Heparin injection into the adult rat hippocampus induces seizures in the absence of macroscopic abnormalities

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Neuroscience Vol. 89, No. 2, pp. 329 333, 1999 Copyright © 1998IBRO. Publishedby ElsevierScienceLtd Printed in Great Britain.All rights reserved PII: S0306-4522(98)00533-8 03064522/99 $19.00+0.00

Letter to Neuroscience H E P A R I N I N J E C T I O N INTO THE A D U L T RAT H I P P O C A M P U S I N D U C E S SEIZURES IN THE ABSENCE OF MACROSCOPIC ABNORMALITIES A. K. MUDHER,*:~ J. MELLANBY,] H. McMATH,*§ V. H. PERRY*¶ and J. R. T. GREENE*II *University Department of Pharmacology, Mansfield Road, Oxford OX1 3QT, U.K. tDepartment of Experimental Psychology, South Parks Road, Oxford OX1 3PS, U.K. K e y words:

Alzheimer's disease, electrophysiology, epilepsy, extracellular matrix, glycosaminoglycans,

subiculum.

The pathological hallmarks of Alzheimer's disease include neurofibrillary tangles, neuropil threads and neuritic plaques. Neurofibrillary tangles and neuropil threads are comprised of paired helical filaments which are themselves composed of a hyperphosphorylated form of the microtubule-associated protein tau. 1°'~3 Neuritic plaques are extracellular deposits of aggregated beta amyloid associated with neurites containing hyperphosphorylated tau. 3 The mechanisms by which the neurofibrillary tangles and neuritic plaques develop in Alzhemier's disease are not clear but it is hypothesized that sulphated glycosaminoglycans are important in their formation. This impression is based on the finding that the glycosaminoglycan, heparan sulphate, is found associated with neurofibrillary tangles, neuritic plaques and neuropil threads s'16'ls while dermatan sulphate, chondroitin sulphate and keratan sulphate immunoreactivity is found around neuritic plaques ~7 in brains of Alzheimer's disease patients. Furthermore, in vitro studies demonstrate that sulphated glycosaminoglycans such as heparan sulphate and the closely related molecule heparin interact with tau and potentiate its phosphorylation by a number of ~:To whom correspondence should be addressed at her present address: The Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, U.K. §Present address: Bayer PLC, Stoke Court, Stoke Poges, Slough SL2 4LX, U.K. ~[Present address: CNS Inflammation Group, School of Biological Sciences, University of Southampton, Basset Crescent East, Southampton SO16 7PX, U.K. HPresent address: Department of Biomedical Science, Alfred Denny Building. The University of Sheffield, Western Bank, Sheffield S10 2TN, U.K. Abbreviations: AD, Alzheimer's disease; CA1, cornu a m m o nis field 1; DG, dentate gyrus; ECM, extracellular matrix; FS, facial spasms; GS, generalized seizures; NBQX, 6-nitro-7-sulphamoylbenzol[f]quinoxaline-2,3-dione disodium; WDS, wet dog shakes.

serine/threonine kinases, reduce its ability to bind to microtubules and induce paired helical filament formation, all properties associated with tau isolated from Alzheimer's disease brain, s'12 Thus, we were interested to learn whether intracerebral injection of the sulphated glycosaminoglycan heparin would give rise to alterations in the cytoskeletal protein tau in the rat brain. Although no cytoskeletal changes were observed, to our considerable surprise we found that the intrahippocampal injection of heparin gave rise to seizures. We have investigated this unexpected effect further in vivo and by using in vitro electrophysiological techniques. © 1998 IBRO. Published by Elsevier Science Ltd. All experiments were conducted in accordance with the Animals (Scientific Procedures) Act 1986 of England and Wales. The stereotaxic surgery, clinical observations and histological examinations were carried out blind. Adult male Wistar rats (250 g, Harlan Olac, Bicester, U.K.) were anaesthetized with halothane and injections of 32 units (1 rag= 172 units, mol. wt=9-12,000; Sigma, Poole, U.K.) of porcine grade I heparin in 1 gl of endotoxin free saline (n=5) (Sigma), or 1 gl of endotoxin free saline (n= 5) were made, using glass micropipettes (15-20gm in diameter), into the CA1/alveus region of the left hippocampus (anterior posterior - 4 ; lateral +4; depth from dura - 2 . 8 ) over 1 min. All rats began to recover from anaesthesia within 15 min of surgery and were watched for up to 2 h thereafter by at least two independent observers. Within 15-20 min of recovery, vigorous and frequently repeated wet dog shakes ( W D S ) 7 w e r e seen in the rats which had received heparin. The form of the seizures was variable between rats though they always involved brief spasms of the facial musculature, rearing up and stiffening of the whole body

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nature o f seizure activity Fig. 1. Number of rats displaying different types of seizure activity after injection of saline or heparin. Wistar rats were injected with 1 i,tl of saline (n=5) or 32 units of heparin in 1 gl of saline (n=5) in the hippocampus. They were observed by two or three assessors who were blind to the agent injected, for up to 2 h after injection. Seizures were characterized by wet dog shakes (WDS) and generalized seizures (GS) that involved the limbs, and facial spasms (FS). **P<0.01, *P<0.05; Fisher's test. and this was usually followed by falling. Some rats exhibited paroxysmal running behaviour and in one of these this was followed by a full tonic~clonic seizure. Saline injected rats behaved normally once they had recovered from the anaesthesia (Fig. 1). To exclude the possibility that seizures were caused by an impurity in the porcine intestinal heparin, an additional three rats were injected with heparin from bovine lung (Sigma, Poole, U.K.). These rats also exhibited seizures like those seen following injections of porcine heparin. To exclude the possibility that the seizure activity was dependent upon the type of anaesthetic used, seven rats were anaesthetized with rat avertin (2,2,2tri-bromo-ethanol in tertiary amyl alcohol) before injection with heparin. These rats took longer to recover (typically 40 min) but then exhibited seizure behaviour similar to that seen in the rats that had been anaesthetized with halothane. To exclude any possible influence of surgical technique, injections were made by two different experimenters and in both cases rats injected with heparin exhibited similar seizure behaviour. The brains from all of the rats were examined histologically. Two observers, blinded to the agent injected, did not distinguish between the heparin- and saline-injected brain sections. The sections were examined initially with the microscope condenser defocused to reveal red blood cells more easily. No

evidence of extravasated red blood cells was found. Using normal bright field illumination, sections stained with anti-IgG antibody were examined and again there was no evidence of intracerebral haemorrhage or of breakdown of the blood brain barrier around the injection site, to which the seizures could be attributed (Fig. 2). lntracellular electrophysiological recordings were made from 16 intrinsically burst firing subicular pyramidal neurons ~s'9 in horizontal slices of hippocampus from eight male Wistar rats, 180 240 g. Slices were maintained in an interface-type recording chamber at 36°C and superfused with artificial cerebrospinal fluid. 9 Eight neurons were exposed to heparin and eight neurons were exposed to heparin in the presence of various neurotransmitter receptor antagonists. In all eight neurons exposed to heparin alone (100-1200units/ml), but not in any of the eight neurons exposed to heparin in the presence of bicuculline ( 2 0 g M ) and 6-nitro-7sulphamoylbenzol[f]quinoxaline-2,3-dione disodium (NBQX) (5 ~tM) or N B Q X alone, there was a marked increase in synaptic activity (Fig. 3). This increase occurred gradually within 10 min of the start of heparin application and subsided within 10 15 min of the start of heparin wash-out. In three out of five neurons exposed to a concentration of heparin of 800 units/ml or more, the increase in synaptic activity was followed by action potential discharge. In six

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Fig. 2. IgG (A, B) and Cresyl Violet (C, D) staining around injection site (arrows) after injection of saline (A, C) or heparin (B, D). There is no evidence of an intracerebral haemorrhage nor of a major breakdown of the blood-brain barrier, or any other macroscopic lesion in any of the brain sections. Rats were observed for up to 2 h after injection and then re-anaesthetized and transcardially perfused with 4% paraformaldehyde. The brains were then removed and sunk in 30% sucrose before being frozen in Tissue Tek (Bayer Diagnostics, Slough, U.K.)• Ten-micrometre-thick cryostat sections were cut and quenched in 10% methanol peroxidase for 20 min and then blocked in 10% normal rabbit serum (Vector Immunochemicals, Peterborough, U.K.) for 30 rain. Intracerebral bleeding was ascertained by detecting rat IgG staining around the injection site using a biotinylated rabbit anti-rat antibody (Vector Immunochemicals); sections were incubated in the rabbit anti-rat antibody (1:100 in phosphate-buffered saline) for 45 min followed by an avidin-biotin complex (Vector Immunochemicals) for 45 min. All incubations were done at room temperature. The chromogen diaminobenzidine was used to visualize the antibody binding. Sections were counter-stained with Cresyl Violet. Sections in C and D were stained with Cresyl Violet alone. Photomicrographs were prepared using Image Grabber and Adobe Photoshop. Scale bar= 100 gin. cells heparin was applied in a range of concentrations in a random order and the increase in synaptic activity was seen to be concentration dependent (Fig. 3). The present study is the first to demonstrate that heparin, a glycosaminoglycan, causes seizures when injected into adult rat brain. We found no evidence to suggest that the seizures were secondary to an intracerebral haemorrhage: the IgG/Cresyl Violet staining did not indicate extravasation of red blood cells or of b l o o d - b r a i n barrier breakdown around the injection site, and two independent observers, blinded to the agent injected did not distinguish between the heparin and saline injected animals in the histological analysis• Furthermore, although we have no conclusive p r o o f of such a link, the electrophysiological p h e n o m e n a seen in brain slices, where bleeding would have been impossible at the time of heparin application, could form the basis of the seizures seen

in vivo. Application of heparin to brain slices caused an increase in synaptic activity which was at least in part excitatory in nature, and was in some cases followed by action potential discharge. These phenomena occurred over a relatively short period and it is interesting to note that the seizures seen in vivo occurred over a similarly short period. The mechanism by which heparin increased synaptic activity is unclear, but known actions of heparin such as inhibition of intracellular IP 3 receptors 14 seem unlikely given that heparin does not easily enter cells and reduction of intracellular calcium would reduce synaptic activity• A direct action on glutamate receptors is possible given that heparin is reported to bind solubilized glutamate receptors in v i t r o ] ~ Another possible mechanism is that heparin's highly sulphated, negatively charged backbone ~9 binds divalent cations thereby reducing concentrations around neurons and resulting in an increase in neuronal

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excitability, s'6 Localized deposits of heparin m a y act as epileptic foci, a n d it is interesting t h a t the amyloid plaques f o u n d in Alzheimer's disease c o n t a i n glycosaminoglycans similar to h e p a r i n a n d t h a t fitting occurs in a b o u t 10 20% o f A l z h e i m e r ' s disease patients. 4 These experiments also provide insights into the possible physiological role o f glycosaminoglycans. N e u r o n s are s u r r o u n d e d by perineuronal nets of the extracellular m a t r i x ( E C M ) comprised o f glyc o s a m i n o g l y c a n s a n d o t h e r glycoproteins 1 a n d perh a p s these regulate n e u r o n a l excitability by buffering the c o n c e n t r a t i o n o f divalent cations immediately adjacent to the cell m e m b r a n e . The i n v o l v e m e n t o f

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the E C M s u r r o u n d i n g n e u r o n s in electrical excitability has recently come to the fore in the work of Strickland a n d colleagues. 2 These a u t h o r s have demonstrated t h a t excitotoxicity caused by kainic acid in m o u s e b r a i n is preceded by loss o f the E C M glycoprotein laminin. 2 I f l a m i n i n b r e a k d o w n does n o t take place, as is the case in the tissue plasminogen activ a t o r k n o c k out mouse, n e u r o n a l degeneration does not occur, but infusion of anti-laminin antibodies into the tissue p l a s m i n o g e n activator k n o c k o u t mice restores the excitotoxic effects of kainic acid. These findings a n d ours raise interesting questions a b o u t the role o f the E C M , which m a y n o t only be structural b u t m a y also play a crucial role in the regulation of n e u r o n a l excitability in b o t h physiological and pathological conditions.

Acknowledgements--This work was funded by a grant from

Bristol-Myers Squibb. J. R. T. G. is an MRC Career Development Award holder.

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Fig. 3. Effect of heparin on non-evoked synaptic activity in a horizontal slice (400 ~tm thick) of rat hippocampal formation. Intracellular electrophysiological recordings, made using a 3-M potassium acetate-filled electrode of 80 Mr2 resistance, from a single subicular neuron of intrinsically burst-firing type are shown. Heparin (Grade 1 Porcine intestinal, supplied by Sigma, Poole, U.K.) was added to artificial cerebrospinal fluid (composition in raM: NaC1, 124; KCI, 123; MgSO 4, 2.0; KH2PO4, 1.3; CaCI> 2.0; NaHCO3, 26; glucose, 10; gassed with 95% 02 +5% CO2; pH 7.4) in an interface-type recording chamber at 36°C. Concentrations used were 300, 600 and 1200units/ml. Records were made at resting membrane potential (in this case - 6 8 mV) 10 min after the start of heparin application. Heparin was washed off and the synaptic activity allowed to return to control values between successive heparin application; this took about 10rain. Heparin caused a concentration-dependent increase in synaptic activity. In the bottom trace, the non-N-methyl-D-aspartate glutamate receptor antagonist NBQX (supplied by Tocris Cookson, Bristol) was added at the same time as heparin and can clearly be seen to have altered the response to heparin, suggesting that at least some of the increased activity caused by heparin was non-N-methyl-D-aspartate receptor dependent. Recordings were filtered at 2 kHz and displayed on a Gould TA240 chart recorder.

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