Input-specific control of reward and aversion in the ventral tegmental area

Ventral tegmental area (VTA) dopamine neurons have important roles in adaptive and pathological brain functions related to reward and motivation. However, it is unknown whether subpopulations of VTA dopamine neurons participate in distinct circuits that encode different motivational signatures, and whether inputs to the VTA differentially modulate such circuits. Here we show that, because of differences in synaptic connectivity, activation of inputs to the VTA from the laterodorsal tegmentum and the lateral habenula elicit reward and aversion in mice, respectively. Laterodorsal tegmentum neurons preferentially synapse on dopamine neurons projecting to the nucleus accumbens lateral shell, whereas lateral habenula neurons synapse primarily on dopamine neurons projecting to the medial prefrontal cortex as well as on GABAergic (γ-aminobutyric-acid-containing) neurons in the rostromedial tegmental nucleus. These results establish that distinct VTA circuits generate reward and aversion, and thereby provide a new framework for understanding the circuit basis of adaptive and pathological motivated behaviours.National Institutes of Health (U.S.) (Grant NIH NS069375)JPB FoundationNational Institute of Mental Health (U.S.

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

Striatal-projecting axons from <i>PV</i>⁺ GP neurons primarily innervate fast-spiking (FS) and low-threshold spiking (LTS) interneurons.

<p>(A) Schematic of strategy to target ChR2 to <i>PV</i>⁺ GP neurons. DIO rAAVs expressing Cre-On ChR2 fused to either mCherry or EYFP were injected into the GP of <i>PV</i> ^i-Cre mice, leading to ChR2⁺ axons in the striatum, STN and SNr. Dashed box shows approximate location of <i>PV</i>⁺ GP-striatal axon in (B). (B) Maximum projection 2-photon stacks of a ChR2-EYFP⁺ axon (white) imaged from an acute slice of <i>PV</i> ^i-Cre;Rosa26^lsl-tdTomato striatum following whole-cell recording and dialysis of tdTomato⁺ (red) interneuron with Alexa Fluor 594 (purple). Insets show high-magnification views of putative ChR2⁺ pre-synaptic terminals (yellow arrowheads) onto proximal dendrites and somata of un-recorded (i) and recorded (ii) tdTomato⁺ interneuron. (C) Light-evoked post-synaptic current (PSC) recorded in voltage-clamp (V_hold = -77 mV) from interneuron shown in (B). Arrows indicate onsets for the 1^st and 2^nd PSC peaks. (D) Example of light-evoked PSCs from a <i>PV</i>⁺ fast-spiking interneuron. This current has both a fast and slow peak and is blocked by bath application GABA_A receptor antagonist SR95331 (50 μM). (E) Maximum projection 2-photon stacks of five striatal cell types following whole-cell recording and dialysis of Alexa Fluor 594. Cell types are identified through the expression of molecular markers and/or the morphology of somata and dendrites. (F) Onset latencies for all recorded light-evoked PSC peaks color-coded by cell type. Open circles represent PSCs with a single peak. Filled circles represent the second PSC peak in cells with two peaks. PSCs that occurred ≤7 ms were classified as “fast”, >7 ms as “slow”. 7 ms represents the fastest “slow” (putative polysynaptic) response observed (green filled circle). (G) Examples of light-evoked PSCs by cell type. (H) Pie-charts illustrating the percentage of recorded neurons by cell type exhibiting either fast or slow PSC. Fractions illustrate the number of cells in which a PSC was detected over the total number of cells recorded. (I) Peak amplitudes for all fast PSCs by cell type. Black bars indicate mean values. Mean (± s.e.m) PSC peaks are larger in PV⁺ interneurons (-384 ± 156 pA) than NPY⁺ interneurons (-46 ± 12 pA). Asterisk, P < 0.05 (Mann-Whitney Test).</p

PV⁺ GP neurons innervate the striatum in addition to the STN and SNr.

<p>(A) Sagittal brain section from a <i>PV</i> ^i-Cre mouse injected in the GP with rAAVs DIO-EGFP (Cre-On, green) and FAS-tdTomato (Cre-Off, magenta). <i>Left</i>, injection site in GP. <i>Right</i>, low-magnification view. Look up table was changed to illustrate axonal projection patterns in low-magnification view. (B) Quantification of co-localization between GFP (green) and tdTomato (magenta) expressing GP neurons from confocal microscopy. <i>Top</i>, a single confocal plane. <i>Bottom</i>, summary graph of co-localization. All GFP expressing neurons are Cre⁺ (n = 76/285 cells tdTomato⁺ only; n = 134/285 cells GFP⁺ only; n = 75/385 cells double-positive, from n = 3 injections, from 2 mice). (C) Overlay of low-magnification sagittal sections from (A). <i>Top</i>, boxes indicate approximate regions of the frontal cortex (FC), striatum (Str) or subthalamic nucleus (STN, from an adjacent section) where GFP and/or tdTomato axonal fluorescence was quantified. <i>Bottom</i>, high magnification view of the striatum. (D) Relative expression of Cre⁺ and Cre^- axons in the FC, Str and STN following pixel binarization of GFP⁺ and tdTomato⁺ fluorescence. Since all GFP⁺ axons are Cre⁺, the relative abundance of Cre^-/Cre⁺ fibers is plotted as (tdTomato-GFP)/GFP pixels. The dotted line shows the relative proportion of Cre^-/Cre⁺ axons predicted for no selective innervation of different brain areas based on somatic co-localization quantified from the injection site (tdTomato only cells / (GFP only + tdTomato and GFP double-positive cells), from data in (B): 76/(134+76) = 0.364). Solid lines represent geometric means (n = 3 injections, from 2 mice). Innervation ratios were different across regions (P < 0.05, Kruskall-Wallis test). (E) Retrograde labeling of PV^- and PV⁺ GP somata. Sagittal section of wild-type mouse following injection of retro beads (magenta) in dorsal striatum and immunolabeling of PV (green). (F) Quantification of co-localization between retro bead⁺ (magenta) and PV⁺ (green) cells in the GP using confocal microscopy. <i>Left</i>, a single confocal plane. Bead⁺ cells are highlighted with circles. <i>Right</i>, summary graph of mean co-localization (from 4 mice). Error bars denote s.e.m. (G-I) Full anatomical visualization of PV⁺ GP-Str neurons following rabies-based retrograde labeling in <i>PV</i> ^i-Cre mice. (G) Experimental strategy to test whether PV⁺ GP-Str neurons also innervate the STN and SNr. First, rAAV DIO-TC^B is injected into the GP of <i>PV</i> ^i-Cre, allowing TC^B+ axons to be selectively transduced by EnvA pseudo-typed recombinant rabies virus (rRV). Second, replication-incompetent rRV EnvA-SADdG-EGFP is injected into the striatum 2 weeks after the rAAV injection. GFP⁺ axons in the STN and SNr indicate innervation by PV⁺ GP-Str neurons. (H) <i>Top</i>, low magnification sagittal section from an example double-recombinant viral experiment described above. TC^B+ expression is restricted to Cre⁺ neurons of the GP and reticular nucleus of the thalamus (RT). The vast majority of GFP⁺ somata were observed in the GP. Additionally, a sparse number of GFP⁺ somata with glial morphology or aspiny dendrites were also present in the striatum. GFP⁺ axons were clearly visible in the striatum, as well as the STN and SNr. <i>Below</i>, higher magnification view of the STN and SNr from the section above. Similar labeling was observed in n = 6/6 injections from 3 mice and n = 0/4 control injections from 2 mice in which DIO-TC^B was replaced by DIO-mCherry. (I) A single confocal plane of the GP from the experiment shown, demonstrating that TC^B+/GFP⁺ GP neurons are also immuno-positive for PV.</p

<i>Parvalbumin</i> (<i>PV</i>) expression defines a physiologically distinct cell type in the GP.

<p>(A) <i>Left</i>, sagittal brain section from a <i>PV</i> ^i-Cre;Rosa26^tdTomato mouse where Cre expression is reported by tdTomato (magenta) followed by immunolabeling for PV (green). <i>Right</i>, high magnification of boxed area containing the GP. (B) Quantification of co-localization between tdTomato (magenta) and immunolabeled PV (green) in the GP using confocal microscopy. <i>Left</i>, a single confocal plane. <i>Right</i>, summary graph of co-localization results (n = 24/679 cells tdTomato⁺ only; n = 184/679 cells PV⁺ only; n = 471/679 cells double-positive, from 2 mice). (C) Horizontal section of <i>PV</i> ^i-Cre;Rosa26^tdTomato mouse where Cre expression is reported by tdTomato (magenta) followed by immunolabeling for NeuN (green). (D) Quantification of the percentage of NeuN⁺ neurons with tdTomato reporter expression along the medial-lateral axis of the GP. <i>Left</i>, schematic of GP in the horizontal plane. Dashed lines delineate approximate groupings of medial, central and lateral sagittal sections. RT, reticular nucleus of the thalamus. <i>Right</i>, summary graph of mean tdTomato/NeuN percent co-localization based on sagittal sections (from 3 mice, 3 replicates/mouse). Error bars denote s.e.m. (E) Acute sagittal slice of <i>PV</i> ^i-Cre;Rosa26^tdTomato mouse brain showing cell-attached recordings from tdTomato⁺ and tdTomato^- GP neurons. DIC image (gray) overlaid with tdTomato fluorescence (magenta). (F) Example cell-attached recordings showing action current deflections indicative of spontaneously active (<i>top</i>) and non-spontaneously active (<i>bottom</i>) GP neurons. (G) Summary graph showing the percent tdTomato⁺ and tdTomato^- GP neurons exhibiting cell-attached spontaneous activity (n = 29/30 <i>PV</i>⁺, n = 15/41 <i>PV</i>^- cells; from 3 mice). Asterisk, <i>P</i><0.05 (Fisher’s Exact test). (H) Cumulative frequency distribution plot of firing rates from spontaneously active tdTomato⁺ and tdTomato^- GP neurons. Active tdTomato⁺ exhibit faster firing rates than tdTomato^- neurons (tdTomato⁺: mean_FR = 54 Hz, median_FR = 50; TdTomato^-: mean_FR = 27 Hz; P < 0.05 Mann-Whitney Test).</p

Molecular markers distinguish low-threshold spiking (LTS) and fast-spiking (FS) striatal interneurons from spiny projection neurons (SPNs).

<p>(A) <i>Top</i>, sagittal brain sections from transgenic mice used to distinguish striatal cell types through selective expression of fluorescent proteins. The dopamine 2 receptor BAC (<i>D2R</i>-EGFP) drives EGFP selectively iSPNs, while the Neuropeptide Y BAC (<i>NPY</i>-EGFP) drives EGFP selectively in putative NPY⁺ LTS interneurons and a smaller number of NPY⁺ neurogliaform interneurons[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149798#pone.0149798.ref051" target="_blank">51</a>]. The <i>PV</i> ^i-Cre;Rosa26^lsl-tdTomato mouse expresses tdTomato in <i>PV</i>⁺ putative FS interneurons. <i>Bottom</i>, example membrane potential recordings for each fluorescently labeled cell type in response to current injections (500 ms, SPN: 200 pA; LTS: 400 pA; FS: 1500 pA). (B) Three-dimensional scatter plot of membrane capacitance (C_m), maximum firing rate (Max FR) and resting membrane potential (V_rest) for striatal cell types color-coded by expressed molecular marker.</p

Electrophysiological properties of striatal cell types.

<p>Electrophysiological properties of striatal cell types.</p

Social isolation uncovers a circuit underlying context-dependent territory-covering micturition.

The release of urine, or micturition, serves a fundamental physiological function and, in many species, is critical for social communication. In mice, the pattern of urine release is modulated by external and internal factors and transmitted to the spinal cord via the pontine micturition center (PMC). Here, we exploited a behavioral paradigm in which mice, depending on strain, social experience, and sensory context, either vigorously cover an arena with small urine spots or deposit urine in a few isolated large spots. We refer to these micturition modes as, respectively, high and low territory-covering micturition (TCM) and find that the presence of a urine stimulus robustly induces high TCM in socially isolated mice. Comparison of the brain networks activated by social isolation and by urine stimuli to those upstream of the PMC identified the lateral hypothalamic area as a potential modulator of micturition modes. Indeed, chemogenetic manipulations of the lateral hypothalamus can switch micturition behavior between high and low TCM, overriding the influence of social experience and sensory context. Our results suggest that both inhibitory and excitatory signals arising from a network upstream of the PMC are integrated to determine context- and social-experience-dependent micturition patterns

Central Control Circuit for Context-Dependent Micturition

Summary Urine release (micturition) serves an essential physiological function as well as a critical role in social communication in many animals. Here, we show a combined effect of olfaction and social hierarchy on micturition patterns in adult male mice, confirming the existence of a micturition control center that integrates pro- and anti-micturition cues. Furthermore, we demonstrate that a cluster of neurons expressing corticotropin-releasing hormone (Crh) in the pontine micturition center (PMC) is electrophysiologically distinct from their Crh-negative neighbors and sends glutamatergic projections to the spinal cord. The activity of PMC Crh-expressing neurons correlates with and is sufficient to drive bladder contraction, and when silenced impairs micturition behavior. These neurons receive convergent input from widespread higher brain areas that are capable of carrying diverse pro- and anti-micturition signals, and whose activity modulates hierarchy-dependent micturition. Taken together, our results indicate that PMC Crh-expressing neurons are likely the integration center for context-dependent micturition behavior