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. 2018 Aug;15(145):20180124.
doi: 10.1098/rsif.2018.0124.

Lidocaine Is a Nocebo Treatment for Trigeminally Mediated Magnetic Orientation in Birds

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Free PMC article

Lidocaine Is a Nocebo Treatment for Trigeminally Mediated Magnetic Orientation in Birds

Svenja Engels et al. J R Soc Interface. .
Free PMC article

Abstract

Even though previously described iron-containing structures in the upper beak of pigeons were almost certainly macrophages, not magnetosensitive neurons, behavioural and neurobiological evidence still supports the involvement of the ophthalmic branch of the trigeminal nerve (V1) in magnetoreception. In previous behavioural studies, inactivation of putative V1-associated magnetoreceptors involved either application of the surface anaesthetic lidocaine to the upper beak or sectioning of V1. Here, we compared the effects of lidocaine treatment, V1 ablations and sham ablations on magnetic field-driven neuronal activation in V1-recipient brain regions in European robins. V1 sectioning led to significantly fewer Egr-1-expressing neurons in the trigeminal brainstem than in the sham-ablated birds, whereas lidocaine treatment had no effect on neuronal activation. Furthermore, Prussian blue staining showed that nearly all iron-containing cells in the subepidermal layer of the upper beak are nucleated and are thus not part of the trigeminal nerve, and iron-containing cells appeared in highly variable numbers at inconsistent locations between individual robins and showed no systematic colocalization with a neuronal marker. Our data suggest that lidocaine treatment has been a nocebo to the birds and a placebo for the experimenters. Currently, the nature and location of any V1-associated magnetosensor remains elusive.

Keywords: Egr-1; Prussian blue; TUBB3; immediate early genes; magnetoreception; navigation.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.
Magnetic field-induced expression of Egr-1 in the trigeminal brainstem. (a,c,e) Frontal sections through PrV in ‘Sham’ (a), ‘Lido’ (c) and ‘Sect’ (e) birds. (b,d,f) Frontal sections through SpV in ‘Sham’ (b), ‘Lido’ (d) and ‘Sect’ (f) birds. Rostral is up, lateral is left. Note the strongly increased nuclear Egr-1 expression under ‘Sham’ (a,b; green) conditions. Egr-1 expression in PrV is confined to the crescent-shaped ventral PrVv part. (g) Quantitative analysis of Egr-1 expression. Boxes represent the upper/lower quartile, black line depicts the median; whiskers show a greatest value excluding outliers; outliers (black dots), defined as a value deviating more than 3/2 times the upper quartile. The number of Egr-1-expressing nuclei in PrV and SpV is significantly decreased only when the ophthalmic branch of the trigeminal nerve (V1) was cut (e,f; red) but is not significantly affected after lidocaine application to the upper beak (c,d; yellow). N.V, trigeminal nerve; N.VIII, vestibulo-cochlear nerve; PrVd, principal sensory nucleus of the trigeminal nerve; PrVv, ventral part of the nucleus of the trigeminal nerve; RF, reticular formation; SpVl, lateral part of the spinal trigeminal nucleus; SpVm, medial part of the spinal trigeminal nucleus; 5M, motor nucleus of the trigeminal nerve. **p < 0.01; ***p < 0.001. Scale bars: 200 µm (a, for a,c,e; b, for b,d,f).
Figure 2.
Figure 2.
Anatomical landmarks for the robin beak. (a–d) Coronal views of the four anatomical landmarks employed to normalize the spatial distribution of PB-positive cell counts between birds. Landmark 1 (a) was defined as the region where the central septum of the olfactory epithelium changed from a ‘U’ to a ‘V’ shape. Landmark 2 (b) was defined as the region where the central septum joined the ventral nasal cavity. Landmark 3 (c) was defined as the location where the small lateral buds of the nasal cavity disappear. Landmark 4 (d) was defined as the position where the nasal cavity is no longer visible. The fifth landmark (not shown) marks the rostral end of the beak. (e) Schematic top view of a beak showing the approximate locations of landmarks (a–d). Scale bar: 300 µm in (e).
Figure 3.
Figure 3.
Distribution of PB-positive cells. (a–h) Graph showing the distribution of PB-positive cells along the rostro-caudal axis of the beak for eight individual European robins in 240 µm increments. Approximate total cell counts are shown in the bottom right-hand corner, along with the bird number. Note the wide variation in the distribution and number of PB-positive cells per bird, and the different y-axis, e.g. for robin 19 (h).
Figure 4.
Figure 4.
Iron-rich cells in the ventral subepidermis of robins. (a–c) Representative images of PB-positive cells found in the ventral subepidermis in European robins. Note the presence of punctate spheres (highlighted with arrows), as well as light blue cytoplasmic staining. (d–f) Representative images of PB-positive cells stained with nuclear fast red, showing that iron-rich cells in the ventral subepidermis are nucleated. Scale bar: 10 µm (f, for a–f).
Figure 5.
Figure 5.
Neuronal immunostaining. (a,b) Representative images of the ventral subepidermis stained with PB, nuclear fast red (pink) and TUBB3 (brown). We found less than 0.6% of PB-positive cells colocalized with TUBB3 (n = 735 cells in eight birds). Arrows in (a) highlight PB-positive cells. (c) A positive control, TUBB3 staining of the ophthalmic branch of the trigeminal nerve. (d) A negative control (no primary antibody) when staining the ophthalmic branch of the trigeminal nerve. Scale bar: 10 µm (d, for a–d).

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