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Final Report Summary - TOPO_HOX (Mapping the face in the somatosensory brainstem: genetic and activity-dependent mechanisms.)

The aim of the project is to enhance our understanding of the fundamental mechanisms by which the somatosensory topographic connectivity neural map is established during early development in mammals. It is well known that the blockage of afferent sensory activity, such as after a limb amputation, can lead to neurons to develop an increase sensitivity to tactile stimulation from ectopic sites outside their receptive fields, giving rise to the phenomenon of “phantom limb” sensation (1). Unraveling the basic molecular mechanisms at the bases of the mammalian somatosensory map formation is essential for the discovery of new therapeutic applications in order to mitigate the side effects of a limb amputation, from pain to psychological form of stress. In addition, the knowledge of biological basic mechanism is fundamental for the development of appropriate “ad hoc” neuroprostethic device supported Brain Machine Interface (BMI).
We use as model the rodent whisker-to-barrel system, a well-established model in which input coming from a single whisker make an ascending pathway, which clusters into discrete anatomical and functional units within different brain relay stations (barrelettes, barreloids and barrels within the brainstem, thalamus and cortex, respectively) (2). It is well known that sensory driven neuronal activity coming from the whiskers already plays a central role at birth in developing the whisker-to-barrel map, which is considered fully completed one week after birth. However, it is not known if the prenatal neural activity, which role has been already observed for the correct establishment of the sensory developmental connectivity in the visual, olfactory and auditory systems, has also a role during the development of the whisker-to-barrel map.
Using transgenic mice we silenced the early neural activity of whisker related modules at the brainstem level (barrelettes) at prenatal stages. Then, applying different neural tracing techniques and the rabies virus technology, we are able to visualize at a single cell level the impact on the whisker to barrel connectivity made by a perturbed prenatal neuronal activity.
Similar to previous reports in which activity was perturbed only postnatally by infraorbital nerve (ION) crush (3) or prenatally (but in the whole animal), when we impaired neuronal activity in the developing barrelette neurons we observed a perturbation of the whisker-to-barrel map using different immunostaining techniques. We found that perturbation of the neuronal activity in neurons located within the first relay station (i.e. barrelettes) impairs the map formation within the whole whisker-to-barrel map in the thalamus and in the cortex (fig. 1). However the main connectivity and gross topography did not result perturbed. Using different neural tracing techniques, we dissected the effects of the perturbation of prenatal neuronal activity. In particular we observed that the topography and refinement of the whisker-related afferent axonal fibers coming from the trigeminal ganglion were not affected when neuronal activity was perturbed prenatally in their post-synaptic target (i.e. the barrelette neurons). On the other hand, activity perturbed barrelette neurons show a symmetric dendritic arbor pattern due to an altered maturation process. The analysis was performed making a specific sparse labeling of barrelette neurons using pseudotyped rabies virus injected in their thalamic target area. After imaging, the 3D dendritic pattern was analyzed upon the creation of specific codes in MATLAB®. This allowed a quantitative measurement of the barrelette neurons dendritic arbors in wild type and activity perturbed animals. Finally, using transynaptic rabies viruses, we observed that upon prenatal activity impairment barrelette neurons were still monosynaptically connected with trigeminal axons, whose synaptic outputs deliver whisker information from the periphery. In summary these data showed that, following hyperpolarization of their membrane using transgenic mice through an intersectional approach, barrelette neurons still retain their potential for a sensory-driven dendritic orientation but they are incapable to enforce sensory information coming from the periphery (fig.5).
Next, we investigated if prenatal impairment of neuronal activity in barrelette neurons can alter the size of the whole affected nucleus in the brainstem, therefore also changing the proportions of somatosensory map within the brainstem. This analysis was performed using transgenic mice allowing the visualization of the brainstem nucleus that relays somatosensory information of the face and whiskers (i.e. the Principal Nucleus, PrV). We found that early perturbation of neuronal activity does not affect the size and the boundaries of the somatosensory brainstem nuclei. The same transgenic mice allowed us to observe if somatosensory target thalamic nuclei were affected when neuronal activity was prenatally perturbed in the barrelette neurons. In particular we found that the thalamic nuclei relaying the whisker information (i.e. dorsal VPM) was significantly shrunk at end of the critical period when activity was prenatally perturbed in the corresponding brainstem nuclei (ventral PrV). We found that the mutant animals already showed a significant reduction of the dorsal VPM volume before birth, when compared with wild type animals. These results are different from deprivation of sensory neuronal activity right after birth, which does not affect the volume of the dorsal VPM (4). Taken together, these results show that prenatal neuronal activity perturbation in the barrelette neurons is fundamental for the development of the whisker-to-barrel map and the size of target somatosensory thalamic nuclei. These experiments also show that the size and the different proportion of the different somatosensory thalamic nuclei are, at least partially, defined by the prenatal neuronal activity in the brainstem nuclei.
A draft for publication in peer-review journal has been prepared and will be soon submitted.
While the research activities performed are aimed to understand basic biological mechanisms, the research outputs allow to significantly advancing the state of the art in the field of developmental neuroscience. As initially described, the same basic mechanisms establishing the somatosensory topographic map and neuronal connections during development might be applied to the plastic events taking place during amputations and appearance of the phantom limb phenomena.
To be also noticed that during the research several cutting-edge technologies have been used (i.e. transynaptic rabies viruses, analysis of brain nuclei reconstructed in 3D using specific software). The protocols and the methods will be also soon available upon the publication of the research on specialized scientific journal.

Bibliography:

1) Schaefer M, et al. My third arm: shifts in topography of the somato-sensory homunculus predict feeling of an artificial supernumerary arm. Hum Brain Mapp. 2009 May;30(5):1413-20.
2) Erzurumlu RS, Murakami Y, Rijli FM. Mapping the face in the somatosensory brainstem. Nat Rev Neurosci. 2010 Apr;11(4):252-63. doi: 10.1038/nrn2804. Review.
3) Lo FS, Erzurumlu RS. Sensory Activity-Dependent and Sensory Activity-Independent Properties of the Developing Rodent Trigeminal Principal Nucleus. Dev Neurosci. 2016;38(3):163-170
4) Bechara A et al. Hoxa2 Selects Barrelette Neuron Identity and Connectivity in the Mouse Somatosensory Brainstem. Cell Rep. 2015 Oct 27;13(4):783-97. doi: 10.1016/j.celrep.2015.09.031.

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FRIEDRICH MIESCHER INSTITUTE FOR BIOMEDICAL RESEARCH
Switzerland
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