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

Roland. B., R. Jordan, D.L. Sosulski, A. Diodato, I. Fukunaga, I. Wickersham, K.M. Franks, A.T. Schaefer, A. Fleischmann. 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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Transgenic targeting of recombinant rabies virus reveals monosynaptic connectivity of specific neurons

Weible AP, Schwarcz L, Wickersham IR, Deblander L, Wu H, Callaway EM, Seung HS, Kentros CG.
J Neurosci. 2010 Dec 8;30(49):16509-13. doi: 10.1523/JNEUROSCI.2442-10.2010.

Understanding how neural circuits work requires a detailed knowledge of cellular-level connectivity. Our current understanding of neural circuitry is limited by the constraints of existing tools for transsynaptic tracing. Some of the most intractable problems are a lack of cellular specificity of uptake, transport across multiple synaptic steps conflating direct and indirect inputs, and poor labeling of minor inputs. (more…)

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Production of glycoprotein-deleted rabies viruses for monosynaptic tracing and high-level gene expression in neurons

Wickersham IR, Sullivan HA, Seung HS.
Nat Protoc. 2010 Mar;5(3):595-606. doi: 10.1038/nprot.2009.248. Epub 2010 Mar 4.

Recombinant rabies viruses rendered replication-deficient by the deletion of their envelope glycoprotein gene are useful tools for neuroscientists, permitting (1) extraordinarily high transgene expression levels within neurons, (2) retrograde infection of projection neurons through their axon terminals, (3) targeted infection of genetically specified neurons and (4) monosynaptic tracing of neuronal inputs. (more…)

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Retrograde tracing with recombinant rabies virus reveals correlations between projection targets and dendritic architecture in layer 5 of mouse barrel cortex

Larsen DD, Wickersham IR, Callaway EM.
Front Neural Circuits. 2008 Mar 28;1:5. doi: 10.3389/neuro.04.005.2007. eCollection 2007.

A recombinant rabies virus was used as a retrograde tracer to allow complete filling of the axonal and dendritic arbors of identified projection neurons in layer 5 of mouse primary somatosensory cortex (S1) in vivo. Previous studies have distinguished three types of layer 5 pyramids in S1: tall-tufted, tall-simple, and short. Layer 5 pyramidal neurons were retrogradely labeled from several known targets: contralateral S1, superior colliculus, and thalamus. (more…)

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Suitability of hCMV for viral gene expression in the brain (reply)

Kasparov S.
Nat Methods. 2007 May;4(5):379; author reply 379.

Viral gene delivery is a powerful tool that is rapidly gaining popularity in experimental neuroscience. I believe that the near future will see rapid expansion of this remarkably versatile and adaptable method. Recent communication by Wickersham et al.1 describes yet another interesting implementation of this technology for retrograde tracing of neurons from their target areas. The key point of the paper is demonstration of (more…)

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