The BRAIN Initiative Cell Census Consortium: Lessons Learned toward Generating a Comprehensive Brain Cell Atlas.

Ecker JR, Geschwind DH, Kriegstein AR, Ngai J, Osten P, Polioudakis D, Regev A, Sestan N, Wickersham IR, Zeng H.
Neuron. 2017 Nov 1;96(3):542-557.

A comprehensive characterization of neuronal cell types, their distributions, and patterns of connectivity is critical for understanding the properties of neural circuits and how they generate behaviors. Here we review the experiences of the BRAIN Initiative Cell Census Consortium, ten pilot projects funded by the U.S. BRAIN Initiative, in developing, validating, and scaling up emerging genomic and anatomical mapping technologies for creating a complete inventory of neuronal cell types and their connections in multiple species and during development. These projects lay the foundation for a larger and longer-term effort to generate whole-brain cell atlases in species including mice and humans.

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Reversing behavioural abnormalities in mice exposed to maternal inflammation

Shin Yim Y, Park A, Berrios J, Lafourcade M, Pascual LM, Soares N, Yeon Kim J, Kim S, Kim H, Waisman A, Littman DR, Wickersham IR, Harnett MT, Huh JR, Choi GB
Nature. 2017 Sep 28;549(7673):482-487.

Viral infection during pregnancy is correlated with increased frequency of neurodevelopmental disorders, and this is studied in mice prenatally subjected to maternal immune activation (MIA). We previously showed that maternal T helper 17 cells promote the development of cortical and behavioural abnormalities in MIA-affected offspring. Here we show that cortical abnormalities are preferentially localized to a region encompassing the dysgranular zone of the primary somatosensory cortex (S1DZ). Moreover, activation of pyramidal neurons in this cortical region was sufficient to induce MIA-associated behavioural phenotypes in wild-type animals, whereas reduction in neural activity rescued the behavioural abnormalities in MIA-affected offspring. Sociability and repetitive behavioural phenotypes could be selectively modulated according to the efferent targets of S1DZ. Our work identifies a cortical region primarily, if not exclusively, centred on the S1DZ as the major node of a neural network that mediates behavioural abnormalities observed in offspring exposed to maternal inflammation.

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Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep.

Liu K, Kim J, Kim DW, Zhang YS, Bao H, Denaxa M, Lim SA, Kim E, Liu C, Wickersham IR, Pachinis V, Hattar S, Song J, Brown SP, Blackshaw S.
Nature. 2017 Aug 31;548(7669):582-587.

Multiple populations of wake-promoting neurons have been characterized in mammals, but few sleep-promoting neurons have been identified. Wake-promoting cell types include hypocretin and GABA (γ-aminobutyric-acid)-releasing neurons of the lateral hypothalamus, which promote the transition to wakefulness from non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. Here we show that a subset of GABAergic neurons in the mouse ventral zona incerta, which express the LIM homeodomain factor Lhx6 and are activated by sleep pressure, both directly inhibit wake-active hypocretin and GABAergic cells in the lateral hypothalamus and receive inputs from multiple sleep-wake-regulating neurons. Conditional deletion of Lhx6 from the developing diencephalon leads to decreases in both NREM and REM sleep. Furthermore, selective activation and inhibition of Lhx6-positive neurons in the ventral zona incerta bidirectionally regulate sleep time in adult mice, in part through hypocretin-dependent mechanisms. These studies identify a GABAergic subpopulation of neurons in the ventral zona incerta that promote sleep.

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Combining Optogenetics and Electrophysiology to Analyze Projection Neuron Circuits

Yamawaki N, Suter BA, Wickersham IR, Shepherd GM.
Cold Spring Harb Protoc. 2016 Oct 3;2016(10)

A set of methods is described for channelrhodopsin-2 (ChR2)-based synaptic circuit analysis that combines photostimulation of virally transfected presynaptic neurons’ axons with whole-cell electrophysiological recordings from retrogradely labeled postsynaptic neurons. The approach exploits the preserved photoexcitability of ChR2-expressing axons in brain slices and can be used to assess either local or long-range functional connections. Stereotaxic injections are used both to express ChR2 selectively in presynaptic axons of interest (using rabies virus [RV] or adeno-associated virus [AAV]) and to label two types of postsynaptic projection neurons of interest with fluorescent retrograde tracers. In brain slices, tracer-labeled postsynaptic neurons are targeted for whole-cell electrophysiological recordings, and synaptic connections are assessed by sampling voltage or current responses to light-emitting diode (LED) photostimulation of ChR2-expressing axons. The data are analyzed to estimate the relative amplitude of synaptic input and other connectivity parameters. Pharmacological and electrophysiological manipulations extend the versatility of the basic approach, allowing the dissection of monosynaptic versus disynaptic responses, excitatory versus inhibitory responses, and more. The method enables rapid, quantitative characterization of synaptic connectivity between defined pre- and postsynaptic classes of neurons.

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Massive normalization of olfactory bulb output in mice with a ‘monoclonal nose’

Roland B, Jordan R, Sosulski DL, Diodato A, Fukunaga I, Wickersham I, Franks KM, Schaefer AT, Fleischmann A.
Elife 5. pii: e16335 (2016).

Perturbations in neural circuits can provide mechanistic understanding of the neural correlates of behavior. In M71 transgenic mice with a “monoclonal nose”, glomerular input patterns in the olfactory bulb are massively perturbed and olfactory behaviors are altered. To gain insights into how olfactory circuits can process such degraded inputs we characterized odor-evoked responses of olfactory bulb mitral cells and interneurons. (more…)

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Assembly and operation of the autopatcher for automated intracellular neural recording in vivo

Kodandaramaiah SB, Holst GL, Wickersham IR, Singer AC, Franzesi GT, McKinnon ML, Forest CR, Boyden ES.
Nat Protoc. 2016 Apr;11(4):634-54. doi: 10.1038/nprot.2016.007. Epub 2016 Mar 3.

Whole-cell patch clamping in vivo is an important neuroscience technique that uniquely provides access to both suprathreshold spiking and subthreshold synaptic events of single neurons in the brain. This article describes how to set up and use the autopatcher, which is a robot for automatically obtaining high-yield and high-quality whole-cell patch clamp recordings in vivo. By following this protocol, a functional experimental (more…)

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A circuit mechanism for differentiating positive and negative associations

Namburi P, Beyeler A, Yorozu S, Calhoon GG, Halbert SA, Wichmann R, Holden SS, Mertens KL, Anahtar M, Felix-Ortiz AC, Wickersham IR, Gray JM, Tye KM.
Nature. 2015 Apr 30;520(7549):675-8. doi: 10.1038/nature14366.

The ability to differentiate stimuli predicting positive or negative outcomes is critical for survival, and perturbations of emotional processing underlie many psychiatric disease states. Synaptic plasticity in the basolateral amygdala complex (BLA) mediates the acquisition of associative memories, both positive and negative. Different populations of BLA neurons may encode fearful or rewarding associations, (more…)

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Concentration and purification of rabies viral and lentiviral vectors

Sullivan HA, Wickersham IR.
Cold Spring Harb Protoc. 2015 Apr 1;2015(4):386-91. doi: 10.1101/pdb.prot075887.

Rabies viral and lentiviral vectors are very useful tools for neuroscientists, but high titer and purity are critical for in vivo applications. Here we present a protocol for concentration and purification of viral stocks by ultracentrifugation on a sucrose step gradient to remove impurities of both higher and lower densities than the virus itself, with sucrose removed by a subsequent pelleting step. (more…)

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Rabies viral vectors for monosynaptic tracing and targeted transgene expression in neurons

Wickersham IR, Sullivan HA.
Cold Spring Harb Protoc. 2015 Apr 1;2015(4):375-85. doi: 10.1101/pdb.prot072389.

Deletion-mutant rabies viral (RV) vectors are powerful tools for neuroscience, allowing monosynaptic tracing of inputs to defined populations and rapid, high-level transgene expression in neurons targeted by multiple routes. High titers and high purity are critical for the successful use of RV vectors in vivo. Here we present a protocol for producing high-quality viral stocks that can be concentrated by ultracentrifugation (more…)

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Lentiviral vectors for retrograde delivery of recombinases and transactivators

Wickersham IR, Sullivan HA, Pao GM, Hamanaka H, Goosens KA, Verma IM, Seung HS.
Cold Spring Harb Protoc. 2015 Apr 1;2015(4):368-74. doi: 10.1101/pdb.prot075879.

Lentiviral vectors pseudotyped with the rabies virus (RV) envelope glycoprotein efficiently infect via axon terminals to stably deliver transgenes to distant neurons projecting to an injection site, but the resulting expression levels are too low and variable for most neuroscientific applications. If used to deliver recombinases or transactivators, however, lentiviral vectors are excellent means of targeting projection neurons when (more…)

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The stimulus selectivity and connectivity of layer six principal cells reveals cortical microcircuits underlying visual processing

Vélez-Fort M, Rousseau CV, Niedworok CJ, Wickersham IR, Rancz EA, Brown AP, Strom M, Margie TW.
Neuron. 2014 Sep 17;83(6):1431-43. doi: 10.1016/j.neuron.2014.08.001. Epub 2014 Aug 28.

Sensory computations performed in the neocortex involve layer six (L6) cortico-cortical (CC) and cortico-thalamic (CT) signaling pathways. Developing an understanding of the physiological role of these circuits requires dissection of the functional specificity and connectivity of the underlying individual projection neurons. By combining whole-cell recording from identified L6 principal cells in the mouse primary visual cortex (V1) (more…)

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Cell type-specific genetic and optogenetic tools reveal hippocampal CA2 circuits

Kohara K, Pignatelli M, Rivest AJ, Jung HY, Kitamura T, Suh J, Frank D, Kajikawa K, Mise N, Obata Y, Wickersham IR, Tonegawa S.
Nat Neurosci. 2014 Feb;17(2):269-79. doi: 10.1038/nn.3614. Epub 2013 Dec 15.

The formation and recall of episodic memory requires precise information processing by the entorhinal-hippocampal network. For several decades, the trisynaptic circuit entorhinal cortex layer II (ECII)→dentate gyrus→CA3→CA1 and the monosynaptic circuit ECIII→CA1 have been considered the primary substrates of the network responsible for learning and memory. Circuits linked to another hippocampal region, CA2, (more…)

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Transgenically targeted rabies virus demonstrates a major monosynaptic projection from hippocampal area CA2 to medial entorhinal layer II neurons

Rowland DC, Weible AP, Wickersham IR, Wu H, Mayford M, Witter MP, Kentros CG.
J Neurosci. 2013 Sep 11;33(37):14889-98. doi: 10.1523/JNEUROSCI.1046-13.2013.

The enormous potential of modern molecular neuroanatomical tools lies in their ability to determine the precise connectivity of the neuronal cell types comprising the innate circuitry of the brain. We used transgenically targeted viral tracing to identify the monosynaptic inputs to the projection neurons of layer II of medial entorhinal cortex (MEC-LII) in mice. These neurons are not only major inputs to the hippocampus, the structure (more…)

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Convergent cortical innervation of striatal projection neurons

Kress GJ, Yamawaki N, Wokosin DL, Wickersham IR, Shepherd GM, Surmeier DJ.
Nat Neurosci. 2013 Jun;16(6):665-7. doi: 10.1038/nn.3397. Epub 2013 May 12.

Anatomical studies have led to the assertion that intratelencephalic and pyramidal tract cortical neurons innervate different striatal projection neurons. To test this hypothesis, we measured the responses of mouse striatal neurons to optogenetic activation of intratelencephalic and pyramidal tract axons. Contrary to expectation, direct and indirect pathway striatal spiny projection neurons responded to both intratelencephalic and (more…)

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Axonal and subcellular labelling using modified rabies viral vectors

Wickersham IR, Sullivan HA, Seung HS.
Nat Commun. 2013;4:2332. doi: 10.1038/ncomms3332.

An important aspect of any neural circuit is the placement of its output synapses, at levels ranging from macroscopic to subcellular. The many new molecular tools for locating and manipulating synapses are limited by the viral vectors available for delivering them. Adeno-associated viruses are the best current means of labelling and manipulating axons and synapses, but they have never expressed more than one transgene (more…)

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Laminarly orthogonal excitation of fast-spiking and low-threshold-spiking interneurons in mouse motor cortex

Apicella AJ, Wickersham IR, Seung HS, Shepherd GM.
J Neurosci. 2012 May 16;32(20):7021-33. doi: 10.1523/JNEUROSCI.0011-12.2012.

In motor cortex, long-range output to subcortical motor circuits depends on excitatory and inhibitory inputs converging on projection neurons in layers 5A/B. How interneurons interconnect with these projection neurons, and whether these microcircuits are interneuron and/or projection specific, is unclear. We found that fast-spiking interneurons received strong intralaminar (horizontal) excitation from pyramidal neurons in layers 5A/B including (more…)

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Hierarchical connectivity and connection-specific dynamics in the corticospinal-corticostriatal microcircuit in mouse motor cortex

Kiritani T, Wickersham IR, Seung HS, Shepherd GM.
J Neurosci. 2012 Apr 4;32(14):4992-5001. doi: 10.1523/JNEUROSCI.4759-11.2012.

The generation of purposive movement by mammals involves coordinated activity in the corticospinal and corticostriatal systems, which are involved in different aspects of motor control. In the motor cortex, corticospinal and corticostriatal neurons are closely intermingled, raising the question of whether and how information flows intracortically within and across these two channels. To explore this, we developed an (more…)

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New technologies for imaging synaptic partners

Wickersham IR, Feinberg EH.
Curr Opin Neurobiol. 2012 Feb;22(1):121-7. doi: 10.1016/j.conb.2011.12.001. Epub 2012 Jan 3.

Understanding the brain will require unraveling its synaptic circuitry, but methods that can reliably identify connected neurons are often excruciatingly slow. Although light microscopy can provide much higher throughput, synapses are smaller than the diffraction limit and cannot readily be assigned to particular presynaptic and postsynaptic cells without specialized labeling methods. Here we review the ongoing development of (more…)

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Cortical representations of olfactory input by trans-synaptic tracing

Miyamichi K, Amat F, Moussavi F, Wang C, Wickersham IR, Wall NR, Taniguchi H, Tasic B, Huang ZJ, He Z, Callaway EM, Horowitz MA, Luo L.
Nature. 2011 Apr 14;472(7342):191-6. doi: 10.1038/nature09714. Epub 2010 Dec 22.

In the mouse, each class of olfactory receptor neurons expressing a given odorant receptor has convergent axonal projections to two specific glomeruli in the olfactory bulb, thereby creating an odour map. However, it is unclear how this map is represented in the olfactory cortex. Here we combine rabies-virus-dependent retrograde mono-trans-synaptic labelling with genetics to control the location, (more…)

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Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus

Wall NR, Wickersham IR, Cetin A, De La Parra M, Callaway EM.
Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21848-53. doi: 10.1073/pnas.1011756107. Epub 2010 Nov 29.

We describe a powerful system for revealing the direct monosynaptic inputs to specific cell types in Cre-expressing transgenic mice through the use of Cre-dependent helper virus and a modified rabies virus. We generated helper viruses that target gene expression to Cre-expressing cells, allowing us to control initial rabies virus infection and subsequent monosynaptic retrograde spread. (more…)

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