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Nature Neuroscience (ISSN 1097-6256) is published monthly by Nature Publishing Group, a trading name of Nature America Inc. located at 75 Varick Street, Fl 9, New York, NY 10013-1917. Periodicals postage paid at New York, NY and additional mailing post offices. Editorial Office: 75 Varick Street, Fl 9, New York, NY 10013-1917. Tel: (212) 726 9319, Fax: (212) 696 0978. Annual subscription rates: USA/Canada: US$225 (personal), US$3,060 (institution). Canada add 7% GST #104911595RT001; Euro-zone: 287 (personal), 2,430 (institution); Rest of world (excluding China, Japan, Korea): 185 (personal), 1,570 (institution); Japan: Contact NPG Nature Asia-Pacific, Chiyoda Building, 2-37 Ichigayatamachi, Shinjuku-ku, Tokyo 162-0843. Tel: 81 (03) 3267 8751, Fax: 81 (03) 3267 8746. POSTMASTER: Send address changes to Nature Neuroscience, Subscriptions Department, 342 Broadway, PMB 301, New York, NY 10013-3910. Authorization to photocopy material for internal or personal use, or internal or personal use of specific clients, is granted by Nature Publishing Group to libraries and others registered with the Copyright Clearance Center (CCC) Transactional Reporting Service, provided the relevant copyright fee is paid direct to CCC, 222 Rosewood Drive, Danvers, MA 01923, USA. Identification code for Nature Neuroscience: 1097-6256/04. Back issues: US$45, Canada add 7% for GST. CPC PUB AGREEMENT #40032744. Printed by Publishers Press, Inc., Lebanon Junction, KY, USA. Copyright 2009 Nature Publishing Group. Printed in USA.

e d i to r i A l

99 Connecting the dots

b o o k r e v i e w

101 Mirroring People by Marco Iacoboni Reviewed by Ullrich Wagner & Patrik Vuilleumier

n e w s A n d v i e w s

103 Making scents out of how olfactory neurons are ordered in spaceNathan E Schoppa see also p 210

105 Whither proBDNF?Philip A Barker see also p 113

107 Too much Sonic, too few neuronsChristopher A Fasano & Lorenz Studer see also p 125

108 PML: a tumor suppressor essential for neocortical developmentKarisa C Schreck & Nicholas Gaiano see also p 132

110 Sleep on itCharvy Narain see also p 122

111 Exit chloride, enter glutamateFelix E Schweizer see also p 156

b r i e F com m u n i c At i o n s

113 Neuronal release of proBDNFJ Yang, C-J Siao, G Nagappan, T Marinic, D Jing, K McGrath, Z-Y Chen, W Mark, L Tessarollo, F S Lee, B Lu & B L Hempstead see also p 105

116 Laminar and compartmental regulation of dendritic growth in mature cortexD K Chow, M Groszer, M Pribadi, M Machniki, S T Carmichael, X Liu & J T Trachtenberg

119 Collagen VI protects neurons against A toxicityJ S Cheng, D B Dubal, D H Kim, J Legleiter, I H Cheng, G-Q Yu, I Tesseur, T Wyss-Coray, P Bonaldo & L Mucke

122 Sleep benefits subsequent hippocampal functioningY D Van Der Werf, E Altena, M M Schoonheim, E J Sanz-Arigita, J C Vis, W De Rijke & E J W Van Someren see also p 110

Dopamine sensitive mGluR5-modulated prefrontal plasticity

(p 190)

Categorical perception is critical for processing vocal communication. In

this issue, Prather and colleagues show that individual swamp sparrow

sensorimotor neurons exhibit categorical responses to the features of their songs. They also find that the neuronal response boundary predicts the categorical perceptual boundary

for the birds own song dialect. The cover is a photograph of a swamp

sparrow courtesy of Rob Lachlan.(p 221)

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125 Wnt antagonism of Shh facilitates midbrain floor plate neurogenesisM Joksimovic, B A Yun, R Kittappa, A M Anderegg, W W Chang, M M Taketo, R D G McKay & R B Awatramani see also p 107

132 The tumor suppressor Pml regulates cell fate in the developing neocortexT Regad, C Bellodi, P Nicotera & P Salomoni see also p 108

141 Identification of distinct telencephalic progenitor pools for neuronal diversity in the amygdalaT Hirata, P Li, G M Lanuza, L A Cocas, M M Huntsman & J G Corbin

150 UNC-129 regulates the balance between UNC-40 dependent and independent UNC-5 signaling pathwaysL T MacNeil, W R Hardy, T Pawson, J L Wrana & J G Culotti

156 A chloride conductance in VGLUT1 underlies maximal glutamate loading into synaptic vesiclesS Schenck, S M Wojcik, N Brose & S Takamori see also p 111

163 Synaptotagmin IV: a multifunctional regulator of peptidergic nerve terminalsZ Zhang, A Bhalla, C Dean, E R Chapman & M B Jackson

172 A critical role for PSD-95/AKAP interactions in endocytosis of synaptic AMPA receptorsS Bhattacharyya, V Biou, W Xu, O Schlter & R C Malenka

182 N-Acetylcysteine reverses cocaine-induced metaplasticityK Moussawi, A Pacchioni, M Moran, M F Olive, J T Gass, A Lavin & P W Kalivas

190 Dopamine modulates an mGluR5-mediated depolarization underlying prefrontal persistent activityK Sidiropoulou, F-M Lu, M A Fowler, R Xiao, C Phillips, E D Ozkan, M X Zhu, F J White & D C Cooper

200 CREB regulation of nucleus accumbens excitability mediates social isolationinduced behavioral deficitsD L Wallace, M-H Han, D L Graham, T A Green, V Vialou, S D Iiguez, J-L Cao, A Kirk, S Chakravarty, A Kumar, V Krishnan, R L Neve, D C Cooper, C A Bolaos, M Barrot, C A McClung & E J Nestler

210 Precision and diversity in an odor map on the olfactory bulbE R Soucy, D F Albeanu, A L Fantana, V N Murthy & M Meister see also p 103

221 Neural correlates of categorical perception in learned vocal communicationJ F Prather, S Nowicki, R C Anderson, S Peters & R Mooney

t e c H n i c A l r e P o r t

229 Bi-stable neural state switchesA Berndt, O Yizhar, L A Gunaydin, P Hegemann & K Deisseroth

n At u r e n e u r o s c i e n c e c l A s s i F i e d

See back pages.

Collagen VI protects against A toxicity(p 119)

Wnt facilitates floor plate neurogenesis(pp 107 and 125)

Pml regulates neocortical cell fate(pp 108 and 132)

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One such study2 used dual microelectrodes to simultaneously record colocalized tissue oxygen measurements (a proxy for the BOLD signal), spiking and LFP activity in the cat visual cortex. Changes in tissue oxygen were more closely coupled to LFPs than spiking activity, leading to the conclusion that the BOLD signal represents perisynaptic, rather than spiking, activity.

These results are by no means universally accepted, however, and there has been debate about them, including in the pages of this journal3. This is partially because it is difficult to ensure that the effects of multi-unit spiking and LFPs are directly comparable and also partially because it is difficult to ensure that these effects are completely separable. Neither LFPs nor multi-unit spiking may completely account for the BOLD signal, but it should be possible to discover which of them makes the greater contribution to the BOLD signal, and the resolution to this debate will be interesting.

Another major issue that arises when connecting neural activity to fMRI maps is the question of how the activity of a heterogeneous population of neurons is reflected in the BOLD signal. For example, the release of inhibitory neurotransmitters at synapses is still energy consuming and inhibitory neural activity could therefore still result in a localized increase in BOLD signal at the site of inhibition, despite a decrease in the responses of excitatory neurons. If the BOLD signal is more affected by perisynaptic than firing activity, as suggested by the study described above2, inhibitory neural activity may well show up as positive activation in fMRI maps, indistinguishable from that caused by excitatory activity. The link between inhibitory neural activity and BOLD also seems to be modulated by the exact brain area being scrutinized and is probably affected by the net energy consumption resulting from the combination of inhibitory and excitatory processes.

The mystery of what information about neural activity is encoded by the BOLD signal is only deepened by reports of a negative BOLD signal, but the discussion about the interpretation of negative BOLD signals and how inhibitory neural activity relates to the BOLD signal has been muted in comparison with the lively debate on the relationship between electrophysiological and blood oxygenation measures.

It has now been nearly two decades since the first fMRI paper was published, and despite a huge increase in the number of papers using fMRI as a tool (from just four in all of 1992 to eight in one day in 2007), only 5% of the over 19,000 fMRI papers published since the 1990s have looked at the neural basis of the fMRI signal4. A mechanistic understanding of the BOLD signal is essential if we are ever to connect up the dots between systems and cellular neuroscience. L

1. Weisberg, D.S. et. al. J. Cogn. Neurosci. 20, 470477 (2008).2. Viswanathan, A. & Freeman, R.D. Nat. Neurosci. 10, 13081312 (2007).3. Nir, Y., Dinstein, I., Malach, R. & Heeger, D.J. Nat. Neurosci. 11, 523524 (2008).4. Logothetis, N.K. Nature 453, 869878 (2008).

an image is worth a thousand words, which may explain why the images produced by techniques such as fMRI have been so effective at capturing the imagination of the public and of neuroscientists. A recent study quantified just how convincing explanations derived from neuroimaging can be: non-experts were more likely to believe a bad explanation for a phenomenon when it was accompanied by an extra few lines saying that the effect was localized to a certain brain area, even though this additional information was entirely irrelevant1. However, these images are the output of an extended analysis and several steps removed from neural activity, so much so that many scientists who study activity at the level of the cell feel that fMRI studies have little that is of interest to them. With the increasing complexity and subspecialization in the techniques now required of modern neuroscience, this ghettoization is perhaps inevitable. To really understand how the brain works, however, it is important that scientists working at the levels of the cell and of the system communicate with each other and that findings at one level can be translated and understood at the other. In particular, an understanding of how the signal tracked in brain imaging studies relates to neural activity is crucial.

Specifically, fMRI measures changes in the blood oxygenation leveldependent (BOLD) signal. The relationship between the BOLD signal and neural activity is necessarily indirect: because neurons do not have internal energy reserves in the form of glucose and oxygen, their firing causes more energy uptake. Oxygen release from blood is therefore greater for active than for inactive neurons and this difference in levels of oxygenated and deoxygenated blood is what drives the BOLD signal.

Of course, fMRI is not the only method to track the activity of groups of neurons in behaving animals. Electrophysiology studies, measuring changes in multi-unit spiking activity and local field potentials (LFPs), fulfill a similar purpose. However, the results from electrophysiology and imaging studies are clearly not equivalent, even though they are often treated as such. For one thing, the link between electrophysiological measures and neural activity is much better understood, with LFPs being thought to reflect perisynaptic activity and spiking activity being thought to reflect the firing of action potentials.

In contrast, the link between the BOLD signal and neural activity is much less clear. One way to get around this problem is to work out how electrophysiological measures such as spiking activity and LFPs correlate with BOLD activity. Because the relationship between these electrophysiological measures and underlying neural activity is relatively clear, tying BOLD activity to either LFPs or multi-unit spiking would be an indirect, but valid, way of connecting fMRI results to activity at the level of the neuron. Although there are other issues that affect the interpretation of the BOLD signal, this has been an area of much recent interest, with some studies attempting to link hemodynamic changes to electrophysiological measures.

Connecting the dotsUnderstanding the exact link between functional magnetic resonance imaging (fMRI) and neural activity is critical to bridge the widening gap between neuroimagers and cellular neuroscientists.

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key to answering many such questions. For example, mirror neurons may lend support to psychological theories that regard imitation as the primary mechanism underlying our understanding of the inner mental states of other people. If mirror neurons represent an immediate, covert imitation of what others do, this could be a way in which we directly understand what they feel and what they intend. Mirror neurons might also address the philosophical problem of intersubjectivity, the sharing of meaning between people, as they could provide us with a direct representation of what others mean. Thus, in line with existential phenomenologists such as Merleau-Ponty or Wittgenstein, we immediately perceive emotions, not muscle movements, when we observe a face. The critical point is that the understanding supported by mirror neurons is fast and intuitive, and needs no further inferential processing. The brain is literally reflecting the other, not reflecting on the other. Or, as Iacoboni says, the mirror neuron system seems to project internally [. . .] other people into our own brains (p.260).

This is in fact the deeper central theme of the book: humans are socially defined in that the self and the other are closely interdependent, connected by their mirror neurons (hence the subtitle: The new science of how we connect with others). Mirror neurons allow us not only to understand others, but also to empathize or even identify with them. They are the foundation of most human social capabilities. Following his interdisciplinary interest, Iacoboni applies this idea to a variety of different areas including, among others, language acquisition, autism, media violence and politics. For the more broadly interested reader, this journey through different domains provides a fascinating perspective on theoretical discussions outside neuroscience.

However, not every one of Iacobonis assumptions about a critical role for mirror neurons is equally convincing. His obvious enthusiasm about this research sometimes leads him to overinterpretations that are difficult to comprehend, particularly in the field he calls neuropolitics. For example, during the US presidential election year of 2004, Iacobonis group found that Democratic and Republican partisans who watched their own candidate, as compared to other candidates, showed relative activation of the middle orbitofrontal cortex (a region containing what Iacoboni calls super mirror neurons) when scanned in spring, but not when scanned in late summer. Iacoboni interprets this change as a reduction of identification with their own candidate because of negative information accumulated during the political campaigns. Of course, many other factors not relating to mirror processes could also account for such a change. But Iacoboni, as one of the driving forces in this field of research, seems inclined to see mirror neurons at work even in cases where the evidence for their effects is quite indirect. The reader who expects to get an experts view of certain shortcomings or limitations of mirror neuron research will search in vain for it in this book.

Still, Mirroring People is highly recommendable both to neuroscientists (particularly those working in social neuroscience) and to a broader audience. Its clear structure and simple language, avoiding technical terms, make it easy to understand even for nonexperts. The concept of mirror neurons is a popular topic today, and many scientifically interested people will have heard of it in one way or another. This book gives them the opportunity to learn more about the specific results and ideas that lie behind this concept. L

Sometimes scientific progress emerges from accidental findings. In the 1980s a group of Italian neuroscientists in the laboratory of Giacomo Rizzolatti, in Parma, implanted microelectrodes into motor areas of monkey brains to record single-cell activity related to movements performed by the animals. They found that some of the neurons were highly specialized for certain actions. For example, some neurons fired only when the monkey grasped a big object such as an apple, but not when it grasped a small object such as a peanut. This was already an interesting feature, but then the researchers found another totally unexpected property: the same neurons that were activated when the monkey grasped an apple also fired when the monkey merely observed another monkey (or the experimenter) grasping an apple, without performing any overt movement itself. Because it seemed that the monkey internally imitated, or mirrors, the action that it observed in others, the term mirror neurons was coined for these cells. With this discovery, a new research direction was born, which ever since has provided fascinating insights into a variety of behaviors in both monkeys and humans.

In his new book Mirroring People, Marco Iacoboni provides a comprehensive account of what this research has revealed so far. Iacoboni, a neurologist at the University of California Los Angeles, is a leading expert on this topic, whose research group has performed many of the human brain imaging studies in the field and whohaving Italian roots himselfhas tight links with Rizzolattis group in Parma. He tells the story systematically, beginning with the initial discovery of mirror neurons in monkeys and turning subsequently to human research, with each chapter focusing on a new empirical question and the relevant results.

What makes this book particularly interesting is that it does not simply summarize the current state of the research itself; the author also uses the existing findings to draw more general conclusions about human nature. According to Iacoboni, the data are pertinent to essential philosophical, psychological and even political questions, and he convincingly argues that the mirror neuron system can be a

brain reflectionsMirroring People

by Marco Iacoboni

Farrar, Straus & Giroux, 2008 320 pp, hardcover, $25 ISBN 0374210179

Reviewed by Ullrich Wagner & Patrik Vuilleumier

Ullrich Wagner and Patrik Vuilleumier are at the Department of Neuroscience,

University Medical School, at the Center for Neuroscience, and at the Swiss

Center for Affective Sciences, University of Geneva, Geneva, Switzerland.

e-mail: ullrich.wagner@unige.ch

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between glomeruli depended on their physical separation.

What they found was quite striking. When odor spectra similarity was plotted as a function of glomerular separation, there was a small tendency (only about 23% of all glomerular pairs tested) for pairs separated by less than approximately 1 mm to have more similar spectra. However, there was little evidence for a fine-scale chemotopic map. Glomeruli that were immediately adjacent to each other were not much more likely to have

and hence the population of glomeruli across the bulb reflects the population of odorant receptors in the nose (typically 2 glomeruli per odorant receptor). To test for the presence of a chemotopic map, or chemotopy, the experimenters exposed rodents (mice or rats) to a battery of ~100 diverse odors and developed an odor spectrum (the pattern of responses to a range of odors) for each glomerulus. They then performed pair-wise comparisons across all tested glomeruli to address how the similarity in odor spectra

The way our mind senses the world around us depends a lot on how neurons are organized in space. For example, in the visual system, retinal ganglion cells are ordered according to the visual field, with neighboring cells being responsive to neighboring parts of the visual field. This organization, known as the retinotopic map, is believed to promote the sharpening of images through center-surround contrast enhancement1. A similar spatial order is seen in the auditory system, where the organization of neurons by sound frequency (a tonotopic map) is believed to narrow frequency tuning. Likewise, a somatotopic map exists in the somatosensory system. For olfaction, however, the question remains as to whether there is a chemotopic map. Are neurons that prefer similar chemical odor molecules located close together in space?

In this issue, Soucy and colleagues2 provide evidence that, surprisingly, there is not much of a chemotopic map, or at least certainly nothing like the order that is seen in other sensory systems. They specifically examined the positions of many of the ~2,000 glomeruli that line the outer surface of the olfactory bulb. Glomeruli are neuropil structures, ~50100 m in diameter, that contain both the axon terminals of olfactory receptor neurons (ORNs) arriving from the nose and dendrites of a number of neuron-types including output mitral cells. Each glomerulus receives inputs from ORNs that express only one type of odorant receptor,

Making scents out of how olfactory neurons are ordered in spaceNathan E Schoppa

Many sensory brain areas are characterized by a specific spatial organization, with neurons being ordered according to their similarity in receptive field properties. A surprising new study provides evidence that the organization of glomeruli in the olfactory bulb violates this anatomical principle, suggesting that olfaction might work by a different set of rules.

The author is in the Department of Physiology

and Biophysics, University of Colorado at Denver,

Anschutz Medical Campus, Mail Stop 8307,

P.O. Box 6511, Aurora, CO 80045 USA. e-mail: nathan.schoppa@uchsc.edu

Input

Lateralinhibition

Chemotopicmap

Nonchemotopicmap

Outputa

b

Figure 1 Effect of the lack of chemotopy on lateral inhibition during presentation of a simple odor. (a) When inputs into the olfactory bulb are ordered chemotopically, lateral inhibition generates a center-surround effect, whereby the activity of the most strongly activated glomerulus (shown as darkest red) dominates the output. In the diagram, each circle for the input is meant to reflect a glomerulus specific for one odorant receptor in the nose, whereas each circle for the output reflects the population of mitral cells associated with each glomerulus. Color reflects the similarity in odorant receptors and the darkness of the filled-coloring reflects the degree of activation. (b) In the absence of chemotopy, lateral inhibition causes a more uniform reduction in activity across all glomeruli, including the most strongly active glomerulus.

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Lateral inhibition is, of course, only one of many functional possibilities to consider when searching for the meaning of a nonchemotopic map, although it should be pointed out that there are other independent lines of evidence that support the notion that lateral inhibition in the bulb functions in a somewhat atypical fashion. For example, two studies examining the spatial relationship between active mitral cells and active glomeruli during odor-evoked responses13,14 found that the sphere of influence onto mitral cells was quite dispersed. Mitral cells were not substantially more influenced by nearby glomeruli, as might be expected if lateral inhibition were operating on a local scale, as in the visual system.

There are, of course, other unresolved issues that will need to be tackled in the future, in addition to trying to understand the effect of lateral inhibition. For example, the just- described gain-control mechanism suggests one possible advantage of having no fine-scale chemotopy in the bulb, yet it does not at all explain the animal-to-animal precision in the spatial glomerular maps, which implies that glomeruli need to be unordered in a very specific way. In addition, there remains the issue of contrast enhancement. A previous study15 showed more than a decade ago that the olfactory bulb circuitry can introduce a form of olfactory contrast enhancement, making neurons more finely tuned to specific odors. If local lateral inhibitory mechanisms are unable to mediate contrast enhancement in the bulb, owing to the lack of fine-scale chemotopy, what are possible alternate mechanisms?

1. Kuffler, S.W. J. Neurophysiol. 16, 3668 (1953).2. Soucy, E.R., Albeanu, D.F., Fantana, A.L., Murthy, V.N.

& Meister, M. Nat. Neurosci. 12, 210220 (2009).3. Miesenbock, G., De Angelis, D.A. & Rothman, J.E.

Nature 394, 192195 (1998).4. Bozza, T., McGann, J.P., Mombaerts, P. & Wachowiak, M.

Neuron 42, 921 (2004).5. Friedrich, R.W. & Korsching, S.I. Neuron 18, 737752

(1997).6. Uchida, N., Takahashi, Y.K., Tanifuji, M. & Mori, K.

Nat. Neurosci. 3, 10351043 (2000).7. Mori, K., Takahashi, Y.K., Igarashi, K.M. & Yamaguchi, M.

Physiol. Rev. 86, 409433 (2006).8. Johnson, B.A. & Leon, M. J. Comp. Neurol. 503,

134 (2007).9. Laurent, G. et al. Annu. Rev. Neurosci. 24, 263297

(2001).10. Aungst, J.L. et al. Nature 426, 623629 (2003).11. Arevian, A.C., Kapoor, V. & Urban, N.N. Nat. Neurosci.

11, 8087 (2008).12. Cleland, T.A. & Sethupathy, P. BMC Neurosci. 7,

7 (2006).13. Luo, M. & Katz, L.C. Neuron 32, 11651179

(2001).14. Fantana, A.L., Soucy, E.R. & Meister, M. Neuron 59,

802814 (2008).15. Yokoi, M., Mori, K. & Nakanishi, S. Proc. Natl. Acad.

Sci. USA 92, 33713375 (1995).

bulb processing does not depend on the spatial order of neurons; for example, the bulb might be more focused on introducing a timing component to an odorant response9. However, such a conclusion is inconsistent with experiments that Soucy et al.2 report in much of the earlier part of their study, where comparisons of glomerular maps were made between olfactory bulbs of different animals. A prediction of a model in which space does not matter is that there might be substantial developmental noise in the positioning of glomeruli. In fact, glomerular maps were found to be highly precise between animals, with deviations of only 12 glomerular spacings. Similarities in glomerular maps were even found when interspecies comparisons were made between rats and mice. The observed precision in the glomerular maps suggests that space does matter, even if glomeruli do not form an ordered chemotopic map on the basis of receptive field.

What might then be the implications of having a nonchemotopic order in glomeruli? Soucy et al.2 argue that it might be important for lateral inhibition. Lateral inhibition is the circuit mechanism that underlies center-surround contrast enhancement in the visual system and there are extensive networks of GABAergic interneurons in the olfactory bulb that appear to be capable of driving lateral inhibition between different glomeruli10,11. With chemotopy, lateral inhibition might do exactly what you would expect on the basis of the example of the visual system. The preferred glomerulus, reflecting the most strongly active odorant receptor, would laterally inhibit surrounding weakly activated glomeruli, such that the output of the bulb would be dominated by the preferred glomerulus (Fig. 1). In contrast, lateral inhibition in the absence of chemotopy might cause a more uniform reduction in activity across all glomeruli. Such a mechanism, which has been shown to be at least feasible in modeling studies12, might act as a form of gain control, preserving information about odor identity with varying odor concentration. One way of understanding the different effects of lateral inhibition in the two schemes is to assume that the strength of lateral inhibition between glomeruli is dependent on physical separation. In the case of chemotopy, the preferred glomerulus is well positioned to turn off near-neighbor weakly- activated glomeruli, whereas in the nonchemotopic situation, it is not. Because weakly active glomeruli are less inhibited, they can in turn inhibit the preferred glomerulus, thus leading to a more uniform activity reduction.

similar odor spectra as compared with those located several hundred microns apart.

These were not easy studies. Besides being computationally intensive, tens of thousands of glomerular pair-wise calculations were required; the experimenters had to overcome one factor that has confounded olfactory physiologists: the difficulty of controlling a chemical odor stimulus. Soucy et al.2 sampled many odors by using a custom-made machine in which odorized air could be quickly applied and switched between odors. An additional advantage was offered by the two different optical probes that were used in their measurements of glomerular activity. One of these, called an intrinsic signal, reflects activity- dependent changes in the optical properties of tissue, probably the result of factors such as changes in hemoglobin absorption. The second probe was genetically encoded synaptopHluorin, a protein associated with synaptic vesicles that changes fluorescence as a function of activity-dependent neurotransmitter release3,4. SynaptopHluorin was specifically targeted to mouse ORNs that expressed olfactory marker protein. Notably, neither of the optical probes require an exogenous indicator and thus are less susceptible to stimulus-dependent signal degradation, a factor that greatly facilitated the long experiments required for developing the complete glomerular odor spectra.

Several groups have performed similar experiments in the past58, finding evidence for clustering of glomeruli on the basis of particular chemical features of odor molecules. This clustering, a form of chemotopy, can be seen, for example, in the positioning of glomeruli responsive to aliphatic aldehydes of different carbon chain length. Soucy et al.2 analyzed glomerular receptive fields in a somewhat different way, examining responses to a large set of diverse odors rather than focusing on particular molecular features. Nevertheless, as they point out, the loose chemotopic map that they saw on a ~1-mm spatial scale may very well reflect the glomerular clusters that were seen previously. Their most interesting result is the near- complete absence of chemotopy at a fine scale. Soucy et al.2 are the first, to the best of our knowledge, to systematically examine the relationship between odor response similarity and glomerular spatial separation, which is the key analysis that allowed them to test for fine-scale chemotopy.

If olfactory bulb glomeruli do not show fine-scale chemotopy, then what makes olfaction different from other sensory systems? One explanation is that olfactory

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Philip A. Barker is at the Montreal Neurological

Institute, McGill University, Montreal,

Quebec H3A 2B4, Canada.

e-mail: phil.barker@mcgill.ca

reported three key findings. First, using a knock-in mouse expressing Myc-tagged BDNF, they found that 90% of the BDNF present in adult brain was the mature, fully processed form. Second, they demonstrated

tPA regulates LTP and showed that proteolytic processing of a single gene product could differentially regulate synaptic plasticity.

Matsumoto et al., however, threw a large wrench into this enticing model1. They

Neurotrophins are produced as pro-isoforms that are cleaved to release mature, secreted ligands that activate Trk receptors and thereby promote neuronal survival and growth. Brain-derived neurotrophic factor (BDNF) exists as pro- and fully processed forms, but the question of whether proBDNF has a physiological function has been controversial. In a recent issue of Nature Neuroscience, Matsumoto et al.1 argued that proBDNF is a transient synthetic intermediate that is unlikely to be secreted or to have a physiological role as an extracellular ligand. In this issue, Yang et al. provide new data indicating that proBDNF is secreted and thereby able to function as a ligand to regulate neuronal function2.

Several studies have previously suggested that proneurotrophins can act as ligands. For example, a landmark study published by the Hempstead group in 2001 showed that proNGF binds the p75 neurotrophin receptor (p75NTR) to induce neuronal apoptosis3. Subsequent work showed that secretion of the uncleaved proform of NGF is induced by neural trauma4 and demonstrated that proNGF-dependent cell death requires the activation of a receptor complex containing p75NTR and sortilin, a VPS10 family member5. Thus, NGF functions as pro- survival or pro-apoptotic ligand depending on whether or not it is fully processed.

The initial studies suggesting that proBDNF plays a role in hippocampal plasticity emerged from Bai Lus group in 2004 (ref. 6). It is generally accepted that the fully processed form of BDNF has a critical role in late-phase long-term potentiation (LTP) in the hippocampus, but Pang et al. showed that proBDNF binding to p75NTR is required for long-term depression (LTD)6. In addition, they reported that the extracellular protease plasmin is activated by tissue plasminogen activator (tPA) to cleave proBDNF and thereby produce BDNF, which then contributes to LTP. This study was important because it provided an answer for the long-standing question of how

Whither proBDNF?Philip A Barker

ProBDNF has been proposed to alter synaptic plasticity, but whether it is normally released from neurons has been a matter of contention. New work suggests that proBDNF is indeed secreted from central neurons.

Furin

a

b

1Plasmin3

5

4

6

Regulatedsecretory vesicles

BDNF mRNAwith short

3 UTR

Regulatedsecretoryvesicles

DendritesAxons

BDNF mRNAwith long3 UTR

Nucleus

ProBDNFp75NTR

MatureBDNF

TrkB

TrkB

ActivatedTrkB

2

AAA

Figure 1 BDNF production and processing in the central nervous system. (a) ProBDNF may be processed to mature BDNF by several cellular mechanisms. ProBDNF can be cleaved within the endoplasmic reticulum by furin (1) and in regulated secretory vesicles by proconvertase enzymes (2). If proBDNF reaches the extracellular milieu, it can be processed by plasmin, and the mature BDNF produced can then activate cell surface TrkB receptors (3). Alternatively, extracellular proBDNF can bind p75NTR and become endocytosed and then cleaved to produce mature BDNF that either activates TrkB within endosomes (5) or is recycled to the cell surface (6). (b) The site of BDNF translation within the neuron may determine the form of BDNF released. BDNF mRNA with a short 3 UTR accumulates in the neuronal soma, whereas BDNF mRNA with a long 3 UTR is trafficked to dendrites. The soma supports BDNF cleavage within the Golgi, but the majority of dendrites lack Golgi elements necessary for processing of proBDNF, and therefore proBDNF may be the predominant form released.

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endoplasmic reticulum14, many dendrites lack the Golgi elements required for secretory protein processing15. Thus, An et al.12 have proposed that BDNF translated in dendrites may evade processing and be secreted as a proform, whereas BDNF produced in the soma undergoes typical processing events. Interestingly, Yang et al.2 reported that proBDNF staining is highest in mossy-fiber axonal projections. Determining the precise mechanisms that allow proBDNF to accumulate in these subcellular compartments will be an important topic for future studies.

Matsumoto et al.1 and Yang et al.2 have gone to extraordinary lengths to determine whether proBDNF is actually produced and secreted by neurons. Taken together with recent advances by other groups, it seem certain that the tools they have developed can be used to resolve the outstanding questions remaining in this area. It will be important to determine whether the BDNF mRNA transcripts produced in the knock-in mice created by both groups show alternative splicing akin to that observed for the normal transcript and, if so, whether they are appropriately targeted to dendrites. If this can be established, developing techniques to show that proBDNF is selectively produced and secreted from dendrites will be a priority. It seems likely that proBDNF secreted from dendrites will have a short half-life in the extracellular milieu, and resolving these issues is likely to require genetic or chemical techniques that block extracellular proBDNF processing.

Perhaps the most important conclusion that emerges from these many recent advances and controversies is that vigorous, productive and congenial scientific debate is a hallmark of the neurotrophin field. These characteristics reflect the robust health of this exciting scientific area and bode well for resolving this controversy in the future.

1. Matsumoto, T. et al. Nat. Neurosci. 11, 131133 (2008).

2. Yang, J. et al. Nat. Neurosci. 12, 113115 (2009).3. Lee, R., Kermani, P., Teng, K.K. & Hempstead, B.L.

Science 294, 19451948 (2001).4. Beattie, M.S. et al. Neuron 36, 375386 (2002).5. Nykjaer, A. et al. Nature 427, 843848 (2004).6. Pang, P.T. et al. Science 306, 487491 (2004).7. Rosch, H., Schweigreiter, R., Bonhoeffer, T.,

Barde, Y.A. & Korte, M. Proc. Natl. Acad. Sci. USA 102, 73627367 (2005).

8. Woo, N.H. et al. Nat. Neurosci. 8, 10691077 (2005).

9. Mowla, S.J. et al. J. Biol. Chem. 276, 1266012666 (2001).

10. Boutilier, J. et al. J. Biol. Chem. 283, 1270912716 (2008).

11. Tongiorgi, E. Neurosci. Res. 61, 335346 (2008).12. An, J.J. et al. Cell 134, 175187 (2008).13. Zagrebelsky, M. et al. J. Neurosci. 25, 99899999

(2005).14. Aridor, M., Guzik, A.K., Bielli, A. & Fish, K.N.

J. Neurosci. 24, 37703776 (2004).15. Horton, A.C. et al. Neuron 48, 757771 (2005).

However, proBDNF is not a particularly good substrate for furin, and when BDNF is overexpressed, much of the secreted protein is the proform9. Therefore, when using knock-in models to follow BDNF fate in vivo, it is critically important to ensure that the levels of tagged BDNF that are produced match those produced normally, as was done by both Matsumoto et al.1 and Yang et al. 2. Once proBDNF leaves the cell, it can be cleaved by extracellular proteases. The best characterized of these is plasmin, which is an efficient protease for proBDNF6. There is also evidence to suggest that proBDNF may be cleaved during endocytosis and that the mature ligand may then bind and activate Trk receptors, either within endosomes or after being recycled to the cell surface10.

A second important factor to consider concerns the site of BDNF protein production within central neurons. Key experiments addressing the production and release of proBDNF by Matsumoto et al.1 and Yang et al.2 are, by necessity, performed in dissociated neuronal cultures and measure BDNF produced throughout the cell. However, the cellular sites of neuronal BDNF release that are relevant for synaptic plasticity in vivo may not be well represented in these systems. It is well established that BDNF mRNA not only is present within the neuronal cell soma but also is localized to dendrites, where its local translation may facilitate synaptic effects11.

Notably, recent studies have shown that alternative splicing of BDNF mRNA plays a crucial role in the targeting of BDNF transcripts12 (Fig. 1b). For example, An et al. showed that alternative polyadenylation of BDNF mRNA functions as a switch to regulate subcellular targeting of BDNF mRNA under physiological conditions, with the long 3 UTR BDNF mRNA being selectively targeted to dendrites12. Using a knockout strategy, they showed that mice that did not produce the long 3 UTR BDNF mRNA isoform had reduced dendritic BDNF protein levels, deficits in developmental dendritic pruning, and defects in dendritic LTP. Previous studies from the Barde group have shown that p75NTR negatively modulates dendritic morphology in adult hippocampal neurons13, and therefore one plausible model is that proBDNF produced in dendrites mediates local dendritic pruning and that BDNF-dependent dendritic LTP is a consequence of tPA- dependent cleavage of locally produced proBDNF12.

Targeting of BDNF mRNA not only may affect where BDNF is produced, but also may influence the BDNF form that is secreted. Although all dendrites possess

through pulse-chase experiments that proBDNF produced in neurons was rapidly converted to mature BDNF and that proBDNF did not accumulate in conditioned medium. Third, although there is agreement that hippocampal LTD is p75NTR dependent7,8, Matsumoto et al. found that LTD occurred in mice lacking neuronal BDNF expression1. Together, these studies indicated that proBDNF is not released from neurons in significant quantities and is not required for p75NTR-dependent synaptic changes.

A study in this issue of Nature Neuroscience now responds with new data indicating that proBDNF is produced and released by central neurons2. Using a knock-in mouse expressing hemagglutinin-tagged BDNF together with a proBDNF-specific antibody, Yang et al. find that levels of proBDNF rise during the first few postnatal weeks, in concert with developmental p75NTR expression, such that in juvenile mice, proBDNF is more abundant than mature BDNF. To determine whether proBDNF is actually released by neurons, the authors assessed levels of proBDNF released into medium conditioned by primary cultured hippocampal neurons. Surprisingly, they report that proBDNF, but not mature BDNF, accumulates in the conditioned medium of these cells, in direct conflict with the results reported by Matsumoto et al.1. Yang et al.2 indicate that the observation of secreted proBDNF in this system requires the use of primary cultures that are essentially free of glia, and in addition requires the presence of a plasmin inhibitor to block proBDNF proteolysis. The lack of mature BDNF accumulation in their experiments seemed to reflect rapid turnover of this factor by the TrkB-expressing neurons, as a TrkB-Fc fusion protein added to the medium succeeded in capturing mature BDNF produced by these cells.

Thus, Matsumoto et al.1 demonstrated that proBDNF produced by neurons is rapidly converted to mature BDNF and proposed that proBDNF is a transient biosynthetic intermediate, whereas Yang et al.2 showed that neurons produce and secrete proBDNF and also found that high levels of proBDNF exist in the postnatal CNS. So, how do we interpret these two important and conflicting studies from leading groups in the field? First, it should be noted that there are several enzymes that cleave BDNF and that these function within distinct subcellular and extracellular locations (Fig. 1a); because of this, it can be difficult to distinguish when and where proBDNF is processed. BDNF can be cleaved by furin within the Golgi and by proconvertases within secretory vesicles.

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the spinal cord. Ectopic hindbrain (but not spinal cord) neurons expressed several features of midbrain dopamine neurons.

One of the key signaling factors specifying midbrain versus hindbrain identity is Wnt1 (ref. 7). The authors therefore also investigated whether differences in Wnt signaling could be responsible for differential regulation of Shh between midbrain and hindbrain floor plate. To test whether canonical Wnt signaling is necessary and sufficient to repress Shh expression and confer neurogenic potential to the midbrain floor plate, the authors conditionally removed -catenin (encoded by Ctnnb1), a critical component and major effector of the canonical Wnt signaling pathway, from cells expressing Shh. In this mutant, the midbrain- hindbrain boundary was not

involved in the apparent regionalization of floor plate neurogenesis.

Using a mouse strain in which Cre recombinase is expressed from the Shh locus, the authors performed both lineage tracing and Shh loss-of-function studies in vivo. The lineage-tracing data confirmed that the midbrain, but not the hindbrain, floor plate gives rise to neurons. However, upon Cre-mediated excision of Shh in the Shh::cre mice, neurons were ectopically produced in the hindbrain floor plate. Consistent with this ectopic neuronal production, in these mutants the proneural gene Ngn2 was also upregulated, and cell proliferation was increased. Remarkably, a neurogenic response was observed in the floor plate throughout the anterior- posterior extent of the neuraxis, including

Neural development is orchestrated in time and space by a complex set of signals, which specify neural precursor identity. One of the most important signaling centers in neural development is the floor plate, located along the ventral midline of the embryo. Classically, the floor plate has been credited with two major functions: secretion of the morphogen Sonic hedgehog (Shh) gives floor plate cells the ability to influence the fate of neural precursor cells1, and expression of Netrin is critical for the guidance of developing axons across the midline2. However, the floor plate is not uniform1. Just as the neural tube has distinct regions along the anterior- posterior axis, as indicated by histology and gene expression, defined regions of the floor plate can be distinguished along the anterior- posterior axis, for example those underlying the midbrain and hindbrain1. In complement to the anatomical data, recent studies have revealed functional differences along the anterior-posterior axis. Perhaps the most surprising such difference is the recently discovered ability of midbrain floor plate cells to undergo neurogenesis3,4. Floor plate cells were long believed to be a non-neurogenic population, and the neurogenic potential of the midbrain floor plate remains a poorly understood phenomenon, but it is one that is of critical importance for the production of midbrain dopamine neurons.

In this issue of Nature Neuroscience, Joksimovic et al.5 use both transgenic mouse models and primary neuronal cultures to define the signals controlling floor plate neurogenesis. Previous studies have shown that Shh expression along the anterior- posterior neuraxis is regulated by specific enhancer elements that drive region- specific expression6. Therefore, because of the regionalization of Shh expression within the floor plate, the authors examined whether differences in Shh expression could be

Too much Sonic, too few neuronsChristopher A Fasano & Lorenz Studer

The floor plate can generate neurons, but does so only in the midbrain. New work shows that Shh suppresses floor plate neurogenesis, and that in the midbrain, Wnt downregulates Shh expression via canonical signaling through -catenin.

The authors are at the Sloan-Kettering Institute for

Cancer Research, Developmental Biology Program

and Department of Neurosurgery, 1275 York

Avenue, New York, New York 10065, USA.

e-mail: fasanoc@mskcc.org or studerl@mskcc.org

Hindbrain Midbrain

Floor plate induction

Floor plate maintenance Floor plate neurogenesis

Floor plate induction

Shh

Shh Shh

Wnt

Figure 1 Both hindbrain and midbrain floor plate are dependent on Shh for floor plate induction. Whereas Shh expression is maintained in the hindbrain floor plate, Wnt signaling suppresses Shh in the midbrain, causing a neurogenic response in the midbrain floor plate and the production of floor platederived dopamine neurons.

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cultures strikes a cautionary note about the prolonged use of Shh in midbrain dopamine neuron induction protocols. The ectopic induction of dopamine-like neuron cells in Wnt-treated primary hindbrain cultures further suggests that these data may have direct relevance for the engineering of midbrain dopamine neurons.

The genetic approaches used in the current study should be valuable for further refining our understanding of the interplay of Shh and Wnt signaling with the downstream transcriptional cascades defining midbrain dopamine neuron identity. One important hint could come from studies comparing the region- specific differences in floor plate competency. The current study by Joksimovic et al.5 demonstrates that despite decades of research, the floor plate continues to reveal new secrets important for basic neurobiology with direct relevance for neurodegeneration and stem cell engineering. These secrets may also turn out to provide useful tricks when devising stem cell therapies for Parkinson disease in the future.1. Placzek, M. & Briscoe, J. Nat. Rev. Neurosci. 6,

230240 (2005).2. Charron, F., Stein, E., Jeong, J., McMahon, A.P. &

Tessier-Lavigne, M. Cell 113, 1123 (2003).3. Kittappa, R., Chang, W.W., Awatramani, R.B. &

McKay, R.D. PLoS Biol. 5, e325 (2007).4. Ono, Y. et al. Development 134, 32133225 (2007).5. Joksimovic et al. Nat. Neurosci. 12, 125131 (2009).6. Epstein, D.J., McMahon, A.P. & Joyner, A.L. Development

126, 281292 (1999).7. Prakash, N. et al. Development 133, 8998 (2006).8. Falk, S. et al. Cell Stem Cell. 2, 472483 (2008).

along with increases in BrdU incorporation and Ngn2-positive cells in the hindbrain, spinal cord and floor plate. This indicates that canonical Wnt signaling is sufficient to induce floor plate neurogenesis (Fig. 1).

However, even though the progenitors were able to acquire some markers of dopaminergic neurons, they did not develop into proper midbrain dopamine neurons and appeared to be lost over time. To complement these in vivo data, the authors showed that when exogenous Wnt1 was added to hindbrain cultures, production of dopaminergic (tyrosine hydroxylase and Foxa2-positive) neurons was increased in a dose-dependent manner.

These data may therefore have important implications for studies aimed at directing midbrain dopamine neuron cell fates in vitro. The lineage-tracing data clearly demonstrate that midbrain dopamine neurons are derived from Foxa2-positive floor plate precursors in the midbrain. The intricate regulation of Wnt and Shh signals revealed in the current study is highly specific to the floor plate. Although Wnt signaling in the floor plate was here shown to be critical for suppression of Shh and induction of neurogenesis, Wnt signals in neural precursors may have a very different function controlling the proliferation of dorsal midbrain neural stem cell populations8. The in vitro finding that high doses of Shh reduce dopamine neuron yield in primary midbrain precursor

altered; however, the removal of -catenin resulted in persistent expression of Shh in the midbrain, which led to a decrease in neurogenesis. Examining embryos after several days showed reduced numbers of dopamine neurons. Thus, prolonging Shh signaling (by removing -catenin) results in decreased neurogenesis and defective midbrain development. Indeed, staining for markers showed a pattern similar to that in the hindbrain.

Importantly, performing the same manipulations in Nestin::cre mice, marking precursors at later stages did not affect Shh expression or floor plate neurogenesis in the midbrain. This suggests that there is a narrow temporal window from 9.5 to 11.5 days post conception for Wnts action on Shh and thus its influence on dopaminergic neurogenesis.

Lastly, the authors performed an interesting proof-of-principle experiment to test whether canonical Wnt signaling is sufficient to repress Shh and induce dopamine neuron markers. To this end, they used a mutant mouse in which -catenin is permanently stabilized along the entire neuraxis. This, of course, creates a highly artificial model, but it also allowed the authors to arrive at several conclusions with exciting implications. First, these floor platespecific -catenin gain-of-function mutants had a phenotype very similar to that of the Shh loss-of-function mutants, in that there was a decrease in Shh signaling

regulates the retinoblastoma protein (pRb), a key component of the cell cycle machinery.

PML was identified at a chromosomal translocation breakpoint responsible for promyelocytic leukemia, a subtype of acute myeloid leukemia4. The translocation, between chromosomes 15 and 17, resulted in the generation of an oncogenic fusion protein between PML and retinoic acid receptor- (RAR). Subsequent studies have revealed that PML is a tumor suppressor and that the PML-RAR exerts its oncogenic effects at least in part by blocking normal PML function5.

one hand, and the differentiation of daughter cells into neurons and glial cells on the other. Although many signaling pathways have been shown to regulate this process (for example, Notch, Wnt, FGF), relatively little is known about the role of cell cycle regulators1,2. In this issue, Regad et al. report a previously unsuspected function for the tumor suppressor promyelocytic leukemia protein (PML) in neocortical NPC biology3. In addition to characterizing the phenotypic effects of PML disruption on neocortical development, the authors show that PML

Construction of the neocortex requires a remarkably complex balancing act between the maintenance and proliferation of the neural progenitor cell (NPC) pool, on the

PML: a tumor suppressor essential for neocortical developmentKarisa C Schreck & Nicholas Gaiano

Brain development requires precise control of progenitor proliferation and differentiation. PML appears to be a crucial regulator of cortical progenitors, limiting proliferation and promoting the generation of committed neuronal precursors.

Karisa C. Schreck is at the Institute for

Cell Engineering and the Department of

Neuroscience, and Nicholas Gaiano is at the

Institute for Cell Engineering and the Departments

of Neurology, Neuroscience and Oncology,

Johns Hopkins University School of Medicine,

Baltimore, Maryland 21205, USA.

e-mail: kschreck@jhmi.edu or gaiano@jhmi.edu.

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expression) and the number of cells in M phase or S phase (marked by phosphorylated histone H3 and BrdU incorporation, respectively), the authors showed that PML mutants had more cell proliferation and less cell cycle exit in the neocortical germinal zone during development. At first glance, the increase in cell proliferation may seem to be inconsistent with the reduction in neocortical thickness. However, if increased progenitor proliferation resulted in decreased neurogenesis, a thinner neocortex could ultimately result.

Two distinct proliferative cell types have been described during neocortical development: radial glial cells (RGCs), which reside in the VZ and possess stem cell character, and intermediate progenitor cells (IPCs), which reside primarily in the SVZ and are neurogenic progenitors7. During neurogenesis, RGCs can give rise either to neurons directly, or to IPCs that generate neurons through subsequent rounds of cell division. To characterize the effect of PML disruption on RGCs versus IPCs, the authors used the molecular markers Pax6 and Tbr2 (expressed in RGCs and IPCs, respectively). Interestingly, they found an increase in the number of Pax6+ cells and a decrease in the number of Tbr2+ cells, suggesting an overabundance of RGCs and a paucity of IPCs (Fig. 1). Consistent with a reduction in the IPC pool, the authors observed a decrease in the number of M-phase (phosphorylated histone H3 positive) cells located in the SVZ. These data indicate that PML is needed for IPC generation, maintenance or both, and they suggest that disruption of PML results in increased RGC proliferation at the expense of IPC specification. This is particularly interesting because currently little is known about the molecular mechanisms regulating the generation of IPCs from RGCs. The observation that PML is required for that process provides a nice foothold to pursue this topic further.

The finding that loss of PML led to defects in IPC generation suggested that PML mutants were likely to have impaired neuronal differentiation. Such an impairment would be expected because IPC divisions provide an amplifying function that is thought to increase overall neuronal output from the germinal area7. Consistent with such impairment, and with the observed reduction in neocortical thickness, the number of neurons (MAP2-positive cells) present during development and after birth was significantly decreased. Interestingly, the number of cells expressing astrocytic (GFAP) and oligodendrocytic (MBP) markers was

regions contain different neural progenitor subtypes (as discussed later). Then, by comparing mouse embryos homozygous for a mutant version of the gene encoding PML (Pml/)5 to heterozygotes (Pml+/), Regad et al. observed that the brains of the Pml/ mutant mice were smaller, with a prominent reduction in neocortical wall thickness. The decrease in mutant brain size persisted into adulthood, indicating that no compensatory mechanisms could circumvent the need for PML during brain development.

A reduction in cortical wall thickness could be caused by either decreased cell division or increased apoptosis; therefore, the authors characterized cell proliferation and cell death in PML mutants. No increase in apoptosis was observed in mutants during mid- neurogenesis (embryonic day 15.5), consistent with previous findings that PML-null cells are resistant to apoptosis5. However, by examining the total number of proliferating cells (marked by Ki67

PML is thought to function primarily in the context of so-called PML nuclear bodies (PML-NBs), protein aggregates that are important in the DNA damage response, apoptosis and tumor suppression6. The mechanism of PML action remains an area of ongoing investigation, and PML has been found to interact with numerous proteins, including the tumor suppressors p53 and pRb and the pro- apoptotic factors c-jun and Daxx. Lagging behind our understanding of PMLs function in disease is our understanding of its function during normal development. The work of Regad et al. now makes a major contribution to that understanding, in particular with respect to the brain3.

The authors began their evaluation of PML in neocortical development by showing that during neurogenesis, PML is expressed primarily in the germinal area, which comprises two spatially distinct regions, the ventricular zone (VZ) and the subventricular zone (SVZ). Both

Figure 1 PML regulates neocortical progenitors through interactions with pRb. In the wild-type neocortex (upper left), radial glial cells (RGCs, blue) are in the ventricular zone (VZ), intermediate progenitor cells (IPCs, purple) are primarily in the subventricular zone (SVZ), and newly generated neurons (brown) are in the cortical plate (CP). PML mutants (upper right) have reduced neocortical thickness, with fewer neurons in the CP, and show both increased RGC proliferation and decreased IPC generation. The work by Regad et al.3 suggests that PML functions to promote the PP1-mediated dephosphorylation of pRb, which can then block the pro-proliferative effects of E2F transcription factors (lower left). In the absence of PML, pRb is phosphorylated and does not inhibit E2F (lower right).

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and that a limited number of IPCs are still generated in these impaired animals, suggests that some functional redundancy, on the molecular and/or cellular levels, exists during neocortical development. However, by showing that the PML plays an important role in IPC generation and pRb regulation during neocortical development, Regad et al.3 have identified a new avenue connecting the somewhat disparate fields of cell cycle control and neural progenitor regulation. In addition, this work is pertinent to the biology of brain tumors, in which the balance between cancer stem cells and more restricted proliferative cell types15 may be regulated by mechanisms similar to those controlling the balance between RGCs and IPCs during development. Determining how PML works in concert or in parallel with other signaling pathways will contribute to a comprehensive understanding of neural progenitor regulation and brain development, and it may add to our understanding of the causes and potential treatment of brain cancer.

1. McClellan, K.A. & Slack, R.S. Cell Cycle 5, 15061513 (2006).

2. Dehay, C. & Kennedy, H. Nat. Rev. Neurosci. 8, 438450 (2007).

3. Regad, T., Bellodi, C., Nicotera, P. & Salomoni, P. Nat. Neurosci. 12, 132140 (2009).

4. Bernardi, R., Papa, A. & Pandolfi, P.P. Oncogene 27, 62996312 (2008).

5. Wang, Z.G. et al. Science 279, 15471551 (1998).6. Dellaire, G. & Bazett-Jones, D.P. Bioessays 26,

963977 (2004).7. Pontious, A., Kowalczyk, T., Englund, C. & Hevner, R.F.

Dev. Neurosci. 30, 2432 (2008).8. Labbaye, C. et al. Oncogene 18, 35293540

(1999).9. Ferguson, K.L. & Slack, R.S. Neuroreport 12, A55A62

(2001).10. Ferguson, K.L. et al. EMBO J. 21, 33373346

(2002).11. MacPherson, D. et al. Mol. Cell. Biol. 23, 10441053

(2003).12. Mu, Z.M., Le, X.F., Vallian, S., Glassman, A.B. &

Chang, K.S. Carcinogenesis 18, 20632069 (1997).13. Arnold, S.J. et al. Genes Dev. 22, 24792484

(2008).14. Sessa, A., Mao, C.A., Hadjantonakis, A.K., Klein, W.H.

& Broccoli, V. Neuron 60, 5669 (2008).15. Stiles, C.D. & Rowitch, D.H. Neuron 58, 832846

(2008).

of pRb. Consistent with the idea that pRb and PP1 functionally interact with PML, immunoprecipitation studies showed that both physically interact with PML. All told, the work of Regad et al. suggests that PML regulates pRb in neocortical progenitors, through direct protein-protein interaction and in a PP1-dependent fashion (Fig. 1).

This work raises many interesting questions about the role of PML, also a tumor suppressor, in normal neural development. For example, is PML function temporally regulated and, if so, how? The balance between RGCs and IPCs in the neocortex is fundamental to proper development, and the temporal regulation of PML could promote a gradual shift from RGC to IPC identity. As the work by Regad et al.3 focused primarily on neurogenesis at embryonic day 15, future studies should consider how PML functions at other time points. Another interesting question raised by Regad et al. is, how does PML, and how do cell cycle regulators in general, interact with the many pathways and genes known to control neocortical progenitors? For example, several recent reports showed that disruption of Tbr2 greatly reduced the number of IPCs in the neocortex13,14, producing a phenotype similar to that observed with PML disruption. This similarity suggests that it would be worthwhile, and potentially very interesting, to determine whether overexpression of Tbr2 in PML mutants promotes IPC character. The mechanistic connection of cell cycle regulators to transcription factors such as Tbr2 is likely to create new avenues of pursuit for the field. It will be especially interesting to determine the extent to which cell cycle regulators influence neural development not only through the direct control of cell division, but also through novel interactions with other pertinent signaling cascades and regulatory molecules1.

That Pml/ mice survive to maturity without any gross neurological defects5,

also reduced, suggesting that the role of PML in neural progenitor proliferation and differentiation is not specific to neurogenesis but affects gliogenesis as well. The authors used an in vitro differentiation paradigm to corroborate their in vivo findings, and showed that reintroduction of PML into PML mutant cells in vitro reversed the decrease in neuronal and glial cell differentiation.

To probe PML function in neocortical progenitors on a molecular level, the authors examined interactions between PML and pRb, as these proteins are known to interact in other settings8 and pRb has been shown to play an essential role during neocortical development911. pRb is expressed at high levels in the developing neocortex where it is involved in cell cycle regulation, differentiation, apoptosis and even migration9. The primary mechanism by which pRb regulates cell cycle progression is inhibition of EF2 transcription factors. When phosphorylated, pRb is unable to bind E2F proteins, allowing them to promote the transition from G1 to S phase1.

Investigation of the putative interactions between PML and pRb in neocortical progenitors led Regad et al. to examine pRb expression and subcellular localization in PML mutants3. As part of this analysis, they also considered the expression of protein phosphatase 1 (PP1), which dephosphorylates pRb, thereby permitting it to inhibit E2F. Normally, pRb and PP1 are expressed in nuclear granules whose expression partially overlaps with those of each other and PML-NBs; however, in Pml/ neocortical progenitors, both pRb and PP1 were dispersed throughout the nucleoplasm and cytoplasm. In addition, pRb was hyperphosphorylated in PML mutants, consistent with previous work showing that PML overexpression led to pRb hypophosphorylation12. Re-introduction of PML into Pml/ neocortical progenitors rescued the subcellular localization of both PP1 and pRb and the phosphorylation state

Sleep on it

A period of sleep is known to benefit performance in memory tasks, but a study on page 122 of this issue suggests that it is not just the amount, but the kind, of sleep that is important.

This study recorded electroencephalograms from people as they slept and set off a beeping sound when the electroencephalograms were consistent with a sleep stage known as slow-wave sleep. Slow-wave sleep is a state of deeper sleep, so although the beep did not awaken the subjects, they slid out of slow-wave sleep into a different, shallower sleep stage.

Although the total amount of sleep that subjects got was unchanged, these people did worse on a later test of scene recall than subjects who had slept normally. Moreover, when the subjects were later scanned in a functional magnetic resonance imaging scanner, they also showed reduced hippocampal activation while they were encoding the to-be-remembered scenes. These results suggest that hippocampus-dependent memory is particularly affected by shallow sleep. Charvy Narain

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So what is the molecular nature of the chloride conductance on glutamatergic vesicles? The cloning of VGLUT1 (ref. 7,8) and its expression in heterologous systems suggested that VGLUTs exhibit an intrinsic chloride permeability7. In addition, a family member of the ClC chloride channels, ClC-3, appeared to be an attractive candidate10. Schenck et al.4 tackled this question at the outset of their study by isolating synaptic vesicles from mice that lack either the predominant VGLUT, VGLUT1, or ClC-3. Exposure of vesicles isolated from wild-type animals to ATP and chloride led to a marked acidification of the lumen, indicating that chloride can enter to dissipate the electrical gradient generated by the ATPase. In vesicles isolated from Clc-3 (also known as Clcn3) null mutant mice, the acidification was unperturbed, indicating that the important chloride conductance is still present and thus cannot be ClC-3. On the other hand, vesicles from Vglut1 (also known as Slc17a7) null mutant mice showed an 80% reduction in acidification, suggesting the absence of a major vesicular chloride conductance. The authors further used an immunological approach with excellently calibrated antibodies to estimate that ClC-3 is found only in a very small subfraction of vesicles (

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that pH gives an additional boost to glutamate uptake, especially at moderate extravesicular chloride concentration. In the presence of a high vesicular chloride concentration, VGLUT1 thus appears to act as a glutamate-chloride antiporter. In the antiporter mode, VGLUT1 can fill vesicles without collapsing the electrical gradient and without increasing the osmotic pressure, two factors that would slow vesicle filling. The filling rate of a newly endocytosed vesicle, full of chloride taken up from the extracellular solution, is therefore particularly fast, and as the vesicular chloride is exchanged for glutamate, glutamate uptake will slow and eventually stop. The level of extracellular chloride might thus represent the long-sought check that limits vesicle filling and contributes to a constant quantal size2.

The work obviously leaves many questions unanswered. Do the other VGLUT family members share this chloride conductance? What is the mode of permeation for chloride and glutamate? What is the stoichiometry of transport for glutamate, chloride, protons and other potential ions? And most excitingly, can external chloride concentrations really govern the refilling rate of vesicles and could this have happened under physiological conditions? If so, then vesicles endocytosed under conditions of low extracellular chloride would fill more slowly, contain less glutamate and thus show a reduced quantal amplitude (Fig. 1d). In neuronal tissue, GABA currents at neighboring inhibitory synapses or tonically active extrasynaptic GABA receptors might affect a local decrease in extracellular chloride. Such regulation of presynaptic quantal size by extracellular chloride concentration, potentially regulated through postsynaptic chloride channels, would represent a previously unknown form of short-term synaptic plasticity. Clearly, the detailed investigation of vesicular glutamate transporters will remain exciting for a long time!

1. Katz, B. The Release of Neural Transmitter Substances (Liverpool University Press, Liverpool, 1969).

2. Edwards, R.H. Neuron 55, 835858 (2007).3. Takamori, S. et al. Cell 127, 831846 (2006).4. Schenck, S., Wojcik, S.M., Brose, N. & Takamori, S.

Nat. Neurosci. 12, 156162 (2009).5. Maycox, P.R., Deckwerth, T., Hell, J.W. & Jahn, R.

J. Biol. Chem. 263, 1542315428 (1988).6. Naito, S. & Ueda, T. J. Neurochem. 44, 99109

(1985).7. Bellocchio, E.E., Reimer, R.J., Fremeau, R.T., Jr &

Edwards, R.H. Science 289, 957960 (2000).8. Takamori, S., Rhee, J.S., Rosenmund, C. & Jahn, R.

Nature 407, 189194 (2000).9. Hartinger, J. & Jahn, R. J. Biol. Chem. 268, 2312223127

(1993).10. Stobrawa, S.M. et al. Neuron 29, 185196

(2001).

show that a vesicle filled with chloride takes up more glutamate than a vesicle devoid of chloride (Fig. 1). In the presence of intravesicular chloride, glutamate uptake was mainly governed by . However, using the pH gradient collapsing drug nigericin (an ionophore allowing passage of H+ and K+ ions), the authors were able to demonstrate

pump showed the biphasic dependence on extravesicular chloride characteristic of native synaptic vesicles. The authors also showed that, although extravesicular chloride is important, it is actually the intravesicular chloride concentration that determines both the speed and the extent of glutamate loading. Their results clearly

Figure 1 Intravesicular chloride determines the amount and speed of glutamate uptake by synaptic vesicles. (a) In the absence of chloride ions on either side of the vesicle, the proton pump establishes an electrical potential and VGLUT transports glutamate against an osmotic gradient, possibly in exchange for protons. (b) A moderate amount of extravesicular chloride triggers influx of chloride through VGLUT into the vesicle, thus collapsing much of the electrical gradient. Weak glutamate transport depends mainly on the pH gradient. (c) In the presence of high intravesicular chloride concentrations, VGLUT allows for the exit of chloride and the entry of glutamate into the vesicle. The electrical gradient established by the proton pump serves as the driving force. This allows for rapid and efficient filling of the vesicle indicated by the high reading on the filling gauge. (d) Local changes in extracellular chloride concentrations (indicated by saturation of blue color) might be caused by activation of GABA receptors or through Cl exchangers. Vesicles endocytosed in the presence of low chloride concentrations will fill slower and/or with less glutamate (light red) than vesicles endocytosed in high chloride (dark red; red boxes indicate glutamate receptors). Glu, glutamate.

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Neuronal release of proBDNFJianmin Yang1, Chia-Jen Siao1, Guhan Nagappan5, Tina Marinic1,Deqiang Jing2, Kelly McGrath1, Zhe-Yu Chen2, Willie Mark3,Lino Tessarollo4, Francis S Lee2, Bai Lu5 & Barbara L Hempstead1

Probrain-derived neurotrophic factor (proBDNF) and matureBDNF utilize distinct receptors to mediate divergent neuronalactions. Using new tools to quantitate endogenous BDNFisoforms, we found that mouse neurons secrete bothproBDNF and mature BDNF. The highest levels of proBDNFand p75 were observed perinatally and declined, but werestill detectable, in adulthood. Thus, BDNF actions aredevelopmentally regulated by secretion of proBDNF ormature BDNF and by local expression of p75 and TrkB.

BDNF critically regulates neuronal function by promoting survival,enhancing synaptic plasticity and altering spine morphology viaTrkB1,2. However, BDNF is initially synthesized as a precursor,

proBDNF, which is trafficked to the regulated secretory pathway bychaperone proteins, including sortilin3. Although proBDNF may becleaved intracellulary to release mature BDNF in an activity-depen-dent manner4,5, it is not clear how efficient this processing is andhow much proBDNF is secreted by neurons. Because recombinantproBDNF does not activate TrkB, but rather activates p75 topromote cell death and attenuate synaptic transmission6,7, this isan important question.

To reliably measure the very low levels of endogenous proBDNF, wegenerated a monoclonal antibody (mAb287) that was specific for theprodomain. This antibody detected a 32-kDa immunoreactive speciesin hippocampal lysates from Bdnf+/+, but not Bdnf /, littermates(Supplementary Fig. 1 and Supplementary Methods online), thatcorresponded in molecular mass to recombinant proBDNF; however,we did not observe a cleaved prodomain (of 1416 kDa; Supplemen-tary Fig. 1). BDNF prodomain immunoreactivity was present incultured neurons from Bdnf+/+, but not Bdnf /, mice hippocampalgranule neurons of the dentate gyrus and most prominently in theirmossy fiber projections from tissue sections of Bdnf+/+, but notBdnf /, mice (cultured neurons from Bdnf+/+, but not Bdnf /, mice

37 kDa

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Figure 1 Detection of proBDNF and mature BDNF in mouse hippocampus and secretion of proBDNF and mature BDNF by cultured hippocampal neurons.(a) Hippocampal lysates of 4-week-old mice of the indicated genotype were immunoprecipitated with antibody to HA and immunoblotted as indicated.

mAb287 is the monoclonal antibody to the BDNF prodomain that we characterized in Supplementary Figure 1. (b,c) Neurons were cultured from P0 mice of

the indicated genotype for 7 d in the presence (b) or absence (c) of alpha 2 anti-plasmin. Media (M) and cell lysates (L) were immmunoprecipitated (IP) with

antibody to HA. proBDNF and mature BDNF were detected with antibody to HA.11. (d,e) Neuronal cultures were established as in b. We added TrkB-Fc (d) or

TrkB-Fc or TrkA-Fc (e) to the media 3 d before harvest. Neurons cultured in the absence of TrkB-Fc were analyzed as controls. Lysates were immunoprecipi-

tated; Fc proteins were captured by Protein A-Sepharose (capture). (f) Embryonic day 16.5 (E16.5) neurons were cultured as in b and stimulated with 56 mM

KCl for 90 min. The media and cell lysates were processed as indicated. (g) E16.5 hippocampal neurons were cultured as described previously9, with or

without fluorouracil for 2 d. At 7 d in vitro, cells were stimulated with 56 mM KCl for 90 min, and immunoprecipitation/western blot analysis was carried

out as indicated. All of our animal studies were approved by the Institutional Animal Care and Use Committee of Weill Cornell Medical College.

Received 24 October 2008; accepted 24 November 2008; published online 11 January 2009; doi:10.1038/nn.2244

1Departments of Medicine and 2Psychiatry, Weill Cornell Medical College, 1300 York Ave., New York, New York 10065, USA. 3Developmental Biology, Memorial SloanKettering Cancer Center, 1275 York Ave., New York, New York 10065, USA. 4National Cancer Institute Frederick, Building 560, Frederick, Maryland 21702, USA. 5NationalInstitute of Child Health and Human Development, MSC 3714, Bethesda, Maryland 20892-4480, USA. Correspondence should be addressed to B.L.H.(blhempst@med.cornell.edu).

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(Supplementary Fig. 1). We also observed prodomain immuno-reactivity in pyramidal cell bodies of CA2 and CA3 and the stratumlucidum, consistent with prior immunolocalization of BDNF in rats8.These results indicated that intact proBDNF was regionally expressedat substantial levels in the adult brain, particularly in hippocampalmossy fibers.

To facilitate quantitative detection of proBDNF and mature BDNF,we generated a knock-in mouse in which the endogenous Bdnf codingexon was replaced with the murine Bdnf sequence with a C-terminalhemagglutinin (HA) epitope tag (Bdnf-HA; Supplementary Fig. 2online). Both homozygous (Bdnf-HA/Bdnf-HA) and heterozygous(Bdnf-HA/+) mice were viable, fertile and indistinguishable fromwild-type littermates. Regional expression of BDNF by ELISA docu-mented comparable levels of BDNF in cortex and hippocampus inBdnf-HA/+ mice and wild-type littermates (Supplementary Fig. 2). Inaddition, the levels of proBDNF in the hippocampus of wild-type andBdnf-HA/Bdnf-HA mice were comparable by western blot (Supple-mentary Fig. 1).

To quantitate proBDNF and mature BDNF,we immunoprecipitated hippocampal lysatesfrom wild-type, Bdnf-HA/+ and Bdnf-HA/Bdnf-HA with antibodies to HA and probedfor HA immunoreactivity. Mature BDNF-HA (B14.2 kDa B13.5-kDa matureBDNF + B0.7-kDa HA) and proBDNF-HA (B32.7 kDa B32-kDa proBDNF +B0.7-kDa HA) were readily detectable fromBdnf-HA/+ and Bdnf-HA/Bdnf-HA, but notwild-type hippocampal lysates (Fig. 1a); theB32.7-kDa proBDNF band was confirmed byprobing with antisera to proBDNF (Fig. 1a)and the B14.2-kDa mature BDNF wasalso detected with antibody to matureBDNF (Fig. 1a). Our analysis confirmedthat BDNF-HA was quantitatively precipi-tated, with BDNF-HA isoforms in Bdnf-HA/+ being detected at approximately 50% of thelevel of BDNF-HA isoforms in Bdnf-HA/Bdnf-HA mice (Fig. 1a).

To determine whether proBDNF wasindeed secreted, we cultured hippocampalneurons from Bdnf-HA/+ or wild-type micein conditions to reduce glia contamination,using alpha 2 anti-plasmin to prevent cleavageof secreted proBDNF. Following neuronalmaturation in vitro, BDNF isoforms werecollected from media and cell lysates usingimmunoprecipitation/western blot analysiswith antibodies to HA. Both proBDNF an