Cellular and synaptic organization of the Octopus vertical lobe

Reviewer #1 (Public review):

The authors identified five complex amacrine cell (CAM) subtypes based on their morphology and synaptic connectivity. It's suggested that the differences in structure may be directly correlated with different functional roles. The authors also describe synaptic compartmentalization in the SFL tract relating to three types of CAM input regions, again implying a specialized role for these cells. The authors also identified neural progenitor cells, which suggests that the octopus's vertical lobe can undergo neurogenesis throughout its life.

The work presented here is valuable and convincing. Below are some suggestions the authors may wish to incorporate:

a) Quantitative measurements to define the CAM subtypes
I think the categorization of the CAMs into five subtypes is convincing, however, I wonder how easily these categories could be identified by other researchers. Would it be possible for the authors to include additional quantitative measurements of these cell types to make their categorization less qualitative and more quantitative? For example, density, volume, and orientation of their dendritic fields?

b) The definition of the neuritic backbone is included in the methods, but I found the term confusing when I first encountered it in the results, so I would suggest adding the definition to the results too.

c) The authors wrote, 'Note that given the pronounced difference in diameters between the neuritic backbones (208.27 +/-87.95 nm) and axons (121.55 +/- 21.28 nm)'. What figure is this in?

d) I am slightly confused about how the authors decided on the specific cubes to reflect the different synaptic compartments in the SFL tract. Is this organisation arranged/repeated vertically or horizontally throughout the SFL tract? The location of the cubes looks to me to be chosen at random, so more information here would be helpful.

e) In Figure 2, could the authors plot the number of synapses per cube to make the result clearer, so that cube 1 has the lowest synaptic density and cube 2 has the highest?

f) SAMs are ACh and excitatory
The authors refer to SAMs as excitatory cholinergic. They should provide more detailed explanations/citations to back up this claim. Could SAMs be synthesizing any other neurotransmitters? Could there be a subpopulation of inhibitory SAMs?

g) CAMs are GABA and inhibitory

The 5 subtypes of CAMs described here have never been directly confirmed to be GABAergic. Could CAMs be synthesizing any other neurotransmitters? Could a subpopulation of CAMs be excitatory? I believe the authors should make this clearer to readers when referring to CAMs, perhaps by saying, 'hypothesized to be inhibitory neurons', or 'putative inhibitory neurons'.

h) Fast neurotransmitters and neuromodulators
The authors refer to neuromodulatory connections in their summary in Figure 4, however, cephalopod receptors have yet to be extensively functionally characterized, therefore, the role different molecules play as neurotransmitters or neuromodulators is not yet known. For example, many invertebrates are known to have functional diversity in their receptors: C. elegans has both excitatory and inhibitory receptors for a range of neurotransmitters, anionic ACh- and glutamate-gated channels, and cationic peptide-gated channels have also been identified in some molluscs. So, probably the authors should be cautious in speculating about how a particular transmitter/modulator acts in the octopus brain.

i) In the methods, the authors refer to "an adult Octopus", what age and size was it? I also know this is Octopus vulgaris, but it would be good to specify it here.

j) A general comment about all figures. All panels should have a letter associated with them to make it easier to refer to them in the text. For example, in Figure 4, please also add letters to the main schematic, the CAM subtypes, and the VL wiring diagram. In addition, D and E are missing boxes on the main schematic. It's also not immediately obvious that A-E are zooms of the larger schematic; perhaps this could be made clearer with colours or arrows. Please also add names to the CAM subtypes.

a) Typo: 'Additionally, the unique characteristics of LTP in the octopus VL, such as its reliance on a NO-dependent mechanism, independent of de novo protein synthesis, persistent activation of (Turchetti-Maia et al., 2018).'

Reviewer #2 (Public review):

Summary:

The paper examines the diversity of complex amacrine neurons in the ventral lobe of the adult octopus brain, a structure involved in learning and memory. The work builds on a recent paper by the authors that described the connectivity of the much larger population of simple amacrine (SAM) interneurons from the same pioneering EM volume.

Strengths:

While the EM volume only provides a snapshot of a tiny fraction of an adult octopus' brain, the authors can make specific conclusions and formulate precise hypotheses about neuron function, synaptic pathways, and developmental trajectories. One example is the reconstruction of a putative maturation sequence for the SAM neuronal lineage, based on the correlation of soma position and the number of synapses, uncovering a plausible developmental sequence of cell morphologies, with interesting parallels to vertebrate neurogenesis.

Weaknesses:

The weakness of the study is that it is examining a relatively small volume (260 × 390 × 27 µm), and several neurons are only incompletely reconstructed. It also remains unclear approximately how many neurons remain to be reconstructed from this volume.

To improve the presentation, the authors should consider showing videos with the volumetric reconstructions of the different types with their partners/synapses and their relation to the SFL track and SAMs. Such videos would help the reader to appreciate the morphological differences between the cell types. The authors could also consider carrying out further morphological analyses to strengthen their cell-type classification, including Sholl value, radial density of input and output synapses, the number of branch nodes, and similar measures.

Reviewer #3 (Public review):

(1) The authors described "the excitatory glutamatergic SFL axons and cholinergic SAM inputs". However, the evidence of their transmitter specificity has not been provided. Compelling evidence was neither provided nor discussed in the context of the study.

(2) Specific interference for inhibitory or excitatory synapses based on EM or other studies must be detailed and elaborated

(3) Different local microcircuits (submodules) referred to in the text should be better described and more specifically defined.

(4) I would recommend incorporating a more detailed description of synapses and, especially, synaptic vesicles, clarifying their diversity and similarity across cell subtypes. Are there any differences between cholinergic and glutamatergic synaptic vesicles, postsynaptic densities, or other features...? It would be good, if possible, to explicitly clarify: how many vesicles per different types of synapses? How many synapses per neuron of different types? How many inputs and outputs per a given neuron?

(5) Authors discuss retrograde messengers like NO? Is there any identifiable morphological type of neuron(s) or synapses that might be nitrergic?

(6) It would be good to provide separate illustrations showing the detailed organization of any glial cell or different types of glial cells they identified in this study. Authors mainly discuss glial processes but refer to "recognized glial types, such as radial glia and astrocyte-like glia" without specific illustrations, which can be deciphered from their EM data. What are vesicular organizations within different types of glial cells?

(7) The authors also discuss "supervising inputs of inhibitory (pain) and neuromodulatory (supervising) signals", without any details. It would be important to provide these details in the discussion. Specifically, I suggest incorporating comments about differences/similarities of transmitters and morphology between pain and modulatory pathways/signaling/circuits.

Reviewer #4 (Public review):

Summary:

The authors present a follow-up to their initial publication of a volume EM reconstruction of a part of the Octopus vulgaris vertical lobe (VL) (Bidel, Meirovitch et al., eLife 2023). In their previous study, they presented a swath of novel observations pertaining to the neuron types making up the VL and their synaptic connectivity. Here, the authors present an extension of those findings in which they (1) demonstrate that the Complex Amacrine cells (CAMs), which they identified previously, can be grouped into at least 5 distinct subclasses; (2) show that there appears to be distinct compartments in the SFL tract that contain specific synapse types; and (3) present morphological evidence that there may be a neurogenic niche in the VL. The findings are intriguing, advance our understanding of memory circuitry in octopus and across the phylogenetic tree, and open new avenues for deeper investigation.

Strengths:

A deeper dissection of the morphologies of CAMs and their distinct complements of synapse types is valuable. The identification of multiple categories of CAMs makes it clearer how the very simple SFL-to-SAM connectivity is likely enriched by a population of diverse interneurons.

The observation that synapse types may be compartmentalized in the superior frontal lobe tract is an intriguing one, and invites more extensive segmentation and future anatomical studies to further characterize the precise architecture of these compartments.

Finally, the evidence of the possibility of a neurogenic niche in the VL is exciting as it suggests that ongoing neurogenesis may be a common feature of memory circuitry, perhaps contributing to keeping the representation space of the circuit flexible and adequately sparse.

Weaknesses:

A key weakness is the reconstruction and grouping of the CAMs:

(1) CAMs are relatively few in number compared to SAMs, and as such, only 53 are reconstructed in this study. Of those 53 cells, 18 were not classified into one of the 5 categories the authors designate, begging the question of how robust those categories are.

(2) Related to (1), in Figure 1B, the proportions given in the bar graph are given cumulatively across the entire population of each category. The proportions should be presented as means within each category to adequately capture the variability of the small sample sizes.

(3) While the xy dimensions of the serial section EM volume are adequate to capture relatively whole cells and neuronal arbors, the volume is only 27µm thick. Thus, many neurite branches are likely truncated in the z-dimension. This may have contributed to ~1/3 of CAMs eluding categorization. However, it is hard to estimate the effect this may have had without knowing the extent of the truncation. It may be worth the authors' time to count the proportion of CAM neurites that are cut off at the edges of the volume.

(4) The authors state that CAMs appear to have axons and dendrites based on neurite widths. This is an interesting finding, given that amacrine cells are generally thought to possess only one type of neurite, which both send and receive synaptic potentials, and therefore deserves more attention. Is the distribution of neurite widths indeed bimodally distributed? Can the axons and dendrites be differentiated by examining the presence and absence of synaptic vesicle pools, respectively?

In Figure 2, the compartmentalization of synapse types is intriguing; however, due to the 3D nature of the data, it is difficult to appreciate clearly from the panels presented. This is particularly true for the suggestion that glia may be forming a barrier around these compartments. This could be rectified by providing Neuroglancer links for these specific reconstructions (neurites, synapses, and glia).

Lastly, although the identification of a putative neurogenic niche is tantalizing, morphological data alone is only an initial hint. Although the chances are slim, it would be more convincing if the authors could identify any actively dividing cells in the proposed niche. More likely, further work, for instance, immunofluorescence, which the lab has previously shown to be viable in octopus, will be needed to add weight to the claim.