Super-resolution 2-photon microscopy reveals that the morphology of each dendritic spine correlates with diffusive but not synaptic properties

The structure of dendritic spines suggests a specialized function in compartmentalizing synaptic signals near active synapses. Indeed, theoretical and experimental analyses indicate that the diffusive resistance of the spine neck is sufficient to effectively compartmentalize some signaling molecules in a spine for the duration of their activated lifetime. Here we describe the application of 2-photon microscopy combined with stimulated emission depletion (STED-2P) to the biophysical study of the relationship between synaptic signals and spine morphology, demonstrating the utility of combining STED-2P with modern optical and electrophysiological techniques. Morphological determinants of fluorescence recovery time were identified and evaluated within the context of a simple compartmental model describing diffusive transfer between spine and dendrite. Correlations between the neck geometry and the amplitude of synaptic potentials and calcium transients evoked by 2-photon glutamate uncaging were also investigated

A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging

Neuromodulators have profound effects on behavior, but the dynamics of their intracellular effectors has remained unclear. Most neuromodulators exert their function via G-protein-coupled receptors (GPCRs). One major challenge for understanding neuromodulator action is the lack of dynamic readouts of the biochemical signals produced by GPCR activation. The adenylate cyclase/cyclic AMP/protein kinase A (PKA) module is a central component of such biochemical signaling. This module is regulated by several behaviorally important neuromodulator receptors. Furthermore, PKA activity is necessary for the induction of many forms of synaptic plasticity as well as for the formation of long-term memory. In order to monitor PKA activity in brain tissue, we have developed a 2-photon fluorescence lifetime imaging microscopy (2pFLIM) compatible PKA sensor termed FLIM-AKAR, which is based on the ratiometric FRET sensor AKAR3. FLIM-AKAR shows a large dynamic range and little pH sensitivity. In addition, it is a rapidly diffusible cytoplasmic protein that specifically reports net PKA activity in situ. FLIM-AKAR expresses robustly in various brain regions with multiple transfection methods, can be targeted to genetically identified cell types, and responds to activation of both endogenous GPCRs and spatial-temporally specific delivery of glutamate. Initial experiments reveal differential regulation of PKA activity across subcellular compartments in response to neuromodulator inputs. Therefore, the reporter FLIM-AKAR, coupled with 2pFLIM, enables the study of PKA activity in response to neuromodulator inputs in genetically identified neurons in the brain, and sheds light on the intracellular dynamics of endogenous GPCR activation

Dopaminergic neurons inhibit striatal output via non-canonical release of GABA

The substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) contain the two largest populations of dopamine (DA)-releasing neurons in the mammalian brain. These neurons extend elaborate projections in striatum, a large subcortical structure implicated in motor planning and reward-based learning. Phasic activation of dopaminergic neurons in response to salient or reward-predicting stimuli is thought to modulate striatal output via the release of DA to promote and reinforce motor action1–4. Here we show that activation of DA neurons in striatal slices rapidly inhibits action potential firing in both direct-and indirect-pathway striatal projection neurons (SPNs) through vesicular release of the inhibitory transmitter γ-aminobutyric acid (GABA). GABA is released directly from dopaminergic axons but in a manner that is independent of the vesicular GABA transporter VGAT. Instead GABA release requires activity of the vesicular monoamine transporter VMAT2, which is the vesicular transporter for DA. Furthermore, VMAT2 expression in GABAergic neurons lacking VGAT is sufficient to sustain GABA release. Thus, these findings expand the repertoire of synaptic mechanisms employed by DA neurons to influence basal ganglia circuits, reveal a novel substrate whose transport is dependent on VMAT2, and demonstrate that GABA can function as a bona fide co-transmitter in monoaminergic neurons

A Direct Projection from Mouse Primary Visual Cortex to Dorsomedial Striatum

The mammalian striatum receives inputs from many cortical areas, but the existence of a direct axonal projection from the primary visual cortex (V1) is controversial. In this study we use anterograde and retrograde tracing techniques to demonstrate that V1 directly innervates a topographically defined longitudinal strip of dorsomedial striatum in mice. We find that this projection forms functional excitatory synapses with direct and indirect pathway striatal projection neurons (SPNs) and engages feed-forward inhibition onto these cells. Importantly, stimulation of V1 afferents is sufficient to evoke phasic firing in SPNs. These findings therefore identify a striatal region that is functionally innervated by V1 and suggest that early visual processing may play an important role in striatal-based behaviors

CRISPR/Cas9-Mediated Gene Knock-Down in Post-Mitotic Neurons

The prokaryotic adaptive immune system CRISPR/Cas9 has recently been adapted for genome editing in eukaryotic cells. This technique allows for sequence-specific induction of double-strand breaks in genomic DNA of individual cells, effectively resulting in knock-out of targeted genes. It thus promises to be an ideal candidate for application in neuroscience where constitutive genetic modifications are frequently either lethal or ineffective due to adaptive changes of the brain. Here we use CRISPR/Cas9 to knock-out Grin1, the gene encoding the obligatory NMDA receptor subunit protein GluN1, in a sparse population of mouse pyramidal neurons. Within this genetically mosaic tissue, manipulated cells lack synaptic current mediated by NMDA-type glutamate receptors consistent with complete knock-out of the targeted gene. Our results show the first proof-of-principle demonstration of CRISPR/Cas9-mediated knock-down in neurons in vivo, where it can be a useful tool to study the function of specific proteins in neuronal circuits

Midbrain dopamine neurons sustain inhibitory transmission using plasma membrane uptake of GABA, not synthesis

Synaptic transmission between midbrain dopamine neurons and target neurons in the striatum is essential for the selection and reinforcement of movements. Recent evidence indicates that nigrostriatal dopamine neurons inhibit striatal projection neurons by releasing a neurotransmitter that activates GABAA receptors. Here, we demonstrate that this phenomenon extends to mesolimbic afferents, and confirm that the released neurotransmitter is GABA. However, the GABA synthetic enzymes GAD65 and GAD67 are not detected in midbrain dopamine neurons. Instead, these cells express the membrane GABA transporters mGAT1 (Slc6a1) and mGAT4 (Slc6a11) and inhibition of these transporters prevents GABA co-release. These findings therefore indicate that GABA co-release is a general feature of midbrain dopaminergic neurons that relies on GABA uptake from the extracellular milieu as opposed to de novo synthesis. This atypical mechanism may confer dopaminergic neurons the flexibility to differentially control GABAergic transmission in a target-dependent manner across their extensive axonal arbors. DOI: http://dx.doi.org/10.7554/eLife.01936.00

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 stimuli1,2. 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 learning3,4. These nuclei lack direct sensory input and are only loosely topographically organized5,6, 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 among 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