Probes for INS

The periaqueductal gray (PAG) is a significant modulator of both analgesic and fear behaviors in both humans and rodents, but the underlying circuitry responsible for these two phenotypes is incompletely understood. Importantly, it is not known if there is a way to produce analgesia without anxiety by targeting the PAG, as modulation of glutamate or GABA neurons in this area initiates both antinociceptive and anxiogenic behavior. While dopamine (DA) neurons in the ventrolateral PAG (vlPAG) /dorsal raphe display a supraspinal antinociceptive effect, their influence on anxiety and fear are unknown. Using DAT-cre and Vglut2-cre male mice, we introduced Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to DA and glutamate neurons within the vlPAG using viral-mediated delivery and found that levels of analgesia were significant and quantitatively similar when DA and glutamate neurons were selectively stimulated. Activation of glutamatergic neurons, however, reliably produced higher indices of anxiety, with increased freezing time and more time spent in the safety of a dark enclosure. In contrast, animals in which PAG/dorsal raphe DA neurons were stimulated failed to show fear behaviors. DA-mediated antinociception was inhibitable by haloperidol and was sufficient to prevent persistent inflammatory pain induced by carrageenan. In summary, only activation of DA neurons in the PAG/dorsal raphe produced profound analgesia without signs of anxiety, indicating that PAG/dorsal raphe DA neurons are an important target involved in analgesia that may lead to new treatments for pain.

Significance Statement Clinicians have long had the goal of separating analgesia from anxiety when using deep brain electrical stimulation of the periaqueductal gray (PAG) for difficult to treat pain. Here we show that selective activation of dopamine neurons within the PAG produces analgesia without other behavioral effects, while stimulating glutamate neurons mediates stress-induced anxiety and analgesia. Our results suggest that dopamine agonists may represent a novel class of analgesic drugs and elucidate target neurons that could mediate their effect.

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.

The median preoptic nucleus (MnPO) is a putative integrative region that contributes to body fluid balance. Activation of the MnPO can influence thirst but it is not clear how these responses are linked to body fluid homeostasis. We used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to determine the role of the MnPO in drinking behavior and vasopressin release in response to peripheral angiotensin II (ANG II) or 3% hypertonic saline in adult male Sprague-Dawley rats (250-300g). Rats were anesthetized with isoflurane and stereotaxically injected with an inhibitory DREADD (rAAV5-CaMKIIa-hM4D(Gi)-mCherry) or control (rAAV5-CaMKIIa-mCherry) virus in the MnPO. After 2 weeks’ recovery, a subset of rats were used for extracellular recordings to verify functional effects of ANG II or hyperosmotic challenges in MnPO slice preparations. Remaining rats were used in drinking behavior studies. Each rat was administered either 10mg/kg of exogenous clozapine-N-oxide (CNO) to inhibit DREADD-expressing cells or vehicle ip followed by a test treatment with either 2mg/kg ANG II or 3% hypertonic saline (1mL/100g bw) sc, twice per week for two separate treatment weeks. CNO-induced inhibition during either test treatment significantly attenuated drinking responses compared to vehicle treatments and controls. Brain tissue processed for cFos immunohistochemistry showed decreased expression with CNO-induced inhibition during either test treatment in the MnPO and downstream nuclei compared to controls. CNO-mediated inhibition significantly attenuated treatment-induced increases in plasma vasopressin compared to controls. The results indicate inhibition of CaMKIIa-expressing MnPO neurons significantly reduces drinking and vasopressin release in response to ANG II or hyperosmotic challenge.

Significance Statement The MnPO is an important regulatory center that influences thirst, cardiovascular and neuroendocrine function. Activation of different MnPO neuronal populations can inhibit or stimulate water intake. However, the role of the MnPO and its pathway-specific projections during ANG II and hyperosmotic challenges still have not yet been fully elucidated. These studies directly address this by using DREADDs to acutely and selectively inhibit pathway-specific MnPO neurons, and uses techniques that measure changes at the protein, neuronal, and overall physiological and behavioral level. More importantly, we have been able to demonstrate that physiological challenges related to extracellular (ANG II) or cellular (hypertonic saline) dehydration activate MnPO neurons that may project to different parts of the hypothalamus.

The release of dopamine (DA) regulates rewarding behavior and motor actions through striatum-targeting efferents from ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Here, we map and functionally characterize axonal projections from oxytocin neurons in the hypothalamic paraventricular nucleus to midbrain DA regions. Electrophysiological recordings of DA neurons reveal that both the application of oxytocin and optogenetic stimulation of oxytocinergic terminals suffice to increase DA neuron activity in the VTA but downregulate it in SNc. This biased modulation is mediated by oxytocin and vasopressin G-protein-coupled receptors. Oxytocin release directly activates DA neurons and indirectly inhibits them through local GABA neurons, but the relative magnitudes of the two mechanisms differ in VTA and SNc. Oxytocin-modulated DA neurons give rise to canonical striatal projections. Since hypothalamic oxytocinergic projections also target the striatum, oxytocin is poised to bias the balance of DA tone through multiple sites in vertebrate reward circuits.

Sensory experience influences the establishment of neural connectivity through molecular mechanisms that remain unclear. Here, we employ single-nucleus RNA sequencing to investigate the contribution of sensory-driven gene expression to synaptic refinement in the dorsal lateral geniculate nucleus of the thalamus, a region of the brain that processes visual information. We find that visual experience induces the expression of the cytokine receptor Fn14 in excitatory thalamocortical neurons. By combining electrophysiological and structural techniques, we show that Fn14 is dispensable for early phases of refinement mediated by spontaneous activity but that Fn14 is essential for refinement during a later, experience-dependent period of development. Refinement deficits in mice lacking Fn14 are associated with functionally weaker and structurally smaller retinogeniculate inputs, indicating that Fn14 mediates both functional and anatomical rearrangements in response to sensory experience. These findings identify Fn14 as a molecular link between sensory-driven gene expression and vision-sensitive refinement in the brain.