and PND 58 Expressed in nmol/h/mg protein + S.E.M. AF64A dose (nmol/ventricle) Vehicle

O.I

0.5

2.0

PND 16 Hippocampus (n) Caudate (n) Cortex (n)

55.6 + 5.9 (15) 158.2 + 18.9 (15) 49.0 4- 3.5 (15)

36.3 +_2.7* (8) 100.1 + 9.7 (8) 42.6 ___4.0 (7)

29.5 + 4.3* (7) 105.9 + 24.2 (7) 30.4 4- 7.8 (7)

30.6 4- 5.1" (9) 158.9 + 33.3 (9) 52.1 + 10.7 (9)

PND 58 Hippocampus

80.1 + 7.4

70.8 + 4.0

82.4 + 4.0

75.4 + 6.5

(n)

(8)

(8)

(8)

(8)

Caudate

230.7 _ 16.9

280.3 4- 22.8

218.8 _ 20.6

231.9 + 17.9

(n)

(8)

(8)

(8)

(8)

Cortex (n)

75.9 _ 5.3 (7)

78.7 4- 3.1 (7)

70.5 _ 3.1 (7)

72.2 +_ 4.8 (8)

* Significantly different from control (Newman-Keuls, P < 0.05).

252 TABLE II Catecholamine levels at PND 58

Expressed in pmol/mg protein + S.E.M. AF64A dose (nmol/ventricle) Vehicle

Hippocampus NE (n) Caudate DA

(n) Cortex NE (n) DA (n)

0.1

0.5

2.0

16.0 + 1.5 (7)

13.6 + 1.2 (8)

15.0 + 2.1 (8)

17.2 + 3.6 (7)

1207.5 _+ 101,5

(7)

1448.9 + 108.6 (8)

1080.2 + 90.4 (7)

1136.1 + 119.6 (6)

19.5 + 3.1 (8) 40.9 _+7.8 (8)

20.7 + 2.0 (8) 33.8 + 5.9 (8)

20.7 + 3.8 (8) 26.6 + 4.6 (8)

19.5 + 3.9 (7) 19.4 _+5.5 (7)

t r e a t e d animals where the 2.0 nmol animals displayed a 50% d e p l e t i o n when c o m p a r e d to controls. Quantitative histology

The results of this assessment are shown in Table III. Only areas where there were statistically significant group differences are shown to simplify the data presentation. The findings are summarized below for each brain area assessed. Cortex. A n a l y s e s o f variance followed by N e w m a n Keuls post hoc tests r e v e a l e d that the left dorsal cortex was significantly t h i n n e r at sections A C (F3,zs = 4.69, P < 0.05) and C C (Fa,25 = 3.57, P < 0.05) in rats t r e a t e d with 2.0 nmol/ventricle A F 6 4 A (see Table III). A l l o t h e r analyses of the left cortex thickness were not significant. Similar analyses of the right cortex showed that the dorsal cortex was significantly thinner at section A C in rats t r e a t e d with 2.0 nmol/ventricle A F 6 4 A (F3,25 = 3.19, P < 0.05). A l l o t h e r analyses of the right cortex were nonsignificant.

Hippocampus. S e p a r a t e A N O V A s of the left a n d right hemispheres carried out on section PC indicated that there were no differences between the groups for the (1) a r e a of C A 1 / C A 2 , (2) a r e a of C A 3 , (3) a r e a of the dentate gyrus and (4) thickness o f the e n t o r h i n a l cortex (all F ' s < 1.2, ns). T h e a r e a of the dorsal h i p p o c a m p u s was significantly r e d u c e d h o w e v e r (/73,26 = 5.04, P < 0.01), with the 2.0 nmol injected rats showing significantly less area when c o m p a r e d to all o t h e r groups ( P ' s < 0.05). Qualitative assessment o f all available sections for each rat showed seven out o f eight animals injected with 2.0 nmol/ ventricle A F 6 4 A had extensive tissue d a m a g e t o the dorsal h i p p o c a m p u s which was most a p p a r e n t in the anterior sections. In 4 of these cases the d a m a g e was bilateral and the dorsal h i p p o c a m p u s was extensively destroyed. In three of these cases the d a m a g e was primarily unilateral and most severe in the left hemisphere. Caudate, corpus callosum and ventricles. A n a l y s e s of

TABLE III Thickness (ram, dorsal cortex) and area (rama, all brain structures) in control and AF64A-treated rats AF64A dose (nmol/ventricle) Vehicle

Dorsal cortex

AC CC

Dorsal hippocampus Ventricles Caudate

AC AC

0.1

0.5

2.0

0.03 0.14 0.03 0.44

1.86 ~ 0.04 1.85 + 0.03 1.84 + 0.03 11.55 + 0.34

1.83 + 0.03 1.85 -+ 0.03 1.88 + 0.02 11.02 + 0.07

1.70 + 0.10" (right) 1.55 + OA4* (left) 1.58 + 0.14" (left) 8.36 _+ 1.02"

0.19 + 0.05 8.99 + 0.27 9.22 + 0.21

0.18 _+0.06 8.88 + 0.30 9.03 + 0.41

0.22 + 0.06 8.70 + 0.42 9.29 + 0.45

2.70 + 1.83" (left) 7.54 :t: 0.69* (right) 7.44 ± 0.88* (left)

1.84 ± 1;86 + 1.81 + 10.58 +

* Significantly different from control (Newman-Keuls, P < 0.05).

253 variance followed by Newman-Keuls post hoc tests (P < 0.05) revealed that the rats treated with 2.0 nmol AF64A had significantly smaller left (F3,24 = 8.69, P < 0.05) and right (/73,24 - 4.79, P < 0.05) caudates when compared to all other groups at section AC. No differences between the groups were observed for the area of the caudate at section CC. Separate ANOVAs were performed on measurements of the right and left ventricles at section AC. Analysis revealed that the left ventricle was significantly affected by drug treatment (F3,24 = 3.74, P < 0.05). N e w m a n Keuls post hoc analyses revealed that the animals treated with 2.0 nmol/ventricle AF64A had approximately 10fold larger left ventricles when compared to animals of all other groups. No differences were apparent in measurements taken from the right ventricle (F3,24 = 1.86, P > 0.05).

Behaviour Spatial learning. All animals swam effectively, using primarily their hindlegs to propel them forwards, As shown in Fig. 1, a repeated measures A N O V A using five blocks of four daily trials as the within-subject factor revealed that place navigation was affected by drug treatment (F12,112 = 3.48, P < 0.001). Newman-Keuls post hoc analyses (P < 0.05) revealed that the animals treated with 2.0 nmol/ventricle AF64A performed similar to controls on day 1, but failed to locate the hidden platform as quickly as controls on days 2, 3 and 4.

120 -

However, by day 5 this group was performing at a level comparable to controls. Two separate A N O V A s were used to evaluate performance during the spatial probe trial. The first compared the groups on the total distance swum during 1 rain exposure to the platformless pool. All groups swam approximately the same distance (F3,28 = 0.096, P > 0.05). The second compared the groups on the ratio between the distance swum in the quadrant formerly containing the platform, and the total distance swum. Again, no differences were found. This was not surprising, as all animals performed similarly to controls on the last day of testing during place navigation. A repeated measures A N O V A was used to evaluate performance in the cued navigation trials. As shown in Fig. 1, all the groups improved their performance from trial 1 to trial 4 and no differences were apparent between the groups (F3,2s = 0.45, P > 0.05). Elevated-plus maze. Two separate ANOVAs were used to evaluate performance in the elevated-plus maze. The first analysis evaluated the total number of arm entries made by each animal during five min exposure to the maze. There was a nearly significant affect of drug treatment on total arm entries (F3,30 = 2.67, P = 0.06). As shown in Fig. 2, Newman-Keuls post hoc analyses (P < 0.05) revealed that the animals injected with 2.0 nmol/ventricle AF64A made more arm entries than controls during the given time period. The second analysis compared group performance on the ratio

120 0.1 nmol

100'

100

80'

•

0.5 nmol

m

2.0 nmol

80 vehicle

@

,.d

60'

60

40"

40

20

20

0

I

1

I

I

i

!

1

2

3

4

5

6

Spatial navigation days

0

0

a

I

1

2

i

I

3

i

I

4

i

1

5

Cued navigation trials

Fig. 1. Mean escape latencies (seconds) during spatial navigation (left) and cued navigation (right) in the Morris water maze. The animals treated neonatally with 2.0 nmol AF64A were slower to escape on days 2, 3 and 4 when compared to vehicle-treated controls. This impairment was observed only in the spatial navigation task. Bars indicate standard errors. *Significant differences from corresponding point for vehicle controls (P < 0.05, Newman-Keuis).

254

it

T

0.4{ =

*~

0.3(

3

"

E 2

0

0

0

o.l~

0.~1

Veh

0.i

0.5

2.0

Dose AF64A (nmoi/ventriele)

Veh

0.1

0.5

2.0

Dose AF64A (nmol/ventriele)

Fig. 2. The mean number of total arm entries (left) and the mean ratio of open to total arm entries (right) during the 5 rain exposure to the elevated,plus maze. The animals treated neonatally with 2.0 nmol AF64A made more arm entries than any other ~ . However they displayed the normal aversion to the open arm. Bars indicate standard errors. *Significant difference from the vehicle controls (P < 0.05, Newman-Keuls).

between open and total arm entries. No differences were apparent between groups (F3,27 = 0.313, P > 0.05), suggesting that the neonatally drug-treated animals showed the normal aversion towards the open arms. DISCUSSION Neonatal i.c.v, injection of 2.0, 0.5 and 0.1 nmol/ ventricle AF64A reduced ChAT activity in the hippocampus of animals that were assayed 14 days post-lesion, on PND 16. However, ChAT activity in all 3 drug groups, assayed 56 days post-lesion on PND 58, was similar to that of controls. The observed decrease in enzyme activity at PND 16 supports the earlier results of Speiser et al. 31 who reported a more moderate 17% decrease of ChAT activity in the frontal cortex and 10% decrease in the caudate 14 days after 1 day old rats were injected with 2.0 nmol/ventricle AF64A. These investigators did not, however, assess the duration of this effect. The present results indicate that some form of compensatory enzyme recovery takes place by the time these AF64A-injected animals reach sexual maturity. This recovery could arguably be the result of at least two mechanisms. First, the remaining ACh neurons may sprout compensatory axons in response to destruction of nearby cells. This is known to be the case in adult rats that display 2- to 6-fold increases in brainstem NE levels following neonatal lesion of noradrenergic projections to the cerebral cortex ~9. Alternatively, undamaged cholinergic cells may compensate by increasing their production of enzyme

molecules. This method of recovery has also been described following 6-hydroxydopamine lesion. Degeneration of catecholaminergic nerve terminals in the hippocampus, resulting in a 85-90% decrease in NE levels causes activation of tyrosine hydroxylase in the rat s. Tyrosine hydroxylase is the r a t e - l i m i n g enzyme in the production of NE, and this phenomenon is thought to be an adaptive response to the lesion. Based on the present results, it is clear that the nervous system can recover biochemically after neonatal administration of AF64A. However, the mechanism of this recovery remains unspecified. There was evidence of non-specific tissue necrosis in the rats who had received 2.0 nmol of AF64A. Quantitative histological analysis of coronal sections at fixed landmarks revealed that the 2.0 nmol group had bilateral tissue damage extending along the dorsal a n d ventral walls of the lateral ventricles. This d ~ a g e included a reduction in both left and right caudate size, enlargement of the left lateral ventricle and a subsequent reduction in cortical thickness in the left cortex at the dorsal and lateral segments in which corresponded to frontal and parietal cortex 29. The area of the dorsal hippocampus was also reduced. Qualitative assessment indicated that seven out of eight animals injected with 2.0 nmoFventricle AF64A had damage to the dorsal hippocampus. In four of these cases there was bilateral damage which was severe - - in the remaining three cases the damage was primarily unilateral, usually to the left hem'mphere, It is not clear why more extensive damage was generally observed in

255 the left hemisphere, since the initial injection of AF64A into the right or left ventricle was counterbalanced within each group. It is not likely that the tissue necrosis observed in the 2.0 nmol group was the result of damage to ACh neurons. There are two lines of evidence to support this claim. First, we have shown that the animals injected with 0.5 or 0.1 nmol AF64A failed to exhibit pathology despite showing reductions in hippocampal ChAT that were similar to those found in the 2.0 nmol group. If the damage was the result of neonatal cholinergic lesion in the hippocampus these other two groups would also have shown this pathology. Second, the damage observed in the 2.0 nmol group was extensive but limited to the perimeters of the lateral ventricles rather than subtle and widespread as would be hypothesized after denervation of cholinergic projections to hippocampus or cerebral cortex 14. Therefore, we conclude that damage to cholinergic neurons is not required for this pathology and that it is evidence of AF64A's non-specific effects. More direct evidence for non-specificity would be provided if it could be shown that treatment prior to AF64A infusion with hemicholinium, an inhibitor of H A C U , did not eliminate this effect. Neonatal injection of 2.0 nmol/ventricle AF64A impaired spatial learning in the Morris water maze. The 2.0 nmol animals were slower to learn the location of the hidden platform as seen in their longer daily latency to escape on days 2, 3 and 4. By day 5, however, these animals were performing at a level comparable to controls. This impairment seems unlikely to be the result of neonatal cholinergic dysfunction since the 0.5 and 0.1 nmol animals, who suffered a similar cholinergic deficit, performed like controls throughout testing. It is more likely that the deficit observed in the 2.0 nmol group was the result of damage to the dorsal hippocampus. The neocortex plays an important role in spatial learning, but only medial frontal and orbital frontal lesions are associated with profound deficits in the Morris water maze 17. In the present study little damage was observed in the frontal cortex of rats treated neonatally with 2.0 nmol AF64A. Rather, damage was restricted primarily to parietal and forelimb/frontal cortical areas. Lesions to these areas result in only minor place learning impairments 17. Furthermore, damage was induced on PND 2, while these animals were still neonates. Neonatal cortical damage is associated with significant sparing of function when compared to damage sustained in adulthood. This phenomenon extends to include spatial learning in the Morris water m a z e 34. Therefore, damage to the neocortex was unlikely to cause the profound impairment in spatial learning observed in the present study.

On the other hand, extensive tissue necrosis was observed in the dorsal hippocampus of animals treated neonatally with 2.0 nmol AF64A. There are two lines of evidence to support the role of this damage in the Morris water maze impairment. First, the dorsal hippocampus may be specifically involved in learning. Bilateral electrical stimulation to induce a localized blockade of function in the dorsal hippocampus prevents rats from learning a position reversal task 26, and a visual discrimination task 2°. Second, the hippocampus plays a vital role in spatial localization 25. Damage to this structure impairs performance in spatial localization tasks such as the Morris water maze 22'33 and the 8-arm radial m a z e 27'28. The longer escape latencies of the 2.0 nmol animals cannot be ascribed to motor impairments or lack of motivation to find the hidden platform because no group differences were apparent during the cued navigation portion of testing in the water maze. When behaviour in the elevated-plus maze was evaluated, the 2.0 nmol group was found to be hyperactive. They entered more arms of the maze than controls during the five min testing period. Previously, Speiser et al. 32 have shown that neonatal bilateral i.c.v, injection of 2.0 nmol/ventricle AF64A caused elevated activity of rats that persisted until the animals were 120 days of age. The decrease in activity observed in young rats at about 30 days of age has been attributed to the development of an inhibitory cholinergic system 2. Speiser et al. 32 argued that partial impairment of this system early in development may cause persistence of hyperactivity. However, it is clear from these results, that neonatal reductions in ChAT are not sufficient to cause this persistence. In this study, the 0.5 and 0.1 nmol groups failed to exhibit hyperactivity even when they showed reductions in ChAT at 16 days of age that were similar to those found in the 2.0 nmol group. Perhaps the persistance of hyperactivity in the 2.0 nmol group reflects damage to the dorsal hippocampus. A second possible explanation for the hyperactivity of the 2.0 nmol animals is the 50% depletion of cortical DA. Previously, Heffner et al. 11 have demonstrated that neonatal depletion of cortical D A by destruction of DA-containing cells in the ventral tegmental area caused increased motor activity at PND 38 in the animals injected with 2.0 nmols AF64A. Future research should re-examine the possibility that neonatal i.c.v, administration of AF64A depletes the cortex of DA. In summary, i.c.v, administration of 2.0 nmol/ventricle AF64A resulted in a significant reduction of ChAT activity in the hippocampus of sexually immature rats assayed on PND 16. Animals of this group showed normal ChAT activity in the hippocampus when assayed at PND 58. Reduction in hippocampal cholinergic activity

256 was a c c o m p a n i e d by extensive tissue necrosis along the dorsal and ventral p e r i m e t e r s of the lateral ventricles when these animals underwent histological analysis at P N D 40. This tissue d a m a g e was most evident in the frontal and parietal areas of the left cortex, in both the right and left caudate in the area of the brain containing the decussation of the anterior commissure and finally, at the dorsal h i p p o c a m p u s just anterior to area of the brain containing the decussation of the posterior commissure. We suspect this d a m a g e to be evidence of A F 6 4 A ' s non-specific toxicity. Behaviourally, the 2.0 nmol animals displayed a spatial learning i m p a i r m e n t on days 2, 3 and 4 of testing in the Morris water maze. By day 5 however, they were p e r f o r m i n g like controls. Analysis of spontaneous b e h a v i o u r in the elevated-plus maze at P N D 58 r e v e a l e d that these animals e n t e r e d m o r e arms than controls during the given time. F u r t h e r m o r e , the 2.0 nmol animals displayed the n o r m a l aversion towards the o p e n arm. We suspect that both the hyperactivity and the i m p a i r m e n t in spatial learning o b s e r v e d in the 2.0 n m o l animals are evidence of A F 6 4 A ' s non-specific

effect to the dorsal hippocampus. I.c.v. administration of 0.5 and 0.1 nmol/ventricle A F 6 4 A resulted in a specific reduction in h i p p o c a m p a l C h A T at P N D 16. H o w e v e r , by P N D 58, animals injected with 0.5 and 0.1 nmols A F 6 4 A showed n o r m a l enzyme activity, indicating that C h A T in the h i p p o c a m p u s returns to n o r m a l by the time these neonatally d e p l e t e d animals are sexually mature. F u r t h e r m o r e , there were no behavioural differences between these animals and vehicle administered controls in place navigation, cued navigation o r spatial p r o b e portions o f testing in the Morris water maze, beginning at P N D 38. In addition to this normal spatial learning ability, the 0.5 and 0.1 nmolinjected animals failed to differ from controls on all aspects of s p o n t a n e o u s b e h a v i o u r in the elevated-plus maze. T h e y e n t e r e d the same n u m b e r o f arms as controls and displayed the n o r m a l aversion towards the open arm.

Acknowledgement. This research was supported by a Natural Sciences and Engineering Research Council operating grant to B.A.P.

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