Mapping of Neuropeptide Y Expression inOctopusBrains

Abstract

15Neuropeptide Y (NPY) is an evolutionarily conserved neurosecretory molecule implicated 16 in a diverse complement of functions across taxa and in regulating feeding behavior and 17 reproductive maturation in Octopus. However, little is known about the precise molecular 18 circuitry of NPY-mediated behaviors and physiological processes, which likely involve a 19 complex interaction of multiple signal molecules in specific brain regions. Here we examined 20 the expression of NPY throughout the Octopus central nervous system. The sequence 21 analysis of Octopus NPY precursor confirmed the presence of both signal peptide and 22 putative active peptides, which are highly conserved across bilaterians. In situ hybridization 23 revealed distinct expression of NPY in specialized compartments, including potential 24 "integration centers," where visual, tactile, and other behavioral circuitries converge. These 25 centers integrating separate circuits may maintain and modulate learning and memory or 26 other behaviors not yet attributed to NPY-dependent modulation in Octopus. Extrasomatic 27 localization of NPY mRNA in the neurites of specific neuron populations in the brain 28 suggests a potential demand for immediate translation at synapses and a crucial temporal role 29 for NPY in these cell populations. We also verified the presence of NPY mRNA in a small 30 cell population in the olfactory lobe, which is a component of the Octopus feeding and 31reproductive control centers. However, the molecular mapping of NPY expression only 32 partially overlapped with that produced by immunohistochemistry in previous studies. Our 33 study provides a precise molecular map of NPY mRNA expression that can be used to design 34 and test future hypotheses about molecular signaling in various Octopus behaviors. 35 reproduction 37 38 Neuropeptide Y in Octopus 3 Research Highlights/Graphical Abstract text: 39Neuropeptide Y (NPY), an evolutionarily conserved neurosecretory molecule, is expressed 40 in specialized regions of the Octopus brain controlling feeding, reproduction, and visual and 41 tactile memory circuits. Extrasomatic mRNAs were found in neurites, suggesting synaptic 42 synthesis of NPY. 43 44

Connectomics of the Octopus vulgaris vertical lobe provides insight into conserved and novel principles of a memory acquisition network

“…Their exceptionally large boutons, frequently lacking the typical release sites, are filled with a variety of large dense-core vesicles of the type usually storing and releasing neuropeptides and non-classical neurotransmitters (Zhu et al 1986; Leng and Ludwig 2008; van den Pol 2012; Witvliet et al 2021). A variety of neuropeptides and hormones have been identified in the Octopus vulgaris VL - serotonin, dopamine, octopamine, FMRFamide-related peptides, vasopressin, oxytocin, octopressin, Y-peptide and more (Shomrat et al 2010; Shigeno and Ragsdale 2015; Winters et al 2020; Stern-Mentch et al 2022). Tyrosine hydroxylase (TH) - labeled processes show a wide distribution in the VL neuropil similar to that of the NM processes, suggestive of a neuromodulatory catecholamine innervation (Stern-Mentch et al 2022).…”

Three-Dimensional Molecular Atlas of Octopus Arm Neuroanatomy Highlights Spatial and Functional Complexity

“…Upon confirming that the octopus had no reflexive movement to a noxious stimulus (arm pinch) it was dissected into individual arms and neural tissues. Initial fixation and sample preparation were modified from established mollusc and octopus colorimetric in situ hybridization protocols 28,34,35 and fixed overnight in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at 4°C. The following day tissues were rinsed in PBS x3, followed by 100% PTW (1xPBS, 0.1% Tween 20) for 10 minutes.…”

“…Protocols for chromogenic in situ hybridization were adapted from previous studies on Octopus 14,28,35 and Aplysia 34,3634,37 nervous systems. After fixation, slicing, and initial PTW washes (see "Animal dissection and tissue preparation"), and subsequent permeabilization steps 34 , probes were dissolved in hybridization buffer at 1 μg/μL and enough of this solution was added to slides to cover tissues.…”

“…FLRIamide-positive neuron density varies significantly between base and tip (Table S3), but these neurons are exceptionally abundant in the base and the tip, suggesting an important role for FLIRamide peptide throughout the physical length and temporal lifetime of an octopus's arm. This secretory molecule is also expressed in numerous morphologically diverse neurons in the brain 14,28 , notably in the large efferent neurons of the vertical lobe, which are GABAergic inhibitory neurons that receive excitatory cholinergic input from the amacrine interneurons and project from the memory circuit to premotor centers 14 . FLRIamide-positive neurons are clearly not all GABAergic in the ANC, but it is plausible that their role in modulating motor control is conserved in the arm.…”