We present the first analysis of the connectome of the vertical lobe (VL) of Octopus vulgaris, a brain structure mediating the acquisition of long-term memory in this behaviorally advanced mollusk. Serial section electron microscopy revealed new types of interneurons, cellular components of extensive modulatory systems, and multiple synaptic motifs. The sensory input to the VL is conveyed via ≈1,800,000 axons that sparsely innervate two parallel and interconnected feedforward networks formed by the two AM types, simple AMs (SAMs) and complex AMS (CAMs). SAMs make up 89.3% of the 25,000,000 AMs, each receiving synaptic input from only a single input neuron on its non-bifurcating primary neurite, suggesting that each input neuron is represented in only ≈12 SAMs. The CAMs, a newly described amacrine type, comprise 1.6% of the amacrine population. Their bifurcating neurites integrate multiple inputs from the input axons and SAMs. While the SAM network appears to feedforward sparse memorizable sensory representations into the VL output layer, the CAMs appear to monitor global activity and feedforward a balancing inhibition for sharpening the stimulus-specific VL output. While sharing morphological and wiring features with circuits supporting associative learning in other animals, the VL has evolved a unique circuit that enables associative learning based strictly on feedforward information flow.

Neural activity is increasingly recognized as a critical regulator of cancer growth. In the brain, neuronal activity robustly influences glioma growth both through paracrine mechanisms and through electrochemical integration of malignant cells into neural circuitry via neuron-to-glioma synapses, while perisynaptic neurotransmitter signaling drives breast cancer brain metastasis growth. Outside of the CNS, innervation of tumors such as prostate, breast, pancreatic and gastrointestinal cancers by peripheral nerves similarly regulates cancer progression. However, the extent to which the nervous system regulates lung cancer progression, either in the lung or when metastatic to brain, is largely unexplored. Small cell lung cancer (SCLC) is a lethal high-grade neuroendocrine tumor that exhibits a strong propensity to metastasize to the brain. Here we demonstrate that, similar to glioma, metastatic SCLC cells in the brain co-opt neuronal activity-regulated mechanisms to stimulate growth and progression. Optogenetic stimulation of cortical neuronal activity drives proliferation and invasion of SCLC brain metastases. In the brain, SCLC cells exhibit electrical currents and consequent calcium transients in response to neuronal activity, and direct SCLC cell membrane depolarization is sufficient to promote the growth of SCLC tumors. In the lung, vagus nerve transection markedly inhibits primary lung tumor formation, progression and metastasis, highlighting a critical role for innervation in overall SCLC initiation and progression. Taken together, these studies illustrate that neuronal activity plays a crucial role in dictating SCLC pathogenesis in both primary and metastatic sites.

The connections between motor neurons and muscle fibers are dramatically reorganized in early postnatal life. This work attempts to better understand this synaptic rewiring by using a connectomic approach, i.e., tracing out all the connections between motor neurons and muscle fibers, at successive ages in a small mouse muscle. We reconstructed 31 partial-complete neuromuscular connectomes, using serial section scanning electron microscopy in a neonatal mouse and Brainbow-based and XFP-based fluorescent reconstructions in older animals. Our data included a total of more than 6000 neuromuscular junctions (NMJs), including complete connectomes from one newborn, seven developmental ages (P6-P9), and two adults. Analysis confirmed the massive rewiring that takes place as axons prune their motor units but add more synaptic areas at the NMJs with which they remain in contact. Interestingly, we found synaptic ordering rules that likely underlie this circuit maturation and yield the resulting adult neuromuscular pattern, as manifest in Henneman's size principle. In particular, by analyzing both the identities of axons sharing NMJs at developing ages and muscle fibers with multiple endplates, we found evidence suggesting an activity-based linear ranking of motor neurons such that neurons co-innervated the same endplates and same muscle fibers (if there were more than one endplate) when the axons were similar in activity and hence rank. In addition, this ranking provided a means for understanding action at a distance in which the activity at one neuromuscular junction can impact the fate of the axons at another junction at a different site on the same muscle fiber. These activity-dependent mechanisms provide insight into the means by which timing of activity among different axons innervating the same population of cells, that start out with nearly all-to-all connectivity, can produce a well-organized system of axons, a system that is necessary for the recruitment order of neurons during a graded behavior like muscle contraction.

Specialized mechanosensory end organs within mammalian skin -- hair follicle-associated lanceolate complexes, Meissner corpuscles, and Pacinian corpuscles -- enable our perception of light, dynamic touch. In each of these end organs, fast-conducting mechanically sensitive neurons, called Aβ low-threshold mechanoreceptors (Aβ LTMRs), associate with resident glial cells, known as terminal Schwann cells (TSCs) or lamellar cells, to form complex axon ending structures. Lanceolate-forming and corpuscle-innervating Aβ LTMRs share a low threshold for mechanical activation, a rapidly adapting (RA) response to force indentation, and high sensitivity to dynamic stimuli. How mechanical stimuli lead to activation of the requisite mechanotransduction channel Piezo2 and Aβ RA-LTMR excitation across the morphologically dissimilar mechanosensory end organ structures is not understood. Here, we report the precise subcellular distribution of Piezo2 and high-resolution, isotropic 3D reconstructions of all three end organs formed by Aβ RA-LTMRs determined by large volume enhanced Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) imaging. We found that within each end organ, Piezo2 is enriched along the sensory axon membrane and is minimally or not expressed in TSCs and lamellar cells. We also observed a large number of small cytoplasmic protrusions enriched along the Aβ RA-LTMR axon terminals associated with hair follicles, Meissner corpuscles, and Pacinian corpuscles. These axon protrusions reside within close proximity to axonal Piezo2, occasionally contain the channel, and often form adherens junctions with nearby non-neuronal cells. Our findings support a unified model for Aβ RA-LTMR activation in which axon protrusions anchor Aβ RA-LTMR axon terminals to specialized end organ cells, enabling mechanical stimuli to stretch the axon in hundreds to thousands of sites across an individual end organ and leading to activation of proximal Piezo2 channels and excitation of the neuron.