Since first manual experiments on DNA sequencing and PCR were reported, industries developed automatic-machines based on original principal (Supplementary Fig.S1). Thus, we will mainly discuss four perspectives; (1) How to develop machine based on the results of Fig. 3 (Fig. 4), (2) What sort of inoculatable devices to be developed (Fig. 2), (3) Application of automatic-brain research to a variety of vertebrates (Fig. 5), (4) Facilitation of study on animal behavior via automatic-brain research. Perspectives, (1)(2)(3) will be developed by industries, just like automatic-DNA sequencing. Achievement of (1)(2)(3) will lead us to legendary item, “King Solomon’s Ring” [10], which enables us to communicate with a variety of vertebrates. Thus, individual researchers could study animal behavior using such symbolic Ring in future.
First, we propose how to automatically inoculate probes into chick brains. Although we succeeded in non-invasively generating animals whose brains contain multiple beads at birth, accurately embedding the beads in specific regions of the brain was not achieved. To overcome this problem, it is essential to develop automatic robotic inoculation systems (Fig. 2A(b)). The following four different industrial systems (Fig. 4(a–d)) exist: (a) the traditional pharmaceutical vaccine production line (which automatically sterilizes fertilized chick eggs and bores a hole in the eggshell), an automatic precise probe inoculation system combining (b) an integrated-circuit electronic production line and (c) an automatic staging and recording stereomicroscope system (utilized in various biological and medical sciences to record digital information of the x_y_z position of the object), and (d) poultry farming for the massive production of chicks. Combining these industries will enable a mass production line of chicks whose brains contain hundreds of beads (probes) (Fig. 2A(b), Fig. 4). Moreover, (e) computed tomography scanning of chick brains could individually, precisely, and digitally record the x_y_z position of each embedded bead in the brain. Finally, (f) artificial intelligence-mediated deep learning of the inoculation position in the embryo (c, g) and the real position of the beads embedded in the brain (e) guarantees an optimized program that inoculates probes into pre-defined brain regions.
Since existing surgical robots have names such as “da Vinci”, the entire system of this putative automatic robot (Fig. 4) could be called “Spemann”, referring to the developmental biologist who manipulated vertebrate embryos and found “the organizer” [11]. “Spemann” could meet the academic and industrial demand for brain probes, devices, and electrodes that are controlled via NIR from outside the skull (Fig. 2A(a) and Fig. 4(h)).
Second, we propose candidate probes and integrating whole brain research. Some existing methods in brain research (Fig. 1) can be replaced with the technology of non-invasively embedding brain probes that are individually driven by NIR (Fig. 2A(0)). Such technologies (Fig. 2A(1)–(13)) include probes that (1) destroy the surrounding neurons in a temporally and spatially specific manner, like surgical resection; (2) sense oxygen or glucose, like fMRI or PET; (3) sense a variety of molecules including neurotransmitters, (4) simultaneously or individually stimulate or suppress neuronal activities, like MEA; (5) simultaneously or individually record neuronal activities, like MEA; and (6) deliver drugs in a temporally and spatially specific manner, instead of drug injection. Furthermore, (7) massive digital signals of a particular animal that are interconnected with machines, robots, and other animals via the internet according to the brain-net concept could be employed. Upon such detailed in vivo top-down probe analyses (1–7), (8) biopsies of the corresponding probes can enable a variety of biochemical analyses, and (9) the neuronal network as a connectome [12] can be visualized by neurons that take up retrograde-type dyes released from the probes. Furthermore, after euthanizing animals, (10–11) neurons and brain sections can be analyzed in vitro by stimulating, suppressing, and recording their activity via the probe; (12) and the signal transduction of neurons and brain sections can be analyzed in vitro around the probe. These bottom-up analyses (8–12) could be functionally and mutually linked to top-down analyses (1–7), integrating all brain research. Finally, when using transgenic animals or recombinant virus-infected animals for optogenetics, (13) blue light can be supplied from the NIR-driven probe [2].
Importantly, such a putative “Spemann” system (Fig. 4) could reproducibly and simultaneously embed multiple probes in the brain, enabling the reproduction of any experiment, anytime and anywhere. Furthermore, all digital data could be opened, shared, analyzed, and utilized by everyone, similar to the free accessibility of existing DNA sequence data banks (Supplementary Fig.S1). In a similar manner to the recent establishment of automatic DNA sequencing (Supplementary Fig.S1(b)(c)), positive feedback between continuous technological improvements that accurately inoculate brain probes and the development of various functional probes (Fig. 4(h)) will synergistically accelerate and integrate a variety of top-down and bottom-up brain research (Fig. 2A). Once such positive feedback begins (Fig. 4(h)), big data and the industrialization of neuroscience [13] will be accelerated under Moore’s law. Thus, the experimental results in Fig. 3 will be a key foundation for the transition from the brain research in Fig. 1 to that in Fig. 2A, via both the development of “Spemann” (Fig. 4) and new functional probes (Fig. 2A and Fig. 4(h)). If so, we predict that the “emperor” of neuroscience research [14] will wear a “new wardrobe” (i.e., the chick as a model [15]) in the near future.
Third, we propose application of the technology on chicks toward studies on a variety of vertebrate behavior. This method of producing chicks with multiple probes embedded in the brain in a non-invasive manner could be applied to any amniote. In chick embryos, stage 18 (Figs. 3A(b) and 5(a)) is similar to the pharyngula stage (Fig. 5(b)), sketched by Haeckel nearly 150 years ago [16]. The pharyngula embryonic stage of reptiles and mammals is remarkably similar to the embryo in Fig. 5(b), noted by Haeckel [16]. Thus, the proposed automatic “Spemann” system (Fig. 4) could be applied to any chick-type eggs derived from birds and reptiles. Thus, the behaviors and brain activities of many animals, such as penguins, hummingbirds, chameleons, and turtles (Fig. 5(c)–(f)), could be analyzed (Fig. 2A), tremendously facilitating comparative brain research. A robot embedding multiple electrodes in the rat brain has been developed (Fig. 1(a)) [1]. Thus, a robot that could operate small mammals (e.g., mice and rats) and embed probes into the head region of embryos in utero could be developed (Fig. 5(g)) using the “Spemann” method, yielding mice or rats with probes inside their brains at birth (Fig. 5(h)).