INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Nicotinic acetylcholine receptors (AChRs) are a diverse family of pentameric proteins assembled from more than a dozen different subunits. Individual neurons may express several types, and the extent of diversity of AChR types among neurons is substantial. This diversity has the potential of allowing AChRs to serve several functions. Currently, nicotinic receptors in the CNS are thought to be important for memory, cognition, regulation of excitability, plasticity, and development (reviewed in Broide and Leslie 1999; Clementi et al. 2000; Cordero-Erausquin et al. 2000; Dani 2001; Jones et al. 1999; Levin and Simon 1998; Paterson and Nordberg 2000; Vizi and Lendvai 1999).

Neuronal AChRs were identified very early as the ionotropic receptors underlying rapid excitatory transmission at ganglionic synapses in the peripheral nervous system. While neuronal AChRs are expressed widely in the brain and spinal cord, their chief functional role in the CNS is not as the principal excitatory transmitter. The discovery that central nicotinic AChRs are often located presynaptically has led to the suggestion that they modulate synaptic transmission (reviewed in Wonnacott 1997), and recently much interest has been focused on "nonclassical" roles for central nicotinic receptors. We have chosen to return to the first-recognized function for central nicotinic receptors as mediators of rapid excitatory synaptic transmission.

The first demonstration that central nicotinic receptors can mediate fast excitatory synaptic transmission was made on Renshaw cells of the cat spinal cord (Curtis and Ryall 1966a,b; Eccles et al. 1954; Ryall 1981). Renshaw cells are inhibitory interneurons that fire bursts of action potentials in response to motor neuron stimulation. Activation of Renshaw cells depresses -motor neuron firing as a result of chloride-dependent inhibitory currents induced by both GABA and glycine (Schneider and Fyffe 1992) possibly released by the same cell (Jonas et al. 1998). Renshaw cells remain one of the few examples in the CNS where nicotinic AChRs are known to underlie rapid synaptic transmission (see also Alkondon et al. 1998; Frazier et al. 1998; Hefft et al. 1999; Nong et al. 1999; Roerig et al. 1997; Zhang et al. 1993). Most of the functional studies of nicotinic receptors on Renshaw cells were done 30-40 yr ago in the intact cat spinal cord using a combination of extracellular and intracellular (sharp electrode) recording techniques. We wished to extend these studies to rat spinal cord, where transmission is also nicotinic (Headley et al. 1975), by using whole cell recording in slices and by examining the sensitivity of -motor neuron-Renshaw cell transmission with AChR subunit-specific antagonists.

One useful approach to studying native AChRs is to compare their functional properties with AChRs of known subunit composition expressed heterologously. Expressed AChRs fall into two broad classes: hetero-oligomeric receptors containing both alpha (2-6) and beta (2-4) subunits, and homomeric receptors (7-10) (see Yu and Role 1998), of which the 7 AChR is the only known member with widespread distribution in brain. 7 AChRs are functionally different from alpha-beta receptors; for example, they have a greater calcium permeability and can be gated either by ACh or by choline. cDNA hybridization studies (Wada et al. 1989) in rat CNS suggest that both 7- and non-7-containing receptors are expressed in the spinal cord. The immediate purpose of this study was to determine whether nicotinic receptors underlying excitatory transmission onto Renshaw cells are 7 or non-7. Our results show that Renshaw cells display 4-like immunoreactivity (4-LI) but not 7-LI and that excitatory postsynaptic currents (EPSCs) evoked in Renshaw cells by antidromic stimulation of -motor neurons are sensitive to low concentrations of dHE but not MLA. These findings suggest that excitatory synaptic transmission between -motor neurons and Renshaw cells is mediated by a non-7 AChR, possibly 42.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Immunolabeling

Spinal tissue was prepared for immunocytochemistry using standard procedures (Alvarez et al. 1999). Briefly, 2-wk-old Sprague-Dawley rats were deeply anesthetized with sodium pentobarbital (50 mg/kg ip) and perfused transcardially with warm (37°C) PBS followed by ice-cold PBS containing 1-2% formaldehyde. The spinal cord was dissected out and placed in freshly prepared 1% formaldehyde in PBS for 1 h at room temperature. The tissue was then washed in PBS, placed in 30% sucrose in PBS at 4°C overnight, and mounted in TissueTek (Triangle Biomedical Sciences, Durham, NC). Transverse sections (30 µm) were cut on a cryostat and mounted on gelatin-coated slides.

The following monoclonal antibodies (mAbs) were tested: 299 (anti-rat 4), 270 (anti-rat 2), 306 and 307 (anti-mouse-7), 318 and 319 (anti-rat 7), and 35 and 210 (assumed to be anti-1, 3, and 5). All of these antibodies stained sets of neurons in either rat brain or chicken ciliary ganglia. The following goat polyclonal anti-AChR antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): anti-3 (A-20), anti-4 (A-20), anti-7 (SC-20), and anti-2 (C-20). Only one of these four stained sets of neurons in slices of rat spinal cord or brain (anti-4), and negative results from the remaining three are not considered to be meaningful. Anti-gephyrin antibodies were generously provided or were purchased from Alexis Biochemicals (San Diego, CA). Alexa 488-labeled -bungarotoxin (-BuTx) was purchased from Molecular Probes (Eugene, OR).

Tissue sections were preincubated in a blocking solution of 5% normal donkey serum in PBS for 1 h at room temperature. Primary antibodies were used at concentrations of 5-50 nM. When primary antibodies against rat subunits were tested, we preincubated the sections with goat anti-rat antibody IgG to block nonspecific labeling by anti-rat labeled secondary antibodies. When permeabilization was required, the blocking solution contained 0.1% (vol/vol) Triton X-100. Sections were washed with PBS following block and incubated with primary antibody in PBS overnight at 4°C followed by Cy3- (Amersham Life Sciences, Pittsburgh, PA) or Alexa 488-labeled secondary antibodies (Molecular Probes, Eugene, OR) for 1-2 h at room temperature. All labeled anti-rat and anti-mouse antibodies were obtained from Jackson Immunoresearch (West Grove, PA) and were preadsorbed by the manufacturer to remove conspecific cross-reactivity. Texas Red-avidin was purchased from Molecular Probes (Eugene, OR). Fluorescent labeling was visualized using a confocal imaging system (BioRad 1024), as described previously (Wilson Horch and Sargent 1995).

Spinal cord slice preparation

Six- to 10-day-old Sprague-Dawley rats were anesthetized with 50 mg/kg sodium pentobarbital (ip) and decapitated. A section of the lumbar spinal cord was dissected in ice cold low-sodium Ringer containing (in mM) 240 sucrose, 2.5 KCl, 1 CaCl₂, 3 MgCl₂, 10 glucose, 25 NaHCO₃, 1.25 NaH₂PO₄ at pH 7.4 when bubbled with carbogen gas (95% O₂/5% CO₂). The dissected lumbar cord was immobilized in low-melting point agarose and immersed in ice-cold Ringer solution for sectioning. Slices of 300-350 µm thickness were obtained by using a Lancer Vibratome Series 1000 and were incubated in extracellular recording solution at room temperature while being continuously bubbled with carbogen gas. Extracellular recording saline contained (in mM) 125 NaCl, 2.5 KCl, 3 MgCl₂, 2 CaCl₂, 25 glucose, 25 NaHCO₃, 1.25 NaH₂PO₄ at pH 7.4 when bubbled with carbogen gas. Cells in the slices maintained in this condition remained viable for approximately 5-6 h.

Electrophysiological recording

Lumbar L3-L5 slices were immobilized in a recording chamber by using a fine nylon mesh and were continuously perfused with extracellular recording solution at 2 ml/min. Neurons in the slice were visualized using infrared differential interference contrast optics produced by an Olympus BX50WI microscope with a 40× water-immersion lens. Patch pipettes for whole cell recording were pulled from 1.2 mm diameter borosilicate glass using a P-87 Flaming-Brown micropipette puller (Sutter Instrument Co., Novato, CA). Initial pipette resistances were 3-5 M.

Whole cell patch clamp recordings were made at room temperature (21-23°C). A drug cocktail consisting of 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 10 µM D,L-2-amino-5-phosphonovaleric acid (APV), 5 µM bicuculline and 1 µM atropine was used to isolate nicotinic currents. All drugs were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise noted. Chloride currents were minimized by using a low-chloride intracellular saline, consisting of (in mM) 125 Cs-gluconate, 15 CsCl, 10 HEPES, 2 EGTA, 2 ATP, 0.2 GTP, and 0.1 glucose, pH 7.3. QX-314 at 0.5 mM was added to the pipette solution to decrease voltage-activated sodium channel activity. Cells were selected for recording from lumbar interneurons in the ventral part of Rexed's lamina VII, where Renshaw cell density is highest (Fyffe 1990; Geiman et al. 2000; Thomas and Wilson 1965). Excitatory postsynaptic currents (EPSCs) were evoked in interneurons by means of a 0.05 ms stimulus which was delivered through a bipolar electrode (FHC Inc., Bowdoinham, ME) that was placed over the clearly visible motor nerve tracts at the ventral margin of the cord. While the intent of this procedure was to stimulate selectively -motor neuron axons, we clearly stimulated other white matter axons as well, since the majority of neurons responded to stimulation with glutamatergic EPSCs, not nicotinic ones (see RESULTS).

Currents were filtered at 2 kHz (Axopatch 200B) and digitized at 10 kHz. Data acquisition and analysis was performed using pClamp 7 (Axon Instruments, Union City, CA) and Origin (OriginLab, Northampton, MA) software.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Renshaw cells display 4-like immunoreactivity

To distinguish Renshaw cells from other neurons, we used an antibody to gephyrin, a glycine receptor clustering protein (Kneussel and Betz 2000). Renshaw cells in rats display stronger gephyrin immunoreactivity than other interneurons with a distinctive staining pattern (Alvarez et al. 1997). Figure 1A1 shows a low power view of the ventral horn of the spinal cord visualized for gephyrin. Gephyrin immunoreactivity is present widely but is especially pronounced on two neurons, identified as Renshaw cells, whose cell bodies and proximal dendrites are outlined by the label. Both of these neurons are stained by an antibody to neuronal AChRs (Fig. 1, A2 and A3).

View larger version (130K): [in this window] [in a new window]   Fig. 1. Renshaw cells display AChR immunoreactivity corresponding to a subset of AChR reactive antibodies. Images are merged stacks of confocal sections. Left: (red) gephyrin immunoreactivity, whose prominent staining is characteristic for Renshaw cells (Alvarez et al. 1997), middle: immunoreactivity for mAb 299 (4, A2, B2) or mAb 319 (7, C2), and right: merges. Top: (A) images illustrate a low power view of a field of interneurons in the ventral horn near the anterior border of the white matter (below). Two of the neurons (arrows) have strong gephyrin immunoreactivity on the soma and proximal dendrites, while other neurons in the field do not. The two neurons strongly reactive for gephyrin are judged to be Renshaw cells. Higher power views of individual Renshaw cells (B and C) show them to be immunopositive for mAb 299 but not mAb 319. Images in A2 and B2 were collected on the confocal microscope using identical gain and black level settings. Fluorescent structure at the base of B2 and B3 represents edge of a presumptive motor neuron, which is immunoreactive for 4. Scale bars represent 50 µm (A) and 17 µm (B and C). No modification was made in look-up tables during processing and printing.

Renshaw cells were immunoreactive for the 4 AChR subunit but not for the 7 AChR subunit. In Fig. 1B, a Renshaw cell, having characteristic gephyrin immunoreactivity (Fig. 1B1), also stains with anti-AChR mAb 299 (Fig. 1, B2 and B3), which is selective for rat 4 subunits (Whiting and Lindstrom 1988). The immunoreactivity is distributed widely within the cell body with no obvious clustering on the surface and with considerably less staining in the dendrites than in the cell body. In Fig. 1C a Renshaw cell from a different section is shown not to react with anti-AChR mAb 319 (Fig. 1C2), which is selective for the rat 7 subunit (Schoepfer et al. 1990). In parallel experiments, mAb 319 did label subsets of neurons within the brain (not shown; see Dominguez del Toro et al. 1994). Anti-7 mAbs 307 and 318 (Schoepfer et al. 1990) also failed to label Renshaw cells or any other cells within spinal cord slices. However, anti-7 mAb 306 (Schoepfer et al. 1990) labeled all cell bodies within the gray matter and, as well, cell bodies and fibers in the white matter (not shown). The finding that only one of four anti-7 mAbs was reactive in the spinal cord raises the possibility that the reactivity of mAb 306 does not result from its binding to an 7-like component. We also examined staining of spinal cord sections with -BuTx, which is specific among known CNS AChR subunits for 7. We found no detectable staining with Alexa 488--BuTx to either fixed or unfixed spinal cord slices (not shown). In parallel experiments, Alexa 488--BuTx labeled endplates in frozen sections of chick or rat muscle and also selected populations of neurons in fixed slices from rat brain (not shown). We were therefore unable to confirm the suggestion, based on the pattern of mAb 306 staining, that 7-LI is present widely in neonatal rat spinal cord. Finally, we found that neither mAb 35 (Tzartos et al. 1982) nor mAb 210 (Whiting and Lindstrom 1986), which recognize both 3 and 5 subunits in chicken (Conroy et al. 1992) and human (Wang et al. 1996), labeled Renshaw cells in the spinal cord, but they did label chicken ciliary ganglion neurons in sections run in parallel (not shown, Wilson Horch and Sargent 1995).

The presence of 4-like immunoreactivity (4-LI) within Renshaw cells was confirmed by showing that these cells were stained by two anti-4 antibodies with different specifics: mAb 299 (Whiting and Lindstrom 1988), which is directed against an extracellular epitope, and polyclonal antibody A-20, which is directed against the carboxyl terminus and likely to recognize an intracellular epitope (Corringer et al. 2000). The two antibodies produced a similar staining pattern, which is illustrated in Fig. 2A (mAb 299) and 2B (antibody A-20). Each pair of images shows a lower (left) and higher power view of merged stacks of optical sections, with 4-LI rendered green and gephyrin immunoreactivity rendered red. In low power images it is clear that 4-LI is present not only in Renshaw cells (arrows) but also in other small neuronal cell bodies, which are presumably other interneurons. -Motor neurons, which are considerably larger than Renshaw cells, also displayed 4-LI (Fig. 2A1). Because 4 and 2 subunits combine to form an AChR that accounts for much of the high-affinity nicotine binding in brain (Flores et al. 1992; Shafaee et al. 1999; Whiting and Lindstrom 1986), we were interested in learning whether Renshaw cells would display immunoreactivity for 2. Figure 2C shows that anti-2 mAb 270 indeed recognizes Renshaw cells (arrows, left panel) and that, like anti-4 antibodies, it binds to non-Renshaw interneurons (note gephyrin-negative cell to left of two gephyrin-positive cells) and presumptive motor neurons. A commercially available antibody to the 2 subunit did not label Renshaw cells, but this antibody also did not label neurons within brain sections.

View larger version (76K): [in this window] [in a new window]   Fig. 2. Renshaw cells display AChR 4-like and 2-like immunoreactivity. All panels represent merged confocal images with anti-AChR immunoreactivity rendered green and anti-gephyrin immunoreactivity rendered red. Left and right panels represent lower and higher power views. A and B: immunoreactivity for anti-4 mAb 299 and anti-4 polyclonal antibody A20, respectively. Both antibodies stain Renshaw cells (arrows in A1, B1) and also larger neurons, which are presumably motor neurons and which are not strongly reactive for anti-gephyrin. Renshaw cells are also immunoreactive for anti-2 mAb 270 (C) as are the larger neurons. Scale bar represents 50 µm in left panels and 17 µm in right panels. Image contrast was enhanced in A2 and C2 prior to printing using Adobe Photoshop.

Renshaw cells display nicotinic EPSCs

We recorded from interneurons located along the ventral margin of the ventral horn in search of Renshaw cells, which should display nicotinic currents in response to ventral root stimulation. The majority of neurons from which we recorded, responded to stimulation with short latency EPSCs, but most of these neurons (>90%) displayed EPSCs that were glutamatergic in character (Fig. 3). These currents appeared reliably and with short latency, had fast rise and decay times, and were reversibly blocked by 10 µM CNQX (Fig. 3). CNQX (10 µM) did not partially block EPSCs in any of the cells we examined: it either blocked currents nearly completely or not at all.

View larger version (9K): [in this window] [in a new window]   Fig. 3. AMPA receptor antagonist CNQX reversibly blocks inward synaptic currents elicited from some ventral horn interneurons by stimulation of the ventral surface of the spinal cord. Six superimposed EPSCs are shown in extracellular PBS (left), in 10 µM CNQX, and after washout. Stimulus intensity and duration were kept constant.

A small fraction of the cells from which we recorded EPSCs (8 of 150, 6%) displayed currents that were insensitive to CNQX but were sensitive instead to the nicotinic antagonist d-tubocurarine (10 µM d-TC, Fig. 4A). These currents had a latency of 2.2 ± 0.6 ms and a 10-90% rise time of 1.0 ± 0.2 ms (n = 8 cells). The averaged current obtained from control traces in Fig. 4A decayed with a single exponential function having a time constant of 4.9 ms (Fig. 4B). Overall, nicotinic currents decayed with a time constant of 5.0 ± 1.6 ms (n = 8 cells).

View larger version (17K): [in this window] [in a new window]   Fig. 4. The nicotinic antagonist d-TC reversibly blocks inward synaptic currents elicited from some ventral horn interneurons. A: each record has 7-9 superimposed EPSCs, which were elicited at 0.2 Hz and at constant stimulus intensity. All records were obtained in the presence of CNQX, APV, bicuculline, and atropine (see METHODS), which did not affect EPSC amplitude for this neuron (not shown). At the perfusion rate used, full effect of d-TC was observed within 2 min and washout was complete within 5 min. B: average of 8 traces from the cell same cell as depicted in A. The average EPSC decayed (90-10%) with a single time constant of 4.9 ms (F test used to distinguish between biexponential and monoexponential fit).

Nicotinic EPSCs elicited from Renshaw cells were typically small, on the order of 100 pA in amplitude, and displayed noticeable amplitude fluctuations when elicited at low frequency (0.2 Hz) and fixed stimulus strength. This fluctuation is illustrated in Fig. 5A (left) for a set of EPSCs to a stimulus just sufficient to elicit a response. At a stimulus intensity of 31 or 40 V, about 20% of the responses were "failures" and the average response, including failures, was about 25 pA. The failures might represent instances where no quanta were released from an afferent having a single release site; alternatively, they might represent instances where an action potential failed to reach the terminal. At stimulus intensities of 50 V and 70 V, the average response was larger and amplitude fluctuation was pronounced (Fig. 5A). Overall, the relationship between stimulus intensity and peak response amplitude (Fig. 5B) suggests that Renshaw cells are multiply innervated, which is consistent with the findings of Ryall (1981) for innervation of Renshaw cells in cat. At constant stimulus strength, we noted fluctuations in response latency as well as in amplitude (Fig. 5A, inset). This fluctuation cannot be explained by trial-to-trial variations in the number of released quanta; it may instead result from trial-to-trial variations in the number of afferents that fire. Within a set of responses at constant stimulus intensity, larger responses had a briefer latency (P < 0.05 by Pearson product moment correlation, n = 4), which suggests that individual afferents elicit responses with different latencies and that the latency for the response to activation of a population of these afferents will be briefer, on average, if more afferents are activated. If this interpretation is correct, then the average response latency should be briefer when we recruit more afferents by raising the stimulus strength, and this was observed (Fig. 5C). These results suggest that at least one source of fluctuation in response amplitude and latency is the trial-to-trial variation in the number of -motor neuron collaterals that "fire" in response to stimulation.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Renshaw cells receive convergent innervation. A: 5-6 superimposed EPSCs elicited by stimulation at three different intensities, as shown; B: at higher stimulation, intensities average EPSC size is increased; and C: latency is decreased. Error bars represent ±SE. Latencies were measured from the start of the stimulus artifact to the start of the response.

We examined the sensitivity of -motor neuron-Renshaw cell synaptic currents to nicotinic antagonists to make inferences about the subunit composition of nicotinic receptors that underlie these currents. d-TC, while useful as a diagnostic tool for detecting nicotinic currents, is not sufficiently selective to discriminate among different AChRs. However, we found clear results from two more selective antagonists, dHE and MLA. dHE at 10 µM reversibly blocked most of the -motor neuron-Renshaw cell EPSC, as is illustrated for one cell in Fig. 6A. Overall, 10 µM dHE reduced peak EPSCs by 87 ± 11% (n = 5 cells, P < 0.0001). We were unable to block significantly more of the EPSC with a 10-fold higher concentration of dHE or with 10 µM d-TC (P > 0.5 by ANOVA). dHE is relatively selective among expressed AChRs for those containing 4 subunits (Gopalakrishnan et al. 1996), which suggests that 4-containing receptors mediate Renshaw cell responses to -motor neuron stimulation. MLA at a concentration of 50 nM, which selectively blocks 7 containing receptors (Palma et al. 1996), had no effect on -motor neuron-Renshaw EPSCs (Fig. 6B, P > 0.5 by students' t-test; similar results were obtained in two other experiments).

View larger version (16K): [in this window] [in a new window]   Fig. 6. Nicotinic EPSCs evoked from Renshaw cells are reversibly blocked by 10 µM dHE but not by MLA. Each record shows 8-10 superimposed EPSCs, which were elicited at 0.2 Hz and at constant stimulus intensity through out each experiment. A: all records were obtained in the presence CNQX, APV, bicuculline, and atropine (see METHODS), which did not affect EPSC amplitude for this neuron (not shown). dHE blocked EPSCs within 2 min, and the effect was reversed within 2 min of washout. B: EPSCs recorded 9 min after the addition of 50 nM MLA (right) were not reduced when compared with control (left). This EPSC was reversibly and nearly completely blocked by 10 µM dHE (not shown), which took <2 min to take full effect.

We studied the morphology of cells having nicotinic EPSCs by filling recording pipettes with neurobiotin, and visualizing neurobiotin-filled cells with Texas Red-avidin after fixation. Cells displaying dHE-sensitive currents were situated in layer VII of the lumbar spinal cord and had fusiform- or stellate-shaped cell bodies 15-25 µm in diameter with large dendrites that ran along the boundary between the gray and white matter orthogonal to motor axons as they leave the ventral horn (Fig. 7). These features are characteristic of Renshaw cells (Fyffe 1990; Lagerback and Kellerth 1985). We attempted to visualize gephyrin immunoreactivity in these cells, to confirm that they display the staining pattern characteristic of Renshaw cells (Alvarez et al. 1997). Unfortunately, gephyrin staining was not successful on cells from which whole cell recordings were made, even though neighboring cells in the slice were often gephyrin-immunopositive (not shown; see also Oleskevich et al. 1999).

View larger version (84K): [in this window] [in a new window]   Fig. 7. Neurons with dHE-sensitive currents have the morphology expected of Renshaw cells. Image is of a neurobiotin-filled neuron situated close to the boundary of the gray matter in lamina VII of the ventral horn. See text for additional details. Scale bar =17 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

With the aid of infra-red differential interference contrast optics in spinal cord slices, we have confirmed earlier findings, based largely on extracellular recordings, that Renshaw cell activation by -motor neurons is nicotinic. We found also that the synaptic currents elicited from Renshaw cells by stimulation of -motor neurons are reversibly blocked by dHE, but not MLA, and that Renshaw cells display AChR 4 and 2 immunoreactivity. These results are consistent with the hypothesis that an 42-containing AChR underlies transmission between -motor neuron collaterals and Renshaw cells.

Double labeling with anti-gephyrin antibodies, which produce a highly characteristic pattern and intensity of staining on Renshaw cells, shows that 4-LI is present there but also on other interneurons in the ventral horn and on -motor neurons as well. Since we used two different antibodies with different specificity, one against the C-terminus and one an extracellular epitope, we suspect strongly that the staining is evidence for the presence of 4-containing nAChRs. Staining with the anti-2 mAb 270 suggests that 2-LI is present on the same wide spectrum of neurons that express 4-LI. Because 2 subunits form functional AChRs with 4 subunits (Flores et al. 1992; Whiting and Lindstrom 1986), we accept mAb 270 staining as evidence for the presence of 2 subunits on these neurons, although the result could not be verified with a second antibody to 2. Relatively little staining was noted at the neuronal surface, where AChRs must be located if they are to underlie synaptic transmission from motor neuron collaterals. This lack of significant staining may be a consequence of low sensitivity of the fluorescent technique used to visualize AChRs and/or to steric hindrance resulting from the presence of AChR-associated proteins (see Sorenson et al. 1998). The presence of 4-LI and 2-LI on -motor neurons is consistent with recent results demonstrating that -motor neurons express functional AChRs and that they are contacted by cholinergic boutons (Ferreira et al. 2001; Messi et al. 1997; Zaninetti et al. 1999).

A majority of interneurons along the ventral margin of the ventral horn responded to stimulation at the ventral surface of the spinal cord with short latency EPSCs. Of these, only 6% had currents with nicotinic pharmacology. This frequency is similar to the proportion of ventral horn interneurons found by Oleskevich et al. (1999) that had nicotinic mEPSCs (2 of 26 neurons). Carr et al. (1998) have reported that in adult rat there are only about 20 Renshaw cells for every 100 µm of rat lumbar spinal cord. The density of Renshaw cells is greater in neonatal rat than in adult (F. J. Alvarez, personal communication), but Renshaw cells nonetheless account for only a modest fraction of the interneurons in the ventral horn. It is striking that virtually all ventral horn neurons that we encountered had inward currents either largely glutamatergic or nicotinic in character: this finding suggests that Renshaw cells receive few glutamatergic inputs that can be activated by stimulation at the ventral margin of the cord.

The pharmacological sensitivity of the nicotinic synaptic currents recorded from Renshaw cells allows us to postulate which AChR(s) underlie these currents. dHE at 10 µM blocked nearly 90% of the synaptic current, and the fraction of current blocked at 100 µM was not significantly different. Moreover, we were unable to block significantly more of the current than this with 10 µM d-TC. Thus 10 µM dHE blocks virtually all of the nicotinic current elicited from Renshaw cells, which suggests that the IC₅₀ for dHE is likely to be at or below 1 µM. dHE is a competitive antagonist of nicotinic receptors whose specificity has been tested on a number of expressed and native AChRs. Among expressed human AChRs, Chavez-Noriega et al. (1997) found that sensitivity to dHE was greatest for 44 (K_d  0.01 µM), and 42 receptors (0.1 µM; see also Sabey et al. 1999), less so for 22 and 32 receptors (K_d  1 µM; see also Harvey and Luetje 1996), and least for 24 and 34 receptors (K_d > 1 µM; see also Harvey and Luetje 1996). We cannot say whether the native AChR(s) that underlie synaptic responses of Renshaw cells correspond to expressed AChRs tested for sensitivity to dHE. However, for the following three reasons we favor the hypothesis that 42 receptors are involved: 4- and 2-LI are expressed by Renshaw cells (Fig. 2), 2 (Wada et al. 1989) but not 4 (Dineley-Miller and Patrick 1992) transcripts are present in rat spinal cord, and Alkondon and Albuquerque (1993) have described a nicotinic response with high dHE sensitivity that they attribute to 42 AChRs on the basis of a more complete pharmacological analysis than we have performed here. The affinity of expressed 7 AChRs for dHBE is too low to explain our results (Chavez Noriega et al. 1997; see also Bertrand et al. 1992), and these AChRs are therefore not likely to play a role in -motor neuron-Renshaw cell transmission. The kinetics of decay of synaptic currents elicited from Renshaw cells is also consistent with an 42 AChR-mediated mechanism. If the decay of these currents is attributable to deactivation, then we would expect the time constant for decay to be similar to that describing the distribution of channel bursts for 42 channels. Charnet et al. (1992) have recorded open times for rat 42 channels expressed in Xenopus laevis oocytes in the range of 6-8 ms (at 150 mV), which are similar to the average EPSC decay time constant we record from Renshaw cell of 5 ms (see also Papke et al. 1989). Figl and Cohen (2000) found that the identity of the beta subunit strongly influences the kinetics of expressed neuronal nicotinic receptors, with the 2 subunit conferring a rapid voltage jump relaxation rate of about 5 ms and the 4 subunit conferring a slower rate of about 50 ms.

dHE at 10-100 µM blocked about 90% of the peak synaptic currents elicited from Renshaw cells, and 50 nM MLA did not reduce the current detectably, which suggests that the current does not have an 7-mediated component. The dHE-resistant current is likely to be non-nicotinic, since the nonspecific AChR antagonist d-TC failed to block any more of the current than 10-100 µM dHE. The dHE-resistant current is unlikely to be glutamatergic, since recordings contained the glutamate receptor antagonists CNQX (10 µM) and APV (10 µM), and it is unlikely to be GABAergic or glycinergic, since we recorded currents under conditions where the driving force on chloride ions is minimized (see METHODS). One remaining possibility is that the currents are purinergic, since -motor neurons release ATP (Redman and Silensky 1994) and since P2X receptors are present in the ventral horn (Kanjhan et al. 1999).

The possibility that the -motor neuron-Renshaw cell connections are mediated via the action of ACh on 42 AChRs is intriguing, since these receptors represent the principal source of high affinity nicotine binding sites in the CNS (Marubio et al. 1999; Picciotto et al. 1995; Whiting and Lindstrom 1986; Zoli et al. 1998). In addition, 42 AChRs are regulated in response to chronic nicotine exposure (Buisson and Bertrand, 2001; Flores et al. 1992, 1997; Fenster et al. 1999; Peng et al. 1994). It would be interesting to know if the functional properties of -motor neuron-Renshaw cell synapses are altered in animals or humans exposed chronically to nicotine.