Neural circuits are made of a vast diversity of neuronal cell types. While immense progress has been made in classifying neurons based on morphological, molecular, and functional properties, understanding how this heterogeneity contributes to brain function during natural behavior has remained largely unresolved. In the present study, we combined the juxtacellular recording and labeling technique with optogenetics in freely moving mice. This allowed us to selectively target molecularly defined cell classes for in vivo single-cell recordings and morphological analysis. We validated this strategy in the CA1 region of the mouse hippocampus by restricting Channelrhodopsin expression to Calbindin-positive neurons. Directly versus indirectly light-activated neurons could be readily distinguished based on the latencies of light-evoked spikes, with juxtacellular labeling and post hoc histological analysis providing ‘ground-truth’ validation. Using these opto-juxtacellular procedures in freely moving mice, we found that Calbindin-positive CA1 pyramidal cells were weakly spatially modulated and conveyed less spatial information than Calbindin-negative neurons – pointing to pyramidal cell identity as a key determinant for neuronal recruitment into the hippocampal spatial map. Thus, our method complements current in vivo techniques by enabling optogenetic-assisted structure–function analysis of single neurons recorded during natural, unrestrained behavior.

Pain perception is decreased by shifting attentional focus away from a threatening event. This attentional analgesia engages parallel descending control pathways from anterior cingulate (ACC) to locus coeruleus, and ACC to periaqueductal grey (PAG) - rostral ventromedial medulla (RVM), indicating possible roles for noradrenergic or opioidergic neuromodulators. To determine which pathway modulates nociceptive activity in humans we used simultaneous whole brain-spinal cord pharmacological-fMRI (N=39) across three sessions. Noxious thermal forearm stimulation generated somatotopic-activation of dorsal horn (DH) whose activity correlated with pain report and mirrored attentional pain modulation. Activity in an adjacent cluster reported the interaction between task and noxious stimulus. Effective connectivity analysis revealed that ACC interacts with PAG and RVM to modulate spinal cord activity. Blocking endogenous opioids with Naltrexone impairs attentional analgesia and disrupts RVM-spinal and ACC-PAG connectivity. Noradrenergic augmentation with Reboxetine did not alter attentional analgesia. Cognitive pain modulation involves opioidergic ACC-PAG-RVM descending control which suppresses spinal nociceptive activity.