Silencing Synapses with DREADDs
2014; Cell Press; Volume: 82; Issue: 4 Linguagem: Inglês
10.1016/j.neuron.2014.05.002
ISSN1097-4199
Autores Tópico(s)Neuroscience and Neuropharmacology Research
ResumoIn this issue of Neuron, Stachniak et al., 2014Stachniak T.J. Ghosh A. Sternson S.M. Neuron. 2014; 82 (this issue): 797-808Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar determine that the chemogenetic silencer hM4Di-DREADD suppresses presynaptic glutamate release, and by generating an axon-targeted hM4Di variant they demonstrate that it can be used to locally silence synaptic transmission in neural circuits. In this issue of Neuron, Stachniak et al., 2014Stachniak T.J. Ghosh A. Sternson S.M. Neuron. 2014; 82 (this issue): 797-808Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar determine that the chemogenetic silencer hM4Di-DREADD suppresses presynaptic glutamate release, and by generating an axon-targeted hM4Di variant they demonstrate that it can be used to locally silence synaptic transmission in neural circuits. "Silence is all we dread.There's Ransom in a Voice —But Silence is Infinity." (Emily Dickinson, Poem 1251, Complete Poetry of Emily Dickenson) Some decades ago, Francis Crick presciently predicted that in order for scientists to elucidate the "neuronal codes" that specify behavior, perception, and consciousness, "a method (is needed) by which all neurons of just one type could be inactivated, leaving the others more or less unaltered" (Crick, 1979Crick F.H. Sci. Am. 1979; 241: 219-232Crossref PubMed Scopus (164) Google Scholar). Now, of course, both optogenetic (Zhang et al., 2007Zhang F. Wang L.P. Brauner M. Liewald J.F. Kay K. Watzke N. Wood P.G. Bamberg E. Nagel G. Gottschalk A. Deisseroth K. Nature. 2007; 446: 633-639Crossref PubMed Scopus (1366) Google Scholar) and chemogenetic (Armbruster et al., 2007Armbruster B.N. Li X. Pausch M.H. Herlitze S. Roth B.L. Proc. Natl. Acad. Sci. USA. 2007; 104: 5163-5168Crossref PubMed Scopus (1277) Google Scholar) technologies are widely available for silencing neurons "of just one type." Simply inhibiting cell body firing and observing the resulting behavioral phenotype, unfortunately, does little to elucidate which specific projections or target areas might be responsible for the observed effects (see Figure 1A). Ideally, what is needed is a technology that can specifically and reversibly silence presynaptic nerve terminals, thereby "silencing synapses" projecting to distinct neuronal populations (Figure 1B). A technological leap forward is now reported by Stachniak et al., 2014Stachniak T.J. Ghosh A. Sternson S.M. Neuron. 2014; 82 (this issue): 797-808Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, who via clever modifications of the DREADD chemogenetic platform achieved synaptic silencing. As first described, the hM4Di-DREADD, when stimulated by clozapine-N-oxide (CNO), activates G protein inwardly rectifying potassium (GIRK) channels, thereby hyperpolarizing and attenuating neuronal activity (Armbruster et al., 2007Armbruster B.N. Li X. Pausch M.H. Herlitze S. Roth B.L. Proc. Natl. Acad. Sci. USA. 2007; 104: 5163-5168Crossref PubMed Scopus (1277) Google Scholar). hM4Di is now routinely used as a tool to diminish the activity of genetically defined neurons in vitro and in vivo (Atasoy et al., 2012Atasoy D. Betley J.N. Su H.H. Sternson S.M. Nature. 2012; 488: 172-177Crossref PubMed Scopus (644) Google Scholar, Ferguson et al., 2011Ferguson S.M. Eskenazi D. Ishikawa M. Wanat M.J. Phillips P.E. Dong Y. Roth B.L. Neumaier J.F. Nat. Neurosci. 2011; 14: 22-24Crossref PubMed Scopus (333) Google Scholar, Krashes et al., 2011Krashes M.J. Koda S. Ye C. Rogan S.C. Adams A.C. Cusher D.S. Maratos-Flier E. Roth B.L. Lowell B.B. J. Clin. Invest. 2011; 121: 1424-1428Crossref PubMed Scopus (897) Google Scholar, Ray et al., 2011Ray R.S. Corcoran A.E. Brust R.D. Kim J.C. Richerson G.B. Nattie E. Dymecki S.M. Science. 2011; 333: 637-642Crossref PubMed Scopus (255) Google Scholar, Carter et al., 2013Carter M.E. Soden M.E. Zweifel L.S. Palmiter R.D. Nature. 2013; 503: 111-114Crossref PubMed Scopus (393) Google Scholar). The robust effects of hM4Di activation on physiology (see Ray et al., 2011Ray R.S. Corcoran A.E. Brust R.D. Kim J.C. Richerson G.B. Nattie E. Dymecki S.M. Science. 2011; 333: 637-642Crossref PubMed Scopus (255) Google Scholar for example) and behavior (see Carter et al., 2013Carter M.E. Soden M.E. Zweifel L.S. Palmiter R.D. Nature. 2013; 503: 111-114Crossref PubMed Scopus (393) Google Scholar for instance) have been difficult to reconcile with the relatively modest ability of hM4Di to hyperpolarize and attenuate neuronal firing in vitro (see, for instance, Ferguson et al., 2011Ferguson S.M. Eskenazi D. Ishikawa M. Wanat M.J. Phillips P.E. Dong Y. Roth B.L. Neumaier J.F. Nat. Neurosci. 2011; 14: 22-24Crossref PubMed Scopus (333) Google Scholar, Krashes et al., 2011Krashes M.J. Koda S. Ye C. Rogan S.C. Adams A.C. Cusher D.S. Maratos-Flier E. Roth B.L. Lowell B.B. J. Clin. Invest. 2011; 121: 1424-1428Crossref PubMed Scopus (897) Google Scholar, Ray et al., 2011Ray R.S. Corcoran A.E. Brust R.D. Kim J.C. Richerson G.B. Nattie E. Dymecki S.M. Science. 2011; 333: 637-642Crossref PubMed Scopus (255) Google Scholar). Here, Stachniak et al., 2014Stachniak T.J. Ghosh A. Sternson S.M. Neuron. 2014; 82 (this issue): 797-808Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar confirm that hM4Di activation leads to hyperpolarization and attenuation of neuronal firing but also discovered a much more potent action of hM4Di as an effective synaptic silencer in slice preparations and in vivo. It is this silencing of synaptic transmission that will ultimately expand the utility of DREADD-based technology for deconstructing the neuronal code. Using postsynaptic current as the readout, they first tested whether the CNO-induced activation of hM4Di in a presynaptic neuron suppresses synaptic transmission to postsynaptic neurons located in the same or in different layers of the cortex. They discovered that CNO-induced activation of hM4Di L2/3 presynaptic cortical neurons robustly inhibited the postsynaptic current in both L2/3 neurons and L5 neurons. They also demonstrated that this inhibition of the postsynaptic current was not due to blockade of either the initiation or the propagation of the axonal action potential. Instead, hM4Di appeared to act by suppressing L2/3 synaptic glutamate release. Importantly, neither CNO administered in control slices nor basal hM4Di activity had any significant effect on synaptic glutamate release from L2/3 glutamatergic neurons. To determine whether this "synaptic silencing" by hM4Di could be useful for studies in vivo, Stachniak et al., 2014Stachniak T.J. Ghosh A. Sternson S.M. Neuron. 2014; 82 (this issue): 797-808Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar chose a well-characterized and popular neural circuit for food intake and examined synaptic transmission from Agouti-related peptide (AgRP)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC) to paraventricular hypothalamic (PVH) neurons (Stachniak et al., 2014Stachniak T.J. Ghosh A. Sternson S.M. Neuron. 2014; 82 (this issue): 797-808Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Prior studies showed that PVH neurons receive axonal projections from AgRP neurons and mediate food intake evoked by activation of AgRP neurons (Atasoy et al., 2012Atasoy D. Betley J.N. Su H.H. Sternson S.M. Nature. 2012; 488: 172-177Crossref PubMed Scopus (644) Google Scholar). Stachniak et al., 2014Stachniak T.J. Ghosh A. Sternson S.M. Neuron. 2014; 82 (this issue): 797-808Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar then coexpressed channelrhodopsin2 (ChR2) and hM4Di in AgRP neurons and examined the effect of CNO on light-evoked food intake. Astoundingly, they found that microinfusion of CNO above the PVH, but not in an area only 300–500 μm distant, reduced feeding by ∼50% during AgRP neuron photostimulation. Taken together, the results obtained from studies in the cortex and hypothalamus demonstrate that hM4Di can effectively suppress presynaptic transmission both ex vivo and in vivo. In support of this notion, a recent report (Mahler et al., 2014Mahler S.V. Vazey E.M. Beckley J.T. Keistler C.R. McGlinchey E.M. Kaufling J. Wilson S.P. Deisseroth K. Woodward J.J. Aston-Jones G. Nat. Neurosci. 2014; 17: 577-585Crossref PubMed Scopus (245) Google Scholar) showed that microinfusion of CNO suppresses terminal dopamine release in hM4Di-expressing dopaminergic axons. As hM4Di is normally localized to both neuronal cell bodies and axons (Mahler et al., 2014Mahler S.V. Vazey E.M. Beckley J.T. Keistler C.R. McGlinchey E.M. Kaufling J. Wilson S.P. Deisseroth K. Woodward J.J. Aston-Jones G. Nat. Neurosci. 2014; 17: 577-585Crossref PubMed Scopus (245) Google Scholar, Zhu et al., 2014Zhu H. Pleil K.E. Urban D.J. Moy S.S. Kash T.L. Roth B.L. Neuropsychopharmacology. 2014; (Published online February 12, 2014)https://doi.org/10.1038/npp.2014.35Crossref PubMed Scopus (106) Google Scholar), it would be more useful to target it specifically to the axonal compartment to achieve selective suppression of synaptic transmission. To achieve this, Stachniak et al., 2014Stachniak T.J. Ghosh A. Sternson S.M. Neuron. 2014; 82 (this issue): 797-808Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar developed an axon-preferring variant of hM4Di they refer to as hM4DNRXN, as it contained the axonal C-terminal targeting sequence of Neurexin1a. This hM4DiNRXN variant displayed reduced somatic expression and enhanced selective axonal expression. Activation of the hM4DNRXN variant by CNO (1 μM) robustly inhibited synaptic transmission but did not induce somatic hyperpolarization and thereby selectively silenced synaptic transmission. Using this synaptic silencing tool, Stachniak et al., 2014Stachniak T.J. Ghosh A. Sternson S.M. Neuron. 2014; 82 (this issue): 797-808Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar then deconstructed the neural circuit downstream of the PVH to demonstrate the further utility of this improved DREADD. Thus, the hM4DNRXN variant was expressed in PVHSIM1 (single-minded homolog 1 [SIM1]) neurons, and CNO was microinfused into the multiple brain regions targeted by PVHSIM1 axon projections. Stachniak et al., 2014Stachniak T.J. Ghosh A. Sternson S.M. Neuron. 2014; 82 (this issue): 797-808Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar reported that inhibition of the PVHSIM1→NTS/DVC (nucleus of the solitary tract and dorsal vagal complex [NTS/DVC]) axon projection was not sufficient to evoke food intake behavior. However, they identified another "hotspot," the caudal ventrolateral periaqueductal gray and dorsal raphe complex, as a key node downstream of ARCAGRP → PVH neural circuit that controls food intake. The study by Stachniak et al., 2014Stachniak T.J. Ghosh A. Sternson S.M. Neuron. 2014; 82 (this issue): 797-808Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar addresses two important issues related to DREADD technology. First, they provide convincing evidence that activation of hM4Di in presynaptic terminals can suppress synaptic transmission without disturbing somatic or axonal membrane potentials. Therefore, hM4Di probably silences neuronal activity in vivo via both hyperpolarization and suppression of presynaptic neurotransmitter release in a manner analogous to that achieved by presynaptic G-protein-coupled receptors. This makes hM4Di unique among the currently available optogenetic and chemogenetic tools that silence neuronal activity via hyperpolarizing neurons to suppress action potentials. Second, they demonstrate that microinfusion of CNO into discrete brain regions is a reliable way to achieve very precise spatiotemporal control of neuronal activity—in agreement with a recent study (Mahler et al., 2014Mahler S.V. Vazey E.M. Beckley J.T. Keistler C.R. McGlinchey E.M. Kaufling J. Wilson S.P. Deisseroth K. Woodward J.J. Aston-Jones G. Nat. Neurosci. 2014; 17: 577-585Crossref PubMed Scopus (245) Google Scholar). This study also raises an intriguing question regarding the mechanism by which hM4Di inhibits presynaptic neurotransmitter release. Although GPCRs are known to be ubiquitously expressed on presynaptic terminals and to modulate synaptic neurotransmitter release, how hM4Di regulates neurotransmitter release is unknown. It is not likely via GIRKS as GIRK1 is primary localized in postsynaptic rather than presynaptic terminals (Drake et al., 1997Drake C.T. Bausch S.B. Milner T.A. Chavkin C. Proc. Natl. Acad. Sci. USA. 1997; 94: 1007-1012Crossref PubMed Scopus (83) Google Scholar) and GPCR agonist-induced presynaptic inhibition is unchanged in GIRK2 knockout mice (Lüscher et al., 1997Lüscher C. Jan L.Y. Stoffel M. Malenka R.C. Nicoll R.A. Neuron. 1997; 19: 687-695Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). Conceivably, hM4Di could induce presynaptic silencing via inhibition of cAMP-mediated signaling, which has been shown to modulate the activity of voltage-gated calcium channels in a model system (Hilfiker et al., 2001Hilfiker S. Czernik A.J. Greengard P. Augustine G.J. J. Physiol. 2001; 531: 141-146Crossref PubMed Scopus (37) Google Scholar). Alternatively, hM4Di could inhibit the SNARE exocytotic fusion machinery downstream of calcium entry through the action G protein βγ subunits (Gerachshenko et al., 2005Gerachshenko T. Blackmer T. Yoon E.J. Bartleson C. Hamm H.E. Alford S. Nat. Neurosci. 2005; 8: 597-605Crossref PubMed Scopus (138) Google Scholar). Whatever the mechanism, it is clear that hM4Di can effectively suppress presynaptic transmission both ex vivo and in vivo, and this makes it useful for many applications. Further, the axon-selective hM4DNRXN variant developed in this study is an exceedingly useful tool to functionally dissect neuronal circuitry by the targeted inhibition of presynaptic transmission without compromising the activities of other synapses originating from the same neurons. Chemogenetic Synaptic Silencing of Neural Circuits Localizes a Hypothalamus→Midbrain Pathway for Feeding BehaviorStachniak et al.NeuronApril 24, 2014In BriefStachniak et al. provide a NeuroResource for chemogenetic silencing of synaptic transmission. They use this method for functional analysis of mammalian neural circuits that regulate appetite. Full-Text PDF Open Archive
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