Conditional Rhythmicity of Ventral Spinal Interneurons Defined by Expression of the Hb9 Homeodomain Protein

The identity of GFP-expressing neurons

In the mammalian spinal cord, the homeobox gene Hb9 is expressed by embryonic motoneurons (Pfaff et al., 1996; Saha et al., 1997; Tanabe et al., 1998; Thaler et al., 1999). Its encoded homeodomain protein, Hb9, has an essential role in consolidating motoneuron fate (Arber et al., 1999; Thaler et al., 1999).

To investigate the postnatal expression of Hb9 in the mouse, GFP was expressed under the control of an ∼9 kb 5′ flank regulatory region (Wichterle et al., 2002). At postnatal stages, GFP was expressed by many motoneurons in the ventral spinal cord (Fig. 1). In P10 to adult mice, GFP⁺ somatic motoneurons were observed in lateral and medial motoneuron pools in the ventral horns throughout the spinal cord (Fig. 1A,B). To examine whether all motoneurons express GFP, motoneurons were labeled by intraperitoneal injection of Fluorogold, which is taken up by peripheral axons to retrogradely label motoneuronal somata (Leong and Ling, 1990). In one representative animal in which the T12-L2 segments were analyzed, ∼85% (547 of 641) of Fluorogold-positive motoneurons were GFP⁺. Fluorogold also labeled GFP⁺ sympathetic preganglionic neurons (SPNs) (Fig. 1A,C) (243 of 313; 78% between T12-L2). Thus, not all somatic motoneurons or SPNs express GFP at postnatal ages.

We observed that interneurons in Hb9: eGFP mice, defined by the lack of Fluorogold tracer, also expressed GFP. These GFP⁺ interneurons were located throughout the ventral horn, primarily in laminas VII, VIII, and ventral X (Fig. 1A,D). In the low thoracic to upper lumbar regions, an average of 41 ± 5 GFP⁺ interneurons per 30 μm section were observed in laminas VII and VIII and in ventral lamina X. Of note were small (8-10 μm diameter) interneurons (4 ± 2 of these interneurons per 30 μm section) that abutted the ventral commissure in medial lamina VIII or ventral lamina X and often formed discrete clusters comprising two or three interneurons (Fig. 1A,D). Because the majority of these cells were in medial lamina VIII, we will refer to them as such. The intensity of GFP fluorescence in these neurons appeared greater than that seen in the majority of other labeled interneurons.

Previous studies using the Hb9 promoter have reported ectopic GFP expression in embryonic interneurons (Wichterle et al., 2002). To investigate the correspondence of transgene GFP expression with endogenous Hb9 expression in interneurons, Hb9:eGFP mice were crossed with Hb9^nlsLacZ/+ mice (Arber et al., 1999). In the double-transgenic offspring, β-gal expression was detected in the nuclei of large GFP⁺ motoneurons in the ventral horn (Fig. 2A,D) and in the cluster of small GFP⁺ cells abutting the ventral commissure (Fig. 2B). Importantly, the GFP⁺ interneurons scattered throughout the remainder of the ventral horn lacked β-gal expression (Fig. 2C). Somatic motoneurons and the medially located cluster of GFP⁺ interneurons expressed Hb9 immunoreactivity (data not shown). In contrast, GFP⁺ interneurons scattered throughout lamina VII and VIII lacked endogenous Hb9 protein expression. Thus, medial lamina VIII eGFP⁺ interneurons alone correspond to endogenous Hb9⁺ interneurons.

The rostrocaudal distribution of β-gal⁺, GFP⁺ interneurons was investigated in the spinal cord of P10 to adult Hb9:eGFP × Hb9^nlsLacZ/+ double-transgenic mice. At thoracic levels, occasional Hb9⁺ neurons were observed in lamina X, below the central canal abutting the ventral commissure, whereas at upper lumbar levels, these neurons were found in more ventral locations, forming clusters along the commissure (mean, 2.2 per 30 μm section). Hb9⁺ interneurons were rarely observed below midlumbar levels. Although not uniformly distributed in the rostrocaudal axis, the mean distribution of these cells was similar at upper thoracic (2.4 cells per 30 μm section, 43 sections), lower thoracic (2.1 cells, 40 sections), and upper lumbar (2.2 cells, 25 sections) levels.

In motoneurons, the expression of Hb9 is associated with a cholinergic phenotype (Tanabe et al., 1998), prompting us to examine whether GFP⁺ interneurons are also cholinergic. In Hb9:eGFP × Hb9^nlsLacZ/+ double-transgenic mice, Hb9⁺ interneurons lacked ChAT expression (Fig. 2B). We next examined interneuronal expression of Islet class LIM homeodomain proteins, which are expressed in motoneurons (Ericson et al., 1992) and in some Hb9⁺ interneurons in Drosophila (Odden et al., 2002). None of the GFP⁺ interneurons in Hb9:eGFP mice expressed Islet proteins (data not shown). Thus, Hb9⁺ interneurons in mice lack both ChAT and Islet protein expression.

To investigate the transmitter phenotype of the Hb9⁺ neurons, we began by investigating the colocalization of GFP with various transmitter transporters in axonal terminals. Because the majority of GFP⁺ interneuronal terminals (>75%) (data not shown) were VGLUT2-immunoreactive (IR), we tested the hypothesis that Hb9⁺ neurons are VGLUT2⁺ by using combined fluorescent in situ hybridization and immunohistochemistry. VGLUT2 mRNA was expressed in medially located GFP⁺ neurons (Fig. 3Ai-Aiv), most notably the small medial lamina VIII Hb9⁺ interneurons (Fig. 3Av), as well as in some larger GFP⁺ neurons located more laterally (Fig. 3Avi). The similar location of VGLUT2⁺/GFP⁺ neurons (Fig. 3Aiii) and GFP⁺/lacZ⁺ neurons (Fig. 3Avii) provides evidence that GFP⁺ interneurons in this region, which includes endogenous Hb9⁺ interneurons, are glutamatergic.

To determine whether Hb9⁺ interneurons form direct contacts with motoneurons, we studied the distribution of GFP⁺/VGLUT2⁺ terminals in the L2-L4 segments (n = 7 mice). However, it should be noted that some GFP⁺/VGLUT2⁺ terminals originate from Hb9^- neurons (Fig. 3A). GFP⁺/VGLUT2⁺ terminals were the most numerous in lamina VIII and the ventral part of lamina X but also formed a plexus in the central part of laminas V-VI. Scattered GFP⁺/VGLUT2⁺ boutons were seen in the medial part of lamina VII. There were relatively few GFP⁺/VGLUT2⁺ terminals in lamina IX in any of these lumbar segments. In addition, a detailed study of both the medial and lateral motor columns revealed that GFP⁺/VGLUT2⁺ terminals were seldom found in contact with motoneuron somata or proximal dendrites (Fig. 3B). Thus, we did not obtain evidence in support of significant input of Hb9 interneurons to motoneurons. However, it remains possible that the terminals of Hb9 interneurons are restricted to the distal dendrites of motoneurons. We note that the study by Hinckley et al. (2005) reports detection of a neurobiotin-filled GFP⁺ process in proximity to a GFP⁺ (presumed motoneuron) dendrite in the region between the medial and lateral motor columns, although the status of endogenous Hb9 expression in this neuron was not determined. Evaluation of the extent of direct connectivity between Hb9 interneurons and motor neurons will therefore require more detailed study.

We next examined the transmitter phenotype of synaptic terminals that are likely to be presynaptic to Hb9⁺ interneurons. Glutamatergic input to GFP⁺ Hb9⁺ interneuron somata and dendrites was detected with antibodies against VGLUT1 (Fig. 4A) and VGLUT2 (Fig. 4B). Virtually all Hb9⁺ interneurons studied (12 of 13) had VGLUT1-IR boutons (3-5 per 60 μm section) contacting the somata or proximal dendrites. These VGLUT1-IR terminals on Hb9⁺ interneurons likely arise from primary afferents, whereas VGLUT2-IR terminals arise from interneurons (Oliveira et al., 2003; Hughes et al., 2004). VGLUT2-IR GFP⁺ terminals were seen on all GFP⁺ Hb9⁺ interneurons, raising the possibility of self-excitation or mutual re-excitation, a property important in rhythm-generating networks (Roberts and Tunstall, 1990; Rowat and Selverston, 1997). Evidence for inhibitory synaptic input was demonstrated by the presence of both GAD-67⁺ and GlyT2⁺ axons in contact with GFP⁺ Hb9⁺ interneurons (Fig. 4C,D). In addition, because serotonin is important in the generation of locomotor rhythm (MacLean et al., 1998; Jiang et al., 1999; Ribotta et al., 2000), we examined serotonergic input to the GFP⁺ Hb9⁺ interneurons and found 5-HT-IR fibers in apposition to these cells (Fig. 4E). Together, these data suggest that the Hb9⁺ interneurons receive diverse excitatory, inhibitory, and neuromodulatory input.

Electrophysiological properties of medial lamina VIII GFP⁺ interneurons

The intrinsic properties of neurons involved in the production of rhythmic movement play a critical role in establishing network activity (Getting, 1989). We investigated the intrinsic membrane properties of medial lamina VIII Hb9⁺ and other nearby interneurons in vitro using whole-cell patch-clamp recording techniques in acutely prepared spinal cord slices (Carlin et al., 2000) (Figs. 5A, 6A). Current-clamp recordings were obtained from GFP⁺ interneurons located below the central canal and in medial lamina VIII, bordering the ventral commissure.

Based on their size, location, passive membrane properties, and responses to injected current steps, two distinct populations of GFP⁺ interneurons could be identified (Table 1). The majority were identified as endogenous Hb9⁺ interneurons by their size and location (type 1; n = 57). The Hb9⁺ neurons were small (mean whole-cell capacitance, 10.0 ± 1.5 pF) with high input resistances (mean, 903.2 ± 226.9 MΩ). These input resistances were in a range (∼700 MΩ) similar to those reported by Hinckley et al. (2005). Approximately one-third (18 of 57) of these cells were spontaneously active, either with tonic firing or with occasional action potentials. The remaining cells in this population did not fire spontaneously and had a mean resting membrane potential of -51.7 ± 6.4 mV. Recordings were also obtained from more lateral GFP⁺ interneurons (type 2; n = 8). The type 2 ectopic GFP⁺ interneurons were not clustered and appeared morphologically distinct from the Hb9⁺ interneurons. Compared with the type 1 cells, these type 2 cells were larger (mean whole-cell capacitance, 16.8 ± 2.3 pF; p < 0.005) with correspondingly lower input resistances (517.8 ± 169.8 MΩ; p < 0.005) (Table 1). These data suggest that Hb9⁺ interneurons are both molecularly and electrophysiologically distinct from surrounding neurons.

To examine additional electrophysiological characteristics of these neurons, we studied postinhibitory rebound (PIR), a critical feature in establishing an alternating rhythm in many motor systems (Perkel and Mulloney, 1974; Tegner et al., 1997). The endogenous Hb9⁺ interneurons (type 1) (Fig. 5A) exhibited strong PIR that was evoked after the break of a hyperpolarizing current pulse (Fig. 5B,D-F). The PIR reached threshold for action potential generation, followed (within 35 ms) by a second action potential, forming a doublet (Fig. 5B,C,F). Depolarizing steps from -90 mV led to all-or-none spiking, which, in the presence of TTX, resembled a calcium-mediated spike (Fig. 5C). The amplitude of the PIR was dependent on both the voltage (Fig. 5B) and time (Fig. 5D) of previous hyperpolarization, consistent with the PIR being mediated by T-type (Ca_V3) calcium channels (Carbone and Lux, 1984; Aizenman and Linden, 1999). This was confirmed by its block with nickel (100-500 μm; n = 6) (Fig. 5E). In experiments using the Hb9:GFP × Hb9^nlsLacZ/+ double-transgenic mice and intracellular injection of Alexa 594, processing for β-gal immunoreactivity established that interneurons with PIR expressed Hb9 (Fig. 5F). These data indicate that Hb9⁺ interneurons have electrophysiological characteristics important in rhythmogenic networks.

To establish whether this electrophysiological profile was selective for the endogenous Hb9⁺ interneurons, type 2 interneurons that expressed GFP ectopically were also studied (Fig. 6A). Unlike the type 1 neurons, injection of hyperpolarizing current pulses revealed a time-dependent sag that is typical of that mediated by a hyperpolarization-activated mixed cationic conductance (I_h) (Pape, 1996) (Fig. 6B). As a consequence, these GFP⁺ interneurons exhibited a small depolarization after the break of the hyperpolarizing current pulse, which could be associated with single action potentials. Doublet spike firing was never observed. The majority of these type 2 cells (5 of 8; 63%) exhibited small-amplitude (peak to peak, 3.9 ± 1.3 mV) biphasic spontaneous oscillations in their membrane potential that were voltage independent (Fig. 6C, arrows). Such oscillations were not detected in type 1 neurons. Thus, the electrophysiological profile of endogenous Hb9⁺ GFP⁺ neurons appears distinct from other ectopic GFP⁺ interneurons found in close proximity in the ventral spinal cord.

Locomotor-induced Fos expression and conditional bursting of Hb9-positive interneurons

Recent studies by Hinckley et al. (2005) have provided evidence that the activity of a population of GFP⁺ interneurons in lamina VIII is synchronized to rhythmic ventral root output. To assess whether Hb9⁺ interneurons are activated in locomotion, adult mice were subjected to a 90 min locomotor task, and their spinal cords were processed for Fos immunohistochemistry (Barajon et al., 1992; Carr et al., 1995; Herdegen and Leah, 1998; Dai et al., 2005). This activity induced Fos expression in these mice in a distribution similar to that demonstrated recently in cat fictive locomotion (Dai et al., 2005). Single small cells and clusters of GFP⁺ interneurons that were morphologically and anatomically homologous to the Hb9⁺ interneurons expressed Fos under these conditions (1.2 cells per 30 μm section; 15 sections per mouse; n = 2 mice) (Fig. 7A, top panels). In contrast, Fos immunoreactivity was not detected in these neurons in mice that were relatively inactive before perfusion (n = 2) (Fig. 7A, bottom panels), linking Fos expression to locomotor behavior. Thus, endogenous Hb9⁺ neurons are activated during locomotion.

Rhythmic locomotor output can be induced in the in vitro mouse spinal cord preparation by the application of 5-HT (20 μm), dopamine (50 μm), and NMDA (20 μm) (Jiang et al., 1999). To investigate whether endogenous Hb9⁺ interneurons have intrinsic properties to support rhythmic activity, these three receptor agonists were bath applied to spinal cord slices of mice of a developmental stage sufficient to bear weight and walk (more than or equal to P8). In all 19 type 1 neurons studied under these conditions, application resulted in an increase in spontaneous activity. After the addition of TTX (1 μm), oscillations in membrane potential were recorded (11 of 19 neurons), indicating that the membrane potential of these cells can oscillate in the absence of synaptic activation. In 8 of 11 neurons, these oscillations were relatively low amplitude (5.1 ± 1.3 mV) and occurred with a frequency of 0.9 ± 0.4 Hz (Fig. 7B). These oscillation frequencies were voltage independent (Fig. 7D), consistent with the hypothesis that the oscillations reflect activity in electrotonically coupled neurons that have similar in-phase membrane potential oscillations (Sillar and Simmers, 1994). These frequencies are in the range of slow overground locomotion, but faster than the frequencies of fictive stepping illustrated in mouse in vitro spinal cord preparations [∼0.2 Hz (Jiang et al., 1999); <0.1 Hz (Hinckley et al., 2005)]. In the remaining three cells, application of these drugs in the presence of TTX resulted in an initial depolarization (8.9 ± 0.4 mV), followed by the onset of large-amplitude oscillations (42.5 ± 2.9 mV) with a frequency of 0.4 ± 0.1 Hz (Fig. 7C). When hyperpolarized to holding potentials of -70 to -75 mV, the depolarized phase of the oscillations initiated an all-or-none type of spike, peaking at -13.3 ± 0.5 mV (amplitude, 61.6 ± 0.6 mV) and decaying to a plateau at -50.7 ± 2.8 mV before rapid hyperpolarization back to baseline. On additional hyperpolarization, a thresholding effect was seen, in which only some of the depolarizations elicited an all-or-none spike. Additional hyperpolarization eliminated these spikes, leaving smaller-amplitude oscillations (14.0 ± 3.5 mV) of similar frequency (0.4 ± 0.1 Hz) (Fig. 7C). In contrast to the lower-amplitude oscillations demonstrated above, these oscillations were voltage-dependent, with hyperpolarization leading to an increase in oscillation frequency (Fig. 7D), indicating that the frequency of oscillations is determined by intrinsic neuronal properties rather than by the network. The reduction in frequency with depolarization primarily results from a prolongation of the depolarized phase of the oscillation. This likely results from the recruitment of slowly inactivating inward conductances.

To investigate the contribution of the Ca_V3-mediated calcium conductance to this intrinsic oscillatory activity, nickel (100 μm) was added to the bath (n = 2). Nickel blocked the large-amplitude spike, leaving small-amplitude (7.8 ± 1.8 mV) oscillations with waveforms similar to those seen in the cells described above. The frequency was relatively unaffected (0.3 ± 0.1 Hz) (Fig. 7C). The large-amplitude oscillations in these three cells may result from more intact dendritic arborizations with their associated currents. These data show that the Hb9⁺ (type 1) interneurons are capable of endogenous rhythmic oscillatory activity under the same conditions that evoke rhythmic locomotor output in the in vitro spinal cord preparation. The large-amplitude oscillations evoked by the locomotor stimuli depend on the conductance that underlies the PIR (nickel-sensitive calcium conductance). These biophysical studies of endogenous Hb9⁺ interneurons support the idea that phasic activity of these neurons may contribute to rhythmic locomotor output.

A possible role for Hb9⁺ interneurons in locomotor rhythm generation

Several features of Hb9⁺ interneurons suggest their possible involvement in rhythm generation. First, the distribution of these neurons (from cervical to upper lumbar spinal cord with few seen caudal to midlumbar segments) is similar to that shown for hindlimb locomotor rhythm-generating networks (Cazalets et al., 1996; Kjaerulff and Kiehn, 1996; Cowley and Schmidt, 1997; Kiehn and Kjaerulff, 1998; Marcoux and Rossignol, 2000).

Second, they receive diverse types of inputs. Serotonergic input is critical in activating the rhythm generator in mammals including the mature mouse (Jiang et al., 1999), neonatal rat (MacLean et al., 1998), and adult cat (Ribotta et al., 2000). In addition, the presence of VGLUT2⁺ GFP⁺ terminals on the Hb9⁺ interneurons raises the possibility that self-excitation or mutual excitation has a role in their function. Evidence from other rhythm-generating networks has revealed a prominent role for mutual excitation or self-excitation (Rowat and Selverston, 1997), in which the combination of PIR with mutual re-excitation can facilitate a sustained rhythm (Roberts and Tunstall, 1990).

Third, the electrophysiological properties of Hb9⁺ interneurons are also suited for rhythm generation. Hb9⁺ interneurons exhibit prominent PIR, mediated by a low-threshold, nickel-sensitive calcium conductance. In other rhythm-generating networks, it has been established that connectivity alone is insufficient to generate rhythm: the intrinsic properties of the network interneurons determine the rhythm and pattern of output (Perkel and Mulloney, 1974; Hartline and Gassie, 1979; Mulloney et al., 1981; Eisen and Marder, 1984; Harris-Warrick et al., 1995; Roberts et al., 1995; Tegner et al., 1997; Arshavsky et al., 1998; Pena et al., 2004). In particular, PIR has been shown to be an essential property in many rhythm-generating neurons. In different networks, this property may facilitate bursting (Perkel and Mulloney, 1974), ensure that the rhythm is sustained (Roberts and Tunstall, 1990), or improve the stability of the rhythm (Tegner et al., 1997). Thus, Hb9⁺ interneurons have the properties expected for neurons involved in rhythm-generating networks.

It has been demonstrated recently that Hb9⁺ interneurons are rhythmically active during locomotor-like ventral root discharges (Hinckley et al., 2005). We found that Hb9⁺ interneurons are activated during overground locomotion, as shown by Fos expression elicited by a walking task (Fig. 7A) (Carr et al., 1995; Wilson et al., 2003). Neurons in this region have been shown to be recruited during locomotor-like activity (Kjaerulff et al., 1994; Cina and Hochman, 2000; Dai et al., 2005), and medial lamina VII and lamina X interneurons have been shown to oscillate rhythmically in response to NMDA (Hochman et al., 1994; Kiehn et al., 1996). Furthermore, Hb9⁺ interneurons are intrinsic oscillators; in the presence of TTX, they have large-amplitude oscillations in response to serotonin, dopamine, and NMDA, the same combination of transmitters that evoke locomotor activity in the isolated mouse spinal cord (Cowley and Schmidt, 1994; Jiang et al., 1999). In addition, other stimuli that evoke locomotor activity, including increased extracellular K⁺ concentration (Bracci et al., 1998), or the addition of K⁺ channel blockers (Taccola and Nistri, 2004), evoke rhythmic oscillations in Hb9⁺ interneurons (Wilson and Brownstone, 2004) (J. M. Wilson and R. M. Brownstone, unpublished data). Furthermore, the voltage independence of the oscillation frequency is consistent with activity produced in electrotonically coupled oscillating networks (Sillar and Simmers, 1994). Together with data demonstrating that electrotonic coupling can function to synchronize and pattern spinal locomotor network activity (Kiehn and Tresch, 2002; Taccola and Nistri, 2004), these data support the idea that Hb9⁺ interneurons may be involved in generating locomotor activity.

In conclusion, our findings demonstrate that the genetic dissection of spinal interneuron circuitry based on selective profiles of transcription factor expression combined with electrophysiological analysis can provide insight into locomotor function. The biophysical properties of Hb9⁺ interneurons documented in this study lend intriguing, albeit incomplete, support for the view that this set of interneurons has a role in rhythm generation in spinal locomotor circuits.