Phosphorylation of dynamin I and synaptic-vesicle recycling

257, 255-259 22 Inoue, A., Obata, K. and Akagawa, K. (1992) J. BioL Chem. 267, 10613-10619 23 Oyler, G. A. etaL (1989) J. Cell Biol. 109, 3039-3052 24 Hess, D. T., Slater, T. M., Wilson, M. C. and Skene, J. H. P. (1992) J. Neurosci. 12, 4634-4641 25 SGIIner,T., Bennett, M. K., Whiteheart, S. W., Scheller. R. H. and Rothman, J. E. (1993) Cell 75, 409-418 26 Niemann, H., Blasi, J. and Jahn, R. (1994) Trends Ceil BioL 4, 179-185 27 Bennett, M. K. and Scheller, R. H. (1993) Proc. Nat/Acad. Sci. USA 90, 2559-2563 28 Cain, C. C., Trimble, W. S. and Lienharcl, G. E. (1992) J. Biol. Chem. 267, 11681-11684 29 McMahon, H. T. eta/. (1993) Nature 364, 346-349 30 Bennett, M. K. et al. (1993) Cell 74, 863-873 31 Rothman, J. E. and Warren, G. (1994) Curt. Biol. 4, 220-233 32 Calakos, N., Bennett, M. K., Peterson, K. E. and Scheller, R. H. (1994) Science 263, 1146-1149 33 Matthew, W. D., Tsavaler, L. and Reichardt, L. F. (1981)

J. Cell Biol. 91,257-269 34 DeBello, W. M., Betz, H. and Augustine, G. J. (1993) Ce1174, 947-950 35 O'Connor, V. M., Shamotienko, O., Grishin, E. and Betz, H. (1993) FEBS Lett. 326, 255-260 36 Hata, Y., Davletov, B., Petrenko, A. G., Jahr!, R. and S0dhof, T. C. (1993) Neuron 10, 307-315 37 Hat& Y., Slaughter, C. A. and S0dhof, T. C. (1993) Nature 366, 347-351 38 Pevsner, J., Hsu, S-C. and Scheller, R. H. (1994) Proc. Nat/ Acad. Sci. USA 91, 1445-1449 39 Garcia, E. P., Gatti, E., Butler, M., Burton, J. and De Camilli, P. Proc. Nat/Acad. ScL USA (in press) 40 Aalto, M. K., Keranen, S. and Ronne, H. (1992) Ce// 68, 181-182 41 Simons, K. and Zerial, M. (1993) Neuron 11, 789-799 42 Fischer yon Mollard, G. eta/. (1992) Proc. Nat/Acad. ScL USA 87, 1988-1992 43 Dascher, C., Ossig, R., Gallwitz, D. and Schmitt, H. D. (1991) /viol. Cell. Biol. 11, 872-885

Phosphorylationof dynaminI andsynaptic-vesiderecycling P h i l l i p J. R o b i n s o n , J u n - P i n g Liu, K a t e A. P o w e l l , Else M a r i e Fykse a n d T h o m a s

Phfflip .Z Robinson, Jun-Pl?g Liu and Kate A. Powell are at the Endocrine Unit, John Hunter Hospital, Locked Bag 1, Hunter Region Mail Centre, Newcastle, NSW 2310, Australia, and Else Marie Fykseand ThomasC SMhofare at the Dept of Molecular Genetics and Howard Hughes Medical lnstitute, The University of Texas 5outhwestem Medical Center, Dallas, Texas 75235, USA.

348

In nerve terminals, neurotransmitters are packaged in synaptic vesicles, and released by exocytosis. Empty synaptic vesicles are rapidly recycled for reuse by endocytosis. Much progress has been made in identifying the proteins involved in synaptic-vesicle trafficking, but the mechanism and regulation of endocytosis have largely remained an enigma. One approach to defining regulatory proteins that might be involved is to study stimulus-d@endent phosphorylation events in nerve terminals. This has led to the identification of dephosphin, which is quantitatively dephosphorylated by nerve-terminal depolarization. Sequencing reveals that dephosphin is identical with dynamin I, a GTP-binding protein that functions in endocytosis. Phosphorylation and dephosphorylation of nerve-terminal dynamin I/ dephosphin regulates its intrinsic GTPase activity in parallel with the regulation of synaptic-vesicle recycling. Therefore, phosphorylation and dephospho~lation of dynamin I might provide a Cae+-d@endent switch for endocytosis in the synaptic-vesicle pathway.

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ment (or fission) s-s. The clathrin coat is quickly shed and the vesicle is available for recycling. It is widely held that the recycling of synaptic vesicles in neurons represents a specialized form of receptor-mediated endocytosis ~'s. Most of our knowledge of the molecular mechanisms underlying endocytosis comes from studies of receptor-mediated endocytosis, but the pathways in nerve terminals are related. Synaptic-vesicle recycling by c l a t h r i n - c o a t e d

pits

Trafficking synaptic vesicles in nerve terminals are surrounded transiently by a protein coat that is assembled at the plasma membrane at a limited number of sites known as clathrin-coated pits 3. Upon purification of brain coated vesicles, several synapticvesicle-specific proteins have been found, indicating that the coated-vesicle pathway predominates in neural cells 9. A few coated vesicles are observed in dendrites where, presumably, they mediate receptormediated endocytosis 3. There are several distinctions Synaptic-vesicle-membrane trafficking is a continuing between receptor-mediated endocytosis and synapticcycle with individual steps mediated by distinct vesicle recycling. Unlike non-neuronal cells, synapticproteins and enzymes. The cycle begins when an vesicle recycling occurs at sites far from the cell body, action potential depolarizes the nerve terminal, and and all components must be transported to the nerve opens voltage-gated Ca .-,+ channels at the active zone. terminal. Synaptic vesicles are transported by fast The resultant rise in the intracellular concentration of axonal transport, but not as clathrin-coated vesicles a. Ca 2+ triggers the fusion of the synaptic vesicles with Synaptic-vesicle recycling must also be maintained in the presynaptic membrane, and the release of neuro- the presence of active neurotransmitter release to transmitter (exocytosis) 1'2. The empty synaptic conserve and maintain the vesicle population 4. A vesicles are then reclaimed from the plasma mem- single bouton contains only - 2 0 0 synaptic vesicles m, brane (endocytosis) and refilled rapidly 1'a'4. There are and the population must be tightly conserved as at least two forms of endocytosis: fluid-phase endo- resupply from the cell body might take many hours. cytosis is for the uptake of extracellular fluid; and The activation of synaptic-vesicle recycling is rapid receptor-mediated endocytosis is for the uptake of (30 s) and the process is short-lived (1 min) n'12 (see membrane-associated particulate material 5-s. Recep- Fig. 1A), in contrast to non-neuronal cells where the tor-mediated endocytosis occurs in all cells when process might take longer than 30min 15. Synapticcell-surface receptors cluster at specialized clathrin- vesicle endocytosis can continue for 60 s after exocoated pits and are internalized from the plasma cytosis is complete, and such vesicles are competent membrane. There are several recognizable steps: to exocytose again after only 30 s TM. There appears to assembly of a protein coat at the plasma membrane; be no mixing of synaptic-vesicle membrane and coated-pit invagination; and finally, vesicle detach- plasma membrane components 1~. Therefore, it is not © 1994.ElsevierSci. . . .

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TINS, Vol. "17, No. 8, 1994

clear whether recycling occurs at the active zone or after lateral diffusion of the vesicle membrane. This is in contrast to receptor-mediated endocytosis where receptors can move freely through the plasma membrane and become trapped in coated pits < 15. Synaptic-vesicle endocytosis is very tightly coupled with exocytosis, and the initiation of both requires an ') + influx of Ca r , however, the subsequent removal of Ca"* rapidly stops exocytosis~':~'~. The requirement for Ca 2+ in endocytosis is not as clear, but maintenance of synaptic-vesicle recycling does not need extracellular Ca 2+ to be present continuously, and does not require changes in membrane potential le. For example, the neurotoxin o~-latrotoxin causes massive exocytosis in the presence of extracellular Ca z+, and also causes endocytosis, resulting in continuous cycling of vesicles, apparently without depletion. However, in the absence of extracellular Ca e+, toxin-induced exocytosis is even more rapid, endocytosis is blocked, and the nerve terminals become depleted of vesicles it. This supports a role for Ca ~+ in recycling but does not identify its point of action. Synaptic-vesicle endocytosis can occur following depolarization if the extracellular Ca e+ is subsequently removed 12, suggesting that Ca e+ might serve as the initiating event but is not necesary for the process to continue. A variety of reconstitution experiments in vitro also appear to require the addition of Ca e+ to support receptor-mediated endocytosis ~s'>, but others apparently do not e° and the role of Ca ~+ in this process is still unclear.

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of 98 kDa a4. Dynamin I probably has a primary role in nerve-terminal vesicle trafficking, and ubiquitous a 864 dynamin II might have a more lla general function in receptor-meDynamin I R diated endocytosis. J 4 isoforms Like many signalling proteins, i b 8sl lb dynamin is 'modular'- that is, comprises domains of amino acid sequences shared with other llala II proteins involved in intracellular Y ~ . .....I I ~ 871 D_namin B 4 isoforms signalling (Fig. 2). Although these i i :! domains have shared functions lblb they can also have remarkably distinct specificity when placed in a new protein, by virtue of small 849 Dynamin III alterations in the amino acid sequence. The first module on % Identity [ 88% jl.76%j i 79% II 53% j d y n a m i n I & l l dynamin is a GTP-binding domain, the function of which is required for endocytosis. Dynamin is a I J I I I I Functional member of a growing family of GTPase Pleckstrin Phosphortlation large GTP-binding proteins which homology SH3-binding domains are distinguished from small GTPPhospholipid-binding binding proteins (for example, ras, Microtubule-binding rab and aft) and heterotrimeric G proteins (for example, G~, Oi and Fig. 2. Domain structure of the dynamins. The products of three distinct mammalian genes have G,,), by their large size (100 kDa) been characterized, with two of the genes giving rise to multiple proteins that are generated by alternative spficing of the mRNA. Dynamin I is expressed in neural tissue, dynamin II is almost and far greater rates of GTP ubiquitously expressed and dynamin III i~; testis-specific 33 3e Dynamin-isoform nomenclature hydrolysis :~r. The proteins in this makes use of the alternative spfice sites, thus the 96 kDa form of dynamin I is either dynamin laa or family include examples with a wide array of function. For Iba, while the 94 kDa form is dynamin lab or Ibb. example, the interferon-inducible mammalian dynamin that are also deficient in GTPase nuclear Mx proteins are implicated in resistance to activity abolish receptor-mediated endocytosis when influenza :~s, the Vpslp/Spo15p yeast protein is intransiently expressed in COS or HeLa cells, confirm- volved in vacuolar sorting and in meiotic spindle-pole ing the role of mammalian dynamin as functionally separation :~j, and the Mgmlp yeast protein controls equivalent to that of the invertebrate dynamin:~°':~k mitochondrial DNA maintenance m. While the hornThe specific site of action of dynamin in the endo- dogies of these proteins with dynamin do not extend cytotic pathway has not been defined fully, but its to their carboxy termini, there are functional parallels. GTPase activity appears to be required for coated-pit For example, the carboxy-terminal half of Vpslp is required for regulation of its function :~~, as appears to invagination and detachment to proceed :~°. be the case for dynamin. Therefore, the function of Dynamin structure these common GTPase domains nught be differentially In shibire, only a single dynamin gene has been controlled by the differing carboxy-terminal domains. described, giving rise to several isoforms of the The second dynamin region is the middle domain, protein by alternate splicing, and producing at least which contains the first site of alternate splicing, and two size variants of 99 and 94 kDa zs'2~. Mammalian its function is unknown. However, the two splice dynamin I has been cloned from rat and human brains variants are subjected to only very minor and and has been shown to be homologous to that of conservative amino acid substitutions. shibire s°':~'e'x~. However, there are at least three The carboxy-terminal region of dynamin has two dynamin genes in mammals, giving rise to three domains for protein-protein interaction. The first is a protein families: neuronal dynamin I (Ref. 33), the pleckstrin homology (PH) domain, which is common ubiquitous dynamin II (Refs 34 and 35) and testis to at least 16 other unrelated proteins TM ~2. The PH dynamin III (Ref. 36; formerly named dynamin-2) domain is a recently discovered 90-110 amino acid (Fig. 2). These dynamins have high sequence simi- sequence, within proteins, that is functionally similar larity: dynamins I and II are 79% identical and have to the well-known src-homology 2 (SH2) domains equal homology to the shibire gene product (66% which mediate binding to specific phosphorylated identity) :u. Within each gene family there are multiple tyrosine residues on other signalling proteins or splice variants. For example, there are at least four receptors u~. Particularly interesting examples of prodynamin I forms in rat brain, arising from alternate teins that contain a PH domain are ras-GAP and splicing at two sites :~:~and another splice variant at the phospholipase Cy, which also have SH2 and SH3 carboxy-terminal of human dynamin I (Ref. 30). The domains, and would appear to interact with many functional significance of the variants is not yet proteins. The I{-adrenergic receptor kinase ([~ARK) known, but all forms appear to be expressed as two that phosphorylates and desensitizes specific horsize variants of 96 and 94 kDa. Dynamin 11 also has mone receptors has a PH domain that includes its site four splice variants, but has a single size for all forms of interaction with [~y subunits of heterotrimeric O 350

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proteins le. Each PH domain would be expected to have a specific cellular protein target or multiple targets, but the target for dynamin binding has yet to be discovered. The final dynamin domain is a proline-rich tail that is characteristic of an SH3-receptive domain. SH3 domains mediate intracellular signalling by binding to other proteins possessing an SH3-receptive domain, resulting in targeting signalling proteins to their site of action in the plasma membrane or other subcellular compartment ~:~. Four proteins, src, GRB-2 (growth factor receptor bound protein-2), phospholipase C,/ and the p85 subunit of phosphatidylinositol 3-kinase, bind dynamin I through this domain, and activate its intrinsic GTPase activity in wtro TM 1"~'.This is the first demonstration that SH3 proteins might alter the function of a target molecule after protein binding. The proline-rich tail has additional functions: it mediates the ability of the protein to bind to micro- Fig. 3. Distribution of dynamin I immunoreactivity in rat brain. Alkafine tubules:~t; is required for phospholipid activation of phosphatase staining of SOl~m thick vibratome sagittal sections from rat brain. dynamin's GTPase activityU~: and probably contains Intensely immunoreactive dynamin I staining appears black, and is particularly the protein kinase C (PKC) phosphorylation sites :~:~. high in hippocampus, cerebellum and forebrain. Scale bar, 3 mm. The tail contains the greatest degree of sequence divergence between the dynamin families, and can be further proposed to play a role in the distinction dephosphin), which suggested that it is phosphorylbetween regulatory control of each dynamin isoform. ated in vivo 11"~1'~'2. All phosphorylated dynamin I in nerve terminals is cytosolic, while the majority of the Dynamin distribution protein is associated with the particulate fraction 17. There are high levels of dynamin I in brain, where it Dynamin I is phosphorylated by PKC in vitro 1:~':':~'~':~. is present at levels greater than 0.4% of brain Dynamin I! is not phosphorylated by PKC under protein *r and is particularly enriched in nerve ter- conditions where dynamin ! is phosphorylated, minals (K.A.P. and P.J.R., unpublished observations). suggesting differences in regulation between dynamin Dynamin I immunoreactivity appears to be largely I and II (Ref. 34). Purified rat-brain dynamin I has the confined to neural tissue, but it is very low in adrenal highest affinity for PKC of any known protein substrate gland or anterior pituitary >'4~. Highest levels are (K~=0.35,uM)/:. The phosphorylation sites on found in the hippocampus and cerebellum 4~ (Fig. 3). In dynamin I found in intact synaptosomes are located on contrast to studies suggesting that dynamin I does not the same protease-derived peptide fragments as localize to any nerve-cell compartment ~~t, within the those that are phosphorylated by PKC, suggesting a cerebellum, highest levels of immunoreactive dynamin role for the same protein kinase in intact nerve I are found in synaptic terminals (K.A.P. and P.J.R., terminals I t..~:~. The best evidence that PKC might be unpublished observations). The concentration in nerve the m vivo dynamin I kinase came from studies with terminals is likely to he in the range of 20-100 uM ~r. PKC inhibitors, which prevented phosphorylation of Dynamin I is present in highest levels in the mature dynamin after depolarization 1i. Although the effects of rat brain only after neurons have ceased division, the inhibitors were relatively selective for PKC, the migration, axonal outgrowth and synapse formation, use of such inhibitors was not unequivocal. An suggesting a role related to development of mature exciting finding is that phosphorylation of dynamin I synaptic contacts >.:~o. There are two mRNA sizes for increases its GTPase activity, indicating a regulatory dynamin 1, which correspond to the two size variants role for phosphorylation:~:'. This is the only known (94 and 96 kI)a) :~l. The mRNA for the longer dynamin regulation of dynamin GTPase activity in vivo, since I variant is expressed at much higher levels (except in other interactions described have yet to be demonPC12 cells where the short form predominates) than strated under physiological conditions. The PKC the shorter dynamin I, and appears to be absolutely phosphorylation site has been mapped to the prolinebrain-specific. The shorter dynamin I variant is also rich tail domain, the site of interaction with other found in very tow levels in adrenal gland and liver. dynamin-I-GTPase activating factors and binding proDynamin II mRNA is expressed in similar amounts in reins :~:~. However, phosphorylation by PKC in nerve all tissues tested. However, highest concentrations of terminals occurs long after endocytosis has been dynamin II are found in the testis :~I, which possibly activated, and parallels the termination of vesicle represents cross-hybridization with dynamin III which endocytosis ~l.~e. Therefore, phosphorylation can be appears to be testis-specific :'~. The co-expression of proposed as the stop signal for synaptic-vesicle dynamin I and II m PC12 cells indicates that different recycling (Fig. 1). functions for the two forms are possible within the Dynamin I is also subject to phosphorylation by same cell, where both synaptic-vesicle recycling and casein kinase II, although this has no effect on its receptor-mediated endocytosis occur. GTPase activity :~:~. Phosphorylation of dynamin I by casein kinase II prevents phosphorylation by PKC, Phosphorylation providing a model for potential interaction between Dynamin i was first discovered as a phosphoprotein distinct signalling pathways in the regulation of in intact nerve terminals (called P96, and later dynamin function in vivo. However, phosphorylation I

TINS, Vol. 17, No. 8, 1994

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by casein kinase II has yet to be found m nerve terminals or intact neurons.

Dynamin I dephosphorylation in nerve terminals Dynamin 1 first received prominent attention as a result of its unique dephosphorylation upon depolarization, and its activation of exocytosis and endocytosissl'se (Fig. 1B). The dephosphorylation is rapid (< 5 s) and extensive and its onset correlates with the influx of'Ca 2+ (Fig. 1A)3s'52'54. Dephosphorylation of dynarnin I is completely dependent on an influx of extracellnlar Ca2+ through voltage-gated ion channels 52. Influx of Ca~+ mediated by ionophores acting at random sites on the plasma membrane does not initiate dephosphorylation, although it does support phosphorylation of other substrates 5'~'55. After Ca2+ influx is induced by ionophores, dephosphorylation of dynamin I can still be activated by application of a depolarizing stimulus 55. Therefore, the site of Cae+ influx at the active zone, where synaptic vesicles are docked on the plasma membrane, must be of primary importance in activation of the dynamin I phosphatase. This suggests that dephosphorylation of dynamin I is specifically coupled to those voltage-gated Ca z+ channels that are associated with exocytosis and endocytosis, and that phospho-dynamin I resides in a specific subsynaptic localization. Calcineurin, the only known Ca2+-dependent protein phosphatase, might be the dynamin I phos352

phatase since dephosphorylation in intact nerve terminals is insensitive to low concentrations of okadaic acid that effectively inhibit protein phosphatases 1 and 2A (PP1 and PP2A) 55'56. Brain is highly enriched in calcineurin, where the calcineurin is concentrated in nerve terminals, but its function there has been previously undefined 5r. Purified calcineurin dephosphorylates purified phospho-dynanfin with fast kinetics (Jun-Ping Liu, A. Sire and Phillip J. Robinson, unpublished observations). The affinity of phosphodynamin for calcineurin has a Kd of 0.5 UM, which is better than that for described calcineurin substrates, suggesting a tight association between phosphodynamin and calcineurin in vivo. These findings predict that the activation of nerve terminal caMneurin might be the initiating signal for endocytosis of synaptic vesicles (Fig. 4). After the depolarizing stimulus is terminated, dynamin I is relatively slowly phosphorylated, with a half-time for rephosphorylation of 40 s (Fig. 1) 15. This recovery time correlates with that of synaptic-vesicle recycling in nerve terminals 11'1~, supporting the hypothesis that phosphorylation and dephosphorylation might be the control signals for endocytosis. Although rephosphorylation of dynamin 1 appears to be mediated by PKC 15, this occurs long after the intracellular Cae+ signal has been removed (Fig. 1). Therefore, it remains unclear how a Ca"+-dependent PKC isoform might mediate this, unless other events contribute to maintaining activation of PKC, such as a relatively long-lasting PKC-activating signal or second messenger.

How does dynamin regulate endocytosis? Perhaps the most confusing aspect in understanding the function of dynamin is determining the role of its GTPase activity. In vitro, this is stimulated bv microtubule binding xr's~, phospholipid association~g, the many dynamin-binding proteins ~4'~5, or by PKC phosphorylation s:~. Such a diversity of signals, all leading to activation of dynamin's GTPase activity, might seem quite surprising. Only dephosphorylation and phosphorylation have been described under physiological conditions and during synaptic-vesicle recycling. Upon depolarization and exocytosis, dynamin I is rapidly dephosphorylated by calcineurin, and low GTPase activity predominates. Therefore, dephosphorylation might be the 'molecular switch' for endocytosis because it slows down the GTPase activity, and thereby increases the concentration of GTP-bound dynamin. This might be the 'active' dynamin form which controls endocytosis by interaction with a target protein that it activates. This target remains to be identified. The termination signal for dynamin function might reside in its phosphorylation by PKC which 'inactivates' dynamin by increasing its rate of GTP hydrolysis. It remains to be determined whether dynamin-binding proteins might contribute to this in vivo. These investigations on dynamin, dephosphin and the shibire gene product come together to form a new and exciting picture of Ca"+-regnlated synaptic control, and increase our understanding of neurotransmission. Dynamin I seems poised to implicate other proteins and molecular interactions, and is likely to produce further surprises into the molecular mechanisms underlying synaptic-vesicle recycling. TINS, Vol. 17, NO. 8, 1994

Selected references 1 Jahn, R. and SQdhof, T. C. (1993) J. Neurochem. 61, 12-21 2 Kelly, R. B. (1993) Ceil 72 (Suppl.), 43-53 3 Heuser, J. (1989) Cell Biol. Int. Rep. 13, 1063-1076 4 SQdhof, T. C. and Jahn, R. (1991) Neuron 6, 665-677 5 Smythe, E. and Warren, G. (1991) Eur. J. Biochem. 202, 689-699 6 Schmid, S. L. (1992) BioEssays 14, 589-596 7 Trowbridge, I. S., Collawn, J. F. and Hopkins, C. R. (1993) Annu. Rev. Cell BioL 9, 129-161 8 Pley, U. and Parham, P. (t993) Crit. Rev. Biochem. Mol. Biol. 28, 431-464 9 Maycox, P. R., Link, E., Reetz, A., Morris, S. A. and Jahn, R. (1992) J. Cell Biol. 118, 1379-1388 10 Harris, K. M. and Stevens, J. K. (1989) J. Neurosci. 9, 2982 -2997 11 Miller, T. M. and Heuser, J. E. (1984) J. Cell Biol. 98, 685-698 12 Ryan, T. A. eta/. (1993) Neuron 11, 713-724 13 Robinson, P. J. (1992) Mol. NeurobioL 5, 87-142 14 Robinson, P. J. (1992) J. Biol. Chem. 267, 21637-21644 15 Rothman, J. E. and Orci, L. (1992) Nature 355, 409-415 16 Nordmann, J. J. and Artault, J-C. (1992) Neuroscience 49, 201-207 17 Torri-Tarelli, F. eta/. (1990)J. Ceil Biol. 110, 449-459 18 Lin, H. C., 5Lidhof, T. C. and Anderson, R. G. W. (1992) Cell 70, 1-20 19 Lin, H. C., Moore, M. S., Sanan, D. A. and Anderson, R. G. W. (1991) Y. CellBiol. 114, 881-891 20 Schmid, S. L. (1993) Trends Cell Biol. 3, 145-148 21 Ahle, S. and Ungewickell, E. (1986) EMBOJ. 5, 3143-3149 22 Kohtz, D. S. and Puszkin, S. (1989)J. Neurochem. 52, 285-295 23 Jackson, A. P. (1992) Biochem. 5oc. Trans. 20, 653-655 24 Carter, L. L., Redelmeier, T. E., Woolenweber, L. A. and Schmid, S. L. (1993) J. Cell Biol. 120, 37-45 25 Hess, S. D., Doroshenko, P. A. and Augustine, G. J. (1993) Science 259, 1169-1172 26 Ferro-Novick, S. and Novick, P. (1993) Annu. Rev. Cell BioL 9, 575-599 27 Koenig, J. H. and Ikeda, K. (1989) J. Neurosci. 9, 3844-3860 28 van der Bliek, A. M. and Meyerowitz, E. M. (1991) Nature 351,411-414 29 Chen, M. S. eta/. (1991) Nature 351, 583-586 30 van der Bliek, A. M. eta/. (1993)J. Cell Biol. 122, 553-563 31 Herskovits, J. C., Burgess, C. C., Obar, R. A. and Vallee, R. B.

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Epilepsy: Models, Mechanisms and Concepts edited by P. A. Schwartzkroin, Cambridge University Press, 1993. £80.00 (xiv + 544 pages) ISBN 0 521 392985

Research on epilepsy and its underlying mechanisms has played a remarkable role in modern neuroscience. Since the work of the Montreal group, studies on epileptogenesis in human and experimental models of epilepsy have provided a rich source of novel concepts and mechanisms that have played starring roles in advances in brain research. The development of slices and other preparations in vitro combined with cellular morphological and molecular biological research had enormous impact on our understanding of how the neural netTINS, Vol. 17, No. 8, 1994

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work generates hyperactivity, and what its consequence is on neural activity. Considering the multitude of mechanisms that can lead to epileptic activity, the 'epileptologist' must have a solid culture in virtually every field of neuroscience. This book constitutes an attempt to compare the models of epilepsy most used and to evaluate some of the mechanisms most frequently used to explain epileptogenesis. It is composed of 15 chapters divided somewhat arbitrarily into three sections: (1) Chronic models, including the kindling and kainate models but also genetic models and transplantation approaches to epilepsy research. (2) Features of the epileptogenic brain with chapters on neonatal seizures, electrophysi-

Acknowledgements This workwas supportedby the AustrahanNH and MRC (920502)and the NIH (RO1 MH47510). We thankJ. M. Sontag and H. McMahon for their Important contributions to this work.

books

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Y. Ben-Ari Unitede Neurobiologieet Physiopathologiedu Developpement, INSERM-U29,H6pital de PortRoyaL 123 Bd de Port Royal, Paris, CEDEX14, France.

In his introduction, the Editor analyzes the various levels of integration at which abnormal activities can be generated. For example, he analyzes changes in receptors and voltage-gated channels, local interaction (role of GABA and excitatory amino acid receptor-driven synapses) and spread of epileptiform activity (integration between neuronal aggregate, preferential involvement of limbic structures and so on). Though our understanding of these various levels has greatly 353