20 research outputs found
Dorsal Raphe Dopamine Neurons Modulate Arousal and Promote Wakefulness by Salient Stimuli
Ventral midbrain dopamine (DA) is unambiguously involved in motivation and behavioral arousal, yet the contributions of other DA populations to these processes are poorly understood. Here, we demonstrate that the dorsal raphe nucleus DA neurons are critical modulators of behavioral arousal and sleep-wake patterning. Using simultaneous fiber photometry and polysomnography, we observed time-delineated dorsal raphe nucleus dopaminergic (DRNDA) activity upon exposure to arousal-evoking salient cues, irrespective of their hedonic valence. We also observed broader fluctuations of DRNDA activity across sleep-wake cycles with highest activity during wakefulness. Both endogenous DRNDA activity and optogenetically driven DRNDA activity were associated with waking from sleep, with DA signal strength predictive of wake duration. Conversely, chemogenetic inhibition opposed wakefulness and promoted NREM sleep, even in the face of salient stimuli. Therefore, the DRNDA population is a critical contributor to wake-promoting pathways and is capable of modulating sleep-wake states according to the outside environment, wherein the perception of salient stimuli prompts vigilance and arousal
Cholinergic Mesopontine Signals Govern Locomotion and Reward through Dissociable Midbrain Pathways
The mesopontine tegmentum, including the pedunculopontine and laterodorsal tegmental nuclei (PPN and LDT), provides major cholinergic inputs to midbrain and regulates locomotion and reward. To delineate the underlying projection-specific circuit mechanisms, we employed optogenetics to control mesopontine cholinergic neurons at somata and at divergent projections within distinct midbrain areas. Bidirectional manipulation of PPN cholinergic cell bodies exerted opposing effects on locomotor behavior and reinforcement learning. These motor and reward effects were separable via limiting photostimulation to PPN cholinergic terminals in the ventral substantia nigra pars compacta (vSNc) or to the ventral tegmental area (VTA), respectively. LDT cholinergic neurons also form connections with vSNc and VTA neurons; however, although photo-excitation of LDT cholinergic terminals in the VTA caused positive reinforcement, LDT-to-vSNc modulation did not alter locomotion or reward. Therefore, the selective targeting of projection-specific mesopontine cholinergic pathways may offer increased benefit in treating movement and addiction disorders
Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing
Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies
Whole-Brain Analysis of Cells and Circuits by Tissue Clearing and Light-Sheet Microscopy
In this photo essay, we present a sampling of technologies from laboratories at the forefront of whole-brain clearing and imaging for high-resolution analysis of cell populations and neuronal circuits. The data presented here were provided for the eponymous Mini-Symposium presented at the Society for Neuroscience's 2018 annual meeting
Whole-Brain Analysis of Cells and Circuits by Tissue Clearing and Light-Sheet Microscopy
In this photo essay, we present a sampling of technologies from laboratories at the forefront of whole-brain clearing and imaging for high-resolution analysis of cell populations and neuronal circuits. The data presented here were provided for the eponymous Mini-Symposium presented at the Society for Neuroscience's 2018 annual meeting
Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping
To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks
An Antidote for Acute Cocaine Toxicity
Not only has immunopharmacotherapy grown into a field
that addresses
the abuse of numerous illicit substances, but also the treatment methodologies
within immunopharmacotherapy have expanded from traditional active
vaccination to passive immunization with anti-drug monoclonal antibodies,
optimized mAb formats, and catalytic drug-degrading antibodies. Many
laboratories have focused on transitioning distinct immunopharmacotherapeutics
to clinical evaluation, but with respect to the indication of cocaine
abuse, only the active vaccine TA-CD, which is modeled after our original
cocaine hapten GNC, has been carried through
to human clinical trials. The successful
application of murine mAb GNC92H2 to the reversal of cocaine overdose
in a mouse model prompted investigations of human immunoglobulins
with the clinical potential to serve as cocaine antidotes. We now
report the therapeutic utility of a superior clone, human mAb GNCgzk
(<i>K</i><sub>d</sub> = 0.18 nM), which offers a 10-fold
improvement in cocaine binding affinity. The GNCgzk manifold was engineered
for rapid cocaine clearance, and administration of the FÂ(ab′)<sub>2</sub> and Fab formats even after the appearance of acute behavioral
signs of cocaine toxicity granted nearly complete prevention of lethality.
Thus, contrary to the immunopharmacotherapeutic treatment of drug
self-administration, minimal antibody doses were shown to counteract
the lethality of a molar excess of circulating cocaine. Passive vaccination
with drug-specific antibodies represents a viable treatment strategy
for the human condition of cocaine overdose
Whole-Brain Analysis of Cells and Circuits by Tissue Clearing and Light-Sheet Microscopy
In this photo essay, we present a sampling of technologies from laboratories at the forefront of whole-brain clearing and imaging for high-resolution analysis of cell populations and neuronal circuits. The data presented here were provided for the eponymous MiniSymposium presented at the Society for Neuroscience’s 2018 annual meeting.NIH (Grant 1-DP2-ES027992