Probes for TDTOMATO

Intermale aggression is used to establish social rank. Several neuronal populations have been implicated in aggression, but the circuit mechanisms that shape this innate behavior and coordinate its different components (including attack execution and reward) remain elusive. We show that dopamine transporter-expressing neurons in the hypothalamic ventral premammillary nucleus (PMvDAT neurons) organize goal-oriented aggression in male mice. Activation of PMvDATneurons triggers attack behavior; silencing these neurons interrupts attacks. Regenerative PMvDAT membrane conductances interacting with recurrent and reciprocal excitation explain how a brief trigger can elicit a long-lasting response (hysteresis). PMvDAT projections to the ventrolateral part of the ventromedial hypothalamic and the supramammillary nuclei control attack execution and aggression reward, respectively. Brief manipulation of PMvDAT activity switched the dominance relationship between males, an effect persisting for weeks. These results identify a network structure anchored in PMvDAT neurons that organizes aggressive behavior and, as a consequence, determines intermale hierarchy.

Past studies have suggested that non-neuronal brain cells express the leptin receptor. However, the identity and distribution of these leptin receptor-expressing non-neuronal brain cells remain debated. This study assessed the distribution of the long form of the leptin receptor (LepRb) in non-neuronal brain cells using a reporter mouse model in which LepRb-expressing cells are permanently marked by tdTomato fluorescent protein (LepRb-CretdTomato). Double immunohistochemistry revealed that, in agreement with the literature, the vast majority of tdTomato-tagged cells across the mouse brain were neurons (i.e., based on immunoreactivity for NeuN). Non-neuronal structures also contained tdTomato-positive cells, including the choroid plexus and the perivascular space of the meninges and, to a lesser extent, the brain. Based on morphological criteria and immunohistochemistry, perivascular cells were deduced to be mainly pericytes. Notably, tdTomato-positive cells were immunoreactive for vitronectin and platelet derived growth factor receptor beta (PDGFBR). In situ hybridization studies confirmed that most tdTomato-tagged perivascular cells were enriched in leptin receptor mRNA (all isoforms). Using qPCR studies, we confirmed that the mouse meninges were enriched in Leprb and, to a greater extent, the short isoforms of the leptin receptor. Interestingly, qPCR studies further demonstrated significantly altered expression for Vtn and Pdgfrb in the meninges and hypothalamus of LepRb-deficient mice. Collectively, our data demonstrate that the only intracranial non-neuronal cells that express LepRb in the adult mouse are cells that form the blood-brain barrier, including, most notably, meningeal perivascular cells. Our data suggest that pericytic leptin signaling plays a role in the integrity of the intracranial perivascular space and, consequently, may provide a link between obesity and numerous brain diseases.

The relationship between neuronal impulse activity and neurotransmitter release remains elusive. This issue is especially poorly understood in the neuroendocrine system, with its particular demands on periodically voluminous release of neurohormones at the interface of axon terminals and vasculature. A shortage of techniques with sufficient temporal resolution has hindered real-time monitoring of the secretion of the peptides that dominate among the neurohormones. The lactotropic axis provides an important exception in neurochemical identity, however, as pituitary prolactin secretion is primarily under monoaminergic control, via tuberoinfundibular dopamine (TIDA) neurons projecting to the median eminence (ME). Here, we combined optogenetic stimulation and fast-scan cyclic voltammetry to address dopamine release dynamics in the male mouse TIDA system. Imposing different discharge frequencies during brief (3 sec) stimulation of TIDA terminals in the ME revealed that dopamine output is maximal at 10 Hz, which was found to parallel the TIDA neuron action potential frequency distribution. Over more sustained stimulation periods (150 sec), maximal output occurred at 5 Hz. Application of the dopamine transporter blocker, methylphenidate, significantly increased dopamine levels in the ME, supporting a functional role of the transporter at the neurons' terminals. Lastly, TIDA neuron stimulation at the cell body yielded perisomatic release of dopamine, which may contribute to an ultra-fast negative feedback mechanism to constrain TIDA electrical activity. Together, these data shed light on how spiking patterns in the neuroendocrine system translate to vesicular release towards the pituitary and identify how dopamine dynamics are controlled in the TIDA system at different cellular compartments.SIGNIFICANCE STATEMENTA central question in neuroscience is the complex relationship between neuronal discharge activity and transmitter release. By combining optogenetic stimulation and voltammetry, we address this issue in dopamine neurons of the neuroendocrine system, which faces particular spatiotemporal demands on exocytotic release; large amounts of neurohormone need to be secreted into the portal capillaries with precise timing to adapt to physiological requirements. Our data show that release is maximal around the neurons' default firing frequency. We further provide support for functional dopamine transport at the neurovascular terminals, shedding light on a long-standing controversy about the existence of neuroendocrine transmitter reuptake. Finally, we show that dopamine release occurs also at the somatodendritic level, providing a substrate for an ultra-short autoregulatory feedback loop.