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1. Probing Neuropeptide Volume Transmission In Vivo by Simultaneous Near‐Infrared Light‐Triggered Release and Optical Sensing**

   https://doi.org/10.1002/ange.202206122  

   Xiong, Hejian ; Lacin, Emre ; Ouyang, Hui ; Naik, Aditi ; Xu, Xueqi ; Xie, Chen ; Youn, Jonghae ; Wilson, Blake A. ; Kumar, Krutin ; Kern, Tyler ; et al ( July 2022 , Angewandte Chemie)

   Neuropeptides are abundant signaling molecules in the central nervous system. Yet remarkably little is known about their spatiotemporal spread and biological activity. Here, we developed an integrated optical approach usingPlasmonic nAnovesicles and cell‐based neurotransmitter fluorescent engineered reporter (CNiFER), or PACE, to probe neuropeptide signaling in the mouse neocortex. Small volumes (fL to pL) of exogenously supplied somatostatin‐14 (SST) can be rapidly released under near‐infrared light stimulation from nanovesicles implanted in the brain and detected by SST2 CNiFERs with nM sensitivity. Our measurements reveal reduced but synchronized SST transmission within 130 μm, and markedly smaller and delayed transmission at longer distances. These measurements enabled a quantitative estimation of the SST loss rate due to peptide degradation and binding. PACE offers a new tool for determining the spatiotemporal scales of neuropeptide volume transmission and signaling in the brain.

    

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2. Neuropeptide Y Signaling Regulates Recurrent Excitation in the Auditory Midbrain

   https://doi.org/10.1523/JNEUROSCI.0900-23.2023  

   Silveira, Marina A. ; Drotos, Audrey C. ; Pirrone, Trinity M. ; Versalle, Trevor S. ; Bock, Amanda ; Roberts, Michael T. ( September 2023 , The Journal of Neuroscience)

   Neuropeptides play key roles in shaping the organization and function of neuronal circuits. In the inferior colliculus (IC), which is in the auditory midbrain, Neuropeptide Y (NPY) is expressed by a class of GABAergic neurons that project locally and outside the IC. Most neurons in the IC have local axon collaterals; however, the organization and function of local circuits in the IC remain unknown. We previously found that excitatory neurons in the IC can express the NPY Y₁receptor (Y₁R⁺) and application of the Y₁R agonist, [Leu³¹, Pro³⁴]-NPY (LP-NPY), decreases the excitability of Y₁R⁺neurons. As NPY signaling regulates recurrent excitation in other brain regions, we hypothesized that Y₁R⁺neurons form interconnected local circuits in the IC and that NPY decreases the strength of recurrent excitation in these circuits. To test this hypothesis, we used optogenetics to activate Y₁R⁺neurons in mice of both sexes while recording from other neurons in the ipsilateral IC. We found that nearly 80% of glutamatergic IC neurons express the Y₁receptor, providing extensive opportunities for NPY signaling to regulate local circuits. Additionally, Y₁R⁺neuron synapses exhibited modest short-term synaptic plasticity, suggesting that local excitatory circuits maintain their influence over computations during sustained stimuli. We further found that application of LP-NPY decreased recurrent excitation in the IC, suggesting that NPY signaling strongly regulates local circuit function in the auditory midbrain. Our findings show that Y₁R⁺excitatory neurons form interconnected local circuits in the IC, and their influence over local circuits is regulated by NPY signaling.

   SIGNIFICANCE STATEMENTLocal networks play fundamental roles in shaping neuronal computations in the brain. The IC, localized in the auditory midbrain, plays an essential role in sound processing, but the organization of local circuits in the IC is largely unknown. Here, we show that IC neurons that express the Neuropeptide Y₁receptor (Y₁R⁺neurons) make up most of the excitatory neurons in the IC and form interconnected local circuits. Additionally, we found that NPY, which is a powerful neuromodulator known to shape neuronal activity in other brain regions, decreases the extensive recurrent excitation mediated by Y₁R⁺neurons in local IC circuits. Thus, our results suggest that local NPY signaling is a key regulator of auditory computations in the IC.

    

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3. Gene Delivery and Analysis of Optogenetic Induction of Lytic Cell Death

   https://doi.org/10.1002/cpz1.1023  

   Oh, Teak‐Jung ; Gworek, Bryan ; Mehfooz, Amna ; Zhang, Kai ( April 2024 , Current Protocols)

   Necroptosis is a form of inflammatory lytic cell death involving active cytokine production and plasma membrane rupture. Progression of necroptosis is tightly regulated in time and space, and its signaling outcomes can shape the local inflammatory environment of cells and tissues. Pharmacological induction of necroptosis is well established, but the diffusive nature of chemical death inducers makes it challenging to study cell‐cell communication precisely during necroptosis. Receptor‐interacting protein kinase 3, or RIPK3, is a crucial signaling component of necroptosis, acting as a crucial signaling node for both canonical and non‐canonical necroptosis. RIPK3 oligomerization is crucial to the formation of the necrosome, which regulates plasma membrane rupture and cytokine production. Commonly used necroptosis inducers can activate multiple downstream signaling pathways, confounding the signaling outcomes of RIPK3‐mediated necroptosis. Opsin‐free optogenetic techniques may provide an alternative strategy to address this issue. Optogenetics uses light‐sensitive protein‐protein interaction to modulate cell signaling. Compared to chemical‐based approaches, optogenetic strategies allow for spatiotemporal modulation of signal transduction in live cells and animals. We developed an optogenetic system that allows for ligand‐free optical control of RIPK3 oligomerization and necroptosis. This article describes the sample preparation, experimental setup, and optimization required to achieve robust optogenetic induction of RIPK3‐mediated necroptosis in colorectal HT‐29 cells. We expect that this optogenetic system could provide valuable insights into the dynamic nature of lytic cell death. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

   Basic Protocol 1: Production of lentivirus encoding the optogenetic RIPK3 system

   Support Protocol: Quantification of the titer of lentivirus

   Basic Protocol 2: Culturing, chemical transfection, and lentivirus transduction of HT‐29 cells

   Basic Protocol 3: Optimization of optogenetic stimulation conditions

   Basic Protocol 4: Time‐stamped live‐cell imaging of HT‐29 lytic cell death

   Basic Protocol 5: Quantification of HT‐29 lytic cell death

    

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4. To hum or not to hum: Neural transcriptome signature of male courtship vocalization in a teleost fish

   https://doi.org/10.1111/gbb.12740  

   Tripp, Joel_A ; Feng, Ni_Y ; Bass, Andrew_H ( June 2021 , Genes, Brain and Behavior)

   For many animal species, vocal communication is a critical social behavior and often a necessary component of reproductive success. Additionally, vocalizations are often demanding motor acts. Wanting to know whether a specific molecular toolkit might be required for vocalization, we used RNA‐sequencing to investigate neural gene expression underlying the performance of an extreme vocal behavior, the courtship hum of the plainfin midshipman fish (Porichthys notatus). Single hums can last up to 2 h and may be repeated throughout an evening of courtship activity. We asked whether vocal behavioral states are associated with specific gene expression signatures in key brain regions that regulate vocalization by comparing transcript expression levels in humming versus non‐humming males. We find that the circadian‐related genesperiod3andClockare significantly upregulated in the vocal motor nucleus and preoptic area‐anterior hypothalamus, respectively, in humming compared with non‐humming males, indicating that internal circadian clocks may differ between these divergent behavioral states. In addition, we identify suites of differentially expressed genes related to synaptic transmission, ion channels and transport, neuropeptide and hormone signaling, and metabolism and antioxidant activity that together may support the neural and energetic demands of humming behavior. Comparisons of transcript expression across regions stress regional differences in brain gene expression, while also showing coordinated gene regulation in the vocal motor circuit in preparation for courtship behavior. These results underscore the role of differential gene expression in shifts between behavioral states, in this case neuroendocrine, motor and circadian control of courtship vocalization.

    

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5. Differentiation of Brain Pericyte‐Like Cells from Human Pluripotent Stem Cell−Derived Neural Crest

   https://doi.org/10.1002/cpz1.21  

   Gastfriend, Benjamin D. ; Stebbins, Matthew J. ; Du, Feifan ; Shusta, Eric V. ; Palecek, Sean P. ( January 2021 , Current Protocols)

   Brain pericytes regulate diverse aspects of neurovascular development and function, including blood‐brain barrier (BBB) induction and maintenance. Primary brain pericytes have been widely employed in coculture‐based in vitro models of the BBB, and a method to generate brain pericytes from human pluripotent stem cells (hPSCs) could provide a renewable, genetically tractable source of cells for BBB modeling and studying pericyte roles in development and disease. Here, we describe a protocol to differentiate hPSCs to NG2⁺PDGFRβ⁺αSMA^lowbrain pericyte‐like cells in 22‐25 days through a p75‐NGFR⁺HNK‐1⁺neural crest intermediate, which mimics the developmental origin of forebrain pericytes. The resulting brain pericyte‐like cells have molecular and functional attributes of brain pericytes. We also provide protocols for maintenance, cryopreservation, and recovery of the neural crest intermediate, and for molecular and functional characterization of the resulting cells. © 2021 Wiley Periodicals LLC.

   This article was corrected on 18 July 2022. See the end of the full text for details.

   Basic Protocol 1: Differentiation of hPSCs to neural crest

   Basic Protocol 2: Differentiation of neural crest to brain pericyte‐like cells

   Support Protocol 1: Flow cytometry analysis of neural crest cells

   Support Protocol 2: Maintenance, cryopreservation, and recovery of neural crest cells

   Support Protocol 3: Molecular characterization of brain pericyte‐like cells

   Support Protocol 4: Cord formation assay with endothelial cells and brain pericyte‐like cells

    

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