Publications by authors named "Ian R Wickersham"

32 Publications

An amygdala circuit that suppresses social engagement.

Nature 2021 May 31;593(7857):114-118. Epub 2021 Mar 31.

Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA, USA.

Innate social behaviours, such as mating and fighting, are fundamental to animal reproduction and survival. However, social engagements can also put an individual at risk. Little is known about the neural mechanisms that enable appropriate risk assessment and the suppression of hazardous social interactions. Here we identify the posteromedial nucleus of the cortical amygdala (COApm) as a locus required for the suppression of male mating when a female mouse is unhealthy. Using anatomical tracing, functional imaging and circuit-level epistatic analyses, we show that suppression of mating with an unhealthy female is mediated by the COApm projections onto the glutamatergic population of the medial amygdalar nucleus (MEA). We further show that the role of the COApm-to-MEA connection in regulating male mating behaviour relies on the neuromodulator thyrotropin-releasing hormone (TRH). TRH is expressed in the COApm, whereas the TRH receptor (TRHR) is found in the postsynaptic MEA glutamatergic neurons. Manipulating neural activity of TRH-expressing neurons in the COApm modulated male mating behaviour. In the MEA, activation of the TRHR pathway by ligand infusion inhibited mating even towards healthy female mice, whereas genetic ablation of TRHR facilitated mating with unhealthy individuals. In summary, we reveal a neural pathway that relies on the neuromodulator TRH to modulate social interactions according to the health status of the reciprocating individual. Individuals must balance the cost of social interactions relative to the benefit, as deficits in the ability to select healthy mates may lead to the spread of disease.
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http://dx.doi.org/10.1038/s41586-021-03413-6DOI Listing
May 2021

Distinct prefrontal top-down circuits differentially modulate sensorimotor behavior.

Nat Commun 2020 11 26;11(1):6007. Epub 2020 Nov 26.

Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Sensorimotor behaviors require processing of behaviorally relevant sensory cues and the ability to select appropriate responses from a vast behavioral repertoire. Modulation by the prefrontal cortex (PFC) is thought to be key for both processes, but the precise role of specific circuits remains unclear. We examined the sensorimotor function of anatomically distinct outputs from a subdivision of the mouse PFC, the anterior cingulate cortex (ACC). Using a visually guided two-choice behavioral paradigm with multiple cue-response mappings, we dissociated the sensory and motor response components of sensorimotor control. Projection-specific two-photon calcium imaging and optogenetic manipulations show that ACC outputs to the superior colliculus, a key midbrain structure for response selection, principally coordinate specific motor responses. Importantly, ACC outputs exert control by reducing the innate response bias of the superior colliculus. In contrast, ACC outputs to the visual cortex facilitate sensory processing of visual cues. Our results ascribe motor and sensory roles to ACC projections to the superior colliculus and the visual cortex and demonstrate for the first time a circuit motif for PFC function wherein anatomically non-overlapping output pathways coordinate complementary but distinct aspects of visual sensorimotor behavior.
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http://dx.doi.org/10.1038/s41467-020-19772-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691329PMC
November 2020

Brainstem neurons that command mammalian locomotor asymmetries.

Nat Neurosci 2020 06 11;23(6):730-740. Epub 2020 May 11.

Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Descending command neurons instruct spinal networks to execute basic locomotor functions, such as gait and speed. The command functions for gait and speed are symmetric, implying that a separate unknown system directs asymmetric movements, including the ability to move left or right. In the present study, we report that Chx10-lineage reticulospinal neurons act to control the direction of locomotor movements in mammals. Chx10 neurons exhibit mainly ipsilateral projection, and their selective unilateral activation causes ipsilateral turning movements in freely moving mice. Unilateral inhibition of Chx10 neurons causes contralateral turning movements. Paired left-right motor recordings identified distinct mechanisms for directional movements mediated via limb and axial spinal circuits. Finally, we identify sensorimotor brain regions that project on to Chx10 reticulospinal neurons, and demonstrate that their unilateral activation can impart left-right directional commands. Together these data identify the descending motor system that commands left-right locomotor asymmetries in mammals.
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http://dx.doi.org/10.1038/s41593-020-0633-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610510PMC
June 2020

Monosynaptic Tracing Success Depends Critically on Helper Virus Concentrations.

Front Synaptic Neurosci 2020 14;12. Epub 2020 Feb 14.

McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, United States.

Monosynaptically-restricted transsynaptic tracing using deletion-mutant rabies virus (RV) has become a widely used technique in neuroscience, allowing identification, imaging, and manipulation of neurons directly presynaptic to a starting neuronal population. Its most common implementation is to use Cre mouse lines in combination with Cre-dependent "helper" adeno-associated viral vectors (AAVs) to supply the required genes to the targeted population before subsequent injection of a first-generation (ΔG) rabies viral vector. Here we show that the efficiency of transsynaptic spread and the degree of nonspecific labeling in wild-type control animals depend strongly on the concentrations of these helper AAVs. Our results suggest practical guidelines for achieving good results.
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http://dx.doi.org/10.3389/fnsyn.2020.00006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033752PMC
February 2020

Monosynaptic tracing: a step-by-step protocol.

J Chem Neuroanat 2019 12 10;102:101661. Epub 2019 Aug 10.

McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, United States. Electronic address:

Monosynaptic tracing using deletion-mutant rabies virus allows whole-brain mapping of neurons that are directly presynaptic to a targeted population of neurons. The most common and robust way of implementing it is to use Cre mouse lines in combination with Cre-dependent adeno-associated viral vectors for expression of the required genes in the targeted neurons before subsequent injection of the rabies virus. Here we present a step-by-step protocol for performing such experiments using first-generation (ΔG) rabies viral vectors.
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http://dx.doi.org/10.1016/j.jchemneu.2019.101661DOI Listing
December 2019

Dichotomous parvalbumin interneuron populations in dorsolateral and dorsomedial striatum.

J Physiol 2018 08 9;596(16):3695-3707. Epub 2018 Jun 9.

McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.

Key Points: There are two electrophysiological dichotomous populations of parvalbumin (PV) interneurons located in the dorsal striatum. Striatal PV interneurons in medial and lateral regions differ significantly in their intrinsic excitability. Parvalbumin interneurons in the dorsomedial striatum, but not in the dorsolateral striatum, receive afferent glutamatergic input from cingulate cortex.

Abstract: Dorsomedial striatum circuitry is involved in goal-directed actions or movements that become habits upon repetition, as encoded by the dorsolateral striatum. An inability to shift from habits can compromise action-control and prevent behavioural adaptation. Although these regions appear to be clearly behaviourally distinct, little is known about their distinct physiology. Parvalbumin (PV) interneurons are a major source of striatal inhibition and are usually considered as a homogeneous population in the entire dorsal striatum. In the present study, we recorded PV interneurons in dorsal striatum slices from wild-type male mice and suggest the existence of two electrophysiological dichotomous populations. We found that PV interneurons located at the dorsomedial striatum region have increased intrinsic excitability compared to PV interneurons in dorsolateral region. We also found that PV interneurons in the dorsomedial region, but not in the dorsolateral striatum region, receive short-latency excitatory inputs from cingulate cortex. Therefore, the results of the present study demonstrate the importance of considering region specific parvalbumin interneuron populations when studying dorsal striatal function.
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http://dx.doi.org/10.1113/JP275936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6092282PMC
August 2018

Nontoxic, double-deletion-mutant rabies viral vectors for retrograde targeting of projection neurons.

Nat Neurosci 2018 04 5;21(4):638-646. Epub 2018 Mar 5.

McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, USA.

Recombinant rabies viral vectors have proven useful for applications including retrograde targeting of projection neurons and monosynaptic tracing, but their cytotoxicity has limited their use to short-term experiments. Here we introduce a new class of double-deletion-mutant rabies viral vectors that left transduced cells alive and healthy indefinitely. Deletion of the viral polymerase gene abolished cytotoxicity and reduced transgene expression to trace levels but left vectors still able to retrogradely infect projection neurons and express recombinases, allowing downstream expression of other transgene products such as fluorophores and calcium indicators. The morphology of retrogradely targeted cells appeared unperturbed at 1 year postinjection. Whole-cell patch-clamp recordings showed no physiological abnormalities at 8 weeks. Longitudinal two-photon structural and functional imaging in vivo, tracking thousands of individual neurons for up to 4 months, showed that transduced neurons did not die but retained stable visual response properties even at the longest time points imaged.
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http://dx.doi.org/10.1038/s41593-018-0091-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503322PMC
April 2018

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

Neuron 2017 Nov;96(3):542-557

Allen Institute for Brain Science, Seattle, WA 98109, USA.

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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http://dx.doi.org/10.1016/j.neuron.2017.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5689454PMC
November 2017

Reversing behavioural abnormalities in mice exposed to maternal inflammation.

Nature 2017 09 13;549(7673):482-487. Epub 2017 Sep 13.

McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

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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http://dx.doi.org/10.1038/nature23909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796433PMC
September 2017

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

Nature 2017 08 23;548(7669):582-587. Epub 2017 Aug 23.

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

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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http://dx.doi.org/10.1038/nature23663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5958617PMC
August 2017

Combining Optogenetics and Electrophysiology to Analyze Projection Neuron Circuits.

Cold Spring Harb Protoc 2016 10 3;2016(10). Epub 2016 Oct 3.

Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611.

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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http://dx.doi.org/10.1101/pdb.prot090084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476926PMC
October 2016

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

Nat Protoc 2016 Apr 3;11(4):634-54. Epub 2016 Mar 3.

Media Lab, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

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 rig for automated whole-cell patch clamping can be set up in 1 week. High-quality surgical preparation of mice takes ∼1 h, and each autopatching experiment can be carried out over periods lasting several hours. Autopatching should enable in vivo intracellular investigations to be accessible by a substantial number of neuroscience laboratories, and it enables labs that are already doing in vivo patch clamping to scale up their efforts by reducing training time for new lab members and increasing experimental durations by handling mentally intensive tasks automatically.
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http://dx.doi.org/10.1038/nprot.2016.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877510PMC
April 2016

A circuit mechanism for differentiating positive and negative associations.

Nature 2015 Apr;520(7549):675-8

The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

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, but the identifying features of these populations and the synaptic mechanisms of differentiating positive and negative emotional valence have remained unknown. Here we show that BLA neurons projecting to the nucleus accumbens (NAc projectors) or the centromedial amygdala (CeM projectors) undergo opposing synaptic changes following fear or reward conditioning. We find that photostimulation of NAc projectors supports positive reinforcement while photostimulation of CeM projectors mediates negative reinforcement. Photoinhibition of CeM projectors impairs fear conditioning and enhances reward conditioning. We characterize these functionally distinct neuronal populations by comparing their electrophysiological, morphological and genetic features. Overall, we provide a mechanistic explanation for the representation of positive and negative associations within the amygdala.
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http://dx.doi.org/10.1038/nature14366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418228PMC
April 2015

Concentration and purification of rabies viral and lentiviral vectors.

Cold Spring Harb Protoc 2015 Apr 1;2015(4):386-91. Epub 2015 Apr 1.

McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.

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. The final stocks are concentrated in volume by a factor of up to 1000, with higher expected purity than is obtained following previously published protocols for preparing G-deleted rabies viral vectors.
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http://dx.doi.org/10.1101/pdb.prot075887DOI Listing
April 2015

Lentiviral vectors for retrograde delivery of recombinases and transactivators.

Cold Spring Harb Protoc 2015 Apr 1;2015(4):368-74. Epub 2015 Apr 1.

Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;

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 used in reporter mice or in combination with a second virus to express "payload" transgenes at high levels. For retrograde infection of significant numbers of neurons, high virus titers are critical. Here we present reagents and a protocol for generating high-titer supernatants that can be concentrated 1000-fold for final titers in excess of 10(10) infectious units per milliliter. We demonstrate the usefulness of these vectors by selectively targeting corticothalamic and corticotectal neurons for high-level expression of a fluorophore in knock-in reporter mice.
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http://dx.doi.org/10.1101/pdb.prot075879DOI Listing
April 2015

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

Cold Spring Harb Protoc 2015 Apr 1;2015(4):375-85. Epub 2015 Apr 1.

Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.

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 for final titers in excess of 10(10) infectious units per milliliter.
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http://dx.doi.org/10.1101/pdb.prot072389DOI Listing
April 2015

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

Neuron 2014 Sep 28;83(6):1431-43. Epub 2014 Aug 28.

The Division of Neurophysiology, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK; Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, UK. Electronic address:

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) with modified rabies virus-based input mapping, we have determined the sensory response properties and upstream monosynaptic connectivity of cells mediating the CC or CT pathway. We show that CC-projecting cells encompass a broad spectrum of selectivity to stimulus orientation and are predominantly innervated by deep layer V1 neurons. In contrast, CT-projecting cells are ultrasparse firing, exquisitely tuned to orientation and direction information, and receive long-range input from higher cortical areas. This segregation in function and connectivity indicates that L6 microcircuits route specific contextual and stimulus-related information within and outside the cortical network.
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http://dx.doi.org/10.1016/j.neuron.2014.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175007PMC
September 2014

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

Nat Neurosci 2014 Feb 15;17(2):269-79. Epub 2013 Dec 15.

RIKEN-MIT Center for Neural Circuit Genetics at the Picower Institute for Learning and Memory, Department of Biology and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

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, have only recently come to light. Using highly cell type-specific transgenic mouse lines, optogenetics and patch-clamp recordings, we found that dentate gyrus cells, long believed to not project to CA2, send functional monosynaptic inputs to CA2 pyramidal cells through abundant longitudinal projections. CA2 innervated CA1 to complete an alternate trisynaptic circuit, but, unlike CA3, projected preferentially to the deep, rather than to the superficial, sublayer of CA1. Furthermore, contrary to existing knowledge, ECIII did not project to CA2. Our results allow a deeper understanding of the biology of learning and memory.
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http://dx.doi.org/10.1038/nn.3614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4004172PMC
February 2014

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

J Neurosci 2013 Sep;33(37):14889-98

University of Oregon, Institute of Neuroscience, Eugene, Oregon 97403, Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, Cambridge, Massachusetts 02139, The Scripps Research Institute, Department of Cell Biology, La Jolla, California 92037, and Norwegian University of Science and Technology, Kavli Institute for Systems Neuroscience and Centre for Neural Computation, 7491 Trondheim, Norway.

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 most clearly implicated in learning and memory, they also are "grid cells." Here we address the question of what kinds of inputs are specifically targeting these MEC-LII cells. Cell-specific infection of MEC-LII with recombinant rabies virus results in unambiguous labeling of monosynaptic inputs. Furthermore, ratios of labeled neurons in different regions are largely consistent between animals, suggesting that label reflects density of innervation. While the results mostly confirm prior anatomical work, they also reveal a novel major direct input to MEC-LII from hippocampal pyramidal neurons. Interestingly, the vast majority of these direct hippocampal inputs arise not from the major hippocampal subfields of CA1 and CA3, but from area CA2, a region that has historically been thought to merely be a transitional zone between CA3 and CA1. We confirmed this unexpected result using conventional tracing techniques in both rats and mice.
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http://dx.doi.org/10.1523/JNEUROSCI.1046-13.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771023PMC
September 2013

Axonal and subcellular labelling using modified rabies viral vectors.

Nat Commun 2013 ;4:2332

Department of Brain & Cognitive Sciences, Massachusetts Institute of Technology, 43 Vassar Street, Cambridge, Massachusetts 02139, USA.

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 highly enough to label fine axonal structure while also labelling or perturbing synapses. Their slow expression also makes them incompatible with retrograde and transsynaptic vectors, preventing powerful combinatorial experiments. Here we show that deletion-mutant rabies virus can be specifically targeted to cells local to an injection site, brightly labelling axons even when coexpressing two other transgenes. We demonstrate several novel capabilities: simultaneously labelling axons and presynaptic terminals, labelling both dendrites and postsynaptic densities, and simultaneously labelling a region's inputs and outputs using co-injected vectors.
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http://dx.doi.org/10.1038/ncomms3332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939574PMC
April 2014

Convergent cortical innervation of striatal projection neurons.

Nat Neurosci 2013 Jun 12;16(6):665-7. Epub 2013 May 12.

Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

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 pyramidal tract activation, arguing that these cortical networks innervate both striatal projection neurons.
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http://dx.doi.org/10.1038/nn.3397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4085670PMC
June 2013

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

J Neurosci 2012 May;32(20):7021-33

Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.

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 corticostriatal and corticospinal neurons, implicating them in mediating disynaptic recurrent, feedforward, and feedback inhibition within and across the two projection classes. Low-threshold-spiking (LTS) interneurons were instead strongly excited by descending interlaminar (vertical) input from layer 2/3 pyramidal neurons, implicating them in mediating disynaptic feedforward inhibition to both projection classes. Furthermore, in a novel pattern, lower layer 2/3 preferentially excited interneurons in one layer (5A/LTS) and excitatory neurons in another (5B/corticospinal). Thus, these inhibitory microcircuits in mouse motor cortex follow an orderly arrangement that is laminarly orthogonalized by interneuron-specific, projection-nonspecific connectivity.
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http://dx.doi.org/10.1523/JNEUROSCI.0011-12.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3377057PMC
May 2012

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

J Neurosci 2012 Apr;32(14):4992-5001

Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.

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 optogenetic technique based on retrograde transfection of neurons with deletion-mutant rabies virus encoding channelrhodopsin-2, and used this in conjunction with retrograde anatomical labeling to stimulate and record from identified projection neurons in mouse motor cortex. We also used paired recordings to measure unitary connections. Both corticospinal and callosally projecting corticostriatal neurons in layer 5B formed within-class (recurrent) connections, with higher connection probability among corticostriatal than among corticospinal neurons. In contrast, across-class connectivity was extraordinarily asymmetric, essentially unidirectional from corticostriatal to corticospinal. Corticostriatal neurons in layer 5A and corticocortical neurons (callosal projection neurons similar to corticostriatal neurons) similarly received a paucity of corticospinal input. Connections involving presynaptic corticostriatal neurons had greater synaptic depression, and those involving postsynaptic corticospinal neurons had faster decaying EPSPs. Consequently, the three connections displayed a diversity of dynamic properties reflecting the different combinations of presynaptic and postsynaptic projection neurons. Collectively, these findings delineate a four-way specialized excitatory microcircuit formed by corticospinal and corticostriatal neurons. The "rectifying" corticostriatal-to-corticospinal connectivity implies a hierarchical organization and functional compartmentalization of corticospinal activity via unidirectional signaling from higher-order (corticostriatal) to lower-order (corticospinal) output neurons.
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http://dx.doi.org/10.1523/JNEUROSCI.4759-11.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3329752PMC
April 2012

New technologies for imaging synaptic partners.

Curr Opin Neurobiol 2012 Feb 3;22(1):121-7. Epub 2012 Jan 3.

Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, 43 Vassar St., 46-5065, Cambridge, MA 02139, USA.

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 techniques that allow direct imaging of neural networks by specifically marking connected cells or their synapses.
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http://dx.doi.org/10.1016/j.conb.2011.12.001DOI Listing
February 2012

Transgenic targeting of recombinant rabies virus reveals monosynaptic connectivity of specific neurons.

J Neurosci 2010 Dec;30(49):16509-13

Department of Psychology, Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403, USA.

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. We used a novel combination of transgenic mouse technology and a recently developed tracing system based on rabies virus (Wickersham et al., 2007a,b) to overcome all three constraints. Because the virus requires transgene expression for both initial infection and subsequent retrograde transsynaptic infection, we created several lines of mice that express these genes in defined cell types using the tetracycline-dependent transactivator system (Mansuy and Bujard, 2000). Fluorescent labeling from viral replication is thereby restricted to defined neuronal cell types and their direct monosynaptic inputs. Because viral replication does not depend on transgene expression, it provides robust amplification of signal in presynaptic neurons regardless of input strength. We injected virus into transgenic crosses expressing the viral transgenes in specific cell types of the hippocampus formation to demonstrate cell-specific infection and monosynaptic retrograde transport of virus, which strongly labels even minor inputs. Such neuron-specific transgenic complementation of recombinant rabies virus holds great promise for obtaining cellular-resolution wiring diagrams of the mammalian CNS.
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http://dx.doi.org/10.1523/JNEUROSCI.2442-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3313909PMC
December 2010

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

Proc Natl Acad Sci U S A 2010 Dec 29;107(50):21848-53. Epub 2010 Nov 29.

Systems Neurobiology Laboratories, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

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. Investigators can use this system to elucidate the connections onto a desired cell type in a high-throughput manner, limited only by the availability of Cre mouse lines. This method allows for identification of circuits that would be extremely tedious or impossible to study with other methods and can be used to build subcircuit maps of inputs onto many different types of cells within the same brain region. Furthermore, by expressing various transgenes from the rabies genome, this system also has the potential to allow manipulation of targeted neuronal circuits without perturbing neighboring cells.
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http://dx.doi.org/10.1073/pnas.1011756107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003023PMC
December 2010

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

Nat Protoc 2010 Mar 4;5(3):595-606. Epub 2010 Mar 4.

Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

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. Here we present a detailed protocol for the production of high-titer and high-purity viral stocks, from initial generation of infectious virus from cDNA through amplification on complementing cell lines, pseudotyping if desired, purification by ultracentrifugation and titering. The procedure requires 3-4 weeks to complete.
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http://dx.doi.org/10.1038/nprot.2009.248DOI Listing
March 2010

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

Front Neural Circuits 2007 28;1. Epub 2008 Mar 28.

Systems Neurobiology Laboratories, The Salk Institute for Biological Studies, Neurosciences Graduate Program, University of California San Diego, La Jolla, CA, USA.

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. The complete dendritic arbors of labeled cells were reconstructed to allow for unambiguous classification of cell type. We confirmed that the tall-tufted pyramids project to the superior colliculus and thalamus and that short layer 5 pyramidal neurons project to contralateral cortex, as previously described. We found that tall-simple pyramidal neurons contribute to corticocortical connections. Axonal reconstructions show that corticocortical projection neurons have a large superficial axonal arborization locally, while the subcortically projecting neurons limit axonal arbors to the deep layers. Furthermore, reconstructions of local axons suggest that tall-simple cell axons have extensive lateral spread while those of the short pyramids are more columnar. These differences were revealed by the ability to completely label dendritic and axonal arbors in vivo and have not been apparent in previous studies using labeling in brain slices.
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http://dx.doi.org/10.3389/neuro.04.005.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2526280PMC
July 2011