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Previous Article | Next Article
The Journal of Neuroscience, December 15, 1999, 19(24):10813-10828
Gap Junctional Coupling and Patterns of Connexin Expression among Neonatal Rat Lumbar Spinal Motor Neurons
Qiang Chang1, Michael Gonzalez1, Martin J. Pinter2, and Rita J. Balice-Gordon1 1 Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6074, and 2 Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Interneuronal gap junctional coupling is a hallmark of neural development whose functional significance is poorly understood. We have characterized the extent of electrical coupling and dye coupling and patterns of gap junction protein expression in lumbar spinal motor neurons of neonatal rats. Intracellular recordings showed that neonatal motor neurons are transiently electrically coupled and that electrical coupling is reversibly abolished by halothane, a gap junction blocker. Iontophoretic injection of Neurobiotin, a low molecular weight compound that passes across most gap junctions, into single motor neurons resulted in clusters of many labeled motor neurons at postnatal day 0 (P0)-P2, and single labeled motor neurons after P7. The compact distribution of dye-labeled motor neurons suggested that, after birth, gap junctional coupling is spatially restricted. RT-PCR, in situ hybridization, and immunostaining showed that motor neurons express five connexins, Cx36, Cx37, Cx40, Cx43, and Cx45, a repertoire distinct from that expressed by other neurons or glia. Although all five connexins are widely expressed among motor neurons in embryonic and neonatal life, Cx36, Cx37, and Cx43 continue to be expressed in many adult motor neurons, and expression of Cx45, and in particular Cx40, decreases after birth. The disappearance of electrical and dye coupling despite the persistent expression of several gap junction proteins suggests that gap junctional communication among motor neurons may be modulated by mechanisms that affect gap junction assembly, permeability, or open state.
Key words: gap junction; motor neuron; connexin; spinal cord; neuromuscular junction; activity
INTRODUCTION
Gap junctional coupling is widespread throughout the developing mammalian nervous system, among neurons as well as among glia (for review, see Dermietzel and Spray, 1993
; Kandler and Katz, 1995
We are particularly interested in understanding the mechanisms that shape motor neuron firing during the perinatal period, when activity-dependent synaptic competition sculpts the innervation of individual muscle fibers from several inputs to a single input (for review, see Thompson, 1985
Using intracellular recordings from identified motor neurons in neonatal rat lumbar spinal cord and iontophoretic injection of Neurobiotin, a low molecular weight compound that passes across gap junctions, into single identified motor neurons, we found that the percentage of dye and electrically coupled motor neurons declines rapidly in the first week after birth. The compact distribution of dye-labeled motor neurons within a cluster suggested that, at the postnatal ages examined, coupling is spatially restricted. We also found that the repertoire and pattern of motor neuronal gap junction protein (connexin) expression is unique compared with those previously reported for other neurons and glia (for review, see Dermietzel and Spray, 1993
Preliminary reports of this work have been published previously in abstract form (Chang and Balice-Gordon, 1997
MATERIALS AND METHODS
Animals and spinal cord preparation. Timed pregnant female Sprague Dawley rats were purchased from Charles River. The day of birth was designated postnatal day 0 (P0). P0-P7 rats were anesthetized on ice and decapitated; P8-P10 rats were injected intraperitoneally with an overdose of sodium pentobarbital and decapitated. The lumbar spinal cord was quickly exposed via a dorsal laminectomy and superfused with cold Ringer's solution containing (in mM): 116 NaCl, 5 KCl, 4 CaCl2, 1 MgSO4, 29 NaHCO3, 1 NaH2PO4, and 11 glucose, bubbled with 95% O2/5% CO2. The meninges were carefully removed, and the cord was hemisected as described previously (Ziskind-Conhaim, 1988
Intracellular recording from identified motor neurons. The L3, L4, and/or L5 ventral roots were placed in suction electrodes so that ~3 mm of root was left between the tip of the suction electrode and the spinal cord surface. Roots were stimulated at 1 Hz with square pulses of 100-200 µV and 100-200 µsec duration. Using sharp high-resistance microelectrodes (80-120 M
) filled with a solution of 4% Neurobiotin (Vector Laboratories, Burlingame, CA) dissolved in 2 M KCl and 10 mM HEPES buffer, motor neuron location was determined by locating extracellular field potentials (Fulton et al., 1980
60 mV or more hyperpolarized and action potentials of 60 mV or higher were characterized further. Data were acquired digitally, stored onto a PC, and analyzed off-line. In most cases, particularly at P3-P4, coupling potentials were measured from averaged records (10-20 sweeps) after subtraction of averaged extracellular antidromic field potential records.
Pharmacological blockade of gap junctions. In some experiments, a gap junction blocker, halothane (Sigma, St. Louis, MO) (Burt and Spray, 1989
Neurobiotin injection and histology. In some experiments, Neurobiotin was iontophoretically injected (0.5-4.0 nA, 400 msec, 1 Hz, 10-20 min) into single, identified motor neurons. To determine whether dye leakage into the extracellular space could spuriously result in the labeling of multiple motor neurons, in some experiments, Neurobiotin was iontophoretically deposited extracellularly (1-5 nA, 400 msec, 1 Hz, 10-20 min).
After a period of at least 2 hr to allow Neurobiotin to diffuse across putative gap junctions, the spinal cords were fixed in 4% paraformaldehyde for 4 hr at room temperature, washed extensively in PBS, cryoprotected in 20% sucrose/PBS at 4°C overnight, embedded in OCT (Sakura), and frozen in an acetone/dry-ice bath. Serial parasagittal sections of spinal cords were cut at 25-35 µm on a cryostat (Leica CM3000) and collected onto slides. Sections were permeablized in methanol at
Motor neuron culture. Motor neurons were isolated from embryonic day 15 (E15) rat spinal cord as previously described (Camu and Henderson, 1992
RT-PCR analysis of connexin expression. Total RNA was extracted from purified E15 rat motor neurons or from E15 or P1 ventral spinal cord using TRIzol (Life Technologies, Gaithersburg, MD) and then treated with RNase-free DNase to remove genomic DNA. Total RNA (1 µg) was reverse-transcribed into first-strand cDNA, using Advantage RT-for-PCR kit (Clontech, Cambridge, UK). Primers were designed for Cx26, Cx30, Cx31, Cx31.1, Cx32, Cx33, Cx36, Cx37, Cx40, Cx43, Cx45, Cx46, and Cx50 to amplify a unique coding region of each gene. The primer sequences for each connexin were as follows: Cx26 5'-CGGAAGTTCATGAAGGGAGAGAT, Cx26 3'-GGTCTTTTGGACTTCCCTGAGCA; Cx30.3 5'-ATGAACTGGGGATTTCTCCAG, Cx30.3 3'-TCATGGATACACACCTGCATC; Cx31 5'-ATGGATTGGAAGAAGCTTCAG, Cx31 3'-TTAAATGGGGGTCAGGCTAGG; Cx32 5'-CTGCTCTACCCGGGCTATGC, Cx32 3'-CAGGCTGAGCATCGGTCGCTTTC; Cx33 5'-GCCAGTGGGGAAAGGCGCTTGCA, Cx33 3'-CCCACCGGGACTACCTGATC; Cx37 5'-GGCTGGACCATGGAGCCGGT, Cx37 3'-TTCTGGCCACCCTGGGGGGC; Cx40 5'-CTGGCCAAGTCACGGCAGGG, Cx40 3'-TTGTCACTGTGGTAGCCCTGAGG; Cx43 5'-TACCACGCCACCACTGGCCCA, Cx43 3'-ATTCTGGTTGTCGTCGGGGAAATC; Cx45 5'-GGGCAAACCAATTCCACCACC, Cx45 3'-CAAGATTAAATCCAGACGGAG; Cx46 5'-GGAAAGGCCACAGGGTTTCCTGG, Cx46 3'-GGGTCCAGGAGGACCAACGG; Cx50 5'-CCTTTGACAGAGGTTGGAATCGTG, Cx50 3'-CCGATTGTCATCGGTTGTCAGCTC. Primers designed to recognize mouse Cx36 as described in Condorelli et al. (1998)
The 25 µl PCR mixture contained 5-10 µl first-strand cDNA, 0.8 µM deoxynucleotide triphosphates, 100 ng of each primer, 3.5 µM magnesium, 2.5 µl 10× PCR buffer (Life Technologies), and 1.25 U Taq polymerase (Life Technologies). The PCR conditions were 94°C for 5 min, 94°C for 45 sec, 55°C for 30 sec, and 72°C for 1 min for 35 cycles followed by 72°C for 10 min. In each case, total RNA was used as a negative control template, and cDNA from tissues known to express a particular connexin(s), such as heart, skin, eye, liver, and testis, were used as positive control templates for the PCR reactions. RT-PCR products from spinal cord, purified embryonic motor neurons, and/or positive control tissues were analyzed using gel electrophoresis. All products were sequenced and compared with sequences in GenBank to determine their identities.
In situ hybridization. E12 and E15 embryos were dissected from time-pregnant females and processed intact. Spinal cords were dissected from E18, P1, P7, P14, and 3-month-old rats under mammalian Ringer's solution. Tissues were fixed in 4% paraformaldehyde, pH 7.4, for 2 hr, rinsed in PBS, cryoprotected in 20% sucrose in PBS overnight at 4°C, embedded in OCT (Tissue-Tek), and frozen in an acetone/dry-ice bath, and 20 µm frozen sections were obtained (Leica CM3000 cryostat). Tissues from P1 mutant mice lacking a connexin were used in some experiments as negative controls. These included Cx37
Each probe was cloned into pGEM3 (Promega, Madison, WI), and cRNA probes for each connexin were transcribed and labeled with digoxigenin-UTP (Boehringer Mannheim, Indianapolis, IN). A typical 50 µl in vitro transcription reaction mixture contained 2 µl (~1 µg) linearized template DNA, 5 µl 10× digoxigenin-NTP mix (Boehringer Mannheim), 80 U RNase inhibitor, 10 mM DTT, 10 µl 5× transcription buffer (Promega), and 95 U T7 RNA polymerase (Promega). The reaction was performed at 37°C for 2 hr.
Frozen tissue sections were rinsed with PBS, incubated with acetylation buffer (0.926 gm triethanolamine, 112 µl 10N NaOH, 125 µl acetic anhydride, and DEPC-H2O to 50 ml), permeabilized with 1% Triton X-100 in PBS for 30 min, and rinsed with PBS. Slides were then incubated with prehybridization buffer (50% formamide, 4× SSC, 1× Denhardt's solution, 10% dextran sulfate, 250 µg/ml Baker's yeast RNA, and 500 µg/ml herring sperm DNA) at 68-72°C for 2 hr. cRNA probe (200-500 ng/ml) was used for hybridization at 68-72°C overnight. Moist chambers and coverslips were used to prevent hybridization buffer from evaporating. The next day, the slides were washed with 0.2× SSC at 68-72°C for 1 hr, rinsed with PBS, blocked with 1% BSA-0.1% Triton X-100 in PBS for 1 hr at room temperature, and incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (1:2000 to 1:5000; Boehringer-Mannheim) overnight at 4°C. Slides were then washed extensively with PBS, equilibrated with 0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 50 mM MgCl2 for 5 min at room temperature. A colorimetric reaction for AP was developed and then stopped after signal was determined to be apparent after examination under a dissecting microscope.
Slides were either photographed onto 35 mm print film and the prints were scanned into a computer, or they were photographed with a Hamamatsu cooled color CCD camera and the images were acquired digitally using a PC-based image processing system (Phase 3 Imaging). Composite images of overlapping fields were made with Adobe Photoshop software.
Northern analysis. Poly(A+) RNAs extracted from E15 rat embryos and spinal cords from P1 wild-type or mutant "knock-out" mice (Cx37
Immunohistochemistry. For some immunostaining experiments, tissues were not fixed before being frozen and sectioned as described above. Frozen tissue sections were picked up on glass slides, rinsed with PBS, blocked with 1% BSA, 0.1% Triton X-100 in PBS, and incubated with anti-Cx antibodies at 1:100-1:500 dilution at 4°C overnight. The antibodies used were anti-Cx26 and anti-Cx43, affinity-purified anti-peptide antibodies derived in rabbit (gift of Dr. Bruce Nicholson); anti-Cx32, mouse monoclonal antibody 7C6C7, raised against amino acid 235-246 in the C terminus of Cx32 [Li et al. (1997)
Slides were washed with PBS, incubated with appropriate fluorescent secondary antibodies at 4°C for 3-4 hr, washed with PBS, and coverslipped in a glycerol-based medium with an anti-fading agent (VectaShield, Vector Labs). At embryonic ages, motor neurons were identified by their location and staining with an antibody against the transcription factor Islet-1 (Tsuchida et al., 1994
Western analysis. Frozen E15 rat embryos and spinal cords from P1 wild-type or knock-out mice (Cx37
RESULTS
Time course of transient electrical coupling among lumbar motor neurons
In hemisected spinal cord preparations from P0-P8 rats, motor neurons were identified by the presence of an antidromic action potential after ventral root stimulation (Fig. 1A). Once a cell was identified, several criteria were used to determine whether the impaled motor neuron might be electrically coupled to other motor neurons. The first of these was a "collision test" (Baker and Llinas, 1971
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Figure 1. Characterization of electrical coupling among developing motor neurons. Motor neurons were identified by intracellular impalement in hemisected spinal cord preparations from P0-P8 rats by the presence of an antidromic action potential after ventral root stimulation. Shown are coupling potentials characterized in P0-P2 motor neurons identified by antidromic ventral root stimulation. A, Intracellular current injection was used to elicit an action potential (a.) in the impaled motor neuron during antidromic ventral root stimulation. By adjusting the timing of the intracellular current pulse (a.) relative to antidromic ventral root stimulation, the antidromic action potential (b.) elicited by ventral root stimulation failed, revealing an initial segment spike (c.). As the interval between the intracellular current pulse and antidromic stimulation was decreased further, the initial segment spike also failed. This failure occurs by collision of the antidromic spike by the intracellularly evoked action potential, which transiently inactivates voltage-gated sodium channels. The remaining depolarizing potential (d.) is a putative coupling potential (5.9 mV amplitude, 1.5 msec rise time; 3.9 msec latency from onset of antidromic stimulation; see Table 1 for summary). Calibration: 20 mV, 10 msec. B, Coupling potential amplitude was graded as the intensity of antidromic stimulation was graded. Partial action potentials, which became apparent as collision tests were performed, occurred in an all-or-nothing fashion as antidromic stimulation was graded. Calibration: 20 mV, 10 msec. C, Intracellular hyperpolarization did not affect the amplitude of the coupling potential, in this case detected by straddling threshold for action potential generation. An electrical potential would be insensitive to changes in membrane potential. Coupling potential amplitude = 1.3 mV; amplitude after hyperpolarization to
At P0-P2, 26 of 35 cells (74%) had coupling potentials identified by this criteria. Coupling potentials were on average 3.2 ± 0.6 mV (mean ± SEM) (Table 1) in amplitude and were detected between 0.15 and 4 msec after the onset of the antidromically evoked action potential (Fig. 1). Coupling potentials had relatively sharp rise times at these ages (1.8 ± 0.1 msec) (Table 1), and their waveforms were relatively similar in duration. At P3-P4, 7 of 10 cells (70%) had coupling potentials. This proportion is not significantly different from that observed at P0-P2 (p > 0.10; Fisher exact test). However, the coupling potentials observed in P3-P4 motor neurons were significantly smaller in amplitude (1.3 ± 0.4 mV) (Table 1) than coupling potentials observed in P0-P2 rats (p < 0.005, Student's t test). The coupling potentials observed at P3-P4 had a similar mean rise time (1.8 ± 0.5 msec; not significantly different; p > 0.5, Student's t test) (Table 1) but a larger range of rise times than those observed at P0-P2. At P7-P8, none of the cells recorded had coupling potentials detectable after collision of antidromic and orthodromic action potentials. In some cases, motor neurons exhibiting coupling potentials were further characterized in one or more of three ways to determine whether these potentials reflected electrical communication among motor neurons. The first characterization involved determining whether grading the intensity of antidromic stimulation resulted in changes in the amplitude of the coupling potential (Fig. 1B). This was performed in most motor neurons with coupling potentials and was necessary because partially blocked action potentials (initial segment or M-spikes) (Llinas and Sasaki, 1989
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Table 1. Characterization of motor neuron electrical and dye coupling The effect of intracellular hyperpolarization on the amplitude of the coupling potential was also evaluated in P0-P2 motor neurons with coupling potentials (n = 7 cells). Electrical potential amplitude would be insensitive to membrane potential, unless the gap junction proteins were strongly gated by transmembrane voltage (cf. Spray et al., 1985
In four P0-P2 motor neurons, the effect of high-frequency stimulation (10-100 Hz) on the coupling potential was also evaluated. Unlike chemical synaptic potentials, electrical potentials do not fail with high-frequency stimulation (Llinas and Sasaki, 1989
Taken together, these data suggest that the prevalence and strength of coupling potentials among rat lumbar spinal motor neurons decreases during the first postnatal week and that electrical coupling potentials are undetectable after this time.
Coupling potentials are abolished by a gap junction blocker
To determine whether coupling potentials could be reversibly abolished by a gap junction blocker such as halothane (Burt and Spray, 1989
In each case, the coupling potential observed during a collision test (Fig. 2A) was observed to steadily decrease in amplitude and become abolished within 10-15 min of perfusion with halothane-saturated Ringer's solution (Fig. 2B). No change in resting membrane potential was observed during perfusion with halothane-saturated Ringer's. In two motor neurons, subsequent washout of halothane with normal Ringer's led to a complete recovery of the coupling potential amplitude (Fig. 2C). This experiment further suggests that electrical coupling potentials observed in motor neurons are mediated by gap junctions.
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Figure 2. Coupling potentials are abolished by halothane, a gap junction blocker. To determine whether coupling potentials could be reversibly abolished by a gap junction blocker, preparations were superfused with halothane-saturated Ringer's solution after characterization of coupling potentials as shown in Figure 1. A, A collision test was initially used to determine whether a coupling potential was present. Coupling potential, 5.6 mV; average of 10 sweeps, P0 spinal cord. B, After several minutes of exposure to halothane-saturated Ringer's, coupling potentials were abolished. Shown are eight successive sweeps illustrating the decrease in coupling potential amplitude until no potential could be detected. No change in resting membrane potential was observed during perfusion with halothane-saturated Ringer's. C, Washout of halothane with normal Ringer's led to a complete recovery of the coupling potential amplitude, 5.6 mV; average of 20 sweeps 15 min after replacement of halothane with normal Ringer's. Calibration: 10 mV, 10 msec.
Extent of dye coupling among lumbar motor neurons
The intracellular recording and pharmacology experiments reported here and previously (Fulton et al., 1980
To address these issues, we used intracellular iontophoretic injection of Neurobiotin, a low molecular weight compound that passes across gap junctions (Kita and Armstrong, 1991
At P0-P2, each injected motor neuron had a coupling potential as determined by one or more of the criteria described above. Injected motor neurons were identified by strong Neurobiotin labeling in the cell body and throughout their extensive dendritic arbor (Fig. 3), and approximately one-third of injected neurons (n = 3 cells) had a Neurobiotin-labeled axon that exited a ventral root. In each case, 1-18 labeled cells were observed surrounding the injected motor neuron (6.1 ± 1.8 mean ± SEM) (Fig. 4; Table 1). These cells had large soma, ranging from 22 to 44 µm in diameter (Table 1), and in many cases primary and secondary dendrites were also labeled. We compared the size distribution of dye-labeled cells with the size distribution of neurons in the ventral horn retrogradely labeled after dye injection into hindlimb muscles of postnatal rats (Swett et al., 1986
motor neurons. There was no relationship between coupling potential amplitude in the injected motor neuron with the number of dye-labeled cells per cluster (r =
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Figure 3. Developing motor neurons are extensively dye coupled around the time of birth. The number of dye-labeled cells was determined 2-4 hr after injection of a single characterized motor neuron with Neurobiotin followed by histology. A, Single plane projection of confocal stack of images from a P2 spinal cord, showing ventral and lateral location of dendrites of injected motor neuron and 10 additional Neurobiotin-labeled cells. Ventral edge of section is shown at top of panel. Scale bar, 25 µm. B, Single plane projection of confocal stack of images of injected motor neuron cell body shown in A (adjacent section), with its proximal dendrites and surrounded by five additional Neurobiotin-labeled cells. A total of 19 labeled cells were in this cluster, spanning five adjacent sections of 20 µm each. Scale bar, 10 µm. C, Single plane projection of confocal stack of images from a P4 spinal cord, showing cell body and proximal dendrites of injected motor neuron. Scale bar, 10 µm. D, Single plane projection of confocal stack of images of an adjacent section, showing one additional Neurobiotin-labeled cell body. Scale bar, 10 µm. E, Single plane projection of confocal stack of images from a P7 spinal cord, showing single cell body and some of the dendritic arbor of the injected motor neuron. Ventral edge of the section is at top of panel. Scale bar, 25 µm. F, Single plane projection of confocal stack of images at higher magnification, confirming that only one Neurobiotin-labeled cell body is present. Scale bar, 10 µm.
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Figure 4. Extent of dye coupling among motor neurons during the first week after birth. The number of cells in each dye-labeled motor neuron cluster is shown plotted against postnatal age in days. Between P0-P2 and P7-P8, there is a sharp decrease in the number of Neurobiotin-labeled motor neurons per cluster.
The distribution of dye-labeled motor neurons was spatially restricted within the medial or lateral columns of the ventral horn. In P0-P2 spinal cords, each dye-labeled cluster of motor neurons occupied a mean volume of 139 × 130 × 96 µm in the rostral-caudal, dorsal-ventral, and medial-lateral dimensions of the ventral horn, respectively (Fig. 5; Table 1). The implications of the compact distribution of dye-labeled motor neurons are considered below.
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Figure 5. Distribution of dye-labeled neurons suggests limited spatial extent of gap junctional coupling after birth. The distribution of dye-labeled motor neurons is shown for each cluster from P0-P2 (A) and P3-P4 (B) spinal cord segments L3, L4, and L5. Each cluster is represented by a different symbol. In P0-P2 spinal cords, each dye-labeled cluster of motor neurons occupied a mean volume of 139 × 130 × 96 µm in the rostral-caudal, dorsal-ventral, and medial-lateral dimensions of the ventral horn, respectively (Table 1). The dimensions of individual motor pools at these ages are more than twice as large. Similarly, at P3-P4, each dye-labeled cluster of motor neurons occupied a mean volume of 70 × 70 × 50 µm in the rostral-caudal, dorsal-ventral, and medial-lateral dimensions of the ventral horn, respectively (Table 1). Given that there is relatively little overlap among motor pools in the rat ventral spinal cord, the distribution of dye-labeled motor neurons strongly suggests that, after birth at least, dye coupling is present among motor neurons that innervate the same skeletal muscle. Scale bar, 100 µm.
At P3-P5, three of five motor neurons injected had a coupling potential as identified by one or more of the criteria described above. In these cases, injection of a single motor neuron resulted in dye labeling of two or three motor neurons (2.3 ± 0.3 mean ± SEM) (Fig. 4; Table 1), identified by their large diameter (26-44 µm) (Table 1) and extensive dendritic arbor (Fig. 3). In two motor neurons, no coupling potential was detected after a collision test. In both of these cases, injection of a single motor neuron resulted in dye labeling of two cells. This suggests that, as has been reported in brainstem and cortex (Mazza et al., 1992
By P7-P8, none of the motor neurons injected (0 of 7) had a coupling potential as identified by collision test or other criteria. In six of these cases, injection of a single motor neuron resulted in dye labeling of a single cell (Figs. 3, 4; Table 1). In one case, no coupling potential was detected, but two dye-labeled neurons were observed after intracellular injection of Neurobiotin. These data suggest that shortly after birth, as observed for electrical coupling, there is a sharp reduction in the extent of dye coupling among lumbar motor neurons.
Two control experiments were performed to rule out dye uptake that might have artifactually resulted in more than one motor neuron becoming labeled by Neurobiotin. In three P0-P2 spinal cords, Neurobiotin was iontophoretically deposited extracellularly into the ventral horn at the location where extracellular field potentials were identified after antidromic ventral root stimulation. In none of these cases was intracellular labeling of motor neurons or other cells observed, although in some sections nonspecific labeling of capillaries was seen.
We also evaluated whether Neurobiotin leaking out of the intracellular electrode, or being inadvertently iontophoresed as intracellular current was injected for physiological characterization of electrical coupling, could result in motor neuron dye labeling, and thus result in a cluster being detected. In three P0-P2 spinal cords, four to five motor neurons were impaled and characterized with a Neurobiotin-filled intracellular electrode, but Neurobiotin was not intentionally iontophoresed. In none of these spinal cords were dye-labeled motor neurons or other cells detected. The results of these control experiments suggest that the clusters of dye-labeled motor neurons that we observed in neonatal spinal cords were caused by intercellular gap junctional communication as opposed to nonspecific dye uptake.
Developing motor neurons express five gap junction proteins
Given that electrical and dye coupling disappear shortly after birth, we reasoned that the temporal and spatial expression patterns of gap junction proteins, called connexins, might also be developmentally regulated. We determined the repertoire of motor neuron connexin expression using RT-PCR, in situ hybridization, and immunostaining. Using primers designed to specifically amplify each of the 13 known rodent connexins, E15 rat motor neurons were purified and analyzed by RT-PCR. Motor neuron preparations were determined to be homogeneous if >98% of the cells were immunopositive after staining with an anti-Islet-1 antibody (Tsuchida et al., 1994
Using primers specific for Cx36, Cx37, Cx40, Cx43, and Cx45, PCR products of the expected size were typically observed in the motor neuron cDNA lanes but not RNA lanes (Fig. 6). Primers specific for Cx36 amplified a 979 bp band in motor neuron and eye cDNA (Condorelli et al., 1998
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Figure 6. RT-PCR analysis of connexins expressed by embryonic motor neurons and in neonatal spinal cord. RT-PCR analysis was performed on motor neuron RNA. PCR products were amplified using primers for each of the 13 known rodent connexins. Bands were typically observed in the motor neuron cDNA lanes, but not RNA lanes, using primers specific for Cx36, Cx37, Cx40, Cx43, and Cx45. Primers specific for Cx36 amplified a 979 bp band in motor neuron and eye cDNA. Primers specific for Cx37, Cx40, Cx43, and Cx45 amplified 422, 308, 292, and 1217 bp bands, respectively, in motor neuron and heart cDNA. In contrast, primers against the other known rodent connexins amplified the predicted size band from tissues known to express that particular connexin (for example, skin, heart, liver, eye, and testis) but failed to amplify the same size band in motor neurons. In each case, PCR products were eluted from gels, cloned, and sequenced to verify their identity. Horizontal lines at left indicate markers (from top to bottom, 1.2, 0.6, and 0.3 kB).
The five connexins identified by RT-PCR from purified embryonic motor neurons were also detected from RNA extracted from P1 spinal cord (data not shown). Cx26 and Cx32, which are known to be expressed in meninges, ependymal cells, and glia, (for review, see Bruzzone and Ressot, 1997
Spatial and temporal patterns of motor neuron connexin expression
To determine the spatial and temporal patterns of connexins expressed by motor neurons that may contribute to the formation of functional gap junctions during development, in situ hybridization was performed in cross sections of lumbar spinal cord from rats ranging from E12 to 3 months of age. cRNA probes against each of the five connexins that were positive in RT-PCR from purified E15 motor neurons were used, as well as cRNA probes against Cx26 and Cx32, identified by RT-PCR from P1 spinal cord. Northern blot analysis showed that each cRNA probe used recognized a single band of the predicted size and, in the case of Cx37, Cx40, and Cx43, did not recognize the same size band from RNA extracted from mutant mice lacking those genes (Fig. 7A). The Cx32 cRNA probe was characterized previously (Bergoffen et al., 1993
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Figure 7. Northern and Western blot analyses of connexin-specific reagents. A, Shown are Northern blots of poly(A+) RNA extracted from E15 rat embryos (lanes a, b, e, h, and k), spinal cords of wild-type P1 mice (c, f, and i), or spinal cords of mutant Cx37
Cx36, Cx37, Cx40, Cx43, and Cx45 were observed to be widely expressed in the neural tube and ventricular zone from E12 to E15 and throughout the spinal cord, particularly in the dorsal horn, at later stages. Although these connexins appear to be differentially developmentally regulated in these structures, here we focus on expression only in ventral horn motor neurons. In experiments in E12-E18 animals, ventral spinal cord regions containing motor neurons were identified by their expression of mRNA for the transcription factor Islet-1 (Tsuchida et al., 1994
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Figure 8. Cx36, Cx37, and Cx43 are expressed by developing and adult motor neurons. Shown are photographs of E15-adult lumbar spinal cord after in situ hybridization for Cx36 (left, left middle), Cx37 (right middle), and Cx43 (right) specific transcripts. Low- and high-power photomicrographs are shown for Cx36 hybridization signal; in left middle column, black arrows indicate examples of the relatively rare motor neurons that appeared negative for Cx36 mRNA. Similar observations were made with Cx37 and Cx43 probes. High-power images are representative fields from ventrolateral spinal cord where motor neurons are located. The temporal and spatial expression patterns of Cx36, Cx37, and Cx43 were similar, in that these connexins were expressed throughout the spinal cord at E15 and in the vast majority of motor neurons from E18 through P14 (see Table 2 for quantification). Cx43, and to a lesser extent Cx37, was expressed in dorsal root ganglia (E15, top row, lateral to spinal cord; data at other ages not shown). Cx36, Cx37, and Cx43 were also expressed in the dorsal horn. Surprisingly, expression was maintained in a substantial proportion of motor neurons in adult animals, despite the lack of functional gap junctional coupling after P3-P4 (also see Table 2). Scale bars: 500 µm (low power Cx36, Cx37, and Cx43); 50 µm (high power Cx36).
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Table 2. Expression of connexin mRNA transcripts in rat lumbar spinal motor neurons The temporal and spatial expression patterns of Cx36, Cx37, and Cx43 were similar, in that these connexins were expressed in the majority of motor neurons from E15 through adulthood (Fig. 8). Expression was also observed in the dorsal root ganglia and in scattered cells in the dorsal horn. Surprisingly, expression was maintained in a substantial population of adult motor neurons (89-93%) (Table 2), despite the lack of functional gap junctional coupling after P3-P4. To determine whether the proportion of motor neurons expressing Cx36, Cx37, and Cx43 transcripts varied among the medial, dorsolateral, or ventrolateral motor columns, the number of positive and negative motor neurons was counted in each of these regions in P1-adult lumbar spinal cord cross sections. No differences were observed in medial compared with dorsolateral or ventrolateral motor columns (data not shown). These data show that the motor neuronal expression of Cx36, Cx37, and Cx43 is relatively homogeneous, spatially as well as temporally, from embryonic life through adulthood.
In contrast, a decrease in the proportion of motor neurons positive for Cx45 and Cx40 transcripts was observed from embryonic to adult life. At E12, Cx45 and Cx40 were expressed relatively uniformly throughout the neural tube (data not shown), and from E15 to E18, these connexins were expressed in most motor neurons (Fig. 9; Table 2). After birth, however, the proportion of motor neurons expressing Cx45 mRNA gradually decreased, from 83% at birth to 48% in adult rat spinal cord (Table 2). Cx40 expression decreased more dramatically. Although at birth 91% of motor neurons were positive for Cx40 transcripts, this decreased to 33% at P7, and only 7% of motor neurons were observed to be positive in adults (Fig. 9; Table 2). As observed for Cx36, Cx37, and Cx43, no differences were observed in the expression of Cx45 or Cx40 transcripts in medial, dorsolateral, or ventrolateral motor columns after birth. Cx45 and Cx40 mRNA was also detected in dorsal root ganglia and in scattered cells in the dorsal horn.
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Figure 9. Motor neuron Cx45 and Cx40 expression decrease after birth. Shown are low- and high-power photographs of E15-adult lumbar spinal cord after in situ hybridization for Cx45-specific (left, left middle) and Cx40-specific (right middle, right) transcripts. High-power images are representative fields in ventrolateral spinal cord where motor neurons are located. Black arrows indicate the motor neurons that appeared negative for a particular connexin mRNA. At E15, Cx45 and Cx40 were expressed relatively uniformly throughout the spinal cord, and from E15 to E18, in most if not all motor neurons. Cx45 mRNA was also detected in dorsal root ganglia (left, E15 panel) and in scattered cells in the dorsal horn. After birth, however, the proportion of motor neurons expressing Cx45 mRNA decreased, with only ~45% of motor neurons remaining positive in P14 and adult rat spinal cord. Cx40 was expressed throughout the spinal cord at E15-P1, including in ventral motor neurons and dorsal root ganglia (right middle, portions shown in E15 panel). Expression decreased after this time, with only ~10% of motor neurons remaining positive at P14 or in adults. No differences were observed in the proportion of motor neurons positive for Cx45 or Cx40 transcripts in medial, dorsolateral, or ventrolateral motor columns at any of the ages examined. Scale bars: 500 µm (low power Cx45 and Cx40); 50 µm (high power Cx45 and Cx40).
The expression patterns of Cx26 and Cx32, identified in RT-PCR analyses from P1 spinal cord, were also examined. These connexins are widely expressed in developing and adult rodent CNS by meningeal and ependymal cells and glia (for review, see Dermietzel and Spray, 1993
Taken together, these results suggest that developing motor neurons express a repertoire of five connexins and that the spatial patterns of expression appear relatively homogeneous across medial, dorsolateral, and ventrolateral motor columns. Two temporal patterns of connexin expression were observed. The proportion of motor neurons expressing of Cx45 and Cx40 mRNA decreased after birth, with Cx40 widely expressed throughout the neural tube at E12 but expressed by <10% of adult motor neurons. The expression of Cx36, Cx37, and Cx43 was widespread throughout development, and expression of each of these connexin transcripts was detected in the majority of adult motor neurons. The implications of the apparent mismatch between electrical and dye coupling and gap junction protein expression are discussed below.
Expression of connexin proteins in developing and adult motor neurons
To compare the spatial and temporal patterns of connexin mRNA expression with that of connexin proteins, spinal cord cross sections were immunostained with anti-connexin antibodies. Motor neurons were identified by Islet-1 immunoreactivity (Fig. 10) (Tsuchida et al., 1994
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Figure 10. Connexin protein expression in developing and adult motor neurons. Confocal microscopic examination of immunostained sections revealed punctate membrane and diffuse cytoplasmic staining for Cx40, Cx43, and Cx45. Shown in each panel is a single plane projection of a confocal stack of images. Top row, At E15, punctate Cx40 immunoreactivity was observed surrounding most Islet-1-positive cells (left panel) in the ventral spinal cord. The inset in second panel from left shows the overlay of Islet-1 and Cx40 immunostaining of this field. More diffuse staining within the cytoplasm was also observed. Scale bars, 100 µm. At P1, few motor neurons with punctate membrane staining and diffuse cytoplasmic staining are observed. In adult spinal cord, few motor neurons are immunopositive for Cx40. Shown is one example of an adult motor neuron with characteristic punctate membrane as well as diffuse cytoplasmic staining; this was one of two positive motor neurons in the section. Middle row, At E15, punctate membrane Cx43 immunoreactivity and more diffuse cytoplasmic staining were observed surrounding most motor neurons. Cx43 immunostaining was sensitive to fixation, and this precluded double-labeling with Islet-1. Scale is same as above. At P1, most motor neurons have both punctate membrane staining and diffuse cytoplasmic staining. In adult spinal cord, dense punctate staining was observed in the neuropil surrounding motor neurons; this reflects Cx43 expression in glia (cf. Theriault et al., 1997
Cx40, Cx43, and Cx45 protein appeared to be expressed in similar temporal and spatial patterns as their mRNA transcripts. From E12-E18, Cx40, Cx43, and Cx45 immunoreactivity was localized to most if not all Islet-1-positive cells in the ventral neural tube or spinal cord (Fig. 10). Cx40, Cx43, and Cx45 immunoreactivity was also observed in the dorsal horn and in the dorsal root ganglia (data not shown). Confocal microscopic examination of immunostained sections revealed punctate staining that was associated with the motor neuron cytoplasm and membrane at these ages. The temporal and spatial expression pattern of each connexin protein was similar in the medial, dorsolateral, and ventrolateral motor columns. From P1 through adulthood, cytoplasmic, perinuclear, and membrane-associated Cx43 immunoreactivity was apparent in >90% of motor neurons. The proportion of Cx45-positive motor neurons decreased from ~90% at birth to ~50% in adult spinal cord, whereas Cx40 immunoreactivity was detected in <10% of motor neurons in adult spinal cord. Thus, for these three connexins, the spatial and temporal patterns of protein expression were similar to those observed for mRNA.
DISCUSSION
We used intracellular recordings to show that motor neurons are extensively electrically coupled at birth and that electrical coupling potentials were reversibly abolished by halothane, a widely used gap junction blocker. Iontophoretic injection of Neurobiotin, a low molecular weight compound that passes across gap junctions, into single motor neurons showed that at P0-P2, dye-labeled clusters contained on average six motor neurons, whereas by P7-P8, only single dye-labeled motor neurons were observed. Thus this work establishes the extent of dye and electrical coupling among motor neurons and a time course of their disappearance after birth. The spatial dimensions of clusters of dye-labeled motor neurons further suggests that, after birth at least, coupling is spatially restricted to small groups of motor neurons. We also report that motor neurons express five connexins: Cx36, Cx37, Cx40, Cx43, and Cx45. This repertoire is distinct from those previously described for cortical and other neurons (Dermietzel and Spray, 1993
Taken together, our results suggest two general mechanisms for the disappearance of electrical and dye coupling. The first is that the downregulation of Cx40 and/or Cx45 may lead to a decrease in functional gap junctions in motor neurons. This seems unlikely, given that this downregulation occurs more slowly than the disappearance of electrical and dye coupling, and that the connexins that continue to be expressed have been shown to form functional gap junctions in various cell types (Haubrich et al., 1996
Neonatal motor neurons are electrically and dye coupled
The results we present here are consistent with and extend previous work from neonatal rat spinal cord (Fulton et al., 1980
We directly compared the extent of electrical coupling with the extent of dye coupling by injecting single motor neurons with and without coupling potentials with Neurobiotin (Kita and Armstrong, 1991
The compact distribution of dye-labeled motor neurons, together with previous electrophysiological characterization (Walton and Navarette, 1991
Repertoire of connexins expressed by motor neurons
The five connexins expressed by motor neurons, Cx36, Cx37, Cx40, Cx43, and Cx45, are distinct from those previously reported for other neurons (Nadarajah et al., 1996
The relatively homogenous patterns of expression for Cx36, Cx37, and Cx43, together with the relatively homogeneous downregulation of Cx45 and Cx40 expression across motor columns, was particularly striking. We looked for, but did not observe, differences in the spatial and temporal aspects of mRNA and protein expression across motor columns and in rostral compared with caudal lumbar spinal segments. Thus the spatial restriction of coupling to small subsets of motor neurons within a pool that was observed after birth does not occur because of distinct spatial patterns of connexin expression among different motor neuron groups.
Given the fact that, around the time of birth, motor neuron cell bodies are displaced from one another as dendritic arbors are elaborated, it seems likely that the location of gap junctions is dendro-dendritic. Two recent ultrastructural studies report extensive gap junctions in spinal cord (Rash et al., 1996
Roles of gap junctional coupling in motor neuron development
Transient gap junctional coupling among developing motor neurons might allow electrical and/or biochemical communication that could serve several roles. Because neural activity plays a critical role in the refinement of synaptic connections throughout the developing nervous system (for review, see Goodman and Shatz, 1993
Gap junctional coupling might also shape the synaptic connections of motor neurons with common muscle targets. Gap junctional coupling is present during the time that motor neurons extend axons and make synapses with skeletal muscle fibers, and temporally correlated motor neuron activity during early phases of synapse formation may allow synapses from several motor neurons to be established and transiently maintained with individual muscle fibers. As electrical coupling disappears, motor neuron activity may become progressively more asynchronous over time. This may be one of several mechanisms that underlie the synaptic competition, which results in the loss of multiple innervation and the establishment of the mature pattern of single innervation of muscle fibers (for review, see Thompson, 1985
Although competition is modulated by the relative activity patterns of convergent inputs, little is known about how motor neuron activity patterns are shaped during development. The role of gap junctions can be evaluated by determining the relative firing of motor neurons to individual muscles in neonatal animals, and whether this firing is altered in animals in which gap junctional communication has been altered either pharmacologically or genetically. Preliminary results from electromyographic recordings of single motor unit activity suggest that motor unit firing is relatively temporally correlated around the time of birth, but temporal correlations are only rarely observed by the end of the second postnatal week (K. Personius and R. Balice-Gordon, unpublished results). It will be of interest to compare motor unit activity, the extent of electrical and dye coupling among motor neurons, and synaptic connectivity in mutant mice lacking one or more connexins.
Although gap junctions mediate electrical communication among some neurons, they also mediate biochemical communication, allowing the exchange of second messengers as well as other factors that may directly or indirectly modulate neuronal activity, as has been recently demonstrated in neocortex (Kandler and Katz, 1998
FOOTNOTES Received Aug. 16, 1999; revised Sept. 23, 1999; accepted Sept. 28, 1999.
This work was supported by grants from National Institutes of Health (NS34373), the Spinal Cord Research Foundation (1472), and the McKnight Foundation to R.B.-G. We thank Drs. R. Kalb and H. Fryer for help with motor neuron purification; Drs. L. Bone, E. Hertzberg, M. Koval, C. Lo, B. Nicholson, D. Paul, and S. Scherer for generously providing connexin-specific PCR primers, cDNAs, antibodies, and/or mutant mice; and Drs. D. Kopp, A. Pereda, and K. Personius for helpful discussions and comments on earlier versions of this manuscript.
Correspondence should be addressed to Rita Balice-Gordon, Department of Neuroscience, University of Pennsylvania School of Medicine, 215 Stemmler Hall, Philadelphia, PA 19104-6074. E-mail: rbaliceg{at}mail.med.upenn.edu.
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