Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose ␣4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased ␣4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change ␣4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional ␣4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases ␣4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated ␣4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional ␣4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain.

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.

Highlights d DRN DA neurons are activated by salient stimuli irrespective of hedonic valence d DRN DA activity fluctuates across sleep-wake states and is highest at wakefulness d Optogenetic activation promotes wakefulness d Chemogenetic inhibition opposes wakefulness, even in the presence of salient stimuli SUMMARY 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 sleepwake patterning. Using simultaneous fiber photometry and polysomnography, we observed time-delineated dorsal raphe nucleus dopaminergic (DRN DA ) activity upon exposure to arousal-evoking salient cues, irrespective of their hedonic valence. We also observed broader fluctuations of DRN DA activity across sleep-wake cycles with highest activity during wakefulness. Both endogenous DRN DA activity and optogenetically driven DRN DA 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 DRN DA 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.

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.

Nicotinic acetylcholine receptors (nAChRs) affect a wide array of biological processes, including learning and memory, attention, and addiction. lynx1, the founding member of a family of mammalian prototoxins, modulates nAChR function in vitro by altering agonist sensitivity and desensitization kinetics. Here we demonstrate, through the generation of lynx1 null mutant mice, that lynx1 modulates nAChR signaling in vivo. Its loss decreases the EC(50) for nicotine by approximately 10-fold, decreases receptor desensitization, elevates intracellular calcium levels in response to nicotine, and enhances synaptic efficacy. lynx1 null mutant mice exhibit enhanced performance in specific tests of learning and memory. Consistent with reports that mutations resulting in hyperactivation of nAChRs can lead to neurodegeneration, aging lynx1 null mutant mice exhibit a vacuolating degeneration that is exacerbated by nicotine and ameliorated by null mutations in nAChRs. We conclude that lynx1 functions as an allosteric modulator of nAChR function in vivo, balancing neuronal activity and survival in the CNS.