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Embryonic entorhinal transplants partially ameliorate the deficits in spatial memory in adult rats with entorhinal cortex lesions
Brain Research 792 Ž1998. 97–104
Research report
Embryonic entorhinal transplants partially ameliorate the deficits in spatial memory in adult rats with entorhinal cortex lesions Wenbo Zhou a , Deming Jiang b , Geoffrey Raisman c , Changfu Zhou
a, )
a
Shanghai Institute of Physiology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai, 200031, China Shanghai Brain Research Institute, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai, 200031, China The Norman and Sadie Lee Research Center, Laboratory of Neurobiology, National Institute for Medical Research, Medical Research Council, The Ridgeway, Mill Hill, London, NW7 1AA, UK b
c
Accepted 27 January 1998
Abstract Our previous studies have demonstrated that axons from grafts of embryonic entorhinal cortex ŽEC. can reinnervate the deafferented zones in the hippocampus and form synaptic connections with the host dentate gyrus in adult mice and rats deprived of their own entorhinal inputs. Here, we have examined the ability of the EC grafts to ameliorate deficits in spatial memory. Three months after transplantation, the grafted rats and control animals were subjected to Morris water maze testing followed by histological examination. According to the exact position of grafts in the host brain, the rats with lesion and EC transplants were divided into two groups, one with EC grafts contacting both the hippocampus and overlying neocortex Ž n s 7, EC1. and another with EC grafts confined within the hippocampus Ž n s 6, EC2.. While EC2 rats were still as impaired as those with lesion and transplants of non-entorhinal cortex Ž n s 10, NEC. or with lesions only Ž n s 7, LES., the EC1 rats performed better than the LES group. In a spatial memory trial, the EC1 group made more crossings over platform site and showed more focused search behavior than EC2, LES, NEC groups. The data suggest that EC grafts could partially ameliorate the deficit in spatial learning behavior in the EC-lesioned adult rats. The requirement for the graft to contact both the neocortex and the hippocampus suggests that the functional effects may be exerted by the formation of new neocortical-EC graft-hippocampal circuits. q 1998 Elsevier Science B.V. Keywords: Hippocampus; Neocortex; Repair; Reinnervation; Grafts; Behavior
1. Introduction The entorhinal cortex ŽEC. is a major relay providing a wide range of cortical input to the hippocampus and transferring the information processed in the hippocampus to the cortices via reciprocal projections both between neocortical multimodal association areas and the EC, and between the EC and hippocampus w1,18,19,32,33,37,40x. The EC has been suggested to play a crucial role in the processing of learning and memory, a proposition that has been supported by abundant evidence from both clinical and experimental neurology. The EC shows some of the earliest and most severe pathological alternations in Alzheimer’s disease ŽAD. w6–8,16,23x. There is a very severe loss in the EC even in very mild AD cases that are )
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0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 1 2 5 - 5
at the threshold for clinical detection of dementia w8,14,18x. The vulnerability of the EC to neuropathological changes in other degenerative disorders with mnemonic malfunction, such as Parkinson’s and Huntington’s diseases, also points to its involvement in learning and memory w6,18,31x. Surgical or electrolytic damage to the EC in laboratory animals has been shown to impair memory, reference memory and memory retention w17,22,28,29,34,39x. Schenk and Morris w30x reported that rats with the EC lesion show severe post-operative impairment in the initial acquisition of the place-navigation task indicating impairment of spatial learning and memory. Transplants of embryonic neuronal tissue are capable of reinnervating denervated areas in the adult mammalian brain, and restoring functions lost as a result of lesions w4,9x. In the septohippocampal and nigrostriatal systems, transplanted embryonic cholinergic and dopaminergic neurons have been shown to send their axons into the dener-
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vated target area at a considerable density, and consequently completely or partially restore functions w3,5,10,11,25,27x. It has been suggested that these graft-derived effects may be exerted by restoration of tonic levelsetting in the deafferented areas due to spontaneous or active transmitter release from the transplanted neurons w2,20,26,35x. We have previously shown that the embryonic entorhinal transplants can survive in the adult rodent hippocampus and specifically reinnervate entorhinally deafferented territory in the outer molecular layer ŽOML. of the dentate gyrus ŽDG. and the stratum lacunosum-moleculare ŽSLM. of CA1 and CA3 w41,42x. Electron microscopy revealed that ingrowing fibers from EC grafts form asymmetric synaptic connections on dendritic spines w42x. We now use the Morris water maze test w24x to explore whether such reconstruction of entorhino-hippocampal projections is able to exert any beneficial effect on the impaired performance of adult rats with complete bilateral transections of perforant paths ŽPP., or destruction of the EC. Here, we report that grafts of fragments of embryonic EC tissue placed as columns extending from the overlying neocortex ventrally through the hippocampus ameliorate the impairment of spatial water maze memory caused by entorhinal lesions; no such effect was found when the grafts were confined to the hippocampus.
perpendicular to the midline was one beginning 2.0 mm caudal, 2.5 mm lateral to the lambda, and extending laterally to the crista frontalis, another was beginning from the end of the first at a frontal dip angle of 308 lateral to the crista frontalis. The microknife was inserted into the brain through two trenches, respectively, and moved down along two curved surfaces just before the medial and lateral angular bundles entering the hippocampus. These perforant path transections completely disrupt the entorhinal efferents to the hippocampus, with little bleeding, and minimal damage to the hippocampus w41,42x. Ten to fourteen days following the bilateral PP lesion, the animals in EC1, EC2 and NEC groups received EC or NEC transplantation. The EC Žthe ventrolateral cortex of the caudal pole of the hemisphere. and NEC Žfrontal cortex. were dissected out from E17 rat embryos Žthe mating day as E0. under dissection microscope, and loaded into an 0.8-mm internal diameter glass tube Žthe length of the transplant mass was either 4.0 mm for EC1 and NEC animals or 2.5 mm for EC2 animals.. Prior to graft preparation, one burr hole Ždiameter of 1 mm. was drilled in each side of the skull Žcoordinates: 4.0–4.2 mm caudal to bregma, 2.2–2.5 mm lateral to midline.. The tube containing the grafts was connected to a metal rod and positioned on the stereotaxic frame and lowered into the brain until the low tip of the implant 4.3 mm below the dura. The grafts were left for 5 min in the brain followed by slow withdrawal of the glass tube and metal rod.
2. Materials and methods 2.3. Morris water-maze test [24] 2.1. Subjects Fifty adult female Sprague–Dawley rats, weighing 220–250 g at the start of experiments, were allocated to five groups: EC1 group Ž n s 10. with bilateral transections of the PP and bilateral EC grafts that were made as a column going through overlying neocortex and hippocampus, EC2 group Ž n s 10. with bilateral transections of the PP and bilateral EC grafts restrictively within the hippocampus, NEC group Ž n s 10. with bilateral transections of the PP and bilateral non-entorhinal cortex ŽNEC. grafts as a column-like EC1 grafts, LES group Ž n s 10. with bilateral transection of PP without grafts, and NOR group Ž n s 10., unoperated controls. 2.2. Lesion and transplantation surgery The general procedure used for producing PP lesions and EC transplantation has been described previously w41,42x. Briefly, animals were anaesthetized with sodium pentobarbital Ž40 mgrkg, i.p.. and placed in a stereotaxic apparatus. After exposing the skull, a specially tooled microknife of silver leaf Ž0.3 mm thick, 2 mm wide and 20 mm long with a radian of 908 at its middle point. was used to cut the perforant paths. Using a small hand drill, two trenches Ž1 mm wide. were made on each side of the skull:
A circular pool Ždiameter 180 cm, height 50 cm. was filled to a depth of 20 cm with water at room temperature Ž20–228C. made opaque by the addition of 30 ml Indian ink. Four equally spaced points around the edge of the pool were designed as four starting positions: east ŽE., south ŽS., west ŽW. and north ŽN.. An escape platform Ždiameter 9 cm. was set 1 cm below the surface of the water and placed in a constant position in the middle of the SW quadrant. The rat in the pool was trained to find the platform using a variety of extramaze cues, including the lamp, wall posters, curtain, window, desk, video recorder, monitor, etc. A video camera was placed directly above the center of the pool. The experimenter always sat at the same position. Three months after transplantation, each rat was given 4 trials on each of 9 consecutive days. They were placed in the water facing the wall from one of four starting sites in a random sequence Ži.e., SENW, so that each site was used once everyday.. The latency to find the escape platform was measured during each trial. Upon finding and climbing onto the platform, the rat stayed there for 30 s. If the rat could not find the platform within 60 s, it was guided by the experimenter and given a maximum score of 60 s. Before the next trial, the rat was allowed to rest in a cage for 60 s. On the fourth trial of training day 9, the platform
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was removed and the swimming behavior of each rat was recorded for 60 s. 2.4. Histology Upon completion of the behavioral testing, animals were perfused transcardially with physiological saline followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer ŽPB, pH 7.4.. The brains were removed and kept in a 20% solution of sucrose in 0.1 M PB ŽpH 7.4. at 48C prior to further processing. Frozen coronal sections of 50 m m thickness were cut from each brain and collected in 0.01 M phosphate buffered saline ŽPBS.. Thionin and acethylcholinesterase ŽAChE. staining were performed on alternative sections to examine the survival of the grafts and extent of lesions as described previously w41,42x. The semi-quantitative measurement of graft size was conducted under microscope on the thionin-stained sections passing through the middle plane of each cylinder-like grafts, and the volume of grafts was calculated by p Ž1r2 width. 2 = length of graft. 2.5. Data statistics Data of Morris water maze test were collected using a path-tracing software ŽShanghai Jiao-Tong University, China. in a computer. All raw data were processed using either SigmaPlot ŽJandel Scientific, V1.0. for behavioral test and GraphPad Instat ŽV2.0. for measurement of graft size. For comparison of the data from Morris water maze test, one-way or two-way analysis of variance ŽANOVA. were performed in Excel ŽMicrosoft Office, V7.0. fol-
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lowed by Student’s t-test at the significance of 0.05. In addition to one-way ANOVA, unpaired Student’s t-test and Tukey–Kramer multiple comparison test were used for analysis of morphological measurement.
3. Results 3.1. Histology Most of the lesioned rats showed substantial bilateral disruptions of the medial and lateral entorhinal cortex throughout its dorsoventral extent ŽFig. 1.. Thus, the transections of bilateral perforant paths were complete. Three of the rats in the LES group had partial sparing of the EC, and were excluded from further analysis. Seven rats in the EC1 group and 10 rats in the NEC group had surviving bilateral columnar grafts ŽFig. 2A,B. that consisted of an overlying intraneocortical portion and a successively intrahippocampal portion. Six EC2 grafted rats had bilateral grafts ŽFig. 2C. that were mainly confined to the host hippocampus. It was noticed that the intraneocortical portions of EC1 and NEC grafts lied passing through over half of the neocortical depth, and all intrahippocampal grafts ran over two-thirds of dorsal hippocampal depth. For the purpose of multiple group comparison, the graft volume was divided into intraneocortical portion and intrahippocampal portion. Semiquantitative measurements ŽTable 1. showed that there was no significant difference in the graft volume of either intraneocortical portion or intrahippocampal portion between EC1 group
Fig. 1. Thionin-stained horizontal sections showing entorhinal cortex ŽEC. of intact ŽA. and lesioned rat ŽB.. Note the subiculums Žsb. intact in the EC-lesioned rat ŽB.. Scale bar: 1000 m m.
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Fig. 2. Representational examples of Nissl-stained coronal sections cut through grafts ŽG. from rats of EC1 ŽA., NEC ŽB. and EC2 ŽC. groups, showing grafts lying in both neocortex and hippocampus ŽA, B. or only within the hippocampus ŽC.. D, E and F are higher magnifications of the areas outlined by the boxes in A, B and C, respectively, showing surviving neurons in the grafts. Arrows and arrowheads, respectively showing well integration of grafts with host neocortex ŽNX., and the outer molecular layer ŽOML. of the host dentate gyrus ŽDG. and the stratum lacunosum-moleculare ŽSLM. of the host field CA1. Scale bars: 1000 m m for A, B,C; 200 m m for D, E and F.
and NEC group Žunpaired Student’s t-test, both p ) 0.05.. Although one-way ANOVA showed that there was a slight difference in the graft volume of intrahippocampal portion among EC1, EC2 and NEC groups Ž FŽ2,39. s 3.39, p s 0.047., the post Tukey–Kramer multiple comparison tests
indicated no significant difference in intrahippocampal graft volume between any two groups ŽEC1 vs. EC2 or EC1 vs. NEC or EC2 vs. NEC, all three p ) 0.05.. The grafts were well integrated with host tissue including neocortex in rats of the EC1 and NEC groups with
Table 1 Measurements of graft size Group
EC1 NEC EC2
n
14 16 12
Intracortical portionrIntrahippocampal portion Length Žmm.
Width Žmm.
Volume Žmm3 .
1.044 " 0.049r1.372 " 0.046 1.088 " 0.046r1.314 s 0.046 r1.245 " 0.051
0.682 " 0.051r0.837 " 0.034 0.663 s 0.049r0.683 s 0.052) r0.699 " 0.044)
0.428 " 0.065r0.774 " 0.072 0.331 " 0.027r0.533 " 0.095 r0.484 " 0.058
Values are mean " S.E.M. of graft sizes in each group. )Significantly different from EC1 Ž P - 0.05. by unpaired Student’s test and post Tukey–Kramer multiple comparison test.
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little indication of gliosis along the local host-graft border. In the host hippocampus, all those grafts showed direct contact with the OML of the DG and the SLM of CA1 and CA3, all of which are entorhinal territories ŽFig. 2D,E,F.. The Nissl stain showed that the grafts contained healthy large pyramidal-type cells ŽFig. 2D,E,F., with no laminar appearance characteristic of normal cortex in situ. AChE staining showed an intense band of AChE positive reaction in the OML of the host DG, further indicating the completeness of the PP lesions Ždata not shown.. In the remaining 3 rats of the EC1 group and 4 rats of the EC2 group, the grafts did not make precise contact with the host hippocampus, and these were excluded from further analysis. 3.2. Performance in the water maze During the 9-day training period, all groups reduced their swimming time Žlatency. significantly to locate the submerged platform ŽFig. 3; repeated measures ANOVA, effect of day on latency, F8,280 s 8.71, p - 0.001.. A significant group effect and group = day effect ŽGroup, F4,35 s 10.2, p - 0.001; Group= Day, F32,280 s 5.43, p 0.001. were also observed, suggesting that different groups learned the task at different rates. The normal rats learned
Fig. 4. ŽA. Average number of annulus crossings during the spatial memory trial. ) p- 0.05; )) p- 0.01 as compared to lesion alone group. ŽB. Relative distance swim in each quadrant during the spatial memory trial, upon removal of the escape platform. ) p- 0.05; )) p0.01 as compared to the distance spent by the same rats in the remaining quadrants.
Fig. 3. Effects of entorhinal transplants on performance of the water maze task. Average latency for the rats of different groups to escape on to the hidden platform during the acquisition of the place navigation task is indicated. Each point represents the mean value for a block of four trials on each of nine consecutive days when tested three months after EC or NEC bilaterally grafting to adult rats with bilateral perforant path transections.
the task much more rapidly than all other animals and reached asymptotic performance at the fourth testing day. The remaining four groups exhibited virtually identical learning rates and did not differ from each other Ž p ) 0.5., except for EC1 group, which showed slightly better performance than EC2, LES and NEC groups in the last four testing days Ž p ) 0.05..
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Fig. 5. Examples of swimming paths taken by representative rats from the different groups during the spatial memory trial on the last day of the training at three months after grafting. The filled circle indicates the former platform site. Note the spatially focused search pattern over the previous platform site displayed by the rats of the normal and EC1 groups.
3.3. Spatial memory After the three trials on day 9, a fourth 1-min memory trial was performed without platform in the tank. The behavior of rats in the spatial memory trial was quantified by counting the number of crossings each rat made over the former platform site Ždefined as an annulus twice the area of the real platform., and measuring the swimming distance in each of 4 quadrants. As illustrated in Fig. 4A,B, the normal rats crossed more times over the previous platform site than all other groups Ž p - 0.01. and swam mainly in the platform quadrant Ž F3,36 s 114, p 0.001., clearly indicating that they had maintained their spatial memory. In contrast, EC2, NEC or LES rats made few crossings over the platform site and showed no preference in distribution of their swim distance ŽEC2, F3,20 s 1.67; NEC, F3,36 s 2.67; LES, F3,24 s 1.77; all n.s... However, the rats in EC1 group showed significantly more crossings over platform site than EC2, NEC and LES group Ž p - 0.05., and they swam preferentially in the platform quadrant Ž F3,24 s 21.9, p - 0.001., indicating these rats partially remembered the previous position of the platform. It was found that only NOR and EC1 rats displayed a spatially focused search behavior ŽFig. 5. and spent most of their swimming over the former platform site Žindicated by the filled circle in the lower left quadrant of the pool., which indicates an intact spatial memory for the original
platform site. In contrast, EC1, NEC and LES rats did not show this behavior, and they swam in a random fashion along the wall of the pool, indicating non-spatial search strategy taken by them in this water maze task.
4. Discussion The present results provide evidence that the entorhinal grafts which contact both the hippocampus and overlying cortex are capable of reducing the spatial learning deficits induced by the perforant path lesions in the adult rat. However, those rats with entorhinal grafts confined within the host hippocampus, and not contacting the neocortex did not exert any positive effects on the behavioral deficits. Histological examination showed that healthy grafts were well integrated with the host in all rats of EC1, EC2 and NEC groups used for the behavioral statistics. During the acquisition period, there was no significant difference in the latency to locate the platform between lesioned rats with or without any types of grafts. However, the different search behavior was observed in the spatial memory test and this reached statistically significant levels. While EC1 group showed marked improvement in the memory of previous platform site, as indicated by making more crossings over platform site, and showing more focused searching in the platform quadrant, the EC2, as well as NEC and LES groups were still impaired and exhibited random
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non-spatial searching behavior. The data suggest that the EC grafts passing through overlying neocortex and the hippocampus of the host can partially ameliorate the deficit of the adult rat in spatial learning and memory caused by lesions disconnecting the host entorhinal area and hippocampus. It has been reported that severe damage to the entorhinal cortex results in a permanent impairment in the spatial and non-spatial learning w22,30,39x. In our material, the extent of recovery in the maze task may be limited by the degree of reinnervation of the host hippocampus by the grafts, andror the degree of innervation of the grafts by host cortical afferents. Similarly, it has been reported that EC grafts located in the damaged cortical area Žhomotypic grafts. were unable to ameliorate the learning ability tested by forced alternation task w13x, although the grafts could both innervate host hippocampus and receive some cortical innervation w12x. In our experiments, direct contact of the grafts with both the hippocampus and the neocortex was crucial for amelioration of the spatial memory deficits. As indicated in our previous studies, the outgrowth of entorhinal axons from embryonic grafts to the host hippocampus only occurs if the grafts are able to make direct contact with the deafferented host territory w41,42x. Thus, both groups EC1 and EC2, which directly contact the host deafferented hippocampus, are in a position to their normal target areas. As all the grafts were more or less like a cylinder, we could use a semi-quantitative method to estimate the volume of the grafts. The measurements showed that all three groups ŽEC1, EC2 and NEC. had a roughly same intrahippocampal grafts volume; therefore, the behavior difference between the EC1 and EC2 groups might be a result of overlying intraneocortical portion of grafts in the EC1 group. Abundant evidence suggests that EC in its intact site receive massive sensory inputs from neocortices Žmay be through multimodal association cortex. w15,21,31,36– 38x. This sensory information is then conveyed from the EC to the hippocampus for further processing during the course of learning and memory. Thus, it is possible that the functional importance of the grafts in the EC1 group is that they are able to receive inputs from the surrounding neocortex. The ineffectiveness of EC2 rats in lessening the behavioral deficits indicated that the presence of efferents from the EC grafts to host hippocampus may not be sufficient to result in beneficial effects for the host. This may reflect the nature of the role of the EC in the neocortex–EC–hippocampal system. As the EC is thought to be involved in bi-directional information transmission, both afferents and efferents are crucial to the cognitive function w18,31x. Compared to other well-characterized system, such as septohippocampal or nigro-striatum, where the grafts appear to be able to exert positive effects without receiving a normal afferent input w2,4,9x, the results of EC2 group suggest a different physiological role for the affected
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system. The present system involves sensory transmission, and requires integration of both afferent and efferent connections, rather than simply restoring a tonic level-setting w20,35x.
Acknowledgements This work was supported by National Natural Science Foundation of China No. 39230140. The authors are grateful to Mr. Zhi-hua Jiang, Tie-feng Zhang, Ms. Zi-mei Ni and Hui Zhu for their technical assistance.
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Research report
Embryonic entorhinal transplants partially ameliorate the deficits in spatial memory in adult rats with entorhinal cortex lesions Wenbo Zhou a , Deming Jiang b , Geoffrey Raisman c , Changfu Zhou
a, )
a
Shanghai Institute of Physiology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai, 200031, China Shanghai Brain Research Institute, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai, 200031, China The Norman and Sadie Lee Research Center, Laboratory of Neurobiology, National Institute for Medical Research, Medical Research Council, The Ridgeway, Mill Hill, London, NW7 1AA, UK b
c
Accepted 27 January 1998
Abstract Our previous studies have demonstrated that axons from grafts of embryonic entorhinal cortex ŽEC. can reinnervate the deafferented zones in the hippocampus and form synaptic connections with the host dentate gyrus in adult mice and rats deprived of their own entorhinal inputs. Here, we have examined the ability of the EC grafts to ameliorate deficits in spatial memory. Three months after transplantation, the grafted rats and control animals were subjected to Morris water maze testing followed by histological examination. According to the exact position of grafts in the host brain, the rats with lesion and EC transplants were divided into two groups, one with EC grafts contacting both the hippocampus and overlying neocortex Ž n s 7, EC1. and another with EC grafts confined within the hippocampus Ž n s 6, EC2.. While EC2 rats were still as impaired as those with lesion and transplants of non-entorhinal cortex Ž n s 10, NEC. or with lesions only Ž n s 7, LES., the EC1 rats performed better than the LES group. In a spatial memory trial, the EC1 group made more crossings over platform site and showed more focused search behavior than EC2, LES, NEC groups. The data suggest that EC grafts could partially ameliorate the deficit in spatial learning behavior in the EC-lesioned adult rats. The requirement for the graft to contact both the neocortex and the hippocampus suggests that the functional effects may be exerted by the formation of new neocortical-EC graft-hippocampal circuits. q 1998 Elsevier Science B.V. Keywords: Hippocampus; Neocortex; Repair; Reinnervation; Grafts; Behavior
1. Introduction The entorhinal cortex ŽEC. is a major relay providing a wide range of cortical input to the hippocampus and transferring the information processed in the hippocampus to the cortices via reciprocal projections both between neocortical multimodal association areas and the EC, and between the EC and hippocampus w1,18,19,32,33,37,40x. The EC has been suggested to play a crucial role in the processing of learning and memory, a proposition that has been supported by abundant evidence from both clinical and experimental neurology. The EC shows some of the earliest and most severe pathological alternations in Alzheimer’s disease ŽAD. w6–8,16,23x. There is a very severe loss in the EC even in very mild AD cases that are )
Corresponding author. [email protected]
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0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 1 2 5 - 5
at the threshold for clinical detection of dementia w8,14,18x. The vulnerability of the EC to neuropathological changes in other degenerative disorders with mnemonic malfunction, such as Parkinson’s and Huntington’s diseases, also points to its involvement in learning and memory w6,18,31x. Surgical or electrolytic damage to the EC in laboratory animals has been shown to impair memory, reference memory and memory retention w17,22,28,29,34,39x. Schenk and Morris w30x reported that rats with the EC lesion show severe post-operative impairment in the initial acquisition of the place-navigation task indicating impairment of spatial learning and memory. Transplants of embryonic neuronal tissue are capable of reinnervating denervated areas in the adult mammalian brain, and restoring functions lost as a result of lesions w4,9x. In the septohippocampal and nigrostriatal systems, transplanted embryonic cholinergic and dopaminergic neurons have been shown to send their axons into the dener-
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vated target area at a considerable density, and consequently completely or partially restore functions w3,5,10,11,25,27x. It has been suggested that these graft-derived effects may be exerted by restoration of tonic levelsetting in the deafferented areas due to spontaneous or active transmitter release from the transplanted neurons w2,20,26,35x. We have previously shown that the embryonic entorhinal transplants can survive in the adult rodent hippocampus and specifically reinnervate entorhinally deafferented territory in the outer molecular layer ŽOML. of the dentate gyrus ŽDG. and the stratum lacunosum-moleculare ŽSLM. of CA1 and CA3 w41,42x. Electron microscopy revealed that ingrowing fibers from EC grafts form asymmetric synaptic connections on dendritic spines w42x. We now use the Morris water maze test w24x to explore whether such reconstruction of entorhino-hippocampal projections is able to exert any beneficial effect on the impaired performance of adult rats with complete bilateral transections of perforant paths ŽPP., or destruction of the EC. Here, we report that grafts of fragments of embryonic EC tissue placed as columns extending from the overlying neocortex ventrally through the hippocampus ameliorate the impairment of spatial water maze memory caused by entorhinal lesions; no such effect was found when the grafts were confined to the hippocampus.
perpendicular to the midline was one beginning 2.0 mm caudal, 2.5 mm lateral to the lambda, and extending laterally to the crista frontalis, another was beginning from the end of the first at a frontal dip angle of 308 lateral to the crista frontalis. The microknife was inserted into the brain through two trenches, respectively, and moved down along two curved surfaces just before the medial and lateral angular bundles entering the hippocampus. These perforant path transections completely disrupt the entorhinal efferents to the hippocampus, with little bleeding, and minimal damage to the hippocampus w41,42x. Ten to fourteen days following the bilateral PP lesion, the animals in EC1, EC2 and NEC groups received EC or NEC transplantation. The EC Žthe ventrolateral cortex of the caudal pole of the hemisphere. and NEC Žfrontal cortex. were dissected out from E17 rat embryos Žthe mating day as E0. under dissection microscope, and loaded into an 0.8-mm internal diameter glass tube Žthe length of the transplant mass was either 4.0 mm for EC1 and NEC animals or 2.5 mm for EC2 animals.. Prior to graft preparation, one burr hole Ždiameter of 1 mm. was drilled in each side of the skull Žcoordinates: 4.0–4.2 mm caudal to bregma, 2.2–2.5 mm lateral to midline.. The tube containing the grafts was connected to a metal rod and positioned on the stereotaxic frame and lowered into the brain until the low tip of the implant 4.3 mm below the dura. The grafts were left for 5 min in the brain followed by slow withdrawal of the glass tube and metal rod.
2. Materials and methods 2.3. Morris water-maze test [24] 2.1. Subjects Fifty adult female Sprague–Dawley rats, weighing 220–250 g at the start of experiments, were allocated to five groups: EC1 group Ž n s 10. with bilateral transections of the PP and bilateral EC grafts that were made as a column going through overlying neocortex and hippocampus, EC2 group Ž n s 10. with bilateral transections of the PP and bilateral EC grafts restrictively within the hippocampus, NEC group Ž n s 10. with bilateral transections of the PP and bilateral non-entorhinal cortex ŽNEC. grafts as a column-like EC1 grafts, LES group Ž n s 10. with bilateral transection of PP without grafts, and NOR group Ž n s 10., unoperated controls. 2.2. Lesion and transplantation surgery The general procedure used for producing PP lesions and EC transplantation has been described previously w41,42x. Briefly, animals were anaesthetized with sodium pentobarbital Ž40 mgrkg, i.p.. and placed in a stereotaxic apparatus. After exposing the skull, a specially tooled microknife of silver leaf Ž0.3 mm thick, 2 mm wide and 20 mm long with a radian of 908 at its middle point. was used to cut the perforant paths. Using a small hand drill, two trenches Ž1 mm wide. were made on each side of the skull:
A circular pool Ždiameter 180 cm, height 50 cm. was filled to a depth of 20 cm with water at room temperature Ž20–228C. made opaque by the addition of 30 ml Indian ink. Four equally spaced points around the edge of the pool were designed as four starting positions: east ŽE., south ŽS., west ŽW. and north ŽN.. An escape platform Ždiameter 9 cm. was set 1 cm below the surface of the water and placed in a constant position in the middle of the SW quadrant. The rat in the pool was trained to find the platform using a variety of extramaze cues, including the lamp, wall posters, curtain, window, desk, video recorder, monitor, etc. A video camera was placed directly above the center of the pool. The experimenter always sat at the same position. Three months after transplantation, each rat was given 4 trials on each of 9 consecutive days. They were placed in the water facing the wall from one of four starting sites in a random sequence Ži.e., SENW, so that each site was used once everyday.. The latency to find the escape platform was measured during each trial. Upon finding and climbing onto the platform, the rat stayed there for 30 s. If the rat could not find the platform within 60 s, it was guided by the experimenter and given a maximum score of 60 s. Before the next trial, the rat was allowed to rest in a cage for 60 s. On the fourth trial of training day 9, the platform
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was removed and the swimming behavior of each rat was recorded for 60 s. 2.4. Histology Upon completion of the behavioral testing, animals were perfused transcardially with physiological saline followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer ŽPB, pH 7.4.. The brains were removed and kept in a 20% solution of sucrose in 0.1 M PB ŽpH 7.4. at 48C prior to further processing. Frozen coronal sections of 50 m m thickness were cut from each brain and collected in 0.01 M phosphate buffered saline ŽPBS.. Thionin and acethylcholinesterase ŽAChE. staining were performed on alternative sections to examine the survival of the grafts and extent of lesions as described previously w41,42x. The semi-quantitative measurement of graft size was conducted under microscope on the thionin-stained sections passing through the middle plane of each cylinder-like grafts, and the volume of grafts was calculated by p Ž1r2 width. 2 = length of graft. 2.5. Data statistics Data of Morris water maze test were collected using a path-tracing software ŽShanghai Jiao-Tong University, China. in a computer. All raw data were processed using either SigmaPlot ŽJandel Scientific, V1.0. for behavioral test and GraphPad Instat ŽV2.0. for measurement of graft size. For comparison of the data from Morris water maze test, one-way or two-way analysis of variance ŽANOVA. were performed in Excel ŽMicrosoft Office, V7.0. fol-
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lowed by Student’s t-test at the significance of 0.05. In addition to one-way ANOVA, unpaired Student’s t-test and Tukey–Kramer multiple comparison test were used for analysis of morphological measurement.
3. Results 3.1. Histology Most of the lesioned rats showed substantial bilateral disruptions of the medial and lateral entorhinal cortex throughout its dorsoventral extent ŽFig. 1.. Thus, the transections of bilateral perforant paths were complete. Three of the rats in the LES group had partial sparing of the EC, and were excluded from further analysis. Seven rats in the EC1 group and 10 rats in the NEC group had surviving bilateral columnar grafts ŽFig. 2A,B. that consisted of an overlying intraneocortical portion and a successively intrahippocampal portion. Six EC2 grafted rats had bilateral grafts ŽFig. 2C. that were mainly confined to the host hippocampus. It was noticed that the intraneocortical portions of EC1 and NEC grafts lied passing through over half of the neocortical depth, and all intrahippocampal grafts ran over two-thirds of dorsal hippocampal depth. For the purpose of multiple group comparison, the graft volume was divided into intraneocortical portion and intrahippocampal portion. Semiquantitative measurements ŽTable 1. showed that there was no significant difference in the graft volume of either intraneocortical portion or intrahippocampal portion between EC1 group
Fig. 1. Thionin-stained horizontal sections showing entorhinal cortex ŽEC. of intact ŽA. and lesioned rat ŽB.. Note the subiculums Žsb. intact in the EC-lesioned rat ŽB.. Scale bar: 1000 m m.
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Fig. 2. Representational examples of Nissl-stained coronal sections cut through grafts ŽG. from rats of EC1 ŽA., NEC ŽB. and EC2 ŽC. groups, showing grafts lying in both neocortex and hippocampus ŽA, B. or only within the hippocampus ŽC.. D, E and F are higher magnifications of the areas outlined by the boxes in A, B and C, respectively, showing surviving neurons in the grafts. Arrows and arrowheads, respectively showing well integration of grafts with host neocortex ŽNX., and the outer molecular layer ŽOML. of the host dentate gyrus ŽDG. and the stratum lacunosum-moleculare ŽSLM. of the host field CA1. Scale bars: 1000 m m for A, B,C; 200 m m for D, E and F.
and NEC group Žunpaired Student’s t-test, both p ) 0.05.. Although one-way ANOVA showed that there was a slight difference in the graft volume of intrahippocampal portion among EC1, EC2 and NEC groups Ž FŽ2,39. s 3.39, p s 0.047., the post Tukey–Kramer multiple comparison tests
indicated no significant difference in intrahippocampal graft volume between any two groups ŽEC1 vs. EC2 or EC1 vs. NEC or EC2 vs. NEC, all three p ) 0.05.. The grafts were well integrated with host tissue including neocortex in rats of the EC1 and NEC groups with
Table 1 Measurements of graft size Group
EC1 NEC EC2
n
14 16 12
Intracortical portionrIntrahippocampal portion Length Žmm.
Width Žmm.
Volume Žmm3 .
1.044 " 0.049r1.372 " 0.046 1.088 " 0.046r1.314 s 0.046 r1.245 " 0.051
0.682 " 0.051r0.837 " 0.034 0.663 s 0.049r0.683 s 0.052) r0.699 " 0.044)
0.428 " 0.065r0.774 " 0.072 0.331 " 0.027r0.533 " 0.095 r0.484 " 0.058
Values are mean " S.E.M. of graft sizes in each group. )Significantly different from EC1 Ž P - 0.05. by unpaired Student’s test and post Tukey–Kramer multiple comparison test.
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little indication of gliosis along the local host-graft border. In the host hippocampus, all those grafts showed direct contact with the OML of the DG and the SLM of CA1 and CA3, all of which are entorhinal territories ŽFig. 2D,E,F.. The Nissl stain showed that the grafts contained healthy large pyramidal-type cells ŽFig. 2D,E,F., with no laminar appearance characteristic of normal cortex in situ. AChE staining showed an intense band of AChE positive reaction in the OML of the host DG, further indicating the completeness of the PP lesions Ždata not shown.. In the remaining 3 rats of the EC1 group and 4 rats of the EC2 group, the grafts did not make precise contact with the host hippocampus, and these were excluded from further analysis. 3.2. Performance in the water maze During the 9-day training period, all groups reduced their swimming time Žlatency. significantly to locate the submerged platform ŽFig. 3; repeated measures ANOVA, effect of day on latency, F8,280 s 8.71, p - 0.001.. A significant group effect and group = day effect ŽGroup, F4,35 s 10.2, p - 0.001; Group= Day, F32,280 s 5.43, p 0.001. were also observed, suggesting that different groups learned the task at different rates. The normal rats learned
Fig. 4. ŽA. Average number of annulus crossings during the spatial memory trial. ) p- 0.05; )) p- 0.01 as compared to lesion alone group. ŽB. Relative distance swim in each quadrant during the spatial memory trial, upon removal of the escape platform. ) p- 0.05; )) p0.01 as compared to the distance spent by the same rats in the remaining quadrants.
Fig. 3. Effects of entorhinal transplants on performance of the water maze task. Average latency for the rats of different groups to escape on to the hidden platform during the acquisition of the place navigation task is indicated. Each point represents the mean value for a block of four trials on each of nine consecutive days when tested three months after EC or NEC bilaterally grafting to adult rats with bilateral perforant path transections.
the task much more rapidly than all other animals and reached asymptotic performance at the fourth testing day. The remaining four groups exhibited virtually identical learning rates and did not differ from each other Ž p ) 0.5., except for EC1 group, which showed slightly better performance than EC2, LES and NEC groups in the last four testing days Ž p ) 0.05..
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Fig. 5. Examples of swimming paths taken by representative rats from the different groups during the spatial memory trial on the last day of the training at three months after grafting. The filled circle indicates the former platform site. Note the spatially focused search pattern over the previous platform site displayed by the rats of the normal and EC1 groups.
3.3. Spatial memory After the three trials on day 9, a fourth 1-min memory trial was performed without platform in the tank. The behavior of rats in the spatial memory trial was quantified by counting the number of crossings each rat made over the former platform site Ždefined as an annulus twice the area of the real platform., and measuring the swimming distance in each of 4 quadrants. As illustrated in Fig. 4A,B, the normal rats crossed more times over the previous platform site than all other groups Ž p - 0.01. and swam mainly in the platform quadrant Ž F3,36 s 114, p 0.001., clearly indicating that they had maintained their spatial memory. In contrast, EC2, NEC or LES rats made few crossings over the platform site and showed no preference in distribution of their swim distance ŽEC2, F3,20 s 1.67; NEC, F3,36 s 2.67; LES, F3,24 s 1.77; all n.s... However, the rats in EC1 group showed significantly more crossings over platform site than EC2, NEC and LES group Ž p - 0.05., and they swam preferentially in the platform quadrant Ž F3,24 s 21.9, p - 0.001., indicating these rats partially remembered the previous position of the platform. It was found that only NOR and EC1 rats displayed a spatially focused search behavior ŽFig. 5. and spent most of their swimming over the former platform site Žindicated by the filled circle in the lower left quadrant of the pool., which indicates an intact spatial memory for the original
platform site. In contrast, EC1, NEC and LES rats did not show this behavior, and they swam in a random fashion along the wall of the pool, indicating non-spatial search strategy taken by them in this water maze task.
4. Discussion The present results provide evidence that the entorhinal grafts which contact both the hippocampus and overlying cortex are capable of reducing the spatial learning deficits induced by the perforant path lesions in the adult rat. However, those rats with entorhinal grafts confined within the host hippocampus, and not contacting the neocortex did not exert any positive effects on the behavioral deficits. Histological examination showed that healthy grafts were well integrated with the host in all rats of EC1, EC2 and NEC groups used for the behavioral statistics. During the acquisition period, there was no significant difference in the latency to locate the platform between lesioned rats with or without any types of grafts. However, the different search behavior was observed in the spatial memory test and this reached statistically significant levels. While EC1 group showed marked improvement in the memory of previous platform site, as indicated by making more crossings over platform site, and showing more focused searching in the platform quadrant, the EC2, as well as NEC and LES groups were still impaired and exhibited random
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non-spatial searching behavior. The data suggest that the EC grafts passing through overlying neocortex and the hippocampus of the host can partially ameliorate the deficit of the adult rat in spatial learning and memory caused by lesions disconnecting the host entorhinal area and hippocampus. It has been reported that severe damage to the entorhinal cortex results in a permanent impairment in the spatial and non-spatial learning w22,30,39x. In our material, the extent of recovery in the maze task may be limited by the degree of reinnervation of the host hippocampus by the grafts, andror the degree of innervation of the grafts by host cortical afferents. Similarly, it has been reported that EC grafts located in the damaged cortical area Žhomotypic grafts. were unable to ameliorate the learning ability tested by forced alternation task w13x, although the grafts could both innervate host hippocampus and receive some cortical innervation w12x. In our experiments, direct contact of the grafts with both the hippocampus and the neocortex was crucial for amelioration of the spatial memory deficits. As indicated in our previous studies, the outgrowth of entorhinal axons from embryonic grafts to the host hippocampus only occurs if the grafts are able to make direct contact with the deafferented host territory w41,42x. Thus, both groups EC1 and EC2, which directly contact the host deafferented hippocampus, are in a position to their normal target areas. As all the grafts were more or less like a cylinder, we could use a semi-quantitative method to estimate the volume of the grafts. The measurements showed that all three groups ŽEC1, EC2 and NEC. had a roughly same intrahippocampal grafts volume; therefore, the behavior difference between the EC1 and EC2 groups might be a result of overlying intraneocortical portion of grafts in the EC1 group. Abundant evidence suggests that EC in its intact site receive massive sensory inputs from neocortices Žmay be through multimodal association cortex. w15,21,31,36– 38x. This sensory information is then conveyed from the EC to the hippocampus for further processing during the course of learning and memory. Thus, it is possible that the functional importance of the grafts in the EC1 group is that they are able to receive inputs from the surrounding neocortex. The ineffectiveness of EC2 rats in lessening the behavioral deficits indicated that the presence of efferents from the EC grafts to host hippocampus may not be sufficient to result in beneficial effects for the host. This may reflect the nature of the role of the EC in the neocortex–EC–hippocampal system. As the EC is thought to be involved in bi-directional information transmission, both afferents and efferents are crucial to the cognitive function w18,31x. Compared to other well-characterized system, such as septohippocampal or nigro-striatum, where the grafts appear to be able to exert positive effects without receiving a normal afferent input w2,4,9x, the results of EC2 group suggest a different physiological role for the affected
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system. The present system involves sensory transmission, and requires integration of both afferent and efferent connections, rather than simply restoring a tonic level-setting w20,35x.
Acknowledgements This work was supported by National Natural Science Foundation of China No. 39230140. The authors are grateful to Mr. Zhi-hua Jiang, Tie-feng Zhang, Ms. Zi-mei Ni and Hui Zhu for their technical assistance.
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