Biology subjects

Sullivan, H. A.

Publications and source records attributed to Sullivan, H. A..

5 recordsLinked to original sources

bioRxiv2022

Single-deletion-mutant, third-generation rabies viral vectors allow nontoxic retrograde targeting of projection neurons with greatly increased efficiency

Rabies viral vectors have become important components of the systems neuroscience toolkit, allowing both direct retrograde targeting of projection neurons and monosynaptic tracing of inputs to defined postsynaptic populations, but the rapid cytotoxicity of first-generation ({Delta}G) vectors limits their use to short-term experiments. We recently introduced second-generation, double-deletion-mutant ({Delta}GL) rabies viral vectors, showing that they efficiently retrogradely infect projection neurons and express recombinases effectively but with little to no detectable toxicity; more recently, we have shown that {Delta}GL viruses can be used for monosynaptic tracing with far lower cytotoxicity than the first-generation system. Here we introduce third-generation ({Delta}L) rabies viral vectors, which, like first-generation vectors, have only a single gene deleted from their genomes (in this case the viral polymerase gene L) but which appear to be as nontoxic as second-generation ones, based on in vivo longitudinal structural and functional two-photon imaging and ex vivo electrophysiology. Although third-generation vectors are therefore phenotypically very similar to second-generation ones, we show that they have the major advantage of growing to much higher titers, and this key difference results in significantly increased numbers of retrogradely labeled neurons in vivo. These {Delta}L rabies viral vectors therefore constitute a new state of the art for minimally perturbative, pathway-specific expression of recombinases and transactivators in mammalian neurons selected on the basis of their axonal projections. Because replication of deletion-mutant rabies viruses within complementing cells is precisely the process that underlies monosynaptic tracing, the higher replication efficiency of this new class of rabies viral vectors furthermore suggests the potential to provide the foundation of an improved nontoxic monosynaptic tracing system.

neuroscience↗

bioRxiv2021

Long-term labeling and imaging of synaptically-connected neuronal networks in vivo using nontoxic, double-deletion-mutant rabies viruses

Monosynaptic tracing is a widely-used technique for mapping neural circuitry, but its cytotoxicity has confined it primarily to anatomical applications. Here we present a second-generation system for labeling direct inputs to targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Spread of the viruses requires expression of both deleted viral genes in trans in postsynaptic source cells; suppressing this expression with doxycycline following an initial period of viral replication reduces toxicity to postsynaptic cells. Longitudinal two-photon imaging in vivo indicated that over 90% of both presynaptic and source cells survived for the full twelve-week course of imaging. Ex vivo whole-cell recordings at 5 weeks postinfection showed that the second-generation system perturbs input and source cells much less than does the first-generation system. Finally, two-photon calcium imaging of labeled networks of visual cortex neurons showed that their visual response properties appeared normal for 10 weeks, the longest we followed them.

neuroscience↗

bioRxiv2021

Cingulate-motor circuits update rule representations for sequential choice decisions

Anterior cingulate cortex mediates the flexible updating of an animals choice responses upon rule changes in the environment. However, how anterior cingulate cortex entrains motor cortex to reorganize rule representations and generate required motor outputs remains unclear. Here, we demonstrate that chemogenetic silencing of the projection terminals of cingulate cortical neurons in secondary motor cortex disrupted sequential choice performance in trials immediately following rule switches, suggesting that these inputs are necessary to update rule representations for choice decisions stored in the motor cortex. Indeed, the silencing of cingulate cortex decreased rule selectivity of secondary motor cortical neurons. Furthermore, optogenetic silencing of cingulate cortical neurons that was temporally targeted to error trials immediately after rule switches exacerbated errors in following trials. These results suggest that cingulate cortex monitors behavioral errors and update rule representations in motor cortex, revealing a critical role for cingulate-motor circuits in adaptive choice behaviors.

neuroscience↗

bioRxiv2020

Dysregulation of hippocampal adult-born immature neurons disrupts a brain-wide network for spatial memory

Mounting evidence suggests that cognitive deficits associated with various neurological disorders may arise in part from a small population of dysregulated adult-born neurons in the dentate gyrus (DG). How these dysregulated adult-born neurons contribute to brain-wide network maladaptation and subsequent cognitive deficits remains unknown. Using an established mouse model with a small number of time-stamped dysregulated adult-born immature neurons and spatial memory deficits, we performed resting state functional magnetic resonance imaging and found that approximately 500 deficient immature neurons (<0.1% of total DG granule neurons) are sufficient to induce a significant decrease in the functional connectivity between DG and insular cortex (IC), two brain regions without direct anatomical connections. Furthermore, using a combination of rabies-based retrograde tracing and in vivo fiber photometry recording, we demonstrated that dysregulated adult-born neurons induce aberrant activity and synchrony in local hippocampal CA3 and CA1 regions, as well as distal medial-dorsal thalamus and IC regions during a spatial memory process. These results suggest that a few hundred dysregulated adult-born immature neurons can impact brain-wide network dynamics across several anatomically discrete regions and collectively contribute to impaired cognitive functions.

neuroscience↗

bioRxiv2019

\"Self-inactivating\" rabies viruses are just first-generation, ΔG rabies viruses

Monosynaptic tracing using rabies virus is an important technique in neuroscience, allowing brain- wide labeling of neurons directly presynaptic to a targeted neuronal population. A 2017 article reported development of a noncytotoxic version - a major advance - based on attenuating the rabies virus by addition of a destabilization domain to the C-terminus of a viral protein. However, this modification did not appear to hinder the ability of the virus to spread between neurons. We analyzed two viruses provided by the authors and show here that both were mutants that had lost the intended modification, explaining the papers paradoxical results. We then made a virus that actually did have the intended modification in at least the majority of virions and found that it did not spread efficiently under the conditions described in the original paper, namely, without an exogenous protease being expressed in order to remove the destabilization domain. We found that it did spread when the protease was supplied, although this also appeared to result in the deaths of most source cells by three weeks postinjection. We conclude that the new approach is not robust but that it could become a viable technique given further optimization and validation. SIGNIFICANCE STATEMENTRabies virus, which spreads between synaptically-connected neurons, has been one of the primary tools used by neuroscientists to reveal the organization of the brain. A new modification to rabies virus was recently reported to allow the mapping of connected neurons without adverse effects on the cells health, unlike earlier versions. Here we show that the conclusions of that study were probably incorrect and based on having used viruses that had lost the intended modification because of mutations. We also show that a rabies virus that does retain the intended modification does not spread efficiently between neurons under the conditions reported previously; however, it does spread between neurons under different conditions, suggesting that the approach may be successful if refined further.

neuroscience↗