Application of Recombinant Rabies Virus to Xenopus Tadpole Brain.

The Xenopus laevis experimental system has provided significant insight into the development and plasticity of neural circuits. Xenopus neuroscience research would be enhanced by additional tools to study neural circuit structure and function. Rabies viruses are powerful tools to label and manipulate neural circuits and have been widely used to study mesoscale connectomics. Whether rabies virus can be used to transduce neurons and express transgenes in Xenopus has not been systematically investigated. Glycoprotein-deleted rabies virus transduces neurons at the axon terminal and retrogradely labels their cell bodies. We show that glycoprotein-deleted rabies virus infects local and projection neurons in the Xenopus tadpole when directly injected into brain tissue. Pseudotyping glycoprotein-deleted rabies with EnvA restricts infection to cells with exogenous expression of the EnvA receptor, TVA. EnvA pseudotyped virus specifically infects tadpole neurons with promoter-driven expression of TVA, demonstrating its utility to label targeted neuronal populations. Neuronal cell types are defined by a combination of features including anatomical location, expression of genetic markers, axon projection sites, morphology, and physiological properties. We show that driving TVA expression in one hemisphere and injecting EnvA pseudotyped virus into the contralateral hemisphere, retrogradely labels neurons defined by cell body location and axon projection site. Using this approach, rabies can be used to identify cell types in Xenopus brain and simultaneously to express transgenes which enable monitoring or manipulation of neuronal activity. This makes rabies a valuable tool to study the structure and function of neural circuits in Xenopus.Significance StatementStudies in Xenopus have contributed a great deal to our understanding of brain circuit development and plasticity, regeneration, and hormonal regulation of behavior and metamorphosis. Here, we show that recombinant rabies virus transduces neurons in the Xenopus tadpole, enlarging the toolbox that can be applied to studying Xenopus brain. Rabies can be used for retrograde labeling and expression of a broad range of transgenes including fluorescent proteins for anatomical tracing and studying neuronal morphology, voltage or calcium indicators to visualize neuronal activity, and photo- or chemosensitive channels to control neuronal activity. The versatility of these tools enables diverse experiments to analyze and manipulate Xenopus brain structure and function, including mesoscale connectivity.

Complementary Genetic Targeting and Monosynaptic Input Mapping Reveal Recruitment and Refinement of Distributed Corticostriatal Ensembles by Cocaine.

Drugs of abuse elicit powerful experiences that engage populations of neurons broadly distributed throughout the brain. To determine how synaptic connectivity is organized to enable robust communication between populations of drug-activated neurons, we developed a complementary targeting system for monosynaptic rabies virus (RV) tracing that identifies direct inputs to activated versus nonactivated neuronal populations. Analysis of over 100,000 synaptic input neurons demonstrated that cocaine-activated neurons comprise selectively connected but broadly distributed corticostriatal networks. Electrophysiological assays using optogenetics to stimulate activated versus nonactivated inputs revealed stronger synapses between coactivated cortical pyramidal neurons and neurons in the dorsal striatum (DS). Repeated cocaine exposure further enhanced the connectivity specifically between drug-activated neurons in the orbitofrontal cortex (OFC) and coactive DS neurons. Selective chemogenetic silencing of cocaine-activated OFC neurons or their terminals in the DS disrupted behavioral sensitization, demonstrating the utility of this methodology for identifying novel circuit elements that contribute to behavioral plasticity.

Brain-Wide Maps of Synaptic Input to Cortical Interneurons.

Cortical inhibition is mediated by diverse inhibitory neuron types that can each play distinct roles in information processing by virtue of differences in their input sources, intrinsic properties, and innervation targets. Previous studies in brain slices have demonstrated considerable cell-type specificity in laminar sources of local inputs. In contrast, little is known about possible differences in distant inputs to different cortical interneuron types. We used the monosynaptic rabies virus system, in conjunction with mice expressing Cre recombinase in either parvalbumin-positive, somatostatin-positive (SST+), or vasoactive intestinal peptide-positive (VIP+) neurons, to map the brain-wide input to the three major nonoverlapping classes of interneurons in mouse somatosensory cortex. We discovered that all three classes of interneurons received considerable input from known cortical and thalamic input sources, as well as from probable cholinergic cells in the basal nucleus of Meynert. Despite their common input sources, these classes differed in the proportion of long-distance cortical inputs originating from deep versus superficial layers. Similar to their laminar differences in local input, VIP+ neurons received inputs predominantly from deep layers while SST+ neurons received mostly superficial inputs. These classes also differed in the amount of input they received. Cortical and thalamic inputs were greatest onto VIP+ interneurons and smallest onto SST+ neurons. SIGNIFICANCE STATEMENT: These results indicate that all three major interneuron classes in the barrel cortex integrate both feedforward and feedback information from throughout the brain to modulate the activity of the local cortical circuit. However, differences in laminar sources and magnitude of distant cortical input suggest differential contributions from cortical areas. More input to vasoactive intestinal peptide-positive (VIP+) neurons than to somatostatin-positive (SST+) neurons suggests that disinhibition of the cortex via VIP+ cells, which inhibit SST+ cells, might be a general feature of long-distance corticocortical and thalamocortical circuits.

Diversity of transgenic mouse models for selective targeting of midbrain dopamine neurons.

Ventral tegmental area (VTA) dopamine (DA) neurons have been implicated in reward, aversion, salience, cognition, and several neuropsychiatric disorders. Optogenetic approaches involving transgenic Cre-driver mouse lines provide powerful tools for dissecting DA-specific functions. However, the emerging complexity of VTA circuits requires Cre-driver mouse lines that restrict transgene expression to a precisely defined cell population. Because of recent work reporting that VTA DA neurons projecting to the lateral habenula release GABA, but not DA, we performed an extensive anatomical, molecular, and functional characterization of prominent DA transgenic mouse driver lines. We find that transgenes under control of the tyrosine hydroxylase, but not the dopamine transporter, promoter exhibit dramatic non-DA cell-specific expression patterns within and around VTA nuclei. Our results demonstrate how Cre expression in unintentionally targeted cells in transgenic mouse lines can confound the interpretation of supposedly cell-type-specific experiments. This Matters Arising paper is in response to Stamatakis et al. (2013), published in Neuron. See also the Matters Arising Response paper by Stuber et al. (2015), published concurrently with this Matters Arising in Neuron.

Characterization of long descending premotor propriospinal neurons in the spinal cord.

The motor function of the spinal cord requires the computation of the local neuronal circuits within the same segments as well as the long-range coordination of different spinal levels. Implicated players in this process are the propriospinal neurons (PPNs) that project their axons across different levels of the spinal cord. However, their cellular, molecular, and functional properties remain unknown. Here we use a recombinant rabies virus-based method to label a specific type of long-projecting premotor PPNs in the mouse upper spinal cord that are monosynaptically connected to the motor neurons in the lumbar spinal cord. With a whole spinal cord imaging method, we find that these neurons are distributed along the entire length of the upper spinal cord with more in the lower thoracic levels. Among them, a subset of thoracic PPNs receive substantial numbers of sensory inputs, suggesting a function in coordinating the activity of trunk and hindlimb muscles. Although many PPNs in the cervical and thoracic spinal cord receive the synaptic inputs from corticospinal tract or serotonergic axons, limited bouton numbers suggested that these supraspinal inputs might not be major regulators of the PPNs in intact animals. Molecularly, these PPNs appear to be distinct from other known premotor interneurons, but some are derived from Chx10+ lineages. This study provides an anatomical basis for further exploring different functions of PPNs.

Differential innervation of direct- and indirect-pathway striatal projection neurons.

The striatum integrates information from multiple brain regions to shape motor learning. The two major projection cell types in striatum target different downstream basal ganglia targets and have opposing effects on motivated behavior, yet differential innervation of these neuronal subtypes is not well understood. To examine whether input specificity provides a substrate for information segregation in these circuits, we used a monosynaptic rabies virus system to generate brain-wide maps of neurons that form synapses with direct- or indirect-pathway striatal projection neurons. We discovered that sensory cortical and limbic structures preferentially innervated the direct pathway, whereas motor cortex preferentially targeted the indirect pathway. Thalamostriatal input, dopaminergic input, as well as input from specific cortical layers, was similar onto both pathways. We also confirm synaptic innervation of striatal projection neurons by the raphe and pedunculopontine nuclei. Together, these findings provide a framework for guiding future studies of basal ganglia circuit function.

Cortical representations of olfactory input by trans-synaptic tracing.

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, number and type of 'starter' cortical neurons, from which we trace their presynaptic neurons. We find that individual cortical neurons receive input from multiple mitral cells representing broadly distributed glomeruli. Different cortical areas represent the olfactory bulb input differently. For example, the cortical amygdala preferentially receives dorsal olfactory bulb input, whereas the piriform cortex samples the whole olfactory bulb without obvious bias. These differences probably reflect different functions of these cortical areas in mediating innate odour preference or associative memory. The trans-synaptic labelling method described here should be widely applicable to mapping connections throughout the mouse nervous system.

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

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.

Genetic dissection of an amygdala microcircuit that gates conditioned fear.

The role of different amygdala nuclei (neuroanatomical subdivisions) in processing Pavlovian conditioned fear has been studied extensively, but the function of the heterogeneous neuronal subtypes within these nuclei remains poorly understood. Here we use molecular genetic approaches to map the functional connectivity of a subpopulation of GABA-containing neurons, located in the lateral subdivision of the central amygdala (CEl), which express protein kinase C-δ (PKC-δ). Channelrhodopsin-2-assisted circuit mapping in amygdala slices and cell-specific viral tracing indicate that PKC-δ(+) neurons inhibit output neurons in the medial central amygdala (CEm), and also make reciprocal inhibitory synapses with PKC-δ(-) neurons in CEl. Electrical silencing of PKC-δ(+) neurons in vivo suggests that they correspond to physiologically identified units that are inhibited by the conditioned stimulus, called CEl(off) units. This correspondence, together with behavioural data, defines an inhibitory microcircuit in CEl that gates CEm output to control the level of conditioned freezing.

Regulation of dendritic protein synthesis by miniature synaptic events.

We examined dendritic protein synthesis after a prolonged blockade of action potentials alone and after a blockade of both action potentials and miniature excitatory synaptic events (minis). Relative to controls, dendrites exposed to a prolonged blockade of action potentials showed diminished protein synthesis. Dendrites in which both action potentials and minis were blocked showed enhanced protein synthesis, suggesting that minis inhibit dendritic translation. When minis were acutely blocked or stimulated, an immediate increase or decrease, respectively, in dendritic translation was observed. Taken together, these results reveal a role for miniature synaptic events in the acute regulation of dendritic protein synthesis in neurons.