281
Functional Neurobiology in Xenopus
FIGURE 19.1 (Continued)
pathfnding (A1); tract-tracing and electrophysiological recordings outlined cerebellar anatomy and function (A2); embryonic removal
of the inner ear demonstrated plasticity-driven reorganization during hindbrain development (A3); the use of mechanosensory stimuli
(water waves) characterized the neuronal activity of lateral line nerve afferents (A4 , top) and the discrimination capabilities underlying
turning behavior (A4 , bottom); the ontogeny of motoneuron activity revealed the network maturation dynamics across developmental
stages (A5). (B) Examples of modern approaches in neurobiology using Xenopus. Embryos can be raised to unmanipulated, wild-type
tadpoles (B1, top) or can be altered by genetic (B1, middle) and/or surgical manipulations (B1, bottom) for subsequent functional prof ling; quantitative behavioral tracking, such as of avoidance responses, permits measurements of learned behavior (B2); single cells can
be recorded in response to sensory stimulation (B3); the transparency of Xenopus tadpoles allows live imaging of selected neurons,
demonstrating the progressive maturation of dendritic arborizations over consecutive days (B4). Cb, cerebellum; CF, climbing f ber; GC,
granule cell; Hb, hindbrain; HC, hair cell; LL, lateral line; MN, motoneuron; Mut, Mutation; OB, olfactory bulb; OT, optic tectum; PC,
Purkinje cell; RGC, retinal ganglion cell; SC, spinal cord; St., stage (Nieuwkoop and Faber, 1994); Te, telencephalon; V, 5th cranial nerve
(trigeminal nerve); VIII, 8th cranial nerve; WT, wild-type.
Source: Panels A 3 , A 4 (bottom), A 4 (top), A 5 , B 3, B 2, B 4 are based on data by Fritzsch, 1990 ; Elepfandt et al., 1985; Kroese et al., 1978; Sillar et al.,
1992; Munz et al., 2014; Blackiston and Levin, 2013; Santos et al., 2018, respectively.
Appreciation of the transformative anuran life cycle,
particularly in Xenopus laevis, is evident from the early
use of this model organism to establish neuroanatomical
principles (e.g. Figure 19.1A 1,2; Nieuwenhuys et al., 1998 )
and to assess questions of plasticity in embryonic circuit
formation, as mentioned previously (e.g. Figure 19.1A3;
Fritzsch, 1990). However, even greater advancements have
been achieved by the use of developmental stage-specif c
analytical instruments and approaches ( Figure 19.1B).
Xenopus embryos in particular are highly suited for a variety of manipulations that infuence the formation of neuronal circuits (Figure 19.1B1). Techniques such as cutting
and pasting of embryonic tissue have continued their use in
modern studies (Figure 19.1B1, lower; Elliott et al., 2015;
Blackiston et al., 2017), with a progressive introduction of
combinatorial genetic and surgical manipulations (Duncan
et al., 2019). Indeed, the ease with which gene expression
can be modulated (Figure 19.1B1, middle; Tandon et al.,
2017; Naert et al., 2020) or molecular pathways perturbed
nowadays (James et al., 2015) offers appealing methods
for targeted disruption of morpho-physiological characteristics that classify specifc neuronal circuits and brain
regions (Figure 19.1B1). These abilities, coupled with the
abundance and size of Xenopus embryos, permit accessibility for various techniques. Such features are also favorable
for reporting on the phenotypic success of a manipulation,
such as genetically encoded fuorescently tagged proteins
in combination with confocal microscopy (Bestman et al.,
2006). Extensive work using these methods has established
Xenopus as a representative model for neurodevelopmental
disorders, where disease modeling is readily approached by
targeting clinically relevant genes and pathways (e.g. Lee et
al., 2010; Pratt and Khakhalin, 2013; Willsey et al., 2020,
2021). Given the effectiveness of modifcations of the early
developing nervous system, functional insight emerging
from such manipulations have been best provided through
the use of tadpoles, where a post-embryonic nervous system
can be readily profled ( Figure 19.1B 1 ).
Xenopus tadpoles express many advantageous features
which facilitate the evaluation of neurobiological principles.
In particular, in vivo profling of tadpole behavior ( Figure
19.1B2) forms an active way of approximating neuronal
function by assessing the execution of motor commands
(Dong et al., 2009; Blackiston and Levin, 2013). Current
methodologies in behavioral assessments are strengthened
by equipment with high-resolution motion tracking abilities (Viczian and Zuber, 2014), such as high-speed cameras
that enable frame-by-frame comparisons of, for example,
swimming-related tail undulations (Lambert et al., 2020) and
head/body turn directions ( Zarei et al., 2017; Gambrill et al.,
2018; Hänzi and Straka, 2017). Such measurements are often
accompanied by automated tracking algorithms, exploiting,
for example, the contrast between the relatively opaque eyes
and the translucent body to demonstrate the developmental
progression of eye movement performance (Lambert et al.,
2020). Behavioral studies in this manner offer the possibility
for non-invasive measurements of functional consequences
of CNS manipulations, such as those following alteration of
gene expression in embryos (Falk et al., 2007; Tandon et al.,
2017; Duncan et al., 2019) or tadpoles (Liu and Haas, 2011;
Bestman and Cline, 2020) (Figure 19.1B1). Thus, conserved
vertebrate genes and molecular pathways can be readily targeted either early in development or after a particular brain
region has fully formed and followed by subsequent evaluation of corresponding phenotypes (e.g. Tosa et al., 2015). Such
assessments thus interrelate to genes and pathways that are
commonly disrupted in disease models.
The ability to profle and quantify tadpole behavior permits assessment of cognitively derived responses (Figure
19.1B2). Learned associations of a visual stimulus with
noxious shocking punishments provides a method to determine the extent of recall performance in Xenopus tadpoles
(Blackiston and Levin, 2013). When limited to visual input
exclusively from a singular grafted eye on the trunk, this
methodology revealed the ability of ectopic sensory input
to be successfully integrated, despite the absence of clearly
defned sensory pathways into the brain (Blackiston and
Levin, 2013). This latter result is particularly striking given
the ability to augment successful integration with pharmacological agents (Blackiston et al., 2017). Such results are
promising for insight into regenerative therapies in humans
and highlight the successful use of Xenopus in this f eld.
While this review has so far made no distinction between
tadpole stages, general behavioral tracking is applicable to
Functional Neurobiology in Xenopus
FIGURE 19.1 (Continued)
pathfnding (A1); tract-tracing and electrophysiological recordings outlined cerebellar anatomy and function (A2); embryonic removal
of the inner ear demonstrated plasticity-driven reorganization during hindbrain development (A3); the use of mechanosensory stimuli
(water waves) characterized the neuronal activity of lateral line nerve afferents (A4 , top) and the discrimination capabilities underlying
turning behavior (A4 , bottom); the ontogeny of motoneuron activity revealed the network maturation dynamics across developmental
stages (A5). (B) Examples of modern approaches in neurobiology using Xenopus. Embryos can be raised to unmanipulated, wild-type
tadpoles (B1, top) or can be altered by genetic (B1, middle) and/or surgical manipulations (B1, bottom) for subsequent functional prof ling; quantitative behavioral tracking, such as of avoidance responses, permits measurements of learned behavior (B2); single cells can
be recorded in response to sensory stimulation (B3); the transparency of Xenopus tadpoles allows live imaging of selected neurons,
demonstrating the progressive maturation of dendritic arborizations over consecutive days (B4). Cb, cerebellum; CF, climbing f ber; GC,
granule cell; Hb, hindbrain; HC, hair cell; LL, lateral line; MN, motoneuron; Mut, Mutation; OB, olfactory bulb; OT, optic tectum; PC,
Purkinje cell; RGC, retinal ganglion cell; SC, spinal cord; St., stage (Nieuwkoop and Faber, 1994); Te, telencephalon; V, 5th cranial nerve
(trigeminal nerve); VIII, 8th cranial nerve; WT, wild-type.
Source: Panels A 3 , A 4 (bottom), A 4 (top), A 5 , B 3, B 2, B 4 are based on data by Fritzsch, 1990 ; Elepfandt et al., 1985; Kroese et al., 1978; Sillar et al.,
1992; Munz et al., 2014; Blackiston and Levin, 2013; Santos et al., 2018, respectively.
Appreciation of the transformative anuran life cycle,
particularly in Xenopus laevis, is evident from the early
use of this model organism to establish neuroanatomical
principles (e.g. Figure 19.1A 1,2; Nieuwenhuys et al., 1998 )
and to assess questions of plasticity in embryonic circuit
formation, as mentioned previously (e.g. Figure 19.1A3;
Fritzsch, 1990). However, even greater advancements have
been achieved by the use of developmental stage-specif c
analytical instruments and approaches ( Figure 19.1B).
Xenopus embryos in particular are highly suited for a variety of manipulations that infuence the formation of neuronal circuits (Figure 19.1B1). Techniques such as cutting
and pasting of embryonic tissue have continued their use in
modern studies (Figure 19.1B1, lower; Elliott et al., 2015;
Blackiston et al., 2017), with a progressive introduction of
combinatorial genetic and surgical manipulations (Duncan
et al., 2019). Indeed, the ease with which gene expression
can be modulated (Figure 19.1B1, middle; Tandon et al.,
2017; Naert et al., 2020) or molecular pathways perturbed
nowadays (James et al., 2015) offers appealing methods
for targeted disruption of morpho-physiological characteristics that classify specifc neuronal circuits and brain
regions (Figure 19.1B1). These abilities, coupled with the
abundance and size of Xenopus embryos, permit accessibility for various techniques. Such features are also favorable
for reporting on the phenotypic success of a manipulation,
such as genetically encoded fuorescently tagged proteins
in combination with confocal microscopy (Bestman et al.,
2006). Extensive work using these methods has established
Xenopus as a representative model for neurodevelopmental
disorders, where disease modeling is readily approached by
targeting clinically relevant genes and pathways (e.g. Lee et
al., 2010; Pratt and Khakhalin, 2013; Willsey et al., 2020,
2021). Given the effectiveness of modifcations of the early
developing nervous system, functional insight emerging
from such manipulations have been best provided through
the use of tadpoles, where a post-embryonic nervous system
can be readily profled ( Figure 19.1B 1 ).
Xenopus tadpoles express many advantageous features
which facilitate the evaluation of neurobiological principles.
In particular, in vivo profling of tadpole behavior ( Figure
19.1B2) forms an active way of approximating neuronal
function by assessing the execution of motor commands
(Dong et al., 2009; Blackiston and Levin, 2013). Current
methodologies in behavioral assessments are strengthened
by equipment with high-resolution motion tracking abilities (Viczian and Zuber, 2014), such as high-speed cameras
that enable frame-by-frame comparisons of, for example,
swimming-related tail undulations (Lambert et al., 2020) and
head/body turn directions ( Zarei et al., 2017; Gambrill et al.,
2018; Hänzi and Straka, 2017). Such measurements are often
accompanied by automated tracking algorithms, exploiting,
for example, the contrast between the relatively opaque eyes
and the translucent body to demonstrate the developmental
progression of eye movement performance (Lambert et al.,
2020). Behavioral studies in this manner offer the possibility
for non-invasive measurements of functional consequences
of CNS manipulations, such as those following alteration of
gene expression in embryos (Falk et al., 2007; Tandon et al.,
2017; Duncan et al., 2019) or tadpoles (Liu and Haas, 2011;
Bestman and Cline, 2020) (Figure 19.1B1). Thus, conserved
vertebrate genes and molecular pathways can be readily targeted either early in development or after a particular brain
region has fully formed and followed by subsequent evaluation of corresponding phenotypes (e.g. Tosa et al., 2015). Such
assessments thus interrelate to genes and pathways that are
commonly disrupted in disease models.
The ability to profle and quantify tadpole behavior permits assessment of cognitively derived responses (Figure
19.1B2). Learned associations of a visual stimulus with
noxious shocking punishments provides a method to determine the extent of recall performance in Xenopus tadpoles
(Blackiston and Levin, 2013). When limited to visual input
exclusively from a singular grafted eye on the trunk, this
methodology revealed the ability of ectopic sensory input
to be successfully integrated, despite the absence of clearly
defned sensory pathways into the brain (Blackiston and
Levin, 2013). This latter result is particularly striking given
the ability to augment successful integration with pharmacological agents (Blackiston et al., 2017). Such results are
promising for insight into regenerative therapies in humans
and highlight the successful use of Xenopus in this f eld.
While this review has so far made no distinction between
tadpole stages, general behavioral tracking is applicable to