Download The horizontal brain slice preparation: a novel approach for

J Neurophysiol 113: 400 – 407, 2015.
First published October 15, 2014; doi:10.1152/jn.00672.2014.
Innovative Methodology
The horizontal brain slice preparation: a novel approach for visualizing and
recording from all layers of the tadpole tectum
Ali S. Hamodi and Kara G. Pratt
Department of Zoology and Physiology, University of Wyoming, Laramie, Wyoming
Submitted 8 September 2014; accepted in final form 12 October 2014
development; electrophysiology; multisensory integration; tadpole;
tectum
THE AMPHIBIAN OPTIC TECTUM, homologous to the mammalian
superior colliculus, is a multisensory processing center that
receives visual input from the retinal ganglion cells (RGCs) in
the eye as well as from nonoptic mechanosensory inputs
originating from the somatosensory, lateral line, auditory, and
vestibular systems (Behrend et al. 2006; Lowe 1986; Munoz et
al. 1995). The neurons that comprise the optic tectum, collectively referred to as tectal neurons, organize into somatic layers
with the deepest layer being the most periventricular and most
medial. In the developing tadpole tectum, the dendrites of these
tectal neurons project laterally from the somatic layer into a
single vast neuropil where they receive strong direct synaptic
visual input from RGCs of the contralateral eye and mechanosensory inputs that arrive at the tectum via the hindbrain (HB).
Ultimately, the tectum of the adult Xenopus frog will organize
into a total of nine layers: cellular layers interleaved with layers
of neuropil or where efferent outputs exit the tectum (Lazar
1973). Based on receptive field properties, seven classes of
Address for reprint requests and other correspondence: K. G. Pratt, Univ. of
Wyoming, Dept. of Zoology and Physiology, 16th and Gibbon St., Laramie,
WY 82071 (e-mail: [email protected]).
400
tectal neurons have been identified in the adult frog (Grusser
and Grusser-Cornehls 1976). The immature tadpole tectum is
considerably less complex compared with the adult but rapidly
developing: by developmental stage 45, which is approximately 7–10 days postfertilization (dpf), the somatic layer of
the tectum appears to be 5–10 somata deep, although at this
early stage of development, defined layers have not been
described. Also by this stage in development, both visual and
mechanosensory inputs have entered the tectum and have
begun forming synapses (Hiramoto and Cline 2009). By developmental stage 49 (⬃16 dpf), the tectal neuron somata have
organized into six compact somatic layers and have begun to
display a range of different morphologies (Lazar 1973). Furthermore, a detailed immunohistochemical study indicates that
even before developmental stage 49, the neurons across the
tectum can be categorized as expressing either GABA or
CaMKII and that the spatial pattern of GABA-expressing
neurons has redistributed from clustered to being more evenly
dispersed throughout the somatic layers (Miraucourt et al.
2012). Although these morphological and immunohistochemical studies indicate that, even at relatively early developmental
stages, tectal neurons are differentiating and reorganizing, the
electrophysiology of the neurons beyond the deepest, most
periventricular somatic layer, henceforth referred to as the
“deep-layer” neurons, has not been described. Whole cell
electrophysiological studies of the tadpole tectum, whether
using the in vivo preparation or the isolated, whole brain
preparation, have focused solely on the deep-layer neurons.
This is because to record from tectal neurons using either the
in vivo or whole brain preparation, the ventricular membrane is
peeled away, making accessible the deep-layer neurons that
line the ventricle. This approach does not permit recording
from neurons of outer layers since there is no obvious way to
circumvent the deep-layer neurons. As a result, whereas the
development and plasticity of these deep-layer neurons has
been well-studied, essentially nothing has been reported about
the electrophysiology of neurons residing beyond this layer.
Hence, there exists a large gap in our understanding about the
functional development of the amphibian tectum as a whole.
To remedy this, we have developed a novel isolated brain
preparation that allows for the visualization and whole cell
recording of individual neurons across the entire medial-lateral
axis of the tectum. This preparation, henceforth referred to as
the “horizontal brain slice preparation,” provides a means to
study readily the physiology of a large population of tectal
neurons that, until now, could not be studied. Also, as with the
original whole brain preparation, the horizontal brain slice
preparation retains the sensory inputs from the RGCs in the
eye and the mechanosensory inputs from the HB. Therefore,
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Hamodi AS, Pratt KG. The horizontal brain slice preparation: a
novel approach for visualizing and recording from all layers of the
tadpole tectum. J Neurophysiol 113: 400 – 407, 2015. First published
October 15, 2014; doi:10.1152/jn.00672.2014.—The Xenopus tadpole
optic tectum is a multisensory processing center that receives direct
visual input as well as nonvisual mechanosensory input. The tectal
neurons that comprise the optic tectum are organized into layers.
These neurons project their dendrites laterally into the neuropil where
visual inputs target the distal region of the dendrite and nonvisual
inputs target the proximal region of the same dendrite. The Xenopus
tadpole tectum is a popular model to study the development of sensory
circuits. However, whole cell patch-clamp electrophysiological studies of the tadpole tectum (using the whole brain or in vivo preparations) have focused solely on the deep-layer tectal neurons because
only neurons of the deep layer are visible and accessible for whole cell
electrophysiological recordings. As a result, whereas the development
and plasticity of these deep-layer neurons has been well-studied,
essentially nothing has been reported about the electrophysiology of
neurons residing beyond this layer. Hence, there exists a large gap in
our understanding about the functional development of the amphibian
tectum as a whole. To remedy this, we developed a novel isolated
brain preparation that allows visualizing and recording from all layers
of the tectum. We refer to this preparation as the “horizontal brain
slice preparation.” Here, we describe the preparation method and
illustrate how it can be used to characterize the electrophysiology of
neurons across all of the layers of the tectum as well as the spatial
pattern of synaptic input from the different sensory modalities.
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NOVEL TADPOLE BRAIN PREPARATION FOR STUDYING THE TECTUM
integration. Here, we illustrate these two experimental attributes.
MATERIALS AND METHODS
All experimental protocols have been approved by the University of
Wyoming’s Institutional Animal Care and Use Committee (IACUC).
Xenopus laevis tadpoles were reared in Steinberg’s solution at 25°C
on a 12:12-h light-dark schedule. Tadpoles were staged according to
the developmental table described by Nieuwkoop and Faber (1994).
The first step for preparing the horizontal brain slice brain preparation
is equivalent to the whole brain preparation described by Wu et al.
(1996) and Pratt and Aizenman (2007). For this, tadpoles are anesthetized in Steinberg’s solution containing 0.02% MS-222, moved to
the recording dish, and pinned to a block of Sylgard silicone elastomer
submerged in external recording solution (in mM: 115 NaCl, 2 KCl,
3 CaCl2, 3 MgCl2, 5 HEPES, and 10 glucose, pH 7.25, osmolarity 255
mosM). Next, using a sterile 26-gauge needle, the skin overlying the
brain is peeled away (Fig. 1, A and B) and the brain and brain stem
filleted by making a shallow incision along the midline such that the
dorsal postoptic commissure is severed, whereas the floor plate is left
intact. The filleted brain and brain stem are then dissected out and
secured to the Sylgard block by placing one insect pin in either
olfactory bulb and one in the brain stem (Fig. 1C). This yields the
whole brain preparation that is commonly used for recording from the
tectum. In this configuration, the somata of the deep-layer neurons are
immediately visible and readily accessible for recording, and their
dendrites are pointing down (parallel with the z-axis) toward the piece
of Sylgard. The second step in this new preparation involves making
a cut parallel to the rostral-caudal plane such that the most lateral
one-fourth (approximately) of the tectum, which in the intact tectum
corresponds to making a cut in the horizontal plane such that the most
dorsal one-fourth of the tectum is excised and the remaining threefourths is kept for recording (Fig. 1C). This cut is made manually
using a razor blade. The removal of this small portion of the tectum
permits access to the most extensive somatic and neuropil layers (i.e.,
where the somatic and neuropil layers are widest). Importantly, slicing
away this small portion of the tectum leaves intact the RGC and
mechanosensory (HB) inputs and hence preserves the capability to
activate these inputs at different stimulation intensities and record the
resulting responses from individual tectal neurons, the major experimental advantage of the traditional whole brain preparation over the in
vivo configuration. Next, the preparation is pinned onto the side of the
aforementioned Sylgard block such that the sliced side is facing up
and the ventricle side is facing away from the Sylgard block (Figs. 1D
and 2). This inside-facing-out orientation allows for a bipolar stimulating electrode to be placed onto the optic chiasm and the HB so that
RGC and mechanosensory inputs, respectively, can be activated (Figs.
1D and 2). Figure 1E shows a portion of the tectum imaged at ⫻60,
the magnification we use when recording. The somatic and neuropil
layers and the border between them can be easily distinguished.
Once mastered, the entire dissection can be completed within 10
min on average. In addition, unlike the whole brain preparation, there
is no ventricular membrane to be removed before neurons can be
recorded. We find that the horizontal brain slice preparation stays
healthy on the rig for ⬃2 h (no perfusion required).
A minor drawback of this preparation is that, even though the
afferent sensory inputs are preserved, removing the most outside edge
of tectum (which corresponds to the most dorsal portion of the intact
tectum) may result in a certain amount of intratectal connectivity to be
lost. This could potentially impact the frequency of spontaneous
synaptic events appearing to be received by a given tectal neuron
during recording. However, compared with deep-layer neurons recorded using the original whole brain preparation, we have observed
no noticeable decrease in the frequency of spontaneous synaptic
events recorded from these deep-layer neurons using the horizontal
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the pattern and strength of the different inputs received by
outer-layer neurons can be explored. Also, this preparation
makes it possible to study the development of local tectal-tectal
connectivity between the different layers. Although it is evident that there exists extensive local connectivity between
tectal neurons (Pratt et al. 2008), there are no reports, to date,
of connections between individual tectal neurons, probably
because the connections exist across layers and so could not be
identified using the whole brain or in vivo preparation.
In addition to being able to record from neurons across
layers, this horizontal brain slice preparation also provides
direct visualization and access to the neuropil where the dendrites of tectal neurons receive synaptic input from the different sensory modalities. With access to the medial-lateral axis
of the neuropil, combined with the ability to activate the two
distinct sensory inputs, this preparation provides a straightforward method to assess the spatial pattern of functional synapses formed onto tectal cell dendrites by the axons of different
sensory modalities. It has previously been established that the
axons of the different sensory modalities segregate to specific
regions of the dendrite: RGC (visual) input targets the most
distal region of the dendrite, whereas the nonvisual mechanosensory targets the proximal region of the same dendrite (Deeg
et al. 2009; Hiramoto and Cline 2009). This suggests that the
functional synapses associated with each modality are spatially
distinct. Using the horizontal brain slice preparation, the spatial
pattern of synaptic input can be measured directly by recording
field potentials (FPs) at equidistant points across the mediallateral axis of the neuropil, which corresponds to the proximalto-distal axis of the dendrites, in response to activation of one,
and then the other, sensory modality. This method of studying
the spatial pattern of synaptic input by recording FPs in
response to sensory stimulation has been used extensively to
characterize organization of retinal (Chung et al. 1974a; Nakagawa et al. 1997; Nakagawa and Matsumoto 1998, 2000)
and somatosensory (Tsurudome et al. 2005) inputs, in vivo, in
the adult frog tectum but has been applied considerably less to
study the juvenile tadpole due to technical difficulties associated with advancing the recording pipette down through the
very thin tadpole tectum as discussed in a FP study using stage
55 Xenopus tadpoles (Chung et al. 1974b). The horizontal brain
slice preparation avoids these technical difficulties because the
recording pipette is moved along the horizontal axis across the
slice surface and never down vertically through the tissue (see
MATERIALS AND METHODS). Therefore, this new preparation provides a means to study the earliest stages of the development of
the spatial pattern of synaptic inputs, how this pattern may
change over development, and the mechanisms and functional
relevance in the context of sensory integration.
In summary, because the horizontal brain slice preparation
allows for the visualization and access across the entire mediallateral axis of the tectum, the experimental attributes of this
horizontal brain slice preparation are twofold: 1) it allows, for
the first time, the systematic characterization of neurons of
different layers of the developing tectum, including the specific
pattern and strength of their sensory inputs; and 2) it provides
a platform to study how the synaptic connections made by the
different sensory afferents are targeted to specific regions of
the dendrite as well as the functional consequence of this
subcellular level of organization in the context of sensory
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NOVEL TADPOLE BRAIN PREPARATION FOR STUDYING THE TECTUM
brain slice preparation. The frequency of spontaneous synaptic events
using either the whole brain preparation or horizontal brain slice
preparation was observed to be in the range of one to seven events per
second, and no preparation-dependent trends in synaptic frequencies
have been observed. A similar concern was that, since the pattern and
strength of evoked polysynaptic activity is determined by local intratectal connectivity, it is conceivable that removing a portion of the
tectum may also stunt the polysynaptic portion of the evoked response. As observed with the frequency of spontaneous synaptic
events, however, polysynaptic responses recorded from deep-layer
neurons using the horizontal brain slice preparation, thus far, appear
quite similar in shape and size to those recorded using the original
whole brain preparation. This is probably because the horizontal brain
slice preparation preserves the local tectal connectivity across layers,
which constitutes the majority of intratectal projections.
Whole cell electrophysiology. We visualized tectal cells using a
Zeiss light microscope with a ⫻60 water-immersion objective in
series with a Hamamatsu infrared CCD camera. Whole cell recordings
were made using borosilicate glass micropipettes filled with K⫹gluconate internal recording saline (in mM: 100 K-gluconate, 8 KCl,
5 NaCl, 1.5 MgCl2, 20 HEPES, 10 EGTA, 2 ATP, and 0.3 GTP, pH
7.2, osmolarity 255 mosM). Pipette resistances were in the range of
8 –12 M⍀. Electrophysiological recordings were done using Axon
Instruments MultiClamp 700B Microelectrode Amplifier (Molecular
Devices, Sunnyvale, CA), digitized at 10 kHz using a Digidata 1322A
digitizer, and acquired using pCLAMP software. Leak current was
subtracted in real-time using the acquisition software. For electrical
stimulation of visual and mechanosensory inputs, we delivered 0.2-ms
pulses using a bipolar stimulating electrode (FHC) placed on the optic
chiasm (to simulate RGC axons) or HB (to stimulate mechanosensory
inputs) with an ISO-Flex stimulator (A.M.P.I.). All data were analyzed using AxoGraph X and graphed using IGOR Pro software
(WaveMetrics).
Extracellular FP recordings. For FP recordings, we used glass
micropipettes that had resistances in the range of 1–2 M⍀. Pipettes
were filled with external recording saline, and evoked FP recordings
were obtained at 10-␮m intervals from the most outer/distal part of the
neuropil (corresponding to the most lateral portion of the intact
tectum) to the deepest somatic layer (corresponding to the most
medial portion of the intact tectum) while stimulating the visual or
mechanosensory inputs. A scale bar was superimposed onto the video
monitor to allow accurate placements of the recording pipette. Importantly, with this horizontal brain slice preparation, the medial-lateral
axis of the neuropil (corresponding to the proximal-distal axis of the
tectal neuron dendritic arbors), and all layers of somata, all lie in the
same horizontal plane or x-coordinate. This means that to record FPs
at different points along this axis, the recording pipette can simply be
raised up off of the tissue, moved 10 ␮m along the x-coordinate, and
lowered back down onto the tectum. Lifting and lowering the pipette
to different points along the dendrites avoids inherent caveats associated with typical FP recordings that require the recording pipette to
be advanced vertically down (along the z-coordinate) and recording at
different depths. Advancing the pipette down to different depths of the
tissue creates depth measurement errors due to dimpling or dragging
of the tissue as the pipette advances. This becomes especially significant when attempting to record FPs from a thin brain structure such
as the tadpole tectum (Chung et al. 1974b). Furthermore, since neither
the pipette nor the tissue is visible below the surface, it is not possible
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Fig. 1. The horizontal brain slice preparation. A: an
overhead view of a stage 49 Xenopus tadpole with the
brain in focus. The skin overlying the intact brain has
been removed, the 1st step in the dissection. B: boxed
area in A with the optic tectum (OT), neuropil, and
soma layers labeled. The dorsal-ventral axis is lying in
the z-coordinate. C: ⫻10 image illustrating the 1st step
in the horizontal brain slice preparation; at this stage in
the dissection, the brain has been isolated, filleted along
the midline (such that the part of tectum that was dorsal
is now lateral), and secured onto the Sylgard by placing
1 insect pin (labeled here with a white asterisk) in the
olfactory bulb (OB) and 1 in the hindbrain (HB; data
not shown). Next, a cut is made along the most lateral
1/4th of the OT (indicated by the dashed line and
scissors) using a razor blade. D: the final step involves
repinning the brain onto the side of the block of Sylgard
such that the sliced side is facing up. Image was
captured using a ⫻5 objective. The sets of double white
lines represent the retinal ganglion cell (RGC) and HB
stimulating electrodes. E: boxed area in D is enlarged
here (60⫻) to show the different somatic layers and the
neuropil of the tectum. Notice how the somatic layers
and the neuropil are visible and readily distinguishable.
The model cell drawn in black depicts the orientation of
the somas and the dendrites. Dashed line indicates the
border between somatic and neuropil layers. OC, optic
chiasm; R, rostral; C, caudal; M, medial; L, lateral; D,
dorsal; V, ventral.
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NOVEL TADPOLE BRAIN PREPARATION FOR STUDYING THE TECTUM
to know whether and to what extent this sort of drag is occurring. In
addition, using the horizontal brain slice preparation described here,
the location of the pipette in relationship to the somatic layers and
neuropil is readily identified. The visual and mechanosensory inputs
were stimulated using bipolar electrodes as described above in the
Whole cell electrophysiology section of MATERIALS AND METHODS. At
the start of each experiment, exploratory FP responses were obtained
from across the rostral-caudal axis in the midneuropil region. Once a
reliable response was identified, FPs were collected from the most
outer (lateral) to the most inner (medial) layer.
Current-source density analysis. We calculated the current-source
density (CSD) profile from the set of FPs according to the method
described by Mitzdorf and Singer (1977) using a spatial differentiation grid of 20 ␮m. For this, we calculate the second spatial derivative
by subtracting twice the voltage measured at a given position [V(x)]
from the sum of the two voltages measured above [V(x⫹n * ⌬x)] and
below [V(x⫺n * ⌬x)] that position and dividing by the square of the
distance between the sites of recording (⌬x). All calculations were
performed using IGOR Pro software.
⭸ 2V
⭸ x2
⫽
V共x ⫹ n ⴱ ⌬x兲 ⫹ V共x ⫺ n ⴱ ⌬x兲 ⫺ 2V(x)
共n ⴱ ⌬x2兲
RESULTS
All of the experiments described here were carried out using
stage 48/49 X. laevis tadpoles. Here, using the horizontal brain
slice preparation combined with a set of whole cell voltageclamp and current-clamp electrophysiological recording protocols, we illustrate how synaptic properties, evoked synaptic
responses, voltage-gated intrinsic currents, and the ability to
fire action potentials (APs) can be quantified and compared
between a deep-layer and an outer-layer tectal neuron. Ultimately, data from these experiments will be used to generate or
piece together a wiring diagram of the tectum. Next, we
demonstrate how this preparation provides a straightforward
way to address the spatial pattern of synapses made by the two
distinct inputs by recording FPs at different points across the
medial-lateral axis of the tectum, from outer, most lateral edge
of the neuropil to the inner, most medial cellular layer, in
response to RGC axon input and mechanosensory input. The
resulting FP traces are converted into CSDs to reveal more
precisely the spatial pattern and magnitude of synaptic current
generated along the distal-to-proximal axis of the dendrites.
Whole cell recordings from individual tectal neurons residing at different levels of the tectum. Using the horizontal brain
slice preparation, neurons across the entire horizontal plane of
the tectum can be readily visualized and recorded in whole cell
configuration (Fig. 1E), making it possible, for the first time, to
characterize many aspects of synaptic and intrinsic properties
displayed by neurons of outer layers of the tectum as well as
the pattern and strength of their RGC and mechanosensory
inputs. Our initial results show that neurons in the outer layers
are, in several ways, electrophysiologically distinct from the
well-studied deep-layer tectal neurons. First, to sample excitatory synaptic currents expressed by individual neurons, spontaneous excitatory postsynaptic currents (sEPSCs) were recorded. For this, whole cell voltage-clamp recordings were
carried out with the potential of the neuron clamped at ⫺60
mV, and excitatory (AMPA-type glutamate receptor-mediated)
currents isolated by blocking spontaneous inhibitory postsynaptic currents (sIPSCs) by including the GABAA antagonist
picrotoxin (100 ␮M) to the external recording solution. Examples of sEPSC recordings from a deep-layer tectal neuron and
a neuron residing at the outer-most somatic layer, at the border
between somatic and neuropil regions, are shown in Fig. 3A.
The outer-layer neuron displays noticeably larger sEPSC amplitudes, a direct measure of synaptic strength. Also, the
synaptic events recorded from the outer-layer neuron display
noticeably faster rates of decay than those displayed by the
deep-layer neuron. This is most likely due to differential
receptor subunit composition and/or differences in synapse
location in relation to the recording pipette (which is in the
soma). Importantly, the synaptic properties displayed by the
outer-layer neuron shown here (Fig. 3A) are very similar to a
large population of outer-layer neurons that we are currently
characterizing and are quite distinct from those expressed by
the deep-layer neurons, indicative of different cell types. In
fact, with the exception of the morphologically distinct mesencephalic V neuron, a large, unipolar trigeminal sensory
neuron that innervates the tentacle (Pratt and Aizenman 2009),
the deep-layer tectal neurons appear to be composed of a
strikingly homologous population of neurons.
In addition to spontaneous synaptic currents, the different
sensory inputs (visual and mechanosensory) can be activated,
and the resultant postsynaptic currents, referred to as evoked
currents, can also be characterized using the horizontal brain
slice preparation. Previously, it had been observed that individual deep-layer tectal neurons receive direct synaptic input
from both RGCs and the mechanosensory HB inputs (Deeg et
al. 2009; Deeg and Aizenman 2011; Pratt and Aizenman 2009).
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Fig. 2. A schematic showing the horizontal brain slice preparation attached to
a Sylgard block and ready for recording. The sliced side of the preparation
(shown in white) is facing up such that tectal neurons and neuropil across the
medial-lateral axis of the tectum (i.e., the horizontal plane, or axis, in vivo) can
be readily visualized and accessed for whole cell patch-clamp or field-potential
(FP) recordings. The ventricle side of the preparation is facing away from the
Sylgard block so that a stimulating electrode (“Stim 1”) can be placed on the
OC and another (“Stim 2”) on the HB so that the RGC axonal inputs and HB
inputs, respectively, can be activated and resulting synaptic responses recorded
from tectal neurons or the neuropil. Dashed line indicates the border between
soma and neuropil layers. This schematic corresponds to Fig. 1D.
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A
Deep-Layer
Outer-Layer
10 pA
B
100 ms
50 pA
50 ms
C
10 pA
50 ms
D
500 pA
2 ms
E
20 mV
50 ms
These experiments were carried out using the original/traditional whole brain preparation that allows for the different
sensory inputs to be controlled by placing one bipolar stimulating electrode onto the RGC axon tracts at the optic chiasm
to activate, at varying intensities, RGC axonal inputs and
placing another one of these electrodes on the HB to activate
the afferent mechanosensory axons. Evoked synaptic responses
are obtained by activating either input while recording in whole
cell mode from a postsynaptic tectal neuron (see MATERIALS AND
METHODS). Since the horizontal brain slice preparation retains
these afferent inputs, and since these inputs remain accessible
to stimulating electrodes, the same experiments can be done
using this horizontal brain slice preparation. Figure 3, B and C,
shows examples of synaptic responses of a deep layer and
outer-layer neuron to RGC input and HB input stimulation,
respectively. Both neurons display a monosynaptic component,
indicating that they both receive synaptic drive from both sets
of sensory inputs, and polysynaptic input, indicating that they
are both integrated into the local tectal-tectal circuitry. Importantly, the RGC evoked responses observed in deep-layer
neurons using the horizontal brain slice preparation were similar in amplitude and pattern to those observed using the whole
brain preparation. Both preparations yield maximum evoked
synaptic amplitudes in the range of 40 – 60 pA (data not
shown). The advantage of carrying out these experiments using
the horizontal brain slice preparation is that only with this
preparation is it possible to characterize the pattern and
strength of the different sensory modalities received as a
function of neuron location within the developing layers of the
tectum as opposed to using the original whole brain preparation
and recording exclusively from the deep-layer neurons. Hence,
data using this new preparation will contribute to a more
comprehensive understanding about how sensory circuits are
built and how they function.
Noticeable differences were also observed in voltage-gated
intrinsic currents expressed by the deep- and outer-layer neuron. (Fig. 3D). Quantifying voltage gated intrinsic currents is
of importance because the combined expression of these currents largely determines the intrinsic excitability of the neuron,
the ease in which the neuron generates APs, and thus determines
the output function. A classic way to characterize voltagegated intrinsic currents expressed by a neuron is to construct
current-voltage plots for the individual currents. For this,
neurons were recorded in whole cell voltage-clamp mode and,
starting from a baseline potential of ⫺60 mV, stepped to
increasingly more depolarized potentials in increments of 10
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Fig. 3. Sample whole cell recordings from a deep-layer
(left) and outer-layer (right) tectal neuron. A: sample
spontaneous excitatory postsynaptic currents (sEPSCs)
from a deep- and outer-layer neuron. Notice that outerlayer neuron sEPSCs have larger amplitude and faster
decay than deep-layer neuron sEPSCs. B and C: sample
evoked synaptic responses recorded from a deep- and
outer-layer neuron elicited by stimulating the RGC
axons (B) and HB axons (C). Both neurons appear to
receive monosynaptic input from both sensory modalities followed in time by polysynaptic activity. D:
sample mixed-current recordings from the deep- and
outer-layer tectal neurons in response to increasingly
depolarizing steps. Downward, inactivating deflections
represent inward Na⫹ current, whereas upward inflections represent outward K⫹ current. Both Na⫹ and K⫹
currents in the outer-layer neuron are bigger and faster
than the deep-layer neuron. Recordings in A–D were
done in voltage-clamp configuration. E: sample current-clamp traces of action potentials (APs) recorded
from a deep- and an outer-layer neuron evoked by
injection of a square 250-ms current pulse. Notice that
the outer-layer neuron can fire many APs and displays
less accommodation compared with the deep-layer
neuron. Also, the spike height of the AP displayed by
the outer-layer neuron is nearly double that of the
deep-layer neuron.
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sinks occur mostly at the inner lamina and essentially not at all
in the outer lamina. Also, notice that the location of the largest
sinks generally corresponds to the location of the largest FP.
We have observed this input-dependent pattern of sinks to be
consistent across preparations. Another recurring pattern we
observed is that the HB sinks are consistently smaller than the
RGC sinks, which is in agreement with our whole cell recordings that indicate that the maximum postsynaptic responses
evoked by RGC axon stimulation are, in general, stronger than
responses evoked by HB stimulation. Finally, the CSD sinks
appearing later in time most likely reflect polysynaptic activity
that is also observed in single-cell recordings.
Interestingly, both the synaptic FP recordings and whole
cell recordings suggest the two sensory inputs evoke different patterns of polysynaptic responses, suggesting that RGC
input and mechanosensory input activate distinct intratectal
networks.
DISCUSSION
The Xenopus tadpole retinotectal projection is a popular
model to study the mechanisms underlying the development of
sensory circuits. This is mainly because the entire development
of the tadpole, from the moment of fertilization on, all takes
place externally (i.e., as opposed to in utero), making it
possible to access and observe readily even the earliest stages
of neural circuit formation without disrupting the natural environment. In combination with this experimental asset, whole
cell recordings of postsynaptic tectal neurons can be readily
carried out in vivo to characterize responses to light or, alternatively, using the whole brain preparation that allows for the
RGC and HB inputs to be activated in a controlled manner. As
a result, the tadpole model has been used to study many aspects
of neural circuit development including the maturation of
synapses (Wu et al. 1996) and intrinsic currents (Hamodi and
Pratt 2014; Pratt and Aizenman 2007), functional refinement
(Tao and Poo 2005), direction-selective plasticity (Engert et al.
2002), visual acuity (Aizenman et al. 2003; Dong et al. 2009;
Schwartz et al. 2011), and spike-timing-dependent plasticity
(Richards et al. 2010; Zhang et al. 1998). This work, however,
does not address the neurons that lie beyond the readily
accessible periventricular layer. To understand the way in
which the tectum functions as a whole, it is necessary to
understand the development of the physiological behavior and
connectivity of all of its different types of neurons. Thus far,
our data from the horizontal brain slice preparation indicate
that neurons of the outer layers are electrophysiologically
distinct from the homogenous population of neurons of the
deep layer and so probably play different roles in sensory
processing.
The pattern of connections formed by a neural circuit is a
major factor in shaping how the circuit functions. In addition to
the well-studied and understood topographic map formed by
the RGC inputs and the mechanosensory inputs onto tectum,
there exists a less-studied subcellular pattern of connectivity in
which RGC inputs target the distal (most lateral) portion of
tectal dendrites, whereas mechanosensory axonal inputs target
the more proximal region (Deeg et al. 2009; Hiramoto and
Cline 2009). Using the horizontal brain slice preparation we
are able to quantify this spatial pattern of synaptic activity. The
FP recordings and CSD analysis shown in Fig. 4 indicate a
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mV. This protocol activates both voltage-gated outward K⫹
and inward Na⫹ currents (Fig. 3D). The representative outerlayer neuron shown in Fig. 3D displays significantly larger
Na⫹ and K⫹ currents compared with the deep-layer neuron.
From these data, current-voltage plots can be constructed by
plotting the maximum (peak) current elicited as a function of
voltage step (data not shown). Another common method to
characterize intrinsic excitability is to quantify the generation
of APs. AP generation can be directly addressed by injecting,
in current-clamp mode, different amounts of current and counting the number of APs elicited by this injection. Typically, the
data from these experiments are plotted as number of APs fired
as a function of the amount of current injected. It is wellestablished that deep-layer tectal neurons exhibit a great
amount of accommodation in response to sustained current
injection (Pratt and Aizenman 2007). This is mainly due to the
inability of these neurons to repolarize sufficiently to allow for
the resetting of Na⫹ channels. Figure 3E shows AP traces
generated by a deep-layer and an outer-layer neuron. Whereas
the deep-layer neuron displays robust accommodation, only
able to fire a few APs in response to a 250-ms sustained current
injection, the representative outer-layer neuron displays essentially no accommodation and fires APs repetitively for the
duration of the current injection. These differences between the
deep-layer and outer-layer neurons likely reflect the medial-tolateral developmental gradient (most immature, medial; most
mature, lateral) formed by the generation of new neurons in the
ventricular proliferative zone that resides at the medial-most
end of the axis (Straznicky and Gaze 1972).
Based on data obtained from this set of experiments using
the horizontal brain slice preparation, at least two distinct
neuron types have been found residing in the outer layers of the
tectum thus far. Studies are underway to identify the neurotransmitters they express.
FPs and CSD profiles evoked by RGC and HB stimulation.
Previously, it has been shown using imaging techniques that
axonal projections from the retina and HB onto the tectum
display laminar specificity such that RGC axons project to a
superficially located, distal lamina in the neuropil, whereas the
mechanosensory inputs arriving via the HB project to a deep
neuropil lamina located proximally to the tectal cell somata
(Fig. 4A; Deeg et al. 2009; Hiramoto and Cline 2009). Although this work demonstrates laminar specificity of sensory
axonal inputs onto the tectal neuropil, it is still not clear where
the major sites of synaptic activity are located. By stimulating
the RGC and HB axonal inputs and recording local FPs from
different locations in the neuropil, the sites of major synaptic
activities can be identified. From these FP recordings, CSD
profiles are derived by calculating the second spatial derivative
(see MATERIALS AND METHODS). Sample FP recordings obtained
from the tectal neuropil in response to RGC and HB stimulation and their corresponding CSD profiles are shown in Fig. 4,
B and C, respectively. For CSD profiles, sinks are upward
inflections (black) and represent excitatory synaptic activity,
whereas sources are downward deflections (white) representing
both the positive current moving back out of the neurons,
“postsink,” and/or GABA-mediated influx of negative current.
Notice the overall pattern of current sinks and sources are
different for RGC and HB inputs and correspond well with the
imaging experiments. For RGC inputs, the majority of current
sinks occur at the outer lamina of the neuropil, whereas HB
405
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406
NOVEL TADPOLE BRAIN PREPARATION FOR STUDYING THE TECTUM
RGC input
Fig. 4. Local FP recordings from the horizontal brain slice preparation. A: simplified schematic of the neuropil showing RGC (distal) and HB (proximal) axonal
inputs onto tectal neuron dendrites. FPs were recorded every 10 ␮m along the distal-proximal axis (indicated by the arrows). B, left: local FPs in response to
RGC stimulation. Right: corresponding current-source density (CSD) profile derived using a spatial differentiation of 20 ␮m. C, left: local FPs in response to
HB stimulation. Right: corresponding CSD profile. Upward inflections are current sinks (black), whereas downward deflections are current sources (white).
Arrowheads indicate time of stimulation.
spatial pattern of visual and mechanosensory driven synaptic
activity that is consistent with the spatial pattern of the axonal
inputs.
In addition to the segregation of visual RGC and mechanosensory (HB) inputs in the tadpole tectum, individual axons
that constitute the RGC input are further organized within the
distal region of dendrite according to the position of the RGC
in the eye: RGCs of the dorsal region of the eye project to the
more medial portion of the distal dendrites, whereas RGCs of
the ventral region of the eye project to the lateral portion
(Bollmann and Engert 2009). Similarly, in zebrafish, it has
been shown that the spatial pattern of visual inputs is arranged
in tectal space according to the direction selectivity of the
individual axons (Gabriel et al. 2012). The mechanism(s)
underlying how different sets of inputs are consistently guided
to specific regions of the dendrite, however, is (are) not
completely clear. It has been suggested that synchronous ac-
tivity of all of the axons associated with specific sensory input
could account for “like” axons converging onto a specific
focused region of the dendrite (Bollmann and Engert 2009).
Consistent with this, in the tadpole tectum, the RGC and
mechanosensory inputs arrive simultaneously (Hiramoto and
Cline 2009), suggesting the possibility that activity-dependent
competition between the different sensory inputs underlies
their segregation. Also not clear is the functional consequence
of this subcellular arrangement of inputs in the context of
sensory integration. In other words, is it important for proper
multimodal sensory integration that, for example, the visual
inputs to be focused on the distal dendrite and mechanosensory
inputs focused closer to the tectal neuron somata? With the
capability to activate the different sensory inputs and quantify,
at high resolution, the spatial pattern of synaptic activity
generated by these inputs in combination with recording responses from individual tectal neurons, the horizontal brain
J Neurophysiol • doi:10.1152/jn.00672.2014 • www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on June 14, 2017
HB input
Innovative Methodology
NOVEL TADPOLE BRAIN PREPARATION FOR STUDYING THE TECTUM
slice preparation provides an improved method to address these
questions.
Overall, the experiments that become possible using the
newly developed and tested tadpole horizontal brain slice
preparation will extend what is known about the functional
development of the amphibian tectum in its entirety.
ACKNOWLEDGMENTS
We thank Drs. Carlos D. Aizenman and Mayank Mehta for their help with
the CSD analysis and Dr. Qian-Quan Sun for helpful comments on the
manuscript.
GRANTS
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
A.S.H. and K.G.P. conception and design of research; A.S.H. and K.G.P.
performed experiments; A.S.H. and K.G.P. analyzed data; A.S.H. and K.G.P.
interpreted results of experiments; A.S.H. and K.G.P. prepared figures; A.S.H.
and K.G.P. drafted manuscript; A.S.H. and K.G.P. edited and revised manuscript; A.S.H. and K.G.P. approved final version of manuscript.
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This work was funded by an Institutional Development Award (IDeA) from
the National Institute of General Medical Sciences of the National Institutes of
Health under Grant P30-GM-32128.
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