The ventral pallidum (VP) lies at the interface between sensory, motor, and cognitive processing—with a particular role in mounting behavioral responses to rewards. Though the VP is predominantly GABAergic, glutamate neurons were recently identified, though their relative abundances and respective roles are unknown. Here, we show that VP glutamate neurons are concentrated in the rostral ventromedial VP and project to qualitatively similar targets as do VP GABA neurons. At the functional level, we used optogenetics to show that activity in VP GABA neurons can drive positive reinforcement, particularly through projections to the ventral tegmental area (VTA). On the other hand, activation of VP glutamate neurons leads to behavioral avoidance, particularly through projections to the lateral habenula. These findings highlight cell-type and projection-target specific roles for VP neurons in behavioral reinforcement, dysregulation of which could contribute to the emergence of negative symptoms associated with drug addiction and other neuropsychiatric disease.

In addition to dopamine neurons, the ventral tegmental area (VTA) contains GABA-, glutamate- and co-releasing neurons, and recent reports suggest a complex role for the glutamate neurons in behavioural reinforcement. We report that optogenetic stimulation of VTA glutamate neurons or terminals serves as a positive reinforcer on operant behavioural assays. Mice display marked preference for brief over sustained VTA glutamate neuron stimulation resulting in behavioural responses that are notably distinct from dopamine neuron stimulation and resistant to dopamine receptor antagonists. Whole-cell recordings reveal EPSCs following stimulation of VTA glutamate terminals in the nucleus accumbens or local VTA collaterals; but reveal both excitatory and monosynaptic inhibitory currents in the ventral pallidum and lateral habenula, though the net effects on postsynaptic firing in each region are consistent with the observed rewarding behavioural effects. These data indicate that VTA glutamate neurons co-release GABA in a projection-target-dependent manner and that their transient activation drives positive reinforcement.

Stopping or pausing in response to threats, conflicting information, or surprise is fundamental to behavior. Evidence across species has shown that the subthalamic nucleus (STN) is activated by scenarios involving stopping or pausing, yet evidence that the STN causally implements stops or pauses is lacking. Here we used optogenetics to activate or inhibit mouse STN to test its putative causal role. We first demonstrated that optogenetic stimulation of the STN excited its major projection targets. Next we showed that brief activation of STN projection neurons was sufficient to interrupt or pause a self-initiated bout of licking. Finally, we developed an assay in which surprise was used to interrupt licking, and showed that STN inhibition reduced the disruptive effect of surprise. Thus STN activation interrupts behavior, and blocking the STN blunts the interruptive effect of surprise. These results provide strong evidence that the STN is both necessary and sufficient for such forms of behavioral response suppression.DOI: http://dx.doi.org/10.7554/eLife.27689.001

Key points• Angiogenic regulators respond to acute exercise with different temporal expression patterns (e.g. 2-4 h versus 12-24 h) creating a complex multifaceted response that must be considered in studies using a single time point for post-exercise analyses.• In response to chronic training there appears to be a complex coordination in the proteomic responses of both positive and negative angiogenic factors that correspond with training-induced muscle capillary adaptation, such that altered basal expression and acute responses to exercise appear to withdraw or reduce the stimulus of angiogenic regulators in an expanding capillary bed with active angiogenesis.• These are the first data to show that nucleolin (a protein responsible for transcriptional processing and transportation of proteins from the cytoplasm to the nucleus) is responsive to acute exercise. We speculate that nucleolin may work in concert with vascular endothelial growth factor-A (VEGF) and endostatin.• Temporal responses observed in mice, particularly for VEGF, MMP-2 and MMP-9, may not be directly comparable to humans.Abstract Angiogenesis is controlled by a balance between positive and negative angiogenic factors, but temporal protein expression of many key angiogenic regulators in response to exercise are still poorly defined. In C57BL/6 mice, we evaluated the temporal protein expression of several pro-angiogenic and anti-angiogenic factors in response to (1) a single acute bout of exercise and (2) chronic exercise training resulting from 3, 5, 7, 14 and 28 days of voluntary wheel running. Following acute exercise, protein levels of vascular endothelial growth factor-A (VEGF), endostatin and nucleolin were increased at 2-4 h (P < 0.05), whereas matrix metalloproteinase (MMP)-2 was elevated within a 12-24 h window (P < 0.05). Training increased muscle capillarity 11%, 15% and 22% starting with 7, 14 and 28 days of training, respectively (P < 0.01). Basal VEGF and MMP-2 were increased by 31% and 22%, respectively, compared to controls (P < 0.05) after 7 days (7d) training, but decreased to back to baseline after 14d training. After 28d training VEGF fell 49% below baseline control (P < 0.01). Basal muscle expression of thrombospondin 1 (TSP-1) was ∼900% greater in 14d-and 28d-trained mice compared to either 5d-and 7d-trained mice (P < 0.05), and tended to increase by ∼180-258% compared to basal control levels (P < 0.10).The acute responsiveness of VEGF to exercise in untrained mice (i.e. 161% increase, P < 0.001) was lost with capillary adaptation occurring after 7, 14 and 28d training. Taken together, these data support the notion that skeletal muscle angiogenesis is controlled by a balance between positive and negative mitogens, and reveals a complex, highly-coordinated, temporal scheme whereby these factors can differentially influence capillary growth in response to acute versus chronic exercise.

Mutations in neurokinin B (NKB) and its receptor, NK3R, were identified in human patients with hypogonadotropic hypogonadism, a disorder characterized by lack of puberty and infertility. Further studies have suggested that NKB acts at the level of the hypothalamus to control GnRH neuron activity, either directly or indirectly. We recently reported that treatment with senktide, a NK3R agonist, induced GnRH secretion and expression of c-fos mRNA in GT1-7 cells. Here, we map the responsive region in the murine c-fos promoter to between -400 and -200 bp, identify the signal transducer and activator of transcription (STAT) (-345) and serum response element (-310) sites as required for induction, a modulatory role for the Ets site (-318), and show that induction is protein kinase C dependent. Using gel shift and Gal4 assays, we further show that phosphorylation of Elk-1 leads to binding to DNA in complex with serum response factor at serum response element and Ets sites within the c-fos promoter. Thus, we determine molecular mechanisms involved in NKB regulation of c-fos induction, which may play a role in modulation of GnRH neuron activation.

Stopping or pausing in response to threats, conflicting information, or surprise is fundamental to behavior. Evidence across species has shown that the subthalamic nucleus (STN) is activated by scenarios involving stopping or pausing, yet evidence that the STN causally implements stops or pauses is lacking. Here we used optogenetics to activate or inhibit mouse STN to test its putative causal role. We first demonstrated that optogenetic stimulation of the STN excited its major projection targets. Next we showed that brief activation of STN projection neurons was sufficient to interrupt or pause a self-initiated bout of licking. Finally, we developed an assay in which surprise was used to interrupt licking, and showed that STN inhibition reduced the disruptive effect of surprise. Thus STN activation interrupts behavior, and blocking the STN blunts the interruptive effect of surprise. These results provide strong evidence that the STN is both necessary and sufficient for such forms of behavioral response suppression.

Vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)‐2 and MMP‐9 are important in extracellular matrix remodeling and skeletal muscle angiogenesis in response to exercise. We evaluated protein expression of VEGF, MMP‐2 and MMP‐9 in mouse gastrocnemius muscle following 0, 3, 5, 7, 14, and 28 (d)ays of voluntary wheel running (n=12 per group). Protein expression was evaluated at either rest (basal, n=6) or after a single 1‐hour treadmill running bout (25 m/min at 10‐degrees, n=6) in each group. Compared to untrained controls, basal VEGF levels were significantly decreased by 36% at 3d, increased by 31% at 7d, and again reduced by 48% at 28d (p<0.05 respectively). Basal MMP‐2 protein expression was only reduced at 3d (36%, p<0.05), while MMP‐9 protein levels were decreased by 43% at 5d, 43% at 7d, and 34% at 14d (p<0.05 respectively). Basal MMP‐9 was not significantly altered at either 3d or 28d. VEGF response to acute exercise was increased in by 61% in control (p<0.05), 33% at 3d (p<0.05), and, to a lesser extent, in 5d (29%, p=0.08) trained mice, but absent after 7d of training. Neither MMP‐2 nor MMP‐9 showed a significant protein response to acute exercise. These data show that the VEGF protein response to acute exercise is blunted after 7 days of training, at a time point when training‐induced capillary adaptation is expected.