Arpiar Saunders

A direct GABAergic output from the basal ganglia to frontal cortex

The basal ganglia are phylogenetically conserved subcortical nuclei necessary for coordinated motor action and reward learning. Current models postulate that the basal ganglia modulate cerebral cortex indirectly via an inhibitory output to thalamus, bidirectionally controlled by direct- and indirect-pathway striatal projection neurons (dSPNs and iSPNs, respectively). The basal ganglia thalamic output sculpts cortical activity by interacting with signals from sensory and motor systems. Here we describe a direct projection from the globus pallidus externus (GP), a central nucleus of the basal ganglia, to frontal regions of the cerebral cortex (FC). Two cell types make up the GP–FC projection, distinguished by their electrophysiological properties, cortical projections and expression of choline acetyltransferase (ChAT), a synthetic enzyme for the neurotransmitter acetylcholine (ACh). Despite these differences, ChAT+ cells, which have been historically identified as an extension of the nucleus basalis, as well as ChAT− cells, release the inhibitory neurotransmitter GABA (γ-aminobutyric acid) and are inhibited by iSPNs and dSPNs of dorsal striatum. Thus, GP–FC cells comprise a direct GABAergic/cholinergic projection under the control of striatum that activates frontal cortex in vivo. Furthermore, iSPN inhibition of GP–FC cells is sensitive to dopamine 2 receptor signalling, revealing a pathway by which drugs that target dopamine receptors for the treatment of neuropsychiatric disorders can act in the basal ganglia to modulate frontal cortices.

Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors.

To understand how the nervous system processes information, a map of the connections among neurons would be of great benefit. Here we describe the use of vesicular stomatitis virus (VSV) for tracing neuronal connections in vivo. We made VSV vectors that used glycoprotein (G) genes from several other viruses. The G protein from lymphocytic choriomeningitis virus endowed VSV with the ability to spread transsynaptically, specifically in an anterograde direction, whereas the rabies virus glycoprotein gave a specifically retrograde transsynaptic pattern. The use of an avian G protein fusion allowed specific targeting of cells expressing an avian receptor, which allowed a demonstration of monosynaptic anterograde tracing from defined cells. Synaptic connectivity of pairs of virally labeled cells was demonstrated by using slice cultures and electrophysiology. In vivo infections of several areas in the mouse brain led to the predicted patterns of spread for anterograde or retrograde tracers.

Recurrent network activity drives striatal synaptogenesis

Neural activity during development critically shapes postnatal wiring of the mammalian brain. This is best illustrated by the sensory systems, in which the patterned feed-forward excitation provided by sensory organs and experience drives the formation of mature topographic circuits capable of extracting specific features of sensory stimuli. In contrast, little is known about the role of early activity in the development of the basal ganglia, a phylogenetically ancient group of nuclei fundamentally important for complex motor action and reward-based learning. These nuclei lack direct sensory input and are only loosely topographically organized, forming interlocking feed-forward and feed-back inhibitory circuits without laminar structure. Here we use transgenic mice and viral gene transfer methods to modulate neurotransmitter release and neuronal activity in vivo in the developing striatum. We find that the balance of activity between the two inhibitory and antagonist pathways in the striatum regulates excitatory innervation of the basal ganglia during development. These effects indicate that the propagation of activity through a multi-stage network regulates the wiring of the basal ganglia, revealing an important role of positive feedback in driving network maturation.

Corelease of acetylcholine and GABA from cholinergic forebrain neurons

Neurotransmitter corelease is emerging as a common theme of central neuromodulatory systems. Though corelease of glutamate or GABA with acetylcholine has been reported within the cholinergic system, the full extent is unknown. To explore synaptic signaling of cholinergic forebrain neurons, we activated choline acetyltransferase expressing neurons using channelrhodopsin while recording post-synaptic currents (PSCs) in layer 1 interneurons. Surprisingly, we observed PSCs mediated by GABAA receptors in addition to nicotinic acetylcholine receptors. Based on PSC latency and pharmacological sensitivity, our results suggest monosynaptic release of both GABA and ACh. Anatomical analysis showed that forebrain cholinergic neurons express the GABA synthetic enzyme Gad2 and the vesicular GABA transporter (Slc32a1). We confirmed the direct release of GABA by knocking out Slc32a1 from cholinergic neurons. Our results identify GABA as an overlooked fast neurotransmitter utilized throughout the forebrain cholinergic system. GABA/ACh corelease may have major implications for modulation of cortical function by cholinergic neurons. DOI: http://dx.doi.org/10.7554/eLife.06412.001

Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in neurons

Understanding the organization of the nervous system requires methods for dissecting the contributions of each component cell type to circuit function. One widely used approach combines genetic targeting of Cre recombinase to specific cell populations with infection of recombinant adeno-associated viruses (rAAVs) whose transgene expression is activated by Cre (“Cre-On”). Distinguishing how the Cre-expressing neurons differ functionally from neighboring Cre-negative neurons requires rAAVs that are inactivated by Cre (“Cre-Off”) and can be used in tandem with Cre-On viruses. Here we introduce two rAAV vectors that are inactivated by Cre and carry different fluorophore and optogenetic constructs. We demonstrate single and dual rAAV systems to achieve Cre-On and Cre-Off expression in spatially-intermingled cell populations of the striatum. Using these systems, we uncovered cryptic genomic interactions that occur between multiple Cre-sensitive rAAVs or between Cre-sensitive rAAVs and somatic Cre-conditional alleles and devised methods to avoid these interactions. Our data highlight both important experimental caveats associated with Cre-dependent rAAV use as well as opportunities for the development of improved rAAVs for gene delivery.

Vesicular stomatitis virus with the rabies virus glycoprotein directs retrograde transsynaptic transport among neurons in vivo

Defining the connections among neurons is critical to our understanding of the structure and function of the nervous system. Recombinant viruses engineered to transmit across synapses provide a powerful approach for the dissection of neuronal circuitry in vivo. We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins. Here we extend the characterization of the transmission and gene expression of recombinant VSV (rVSV) with the rabies virus glycoprotein (RABV-G), and provide examples of its activity relative to the anterograde transsynaptic tracer form of rVSV. rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner. Depending upon how the RABV-G was delivered, VSV served as a polysynaptic or monosynaptic tracer, or was able to define projections through axonal uptake and retrograde transport. In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas. rVSV with RABV-G provides a flexible, rapid, and versatile tracing tool that complements the previously described VSV-based anterograde transsynaptic tracer.

Genetically Distinct Parallel Pathways in the Entopeduncular Nucleus for Limbic and Sensorimotor Output of the Basal Ganglia

The basal ganglia (BG) integrate inputs from diverse sensorimotor, limbic, and associative regions to guide action-selection and goal-directed behaviors. The entopeduncular nucleus (EP) is a major BG output nucleus and has been suggested to channel signals from distinct BG nuclei to target regions involved in diverse functions. Here we use single-cell transcriptional and molecular analyses to demonstrate that the EP contains at least three classes of projection neurons-glutamate/GABA co-releasing somatostatin neurons, glutamatergic parvalbumin neurons, and GABAergic parvalbumin neurons. These classes comprise functionally and anatomically distinct output pathways that differentially affect EP target regions, such as the lateral habenula (LHb) and thalamus. Furthermore, LHb- and thalamic-projecting EP neurons are differentially innervated by subclasses of striatal and pallidal neurons. Therefore, we identify previously unknown subdivisions within the EP and reveal the existence of cascading, molecularly distinct projections through striatum and globus pallidus to EP targets within epithalamus and thalamus.

Heritability enrichment of specifically expressed genes identifies disease-relevant tissues and cell types

We introduce an approach to identify disease-relevant tissues and cell types by analyzing gene expression data together with genome-wide association study (GWAS) summary statistics. Our approach uses stratified linkage disequilibrium (LD) score regression to test whether disease heritability is enriched in regions surrounding genes with the highest specific expression in a given tissue. We applied our approach to gene expression data from several sources together with GWAS summary statistics for 48 diseases and traits (average N = 169,331) and found significant tissue-specific enrichments (false discovery rate (FDR) < 5%) for 34 traits. In our analysis of multiple tissues, we detected a broad range of enrichments that recapitulated known biology. In our brain-specific analysis, significant enrichments included an enrichment of inhibitory over excitatory neurons for bipolar disorder, and excitatory over inhibitory neurons for schizophrenia and body mass index. Our results demonstrate that our polygenic approach is a powerful way to leverage gene expression data for interpreting GWAS signals.A new method tests whether disease heritability is enriched near genes with high tissue-specific expression. The authors use gene expression data together with GWAS summary statistics for 48 diseases and traits to identify disease-relevant tissues.

Globus Pallidus Externus Neurons Expressing parvalbumin Interconnect the Subthalamic Nucleus and Striatal Interneurons

The globus pallidus externus (GP) is a nucleus of the basal ganglia (BG), containing GABAergic projection neurons that arborize widely throughout the BG, thalamus and cortex. Ongoing work seeks to map axonal projection patterns from GP cell types, as defined by their electrophysiological and molecular properties. Here we use transgenic mice and recombinant viruses to characterize parvalbumin expressing (PV+) GP neurons within the BG circuit. We confirm that PV+ neurons 1) make up ~40% of the GP neurons 2) exhibit fast-firing spontaneous activity and 3) provide the major axonal arborization to the STN and substantia nigra reticulata/compacta (SNr/c). PV+ neurons also innervate the striatum. Retrograde labeling identifies ~17% of pallidostriatal neurons as PV+, at least a subset of which also innervate the STN and SNr. Optogenetic experiments in acute brain slices demonstrate that the PV+ pallidostriatal axons make potent inhibitory synapses on low threshold spiking (LTS) and fast-spiking interneurons (FS) in the striatum, but rarely on spiny projection neurons (SPNs). Thus PV+ GP neurons are synaptically positioned to directly coordinate activity between BG input nuclei, the striatum and STN, and thalamic-output from the SNr.

Neuromodulation of excitatory synaptogenesis in striatal development

Dopamine is released in the striatum during development and impacts the activity of Protein Kinase A (PKA) in striatal spiny projection neurons (SPNs). We examined whether dopaminergic neuromodulation regulates activity-dependent glutamatergic synapse formation in the developing striatum. Systemic in vivo treatment with Gαs-coupled G-protein receptors (GPCRs) agonists enhanced excitatory synapses on direct pathway striatal spiny projection neurons (dSPNs), whereas rapid production of excitatory synapses on indirect pathway neurons (iSPNs) required the activation of Gαs GPCRs in SPNs of both pathways. Nevertheless, in vitro Gαs activation was sufficient to enhance spinogenesis induced by glutamate photolysis in both dSPNs and iSPNs, suggesting that iSPNs in intact neural circuits have additional requirements for rapid synaptic development. We evaluated the in vivo effects of enhanced glutamate release from corticostriatal axons and postsynaptic PKA and discovered a mechanism of developmental plasticity wherein rapid synaptogenesis is promoted by the coordinated actions of glutamate and postsynaptic Gαs-coupled receptors. DOI: http://dx.doi.org/10.7554/eLife.10111.001