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Certain homebox transcription factors (HTFs) are
thought to specify different types of spinal interneurons and their
associated motor circuits. We are studying how HTFs specify one
class of spinal interneuron called the R-interneuron. To this end,
we must first identify which HTFs are expressed in R-interneurons.
We are attempting to establish an immunohistochemical marker for
the R-interneuron which could then be compared to the expression
of various HTFs. The calcium binding protein, calbindin D28k, is
a marker for the Renshaw cell – the adult mammalian homologue
of the R-interneuron. In this project we have begun to determine
if calbindin is a marker for the R-interneuron population. We show
that calbindin immunoreactive cells exist in the R-interneuron region,
dorsomedial to motoneurons, and therefore could identify the population.
Secondly, we have physiologically identified R-interneurons and
labeled them with biocytin. Preliminary results suggest that at
least some R-interneurons are calbindin immunoreactive. Further
studies will be necessary to determine if calbindin is a reliable
marker for all R-interneurons.
Early in development, the secreted protein sonic hedgehog
regulates the expression of certain homebox transcription factors
(HTF) that are believed to specify different classes of spinal neurons.
At this early developmental stage, the expression of various HTFs
is localized within distinct regions, or domains (See figure below,
right). HTFs are known to specify certain features that distinguish
different classes of motoneuron, such as axonal trajectory. How
these HTFs specify the many classes of spinal interneurons is unknown.
We are studying how HTFs specify one class of interneuron that we
have characterized in the developing spinal cord of the chick embryo.
These cells, called R-interneurons, are the most easily identified
interneurons because they receive direct input from motoneurons
(see figure below, left). This input can be observed in whole-cell
recordings as a short latency potential following motoneuron stimulation
(Figure 2A). R-interneurons are likely to be the homologue of the
Renshaw cell previously described in the adult cat and like the
Renshaw cell regulate the motoneuronal activity. R-interneurons
are located in a region dorsomedial to the motoneurons (Figure 1A).
Physiological identification of R-interneurons is a relatively slow
process. Therefore, in this study we are attempting to identify
an immunohisotochemical marker for the R-interneuron population
that would provide a quick and reliable means of identifying these
cells. Such a marker could be used in a preliminary screen to identify
which HTFs are expressed in, and therefore specify, R-interneurons.
In adult mammals, the calcium binding protein, calbindin D28k is
a marker of the Renshaw cell. If calbindin is a marker for the R-interneuron
population, then calbindin can be used to identify HTFs in the R-interneuron
population using double-label immunofluorescence. Additionally,
such a marker would provide valuable information about the R-interneuron
population (i.e. distribution, morphology).
Spinal Cord Preparation Chick embryos were sacrificed
at stage 36 (~embryonic day 10, Hamburger and Hamilton). The spinal
cord was isolated from the embryo in recirculating oxygenated Tyrode’s
solution (concentration in mM: NaCl 139, KCl 3, NaHCO3 17, glucose
12, CaCl2 3, MgCl2 1) from thoracic segment 7 (T7) to lumbosacral
segment 4 (LS4) as described previously (Wenner & O'Donovan
2001). Specimens were placed in fixative (4% paraformaldehyde in
PBS, pH 7.4) for 2 hours at room temperature and rinsed in 10% sucrose
in PBS overnight at 4ºC. Specimens were frozen in Tissue Tek, on
dry ice, sectioned on a cryostat at 15 µm, and stored at -20ºC.
Whole-Cell Electrophysiology The spinal cord was isolated together
with ischiadic and crural nerves. The dorsal pia was removed and
a horizontal cut was made using a Leica vibratome at the midpoint
of the dorso-ventral axis, leaving equal dorsal and ventral halves.
The ventral piece, with intact nerves, was then transferred to the
recording chamber and the solution temperature was increased to
27°C for the remainder in the experiment. Nerves were drawn
into suction electrodes for recording and/or stimulating. Whole-cell
electrodes (4-8 M?, with a K-gluconate solution concentration in
mM: NaCl 10, K-gluconate 130, HEPES 10, EGTA 1.1, CaCl2 0.1, MgCl2
1, Na2ATP 1) were driven ventrally through the dorsal aspect of
the ventral piece of cord. The electrode was positioned directly
over the R-interneuron region dorsal to the medial part of the motor
column. Extracellular suction electrode recordings from muscle nerves
were amplified 1,000X and filtered at DC-1KHz. Single-pulses and
stimulus trains (20-50Hz for 0.5ms) of 30 µA were delivered
to nerves to activate R-interneurons (Wenner and O’Donovan,
1999). R-interneurons were identified by the presence of short latency
synaptic input following stimulation of muscle nerves. Cells falling
into this category had latencies to the onset of the earliest synaptic
potential of <= 5ms (see Wenner and O’Donovan, 1999). Immunohistochemistry
Slides containing spinal cord sections were rinsed initially in
0.1% Trtion X-100/PBS overnight. All rinses were performed on an
orbital shaker. Rabbit anti-Calbindin D28k polyclonal antibody (Chemicon,
Temecula, CA) was applied to the slides in a (1:50) dilution, in
PBS with 0.1% Triton X-100 and 10% normal horse serum (Sigma, St.
Louis, MO). Sections were incubated in the anti-calbindin antibody
for a period of 48-72 hours at 4ºC. Prior to the application of
secondary antibodies, slides were rinsed in 0.1% Triton X-100/PBS
3 times for 30 minutes. For double-labeling, Cy-3 conjugated donkey
anti-rabbit IgG (1:250) and fluorescein conjugated streptavidin
(1:100, Jackson ImmunoResearch, West Grove, PA) were applied to
the slides in PBS containing 0.1% Triton X-100. Secondary antibody
were performed over a period of 2 hours and at room temperature.
Slides were then rinsed in 0.1% Trtion X-100/PBS for 20 minutes
and twice in Tris-HCl (diluted at 1:20 in dH2O). Finally, slides
were coversliped with Vectashield (Vector Laboratories, Burlingame,
CA) and viewed under epifluorescence.
1. Do calbindin immunoreactive cells exist in regions
dorsomedial to the motor column? [Figure 1]Calbindin immunoreactivity
was detected in the region of the R-interneuron and in other areas
of the spinal cord. A: Calcium imaging highlights the region dorsomedial
to motoneurons where R-interneurons reside; percentages are of the
distance from the midline (45%) and lateral edge (35%) (adapted
from Wenner & O’Donovan, 2001). Following motoneuron stimulation
R-interneurons are excited and become optically active having been
filled with a calcium-sensitive dye. B: Two calbindin immunoreactive
cells are localized dorsomedial to motoneurons (red arrows), in
a ventral half preparation where the dorsal cord has been removed.
C: Localization of calbindin expression in the chick spinal cord.
Calbindin immunoreactivtiy was detected in cells dorsolateral to
motoneurons (yellow arrowheads) and in the dorsal horn (white arrowheads).
Some calbindin immunoreactive cells were in te R-interneuron region
(red arrowheads). LMC, lateral motor column. Scale bar equals in
B 100 µm. Scale bar in C equals 200 µm. 2. Are calbindin
immunoreactive cells dorsomedial to the LMC R-interneurons? [Figure
2]At least some R-interneurons are calbindin immunoreactive. A:
Recording obtained from a physiologically identified R-interneuron
during motoneuron stimulation (arrowhead represents the stimulus
artifact). Biocytin was included in the patch solution and therefore
allowed immunohistochemical identification of the cell (C &
D). B: Calbindin immunoreactive cells (arrowheads), in a ventral
half preparation. C: Recorded R-interneuron labeled with biocytin.
D: Merged image of the field shows calbindin immunoreactivty and
biocytin labeling in the recorded R-interneuron (arrow). LMC, lateral
motor column. Scale bar equals 100 µm.
1. Localization of calbinidin D28k expression in regions
dorsomedial to motoneurons suggests that calbindin could serve as
a marker for the R-interneuron population. 2. Calbindin immunoreactivity
is not specific to the R-interneuron population. Other spinal classes
of interneuron, in the ventral and dorsal horns, express calbindin
D28k. 3. At least some calbindin immunoreactive cells located in
a region dorsomedial to motoneurons are R-interneurons. 4. Further
studies will determine if all calbindin immunoreactive cells within
a defined region dorsomedial to motoneurons are R-interneurons.
We would like to thank Veronica Barajas for her expert
technical assistance. We would also like to thank Shawn Hochman
and Mike Sawchuk. This study was supported by the laboratory's startup
funds.
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