Phorbol ester binding sites and neuronal plasticity in ... - Science Direct

23

Brain Research, 607 (1993) 23-32 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 18639

[3H]Phorbol ester binding sites and neuronal plasticity in the hippocampus following entorhinal cortex lesions Angble Parent, Doris Dea, R6mi Quirion and Judes Poirier Douglas Hospital Research Centre and Department of Psychiatry, Centre for Studies in Aging, McGill University, Verdun, Qua. (Canada) (Accepted 20 October 1992)

Key words: Entorhinal cortex lesion; [3H]PDBu binding; Protein kinase C; Synaptic plasticity; Hippocampus

Entorhinal cortex lesioning (ECL) produces a loss of more than 80% of the synapses in the outer molecular layer of the hippocampus. However, the loss of synapses is transient. Beginning a few days after denervation, new synapses are formed, virtually replacing the lost inputs within 2 months. Synaptic remodelling induced by ECL is associated with specific modifications of neurotransmitters, hormones and growth factors. Particularly, protein kinase C (PKC) plays important functional roles in receptor-mediated transmembrane signal transduction. PKC is also involved in various aspects of synaptic plasticity, such as cellular growth and differentiation. To investigate further the potential roles of PKC in synaptic plasticity observed in the ECL model, [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding, a putative marker of PKC, was examined at different times post-lesion. [3H]PDBu binding sites transiently decreased bilaterally at 2 and 8 days post-lesion (20%) in different laminae and sub-fields of the rostral hippocampus but returned to control values at 14 and 30 days post-lesion. In caudal portion of the hippocampus, [3H]PDBu binding was also decreased at 2 days post-lesion but only on the contralateral side. Interestingly, [3H]PDBu binding sites in the cortex increased by up to 30% in the contralateral side while no significant change was observed in the ipsilateral side at any time post-lesion. It is known that PKC can be regulated by different systems following alterations of neuronal and glial activity. We suggest that these could be involved in the response of PKC and [3H]PDBu binding sites following ECL. Moreover, PKC seemed to be modified in different brain areas in addition to the hippocampal formation in this model. This can be associated to a rather general reorganization observed following losses of neuronal inputs from the entorhinal cortex and the subsequent reinnervation process.

INTRODUCTION

Entorhinal cortex lesions (ECL) in rats, a model of synaptic plasticity, is known to induce severe losses of hippocampal inputs within 5-6 days post-lesion 26. The proportion of degenerating synapses generally reaches a peak at 2 days post-lesion, to represent up to 80% of the total number of synapses in the molecular layer of the dentate gyrus. However, the percentage of degenerating synapses gradually decreases to only represent a very small portion by 60 days post-ECL 26. In fact, the deafferented zone of the hippocampus is repopulated by newly formed synapses between day 8 and 30. In the molecular layer of the dentate gyrus of lesioned animals, the quantities of synapses greatly exceed that found under normal conditions, and this as early as a few days post-lesion. Therefore, new synapses have formed to replace most of those lost following the lesion 8,a7. It has been proposed that this phenomenon

is due to septo-dentate afferents which can proliferate in response to lesions of the entorhinal cortex, revealing that septal fibers are capable of substantially increasing their arborization, especially within the outer part of the molecular laye r of the dentate gyrus as demonstrated by an intensification of acetylcholinesterase (ACHE) staining 23'43. Synaptic remodelling in the CNS is generally associated with specific modifications of a variety of neurotransmitters, hormones and growth factors. These neuronal factors exert and mediate their effects, including neuronal survival, through various transduction mechanisms 22 such as protein kinase C (PKC). Biochemical and molecular studies have shown that PKC can be divided into two functionally distinct domains: the Nterminal regulatory domain which contains sites able to recognize phospholipids, diacylglycerols and phorbol esters, and the C-terminal portion which contains both the ATP-binding and substrate-binding sites and can

Correspondence: J. Poirier, Douglas Hospital Research Centre, 6875 LaSalle Blvd., Verdun, Qu6bec, H4H 1R3, Canada. Fax: (1) (514) 766-3224.

24 function as a constitutively a c t i v a t e d p r o t e i n kinase in the a b s e n c e of a r e g u l a t o r y d o m a i n 5'6'js'32. P K C is p r o b a b l y involved in m a j o r cellular events u n d e r l y i n g synaptic plasticity by r e g u l a t i n g the p h o s p h o r y l a t i o n state o f d i f f e r e n t s u b s t r a t e p r o t e i n s 3s. It has b e e n shown that P K C can m o d u l a t e t h e g r o w t h of p r e s y n a p tic terminals, i n c r e a s e n e u r o t r a n s m i t t e r releases, play a role in r e c e p t o r m o d u l a t i o n a n d r e g u l a t e various ionic c h a n n e l c u r r e n t s 2'3'18'32'3~. A c t i v a t i o n of P K C has also

P K C in n e u r o n a l plasticity, we have q u a n t i f i e d b i n d i n g sites for [3H]phorbol 12,13-dibutyrate ([3H]PDBu), a p u t a t i v e m a r k e r of PKC, in the rat E C L m o d e l at p e r i o d s which c o r r e s p o n d to b o t h the d e n e r v a t i o n p h a s e ( 0 - 5 days p o s t - l e s i o n ) and the r e i n n e r v a t i o n process ( 6 - 3 0 days post-lesion).

b e e n a s s o c i a t e d with the i n d u c t i o n a n d m a i n t e n a n c e of l o n g - t e r m p o t e n t i a t i o n ( L T P ) in the h i p p o c a m p u s , a well-known e x a m p l e of synaptic plasticity 24'25.

Entorhinal cortex lesions

In o r d e r to f u r t h e r investigate the possible role of

MATERIALS AND METHODS

Young adult Fischer 344 male rats (3-months old) were given multiple electrolytic lesions (30 s) of the entorhinal cortex area according to the methods previously described by Poirier et al. 36'37 a s adapted from Scheff et al.4°. At 2, 4, 8, 14 and 30 days post-ECL,

Fig. 1. Effect of unilateral lesion of the entorhinal cortex on acetylcholinesterase (ACHE; left column), GFAP-positive astrocyte immunostaining (middle column), and [3H]PDBu binding (right column) in the hippocampal formation. The right side of the brain section represented the lesioned (ipsilateral) side and the left side represented the non-lesioned (contralateral) side. Photomicrographs are shown for control (CONT) and at various (2, 4, 8, 14 and 30) days post-lesion. Arrows pointed out the outer molecular layer of the dentate gyrus in the lesion side. Refer to Fig. 4 and Table II for quantitative data regarding alterations in various laminae and sub-fields of the hippocampal formation at these various times, post-lesion.

25 animals were sacrificed for [3H]PDBu autoradiographic studies and glial fibrillary acidic protein (GFAP) immunocytochemistry.

[ 3H]PDBu autoradiography After decapitation, brains were rapidly removed from the skull and immersed in 2-methyl-butane at -40°C during 15 s and then

kept frozen at -80°C until sectioning. 15-/~m thick frozen tissue sections were then cut and mounted onto L-polylysine (Sigma Chemical Co., St-Louis, MO) precoated slides, and air-dried at room temperature for 24 h and subsequently stored at -80°C until use. [3H]PDBu (17 Ci/mmol, New England Nuclear, Boston, MA) binding assay was performed by preincubating sections for 1 h at 4°C in a buffer containing 50 mM Tris-HCl (pH 7.7), 100 mM NaCl, and I

Fig. 2. Photomicrographs of the autoradiographic distribution of [3H]PDBu binding sites at the level of the rostral and caudal hippocampal formation of the rat brain. [3H]PDBu binding is heavily concentrated in various laminae of the hippocampus and the cortex while being much lower in thalamic and mesencephalic areas. Amyg, amygdaloid nucleus; Ent, entorhinal cortex; GrDG, granular layer of the dentate gyrus; Hil, hilus of the hippocampus; Hip, hippocampus; lMol, lacunosum molecular layer of the hippocampus; Mol, molecular layer of the hippocampus; Oc, occipital cortex; Or, oriens layer of the hippocampus; Par, parietal cortex; PRh, pirirhinal cortex; py, pyramidal cell layer of the hippocampus; Rad, radiatum layer of the hippocampus; SN, substantia nigra; Sup Col, superior colliculus; and Te, temporal cortex.

TABLE I

7

5

8

5

6

9

1 752 _+267 1095 ± 110 936_+ 54

1712_+ 95 1099 _+ 86 959.+ 72

1662-+ 168 979.+ 91 851 .+ 64

2107 _+204 1237 -+ 148 1029-+ 77

1930 -+ 282 1072 + 112 907 + 64

2013 .+ 107 1311.+ 99 986 _+ 40

1736.+189 1069.+ 84 953.+ 63

1803_+226 1090-+ 88 932_+ 50

2101.+100 1096_+ 68 906+ 43

2028.+221 1222.+ 92 1004.+ 58

2201_+191 1170+117 908.+ 87

1714.+249 1153.+172 1026.+106

1513-+ 34 1070.+ 71 1033.+ 70

1695.+176 1156.+ 93 1015-+ 80

1748.+221 1205.+128 1022.+ 69

1408-+288 1077.+160 985+ 86

1892 ± 127 1 255 + 64 1111-+ 56

IPSI

IPSI

CONTRALATERAL

Temporal

Parietal

1717.+249 1161.+111 994.+ 56

1857-+158 1174.+ 44 1053.+ 69

2215.+103 ** 1333.+104 1128-+100

2081+241 1374.+ 75 1119.+ 44

2001.+295 1263_+160 1038.+ 92

CONTRALATERAL

1913+353 1283.+211 1158+162

1895+ 55 1201.+ 57 1152.+ 69

2033.+139 1268.+ 98 1153-+ 88

1837.+308 1226.+167 1080.+103

1760.+264 1208_+132 1119+ 78

2 092 ++_145 1 345 + 73 1181.+ 59

IPSI

Occipital

1816.+248 1151.+ 77 1032.+157

1990-+251 1273.+ 93 1131.+ 38

2213.+121 1330.+111 1204.+119

1986.+220 1233.+ 42 1104+ 31

1791.+281 1121-+142 995.+ 69

CONTRALATERAL

1658.+349 1271.+180 1158.+133

1431+ 86 1108_+ 58 1012.+ 78

1519.+167 1207.+ 98 1167.+ 84

1533.+206 1209_+125 1133.+109

1173.+185 1065+108 1006.+102

1655 +_ 109 1283-+ 53 1141.+ 51

IPSI

Pirirhinal

* Rostral part of the entorhinal cortex; ** P < 0.05 compared contralateral side to the ipsilateral side, evaluated by paired Student's t-test.

Control Superficial Middle Deep 2DPL Superficial Middle Deep 4DPL Superficial Middle Deep 8DPL Superficial Middle Deep 14DPL Superficial Middle Deep 30DPL Superficial Middle Deep

n

1846.+288 1363.+133 1151.+ 78

1725_+ 81 1237.+ 65 1133.+ 95

2019.+190 1517.+124"* 1278.+114

2085.+260 1529-+151 1274+ 94

2018.+288 1429_+217 1390.+221

CONTRALATERAL

1179.+136 1054.+ 90 1000.+ 41

1182+ 51 1054_+ 63 977+ 72

1205_+106 1064+ 77 1043.+ 71

1385+127 1166_+117 1014.+ 66

1079+154 996_+ 97 901.+ 60

1434 _+ 108 1288-+ 146 1069.+ 34

IPSI

Entorhinal *

1631+259 1327_+161 1117.+ 89

1642+304 1326+175 1136+122

1818+159 ** 1445_+114"* 1238.+ 7 6 * *

1788+254 1372.+148 1078_+ 75

1634.+236 1300_+180 1034+123

CONTRALATERAL

n number of animals; specific [3H]PDBu binding is expressed as mean+S.E.M, in fmol/mg tissue, wet weight. Superficial corresponds to layer I; Middle corresponds to layer II-IV; Deep corresponds to layer V-VI.

[ ~H]PDBu binding sites in the cortex following a lesion of the entorhinal cortex

|,,j

27 mM CaCI 2 according to the method described by Worley et al. 47. Incubations were then carried out in the same buffer containing bovine serum albumin (0.1%; Boehringer-Mannheim, Laval, Que., Canada) and 2.5 nM [3H]PDBu for 1 h at 33°C. Non-specific labelling was assessed by adding excess of unlabelled PDBu (1 /~M; Sigma) to the incubation solution. Following incubations, sections were washed twice in ice-cold buffer for 2 min, dipped in ice-cold distilled water and immediately dried under a stream of cold air. Under these conditions, specifically bound [3H]PDBu basically represented the totality of bound ligand. Autoradiograms were generated by apposing 3H-Hyperfilm (Amersham, Oakville, Ont., Canada) to slides for 7 days. Binding of [3H]PDBu to sections was quantitated by microcomputer image analysis (MCID system, Ste-Catherines, Ont., Canada), using tritium-labelled standards (Amersham) for film calibration.

GFAP immunocytochemistry GFAP immunoreactive reactive astrocytes, from the same animals, were visualized using 15 tzm sections from ECL and control rats according to the method of Poirier et al. 35. Paraformaldehyde

post-fixed tissue sections were processed with the avidin-biotin-peroxidase method (ABC Kit, Vector Labs, Burlingame, CA) with pre-treatement (1 h, 3% H 2 0 2) to block endogenous peroxidase activity. Following a 60 min incubation with 1% goat serum (Vector Labs) to block non-specific IgG binding, tissues were incubated overnight at 4°C with rabbit polyclonal primary antibodies against rat GFAP (DAKO, Carpinteria, CA; pre-diluted working solution). After rinsing with a phosphate-buffered solution, biotinylated-horseradish peroxidase (Vector Labs) was sequentially applied and visualized by addition of 0.3% H 2 0 2 and 0.1% 3,3'-diaminobenzidine (Sigma) for 15 min at room temperature. Control sections were processed in parallel without primary antibodies.

Acetylcholinesterase staining Reactive synaptogenesis in the dentate gyrus following ECL was confirmed histochemically using AChE staining. After being exposed for autoradiography, slides were stained with a solution containing acetylthiocholine iodide (Sigma) for 5 h, according to the well-known method described by Karnovsky and Roots 19.

Analysis of data [3H]PDBu binding levels of various post-lesion days were expressed as means+S.E.M, and were compared to control non-lesioned brain. Statistical significances ( P < 0.05) were evaluated by one-way analysis of variance, followed by a posteriori Duncan's analysis. Contralateral side was compared to the ipsilateral side using paired Student's t-test ( P < 0.05).

CORTICAL AREAS

A)

Control 2250

RESULTS ~

, ~

1750 AChE As reported previously 23'43, a gradual increase in the AChE staining ipsilateral to the lesion was detected in the outer molecular layer of the dentate between 8 and 30 days post-lesion (Fig. 1, left column).

.~. ~

1250

750 Ent

PRh

Te

Oc

Par

GFAP immunostaining

s days post-lesion

B)

O

Intense bilateral GFAP-like immunoreactivity was evident at 4 days post-lesion and decreased progressively to become restricted to the ipsilateral side of the outer portion of the molecular layer of the dentate gyrus at 14 and 30 days post-lesion (Fig. 1, middle column).

20 2 10"

Z

0

[3H]PDBu binding sites

, -20-

-30 Ent

PRh

Te

Oc

Par

Fig. 3. Quantitative autoradiographic data of cortical [3H]PDBu binding in control (A) and contralateral side of ECL animals at 8 days post-lesion (B). [3H]PDBu binding was quantified using computerized densitometry and is expressed as the mean_+ S.E.M. of at least 5-9 animals (fmol/mg tissue, wet weight). In control brains, binding is greater in superficial (sup) than middle (mid) or deep laminae. Binding in superficial laminae is significantly more pronounced in the temporal (Te), occipital (Oc) and parietal (Par) cortices but not in pirirhinal (PRh) cortices when compared to the entorhinal (Ent) cortices. Alterations in [3H]PDBu binding reached significance at 8 days post-ECL in the intermediate layer of the parietal cortex when compared to the respective layer of the Ent cortex (B). * P < 0.05, one-way analysis of variance.

[3H]PDBu binding sites are particularly abundant in various cortical laminae and the hippocampal formation, especially in its oriens, radiatum and pyramidal layers (Fig. 2). In the cortex, superficial laminae of the parietal, temporal and occipital areas are markedly labelled whereas the amygdaloid bodies and the entorhinal cortex showed intermediate labelling intensity (Fig. 2). The middle and deeper layers of most cortical areas possessed similar amount of binding sites (Fig. 3A, Table I). In ECL rats, the rostral portion of the hippocampus revealed a labelling profile that is different from the caudal region of this area (Fig. 4, Table II). A bilateral decrease (20%) in [3H]PDBu binding was observed in

28 the rostral portion of the hippocampus at 2 and 8 days post-lesion, with all layers of the hippocampus being as affected as the molecular layer and the CA1 and CA3 areas (Fig. 1, right column and Fig. 4). A somewhat similar phenomenon could be observed in the caudal portion of the hippocampus. However, a significant decrease in [3H]PDBu binding was only observed at 2 days post-lesion on the contralateral, but not in the ipsilateral side, revealing differences in response to

ECL in the rostral and caudal portions of the hippocampal formation. Interestingly, increases in [3H]PDBu binding sites were observed on the contralateral side in certain cortical areas (Fig. 3B). This was particularly evident in superficial layers of the rostral portion of the entorhihal cortex at 8 days post-lesion (27%), returning to control values by 14 days (Table I). [3H]PDBu binding was also affected in various cortical areas, but only on

ROSTRAL Molecular layer

A)

CAUDAL Molecular layer

B)

"~

1400

"~

1300

"~

1200 ~

•o

1100

II00'

1000

1000"

e~

900

~

800

1° 1

---0"-- Lesioned

1300 ~

1200

,

900S

0

' 10

' 20

' 30

Oriens layer

c)

800

0

D)

1800

!

I

!

10

20

30

Oriens layer

',=1,

1700 1600

1500

1500

1400

:=tW,f---r-

1300 1200 ~

1101) 0

10

20

30

10

F)

CA3

E)

0

20

30

, 20

, 30

CA3

"-"

1400"

1400

'~

1300 "

1300

E

"~

1200

i

e~

'~ C

1100

i

I000

1100 1000

m

m

900"

900 8O0

1200

, 10 DAYS

o 20 POST-LESION

, 30

800 0

, 10 DAYS

POST-LESION

Fig. 4. Quantitative autoradiographic data of hippocampal [3H]PDBu binding at various days post-ECL lesions. [3H]PDBu binding was quantified using computerized densitometry and is expressed as the mean _+S.E.M. of 4-10 animals per group (fmol/mg tissue, wet weight). Left column, rostral hippocampus; right column, caudal hippocampus. * P < 0.05, one-way analysis of variance.

29 TABLE II [3H]PDBu binding sites in the hippocampus following a lesion of the entorhinal cortex

n number of animals; specific [3H]PDBu binding is expressed as mean ± S.E.M. in fmol/mg tissue, wet weight. Hil, hilus; GrDG, granular cells of dentate gyms; Mol, molecular; Or, oriens; Py, pyramidal; Rad, radiatum; IMol, lacunosum molecular. n

Hil

GrDG

Mol

Or

Py

Rad

IMol

CA3

8 10

1218± 57 1122-+ 50

908+35 822-+56

1200-+ 69 1186± 58

1568± 64 1534± 76

1459_+ 90 1288_+ 96

1481_+ 90 1381_+ 70

1021_+ 99 1013± 36

1161± 51 1065± 33

1005+ 44* 1030± 69

745_+87 * 741+80 *

980_+ 86 988_+ 58 *

1387_+133 1320±104

1258_+129 1257± 63 1170±110 1244_+ 73

863_+ 76 889± 53

1030± 64 1080+_ 84

1102+ 84 953-+ 66

785±52 688±65*

1063_+ 65"* 930-+ 44*

1575±145 1175-+ 52

1324±139 1022± 56

1335+122 1092± 58

952_+ 66** 806± 38*

1029± 58 946± 40

1214± 73 1175± 52

879_+20 868-+18

1153-+ 60 1124+ 42

1630± 96 1486-+ 67

1461+_ 65 1413± 53

1423+ 74 1376± 48

942± 29 978_+ 23

1240+ 73 1270± 50

1113± 84 1098± 75

814_+47 791_+26

1119_+ 74 1138_+ 76

1498_+158 1478_+102

1278±106 1193± 68

1367±120 1302± 88

964± 67 984± 65

1072± 84 1037± 46

1051_+ 46 991_+ 38 *

822_+27 794_+24

1079_+ 56 970-+ 14 *

1396_+ 79 1299_+ 25 *

1240± 80 1239_+ 41

1274± 62 1210± 29

870± 23 870± 7

1053+ 58 1022+ 37

1091_+ 62 1050± 79

784_+36 786±28

1131_+ 61 1078-+ 81

1335_+105 1409±138

1182± 82 1228+ 75 1247+_102 1245±108

840+ 79 910± 66

996± 80 983+ 84

1101± 65 1083_+ 99

897+50 868±76

1167± 76 1115_+ 96

1510±161"* 1607+184

1378±135 1429±154

1262±129 1369±143

974± 68 982± 88

1063± 77 1079± 90

1126± 67 1048_+ 83

833±22 794_+20

1157± 51 1053_+ 41

1382_+ 90 1408±119

1195_+ 81 1201_+ 82

1236± 67 1202± 72

984_+ 47 925± 30

1001± 33 969± 39

1099+ 81 1121± 90

841+53 838+57

1102± 74 1106+ 68

1560+127 1567_+147

1350_+123 1340±107 1387±130 1354±135

928+ 64 969± 86

1093+ 82 1101± 91

1145±113 1036_+ 58

868±81 792_+49

1080± 80** 1218_+111

1513_+200 1390+ 77

1269±175 1156+ 73

1007_+118 935± 68

1036_+ 73 1014+ 71

Control

Rostral CAUDAL 2DPL

Rostral Ipsilateral Contralateral Caudal Ipsilateral Contralateral

6 7

4DPL


7 4

8DPl


6 9

14DPL


5 5

30DPL


7 5 1353±156 1226± 98

* P < 0.05 compared to the control, evaluated by one way analysis of variance, followed by a posterori Duncan's analysis; ** P < 0.05 compared contralateral side to the ipsilateral side, evaluated by paired Student's t-test.

t h e c o n t r a l a t e r a l side. This e x t e n d e d f r o m the r o s t r a l p a r t o f t h e e n t o r h i n a l to the p a r i e t a l cortex; g r e a t e r d i f f e r e n c e s b e i n g o b s e r v e d in m o s t c a u d a l structures. This p h e n o m e n a was e v i d e n t in all l a m i n a e o f the c o r t e x b e i n g especially m a r k e d in m i d d l e layers o f t h e p a r i e t a l c o r t e x (Fig. 3B). DISCUSSION [ 3 H ] P D B u b i n d i n g is m o d i f i e d , in a c o m p l e x fashion, in c e r t a i n r e g i o n s o f t h e h i p p o c a m p u s a n d c o r t e x following a selective u n i l a t e r a l lesion o f t h e e n t o r h i n a l cortex, a m o d e l o f s y n a p t i c plasticity. B i l a t e r a l significant d e c r e a s e s in b i n d i n g w e r e o b s e r v e d in t h e r o s t r a l h i p p o c a m p u s at days 2 a n d 8, post-lesion, with b i n d i n g r e t u r n i n g to n o r m a l v a l u e s by d a y 14 a n d stabilizing at

this level for at least t h e next 30 days.. M o r e caudally, [ 3 H ] P D B u was significantly d e c r e a s e d only on t h e cont r a l a t e r a l side at 2 days post-lesion. Interestingly, cortical [ 3 H ] P D B u b i n d i n g i n c r e a s e d on t h e c o n t r a l a t e r a l side at d i f f e r e n t times p o s t - l e s i o n while no c h a n g e s w e r e d e t e c t e d in t h e lesion side. It is well k n o w n t h a t lesions o f t h e e n t o r h i n a l c o r t e x i n d u c e m a r k e d losses o f i n p u t s in t h e i p s i l a t e r a l hipp o c a m p a l f o r m a t i o n ~6,~7. It is also well e s t a b l i s h e d t h a t t h e c o n t r a l a t e r a l m o l e c u l a r layer o f t h e d e n t a t e gyrus is only m i n i m a l l y d e n e r v a t e d , yet exhibits extensive synaptic losses followed by t h e i r r a p i d h o m o t y p i c rep l a c e m e n t 16'~7. M o r e o v e r , it was o b s e r v e d t h a t t h e neurite g r o w t h - p r o m o t i n g activity was highest in t h e most p o s t e r i o r regions o f b o t h h e m i s p h e r e s , s t r e n g t h e n i n g t h e conclusion t h a t d e n e r v a t i o n is sufficient to i n d u c e

30 the release of trophic factors at distances from the lesion site -~°. Unilateral lesions of the entorhinal cortex produce a mixture of hippocampal denervation and direct damage to the cortex ipsilateral to the wound. While the contralateral side is not directly injured, it loses commissural innervation originating from the damaged area; at least one afferent input has been shown to exist )3. This input is well known to sprout after ipsilateral entorhinal cortex lesion 4s and is likely to be related to alterations in [3H]PDBu binding sites observed in the contralateral hippocampus following denervation. The increase in [3H]PDBu binding seen in the contralateral entorhinal cortex a few days following the lesion could be associated to a compensatory mechanism induced by the loss of neuronal inputs. Other secondary pathways are also probably directly or indirectly affected by ECL as we observed that [3H]PDBu binding was decreased bilaterally (approximately 19%) in the dorsal lateral septum and the substantia nigra at 8 days post-lesion (data not shown). In fact, while the ECL model has been well characterized in relation to the predominant sprouting observed in the molecular layer of the dentate gyrus 8'26'27, other brain areas have not been extensively studied, probably because of limited apparent sprouting in these regions. Our data, using [3H]PDBu as a marker, reveal that, in addition to the molecular layer, certain layers of the entorhinal cortex as well as other sub-fields of the hippocampus, the substantia nigra and the septum, are affected at certain periods post-ECL. This is consistent with the bilateral changes in GFAP-immunoreactivity (see refs. 11,35, and our results) and neurite promoting activity 3° observed in different fields of the brain in this model. Therefore, it appears that ECL can induce alterations of various markers in other brain areas which extend well beyond the major sprouting site associated with this model, the outer molecular layer. The restricted decrease in [3H]PDBu binding observed in the contralateral side of the caudal hippocampus at 2 days post-ECL could be related to glial reactivity since Baudry and Altar have reported rostro-ventral differences in the intensity of a marker of gliosis in lesioned hippocampus 4. In that regard, what is the likelihood that [3H]PDBu is labelling either neuronal or glial (or both) cells in the ECL model? It is well known that marked synaptic losses are observed on the lesioned side during the first few days, post-lesion, and that the reinnervation process precedes and parallels gliosis 11'12. An increase in microglia is observed within the first day in the denervated molecular layer of the dentate gyrus IIA2. This -

increase is already maximal at the end of day 1 and decreases at day 6, post-lesion. The response of reactive astrocytes occured after microglial cell proliferation, their number increasing by 23% of their normal value on the fifth day post-lesion ~. Using GFAP-immunoreactivity as a marker, it has also been reported that reactive hypertrophic gliosis can be observed bilaterally within a few days (2-3) becoming maximal after 4 - 6 days, post-lesion (see ref. 1 1, and our results). Similarly, GFAP mRNA rapidly (day 2) increases bilaterally in the various sub-fields of the hippocampus to gradually become restricted only to the molecular layer of the dentate gyrus of the ipsilateral side 35"44. We observed that hippocampal [3H]PDBu binding was decreased bilaterally for a discrete period (2-8 days) before returning to control values by day 14 post-ECL. It would thus appear unlikely that alterations in [3H]PDBu binding relate to the proliferation of astrocytes, the onset of this phenomenon being observed after the initial changes in [3H]PDBu binding. It would seem that at least a good proportion of [3H]PDBu binding is associated with degenerating neuronal inputs observed on both the ipsilateral and contralateral sides of the hippocampal formation following ECL. This would explain the bilateral decreases detected at early times, post-lesion. It is of interest to note that PKC is enriched 4-fold in neuronal as compared to glial cell populations 2~. One also has to consider the possibility that electrolytic lesion used in our experiments could cause a seizure-dependent alteration of PKC. However, in the case of experimental seizure where a pronouced induction of G F A P mRNA could be observed 45, the PKC activity of the particulate fraction remained increased 9, indicating that seizure activity is unlikely to be the apparent cause of [3H]PDBu binding reduction observed in ECL rats. While the precise nature of the [3H]PDBu binding sites remains to be fully established, it most likely relates to PKC. Using an autoradiographic method similar to ours, it has already been demonstrated that the majority of [3H]PDBu binding sites are membrane bound 39 and that [3H]PDBu binding increases in the membrane fraction upon stimulation m'28"39. This probably reflects the translocation of PKC from the cytosol to the membrane compartment. Moreover, it is known that modifications in the amount of particulate PKC are correlated with changes in PKC activity 7'15'33'46. Therefore, this suggests that decreases in hippocampal [3H]PDBu binding observed as early as 2 days post-lesion correspond to alterations in PKC activity in the ECL model. Interestingly, decrements in [3H]PDBu binding and PKC activity have also been observed in

31 Alzheimer's disease in brain areas TM associated with the most prominent marked synaptic losses 41. In the ECL model, synaptic losses are rapidly compensated by the reinnervation process, this possibly explaining the transient decrease in [3H]PDBu binding observed in the hippocampus. Alternatively, decreases in [3H]PDBu binding can occur following calpain-I-induced proteolysis of various membrane-bound PKC isoenzymes2°'21. In the ipsilateral hippocampus in the ECL model, calpain-I-related spectrin breakdown products are increased very early (4 h) after the lesion, reaching maximal amounts by the second day and rapidly decreasing thereafter, although still at above normal values for at least 27 days4a. Significant increases in spectrin proteolysis are also observed in the contralateral hippocampus. This profile of events is similar to that reported here for [3H]PDBu binding. It is thus possible that alterations in [3H]PDBu binding in the ECL model are probably linked to a balance between the proteolysis of PKC and its translocation to the membrane fraction, at least in the hippocampal formation. This could explain the fact that [3H]PDBu labelling in ECL is affected only at day 2 and 8 but not on day 4, in the rostral portion of the hippocampus. Finally, while early studies have clearly demonstrated that [3H]PDBu binding sites were almost exclusively associated with PKC TM, it has recently been proposed that phorbol esters can also bind to a protein known as n-chimaerin which possesses important sequence homologies with PKC 14. However, the apparent affinity of phorbol esters for the n-chimaerin domain is 10-20-times lower than for PKC x. Moreover, n-chimaerin is more specifically localized to the pyramidal and granular cell layers of the hippocampus and the piriform cortex TM, this being markedly more restricted than the distribution of [3H]PDBu binding detected under our assay conditions. It thus appears that the majority of [3H]PDBu-labelled sites in the model discussed here are PKC-related. Acknowledgements. This research was supported by the Medical Research Council of Canada (MRCC) and the Alcan Corporation. A.P. is holder of a followship from the Alzheimer Society of Canada, while R.Q. is a 'Chercheur boursier' from the 'Fond de Recherche en Sant6 du Qu6bec' and J.P. holds a scholarship from MRCC. The authors are grateful to Dr. Richard Alonso for his helpful comments and discussion, and Mrs. Joan Currie for the secretarial assistance.

REFERENCES 1 Ahmed, S., Kozma, R., Monfries, C., Hall, C., Lim, H.H., Smith, P. and Lim, L., Human brain n-chimaerin cDNA encodes a novel phorbol ester receptor, Biochem. J., 272 (1990) 767-773.

2 Akers, R.F., Lovinger, D.M., Colley, D., Linden, D. and Routtenberg, A., Translocation of protein kinase C activity may mediate hippocampal long-term potentiation, Science, 231 (1986) 587-589. 3 Alkon, D.L. and Rasmussen, H., A spatial-temporal model of cell activation, Science, 239 (1988) 998-1005. 4 Baudry, M. and Altar, A., Entorhinal cortex lesion or intrahippocampal colchicine injection increases peripheral type benzodiazepine binding sites in rat hippocampus, Brain Res. 553 (1991) 215-221. 5 Blumberg, P.M., Complexities of the protein kinase C pathway, Mol. Carcinogen., 4 (1991) 339-344. 6 Burns, D.J. and Bell, R.M., Protein kinase C contains two phorbol ester binding domains, J. Biol. Chem., 266 (1991) 18330-18338. 7 Cole, G., Dobkins, K.R., Hansen, L.A., Terry, R.D. and Saitoh, T., Decreased levels of protein kinase C in Alzheimer brain, Brain Res., 452 (1988) 165-174. 8 Cotman, C.W. and Anderson, K.J., Neural plasticity and regeneration. In Siegel G.J. et al. (Eds), Basic Neurochemistry: Molecular, Cellular and Medical Aspect, 4th edn., Raven Press, New York, 1989 pp. 507-521. 9 Daigen, A., Akiyama, K. and Otsuki, S., Long-lasting change in the membrane-associated protein kinase C activity in the hippocampal kindled rat, Brain Res., 545 (1991) 131-136. 10 Etoh, S., Baba, A. and Iwata, H., NMDA induces protein kinase C translocation in hippocampal slices of immature rat brain, Neurosci. Lett., 126 (1991) 119-122. 11 Fagan, A. and Gage, F.H., Cholinergic sprouting in the hippocampus: a proposed role for IL-1, Exp. Neurol., 110 (1990) 105-120. 12 Gall, C., Rose, G. and Lynch, G., Proliferative and migratory activity of glial cells in the partially deafferented hippocampus, J. Comp. Neurol., 183 (1979) 539-550. 13 Goldowitz, D., White, W.F., Steward, O., Lynch, G. and Cotman, C.W., Anatomical evidence for a projection from the entorhinal cortex to the contralateral dentate gyrus of the rat, Exp. Neurol., 47 (1975) 433-44l. 14 Hall, C., Monfries, C., Smith, P., Lim, H.H., Kozma, R., Ahmed, S., Vanniasingham, V., Leung, T. and Lim, L., Novel human brain cDNA encoding a 34,000 M r protein n-chimaerin, related to both the regulatory domain of protein kinase C and BCR, the product of the breakpoint cluster region gene, J. Mol. Biol., 211 (1990) 11-16. 15 Hara, H., Onodera, H., Kato, H., Araki, T. and Kogure, K., Autoradiographic analysis of second messenger and neurotransmitter system receptors in the gerbil hippocampus following transient forebrain ischemia, Brain Res., 545 (1991) 87-96. 16 Hoff, S.F., Lesion-induced transneuronal plasticity in the adult rat hippocampus, Neuroscience, 19 (1986) 1227-1233. 17 Hoff, S.F., Scheff, S.W., Kwan, A.Y. and Cotman, C.W., A new type of lesion-induced synaptogenesis: I. Synaptic turnover in non-denervated zones of the dentate gyrus in young adult rats, Brain Res., 222 (1981) 1-13. 18 Huang, K.P., The mechanism of protein kinase C activation, Trends Neurosci., 12 (1989) 425-432. 19 Karnovsky, M.J. and Roots, L.A., A 'direct-coloring' thiocholine method for cholinesterases, J. Histochem. Cytochem., 12 (1964) 219-221. 20 Kishimoto, A., Kajikawa, N., Shiota, M. and Nishizuka, Y., Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease, J. Biol. Chem., 258 (1983) 1156-1164. 21 Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda, I., Tanaka, S., Tominaga, M., Kuroda, T. and Nishizuka, Y., Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain), J. Biol. Chem., 264 (1989) 4088-4092. 22 Lipton, S.A. and Kater, S.B., Neurotransmitter regulation of neuronal outgrowth, plasticity and survival, Trends Neurosci., 12 (1989) 265-270. 23 Lynch, G.S., Mattews, D.A., Mosko, S., Parks, T. and Cotman, C.W., Induction of acetylcholine esterase rich layer in dentate gyrus following entorhinal lesions, Brain Res., 42 (1972) 311-318. 24 Malinow, R., Madison, D.V. and Tsien, R.W., Persistent protein

32

25

26

27

28

29

30

31

32

33

34

35

36

kinase activity underlying long-term potentiation, Nature, 335 11988) 820-824. Malinow, R., Schulman, H. and Tsien, R.W., Inhibition of postsynaptic PKC or CaMKII blocks induction but not expression of LTP, Science, 245 (1989) 862-866. Matthews, D.A., Cotman, C.W. and Lynch, G., An electron microscopic study of lesion-induced synaptogenesis in the dentate gyrus of the adult rat. I. Magnitude and time course of degeneration, Brain Res., 115 (1976) 1-21. Matthews, D.A., Cotman, C.W. and Lynch, G., An electron microscopic study of lesion-induced synaptogenesis in the dentate gyrus of the adult rat. II. Reappearance of morphologically normal synaptic contacts, Brain Res., 115 (1976) 23-41. McMillan, M.K., He, X.P., Hong, J.S. and Pennypacker, K.R., Dopamine stimulates [3H]phorbol 12,13-dibutyrate binding in cultured striatal cells, J. Neurochem., 58 (1992) 1308-1312. Mudd, L.M. and Raizada, M.K., Effects of phorbol ester on immunoreactive protein kinase C, insulin binding, and glucose uptake in astrocytic glial and neuronal cells from the brain, Neurochem. Res., 15 (1990) 272-278. Needels, D.L., Nieto-Sampedro, M. and Cotman, C.W., Induction of neurite-promoting factor in rat brain following injury or deafferentation, Neuroscience, 18 (1986)517-526. Niedel, J., Kuhn, L.J. and Vandenbark, G.R., Phorbol diester receptor copurifies with protein kinase C, Proc. Natl. Acad. Sci. USA, 80 (1983) 36-40. Nishizuka, Y., The molecular heterogeneity of protein kinase C and its implications for cellular regulation, Nature, 334 (1988) 661-665. Onodera, H., Araki, T. and Kogure, K., Protein kinase C activity in the rat hippocampus after forebrain ischemia: autoradiographic analysis by [3H]phorbol 12,13-dibutyrate, Brain Res., 481 11988) 1-7. Parent, A., Brooks, W. and Quirion, R., Brain [3H]Phorbol ester binding sites in Alzheimer's and Parkinson's diseases: an autoradiographic study. In K. Iqbal et al. (Eds), Proceedings of 3rd International Symposium in AD and Related Disorders, John Wiley and Sons, UK (in press). Poirier, J., Hess, M., May, P.C. and Finch, C.E., Astrocytic apolipoprotein E mRNA and GFAP mRNA in hippocampus after entorhinal cortex lesioning, Mol. Brain Res., 11 (1991) 97-106. Poirier, J., May, P.C., Osterburg, H.H., Geddes, J., Cotman, C. and Finch, C.E., Selective alterations of RNA in rat hippocampus

37

38 39

40

41

42

43

44

45

46

47

48

after entorhinal cortex lesioning, Proe. NatL Acad. Sci. USA, 87 11990) 303-3117. Poirier, J. and Nichols, N.R., Alteration of hippocampa[ RNA prevalence in response to deafferentation, Methods Neurosci., 7 (1991) 182-199. Routtenberg, A., Synaptic plasticity and protein kinase C, Prog. Brain Res., 69 (1986) 211-234. Scharenberg, A.M., Olds, J.L., Schreus, B.G., Craig, A.M. and Alkon, D.L., Protein kinase C redistribution within CA3 stratum oriens during acquisition of nictitating membrane conditioning in the rabbit, Proc. Natl. Acad. Sci. USA, 88 (1991) 6637-6641. Scheff, S.W., Benardo, SW and Cotman, CW, Decline in reactive fiber growth in the dentate gyrus of aged rats compared to young adult rats following entorhinal cortex removal, Brain Res., 199 (1980) 21-38. Scheff, S.W., DeKosky, S.T. and Price, D.A., Quantitative assessment of cortical synaptic density in Alzheimer's disease, Neurobiol. Aging, 11 (1990) 29-37. Seubert, P., Ivy, G., Larson, J., Lee, J., Shahi, K., Baudry, M. and Lynch, G., Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. I. Biochemical studies, Brain Res., 459 (1988) 226-232. Stanfield, B.B. and Cowan. W.M., The sprouting of septal afferents to the dentate gyrus after lesions of the entorhinal cortex in adult rats, Brain Res., 232 (1982) 162-170. Steward, O., Torre, E.R., Phillips, L.L. and Trimmer, P.A., The process of innervation in dentate gyrus of adult rats: time course of increases in mRNA for glial fibrillary acidic protein, J. Neurosci., 10 (1990) 2373-2384. Steward, O., Torre, E.R., Tomasulo, R. and Lothman, E., Neuronal activity up-regulates astroglial gene expression, Proc. Natl. Acad. Sci. USA, 88 (1991) 6819-6823. Wieloch, T., Cardell, M., Bingren, H., Zivin, J. and Saitoh, T., Changes in the activity of protein kinase C and the differential subcellular redistribution of its isozymes in the rat striatum during and following transient forebrain ischemia, J. Neurochem., 56 (1991) 1227-1235. Worley, P.F., Baraban, J.M. and Snyder, S.H., Heterogeneous localization of protein kinase C in rat brain: autoradiographic analysis of phorbol ester receptor binding, J. NeuroscL, 6 (1986) 199-207. Zimmer, J. and Hjorth-Simonsen, A., Crossed pathways from the entorhinal area to the fascia dentata, J. Comp. Neurol., 161 11975) 71-102.