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From retina to motoneurons: A substrate for visuomotor transformation in salamanders

Abstract The transformation of visual input into motor output is essential to approach a target or avoid a predator. In salamanders, visually guided orientation behaviors have been extensively studied during prey capture. However, the neural circuitry involved is not resolved. Using salamander brain preparations, calcium imaging and tracing experiments, we describe a neural substrate through which retinal input is transformed into spinal motor output. We found that retina stimulation evoked responses in reticulospinal neurons of the middle reticular nucleus, known to control steering movements in salamanders. Microinjection of glutamatergic antagonists in the optic tectum (superior colliculus in mammals) decreased the reticulospinal responses. Using tracing, we found that retina projected to the dorsal layers of the contralateral tectum, where the dendrites of neurons projecting to the middle reticular nucleus were located. In slices, stimulation of the tectal dorsal layers evoked glutamatergic responses in deep tectal neurons retrogradely labeled from the middle reticular nucleus. We then examined how tectum activation translated into spinal motor output. Tectum stimulation evoked motoneuronal responses, which were decreased by microinjections of glutamatergic antagonists in the contralateral middle reticular nucleus. Reticulospinal fibers anterogradely labeled from tracer injection in the middle reticular nucleus were preferentially distributed in proximity with the dendrites of ipsilateral motoneurons. Our work establishes a neural substrate linking visual and motor centers in salamanders. This retino‐tecto‐reticulo‐spinal circuitry is well positioned to control orienting behaviors. Our study bridges the gap between the behavioral studies and the neural mechanisms involved in the transformation of visual input into motor output in salamanders.


INTRODUCTION
Visually guided orientation behaviors allow animals to move toward or away from a stimulus. In salamanders, visually guided orientation behaviors have been extensively studied in the context of prey catching. When a prey appears in the visual field, such as a cricket (Schülert & Dicke 2002) or a worm (Roth, 1987), some salamander species turn their head toward the prey and use full body movements to follow it and eventually snap it (Schülert & Dicke 2002). Other salamander species ambush their prey and use motion extrapolation to catch it by projecting their tongue (Borghuis & Leonardo 2015). When presented with two different quantities of prey on the left and right visual fields, salamanders orient toward the highest number of preys (Uller et al. 2003, Krusche et al. 2010). However, the circuitry underlying asymmetric visually guided orientation behaviors is not fully resolved in salamanders.
A brain region playing a key role in the integration of visual inputs to generate eye, head, or body movements to approach or avoid a sensory object is the optic tectum, also called superior colliculus in mammals (for review Isa et al. 2021, Basso et al. 2021. In salamanders as in other vertebrates, the anatomical organization of the retinal inputs in the tectum follows the topography of the visual field, and therefore builds a map of visual inputs (Grüsser-Cornehls & Himstedt 1973, Margolis 1976a, 1976b, Manteuffel et al. 1989, Jakway & Riss 1972, Gruberg 1973, Guillery & Updyke 1976, Caldwell and Berman 1977, Fritzsch 1980, Fujisawa et al. 1981, Ingham & Güldner 1981, Rettig & Roth 1982, Rettig 1988. Extracellular recordings showed that salamander tectal neurons respond to specific size, contrast, or movement pattern of a stimulus moving in the visual field (Grüsser-Cornehls & Himstedt 1973, Roth 1982, Schuelert & Dicke 2005. Single cell fills and immunohistochemistry provided detailed information about the anatomy and overall projection patterns of salamander tectal neurons (Dicke 1999, Roth et al. 1999, Roth et al. 1990, Landwehr & Dicke 2005). However, the precise downstream targets through which tectal neurons generate asymmetrical body movements are largely unknown in salamanders.
However, the existence of this circuitry was not tested experimentally in salamanders.
Here, we aimed at uncovering the neural circuitry through which visual input is transformed into an asymmetric motor command in salamanders. Using tracing and calcium imaging, we found a retinotecto-reticulo-spinal pathway that constitutes a substrate for visually guided orientation movements in salamanders. Our study opens the door to mechanistic and computational studies of visuomotor transformations in salamanders. It also contributes to our understanding of visuomotor transformations together with other models such as, for example, lamprey, zebrafish, rodent, and primate (Rozenblit & Gollisch 2020). In addition, the regenerative capacities of the salamander make it a unique model to examine how this circuitry is rewired after injury (Joven & Simon, 2018, Rozenblit & Gollisch 2020).

Ethics statement
The procedures were in accordance with the guidelines of the Canadian Council on Animal Care and approved by the animal care and use committees of the Université de Sherbrooke.

Animals
We used juvenile salamanders Pleurodeles waltl (Andras Simon laboratory, Karolinska Institute, Stockholm, Sweden) of either sex with snout-vent length ranging from 50 to 80 mm. The animals were housed in plastic tanks at 17-19 • C and fed with bloodworms, beef meat, and fish pellets. The anatomical experiments were carried out in eight CAG:NucCyt females and one CAG:NucCyt male and three wildtype females. The physiology experiments were carried out in fifteen leucystic males, nine leucystic females and two wild-type females.

Surgical procedures
The animals were anesthetized with tricaine methanesulfonate (MS-

Microscopy
Whole mounts or brain sections were observed using an epifluorescent

Optical density
To estimate the density of the descending RS innervation in the spinal cord, we measured in spinal cord sections the optical density of the fluorescent signal generated by the fibers anterogradely labeled from TRDA injection in the reticular nucleus (Xavier et al. 2005, Perlbarg et al. 2018) as we did in a previous study (Fougère et al. 2021b

Tectum slices
To record the tectum cells projecting to the mRN, the tracer Ca 2+ green was injected at the level of the mRN bilaterally and the preparation was pinned down in a cold (10 • C) oxygenated chamber overnight. The next day, transverse slices were obtained as previously described

Calcium imaging
The procedure was similar to that we previously used in lampreys and salamanders (e.g., Brocard et al. 2010;Ryczko et al. 2016aRyczko et al. , 2016bRyczko et al. , 2016c. The brain rostral to the optic nerve was removed following a
The injected volumes were estimated by measuring the diameter of a droplet ejected in air from the tip of the pipette multiplied by the number of pressure pulses, and the resulting number of moles ejected was calculated (Le Ray et al., 2003;Ryczko et al., 2013Ryczko et al., , 2016aRyczko et al., , 2016bRyczko et al., , 2017Ryczko et al., , 2020aRyczko et al., , 2021).

Electrical stimulation
Glass-coated tungsten microelectrodes (0.7-3.1 MΩ with 10-40 μm exposed tip) and a Grass S88 stimulator coupled to a Grass PSIU6 photoelectric isolation unit for controlling stimulation intensity (Astro Med) were used. The stimulation electrode was placed in the retina or optic tectum. In tectal slices, the electrode was placed in the dorsal layers, ∼100 μm lateral to the level of the recording site of the deeper located tectal output neurons, to elicit action potentials in retinofugal axons contacting the recorded tectal output neurons. The electrical stimulation consisted of square pulses (2 ms duration) applied with a frequency of 10 Hz for 5 s. A pause of at least 2 min was given between two stimulation trains. The stimulation intensities ranged from 1 to 70 μA.

Statistical analysis
Data are presented as mean ± SEM unless stated otherwise. Statistical analyses were done using Sigma Plot 12.0. Parametric analyses were used when assumptions for normality and equal variance were respected, otherwise nonparametric analyses were used. To compare the means between two dependent groups, a two-tailed paired t-test or a nonparametric Wilcoxon signed rank test was used. To compare the means between two independent groups, a two-tailed t-test or a nonparametric Mann-Whitney rank sum test was used. Linear and regressions between variables, their significance, and the confidence intervals were calculated using Sigma Plot 12.0. Statistical differences were assumed to be significant when p < .05.

RESULTS
We examined the existence of a visuomotor pathway that would serially connect the retina, tectum, mRN RS neurons, and motoneurons of the third spinal segment. To visualize the entire visuomotor pathway, the reader is referred to Figure 7.

Retina stimulation evokes RS responses
In salamanders, RS neurons of the mRN evoke ipsilateral steering movements when activated pharmacologically in semi-intact preparations (Ryczko et al. 2016c). We examined whether retina could evoke responses in mRN RS neurons retrogradely labeled by injection of the calcium (Ca 2+ ) sensor Ca 2+ green in the first spinal segment (Ryczko et al. 2016a(Ryczko et al. , 2016b(Ryczko et al. , 2016c). An electrical stimulation electrode was placed in the retina on one side in the isolated brain (Figures 1(a)).

Retina projects to thalamus, pretectum, and tectum
We then examined the circuitry involved in this sensorimotor transformation. To identify the central targets of the retina, we anterogradely labeled the retinofugal projections by injecting a tracer in the optic nerve (biocytin, n = 2 animals; TRDA, n = 1 animal) (Figure 2(a)). These injections anterogradely labeled the optic tract (Figure 2(b)), and fibers in the contralateral lateral geniculate nucleus of the thalamus (corpus geniculatum thalamicum) and in the neuropil of Bellonci (Figure 2(c)).
More caudally in the brain, anterogradely labeled fibers were found in the contralateral pretectum (Figure 2(d)), and a dense innervation was found in the dorsal layers of the contralateral tectum (Figure 2(e)).
Occasionally some fibers were observed in the tectum ipsilateral to the injected optic nerve. Some retrogradely labeled cell bodies projecting to the retina were also observed in the tegmentum ventral to the tectum, as reported in many vertebrate species (e.g., ventral brain in Figure 2(e), for review see Repérant et al. 2006Repérant et al. , 2007. Altogether, this indicates that the retina sends projections to the thalamus, pretectum and tectum, and receives projections from central neurons in the tegmentum, features that are well conserved from basal vertebrates to mammals (see discussion, Suryanarayana et al. 2020, Repérant et al. 2006.

Tectal neurons innervated by retinal fibers project to the mRN
The tectum plays a key role in visuomotor transformation in vertebrates (Basso et al. 2021, Isa et al. 2021). Next, we examined using

Tectal neurons projecting to mRN receive glutamatergic inputs from retina
Next, we examined the transmission involved. In the isolated brain, we stimulated the retina and recorded the Ca 2+ RS responses in the mRN (Figure 3(a)). In each preparation, the Ca 2+ responses were normalized as a function of the maximal response. Responses were stronger . Each trace represents the responses of a single RS cell ipsilateral to stimulation (g) or contralateral to stimulation (h). (i and j) Relationships in six preparations between Ca 2+ response peak (mean ± SEM) and retina stimulation intensity for RS cells located ipsilateral (n = 49 RS cells, 4-11 cells per preparation, (i)) and contralateral (n = 41 RS cells, 4-11 cells per preparation, (j)) to the stimulated retina (0.6-30 μA, 5 s train, 10 Hz, 2 ms pulses). In each preparation, response peaks were expressed in % of the maximal peak recorded, and stimulation intensity in % of the maximal intensity used. (k and l) Relationship between Ca 2+ response peak (mean ± SEM) and stimulation intensity in the same 6 animals as in (i and j). Data were binned with a bin size of 10%. The data followed a linear polynomial function both for ipsilateral RS cells (solid black line, p < .001, R = 0.98, (k)) and contralateral RS cells (solid black line, p < .001, R = 0.91, (l)). Note that RS cells contralateral to retina stimulation generated lower maximal responses. Gray lines illustrate the confidence intervals (+37%) on the ipsilateral side (p < .001, t-test, Figures 3(b) and 3(c)), consistent with the results presented in Figure 1. The retina-evoked Ca 2+ responses were largely decreased after bath application of the glutamatergic antagonists CNQX and AP5 in mRN RS located ipsilaterally (p < .001, paired t-test, n = 30 RS cells) and contralaterally (p < .001, paired t-test, n = 30 RS cells) to the stimulated retina (Figures 3(b) and 3(c), data pooled from four preparations, five to nine trials per drug condition per preparation).
Next, we examined whether local blockade of a tectal glutamatergic synaptic relay contralateral to the stimulated retina was sufficient to abolish the retina-evoked RS responses. In the isolated brain, we stimulated the retina and recorded the Ca 2+ responses in mRN RS neurons  -c), showing the responses of RS cells ipsilateral (N = 29 RS cells pooled from three animals, 7-11 cells per animal) or contralateral (N = 16 RS cells pooled from three animals, 4-8 cells per animal) to retina stimulation (5 s train, 10 Hz, 2 ms pulses, 3-10 μA) before and 4-20 min after local injection of CNQX (0.07 pmol) and AP5 (0.03 pmol) in the tectum contralateral to the stimulated retina using a picospritzer (CNQX 1 mM, AP5 0.5 mM). Each line illustrates the average response of one cell (five to seven trials per cell per drug condition). (g and h) To record tectal neurons projecting to the mRN, Ca 2+ green was injected in the mRN and transverse slices of the tectum were obtained. (i and j) A stimulation electrode was placed laterally (∼100 μm) from the apical dendrites of the labeled tectal neurons, and the Ca 2+ responses of tectal neurons were recorded. (k) Ca 2+ fluorescence at rest of tectal neurons. (l) Ca 2+ response in tectal cells in response to stimulation of the retinal input layer before (single 2 ms pulses, 60-70 μA, five to seven trials per cell per drug condition) and 6-38 min after bath application of CNQX (0.9 μM) and AP5 (3.5 μM). On the color plot, each line illustrates the average response of one cell (five to seven trials per cell per drug condition). (m) Bar charts showing that Ca 2+ responses of tectal neurons decreased in presence of glutamatergic antagonists (n = 4 tectal cells pooled from two slices from two animals). *, p < .05, ***, p < .001, paired t-test; +++ p < .001, t-test ( Figure 3(d)). These responses were again stronger ipsilaterally (+29%), consistent with Figures 1 and 3(a)-3(c). Local microinjections of the glutamatergic antagonists CNQX and AP5 in the tectum located contralateral to the stimulated retina largely decreased Ca 2+ RS responses ipsilaterally to the stimulated retina (n = 29 RS cells pooled from three preparations) and contralaterally to the stimulated retina (n = 16 RS cells) (Figures 3(e)-3(f)). We further tested the presence of a local glutamatergic synapse between retinofugal inputs and tectal output neurons projecting to the RS neurons in another series of experiments.
We retrogradely labeled the tectal output neurons projecting to the mRN using Ca 2+ green injections in the right mRN (Figure 3(g)). Transverse slices of the tectum were obtained (Figures 3(h) and 3(i)) and the retrogradely labeled tectal neurons and their dendritic trees could be observed (Figure 3

Tectum sends bilateral inputs to mRN RS neurons
We then examined whether the tectum could control the activity of mRN RS neurons through such tecto-reticular pathway. In the isolated brain, a stimulation electrode was placed in the tectum on one side and we used Ca 2+ imaging to record mRN RS neurons retrogradely labeled by Ca 2+ green injection in the spinal cord (Figures 4(a) and 4(b)).

The mRN projects to spinal motoneurons
We then looked for an anatomical substrate through which the mRN, which receives innervation of the tectum and therefore relays retinal inputs, could feed such inputs to spinal motor circuits to generate body movements. To do this, we used dual tracing experiments. To retrogradely label motoneurons innervating axial and forelimb muscles on one side, a tracer (biocytin) was injected in the right ventral root of the third cervical segment (Figure 5(a)). These motoneurons were positive for the motoneuronal marker Islet-1/2 (n = 3 preparations,

Tectum evokes spinal motoneuron responses via the mRN
We then examined whether unilateral activation of the tectum trans-

DISCUSSION
In the present study, we identified a neural substrate for visuomotor transformation in salamanders. The circuitry comprises the retina, . Each trace represents the responses of a single RS cell ipsilateral to tectum stimulation (g) or contralateral to stimulation (h). (i and j) Relationships in four preparations between Ca 2+ response peak (mean ± SEM) and tectum stimulation intensity for RS cells located ipsilateral (n = 28 RS cells, 5-8 cells per preparation, (i)) and contralateral (n = 32 RS cells, 7-10 cells per preparation, (j)) to the stimulated tectum (1-11 μA, 5 s train, 10 Hz, 2 ms pulses). In each preparation, response peaks were expressed in % of the maximal peak recorded, and stimulation intensity in % of the maximal intensity used. (k and l) Relationship between Ca 2+ response peak (mean ± SEM) and tectum stimulation intensity in the same 4 animals as in (i and j) Data were binned with a bin size of 10%. The data followed a linear polynomial function both for ipsilateral RS cells (

A conserved visuomotor circuitry
The organization of the retino-tecto-reticulo-motoneuronal pathway we report here is in accordance with the circuitry proposed in salamander modeling studies to implement visually guided orienting behaviors (Ijspeert & Arbib 2000, Petreska 2004). In our experiments, we do not provide a definitive evidence that the retino-tecto-reticulomotoneuronal connections are monosynaptic. However, this is possible since in lamprey, retina send monosynaptic excitation to tectal output neurons (Kardamakis et al. 2015), tectal output neurons send monosynaptic input to RS neurons (Kardamakis et al. 2015), and RS neurons send monosynaptic input to motoneurons , Otah & Grillner 1989, Suzuki et al. 2019. We cannot rule out that spinal premotor interneurons could intervene in the motoneuronal responses. In lamprey, interneurons of the central pattern generator for locomotion are also targeted by RS neurons . In mice, spinal interneurons are targeted by ipsilaterally projecting Chx10 + RS neurons and this contributes to orienting maneuvers (Bouvier et al. 2015, Cregg et al. 2020, Usseglio et al. 2020. Therefore, in salamanders RS connections on central pattern generator neurons could also contribute to tectum-evoked steering as proposed in a salamander modeling studies (Ijspeert & Arbib 2000, Petreska 2004 and in a lamprey modeling study (Kozlov et al. 2014). Future studies should identify the muscles of the motoneurons recorded here, which possibly comprise motoneurons innervating axial or limb muscles (Francis 1934). Previous modeling studies suggested that targeting axial muscles is sufficient to evoke orienting maneuvers during ongoing locomotion (Ijspeert & Arbib 2000). The descending RS projections possibly also innervate neck motoneurons, which are located in the cervical segments in salamanders (Nishikawa et al. 1991) as in birds (Hörster et al. 1990) and mammals (for review Isa & Sasaki 2002). Such possible connection to neck motoneurons would allow the animal to couple an ipsilateral head movement together with ipsilateral body bending toward the visual target, as recently shown in mice (Cregg et al. 2020, Usseglio et al. 2020).
It appears that the retino-tecto-reticulo-motoneuronal pathway is highly conserved in vertebrates. Lamprey and zebrafish tectal output neurons project to RS neurons, which send descending commands to spinal motor circuits (lamprey: Kardamakis et al. 2015, Suzuki et al. 2019zebrafish: Gahtan et al. 2005). In mammals, descending connections from the superior colliculus (the mammalian tectum) to the RS system control turning and orienting movements in vivo (Cregg et al. 2020, Usseglio et al. 2020). These authors found that glutamatergic neurons of the superior colliculus project to contralateral Chx10 + RS neurons that control ipsilateral steering movements via ipsilateral projections to spinal cord neurons (Cregg et al. 2020, Usseglio et al. 2020). In our experiments, whether the RS neurons that responded to stimulation of the contralateral tectum also express Chx10 remains to be determined. This is possible since RS neurons projecting ipsilaterally are Chx10 + positive in mice (see e.g., Bouvier et al. 2015, Cregg et al. 2020, Usseglio et al. 2020) and the Chx10 gene is present in salamanders (Ryczko et al. 2020b).
Our study also highlights the multiple functions of RS neurons. In salamanders mRN RS neurons also receive the locomotor drive generated by the Mesencephalic Locomotor Region (i.e. MLR), a region that controls locomotion initiation and speed in salamanders (Cabelguen et al. 2003, Ryczko et al. 2016a) as in other vertebrates (e.g., lamprey: Brocard et al. 2010, rat: Bachmann et al. 2013, cat: Shik et al. 1966, Opris et al. 2019mouse: Bretzner & Brownstone 2013, Lee et al. 2014, Roseberry et al. 2016, Capelli et al. 2017, Caggiano et al. 2018, Josset et al. 2018, van der Zouwen et al. 2021, Dautan et al. 2021, pig: Chang et al. 2021. In lamprey, RS neurons also relay the MLR drive (Brocard et al. 2010), tactile inputs that evoke locomotion (Di Prisco et al. 1997), but also the olfactory inputs that evoke locomotion (Derjean et al. 2010, Daghfous et al. 2018, Beauséjour et al. 2020, for review Beauséjour et al. 2021). Altogether this strengthens the idea that RS neurons are key command neurons that evoke and adapt locomotion in many circumstances and can be considered the "final common descending pathway" activating the spinal locomotor networks (Dubuc et al. 2008).
contralateral to tectum stimulation generated lower maximal responses. Gray lines illustrate the confidence intervals. From there, RS neurons send a mainly ipsilateral descending excitatory input to motoneurons, which increase muscular contraction on the side of the detected visual input. Here, the recorded motoneurons were retrogradely labeled from the third ventral root. The descending RS input is in a good position to also target other motoneurons in spinal segment three, such as those innervating the neck muscles (Nishikawa et al. 1991, also see discussion). Altogether, the spinal motoneurons activated by the visual stimulus would cause orientation movements toward the visual stimulus. Right, the circuitry could account for prey tracking during ongoing locomotion. When a prey appears in the left visual field (e.g., cricket), the proposed circuitry would allow the animal to orient its body movements toward the prey and track it, to eventually snap it et al. 2019). A lamprey modeling study suggested that asymmetrical activity of tectal output neurons generate steering commands through modulation of RS activity during locomotion (Kozlov et al. 2014).
We were not able to evoke stronger mRN RS responses contralaterally to the stimulated retina, a response that would be interpreted as an escape response according to salamander modeling studies (Ijspeert & Arbid 2000, Petreska 2004) and previous lamprey studies (Kardamakis et al. 2015, Suzuki et al. 2019. A possibility is that our retina stimulations preferentially recruited tectal output neurons projecting to contralateral mRN RS neurons. In lamprey, such neurons are more excitable, with a spiking threshold 8 mV lower than those projecting ipsilaterally (Kardamakis et al. 2015). Future studies should determine whether a similar difference in intrinsic properties is present in salamander tectal output neurons. Another factor that could contribute is that we used electrical stimulation of the retina, which does not activate retinal cells the way a natural visual stimulus, such as a predator, would. In salamander retina slice, whole cell patch-clamp recordings showed that the brief current pulses used here (2 ms) preferentially activate retinal ganglion cells, which send their axons through the optic nerve to the brain, rather than local retinal bipolar or amacrine cells (Margalit & Thoreson 2006, Luo et al. 2012

Visuomotor processing beyond the optic tectum
On top of the retino-tecto-reticulo-motoneuronal pathway we describe here, other circuits likely contribute to visuomotor processing in salamanders. In the tectum, local GABA-immunoreactive neurons are present in all the cellular layers (Franzoni & Morino 1989). In lamprey, tectal GABAergic interneurons play a role in the selectivity of the activated tectal output neurons (Kardamakis et al. 2015). Beyond visual information, the tectum receives other sensory modalities and performs multisensory integration, such as aligning auditory and visual maps in owls (e.g., Knudsen 1982, Reches & Gutfreund 2009, Mysore et al. 2010, modeled in Huo et al. 2012 or integrate visual and electrosensory information in lamprey (Kardamakis et al. 2016). Future studies should explore whether the pathways reported here can be controlled by GABAergic tectal transmission and integrate multisensory information.
The salamander tectum is innervated by neuromodulatory systems as shown by the presence of, for example, serotoninergic fibers (Gruber & Harris 1981, Dicke et al. 1997, cholinergic fibers (Marin & Gonzalez 1999, Sanchez-Camacho et al. 2002 and dopaminergic fibers (Sanchez-Camacho et al. 2002). These inputs likely influence visuomotor processing in salamanders. In other species, if we consider dopaminergic inputs alone for instance, dopaminergic fibers originating from the posterior tuberculum increase the excitability of tectal output neurons via D1 receptor activation and decrease it via D2 receptor activation (Perez-Fernandez et al. 2017, von Twickel et al. 2019). In toads the dopaminergic agonist apomorphine attenuates prey-oriented turning movements (Glagow & Ewert 1997). In mammals, dopaminergic fibers from A13 innervate the superior colliculus and likely influence the ability to allocate attention and orienting movements toward regions of the visual field (Bolton et al. 2015, Woolrych et al. 2021).
The pretectum might play a crucial role in the switch between orienting or avoidance responses. This idea comes from dual lesion paradigms in toads. After a first lesion (bilateral ablation of the telencephalon, i.e., striatum and pallium), no visually guided prey catching occurs (Matsumoto et al. 1991). Strikingly, adding a second lesion in the pretectum results in "disinhibition" of orienting behaviors, that is, increased prey catching toward any moving object. Such effect of pretectum lesion was also observed in salamanders (Finkenstädt 1980). Future studies should study whether and how the pretectum controls the pathway that we report here.
The innervation of the thalamus that we report likely underlies the previously reported responses evoked by optic nerve stimulation in thalamic neurons, that relay visual information higher brain regions in salamanders (Roth & Grunwald 2000). Such retino-thalamo-pallial pathway, well known in mammals (Butler 1994) birds (Butler & Hodos, 1996) and frogs (Neary & Northcutt 1983), was also recently described in the phylogenetically older lamprey (Suryanarayana et al. 2020) and is therefore another conserved component of the visual system. It was proposed to play a role in sensory representation and cognitionbased visuomotor processing in lamprey (Suryanarayana et al. 2020).
In salamanders, the thalamus receives projections from the optic nerve and relay the information to the pallium, septum, striatum, and amygdala (Ruhl & Dicke 2012). The thalamus is also bilaterally connected with the tectum (Ruhl & Dicke 2012). Lesion of the dorsal thalamus in salamanders impairs recognition, evaluation of objects and spatial attention. Salamanders can still snap a prey presented in front of their head, but in the presence of two preys, they are less able to select one and orient toward it (Ruhl & Dicke 2012). Future studies should examine how these higher centers control the pathway reported here.

CONCLUSION
We uncovered a retino-tecto-reticulo-motoneuronal circuitry through which visual input can be transformed into a spinal motor command in salamanders. This pathway is likely involved in orienting behaviors dur-ing prey capture. Our work shows that the organization of visuomotor pathways is largely similar to that of lamprey, zebrafish, and mammals.
Intriguingly, the salamander has the extraordinary capacity to regenerate its nervous system (for review Joven & Simon 2018). The retina of salamanders, which has been extensively studied at the cellular level (for review Thoreson 2021), regenerates and regrows its central projections after lesion (Okamoto et al. 2007). Thus, salamanders provide a unique opportunity to study how visuomotor pathways are repaired after lesion in tetrapods.

ACKNOWLEDGMENTS
We thank Jean Lainé for technical assistance with the microscopy platform, Alberto Joven and Andras Simon for the animals and Jordan Swiegers for the help with some of the experiments.