A GABAergic projection from the zona incerta to cortex promotes … · incerta to cortex promotes...

functional modules that gate different streams ofsomatosensory information.In identifying a previously uncharacterized gate

for low-threshold mechanical itch, this study high-lights a largely overlooked driver of chronic itch—namely, the light-touch pathway that is insensitiveto antihistamine or anti-GRPR drugs (8). Humanpatients with chronic itch (trichoknesis) (25) dis-play a phenotype similar to that seen in the NPY::Cre IN–ablated mice. In both instances, itch sen-sitivity is restricted to the hairy skin. Our findingthat the NPY::Cre-derived INs are innervated bytactile inputs from hairy-skin LTMs (Fig. 4) sug-gests that the NPY::Cre INs are key componentsof a spinal inhibitory circuit by which hairy-skin LTMs gate mechanical itch. We proposethat the NPY::Cre INs function as rheostat forlow-threshold tactile stimuli, suppressing itchand preventing excessive scratching.

REFERENCES AND NOTES

1. T. Akiyama, E. Carstens, Neuroscience 250, 697–714(2013).

2. D. M. Bautista, S. R. Wilson, M. A. Hoon, Nat. Neurosci. 17,175–182 (2014).

3. J. Braz, C. Solorzano, X. Wang, A. I. Basbaum, Neuron 82,522–536 (2014).

4. S. Davidson, G. J. Giesler, Trends Neurosci. 33, 550–558(2010).

5. L. Han, X. Dong, Annu. Rev. Biophys. 43, 331–355 (2014).6. R. H. LaMotte, X. Dong, M. Ringkamp, Nat. Rev. Neurosci. 15,

19–31 (2014).7. J. Jeffry, S. Kim, Z. F. Chen, Physiology 26, 286–292 (2011).8. M. Fukuoka, Y. Miyachi, A. Ikoma, Pain 154, 897–904

(2013).9. C. F. Wahlgren, O. Hägermark, R. Bergström, Acta Derm.

Venereol. 71, 488–494 (1991).10. M. Hosogi, M. Schmelz, Y. Miyachi, A. Ikoma, Pain 126, 16–23

(2006).11. S. E. Ross et al., Neuron 65, 886–898 (2010).12. E. Foster et al., Neuron 85, 1289–1304 (2015).13. D. Bröhl et al., Dev. Biol. 322, 381–393 (2008).14. M. Huang et al., Dev. Biol. 322, 394–405 (2008).15. T. C. Sardella et al., Mol. Pain 7, 76 (2011).16. S. Y. Tiong, E. Polgár, J. C. van Kralingen, M. Watanabe,

A. J. Todd, Mol. Pain 7, 36 (2011).17. A. P. Kardon et al., Neuron 82, 573–586 (2014).18. B. Duan et al., Cell 159, 1417–1432 (2014).19. T. Akiyama et al., J. Invest. Dermatol. 132, 1886–1891

(2012).20. S. G. Shimada, R. H. LaMotte, Pain 139, 681–687 (2008).21. B. N. Armbruster, X. Li, M. H. Pausch, S. Herlitze, B. L. Roth,

Proc. Natl. Acad. Sci. U.S.A. 104, 5163–5168 (2007).22. Y. G. Sun, Z. F. Chen, Nature 448, 700–703 (2007).23. Y. G. Sun et al., Science 325, 1531–1534 (2009).24. L. Li et al., Cell 147, 1615–1627 (2011).25. A. Reich, K. Mędrek, Z. Adamski, J. C. Szepietowski, Acta Derm.

Venereol. 93, 591 (2013).26. S. K. Mishra, M. A. Hoon, Science 340, 968–971 (2013).

ACKNOWLEDGMENTS

This study was supported by grants from the NIH (NS086372to M.G. and Q.M., NS080586 and NS072031 to M.G., andNS072040 to Q.M.) and by the Caterina Foundation (S.B. andS.C.K.). We thank C. Padilla and T. Velasquez for help in generatingthe R26GAGds-hM4D-tdTomato mice. All data are contained in themanuscript and the supplementary materials. A.G. and E.K. weresupported by the Gatsby Charitable Foundation and the NIH(grant R01 NS 067216).

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/350/6260/550/suppl/DC1Material and MethodsFigs. S1 to S8References (27–39)

25 June 2015; accepted 21 September 201510.1126/science.aac8653

BRAIN DEVELOPMENT

A GABAergic projection from the zonaincerta to cortex promotes corticalneuron developmentJiadong Chen* and Arnold R. Kriegstein*

g-Aminobutyric acid (GABA) is the major inhibitory transmitter in the mature brain but isexcitatory in the developing cortex. We found that mouse zona incerta (ZI) projectionneurons form a GABAergic axon plexus in neonatal cortical layer 1, making synapses withneurons in both deep and superficial layers. A similar depolarizing GABAergic plexus existsin the developing human cortex. Selectively silencing mouse ZI GABAergic neurons at birthdecreased synaptic activity and apical dendritic complexity of cortical neurons. The ZIGABAergic projection becomes inhibitory with maturation and can block epileptiformactivity in the adult brain.These data reveal an early-developing GABAergic projection fromthe ZI to cortical layer 1 that is essential for proper development of cortical neurons andbalances excitation with inhibition in the adult cortex.

During embryonic development, neural ac-tivity (1, 2) influences proliferation, migra-tion, and differentiation, as well as circuitrefinement (3–5). In immature brains, theneurotransmitter GABA has excitatory ef-

fects due to high intracellular chloride (6, 7), con-trary to its inhibitory effects in adult brains. GABAin the immature neocortex comes from local in-terneurons and axonal projections from otherbrain regions (8–12). The neonatal rodent brainhas an excitatory GABAergic plexus projectingwidely within cortical layer 1 (13, 14). Here, weshow that the zona incerta (ZI) generates theneurons of this GABAergic plexus.Wemapped the ZI pathway in transgenicmice

by manipulating channelrhodopsin-2 (ChR2) ex-pression in somatostatin (SST)–expressing neu-rons in the ZI of neonatal mice (postnatal dayP0–P1) (15, 16). Labeling was restricted to ZIGABAergic (Fig. 1A), SST+ (Fig. 1C) neurons 1weekafter virus injection, and we observed EYFP+ ZIaxonal projections widely distributed in layer 1 ofsomatosensory and motor cortex (Fig. 1, A and B,and fig. S1). At P7, ChR2 was reliably expressed inZI neurons, and blue light stimulation inducedfiring of EYFP+ ZI neurons (Fig. 1, D to F). Wefilled layer 5 cortical neurons with neurobiotin inacute slices of somatosensory and motor cortexand coimmunostained with the cortical layer 5marker Ctip2 (Fig. 1I, 16/16 neurons). The apicaldendrites of these pyramidal neurons contactedlayer 1 EYFP+ axons (Fig. 1G) and colocalized withthe GABAergic presynaptic marker vGat (vesicu-lar GABA transporter) (Fig. 1H).Blue light stimulation of layer 1 evoked synaptic

responses in layer 4 and layer 5 pyramidal neu-rons. The light-evoked responseswerenot sensitiveto a-amino-3-hydroxy-5-methyl-4-isoxazole propi-

onate (AMPA) or N-methyl-D-aspartate (NMDA)receptor antagonists [6-cyano-2, 3-dihydroxy-7-nitro-quinoxaline (CNQX) and D-2-amino-5-phosphonovalerate (D-APV), respectively] butwereabolished by the GABAA receptor antagonist bicu-culline (BMI) (Fig. 1, J to L) and reversibly blockedby tetrodotoxin (TTX, n = 4; Fig. 1K). Stimulationof layer 1 axons also inducedGABAergic responsesfrom layer 2/3 neurons, supporting observationsthat the layer 1 GABAergic plexus connects withpyramidal neurons from multiple layers in neo-natal mice (13).Morphologically, the human brain has abun-

dant GABAergic synapses in cortical layer 1 asearly as gestational week (GW) 12 (17, 18). In thesecond trimester, subplate neurons show sponta-neous firing and synaptic activity (19, 20). Exam-ining the expression anddistribution ofGABAergicaxons in human cortex at GW 24, we found thatboth the axon marker neurofilament-2H3 andthe GABAergic presynaptic marker vGat wereexpressed in cortical layer 1 (Fig. 2, A to C). Inacute brain slices from GW 22, pyramidal neu-rons in deep cortical layers had high membraneresistance (1.1 ± 0.1 gigaohms, n = 15), low mem-brane capacitance (23.0 ± 2.9 pF, n = 15), andfired only one or two action potentials (Fig. 2, Dand E) (19). Cortical neurons expressed GABAAreceptors (fig. S2) and displayed typical GABAergicminiature synaptic currents (Fig. 2F). Thus, hu-man cortical neurons express functional GABAAreceptors and GABAergic synapses in the late sec-ond trimester.We characterized the morphology and loca-

tion of recorded cortical neurons by filling cellswith neurobiotin and postimmunostaining withstreptavidin-549 and the deep cortical layermarker Ctip2. Ctip2 marker expression (Fig. 2Dand fig. S3) identified layer 5 pyramidal neu-rons with apical dendrites extending to corticallayer 1 (Fig. 2D). We recorded robust evoked syn-aptic responses onlywhen stimulating layer 1 (Fig.2G), but not deep cortical layers (–0.1 ± 0.6 pA,

554 30 OCTOBER 2015 • VOL 350 ISSUE 6260 sciencemag.org SCIENCE

Department of Neurology, Eli and Edythe Broad Center ofRegeneration Medicine and Stem Cell Research, University ofCalifornia, San Francisco, CA 94143, USA.*Corresponding author. E-mail: [email protected] (J.C.);[email protected] (A.R.K.)

RESEARCH | REPORTSon June 4, 2021

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n = 10) or the subplate (–1.6 ± 0.5 pA, n = 10).Evoked responses were insensitive to the AMPAreceptor antagonist CNQX but were blocked byBMI (Fig. 2, G and H). The evoked synapticcurrents reversed at –34.7 mV, close to the Cl–

equilibrium potential [–31.1 mV = –46.0 mV (byNernst equation) + 14.9mV (junction potential)],consistent with mediation through GABAA re-ceptors (Fig. 2, I and J).Neurons in human layer 1 cortex hadGABAergic

responses by GW 20 that were not evident atGW 16 or 18. By GW22, themajority of deep-layerneurons demonstrated GABAergic synaptic re-sponses. We did not detect evoked responses inGW 24 layer 2/3 neurons, which are still very im-mature (fig. S4). To evaluate the potential contri-bution of layer 1 local neurons to the GABAergicresponse of pyramidal neurons, we applied gluta-mate locally in layer 1. Although this induced ro-bust firing of layer 1 neurons, it did not evokesynaptic responses in layer 4 and layer 5 pyramidalneurons (fig. S5). Thus, a GABAergic axon plexusarising from long-projection neurons is present inlayer 1 in second-trimester human cortex.GABA responses are depolarizing in themouse

brain during the first postnatalweek (fig. S6) as aresult of high intracellular chloride ion concen-tration [Cl–] (6, 7). To study GABA responses in

GW22 cortical neurons, we performed gramicidin-perforated patch recordings to avoid perturbingthe intracellular [Cl–]. GABA responses reversedat –44.4 mV (fig. S7) and were depolarizing be-cause the resting membrane potential of corticalneurons at this stage was –64.8 ± 1.6mV (n = 14).We then monitored intracellular Ca2+ in responseto GABA by loading neurons with a membrane-permeable Ca2+ indicator, Oregon Green BAPTA-1AM. Local GABA application induced robust in-tracellular Ca2+ elevation in layer 5 cortical neu-rons (fig. S7). Thus, as in mouse (21–23), GABA isdepolarizing in human fetal cortex when func-tional GABAergic synapses are first established.To explore the effect of the layer 1 GABAergic

axon plexus on cortical development, we selec-tively blocked synaptic GABA release frommouseZI neurons by selective expression of tetanus toxinlight chain (TeLC), which blocks synaptic vesiclerelease (24). At P0–P1, we injected AAV viruscontaining Cre-dependent TeLC fused with 2A–green fluorescent protein under human elongationfactor 1a (EF1a) promoter (AAV1-EF1a-DIO-TeLC-2A-GFP) stereotaxically into the ZI of SST::Cre;Ai14mice.We detected GFP expression in the ZI within1 week (fig. S8). To test the efficiency of TeLCblockade, we first co-injected AAV1-DIO-ChR2with AAV1-DIO-TeLC-2A-GFP unilaterally into the

ZI. Blue light stimulation of cortical layer 1 failedto induce synaptic currents in layer 5 pyramidalneurons from acute brain slices obtained oneweek post viral injection (0.12 ± 0.20 pA, n = 16;Fig. 3, A and B). To evaluate the extent to whichlayer 1 GABAergic synaptic release is impaired byTeLC expression in the ZI, we recordedGABAergicresponses in layer 5 pyramidal neurons evoked byelectrical stimulation of layer 1. We found that theamplitude of evoked GABAergic responses wasreduced by 79.5% in neurons ipsilateral to theTeLC injection site (34.1 ± 1.8 pA,n= 18) relative tothe contralateral hemisphere (166.0±6.4 pA,n= 13,P

dendritic brancheswere unaffected (Fig. 3, E toH).We also observed reduced spine numbers in apicaldendrites after TeLC treatment (Fig. 3, I and J).Thus, layer 1 GABAergic synaptic activity is crucial

for normal development of synapses anddendritesin pyramidal neurons.GABA responses become hyperpolarizing after

the first postnatal week in mice (fig. S6), due to a

lower intracellular [Cl–], and thus become inhib-itory (7, 25–27). To explorewhether a functional ZIGABAergic pathway persists in thematuremousebrain, we used selective labeling of ChR2 layer 1 ZI


Fig. 2. Synaptic responses in developing human cor-tical neurons from layer 1 GABAergic axons. (A to C)Neurofilament-2H3 (NF-2H3) (A) and vGat (B) are co-expressed (C) in human fetal cortical layer 1. (D) Corticalneuron filled intracellularly with neurobiotin. Ctip2, cor-tical layer 5 marker; DAPI, nuclear marker. (E) Actionpotential firing of a layer 5 neuron under a stepped-current protocol ranging from –30 pA to 50 pA. (F) Mini-ature postsynaptic potentials of cortical neurons aresensitive to BMI. (G) Synaptic responses in layer 5 neu-rons evoked from cortical layer 1 were insensitive toCNQX but were reversibly blocked by BMI. (H) Sum-marized results showing average amplitude of evokedPSC responses in the presence or absence of BMI. (I and J)Sample traces (I) and summarized result (J) of evokedsynaptic responses at holding potentials from –65 mV(bottom) to 35 mV (top), with 20 mV interval. Scale bars,50 mm [(A) to (D)]; 25 mV, 200 ms (E); 40 pA, 2 s, 0.2 s(dashed line) (F); 100 pA, 10 ms [(G) and (I)].

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Fig. 3. Reduced synaptic activity and api-cal dendritic complexity after blockingZI GABAergic transmission in neonatalmice. (A) Schematic showing layer 5 neuronsrecorded with light or electrical stimulation oflayer 1. Red cross indicates blocked ZI activity.TH, thalamus. (B) Light stimulation of layer1 fails to induce synaptic responses in P7SST::Cre mice where TeLC and ChR2 werecoexpressed in the ZI (TeLC+ChR2) atP0 (compare to Fig. 1K). (C) Reduced amplitude of layer 1 electrical stimulation–induced GABAergic responses (at 0 mV) in SST::Cre mice expressing TeLC inthe ZI. (D) Reduced frequency of spontaneous GABAergic (at 0 mV) and glutamatergic (at –50 mV) responses in layer 5 neurons (P7–P9). (E and F)Morphology of P7 layer 5 cortical neurons in control-injected (E) or TeLC-injected (F) hemisphere. (G and H) Linear Sholl plots of apical (G) or basal (H)dendritic branches, showing best-fit polynomials (dashed lines) and SEM (shaded regions). Statistical results showmaximum number of intersections. (I and J)Confocal microscopy images (I) and statistical results (J) show decreased spines in apical dendrites (***P < 0.001). Scale bars, 100 pA, 50 ms (B); 20 pA, 50 ms(C); 20 pA, 2s (D); 100 mm (F); 5 mm (I). In (G), (H), and (J), error bars denote SEM.



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axons in P21 mice (Fig. 4A) and found that bluelight stimulation induced robust GABAergicsynaptic currents in pyramidal neurons (Fig. 4, Band C). To evaluate the role of ZI GABAergicprojections in cortical circuit function, we useda slice model in which cortical excitation wasenhanced byMg2+-free artificial cerebrospinal fluid(28). We then used cell-attached recordings ofcortical layer 4 neurons to preserve intracellularCl– integrity. Electrical stimulation of adjacentregions within the same cortical layer induced ep-ileptiform activity in recorded neurons (fig. S10).Coactivation of ZI axons by blue light stimulationof layer 1 ChR2-expressing axons reversibly sup-pressed the induced epileptiform activity (Fig. 4,D and E) but had no effect in slices from the con-tralateral hemisphere lackingChR2 expression (Fig.4F and fig. S10). Thus, ZIGABAergic projections areinhibitory in the mature mouse brain.The circuit identified here is one of the earliest

to appear in cortical development. While the im-portance of cortical interneurons to circuit func-tion has beenwell documented (29, 30), our studyshows that the ZI circuit originating in the di-encephalon supports synaptogenesis, apical butnot basal dendritic branching (Fig. 3, E to H),and spine development (Fig. 3, I and J) in corticalneurons. In the absence of ZI activity, we founddecreased inhibitory postsynaptic current (IPSC)

frequency in upper- and lower-layer neurons; wefound decreased excitatory postsynaptic current(EPSC) frequency only in layer 5 neurons (Fig. 3,E toH, and fig. S11), possibly because they receivedifferent presynaptic inputs that could target dis-tinct dendritic domains (31). GABA signaling ele-vates [Ca2+] (32), which could be restricted to theapical dendritic tuft (33) andmaymodulate apicaldendritic development. Neurotrophic factors suchas reelin and semaphorin 3A modulate apicaldendritic branching and spinedensity in an activity-dependent manner (34, 35); thus, it will be inter-esting to examine whether the developmentaleffects of ZI activity aremediated by neurotrophicfactors (4, 36, 37). Relatively modest structural orfunctional changes in cortical neurons can havesubstantial behavioral impacts. The defects indendritic arborization, spine density, and syn-aptic activity of cortical neurons observed uponblocking ZI activity resemble those implicated in avariety of neurodevelopmental diseases (9, 38–41);thus, the ZI pathway may play a role in diseaseetiology (42).

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16, 6414–6423 (1996).8. M. Demarque et al., Neuron 36, 1051–1061 (2002).9. A. Saunders et al., Nature 521, 85–89 (2015).10. A. Caputi, S. Melzer, M. Michael, H. Monyer, Curr. Opin.

Neurobiol. 23, 179–186 (2013).11. S. Melzer et al., Science 335, 1506–1510 (2012).12. M. A. Picardo et al., Neuron 71, 695–709 (2011).13. R. S. Dammerman, A. C. Flint, S. Noctor, A. R. Kriegstein,

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521–532 (2001).26. H. J. Luhmann, D. A. Prince, J. Neurophysiol. 65, 247–263

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Fig. 4. ZI GABAergic connections remain functionalin mature mouse brain and can suppress epilepti-form activity. (A) Layer 1 ChR2-EYFP axons overlapwith distal dendrites of layer 5 pyramidal neurons in P21SST::Cre mice. (B and C) Representative (B) and sta-tistical (C) results show that layer 1 light stimulation–induced synaptic currents in layer 5 neurons (at +10mV)are GABAergic (***P < 0.001). (D) Epileptiform activity isreversibly blocked by light stimulation of layer 1 ChR2-EYFP+ axons from ZI (50 ms, 10 Hz, 20 pulses, 30 sinterval). (E and F) Both spike frequency and numberswere reversibly reduced upon light stimulation (*P <0.05) (E), but not in controls lacking ChR2 expression(F). Scale bars, 50 mm (A); 50 pA, 100 ms (B); 40 mV,0.2 s (D). In (C), (E), and (F), error bars denote SEM.

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41. J. L. Rubenstein, M. M. Merzenich, Genes Brain Behav. 2,255–267 (2003).

42. J. Mitrofanis, Neuroscience 130, 1–15 (2005).

ACKNOWLEDGMENTS

We thank J. Huguenard, J. Paz, S. H. Wang, W. Walantus, and Y. Y. Wangfor technical assistance; C. R. Yu for TeLC plasmid; G. P. Feng andY. Zhou for TeLC-2A-GFP viral vectors; K. Deisseroth for ChR2vectors; A. Alvarez-Buylla and C. Arnold for the stereotaxic injectionrig; Kriegstein laboratory members for discussions; and W. P. Ge,S. Mayer, C. Gertz, and T. Nowakowski for critical reading of the

manuscript. Supported by National Institute of Neurological Disordersand Stroke grant R37 NS35710 (A.R.K.) and a CARE & CURE PediatricEpilepsy Fellowship from the Epilepsy Foundation of Greater LosAngeles (J.C.). The supplement contains additional data.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/350/6260/554/suppl/DC1Materials and MethodsSupplementary TextFigs. S1 to S11References (43–47)

25 May 2015; accepted 10 September 2015Published online 1 October 201510.1126/science.aac6472

MICROBIOME

Disease tolerance mediated bymicrobiome E. coli involvesinflammasome and IGF-1 signalingAlexandria M. Palaferri Schieber,1* Yujung Michelle Lee,1* Max W. Chang,2

Mathias Leblanc,3 Brett Collins,3 Michael Downes,3

Ronald M. Evans,3,4 Janelle S. Ayres1†

Infections and inflammation can lead to cachexia and wasting of skeletal muscle and fattissue by as yet poorly understood mechanisms.We observed that gut colonization of miceby a strain of Escherichia coli prevents wasting triggered by infections or physical damageto the intestine. During intestinal infection with the pathogen Salmonella Typhimuriumor pneumonic infection with Burkholderia thailandensis, the presence of this E. coli did notalter changes in host metabolism, caloric uptake, or inflammation but instead sustainedsignaling of the insulin-like growth factor 1/phosphatidylinositol 3-kinase/AKT pathway inskeletal muscle, which is required for prevention of muscle wasting. This effect wasdependent on engagement of the NLRC4 inflammasome. Therefore, this commensalpromotes tolerance to diverse diseases.

Infections and inflammation lead to profoundmetabolic alterations that are primarily driv-en by responses of muscle, fat, and liver tis-sues (1, 2). Coupled with loss of appetite,dysregulated metabolism can lead to the

severe metabolic pathology called wasting syn-drome (cachexia). Such wasting constitutes lossof skeletal muscle (with and without adipose tis-sue depletion), resulting in weight loss (2). Tuber-culosis, sepsis, and HIV, as well as inflammatorydiseases including colitis (1, 3, 4), can triggerwasting. Wasting can also interfere with ther-apeutic interventions and cause untreatable mor-bidity and mortality (5).Mammals have coevolved with a complex gut

microbial community, the gut microbiota, and

depend on the metabolic benefits that it conferson the host (6, 7). Here, we have investigatedwhether constituents of the microbiota have anyprotective effect during metabolic dysregulationcaused by gut trauma and/or infection.Intestinal injury and inflammation can cause

muscle wasting and inflammatory bowel disease(IBD), and Crohn’s disease (CD) patients sufferfrom muscle wasting (8). Antibiotics cause eco-logical perturbations in the microbiota (9), andcoupling antibiotics with disease models canreveal how specific constituents of the micro-biota impact disease. The dextran sulfate sodium(DSS) intestinal injury model is one of the best-studied models of IBD/CD–associated patholo-gies, including intestinal inflammation, mucuserosion, and microbiota decompartmentalization(10). C57Bl/6 mice treated with DSS exhibitedmuscle and fat wasting that was associated withanorexia, colonic inflammation, and bloody diar-rhea (Fig. 1, A and B, and fig. S1). Administrationof the broad-spectrum antibiotic cocktail ampi-cillin, vancomycin, neomycin, andmetronidazole(AVNM) had no significant impact on the se-verity of DSS-induced wasting in C57Bl/6 miceobtained from Jackson Laboratories (Jax mice)

(fig. S2). Surprisingly, we found that C57Bl/6 fromthe University of California, Berkeley, colony (CBmice) showed significantly less wasting whenDSS was coupled with AVNM treatment (Fig.1C and fig. S2).We hypothesized that differences in the micro-

biota composition between Jax and CB mice mayaccount for the differences we observed in wast-ing pathogenesis in response to the AVNM cock-tail plus DSS. We cultured an AVNM-resistant(AVNMR) population of bacteria from the ceca ofCBmice that was not present in Jaxmice (fig. S2).On cohousingwithAVNM-treatedCBmice, AVNM-treated Jax mice became colonized with AVNMR

bacteria andwerenowprotected fromDSS-inducedwasting.Hence, protection fromwasting andCB-associated AVNMR bacteria can be horizontallytransferred to otherwise wasting-susceptible an-imals (fig. S2).We performed culture-independent analysis of

amplicons generated by primers specific to theV3 and V4 variable regions of bacterial 16S ribo-somal RNA genes of cecal content samples fromCB and Jax mice treated with AVNM for 5 days(11). The bacterial communities detected werecompared phylogentically (11). We found thatAVNM-treated CB mice hosted large numbers ofEscherichia spp. compared with AVNM-treatedJaxmice (fig. S2). Using culturing techniques, sero-typing, and genetic and molecular characteriza-tion (11), we isolated a single AVNMREscherichiafrom the ceca of CB mice that we classified as anE. coli O21:H+ strain that was absent from Jaxmice (fig. S2) (11). We performed multilocus se-quence typing (MLST) by analyzing seven loci ofE. coli O21:H+ (tables S1 and S2) (11) and foundthis E. coli to be a perfect match at these loci forE. coli of the strain ST101.Oral administration of E. coli O21:H+ resulted

in colonization of the intestine of Jax mice buthad no significant effect on weight, muscle mass,fat mass, or food consumption under homeostaticconditions (fig. S3). However, on DSS treatment,E. coli O21:H+ Jax mice showed significantly lesswasting than did DSS-treated vehicle mice (Fig.1D and fig. S4). Jax mice that were administeredheat-killed E. coli O21:H+ or live doses of thecommensal strain E. coli MG1655 exhibited sim-ilar wasting as that of vehicle-control mice (fig.S4). In accordance with our animal protocol, weused >15%weight loss as a clinical end point. Weterminatedall experimentson thedayafter challenge


1Nomis Center for Immunobiology and MicrobialPathogenesis, The Salk Institute for Biological Studies, LaJolla, CA 92037, USA. 2Integrative Genomics andBioinformatics Core, The Salk Institute for Biological Studies,La Jolla, CA 92037, USA. 3Gene Expression Laboratory, TheSalk Institute for Biological Studies, La Jolla, CA 92037, USA.4Howard Hughes Medical Institute, The Salk Institute forBiological Studies, La Jolla, CA 92037, USA.*These authors contributed equally to this work. †Correspondingauthor. E-mail: [email protected]



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developmentA GABAergic projection from the zona incerta to cortex promotes cortical neuron

Jiadong Chen and Arnold R. Kriegstein

originally published online October 1, 2015DOI: 10.1126/science.aac6472 (6260), 554-558.350Science

, this issue p. 554; see also p. 510Scienceinhibitory functions, helping the brain resist epileptiform activity.dendrites and synapses. Later on, after the fundamentals of brain construction are in place, the circuit switches to the cortex. Early in development, the circuit is excitatory and affects how the cortical neurons it touches build theirdeveloping and mature mouse brains (see the Perspective by Spitzer). This circuit connects the brain's diencephalon to

The brain is made up of circuits. Chen and Kriegstein analyzed how one particular circuit functions differently inCircuits for brain building

ARTICLE TOOLS http://science.sciencemag.org/content/350/6260/554

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2015/09/30/science.aac6472.DC1

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REFERENCES

http://science.sciencemag.org/content/350/6260/554#BIBLThis article cites 47 articles, 10 of which you can access for free

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is a registered trademark of AAAS.ScienceScience, 1200 New York Avenue NW, Washington, DC 20005. The title (print ISSN 0036-8075; online ISSN 1095-9203) is published by the American Association for the Advancement ofScience

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