Parenterally administered GM1 ganglioside prevents retrograde degeneration of cholinergic cells of the rat basal forebrain

Brain Research, 398 (1986) 393 396 Elsevicr

393

BRE 21896

Parenterally administered ganglioside prevents retrograde degeneration of cholinergic cells of the rat basal forebrain M . V . S o f r o n i e w 1, R . C . A . P e a r s o n 2, A . C . C u e l l o 3, P.C. Tagari 3 and P . H . Stephens 4 iA natomy School, Cambridge ( U.K.), "Department of Hurnan Anatomy, Oxford ( U. K. ), ~Department of Pharmacology and Therapeutics, McGill University, Montreal (Canada) and 4Department of Pharmacology, Oxj?~rd ( U. K. )

(Accepted 29 July 1986) Key words: Septal nucleus; Ganglioside: Immunohistochemistry; Choline acetyhransferase: Retrograde degeneration

The effect of daily intraperitoneal injections of GM l ganglioside on retrograde degeneration in the basal forebrain has been examined, using a monoclonal antibody directed against choline acetyltransferase to identify the cholinergic neurones. Rats underwent extensive damage of the cerebral cortex and underlying hippocampus. From the day of operation on, they received daily injections of ganglioside. After a survival of 30 days, the animals were killed and the cholinergic cells of the basal forebrain were examined. These were compared with material treated in the same way from animals who had received the injections of ganglioside but no lesion, animals who had been operated upon but without the ganglioside treatment, and normal animals. Intraperitoneal ganglioside administration markedly reduces the retrograde death of the cholinergic neurones of the medial septal nucleus and abolishes the shrinkage of the remaining neurones following hippocampal damage. Gangliosides are sialic acid containing glycosphingolipids which are normal constistuents of cell membranes, particularly in neurones, where they are located in the outer leaflets of the m e m b r a n e s 9"17. Exogenously administered gangliosides, particularly the monosialoganglioside, GM1, have been shown to stimulate neurite outgrowth in cell culture 6'lj and to enhance reinnervation by increased neuronal sprouting in the peripheral nervous system 3"7's. In the central nervous system, G M 1 has recently been shown to p r o m o t e the reinnervation of the striatum by dopaminergic fibres from the substantia nigra following hemitransection of this pathway in the rat 14, possibly by enhancing collateral sprouting 2. In addition, such treatment also substantially reduces the retrograde death of substantia nigra neurones which normally accompanies such a transection 15. Finally, intraperitoneal injections of ganglioside have been shown to facilitate behavioural recovery in rats following bilateral damage of the striatum ~2, and to accelerate the recovery of choline acetyltransferase (CHAT) and acetylcholine esterase (ACHE) levels in the hippocampus following septal lesions 16. Following damage of the hippocampus in rats, there is m a r k e d retro-

grade cell degeneration in the medial septal nucleus s, and this represents a substantial loss of cholinergic neurones from this nucleus m. Following lesions of the neocortex, the retrograde shrinkage of cholinergic cells of the basal nucleus which occurs and which is not accompanied by cell loss I~ can be p r e v e n t e d by daily intraperitoneal injections of G M l (ref. 4). The aim of the present study was to examine whether a similar regime would prevent or diminish cell loss from the medial septal nucleus following removal of the hippocampus. At 30 days of age, under ether anaesthesia, 3 out of 6 litter-matched male Wistar rats had a large area of the neocortex and underlying hippocampus removed by suction. For 30 days following the operation, all 6 animals received daily intraperitoneal injections of 30 mg/kg G M I (Fidia). A t the end of this time. the animals were deeply anaesthetised and perfused through the heart with a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde. Vibratome sections of 100lira through the forebrain were processed for the immunohistochemical detection of C h A T using a rat monoclonal antibody, as previously described 13. In each rat, the numbers of C h A T - c o n t a i n -

Correspondence: M.V. Sofronicw. Anatomy School. Downing Street, Cambridge CB2 3DY. U.K.

394 ing cells were counted at 5 carefully matched levels

the numbers of ChAT-positive cells in the ipsilatera/

through the medial septal nucleus (MS) and 2

medial septal (Fig. 1 ) and vertical nuclei are dramat-

through the vertical nucleus of the diagonal band of

ically reduced ~mean loss: MS 74';_ VN 46~>; t. and the remaining neurones are smallcr than in the nor-

Broca (VN). The cell counts were corrected for cell size using the method of A b e r c r o m b i e ~. The results

mal animal Imean shrinkage: MS and VN 18',; ~.

were compared with those obtained in 3 naive and 3

These changes are largely prevented b~, the chronic

operated rats without gangtioside administration. Also, in the animals receiving ganglioside. 50 ran-

administration of intraperitoneal (}M~ when the cell loss is small Imean loss: MS 15', VN 13%. Fig. l),

domly selected C h A T - s t a i n e d cells within the central

and the remaining cells are ol normal size i m e a n change: MS + 8 ~ . VN +2f,; I lntraperitoneal G M

portion of each nucleus were drawn at a magnification of x600 and their cross-sectional area was mea-

administration has no appreciable cflect on the num-

sured. These data were compared with mean values

ber or size of these neurones m control animals.

for the cross-sectional areas from 15 naive animals

These observations were confirmed quantltativel)

(MS 170 +_ 41: VN 250 _~ 47/. as it has previously

ITables I and I1). This study has extended previous w,ork on the pre-

been shown that the mean size of cells in both the medial septal m and the vertical (unpublished observations) nucleus does not vary significantly in normal adult Wistar rats. Using the same methodology, the shrinkage of cholinergic neurones remaining in the MS and VN was measured in 7 rats after the removal of the hippocampus but without gangtioside treatment. Following removal of the hippocampus of one side.

vention of retrograde cell degeneration in the cholinergic nuclei of the basal forebrain of the rat with chronic parenteral GM~ treatment +. Not ontv does this procedure prevent retrograde celt shrinkage of these cells, but it also markedly reduces the cell loss which occurs without gangliosides, This protection against cell death following axotomv is similar to that seen in the dopaminergic neurones of the substantia

Fig. 1. Photomicrographs of the medial septal nucleus stained for the immunohistochemical detection of CHAT. A: normal animal. B: following removal of the left hippocampus: note the marked loss of cholinerglc cells from the left MS. C: followmg removal of the left hippocampus in an animal receiving GM ~:the majority of the cholinergic cells in the left MS are preservcd, x 36: L. left.

395

TABLE I Numbers o f ChA T-containing cells in the medial septal and vertical nuclei (A bercrombie corrected)

Mean % difference (left:right) in operated animals without GMI: MS -74%, VN -46%. Mean % difference (left:right) in operated animals receiving GMI: MS - 15%, VN -13%. Rat no

Numbers o f ChA T containing cells" Medial septal nucleus (5 matched levels) Left

Right

783 641 623

743 658 661

Controlanimals + ganglioside 4 691 5 659 6 593 Operated animals 7 8 9

Normal animals 1 2 3

204 199 107

Operated animals + ganglioside 10 569 11 695 12 653

% difference (left:right)

Vertical nucleus (2 matched levels)

c;~ dtflerence (left:right)

Left

Right

+ 5 3 - 6

231 239 2/)5

201 251) 185

+15 - 4 + I1

731 625 580

- 5 + 5 + 2

241 181 17(1

235 167 156

+ 3 + 8 + 9

716 667 529

-72 -7(I -80

169 113 111

279 216 223

-39 -48 -50

788 766 704

-28 9 7

225 184 178

275 208 196

-18 -12 9

TABLE II Mean cross-sectional areas o f ChA T-containing cells in the medial septal and vertical nuclei

Overall mean shrinkage following lesion without GM 1 (7 animals): MS -18%, VN -18%. Overall mean change following lesion with GM~: MS +8%, VN +2%. Rat no.

Medial septal nucleus

Vertical nucleus

(urn ~"+ S.D.; n = 50)

% difference vs normal

(um 2 +_S. D. ,"n = 50)

% di[[erence vs normal

Controls + GM~ 4 5 6

199 _+ 38 172 + 31 168 _+ 35

+ 17 + 1 - l

250 _+ 36 236 __+39 255 _+ 58

0 -6 +2

Operated + GM I 10 11 12

176 _+ 41 18t)_+51 195 _+ 37

+ 4 + 6 + 15

255 _+ 41 258 +_ 48 256 + 42

+2 +3 +2

n i g r a f o l l o w i n g t r a n s e c t i o n of t h e n i g r o s t r i a t a l p a t h -

d u c e d f r o m t h e s e e x p e r i m e n t s , b u t it s e e m s t h a t t h e i r

way in rats ~5. In this s t u d y h o w e v e r , t h e p r e s e r v a t i o n

a c t i o n in p r e s e r v i n g d a m a g e d n e u r o n e s m a y b e i n d e -

of t h e d a m a g e d c h o l i n e r g i c n e u r o n e s is u n l i k e l y to b e

p e n d e n t of a n y s t i m u l a t i o n of a x o n a l r e g e n e r a t i o n o r

d u e to t h e p r o m o t i o n o f r e g e n e r a t i o n o r c o l l a t e r a l

collateral sprouting within their target area.

s p r o u t i n g of c h o l i n e r g i c fibres, at l e a s t w i t h i n t h e p a r t of t h e h i p p o c a m p u s to w h i c h t h e y n o r m a l l y p r o j e c t ,

T h i s w o r k was s u p p o r t e d by t h e W e l l c o m e T r u s t

since this t a r g e t h a s b e e n r e m o v e d . N o i n d i c a t i o n o f

a n d t h e M R C ( U . K . a n d C a n a d a ) . W e are g r a t e f u l to

t h e m e c h a n i s m o f a c t i o n o f t h e g a n g l i o s i d e c a n b e de-

Fidia for t h e p r o v i s i o n of g a n g l i o s i d e .

396

1 Abercrombie, M., Estimation of nuclear populations from microtome sections, Anat. Rec., 94 (1946) 239-247. 2 Agnati, L.F., Fuxe, K., Calza, L., Benfanti, F., CavicchioIf, L., Toffano, G. and Goldstein, M., Gangliosides increase the survival of lesioned dopamine neurons and favour the recovery of dopaminergic synaptic function in striatum of rats by collateral sprouting, Acta Physiol. Scand., 119 (1983) 347-363. 3 Ceccarelli, B., Aporti, F. and Finesso, M., Effects of brain gangliosides on functional recovery in experimental regeneration and reinnervation, Adv. Exp. Med. Biol., 21 (1976) 275-293. 4 Cuello, A.C., Stephens, P.H., Tagari, P.C., Sofroniew, M.V. and Pearson, R.C.A., Retrograde changes in the nucleus basalis of the rat, caused by cortical, are prevented bv exogenous ganglioside GM n, Brain Research, in press. 5 Daitz, H.M. and Powell, T.P.S., Studies on the connexions of the fornix system, J. Neurol. Neurosurg. Psychiato,, 17 (1954) 75-82~ 6 Facci, L., Leon, A., Toffano, G., Sonnino, S., Ghidoni, R. and Tettamanti, G., Promotion of neuritogenesis in mouse neuroblastoma cells in exogenous gangliosides. Relationship between the effect and the cell association of ganglioside GM 1, J. Neurochem., 42 (1984) 299-305. 7 Gorio, A. and Carmignoto, G., Motor nerve sprouting induced by ganglioside treatment. Possible implications for gangliosides on neuronal growth, Brain Research, 197 (1980) 236-241. 8 Gorio, A., Marini, P. and Zanoni, R., Muscle reinnervation III: motoneuron sprouting capacity, enhancement by exogenous gangliosides, Neuroscience, 8 (1983) 417-429. 9 Ledeen, R.W., Ganglioside structure and distribution: are they located at the nerve ending?, J. Supramol. Struct., 8 (1978) 1-17.

10 Pearson. R.C.A.. Sofroniew. M.V and Powell T.P.S Hypertrophy of immunohistochemically identified cholincrgic neurons of the basal nucleus of Mevnert following ablation of the contralateral cortex in the rat. Brain Research 311 ~1984) 194-198. 11 Roisen. F..I.. Batficld. H.. Nagelc. R and Yorke. F. (Janglioside stimulation of axonal sprouting m vitro. Science. 214 (1981) 577-578 12 Sabet. B.A.. Slavin. M.D. and stem D.G._ GM 1 ganglioside treatment facilitates behavioural recovery from bilateral brain damage, Science. 225 ( 1984 34(/- 342. 13 Sofroniew. M.V.. Pearson. R.C.A.. Eckenstem. i-.. CuelIo. A.C. and Powell. T.P.S.. Retrograde changes in cholmergic neurons in the basal forebrain o1 the rat following cortical damage. Brain Research. 289 / 1983) 370-374. 14 Toffano. G. Savoini. G.E.. Moroni. F Lombardi. ().. Calza. L. and Agnati. L.F.. GM~ ganglioside stimulates the regeneration of dopaminergic neurons in the central nervous system. Brain Research. 261 t1983) 163-166. 15 Toffano. G. Savoini. G.E.. Moroni. F. Lombardi. G.. ('alza. L. and Agnatt, L,F.. Chronic G M ganglioside treatment reduces dopamine cell body degcneration in the substantia nigra after unilateral hemitr~msection in rat, Brain Research, 296 [ 1984) 233-239 16 Wojeik. M.. Ulas, J. and Oderfeld-Nowak, B.. Ihe stimulating effect of ganglioside inJections on the recovery ot choline acetvltransferase activities in the hippocampus oI the rat after septal le~sions.Neuroscicnce. "7(1982) 495-499. 17 Zambotti. V.. Tettamanti. G. and Arrigoni, M.. GIvcolipids, G(vcoprotems and Mucopolysaccharides (~f the Nervous System. Plenum Press. New York 1972