Manganese-enhanced magnetic resonance imaging reveals light-induced brain asymmetry in embryo
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This manuscript is important as it showed an establishment of a method for looking a neuronal activity in embryos which can support the previously reported laterality in chick thalamofugal system. However, the evidence the author provided was incomplete as no actual data was provided.
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Abstract
The idea that sensory stimulation to the embryo (in utero or in ovo) may be crucial for brain development is widespread. Unfortunately, up to now evidence was only indirect because mapping of embryonic brain activity in vivo is challenging. Here, we applied for the first time manganese enhanced magnetic resonance imaging (MEMRI), a functional imaging method, to the eggs of domestic chicks. We revealed light-induced brain asymmetry by comparing embryonic brain activity in vivo of eggs that were stimulated by light or maintained in the darkness. Our protocol paves the way to investigation of the effects of a variety of sensory stimulations on brain activity in embryo.
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Author Response
Reviewer #3 (Public Review):
Because of the position of pigeon embryos in eggs, light exposure will only stimulate the right eye, leading to lateralisation of brain responses and behaviour. Lorenzi and colleagues injected manganese chloride into pigeon eggs, to assess neuronal activation in the embryonic brain. While the eggs were placed in the light or dark, manganese ions accumulated in neurons that were activated (in cell bodies and axons), which was then visualized with MRI of the embryos before hatching. The authors report lateralisation of neuronal activity in three brain regions, which could potentially be important for our understanding of experience-dependent development of lateralised neural activation.
The tectofugal pathway in pigeons projects from the retina to the optical tectum, then to the nucleus …
Author Response
Reviewer #3 (Public Review):
Because of the position of pigeon embryos in eggs, light exposure will only stimulate the right eye, leading to lateralisation of brain responses and behaviour. Lorenzi and colleagues injected manganese chloride into pigeon eggs, to assess neuronal activation in the embryonic brain. While the eggs were placed in the light or dark, manganese ions accumulated in neurons that were activated (in cell bodies and axons), which was then visualized with MRI of the embryos before hatching. The authors report lateralisation of neuronal activity in three brain regions, which could potentially be important for our understanding of experience-dependent development of lateralised neural activation.
The tectofugal pathway in pigeons projects from the retina to the optical tectum, then to the nucleus rotundus in the thalamus, and then to the entopallium. The thalamofugal pathway projects from the retina to the GLd in the thalamus, and then to the wulst in the hyperpallium. The two pathways involve different thalamic nuclei (e.g., Deng 2006). In the methods and throughout the manuscript it should be specified which thalamic region is used as ROI.
Here we refer to the Gld in the thalamofugal visual pathway, we did not estimate activity in the n. rotundus. We have now clarified this point in the revised MS (ll. 54, 80, 86).
This manuscript only describes neural activity, but the MEMRI technique should also be used to assess the effect of experimental manipulations on axonal connectivity. It is important to learn about the asymmetry of contralateral projections in the light vs dark groups for answering the research question.
Here we used systemic administration of Mn through the CAM. The Blood Brain Barrier at this embryonic stage is not completely developed and its permeability to ions and small molecules is way higher in embryo than in later stages of development (Engelhardt, B. (2003). Development of the blood-brain barrier. Cell and tissue research, 314(1), 119-129.). Other studies involving direct, local injection in selected brain regions are more apt to investigate connectivity, but this is not the protocol used here. We appreciate the reviewer’s suggestion, and this will be the object of future experiments. However, we would like to disseminate the current protocol and the results it led to at an early stage to enable and encourage its use by other researchers in the field.
There is an overinterpretation of post-hoc statistics that are reported without correction for multiple testing. The wulst light group lateralization is probably not actually different from zero (uncorrected p=0.04).
We considered the reviewer's observation regarding the need for improvements in the statistical methods. In response, we have made amendments to the relevant section of the manuscript, explicitly stating that significant findings were obtained using a two-way ANOVA. For comparisons between conditions within specific brain regions, we conducted two-sample t-tests, and the results were corrected for Type I errors using the false discovery rate (FDR) method. Post-hoc one-sample t-tests were employed to assess lateralization across brain regions and conditions, and the corresponding p-values were reported without correction for multiple comparisons (as explicitly reported in the text, to avoid any confusion).
The first line in the discussion states that there is thalamofugal lateralization, but no lateralization in the tectofugal pathway. To my understanding, previous literature reported it the other way around: in altricial pigeons, light exposure in the egg mainly affected the tectofugal pathway (Deng & Rogers 2002), while the thalamofugal pathway in pigeons was not lateralized (Strockens et al., 2013). The manuscript should compare the current findings with the literature and discuss differences.
We are aware of the substantial differences in brain lateralization of the two visual pathways between pigeons and chicks after embryonic light exposure. However, in the present work we employed chick embryos (Gallus gallus domesticus), and the space limitations of a Brief Communication do not allow for an in-depth discussion of these differences between avian species.
Moreover, the tectum is the only region shown here from the tectofugal pathway. However, lateralization of contralateral connections is expected from tectum to the nucleus rotundus in the thalamus, and thus lateralization of activation may only arise in downstream brain regions from the optical tectum. Therefore, the conclusion that there is no lateralization in the tectofugal pathway is not supported by the data.
In conclusion, I think it is interesting and worthwhile that the authors assessed neural activity in response to visual stimulation in the embryo prior to hatching, but multiple methodological weaknesses and unclarities should be addressed.
The ROI that we here named Thalamus does not include the nucleus rotundus, but is referring to the nucleus geniculatus lateralis (Gld). We have now clarified this point in the revised MS (ll. 54, 80, 86), and we now refer only to the tectum, without generalizing to the entire tectofugal pathway, which will be the subject of future investigations.
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eLife assessment
This manuscript is important as it showed an establishment of a method for looking a neuronal activity in embryos which can support the previously reported laterality in chick thalamofugal system. However, the evidence the author provided was incomplete as no actual data was provided.
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Reviewer #1 (Public Review):
The authors have developed a new method to measure brain activity in the developing chick embryo. Thereby they have provided convincing evidence of asymmetry in the chick embryo and shown how it is influenced by exposure of the embryo to light. This is an important step forward in understanding the development of visual lateralization of behaviour and asymmetry of the thalamofugal visual pathway. Although asymmetry of the thalamofugal visual projections to the Wulst in newly hatched chicks has been well-documented previously, until now, it has not been possible to obtain such direct evidence of lateralized neural activity in the embryo.
The method that the authors have developed has potential for future research. It could now be applied at other times during embryonic development and to other species. In …
Reviewer #1 (Public Review):
The authors have developed a new method to measure brain activity in the developing chick embryo. Thereby they have provided convincing evidence of asymmetry in the chick embryo and shown how it is influenced by exposure of the embryo to light. This is an important step forward in understanding the development of visual lateralization of behaviour and asymmetry of the thalamofugal visual pathway. Although asymmetry of the thalamofugal visual projections to the Wulst in newly hatched chicks has been well-documented previously, until now, it has not been possible to obtain such direct evidence of lateralized neural activity in the embryo.
The method that the authors have developed has potential for future research. It could now be applied at other times during embryonic development and to other species. In fact, since the tectofugal system is asymmetrical in the pigeon, it would be interesting to use the technique in pigeon embryos, as a comparison.
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Reviewer #2 (Public Review):
The study was highly interesting personally as it tries to address a very important question of light induced brain development. The study uses a very efficient model system of birds. Using in-vivo MRI and a contrast agent increases the confidence on the results but also makes the experiments more challenging. I feel that the protocol will help fellow researchers interested in such questions a lot.
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Reviewer #3 (Public Review):
Because of the position of pigeon embryos in eggs, light exposure will only stimulate the right eye, leading to lateralisation of brain responses and behaviour. Lorenzi and colleagues injected manganese chloride into pigeon eggs, to assess neuronal activation in the embryonic brain. While the eggs were placed in the light or dark, manganese ions accumulated in neurons that were activated (in cell bodies and axons), which was then visualized with MRI of the embryos before hatching. The authors report lateralisation of neuronal activity in three brain regions, which could potentially be important for our understanding of experience-dependent development of lateralised neural activation.
The tectofugal pathway in pigeons projects from the retina to the optical tectum, then to the nucleus rotundus in the …
Reviewer #3 (Public Review):
Because of the position of pigeon embryos in eggs, light exposure will only stimulate the right eye, leading to lateralisation of brain responses and behaviour. Lorenzi and colleagues injected manganese chloride into pigeon eggs, to assess neuronal activation in the embryonic brain. While the eggs were placed in the light or dark, manganese ions accumulated in neurons that were activated (in cell bodies and axons), which was then visualized with MRI of the embryos before hatching. The authors report lateralisation of neuronal activity in three brain regions, which could potentially be important for our understanding of experience-dependent development of lateralised neural activation.
The tectofugal pathway in pigeons projects from the retina to the optical tectum, then to the nucleus rotundus in the thalamus, and then to the entopallium. The thalamofugal pathway projects from the retina to the GLd in the thalamus, and then to the wulst in the hyperpallium. The two pathways involve different thalamic nuclei (e.g., Deng 2006). In the methods and throughout the manuscript it should be specified which thalamic region is used as ROI.
This manuscript only describes neural activity, but the MEMRI technique should also be used to assess the effect of experimental manipulations on axonal connectivity. It is important to learn about the asymmetry of contralateral projections in the light vs dark groups for answering the research question.
There is an overinterpretation of post-hoc statistics that are reported without correction for multiple testing. The wulst light group lateralization is probably not actually different from zero (uncorrected p=0.04).
The first line in the discussion states that there is thalamofugal lateralization, but no lateralization in the tectofugal pathway. To my understanding, previous literature reported it the other way around: in altricial pigeons, light exposure in the egg mainly affected the tectofugal pathway (Deng & Rogers 2002), while the thalamofugal pathway in pigeons was not lateralized (Strockens et al., 2013). The manuscript should compare the current findings with the literature and discuss differences.
Moreover, the tectum is the only region shown here from the tectofugal pathway. However, lateralization of contralateral connections is expected from tectum to the nucleus rotundus in the thalamus, and thus lateralization of activation may only arise in downstream brain regions from the optical tectum. Therefore, the conclusion that there is no lateralization in the tectofugal pathway is not supported by the data.
In conclusion, I think it is interesting and worthwhile that the authors assessed neural activity in response to visual stimulation in the embryo prior to hatching, but multiple methodological weaknesses and unclarities should be addressed.
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