influence of the vestibular inputs that transmit phasic/
`signals to the ceiebellum while decreasing the influence of the
`inputs that transmit purely tonic signalsls.
`The recurrent positive feedback pathway in our neural
`network was critical for converting a subtle change in the time-
`course of neuronal responses into a change in the steady-state
`output ofthe system. Feedback connections are a general archi-
`tectural feature of the brain and are found at many different
`levels, including the inputs and outputs of the cerebellum and
`the connections between areas of the cerebral cortex. The
`importance of recurrent connections in the pathways that medi-
`ate the VORe allows us to model how recurrent connections
`could contribute to learning and niemory. The model raises the
`possibility that subtle changes in the function of individual
`cellular mechanisms may have profound eff.ects on the output
`lrom speciûc behavioural systems and emphasizes the import-
`ance of understanding the architecture of the neural networks
`that convert cellular changes into changes in behavioural
`output.
`tr
`
`Recelved 5 Maln æpted 18 SeÞtember 1992.
`1. Chufchland, P. S. & Sejnowsk¡, T. J. fhe Computat¡onl B{a¡n (Mll Press, Cambridge, MA, 1992).
`2. Storm, J. F. ¡Va¿uÆ 3:F, 379-3a1 (1988).
`3. Bradley, K. & Somjen, G. G. J Phys¡o|., Lotú. LA6,75-92 (1961).
`4. Strong, J. A, & Kaczmtrek, L. N. iñ l,leurcmdtlation: 'nE Biæhenacal CØtrol of Newonal
`Ercitùil¡ty leds Kaczmarek, L. K. & Levitân, l- B.) (Oxford th¡versity Press, 1987).
`5. Lisbergq, S. G. Scìerc 212, 728-735 (198{l).
`6. Miles, F. 4., Bra¡tman, D, J. & Dow, B. M. J. Nwophysiol.441477-1493
`Sâìence 242 771-773 (1988).
`7. Lisb€rger, S. G. & Pæelko, T.
`8. Mil€s, F. 4., Fuller. J. H., Braitman, D. J. & DN, B. M. J. Niltophysiol. ß, L437-1476 (79æ1.
`^.
`9. L¡sberger, S. G., i¡lorris, E. J. & Tychsen, L- A- Rev. l¿euñLLq 97-129 {1987).
`10. Fud1s, A. F. & Kimm, J. L lleurophysiol.4 1140-1161 (1975).
`11. Gonshor. A. & Melvill ¡æs, c. I Phys¡d-. Lond.256, 381-414 (1976).
`12. M¡les, F. A. & Lisberget S. G. A. Rev. NwrosL4,273-299 (1981).
`13. lto, M. 8ráin Âes $, A!-U $972],.
`14. Watanôe, E. Btaln Res. 29¡l, 1,69-L7 4 (tú4I
`15. L¡sberger, S. G, & Sejnowski,'1. J. U,liv, Cd¡lon¡ê Sân Diego lnst. Ileúal Comput. fech. Rep.
`920L(7992).
`16. L¡sberger, S. G. & Pavelko, T, A. J NMoæl 6,346-354 (1986).
`
`(1980).
`
`AC]fiOW"E0GEI\,iÉNTS. We thilk numerous ælleagues for their helpful comments. Research was
`supported by a grãnt from the D€fenæ Advâæd Reæarch Proiæt Agercy, awarded through the
`Offìce of Naval Research.
`
`A single am¡no-acid difference
`confers ma¡or pharmacological
`variation between human and
`rodent 5-HTr" receptors
`Donna Oksenberg*f, Scot A. tlarsters*,
`Brian F. O'Dowdf, Hui lint, Sona Havlikf,
`Stephen l. Peroutkat$ & Avi Ashkenazi*
`Departments of * Pulmonary Research and Gene Therapy, and
`$ Neuroscience, Genentech lnc., 460 Point San Bruno Boulevard,
`South San Francisco, California 94080, USA
`t Department of Neurolory, Stanford University School of Medicine,
`Stanford, California 94305, I,JSA
`i Department of Pharmacology, Uñiversity of Toronto, Toronto,
`Ontario MsS 148, Canada
`
`NnunopsvcglATRrc disorders such as anxiet¡r, depressionr.
`migrrine, v¡sospasm and epilepsy may involve different subtypes
`of the Shydroxytryptamine (5-HÐ receptorl'2. The lB subtype,
`whieh has a unique pharmacology, w¡s first identified iu rodent
`br¡in}7. But a similar receptor could not be detected in humrn
`brain6, suggesting the abseuce in man of a receptor with equivalent
`function. Recently a human receptor gene nas isolated (dæigneted
`s-lfTrB receptoPe, 5-HT1DÉ receptorrqlr, or S12 receptorr2)
`'which sh¡res 93% identity of the deduced protein sequence with
`rodent S-HT¡¡ receptorsr!¡!. Although this receptor is identical
`NATURE ' VOL 360 . 12 NOVEMBER 1992
`
`TETTERS TO NATURE
`
`TABLE 1 Ligand-binding properties of the wild-type and T355N mutant human recep-
`tors compared with those of the rat and mouse s-HTß leceptors
`
`/(, (nM)
`
`Human
`(T3s5N)
`
`Ratla
`(5-HI1B)
`
`Mousels
`(5-HT1B)
`
`8+1
`3+1
`2*l
`' 2+l
`200+40
`560+100
`97Oi 130
`25,000 i 1,000
`i8r8
`L7 *L
`20+3
`13+1
`
`16+ 1
`7 +1,
`4*2
`2*I
`129 * 33
`465 + 85
`!823t?97
`>10,000
`13t4
`
`57 14
`153 r 62
`NA
`
`39
`10
`NA
`10
`NA
`NA
`NA
`30,000
`NA
`
`NA
`69
`NA
`
`Human
`(wild type)
`
`10+1
`4+L
`6+1
`44!4
`25+3
`38i3
`130r7
`1,600+100
`L2r1,
`
`8,100+40o
`11,000 * 1,000
`11,000+800
`
`Ligand
`
`Serotonergic
`5-HT
`5-CT
`Dl.C
`RU24969
`Metergoline
`Sumatriptan
`Methyserɡde
`8.OH.DPAT
`Methlothepin
`B-adrenêrg¡c
`(-)Propranolol
`(-)P¡ndolol
`(-)Alprenolol
`
`The values are dep¡cted as mean *s.e.m, from 4 (wild type) and 3 (T355N) indepen-
`dent experiments done in triplicâtes. NA, data not available. Complementary DNAS
`encoding the wildlype and T355l.l mutant human receptors were inserted into the
`mammalian express¡on vector pRKs and introduced by trans¡ent transfection into the
`human embryonic kidney 293 cell line by a modified calcium phosphate precipitation
`method3, The cells were collected by c€ntrifugation 48h after transfection, lysed in
`ice-cold buffer (5OmM Tr¡s-l-lcl, pH 7.4, contain¡ng 5mM EDIA), homogenized, and
`sonicated for 10s. Nuclei ãnd intact cells were removed by centrifugêtion ât 1,0009
`for 10 m¡ñ. The supernatant was spun at 35,0009 for 30 min and the resulting pellet,
`containing the m¡crosomal membrane fraction, wâs resuspended ¡n bind¡ng buffer
`contain¡ng 50 mM Tris-rc| (pH 7.4), 4 m¡¿ cacl2, 0.1% âscrobic acid, l0 mM pargyline
`and 1 ¡rM leupept¡ne. Microsomal membranes (5O p! protein) were incuÞated with the
`ligands in bind¡ng buffer (30m¡n,25qC). Binding was terminated by the add¡tion of
`5 ml ice-cold 50 mM Tris-l-lCl (pH 7.8), rapid vacuum fiftration through glass fibre f¡lters,
`and two subsequent s-ml washes. Spec¡fic bind¡ng wâs def¡ned as the excess over
`blanks taken in the presence of 1O-sM cold s-HT. Scatchard analyses of saturation
`binding of [3H]s-Hf showed two populat¡ons of binding sites; equilibrium d¡ssociation
`constants (KJ for the wíld-type and mutant receptor, respectively, were 4.611.4 and
`3.9 + 1.2 nM (high-âffinity sites);72 + 24and75+L4rì¡(low-affin¡ty sites). The respec-
`t¡ve receptor densit¡es ¡n pmol per gram of protein were 1,000+ 32O and L,440*6LO
`(high-aff¡nity s¡tes), 13,670+1,860 and 25330i4,¿180 (low-aff¡n¡ty sites). Specific
`b¡nd¡ng of f3HlsJ-ff was not detectable in untransfected cells (not shown), ind¡cating
`that these cells do not express significant levels of endogenous s-HT receptors.
`Equilibrium ¡nhibition constanls (K¡)were determined according to thefollowingequat¡on:
`Kt=lcla/í +lT)/ Ko), where lcs is the c$ncentrat¡on of compet¡ng liÉand required for
`5096 inhib¡tion of [3H]5-HT Þ¡nding, [Tl is the concentration of the f3Hls-l{T tracer
`(3nM), and KD is the high-aff¡nity constant of [3H]5-Hf, as determined by saturation
`binding. The deta were analysed by nonlinear least-square fitt¡ng using the EBDA2a
`and LIGAND25 programs.
`
`to rodent 5-HT1s receptors in binding to S-HT, it differs profoundly
`in binding to many drugs. Here we show that replacement of a
`single amino acid in the human receptor (threonine at residue 355)
`with a corresponding asparagine found in rodent S-HT'B receptors
`renders the pharmacology of the receptors essentially identical.
`This demonstrates that the human gene does indeed encode a 1B
`receptor, which is likely to h¡ve the same biological functions as
`the rodent S-HTIB receptor. In addition, these findings show that
`minute sequence differences between homologues of the same
`receptor from different species c¡n cause large pharmacological
`vari¡tion. Thuq drug-receptor interactions should not be extrapo-
`lated from animal.to human species without verific¡tion.
`The human and rodent 5-HTrB receptors bind to 5-HT with
`comparably high affinity. But the human receptor binds with
`much lower affinity to the serotonergic agonist RU 24969 and
`to the B-adrenergic receptor antagonists propranolol, pindolol
`and alprenolol, and with higher affinity to several serotonergic
`drugs, including sumatriptan and 8-hydroxy-2-(di-n-propyl-
`amino)retralin (8-OH-DPAT¡6-I4 lTable 1).
`Tl¡e 32 amino-acid differences between the human and rat
`receptors are scattered throughout the molecule, but only eight
`are found in the transmembrane domains, which are thought to
`contain the ligand-binding pocket (Fig. l). An asparagine
`residue in the seventh transmembrane segment has been impli-
`cated in B-antagonist binding to the B-adrenergic receptorró
`and to human 5-HTrA receptorsrT. Notably, an asparagine
`161
`
`@ f992 NaturêPublishing Group
`
`Page 1 of 3
`
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`

`
`NHr
`
`A
`
`A
`
`LETÎERS TO NATURE
`
`FlG. 1 Seven-transmembrane-segment model of the
`human s-HTß receptor. The amlno acids found in
`the humân rec€ptoÊ12 are circled; residues that
`are d¡fferent or absent in the rat 5-HT1¡ feceÞtorl3aa
`are shown next to the corresponding positions. The
`rat sequence is 98% identical to the mouselu s-Hfr"
`receptor sequence. Tlp dark box ind¡cates position
`355, in which the threon¡ne of the human receptor
`was replaced with the corresponding asparagine
`found in the rat ênd the mouse 5-HTts receptors.
`METHIDS. Threonine codon 355 of the human recep-
`tor cDNAB was replaced w¡th an asparagine codon
`by oligonucleotide-directed mutagenesis. A 39-base
`synthetic ol¡gonucleotide canying the asparâgíne
`codon (contain¡ng two mismatches with the
`threoninc COdON) (GTTGAGATAGCCCAGCCAGTTGAA.
`GAAGTCAAAGATGGC) was used as the mutagenesis
`nrimar
`Thic nrimar wac hvhti.l¡"ê.i
`tô â <iñdlê-
`stranded DNA containing the wild-type sequence
`and extended by T4 DNA polymerase, and the
`double-strânded DNA plasmid was introduced into
`Escherichia coli. Bacterial colonies containing the
`T355N mutation were ident¡fied by two consecutive
`hybridizat¡on analyses and a single clone carrying
`the mutation was selected and confirmed by DNA
`sequefìcinE.
`
`a 120
`
`o---
`-l
`o.-....--.-....'..-o
`
`OWT
`O T355N
`
`100
`
`80
`
`60
`
`40
`
`0
`11
`
`'t0
`
`9
`
`7
`
`Þ
`
`5
`
`sF
`
`.I
`ro
`
`I oo
`
`)
`.GT'c
`-o
`
`oEooo
`
`.
`Ø
`
`TOG
`
`L
`
`YD
`
`H
`
`Extracellular
`
`lntracellular
`
`Ð
`
`E
`
`) P¡ndolol
`) Propranolol
`) Alprenolol
`
`WT
`
`ovo
`
`T355N
`(-) Pindolol
`(-) Propranotol
`(-) Alprenolof
`
`avI
`
`-loo tliqandl lMl
`
`2
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`i
`
`10
`
`b ñl
`
`-
`
`foIo
`
`oo
`
`.çõc
`
`o.
`
`o=ooo
`
`.
`U)
`
`o10
`
`11
`
`a4
`
`o3a
`
`2
`
`a5
`O6
`
`a7
`
`r- ô 1q
`
`5
`7
`B
`Human wild-type recèplor pKl
`
`9
`
`4O
`
`a'
`
`10
`
`alr
`
`/ = 0.86
`
`7
`
`Human T355N receptor p(
`NATURE . VOL 360 . 12 NOVEMBER 1S92
`
`B
`
`6
`
`4
`
`ds
`
`q.
`
`oõ
`
`.o
`
`o @
`
`F+ 6
`
`tc
`
`e9
`
`\l
`a.
`ooo
`
`o Érnd
`
`CC
`
`I
`-lon f6-HTì lMì
`
`WT
`
`O Metergol¡ne
`v Sumamplan
`o Methysergíde
`
`T355N
`
`Metergol¡ne
`Sumatriptan
`Methysergide
`
`ovI
`
`E
`
`I
`
`V o
`
`o
`
`c 120
`F roo
`FI
`ÈBo
`:E
`
`Ç:rD_0
`
`Y
`
`v
`
`360c
`
`EC ç'
`
`õ&
`
`zoø
`
`0
`
`10
`
`86
`-log [Ligandl (M)
`FlG. 2 comparison of the ligand-b¡nding properties of the wild type (WI
`open symbols) and T355N mutant (f¡lled symbols) human receptors
`expressed in human embryonic kidney 293 cells. 4 Competit¡on of 5-Ht
`(O,O) for [3H]5-Hr bind¡ng. b, Compet¡tion of the B-adrenergic receptor
`antagonists (-)pindolol (O,O), (-)propranolol (V,V) and (-)alprenolol
`(D,l) for [3H]5-HT binding. c, competit¡on of the serotonergic ligands
`metergoline (O, O), sumatriptan (V, V) and methysergide (tr,1) for [3H]5-
`HT-binding. d Correlation between pK, values for the human wild-type
`receptor and the rat 5-HT"" receptor. (e) Correlation between pK, values
`for the human T3551{ mutant receptor versus the rat 5-HT1B leceptor. Ligand
`numbers and pKrs are based on the¡t order of appearance in Table 1.
`t62
`
`-\
`
`4
`
`@ 1992 Nature Publishing Group
`
`Page 2 of 3
`
`YEDA EXHIBIT NO. 2033
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`is found at this location in all cloned 5-HT receptors
`r€sidue
`that bind p-antagonists; ratr3'ta and mousels S'HTrB receptors,
`ratrE and humant'''o 5-HT1A receptors' and in B-adrenergic
`receptors2r. In contrast,. a threonine residue- is found at this
`poriiion (355) in the human s-HTrB receptors-r2. To assess the
`contribution of this threonine to the distinct pharmacology of
`the human receptor, we replaced it with the corresponding
`asparagine founã in rodent S-HT,B re€eptors by site-directed
`mutagenesis (Fig. l). Scatchard analysis showed that the wild-
`type ãnd rnot"nút¡ssN) receptors bind f HI5-HT with similar
`afùnity, each with two binding-site populations (Ko:4.6 ¿nd
`72r,M,'wild type; 3.9 and ?5 nM, mutant; data not shown)'
`Competition analysìs also showed similar 5-HT affinities for the
`wild-iype and mutant receptors (Fig. 2a; Table 1). Thus, the
`ß55Ñìubstitution does not afiect binding to the natural ligand
`5-HT.
`To compare the pharmacology of the wild-type and mutant
`human receptors wiih that of rodent 5-HT¡s receptors, we caruied
`out competilion assays with serotonergic and B -adrenergic drugs
`from various chemicàl classes' Like 5'HT, 5-carboxyamidotrypt-
`amine (5-CT) and dihydroergotamine (DHE)' which have
`similar inhibition constants (Kr) with the wild-type human and
`the rodent receptors, showed comparable K¡s for T355N (Table
`l). ln contrasi, tlne B-attagonists propranolol pindolol and
`aiprenolol had markedly reduced K¡s with T355N, which were
`comparable to the Kis seen with the rodent receptors (Fig' 2b;
`Table l). The K¡. of the serotonergic ligand RU 24969 was
`-2O-fold lower with T355N than with the wild type, and similar
`to the K¡s for the rodent receptors (Table 1)' Metergoline,
`sumatriptan, methysergide and 8-OH-DPAT bound to T355N
`with ¡í.s that were 7-15 times higher than those seen with the
`witd type, and more similar to K¡s observed for the rodent
`receptôis (Fig.2c; Table l). Methiothepin, which has similar
`K,s with th" *ild-typ" human and the rodent receptors, showed
`a iourfold higher K¡ with T355N (Table 1)- The Kls of 11 ligands
`showed no cõrrelati,on between the human wild type and T355N
`(r:0.03), or the human wild-type and rat receptors (r:0'18)'
`but showed a significant correlation between T355N and the rat
`receptor (r:0.86; P<0.0025) (Fie.2d, e)-
`Thus, substitution of threonine 355 o[ the human receptor
`with the corresponding asparagine of rodent 5-HTrB receptors
`confers the pharmacology of a I B subtype on the human recep-
`tor. Taken together with the high sequence homology between
`these receptors, this demonstrates that the 5-HT receptor gene
`investigated here does indeed encode the human species variant
`of the lB 5-HT receptor subtype. Therefore, given that the
`human and rodent 5-HT'B receptors bind the natural ligand,
`5-HT, with the same affinity, it is likely that despite their dramati-
`cally different pharmacology, these receptors are equivalent in
`function.
`The identification of a human S-HTrB receptor is of biological
`and clinical significance. Whereas this receptor and the human
`5-HTrD receptor2z share only ó87o amino-acid sequence identity
`an¿ aré encoded on different chromosomes, their pharmacologi-
`cal properties are virtually indistinguishableE-'2. Therefore, bio-
`Iogical functions such as appetite control, migraine reduction
`and regulation of 5-HT release, attributed previously to the
`S-HT,5 receptor on the basis ol drugs presumed to be selective
`for this subtype, indeed rnay be mediated by either the 5-HTtp
`or the 5-HT1¡ receptor, or by both. Understanding the individual
`functions of these receptors thus may help improve the clinical
`application of compounds such as the antimigraine drug
`sumatriptan, which binds to both the 5-HTr¡ and 5-HT¡s
`receptors.
`Finally, our results show that a single arnino-acid difterence
`can cuuJe dramatic pharmacological variation between species
`homologues ol the same receptor. Thus, ligand-binding proper-
`ties of ã given receptor cannot be extrapolated across species
`without independent validation, even in the context ofvery high
`interspecies iequence identity. This has important implications
`NATURE' VOL 360 . 12 NOVEMBERlgg2
`
`LETTERS TO NATURE
`
`for the design and therapeutic use of receptor-selective drugs
`when these are based on measurements ofselectivity and efrEcacy
`n
`in non-human species.
`
`Ræived 19 June; accept€d 28 September 1992-
`1, Hartig. P. R. et al. lþutopsycl",opharmmlogv 3,335-347 (1990).
`2. Perottkâ, s, J. Neurophilmædogy 31, 609-613 (1992)
`3. Pedigo, N. W., Yamamura, H. l & Nelsn' D L ). \deuroc'lgm' æ' 220-226 îæLI
`¿. sctr¡ietiman, R. e., Waters, S. J & Nelson, A.L. J. Neuræhem' 42' 65-70 (1984)'
`5. l-loyer,0., EnÉel, G. & Kâlkman, H. o. Eut' J Philmæ. alq 1 12 {1985)
`ã. Xo!"r, o., en!"r, c. & Kalkman, H. O. Eut. J. Phârnæ 118, 13-23 (1985)'
`Z. fOyer. O- A uticotemis, D. N. frends phamæ. 31¡ 10' 130-132 (1989)'
`8. J¡n, H. el aL l b¡ot. CÌEñ.2€7,5735-5738
`(1992).
`9. Hambi¡n, M. W., Met€lf, M. 4., t¡ccuffin, R. w. & Karpells S Bioehem' b¡qhys Res Cofrmun
`L84,752_759 (1992\.
`10. Weinshar{í R. L et at. Ptæ. natn Aeàd Scr: Us.A 89,3630-3634 (1992)
`11. Demchystryn, L. et at. Ptæ. natn. Acad Sai t-ts¡ 89, 5522-5526 17992)
`12. Olav-Levy, F. et al. ! b¡ot. clÊn.?67,7553-7562
`(1992)
`13. Voigt, M. M., Lilr¡e, D. J., Seeburg, P H. & Bach, A. EMEO J.1O,4017-4023 (1991)'
`14. Adham, N. et d. Molæ Philmæ. 41,1-7 (1992)
`15. Maroteaux, L. et al. Ptæ. natn. Acad, Sc,: USA &t,3020-3024 (1992)
`16.Suryanarayana,A-.Dalnt,D.A,VonzåstroqM.&Nobilka,BK J'Ad'CheñM'7548a-15492
`(1991).
`12. òuan, x. G., Peroutka. S. .1. & Kobilka' a.K. Molæ- Pr¡åmæ.4t" 695-69a (1992)'
`18. Albert, P. R. et a'. J. bio!. chem.85,5825-5832 (1990).
`19. Kobilka, B. K. et aì. I'taturc 3fEt,75-79 (1987)
`20. Fargin, A. et at. J. bid. clem. N,4848-4852 (1989).
`21. Findlay, I & Eliopoulos, E. Ttnds phatmac Sci L7,492 499 (1990)
`22. Hamrriin, ¡r. w. & lvletcalf . M. A. Motec' mamæ. Æ. 143 148 (1991).
`23. Ashkenazi,4.,Peralta,E.G,,W¡nslow.J.W. Ramæhandfan,J.&Câpon D J Ceilt6'ß7-493'
`24. McPherson, G. A. Comp. ProE Siomed L7, IO7-ll4 \7983).
`25. Munson, P. J. & Rocbard D. Analy¿ Biochem. aO?, 220-239 (1989)'
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`ACKNoWLEDGE},ENTS.wethankc.clarkandR.waldforcommeôtsonthemanuscfipt.Thiswork
`was suppoíted by the NlH, the Kleirer Family Foundation and Geneñtech, lnc'
`
`Positive feedback of glutamate
`exocytosis bY metabotropic
`presynaptic receptor stimulation
`lnmaculada Herrero, M. Teresa Miras'Portugal
`& losé Sánchez-Prieto*
`
`Departamento de Bioquimica, Facultad de Veterinaria,
`Universidad Complutense, 28040 Madrid' Spain
`
`GLUTAMATE is important in several forms of synaptic plasticity
`such as long-term potentiation, ¡nd in neuronal ccll degener-
`ationr?. Gluiamate activates several types of receptors, including
`a metabotropic receptor that is sensitive to fiøns'l'^mino-cyclo-
`penthyl-1,3-dicarboxylate, couple$ to G protein(s) and linked to
`inositäl phospholipid metabolismH. The activation of the meta-
`botropicìeceptor in neurons generates inositol 1,4,5-trisphosphate'
`which causeJ the release of Ca2t from intracellular stores and
`rtiacylglycerol, which âctivates protein kinase C7-e' In nerve ter-
`m¡nals, ihe activation of presynaptic protein kinase C with phorbol
`esters enhances glutamaie releasero. But the presynaptic receptor
`involved in this protein kinase C-mediated increase in the release
`of glutamate has not yet been identifierl. Here we demonstrate the
`pruiunce of a presynaptic glutamate receptor of the metabotropic
`þpe that medi¡tes an enhancement of glutamate exocytosis in
`cerebrocortical nerve terminals, Interestingly, this potentiation of
`glutamate release is observed only in the presetrce of erachidonic
`ãcid, which may reflect that this positive feedback control of
`glutamrte exocytos¡s operates in concert with other pre- or post-
`Jynaptic events of the glutamatergic neurotransmission that gener-
`ate arachidonic acid. This presynaptic glutamate receptor mây
`have a physiological role in fhe maintenance of long-term potenti-
`ation where there is an increase in glutamate rele¡se mediated by
`postsynaptically generated arachidonic acidll.
`' fnä aã¿itlott ãf tne selective agonist for the metabotropic
`receptor, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid
`
`* To whom correspondence should be addressed.
`
`163
`
`@ 1992 Nature Publishing GrouP
`
`Page 3 of 3
`
`YEDA EXHIBIT NO. 2033
`MYLAN PHARM. v YEDA
`IPR2015-00644