The study uses a single fly brain for connectome reconstruction. While this offers high resolution data, it cannot account for inter-individual variability in brain wiring or developmental differences between neuron types in various wild-type or genetically modified fly lines.
The connectome used in the study does not capture the full extent of neural connectivity. The resolution is insufficient to detect gap junctions or neuromodulatory connections mediated by neuropeptides. Glial cells and certain brain structures connected to the CX are also not fully segmented or included in the volume.
Synaptic strength and neuron biophysics
While the study assigns function based on neuron type and the location of synapses, the influence of a synaptic connection on the neuron's response is complex and depends on various factors, including the proximity of synapses to the spike initiation zone, unknown for most neuron types, and experience-dependent plasticity of synaptic strengths.
While the study uses directional tuning and location of synapses to build conceptual models of how the CX implements vector-based computations, testing these models necessitates physiological recordings from behaving animals, and the lack of full information from certain brain regions, such as CRE, WED, SMP, limits the exploration of downstream networks.
Unknown function of many neuron types
Many of the identified CX neuron types, especially the tangential FB neurons, and their related pathways lack functional characterization, hindering a comprehensive understanding of the CX's role in various behaviors beyond navigation.