Drooling and stuttering, or do synapses whisper?

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TRENDS in Neurosciences Vol.26 No.1 January 2003

12 D’Amato, C.J. and Hicks, S.P. (1965) Neuropathologic alterations in the ataxia (paralytic) mouse. Arch. Pathol 80, 604 – 612 13 Chung, C.H. and Baek, S.H. (1999) Deubiquitinating enzymes: their diversity and emerging roles. Biochem. Biophys. Res. Commun. 266, 633 – 640 14 Glickman, M.H. and Ciechanover, A. (2002) The ubiquitin–proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, 373–428 15 Hicke, L. (2001) Protein regulation by monoubiquitin. Nat. Rev. Mol. Cell Biol. 2, 195 – 201 16 Wilkinson, K.D. (1997) Regulation of ubiquitin-dependent processes by deubiquitinating enzymes. FASEB J. 11, 1245– 1256 17 Yin, L. et al. (2000) Nonhydrolyzable diubiquitin analogues are inhibitors of ubiquitin conjugation and deconjugation. Biochemistry 39, 10001–10010 18 Burt, A.M. (1980) Morphologic abnormalities in the postnatal differentiation of CA1 pyramidal cells and granule cells in the hippocampal formation of the ataxic mouse. Anat. Rec. 196, 61 – 69 19 Murphey, R. and Godenschwege, T. (2002) New roles for ubiquitin in the assembly and function of neuronal circuits. Neuron 36, 5 20 Freir, D.B. et al. (2001) Blockade of long-term potentiation by b-amyloid peptides in the CA1 region of the rat hippocampus in vivo. J. Neurophysiol. 85, 708– 713 21 Kim, J.H. et al. (2001) Use-dependent effects of amyloidogenic fragments of b-amyloid precursor protein on synaptic plasticity in rat hippocampus in vivo. J. Neurosci. 21, 1327 – 1333 22 Stephan, A. et al. (2001) Generation of aggregated b-amyloid in the rat hippocampus impairs synaptic transmission and plasticity and causes memory deficits. J. Neurosci. 21, 5703– 5714 23 Parent, A. et al. (1999) Synaptic transmission and hippocampal long-term potentiation in transgenic mice expressing FAD-linked presenilin 1. Neurobiol. Dis. 6, 56 – 62

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24 Zaman, S.H. et al. (2000) Enhanced synaptic potentiation in transgenic mice expressing presenilin 1 familial Alzheimer’s disease mutation is normalized with a benzodiazepine. Neurobiol. Dis. 7, 54 – 63 25 Alves-Rodrigues, A. et al. (1998) Ubiquitin, cellular inclusions and their role in neurodegeneration. Trends Neurosci. 21, 516 – 520 26 McNaught, K.S. et al. (2001) Failure of the ubiquitin – proteasome system in Parkinson’s disease. Nat. Rev. Neurosci. 2, 589 – 594 27 Jiang, Y.H. et al. (1998) Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation. Neuron 21, 799– 811 28 Moss, A. et al. (2002) A role of the ubiquitin – proteasome system in neuropathic pain. J. Neurosci. 22, 1363– 1372 29 Fallon, L. et al. (2002) Parkin and CASK/LIN-2 associate via a PDZmediated interaction and are co-localized in lipid rafts and postsynaptic densities in brain. J. Biol. Chem. 277, 486 – 491 30 Zhang, Y. et al. (2000) Parkin functions as an E2-dependent ubiquitinprotein ligase and promotes the degradation of the synaptic vesicleassociated protein, CDCrel-1. Proc. Natl Acad. Sci. U.S.A. 97, 13354 – 13359 31 van Leeuwen, F.W. et al. (1998) Frameshift mutants of b amyloid precursor protein and ubiquitin-B in Alzheimer’s and Down patients. Science 279, 242– 247 32 Lam, Y.A. et al. (2000) Inhibition of the ubiquitin – proteasome system in Alzheimer’s disease. Proc. Natl Acad. Sci. USA 97, 9902 – 9906 33 Kim, T.W. et al. (1997) Endoproteolytic cleavage and proteasomal degradation of presenilin 2 in transfected cells. J. Biol. Chem. 272, 11006– 11010 34 Steiner, H. et al. (1998) Expression of Alzheimer’s disease-associated presenilin-1 is controlled by proteolytic degradation and complex formation. J. Biol. Chem. 273, 32322 – 32331

Drooling and stuttering, or do synapses whisper? Craig E. Jahr Vollum Institute, Oregon Health and Science University, L474, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201-3098, USA

Spillover and multivesicular release are likely to occur at a variety of synapses in the CNS. Although the latter could enhance the former, neither appears to require the other, even though both affect neuronal communication. In two recent papers, new techniques, as well as the careful use of old standards, provide compelling evidence for these formerly heretical mechanisms. The exquisite alignment of pre and postsynaptic specializations suggests that these point-to-point connections between neurons serve as isolated pathways of information transfer. In addition, classical physiological studies indicate that, at each active zone, release is binary: when an action potential invades the presynaptic element, either a single synaptic vesicle is released or no release occurs. However, in recent years, reports of transmitter ‘spillover’ between synapses and of ‘multivesicular release’ (MVR) have suggested that these rules are broken at several synapses, if not in general. In most of these papers, either spillover or MVR could explain at least qualitatively many of the observations but, as is our want, the authors have usually chosen to back one hypothesis or the other. This summer, two Corresponding author: Craig E. Jahr ([email protected]).

compelling papers have appeared that each report one of the phenomena and reject the other. Unfortunately for the goal of parsimony, one paper decides for MVR whereas the other favors spillover. As two very different synapses were examined in the two laboratories, these disparate interpretations do not come as a complete surprise. Stuttering The first paper [1] reports MVR at Schaffer collateral synapses in the hippocampus. Although not the first report of MVR [2 – 5], this study makes use of two-photon microscopy to study synaptic events at individual dendritic spines. As the vast majority of spines in this region receive single presynaptic inputs [6,7], this technique allows the monitoring of the strength of transmission at morphologically defined single synapses. The protocol is conceptually simple: intra-spine Ca2 transients mediated by synaptically activated NMDA receptors are recorded at different release probabilities (Pr). If release at individual synapses is restricted to single vesicles, the size of the Ca2 transients should not be affected by changing Pr. However, if these synapses are not restricted to univesicular release, then MVR might occur more frequently at high Pr and, assuming that NMDA receptors are not saturated by

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single vesicles [8,9], the average Ca2 transient should be larger than that at low Pr. This was, in fact, the finding. As this synapse displays paired-pulse facilitation, the Ca2 transient following a single stimulus was compared to that following a pair of stimuli, the first of which resulted in a failure of transmission. The second, facilitated response was ,1.5 times larger than the response to a single stimulus, indicating that more NMDA receptors were activated and suggesting that the synapse was capable of MVR. Theoretically, this result could also be explained by spillover. At higher Pr, the likelihood of neighboring synapses releasing simultaneously is also elevated and, therefore, pooling of transmitter from adjacent sites could enhance NMDA-receptor activation at the spine under observation. However, these studies were performed at near-physiological temperatures, at which hippocampal spillover is thought to be diminished ([10], but see Refs [11,12] for reports of spillover), and low stimulus intensities were used to restrict activation to a few presynaptic fibers. Drooling The second paper [13] reports spillover of glutamate between synapses of cerebellar mossy fibers and granule cells. Each mossy fiber terminal makes many tens of synapses onto neighboring granule cells in a glomerular structure of tightly packed synapses. However, a given granule cell receives direct input from only a single mossy fiber active zone. The first telling findings of this paper are that individual AMPAreceptor-mediated excitatory postsynaptic currents (EPSCs) from the same cell could have fast or slow rising phases and that the probability of a slow-rising EPSC was greater than that of a fast-rising EPSC. Averages across all EPSCs from a cell, however, had fast-rising phases and usually both fast and slow components to their decay phases. The interpretation is that slow-rising EPSCs are caused solely by spillover of glutamate from nearby synapses within the glomerulus. Because of dilution in the extracellular space, spillover results in lower glutamate concentrations at the postsynaptic receptors than direct release and, therefore, receptor activation is slower. As there are many nearby synapses, this type of event occurs often. Furthermore, the low-affinity AMPA receptor antagonist, kynurenate, was more effective at blocking the slow-rising EPSCs (as well as the slowly decaying phase of averaged EPSCs) than the fastrising portion of averaged EPSCs. Because low-affinity antagonists are more effective at blocking events produced by transients at low glutamate concentrations than those at high-concentrations, these results indicate that the glutamate concentration transients responsible for the slowrising EPSCs were lower than those responsible for the fast components. Large glutamate transients and fast EPSCs are expected from release just across the synaptic cleft. In keeping with the higher incidence of slow-rising EPSCs, the authors estimated that there are 3–4 times as many active zones contributing to the slow portion of the EPSC than to the fast portion. Although some of the results could also be explained by MVR, the existence of the slow-rising EPSCs argues very strongly for spillover as the major contributor to the slow component of the averaged EPSC. Were MVR to occur at these synapses, however, spillover would be enhanced. http://tins.trends.com

Both of these papers provide convincing interpretations but questions (although perhaps nit-picking ones) remain. For example, it was not possible to record Ca2 transients from small spines. About half of the spines in the stratum radiatum of the CA1 hippocampus have dimensions that are smaller [6] than the smallest spines included in the first study [1]. Small spines could be more apt to sense glutamate spillover because of differences in glial investiture [14] and, therefore, glutamate transporter densities. Small spines might also be less likely to exhibit MVR because the Pr of their presynaptic partners could be lower [7]. The second paper [13] does not report the effect of changing Pr on potency (the mean size of EPSCs, excluding failures) or the amount of block of the fast EPSC by kynurenate. These two measures could help determine the degree to which spillover contributes to the amplitude of the fast component of the EPSC or whether there is any contribution of MVR at this synapse. Whispering A final concern is the possibility that the amplitude and time course of the glutamate transient in the cleft can be controlled by altering the rate of glutamate expulsion from exocytotic vesicles, by regulation of fusion-pore opening [15,16]. If rapid exocytosis is more likely to occur in conditions of high Pr, and prolonged, slow release occurs at low Pr, fusion-pore regulation could at least qualitatively account for paired-pulse facilitation of intra-spine Ca2 transients. In addition, slow release also could result in slow-rising and decaying AMPA-receptor EPSCs. Although both papers [1,13] argue that fusion-pore regulation cannot account for their data, the fusion-pore loophole was not entirely closed. The rise-time of the spine Ca2þ transient should, in theory, track changes in the kinetics of the cleft glutamate transient and, indeed, no differences were observed when Pr was altered. It could be argued, however, that the rise of the Ca2þ transient might be controlled by a slower, rate-limiting process that would obscure changes in the glutamate transient. Slow release could also produce slow-rising AMPA-receptor EPSCs at mossy fiber– granule cell synapses; however, such EPSCs are only observed in older animals [13]. Thus, the developmental expression sequence would have to be the opposite of that seen in hippocampal neuronal cultures [16], in which evidence for slow release was observed more often at early times. But this is not impossible. Other than providing physiologists with something interesting to study, what are the consequences of MVR and spillover? If the incidence of MVR increases with Pr [2,4,5], two factors will combine to increase postsynaptic responses: more active synapses and larger contributions from each synapse. In addition to this, the larger bolus of glutamate released into the cleft could have several consequences, including prolonged activation of postsynaptic receptors, and more spillover to extrasynaptic receptors on both pre and postsynaptic elements as well as to receptors at neighboring synapses. Altering Pr is not the exclusive province of the experimenter. Physiological events that alter Pr include changes in presynaptic action potential frequency and the activity of presynaptic receptors, such as metabotropic glutamate, GABAB and

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adenosine receptors. The utility of spillover might depend on the tissue being considered. In the present case of the mossy fiber–granule cell glomerulus, spillover augments excitability by activating receptors at neighboring synapses and, therefore, increases the reliability of transmission. In addition, because adjacent granule cells presumably all sense spillover, it might help to synchronize the activity of multiple granule cells. In other tissues, however, spillover can inhibit neighboring synapses by activating presynaptic metabotropic receptors [17] or can cause prolonged depolarizations of postsynaptic cells by activating postsynaptic metabotropic receptors [18]. The two papers highlighted here illustrate that MVR and spillover can both occur. Which of these two mechanisms dominates at a given synapse will depend on several morphological and physiological characteristics, including release probability, intersynaptic distance and the density of surrounding glutamate transporters. Heresy or not, these mechanisms seem to be here to stay.

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References 1 Oertner, T.G. et al. (2002) Facilitation at single synapses probed with optical quantal analysis. Nat. Neurosci. 5, 657 – 664 2 Auger, C. et al. (1998) Multivesicular release at single functional synaptic sites in cerebellar stellate and basket cells. J. Neurosci. 18, 4532–4547 3 Prange, O. and Murphy, T.H. (1999) Analysis of multiquantal transmitter release from single cultured cortical neuron terminals. J. Neurophysiol. 81, 1810– 1817 4 Tong, G. and Jahr, C.E. (1994) Multivesicular release from excitatory synapses of cultured hippocampal neurons. Neuron 12, 51 – 59 5 Wadiche, J.I. and Jahr, C.E. (2001) Multivesicular release at climbing fiber– Purkinje cell synapses. Neuron 32, 301 – 313 6 Harris, K.M. and Stevens, J.K. (1989) Dendritic spines of CA1

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pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics. J. Neurosci. 9, 2982– 2997 Schikorski, T. and Stevens, C.F. (1997) Quantitative ultrastructural analysis of hippocampal excitatory synapses. J. Neurosci. 17, 5858–5867 Mainen, Z. et al. (1999) Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated. Nature 399, 151 – 155 McAllister, A.K. and Stevens, C.F. (2000) Nonsaturation of AMPA and NMDA receptors at hippocampal synapses. Proc. Natl Acad. Sci. U.S.A. 97, 6173– 6178 Asztely, F. et al. (1997) Extrasynaptic glutamate spillover in the hippocampus: dependence on temperature and the role of active glutamate uptake. Neuron 18, 281– 293 Diamond, J.S. (2001) Neuronal glutamate transporters limit activation of NMDA receptors by neurotransmitter spillover on CA1 pyramidal cells. J. Neurosci. 21, 8328– 8338 Arnth-Jensen, N. et al. (2002) Cooperation between independent hippocampal synapses is controlled by glutamate uptake. Nat. Neurosci. 5, 325 – 331 DiGregorio, D.A. et al. (2002) Spillover of glutamate onto synaptic AMPA receptors enhances fast transmission at a cerebellar synapse. Neuron 35, 521 – 533 Ventura, R. and Harris, K.M. (1999) Three-dimensional relationships between hippocampal synapses and astrocytes. J. Neurosci. 19, 6897– 6906 Choi, S. et al. (2000) Postfusional regulation of cleft glutamate concentration during LTP at silent synapses. Nat. Neurosci. 3, 330–336 Renger, J.J. et al. (2001) A developmental switch in neurotransmitter flux enhances synaptic efficacy by affecting AMPA receptor activation. Neuron 29, 469 – 484 Scanziani, M. et al. (1997) Use-dependent increases in glutamate concentration activate presynaptic metabotropic glutamate receptors. Nature 385, 630 – 634 Bransjo, G. and Otis, T.S. (2001) Neuronal glutamate transporters control activation of postsynaptic metabotropic glutamate receptors and influence cerebellar long-term depression. Neuron 31, 607 – 616

An activity-dependent spermine-mediated mechanism that modulates glutamate transmission Domenico E. Pellegrini-Giampietro Dipartimento di Farmacologia Preclinica e Clinica, Universita` di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy

Intracellular polyamines are responsible for inward rectification of Ca21-permeable AMPA receptors and, hence, exert a voltage-dependent block upon these channels. In a recently described mechanism, neuronal activation modulates the synthesis of polyamines to regulate the amount of Ca21 flux and the excitability threshold at developing synapses that contain polyamine-sensitive AMPA receptors. The polyamines putrescine, spermidine and spermine are present in almost all cells. These organic polycations appear to play important roles in protein synthesis, cell growth and cell differentiation, and their synthesis and degradation are tightly controlled by several enzymes that are regulated by cellular activity [1]. Polyamines are protonated at physiological pH and can interact with several intracellular targets, including nucleic acids and Corresponding author: Domenico E. Pellegrini-Giampietro ([email protected]).

proteins. In the past few years, the specific interactions between polyamines, in particular spermine, and several functionally diverse ion channels have been described [2]. Spermine blocks the channel pore of inward-rectifier Kþ channels from the intracellular side, controlling the resting membrane potential and excitability in neurons and cardiac myocytes. By contrast, spermine acts at extracellular sites in neurons to potentiate the activity of NMDA receptors. Similar to the situation with Kþ channels, intracellular spermine has also been shown to control rectification and the total amount of current flow in some subtypes of AMPA and kainate receptors. Spermine determines inward rectification in Ca21-permeable AMPA receptors AMPA-type glutamate receptors are responsible for fast excitatory neurotransmission in the CNS. They are heteromeric ligand-gated channels composed of four possible subunits (GluR1– GluR4). Most AMPA receptors

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