Prussian blue technique is prone to yield false negative results in magnetoreception research

Abstract

Perls’s Prussian blue staining technique has been used in magnetoreception research to screen tissues for iron-rich structures as proxies for putative magnetoreceptor structures based on magnetic particles. However, seemingly promising structural candidates in the upper beak of birds detected with Prussian blue turned out to be either irreproducible or located in non-neuronal cells, which has spurred a controversy that has not been settled yet. Here we identify possible pitfalls in the previous works and apply the Prussian blue technique to tissues implicated in magnetic-particle-based magnetoreception, in an effort to reassess its suitability for staining single-domain magnetite, i.e., the proposed magnetic substrate for the interaction with the external magnetic field. In the upper beak of night-migratory songbirds, we found staining products in great numbers, but not remotely associated with fiber terminals of the traced ophthalmic branch of the trigeminal nerve. Surprisingly, staining products were absent from the lamina propria in the olfactory rosette of rainbow trout where candidate magnetoreceptor structures were identified with different techniques earlier. Critically, magnetosome chains in whole cells of magnetotactic bacteria remained unstained. The failure to label single-domain magnetite in positive control samples is a serious limitation of the technique and suggests that two most influential but antipodal studies conducted previously stood little chances of obtaining correct positive results under the assumption that magnetosome-like particles were present in the tissues. Nonetheless, the staining technique appears suitable to identify tissue contamination with iron-rich fine dust trapped in epithelia already in vivo.

A magnetic pulse does not affect free-flight navigation behaviour of a medium-distance songbird migrant in spring

“…How birds detect geomagnetic information is still unknown, but is widely believed to be based on small magnetic particles innervated by sensory nerve endings of the trigeminal nerve ( Kishkinev et al, 2013 ; Beason and Semm, 1996 ). Although we still lack direct anatomical evidence for the postulated magnetic particles ( Curdt et al, 2022 ), their existence is supported by a number of behavioural studies that pre-treated birds with a strong magnetic pulse (e.g. Wiltschko and Wiltschko, 1995 ; Holland and Helm, 2013 ).…”

A refined magnetic pulse treatment method for magnetic navigation experiments with adequate sham control: a case study on free-flying songbirds

“…As finding the sensor structure is a tedious and probably lengthy task, e.g. [26], we propose a series of potential future experiments to shed more light on this matter. Methodologically, we propose using a pulser device that enables controlling for potential effects of the electric field component, to conduct experiments with a reasonable high sample size, ideally over more than 1 year, and to collect and analyse data free from any observer bias.We need independent, fully blinded replications of orientation-cage-based pulse experiments under meticulously controlled conditions in the laboratory, to validate the basic hypothesis of magnetic-particle-based magnetoreception [7–16].…”

“…As finding the sensor structure is a tedious and probably lengthy task, e.g. [26], we propose a series of potential future experiments to shed more light on this matter.…”

The cervical and meningeal lymphatic network as a pathway for retrograde nanoparticle transport to the brain

“…However, no staining was observed within the brain parenchyma with the Perls’ Prussian Blue technique. Iron detection through this staining method is prone to yield false negatives, as the detection requires the accumulation of several hundreds of nm in diameter 10 , which could hinder signals from SPIONs smaller than 10 nm diluted in the volume of the brain parenchyma. Combined Perls’ Prussian Blue staining and anti- LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1) immunohistochemistry revealed nanoparticles within the cervical lymphatic vessels towards the meningeal lymphatic vessels in the in vivo retrograde directional flow experiments performed on all C57BL/6 mice (n=3).…”