1National Institutes of Health, National Institute of Neurological Disorders and Stroke, Lab of Neural Control, Bethesda, Maryland 20892-4455; and 2Department of Neuroscience and Cell Biology, UMDNJ/Robert Wood Johnson Medical School, Piscataway, New Jersey 08854
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ABSTRACT |
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Wenner, Peter, Michael J. O'Donovan, and Michael P. Matise. Topographical and Physiological Characterization of Interneurons That Express Engrailed-1 in the Embryonic Chick Spinal Cord. J. Neurophysiol. 84: 2651-2657, 2000. A number of homeodomain transcription factors have been implicated in controlling the differentiation of various types of neurons including spinal motoneurons. Some of these proteins are also expressed in spinal interneurons, but their function is unknown. Progress in understanding the role of transcription factors in interneuronal development has been slow because the synaptic connections of interneurons, which in part define their identity, are difficult to establish. Using whole cell recording in the isolated spinal cord of chick embryos, we assessed the synaptic connections of lumbosacral interneurons expressing the Engrailed-1 (En1) transcription factor. Specifically we established whether En1-expressing interneurons made direct connections with motoneurons and whether they constitute a single interneuron class. Cells were labeled with biocytin and subsequently processed for En1 immunoreactivity. Our findings indicate that the connections of En1-expressing cells with motoneurons and with sensory afferents were diverse, suggesting that the population was heterogeneous. In addition, the synaptic connections we tested were similar in interneurons that expressed the En1 protein and in many that did not. The majority of sampled En1 cells did, however, exhibit a direct synaptic connection to motoneurons that is likely to be GABAergic. Because our physiological methods underestimate the number of direct connections with motoneurons, it is possible that the great majority, perhaps all, En1-expressing cells make direct synaptic connections with motoneurons. Our results raise the possibility that En1 could be involved in interneuron-motoneuron connectivity but that its expression is not restricted to a distinct functional subclass of ventral interneuron. These findings constrain hypotheses about the role of En-1 in interneuron development and function.
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INTRODUCTION |
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The last few
years have witnessed a major advance in our understanding of the
molecular mechanisms controlling the specification of spinal
motoneurons during development. This progress has occurred because it
has been possible to identify the expression of certain transcription
factors in different populations of motoneurons (Lin et al.
1998; Tsuchida et al. 1994
). Two families of
transcription factors have garnered the most attention. These are the
LIM and ETS families (whose names derive from an acronym of the
original three proteins that were identified in each family), which are differentially expressed in motoneurons projecting to different muscles. These correlations have formed the basis for hypotheses about
the roles of transcription factors in determining the phenotype of
motoneuron classes (Pfaff et al. 1996
; Sharma et
al. 1998
; Tanabe et al. 1998
). Extension of this
approach to spinal interneurons has been slow because subpopulations of
interneurons are much harder to identify than motoneurons. Whereas
motoneuron subsets can be labeled through individual muscle nerves, the
same type of identification is not possible for interneurons. Rather,
to determine the synaptic connectivity of interneurons, intracellular recordings are required.
Several studies have demonstrated differential expression of
transcription factors in subsets of spinal interneurons during development (Burrill et al. 1997; Liem et al.
1997
; Matise and Joyner 1997
; Pierani et
al. 1999
). In addition to providing important insights into the
specification and development of interneurons, the ability to identify
unique markers for particular types of interneurons would also be
extremely useful for studies of interneuronal function and anatomy. At
present, in the absence of such markers, interneuronal studies rely
largely on single-cell electrophysiology.
For these reasons, we have examined some of the synaptic connections of
interneurons expressing the Engrailed-1 transcription factor (En1) in
the developing chick spinal cord. We focused on En1 for several
reasons. First, En1 expression has been extensively characterized in
the chick spinal cord (Burrill et al. 1997;
Davidson et al. 1988
; Gardner et al.
1988
). Second, expression of En1 in ventral spinal interneurons
is extremely well conserved across vertebrate species, being found in
the mouse, chick, zebrafish, and Xenopus (Davidson et
al. 1988
; Davis et al. 1991
; Gardner et
al. 1988
; Hatta et al. 1991
), suggesting an
important developmental role. Third, while studies in knockout mice
have suggested that En1 is not required for the survival, migration, or
expression of several other transcription factors normally coexpressed
(Matise and Joyner 1997
), the protein does appear to be
involved in the regulation of axonal pathfinding (Sauressig et
al. 1999
). Furthermore, En1-expressing (En1+) cells are likely
to be GABAergic, and many send their axons into the ventrolateral white
matter and into the motor column (Sauressig et al.
1999
). These and other studies have led to the hypothesis that
En1+ cells make monosynaptic connections with motoneurons
(Sauressig et al. 1999
).
In this paper we have directly tested this hypothesis by examining the
synaptic connections between En1+ cells and motoneurons. We have also
addressed the question of whether En1+ cells constitute a single
functional class (defined by their synaptic connections with
motoneurons and dorsal root afferents) or whether En1 expression occurs
in different interneuronal types. Some of this work has been published
in abstract and in meeting proceedings (Wenner et al.
1998a,b
).
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METHODS |
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Physiology and biocytin labeling
Chick embryos were removed from the egg at embryonic days
10 to 11 (E10-E11; stage 36-37) and staged
according to the criteria of Hamburger (Hamburger and Hamilton
1951). Embryos were decapitated and the spinal cords isolated
as described previously (O'Donovan 1989
;
O'Donovan and Landmesser 1987
) in recirculating cold
(12-15°C) Tyrode's solution (concentration in mM: 139 NaCl, 2.9 KCl, 17 NaHCO3, 12 glucose, 3 CaCl2, and 1 MgCl2). The
spinal cord was isolated together with certain muscle nerves
(adductor = adductors and obturator; femorotibialis = external and medial head). The perfusion solution was slowly brought to
room temperature (~21°C), and the cord was transferred to a
recording chamber for at least 2 h, before raising the temperature
to ~27°C for the remainder of the experiment. For overnight
experiments, where recordings were obtained throughout the next day,
the temperature was brought to 17°C and left for up to 12 h
before beginning the experiment. Afferent muscle nerves (where
motoneuron axons have been cut at the ventral root), ventral roots, and
a strip of the ventrolateral funiculus (VLF) were drawn into suction
electrodes for stimulation and/or recording. Whole cell electrodes
(4-8 M
, with a K gluconate solution concentration in mM: 10 NaCl,
130 K gluconate, 10 HEPES, 1.1 EGTA, 0.1 CaCl2, 1 MgCl2, and 1 Na2ATP) were
driven through the ventral white matter into the ventral horn (after
removal of the pia) as described previously (Sernagor and
O'Donovan 1991
; Wenner and O'Donovan 1999
).
Recordings were made along the anterior lumbosacral spinal cord between
thoracic segment 7 (T7) and lumbosacral segment 5 (LS5). All whole cell
recordings were obtained using an Axoclamp 2B amplifier and custom
written data acquisition software (Labview 4.0). Extracellular suction
electrode recordings from ventral roots were amplified ×10,000 and
filtered at DC-0.1 kHz. Cells were only accepted for further study if
their membrane potential was less than or equal to
40 mV.
Single-pulse stimuli of 30-50 µA (0.5 ms) were delivered to dorsal
or ventral roots. Data on afferent stimulation were only accepted in
cases where an evoked potential was concurrently observed in the
ventral root. Such a response was taken as evidence that the root was
intact and stimulated effectively.
Whole cell recordings were obtained with 0.5% biocytin added to the patch solution to label the recorded cell. No more than two cells per hemisegment were labeled, and their locations were recorded. The low number of labeled cells in each cord allowed unambiguous matching of the biocytin-labeled cell with its physiologically determined synaptic connections.
Spike triggered averaging
To determine whether a particular interneuron projected
monosynaptically to motoneurons, we used the interneuron's spike as a
trigger to average synaptic potentials from the ventral roots. For this
purpose, we depolarized the membrane potential of the interneuron by
~5-10 mV to produce action potentials at a steady rate of
approximately 1-2 Hz. The spikes were used to trigger an averaging
program running on a Macintosh computer that accumulated from 200 to
400 traces of the ventral root recording (Wenner and O'Donovan
1999). Averages were acquired shortly after (~2-5 min) an
episode was evoked by electrode penetration of the ventral cord
surface, and so the interneuronal network was most depressed at this
time minimizing polysynaptic transmission (Fedirchuk et al.
1999
). Such a procedure made it unlikely that a potential observed in the averaged ventral root recording would be mediated polysynaptically. When a spike-triggered potential was not
observed, the data were only included if the ventral root recording
revealed the presence of an evoked potential in response to VLF stimulation.
Immunohistochemistry and cell counts
For antibody staining, embryonic day 10 (E10) chick embryos were fixed for 2 h in 4%
paraformaldehyde/PBS on ice, washed in PBS, sunk in 30% sucrose/PBS,
embedded in Tissue Tek and cut serially in a cryostat. Anti-Engrailed
antibody (Enhb-1) (Davis et al. 1991
) was used as
described previously (Matise and Joyner 1997
). This
antibody detects both En1 and En2 proteins, but since En2 is not
expressed in the lumbosacral region of the embryonic chick spinal cord
at any stage before E10 (Millet and Alvarado-Mallart 1995
and data not shown) this antibody reveals only En1 protein in this tissue.
Fluorescently coupled secondary antibodies were obtained from Jackson Immunoresearch and used as follows. For single-labeling studies, cy3-conjugated goat anti-rabbit IgG was used at 1:250. For double-labeling in experimental E10-11 embryos, cy3-goat anti-rabbit IgG was used at 1:500 to detect En1, and fluorescein-conjugated streptavidin was used at 1:100 to detect biocytin.
En1+ interneurons were counted in every fifth 10 µM section through
the anterior spinal cord segments (thoracic 7 to lumbosacral 3). Spinal
segments were identified as described previously (Matise and
Lance-Jones 1996). Three separate embryos were counted at each
stage, and the counts were averaged.
The schematic in Fig. 4 was created by making camera lucida tracings of biocytin-labeled cells in each preparation. To combine the data onto a single image, traces were aligned using the ventrolateral margin of the gray matter, and the outlines (white matter and cells) were scaled using the central canal as a second reference point. All data are expressed as means ± SD.
Image and data processing
Images were captured on a Princeton Instruments cooled
charge-coupled device (CCD) camera and processed in Metamorph software (Universal Imaging) as previously described (Matise and Joyner 1997).
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RESULTS |
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Distribution of En1+ cells in the ventral cord between E7 and E10
Previous studies in mouse and chick spinal cords have shown
that En1 interneurons migrate ventrally and laterally toward the ventral horn after they are born in the ventricular zone at an intermediate dorsoventral position (Gardner et al. 1988;
Matise and Joyner 1997
). In the present work, we found
that the labeled cells lie dorsal to the lateral motor column with few,
if any, within the motor column at E7. A few days later at
E10, En1 expression was detected in fewer cells per 10 µM
section than at E7 measured between segments T7-LS3
(E10: 99 ± 20 cells/section, mean ± SD, n = 25 sections; E7: 168 ± 26, n = 18 sections; Fig. 1,
A and B). In addition, at E10 the
intensity of En1 labeling varied between cells as illustrated in the
micrographs of Fig. 1C. These observations suggest that En1
expression is extinguishing in some cells by E10.
Alternatively, or in addition, En1 expression might differ between
cells and be extinguished later in development.
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Distribution and morphology of biocytin labeled En1 expressing interneurons
To compare the connectivity of both En1+ and En1 spinal
interneurons, we made whole cell recordings from randomly sampled ventral/intermediate zone neurons. Recording electrodes were filled with a solution containing 0.5% biocytin to label cells for later histological identification. After the recording, the lumbosacral spinal cord was fixed, serially sectioned, and stained for En1 protein
and biocytin (see METHODS).
Using this protocol, we identified 62 biocytin-labeled cells in 9 different embryos. Forty-two of these cells had acceptable physiological recordings (for criteria, see METHODS) and
were examined further. Nineteen of these 42 cells were labeled with biocytin and also expressed En1
(En1+/bio+). Examples of
two double-labeled cells are shown in Fig.
2, B-D (cell
1) and E-G (cell 2). En1+ and
En1 biocytin-labeled cells were in similar positions and were widely
distributed in the ventral horn, both dorsal and medial to motoneurons
(Fig. 4A). No obvious correlation was observed between the
location of En1+/bio+ double-labeled cells and their morphology or
synaptic connections (see next section).
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Physiological examination of the synaptic connections of En1 expressing interneurons
The 19 En1+/bio+ cells could be divided into two groups using the
spike-triggered average ventral root potential as a marker for
monosynaptic connections with motoneurons. According to this criterion,
11 cells made monosynaptic connections with motoneurons (Fig.
3A). Another cell was
identified by its direct recurrent input from motoneurons and was
therefore an R-interneuron (as defined by Wenner and O'Donovan
1999). These cells are known to make depolarizing GABAergic
connections to motoneurons (Wenner and O'Donovan 1999
).
Therefore 63% (12/19) of the sample made monosynaptic connections with
motoneurons confirming the hypothesis, first proposed in the mouse,
that some En1 cells project directly to motoneurons (Sauressig
et al. 1999
).
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We tested 4/12 of these last order interneurons pharmacologically and
determined that all of them were GABAergic because the spike-triggered
ventral root potential disappeared following bath application of the
GABAA antagonist bicuculline (Fig.
3A). Two additional neurons were assumed to be GABAergic
because they were identified as R-interneurons, for a total of six
GABAergic En+ cells. This functional evidence is consistent with
immunocytochemical data from the mouse suggesting that En1+ cells are
GABAergic (Sauressig et al. 1999).
The second group of En1+/bio+ interneurons comprised seven cells (37%; 7/19) in which we were not able to resolve a ventral root potential by spike-triggered averaging. The anatomical distribution of this group was no different from that of the first group (Fig. 4A).
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Twenty-three of the 42 biocytin-labeled cells that we recorded from did
not express the En1 protein. Of these, seven cells (30%; 7/23)
projected monosynaptically to motoneurons as determined by
spike-triggered averaging. Two of seven of these cells appeared to be
GABAergic based on the abolition of the averaged ventral root potential
by bicuculline. Collectively, these results suggest that En1+
interneurons had a greater tendency to project to motoneurons than did
randomly sampled En1 ventral horn interneurons, although the
difference was not statistically significant (P = 0.07;
Z-test). We found that En1
/bio+ and En1+/bio+ cells were
distributed similarly in the intermediate region of the cord, dorsal to
the lateral motor column (LMC) (Fig. 4A).
We also measured the muscle sensory input to spinal interneurons to
determine whether En1+ cells could be distinguished by their inputs
from different muscle nerves (Figs. 3B and 4B).
For this purpose, we stimulated the femorotibialis or adductor muscle afferents, which are known to make connections with different classes
of motoneurons (Lee and O'Donovan 1991;
Mendelson and Frank 1991
; Wenner and Frank
1995
). Such stimuli are likely to activate many different
functionally distinct classes of afferent within a muscle nerve
including those making monosynaptic connections with motoneurons
(Lee et al. 1988
). We found that the En1+ and En1
interneurons received very similar patterns of afferent input (Fig.
4B): Short-latency (~10 ms, probably monosynaptic) input from femorotibialis muscle afferents was observed in 9/15 En1+ neurons
and 9/15 En
cells. Similarly 7/13 En1+ versus 9/13 En1
cells
received short latency adductor input; 6/13 En1+ versus 6/13 En1
cells received inputs from both muscle afferents, and 3/13 En1+ versus
3/13 En1
cells received from neither. We also found similar patterns
of afferent input in last order interneurons and interneurons that did
not make a detectable connection to motoneurons. Thus the expression of
En1 does not distinguish between last order interneurons based on the
muscle afferent inputs we examined. A summary of the above
physiological data is provided schematically in Fig. 4B.
In a few cells, evidence for an axonal projection into the VLF was
obtained by stimulating a strip of VLF drawn into a suction electrode
and evoking a short-latency, all or none, action potential (Fig.
3C). In two of the En1+ and six of the En1 interneurons we
were able to record such a potential. Although these spikes were evoked
in an all-or-none manner, it is difficult to be certain that they
originated antidromically because VLF stimulation subthreshold for the
spike, invariably produced a synaptic potential and often triggered an
episode (Fig. 3C, top panel). However, in five
cells (1 En+, 4 En
), the VLF stimulus evoked action potential
exhibited a clear A/B break consistent with antidromic activation
(arrowhead in Fig. 3C, bottom trace).
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DISCUSSION |
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In this study we have combined physiological and immunohistochemical approaches to examine the characteristics of ventral interneurons that express the En1 homeodomain-containing transcription factor. We have addressed two questions about this population. First, do some, or all, of the cells make monosynaptic connections with motoneurons? Second, does En1 mark a unique interneuronal population? Our results show that many En1+ cells project to motoneurons and that the population is not uniform but instead exhibits a diverse pattern of synaptic connectivity.
En1 expression marks a diverse population of ventral interneurons
Several lines of evidence suggest that En1 expression does not
identify a unique interneuronal class. First, the synaptic connections
of En1+ and En1 interneurons were similar. Second, the connections of
En1+ cells were extremely variable. En1+ cells exhibited a wide range
of muscle afferent input; some received input from muscle afferents
projecting to one muscle (Fig. 3B), some from a functionally
distinct muscle, some from both, and some from neither. While these
findings suggest that specific patterns of muscle afferent input are
not associated with En1 expression, we cannot exclude the possibility
that connections from an untested muscle nerve or different classes of
afferent within a single muscle nerve could be associated with the
expression of En1. Third, there is evidence suggesting that while some
R-interneurons express En1, not all do. We found that two of the En1+
cells in our sample were R-interneurons (Wenner and O'Donovan
1999
). R-interneurons are labeled following injections into the
rostral or caudal VLF (Wenner and O'Donovan 1999
)
indicating that their axons project rostrally and caudally. However, in
the early mouse cord (up to E12) En1+ cells project an axon
rostrally but not caudally in the VLF (Sauressig et al.
1999
).
Finally, we found that En1+ cells were widely distributed within the
ventral horn and were not concentrated in distinct pools, in contrast
to the discrete organization of many functional subclasses of
interneurons. For instance, Ia inhibitory interneurons (Hultborn et al. 1971), Clarke's column (Rethelyi 1968
),
Hoffman's nucleus (Eide and Glover 1996
), and
R-interneurons (Wenner and O'Donovan 1999
) are all
organized in spatially discrete nuclei or columns. It is unlikely,
therefore that En1 expression defines any of these functionally and
anatomically distinct interneuronal subpopulations.
Does En1 expression identify interneurons that form monosynaptic connections with motoneurons?
It had been proposed previously that the En1 population may
consist of interneurons that make direct synaptic connections onto
motoneurons (Sauressig et al. 1999). In this report we
have shown that spike-triggered averaging from 63% of the En1+
population produced a potential in the ventral roots, indicating that
many of these cells did project synaptically to motoneurons. While it
is possible that a polysynaptic connection might be responsible for
some of the potentials recorded by spike-triggered averaging, we
believe this to be unlikely for the following reasons. First, the
spike-triggered ventral root potentials acquired in this study were
obtained shortly after an episode when polysynaptic transmission is depressed (Fedirchuk et al. 1999
). Second, the
potentials were resolvable after only 200 or fewer sweeps, indicating
the strength and reliability of the response. Finally because
developing synapses are immature, polysynaptic connections are
particularly susceptible to low-frequency synaptic fatigue (Lee
and O'Donovan 1991
).
We found that many of the direct connections between motoneurons and
En1+ cells were blocked by the GABAA antagonist
bicuculline, consistent with a previous report in the mouse that the
En1+ cells are GABAergic (Sauressig et al. 1999). Of the
12 En1+ cells that exhibited a spike-triggered potential, four of four
tested were GABAergic (Fig. 3A). It is quite possible that
the other eight last order interneurons were also GABAergic because we
have found that most interneuron spike trigger averaged ventral root
potentials are blocked in the presence of bicuculline (10/12, 83%,
from this study and unpublished observations). It is not yet clear why
we have observed such a predominance of monosynaptic GABAergic
connections, but one possibility is that such connections are somatic,
which would favor their detection in ventral root recordings.
Notwithstanding this bias, our results are consistent with the idea
that a significant proportion of last order En1+ interneurons are
GABAergic at E10/11.
One question that is difficult to answer from our work is whether all En1+ are monosynaptically connected with motoneurons. We found that En1+ cells exhibit a higher preponderance of spike trigger averaged ventral root potentials than cells that did not express the protein (63 vs. 30%, P = 0.07). Although we have argued that such potentials are strong evidence of a direct connection to motoneurons, a negative result is more difficult to interpret. False negatives could arise for several reasons. For example, it is possible that the interneuron's axon was damaged, the synaptic connection was too weak to be resolved by spike-triggered averaging of the ventral root potential, or the interneuron projected to motoneurons in a different segment to that of the ventral root recording. A definitive answer to this question will probably require combined dual intracellular recording from En1+ interneurons and motoneurons.
Finally, it is possible that some cells we identified as En1 at
E10 expressed En1 earlier in development but down-regulated expression by E10. We provided evidence that En1 expression
appears to be in the process of being down-regulated in chick spinal
cord interneurons at the time that the physiological experiments were performed, and this must be considered when interpreting our findings. Preliminary experiments in mice using a lineage tracing technique reveal that En1 is expressed transiently in a larger population of
cells than express the protein at later, mid-gestation stages in mouse
embryos, and is beginning to be down-regulated by E15.5 (M. P. Matise, personal observations). It is possible, therefore that all cells that express the En1 protein at some point in their history make monosynaptic connections to motoneurons. This hypothesis will have to be tested by making more extensive measurements of the
connections of individual interneurons than was possible in the present
study, and by combining genetic techniques that allow permanent marking
of all cells that express En1 at any time in their developmental history.
Conclusions
The results of this paper illustrate the feasibility of combining electrophysiology and immunocytochemistry to identify the synaptic connections of spinal interneurons that express particular transcription factors. Such experiments are essential to our understanding of the role that transcription factors play in the specification and differentiation of spinal interneurons during development. We have demonstrated that a substantial proportion of En1+ cells make monosynaptic connections with motoneurons but that the protein is unlikely to mark a unique, anatomically defined interneuron sub-class. To determine whether the En1 protein is involved in specifying the projections to motoneurons, physiological studies of ventral interneuron connectivity in animals in which En1 function has been eliminated will be necessary.
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ACKNOWLEDGMENTS |
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The authors thank Dr. Alexandra Joyner for generous support of this project.
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FOOTNOTES |
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* P. Wenner and M. P. Matise contributed equally to this work.
Address for reprint requests: M. P. Matise, UMDNJ/Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854 (E-mail: matisemp{at}umdnj.edu).
Received 19 April 2000; accepted in final form 28 July 2000.
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REFERENCES |
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