Parabrachial Calca neurons drive nociplasticity.

Pain that persists beyond the time required for tissue healing and pain that arises in the absence of tissue injury, collectively referred to as nociplastic pain, are poorly understood phenomena mediated by plasticity within the central nervous system. The parabrachial nucleus (PBN) is a hub that relays aversive sensory information and appears to play a role in nociplasticity. Here, by preventing PBN Calca neurons from releasing neurotransmitters, we demonstrate that activation of Calca neurons is necessary for the manifestation and maintenance of chronic pain. Additionally, by directly stimulating Calca neurons, we demonstrate that Calca neuron activity is sufficient to drive nociplasticity. Aversive stimuli of multiple sensory modalities, such as exposure to nitroglycerin, cisplatin, or lithium chloride, can drive nociplasticity in a Calca-neuron-dependent manner. Aversive events drive nociplasticity in Calca neurons in the form of increased activity and excitability; however, neuroplasticity also appears to occur in downstream circuitry.

Parabrachial Calca neurons drive nociplasticity.

Pain that persists beyond the time required for tissue healing and pain that arises in the absence of tissue injury are poorly understood phenomena mediated by plasticity within the central nervous system. The parabrachial nucleus (PBN) is a hub that relays aversive sensory information and appears to play a role in nociplasticity. Here, by preventing PBN Calca neurons from releasing neurotransmitter or directly stimulating them we demonstrate that activation of Calca neurons is both necessary for the manifestation of chronic pain after nerve ligation and is sufficient to drive nociplasticity in wild-type mice. Aversive stimuli such as exposure to nitroglycerin, cisplatin, or LiCl can drive nociplasticity in a Calca-neuron-dependent manner. Calcium fluorescence imaging reveals that nitroglycerin activates PBN Calca neurons and potentiates their responses to mechanical stimulation. The activity and excitability of Calca neurons increased for several days after aversive events, but prolonged nociplasticity likely occurs in downstream circuitry.

Novel genetically encoded tools for imaging or silencing neuropeptide release from presynaptic terminals in vivo.

Neurons produce and release neuropeptides to communicate with one another. Despite their profound impact on critical brain functions, circuit-based mechanisms of peptidergic transmission are poorly understood, primarily due to the lack of tools for monitoring and manipulating neuropeptide release in vivo. Here, we report the development of two genetically encoded tools for investigating peptidergic transmission in behaving mice: a genetically encoded large dense core vesicle (LDCV) sensor that detects the neuropeptides release presynaptically, and a genetically encoded silencer that specifically degrades neuropeptides inside the LDCV. Monitoring and silencing peptidergic and glutamatergic transmissions from presynaptic terminals using our newly developed tools and existing genetic tools, respectively, reveal that neuropeptides, not glutamate, are the primary transmitter in encoding unconditioned stimulus during Pavlovian threat learning. These results show that our sensor and silencer for peptidergic transmission are reliable tools to investigate neuropeptidergic systems in awake behaving animals.

Molecular and anatomical characterization of parabrachial neurons and their axonal projections.

The parabrachial nucleus (PBN) is a major hub that receives sensory information from both internal and external environments. Specific populations of PBN neurons are involved in behaviors including food and water intake, nociceptive responses, breathing regulation, as well as learning and responding appropriately to threatening stimuli. However, it is unclear how many PBN neuron populations exist and how different behaviors may be encoded by unique signaling molecules or receptors. Here we provide a repository of data on the molecular identity, spatial location, and projection patterns of dozens of PBN neuron subclusters. Using single-cell RNA sequencing, we identified 21 subclusters of neurons in the PBN and neighboring regions. Multiplexed in situ hybridization showed many of these subclusters are enriched within specific PBN subregions with scattered cells in several other regions. We also provide detailed visualization of the axonal projections from 21 Cre-driver lines of mice. These results are all publicly available for download and provide a foundation for further interrogation of PBN functions and connections.

Dissociable control of unconditioned responses and associative fear learning by parabrachial CGRP neurons.

Parabrachial CGRP neurons receive diverse threat-related signals and contribute to multiple phases of adaptive threat responses in mice, with their inactivation attenuating both unconditioned behavioral responses to somatic pain and fear-memory formation. Because CGRPPBN neurons respond broadly to multi-modal threats, it remains unknown how these distinct adaptive processes are individually engaged. We show that while three partially separable subsets of CGRPPBN neurons broadly collateralize to their respective downstream partners, individual projections accomplish distinct functions: hypothalamic and extended amygdalar projections elicit assorted unconditioned threat responses including autonomic arousal, anxiety, and freezing behavior, while thalamic and basal forebrain projections generate freezing behavior and, unexpectedly, contribute to associative fear learning. Moreover, the unconditioned responses generated by individual projections are complementary, with simultaneous activation of multiple sites driving profound freezing behavior and bradycardia that are not elicited by any individual projection. This semi-parallel, scalable connectivity schema likely contributes to flexible control of threat responses in unpredictable environments.

Treatment of landfill leachate by white rot fungus in combination with zeolite filters.

This article presents the experimental work for the treatment of landfill leachate in a combined process using the white rot fungus Phanerochaete chrysosporium and the natural zeolite Clinoptilolite. Clinoptilolite was used in a pretreatment step as a sink for ammonia nitrogen and, on average it reduced the levels of ammonia nitrogen, soluble chemical oxygen demand (COD) and color by 72, 4.7, and 25%, respectively. The reductions by fungal treatment alone were 16.6, 21.5, and 31.2%, respectively. However, a reduction in nitrogen loading greatly enhanced fungal treatment efficiency. A high C/N ratio in the leachate was found preferable for the fungal treatment. With the synergy created by pretreatment and fungal growth that was stimulated by the addition of a growth medium, the process could remove ammonia nitrogen, soluble COD (SCOD) and color at levels as high as 81.5, 65, and 59%, respectively. The ratio of SBOD5/SCOD increased from 0.1 to 0.17 upon treatment, indicating that the process rendered the leachate more amenable to the biological process. This result suggested that the preliminary reduction of ammonia nitrogen was essential in making the fungal process practicable for landfill leachate treatment.