Neuroscience Research, 3 (1986) 237-241 Elsevier Scientific Publishers Ireland Ltd.
Electron microscopic study of mossy fiber endings in the hippocampal formation of rats after picrotoxin administration Hiroki Watanabe, Toshikazu Hochi, Kiminao Mizukawa and
Nagayasu Otsuka Department of Anatomy, Okayama UniversityMedical School, 2-5-1 Shikata-cho, Okayama 700 (Japan)
(Received January 21st, 1985; Revised version received August 1st, 1985; Accepted August 27th, 1985) Key words: hippocampus - - mossy fiber ending - - large dense-core vesicles - - picrotoxin - - seizure - electron microscopy - - rat
SUMMARY The hippocampal mossy fiber endings of rats were examined with electron microscopy, following the administration of picrotoxin. In the control group, many small round clear vesicles (40-60 nm in diameter) filled the nerve endings. Further, it was noted that large dense-core vesicles (80-100 nm in diameter) in small numbers were scattered among these small clear vesicles. After administration of picrotoxin, the large dense-core vesicles increased in number and accumulated next to the presynaptic membrane. These vesicles were often fused to the presynaptic membrane, in the form of omega-shaped profiles. In an electron m i c r o s c o p i c study o f the h i p p o c a m p u s , N i t s c h et al.8 r e p o r t e d that m a n y large d e n s e - c o r e vesicles a c c u m u l a t e d on the presynaptic m e m b r a n e at the m o s s y fiber endings following m e t h o x y p y r i d o x i n - i n d u c e d epileptic seizure. In o r d e r to ascertain that this is an effect specifically related to epileptic seizure, it is desirable to test also other epileptogenic drugs. In this study, we a d o p t e d picrotoxin which has been k n o w n to p r o d u c e seizures by blocking G A B A - m e d i a t e d p o s t s y n a p t i c inhibition2.6.7,9-11 a n d to induce a b n o r m a l w a v e f o r m s o f E E G in the h i p p o c a m p u s 9. W e confirmed in the h i p p o c a m p u s o f picrotoxin-treated rats a similar accumulation o f large dense-core vesicles to that r e p o r t e d by N i t s c h et al.8, a n d further found that this change occurred prior to a n d outlasted the seizure. Thirty male W i s t a r strain rats o f 3 0 0 - 4 0 0 g b o d y weight were used. The control rats were anesthetized with intraperitoneal injection o f sodium p e n t o b a r b i t a l (25 mg/kg) and Correspondence: H. Watanabe, Department of Anatomy, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan.
0168-0102/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
238 perfused with 400 ml of fixative containing 1 ~o paraformaldehyde and 10~o glutaraldehyde in 0.1 M phosphate buffer at pH 7.4. The experimental rats were injected in the abdominal cavity with 9 mg/kg picrotoxin dissolved in 0.9% NaC1 solution. The perfusions were started before convulsion, during convulsion and after convulsion, that is: 5 min, 25 min and 45 min after the injection of picrotoxin, respectively. The experimental rats were perfused and fixed as in the case of the control rats. The hippocampus tissues were cut with a vibratome and collected in 0.1 M phosphate buffer at pH 7.4. The blocks were fixed in the same fixative as used for control rats (see above) for 2 h, rinsed with phosphate buffer solution, and postfixed in 1% osmium tetroxide for 2 h. The blocks were dehydrated through a graded series of acetone immersions and finally embedded in an Epon 812 mixture. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a JEM 100CX electron microscope. Hippocampal mossy fibers are axons of the granular cells of the dentate gyrus, which end at the apical dendrites of the pyramidal cells of hippocampal CA3 and CA4 stratum radiatum. Electron micrographs show that the mossy fiber endings surround the dendritic spines of the CA3 and CA4 pyramidal cells, and their endings show the characteristic structure of their relatively large size (3-5 #m in diameter) 3'4. The dendritic spines ramify repeatedly, and one or two mossy fiber endings surround a ramified spine. Mossy fiber endings also form synapses with the shaft of smooth dendrites. These endings are filled with numerous small clear round vesicles, about 40-50 nm in diameter, among which are scattered a few large dense-core vesicles with diameter of 80-100 nm (Fig. 1). Besides these vesicles, there are also scattered relatively numerous mitochondria. Synaptic contacts between mossy fiber endings and spines are asymmetrical, while those between the former and the shaft surface of the dendrites are symmetrical. In the mossy fiber endings of the rats during convulsion, that is, 25 min after the administration of picrotoxin, large dense-core vesicles increased in number in comparison with the case of the control rats. The increase was about 2.5-fold according to measurement of the number of vesicles per unit area (1 #m 2) in 320 endings of control and 320 endings of experimental rats (t-test, P < 0.001). The large dense-core vesicles were either assembled near or attached to the presynaptic membrane. Further, the presynaptic membrane was shaped like an omega profile, hypertrophied in some places and only the large dense-core vesicles showed exocytosis. Neither an increased number nor exocytosis of small clear round vesicles could be observed (Fig. 2a ad b). These changes were mostly noted in synapses between mossy fiber endings and spiny dendrites of pyramidal cells. The number of large dense-core vesicles was also measured in mossy fiber endings obtained before and after convulsion, that is: 5 min and 45 min after the injection of picrotoxin. There was about a 2-fold increase in both groups of experimental animals, as compared with the control rats (t-test, P < 0.001). Presynaptic endings appeared enlarged, and many tubular, vesicular structures were observed in abnormally enlarged endings. Picrotoxin appears to interfere with a chloride channel which in the vertebrate CNS
Fig. 1. Axo-spinodendritic synapse in the stratum radiatum of CA4 from a control rat in the resting status. A giant mossy fiber ending (MFE) forms synaptic contacts with spines (SP). The ending is filled with small clear vesicles among which some large dense-core vesicles (asterisks) are present. M, mitochondrion. Fig. 2. Axo-spinodendritic synapse in the stratum radiatum of CA4 25 min after administration of picrotoxin, i.e. during the convulsion. (a) Increase in number and accumulation near the presynaptic membrane of large dense-core vesicles. Dense-core vesicles are attached to the presynaptic membrane (arrowheads). (b) Omega-shaped fusion of dense-core vesicles with the presynaptic membrane (arrow). Scale bars: 0.5 #m in Figs. 1 and 2a; 0.2 #m in Fig. 2b.
240 is usually associated with the receptor at GABAergic synapses 2,~°,1~. GABAergic synapses are present on initial segments of axons and somatic spines as well as on somata and dendritic shafts of granule cells in the dentate gyrus 5. Picrotoxin may block G A B A action at these synapses, and thereby produce abnormal activity in granule cells and their axons which form hippocampal mossy fibers. The observation o f ( l ) increased number of large dense-care vesicles in mossy fiber endings, (2) their accumulation and attachment to the presynaptic membrane, and (3) the formation of omega-shaped profiles in particular, resembles the changes in the mossy fiber endings witnessed by Nitsch et al. 8 after administration of methoxypyridoxin. It is interesting that seizures caused by different drugs present the same morphological changes. Since hypoxia accompanies convulsion, it may be suspected that the presently observed morphological changes in hippocampal mossy fiber endings are merely due to hypoxia. However, according to Bakay et al. ~, swelling of mitochondria in neurons is the most common abnormality in severe hypoxia. We did not observe such swelling of mitochondria in the present experimental rats, and considered that effects of hypoxia were negligible in terms of ultrastructures of neurons. Functional significance of the presently observed morphological changes in profiles of hippocampal mossy fiber endings is not clear for the present, but these may suggest the importance of substances released from the large dense-core vesicles in seizure activity. The fact that dense-core vesicles increased 5 min after picrotoxin administration, prior to onset of seizure, suggest that this is not a result from seizure, but that it represents a process which precludes the onset of seizure,
ACKNOWLEDGEMENT The authors wish to thank Dr. Steven R. Vincent, Assistant Professor of Neurological Research, The University of British Columbia, for his valuable criticism of this manuscript.
REFERENCES 1 Bakay,L. and Lee, J.C., The effectof acute hypoxiaand hypercapniaon the ultrastructure of the central nervous system, Brain, 9 (1968) 697-706, 2 Bruggencate,G.Ten. and Engberg, I., Iontophoretic studies in Dieters' nucleus of the inhibitory actions of GABA and related amino acid and the interactions of strychnine and picrotoxin,Brain Res., 25 (1971) 431-448. 3 Hamlyn, L.H., The fine structure of the mossy fiber endings in the hippocampus of the rabbit, J. Anat., 96 (1962) 112-126. 4 Ibata, Y., Electron microscopy of the hippocampal formation of the rabbit, J. Hirnforsch., 10 (1968) 451-469. 5 Kosaka, T., Hama, K. and Wu, J.-Y.,GABAergicsynapticboutons in the granulecell layer of rat dentate gyrus, Brain Res., 293 (1984) 353-359.
241 6 L6scher, W., Relationship between GABA concentrations in cerebrospinal fluid and seizure excitability, J. Neurochem., 38 (1982) 293-295. 7 Mori, A., Experimental model of epilepsy, Adv. Neurol. Sci., 23 (1979) 891-903. 8 Nitsch, C. and Rinne, U., Large dense-core vesicles exocytosis and membrane recycling in the mossy fiber synapses of the rabbit hippocampus during epileptiform seizures, J. Neurocytol., 10 (1981 ) 201-219. 9 Saito, S., Biochemistry of Epilepsy, Adv. Neurol. Sci., 12 (1968) 599-609. 10 Simmonds, M.A., A site for the potentiation of GABA mediated responses by benzodiazepines, Nature (London), 284 (1980) 558-560. 11 Simmonds, M.A., Evidence that bicuculline and picrotoxin act at separate sites to antagonize GABA in rat cuneate nucleus, Neuropharrnacology, 19 (1980) 35-45.