INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The pedunculopontine nucleus (PPN), as the cholinergic arm of the reticular activating system (RAS), has been implicated in the modulation of sleep-wake states, the startle response (SR), and of posture and locomotion (reviewed in Garcia-Rill 1991; Reese et al. 1995; Steriade and McCarley 1990). Electrical stimulation of the PPN can induce widespread effects, both ascending and descending, such as cortical electroencephalographic (EEG) desynchronization (Moruzzi and Magoun 1949) [better referred to as synchronization of fast cortical rhythms (Steriade et al. 1996)], changes in muscle tone (Lai and Siegel 1990), and the recruitment of stepping (Garcia-Rill et al. 1983, 1986, 1987; Garcia-Rill and Skinner 1987a,b, 1988, 1991). The PPN therefore appears to be involved in the ascending control of transitions in state from slow-wave sleep to either waking or REM sleep, and in descending functions that involve changes in muscle activity from standing (extensor activation), to the SR (flexor activation, extensor inhibition), to the atonia of REM sleep (flexor and extensor inhibition), and to locomotion (flexor-extensor alternation). PPN neurons are known to increase their firing rates during synchronization of fast rhythms in waking and REM sleep [tonically in waking, bursting during REM sleep, and reduced activity during slow-wave sleep (Steriade and McCarley 1990; Steriade et al. 1990)] and to show both tonic and rhythmic activity in relation to either the duration of stepping episodes, or the rhythmic alternation of locomotor movements (Garcia-Rill et al. 1983; Garcia-Rill and Skinner 1988). The PPN sends diffuse, mostly cholinergic, projections throughout the pontine reticular formation (Garcia-Rill 1991; Garcia-Rill and Skinner 1987b; Garcia-Rill et al. 1986; Jones 1990; Mitani et al. 1988; Rye et al. 1988; Semba et al. 1990; Shiromani et al. 1988). In turn, the induction of REM sleep has been proposed to be facilitated by pontine reticular neurons (Jouvet 1975; Yamamoto et al. 1990).

During REM sleep, the release of acetylcholine in the pontine reticular formation is augmented (Kodama et al. 1990; Leonard and Lydic 1997), and neurons in this region are depolarized (Ito and McCarley 1984). Injections of cholinergic agonists into the pontine reticular formation have been found to depolarize pontine reticular formation neurons (Greene et al. 1989) and to induce REM sleep whether injected into anterodorsal pons (Baghdoyan et al. 1987; Yamamoto et al. 1990), or into more posterior pontine regions, although with lowered effectiveness (Baghdoyan et al. 1987). Electrical stimulation of the PPN increases the release of acetylcholine in the pontine reticular formation (Lydic and Baghdoyan 1993), as well as enhances REM sleep (Thakkar et al. 1996). Interestingly, the parameters of PPN stimulation used to elicit acetylcholine release in the pontine reticular formation are similar to those used for inducing locomotion [i.e., continuous 0.5-ms pulses at 50 Hz (Lydic and Baghdoyan 1993) vs. continuous 0.5-ms pulses at 20-60 Hz (Garcia-Rill 1991; Garcia-Rill et al. 1987), respectively], but different from those used to induce suppression of muscle tone [i.e., short trains of 0.2-ms pulses at 100 Hz (Lai and Siegel 1990)]. These results would at first sight appear contradictory, unless one postulated, among other options, that there is a stimulation frequency-dependent effect at play. A recent study reported the presence, in the decerebrate cat caudal pontine reticular formation, of extracellularly and intracellularly recorded neurons that showed such frequency-dependent responses following PPN stimulation (Garcia-Rill et al. 2001). That study showed that short (1-s) trains of medium frequency (60-Hz) stimulation induced prolonged responses (>12 s) in caudal pontine neurons, whereas low (10-Hz) or high (100-Hz) frequency trains induced much briefer responses, if any. However, to perform a more thorough investigation of the responses and potential mechanisms mediating these responses, intracellular recordings in vitro became essential. The present studies were undertaken to determine the nature of the responses of rat caudal pontine reticular neurons recorded in vitro following PPN stimulation using various frequencies of stimulation. Preliminary findings have been reported in abstract form (Homma et al. 2000).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

Adult timed-pregnant Sprague-Dawley rats (280-350 g) were used and the litters culled to 10. When the pups were 12-21 days old, individual pups were anesthetized using ±2-(2-chlorophenyl)-2-(methylamino)cyclohexanone (70 mg/kg im) until tail pinch and corneal reflexes were absent, then they were rapidly decapitated. The brains were dissected free under cooled (4°C) oxygenated (95% O₂-5% CO₂) artificial cerebrospinal fluid (ACSF), and the brain stem containing the PPN and the caudal pontine reticular nucleus (PnC) was bilaterally blocked so slices could be cut semihorizontally. The block of tissue was glued onto a stage, and 500-µm slices were cut with a Vibroslice (Campden Instruments, London, England) under cooled, oxygenated ACSF, and then allowed to equilibrate for 1 h in oxygenated ACSF at room temperature before recording. The composition of the ACSF was (in mM) 122.8 NaCl, 5 KCl, 1.2 MgSO₄, 2.5 CaCl₂, 1.2 NaH₂PO₄, 25 NaHCO₃, and 10 dextrose. For BaCl₂ superfusion, MgSO₄ was replaced by MgCl₂ to prevent formation of BaSO₄. The slices contained most PnC neurons as well as the direct pathway from the PPN. Only one or two of the 500-µm slices from each brain contained the PnC and PPN. In this study, we used a total of 80 pups. Typically, we recorded from the PnC and stimulated the PPN on one side of the brain. Once a well-studied cell was injected intracellularly, we moved the stimulating electrode to the contralateral PPN and recorded from the contralateral PnC. That is, in some slices, one cell per side per slice was injected intracellularly.

Recording procedures

The recording chamber allowed the slice to be suspended on a nylon mesh so that oxygenated ACSF could flow all around the slice. The gravity-fed ACSF flowed through a sleeve of circulating warmed water so that the temperature of the ACSF in the chamber was 30 ± 1°C. The outflow was removed by suction and flow adjusted to 2-3 ml/min. Microelectrodes were pulled in a Sutter Instruments (Novato, CA) puller using Omega-Dot, thin-wall borosilicate glass and filled with 3 M K⁺ acetate and 1% biocytin, with a resistance of 70-100 M. Signals were amplified with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA) in these current-clamp recordings. Neurons were impaled and allowed to stabilize for about 5 min before testing. Neurons that showed a stable resting membrane potential (RMP) less than or equal to 55 mV and action potentials 40 mV and that had stable, long-term recordings were accepted for data analysis. PnC neurons at the ages studied appeared to be small in size, as evidenced by their high-input resistance (R_in) and morphology. The RMPs were verified and adjusted when the electrode was withdrawn at the end of recordings (usually only 1-2 mV difference, sometimes >5 mV especially after biocytin injection). In bridge mode, a series of hyperpolarizing and depolarizing current steps of 0.1 nA at RMP were applied to determine several membrane properties (see following text). These current steps also allowed the computation of a preliminary current-voltage (I-V) curve during the linear range of voltage deflections using SuperScope software (GW Instruments, Somerville, MA). In some neurons, we also calculated an I-V curve using ramp stimulation, usually from 80 to +30 mV to reveal potential bistable properties. Ramp stimulation I-V curves were calculated before, during, and after tetrodotoxin (TTX) superfusion (see following text). The properties measured included membrane input resistance (R_in, determined using hyperpolarizing 300-ms duration pulses of 0.1-0.3 nA applied at RMP), action potential amplitude and threshold (determined from the beginning of the sodium spike to its peak in action potentials occurring spontaneously at RMP or, if no spontaneous activity was evident, by depolarizing the membrane until individual action potentials were induced, i.e., at action potential threshold), action potential duration at threshold (determined as the duration of the action potential at half-amplitude in spikes recorded at action potential threshold), afterhyperpolarization (AHP) amplitude (determined from action potential threshold to the peak of the AHP in individually occurring spikes) and AHP duration (determined from action potential threshold to the return to prespike membrane potential in individually occurring spikes).

Stimulation procedures

Electrical stimulation of the PPN was carried out using a bipolar concentric electrode (100 µm diam, 100 K resistance) applying currents of 100-500 µA in amplitude using pulses of 0.2-0.5 ms duration, at frequencies of 10-90 Hz, individually and in trains of various durations, usually 1 s. The location of the stimulating electrode was confirmed using NADPH diaphorase histochemistry as described below. In the studies described, all stimulating electrode sites were found within the region of NADPH diaphorase-positive (NADPHd+) cells.

Neuroactive agents were applied via a manifold with six perfusion ports, hence multiple gravity-fed solutions could be applied for pharmacological characterization of neuronal properties. The concentrations of the superfused neuroactive agents in ACSF were as follows: carbachol (CAR; 5 µM), kynurenic acid (KYN; 300 µM), (R,S)-methyl-4-carboxyphenylglycine (MCPG; 300 µM), nifedipine (NIF; 10 µM), scopolamine (SCOP; 10-100 µM), and tetrodotoxin (TTX; 0.3 µM). Direct effects of these agents on recorded PnC neurons were confirmed before, during, and after wash out/recovery from TTX superfusion. Ramp stimulation for I-V curves was carried out during these three conditions. The concentrations of these agents were adjusted so that effects were evident using superfusion times of 1-2 min. CAR was also used for micropressure application using a higher concentration (30 µM) adjusted to elicit a response following 2-5 puffs applied to the surface of the tissue when the pipette was <100 µm from the recording microelectrode. The micropressure system was set at 30 psi, 50-ms duration puffs, and pipette resistance was designed for application of about 100 pl/puff. The micropressure pipettes contained 2% Fast Green dye to visualize flow of the puffed solution across the downstream-located recording microelectrode.

Histological procedures

At the end of the recording period, each neuron was injected with biocytin using intracellular depolarizing pulses adjusted to elicit a train of action potentials (about 0.5-1.0 nA) of 500 ms duration at 1 Hz for 10-15 min. Such injections yielded well-filled neurons. All of the slices were processed for NADPH diaphorase histochemistry for selective labeling of cholinergic mesopontine (PPN) neurons around stimulation sites. Briefly, slices were fixed in 4% buffered paraformaldehyde for 1-2 h, cryoprotected in 20% sucrose, and cut in a cryostat at 50 µm. Sections were incubated in 1 mg/ml NADPH and 0.1 mg/ml nitroblue tetrazolium in PBS at 37°C for 30-60 min (Garcia-Rill and Skinner 1987a,b, 1988; Skinner and Garcia-Rill 1984). For intracellularly labeled PnC neurons, biocytin immunocytochemistry was carried out preceding NADPH diaphorase histochemistry using a Vector ABC kit using the PAP method with diaminobenzidine as the chromogen. Sections were mounted on gelatin-coated slides and coverslipped with Eukitt (Calibrated Inst., Hawthorne, NY) for brightfield optics.

Statistical procedures

STATISTICAL MODELING. For comparison of data between the different groups in each experiment, measures were tested using one-factor, two-factor, or multifactor ANOVA to conclude whether any of the factors had a significant effect on the magnitude of the variable and also whether the interaction of the factors significantly affects the variable. Differences were considered significant at values of P  0.05. If a statistical significance was present, a post hoc test (Scheffe) was used to compare between groups. Each of the measures of intrinsic membrane properties and responses to low versus medium versus high frequency of stimulation were compared using two-factor ANOVA (e.g., response amplitude and stimulation frequency). Comparisons of duration of effect that involved repeated observations on the same neurons, e.g., responses to neuroactive agents over time, were carried out using repeated measures ANOVA since each time sampled could be considered a different "treatment" on each cell. When statistical significance was evident, the post hoc test (Scheffe) was carried out to determine differences between groups of cells across treatment (e.g., duration or frequency of response).

POWER ANALYSIS. Considering, for example, the analysis of AHP amplitude between two groups [prolonged response (PR) vs. nonprolonged response (NPR) cells, Fig. 2C], with an n = 49 for NPR and n = 91 for PR cells, we had in excess of a two standard error difference in group means. Even with an n of about 15 cells per group, we had in excess of 80% power to detect a difference at the 5% level of significance.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Localization

We recorded a total of 140 cells, which met the criteria of stable RMP less than or equal to 55 mV and action potential 40 mV. These neurons were localized in the posterior part of the oral pontine reticular formation (PnO) and throughout the caudal pontine reticular formation (PnC) at the anterior edge of the gigantocellular tegmental field. The cells studied were found between the lateral edge of the midline raphe medially, extending laterally to the medialmost edge of the 7th nerve. Figure 1 is a drawing of a representative histological section of one of the semihorizontal slices used. The localization of a selected sample of the neurons recorded that were well filled with biocytin reflects the distribution described. While only one neuron per side per slice was injected with biocytin, recordings were also carried out in noninjected cells in the vicinity of the injected neurons but still within the boundaries described. Functionally, the region studied appeared to be well posterior and medial to the pontine inhibitory area (Baghdoyan et al. 1984; Yamamoto et al. 1990), to be dorsal to the trapezoid body, and overlap with the distribution of giant PnC neurons known to mediate the SR (Davis 1984; Koch 1999; Swerdlow et al. 1992). The region sampled was intended to be equivalent to that sampled in our previous studies in the decerebrate cat PnC (Garcia-Rill et al. 2001).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Locations of recorded PnC neurons. Diagram of a representative histological section through a semi-horizontal slice of the rat brain stem. The slice was typically through the central gray (CG) and midbrain raphe (DR) in the dorsal midbrain (top) angled toward the anteroventral medulla (bottom), which contained the pedunculopontine nucleus (PPN; NADPH diaphorase-positive cells) located lateral to the decussation of the superior cerebellar peduncle (SCP). The neurons recorded were located in the posterior oral pontine (PnO) and caudal pontine (PnC) reticular formation, medial to the motor nucleus of V (MoV) and 7th nerve (VII), and anterior to the gigantocellular nucleus (Gi), each outlined by dashed lines. The PPN stimulation site is denoted by the large filled circle at right, and a sample of cells (n = 36 for clarity) showing prolonged responses (PR, n = 20) are shown as small filled circles, while those without prolonged responses (NPR, n = 16) are shown as small open circles. Please note that the most medial row of cells were all NPR neurons and could have been raphe nucleus neurons. There were no discernible differences in the properties of these medial NPR neurons compared with more lateral NPR cells. Since no immunocytochemical labeling for serotonergic neurons was undertaken, we cannot speak to the neurochemical nature of these cells.

Electrophysiological properties

We studied a total of 140 PnC neurons, of which 91 (65%) showed PRs and 49 (35%) did not show PRs (NPR). The definition of a PR requires explanation. In our previous study using extracellular recordings in the decerebrate cat brain stem, the PR was defined as the duration of the train of action potentials induced in PnC neurons following PPN stimulation from the beginning of the response until firing had ceased for 1 s (Garcia-Rill et al. 2001). The present intracellular studies revealed that PPN stimulation induced a long-lasting depolarization in PnC neurons on which was superimposed a train of action potentials. For comparative purposes, the duration of the PR herein still is defined as the duration of the action potential train until firing had ceased for 1 s. However, the underlying, and more important, mechanism at play is obviously the much longer-lasting depolarization induced by PPN stimulation. In the sample recordings that follow, the prestimulation membrane potential is denoted by a dotted line to facilitate detection of the depolarization induced in PR cells. We will first describe the general electrophysiological characteristics of PnC cells across age, then compare the properties of PR and NPR cells, and, finally, turn to detailing the features of the PRs.

EFFECTS OF AGE. Of the cells recorded, 67/140 were studied in slices from pups younger than 16 days old and 73/140 in slices from pups 16-21 days old. That is, we compared cells recorded during the first half of the developmental window studied to those recorded during the second half. The rationale behind this division is related to the known change at around 15 days of age in the descending control of locomotor and swimming movements in the rat (Bekoff 1979; Iwahara et al. 1991). For example, if a spinal cord transection is made before 15 days of age, rats recover the capacity for spontaneous locomotion, whereas they do not recover such capacity if the transection is performed after 15 days (Stelzner et al. 1975; Weber and Stelzner 1977). There were no statistically significant differences between PnC cells in these age groups on the basis of mean ± SD RMP (62 ± 7 mV vs. 62 ± 6 mV), action potential threshold (46 ± 9 mV vs. 48 ± 8 mV), action potential amplitude (51 ± 8 mV vs. 53 ± 10 mV), action potential duration at half-amplitude (1.0 ± 0.4 ms vs. 0.7 ± 0.4 ms), AHP amplitude (17 ± 4 mV vs. 18 ± 4 mV) or R_in (105 ± 42 M vs. 99 ± 47 M), regardless of response type. However, there was a significant decrease across these ages in AHP duration for all cells (140 ± 68 ms before vs. 116 ± 58 ms after day 16; P < 0.02).

PR VERSUS NPR CELLS. The electrophysiological characteristics of PR compared with NPR cells are listed in Table 1. The main difference, of course, was the presence of a PR, along with a larger AHP amplitude in PR cells, but there were no significant differences in terms of RMP, R_in, action potential threshold, amplitude or duration, or AHP duration. Figure 2, A and B, shows representative examples of the two cell types that could be divided on the basis of responsiveness to PPN stimulation. PnC neurons that showed PRs (91/140) following PPN stimulation (using paradigms to be discussed in the following text) had AHP durations of 138 ± 70 ms before and 126 ± 58 ms after day 16, i.e., did not change appreciably with age (NS). However, PnC cells without PRs (NPR cells, 49/140) had longer duration AHPs before compared with after day 16 (147 ± 64 ms vs. 102 ± 56 ms; P < 0.01), i.e., the decrease in overall AHP duration across age was due mostly to changes in NPR, not PR, type PnC cells. However, when cells of all ages were combined, AHP duration in PR cells was virtually the same as that of NPR cells (Table 1).

View larger version (32K): [in this window] [in a new window]   Fig. 2. Response types of PnC neurons following PPN stimulation. A: representative PnC neuron [18 day, 60 mV resting membrane potential (RMP)] with a PR (not shown, see subsequent figures) following PPN stimulation showing a lower amplitude, longer duration afterhyperpolarization (AHP; left side, depolarizing current applied sufficiently to induce a single action potential at 50 mV). Depolarizing pulses induced stable, nonadapting firing frequencies (right side). Hyperpolarizing pulses showed the presence of an Ih current in this cell. B: representative PnC neuron (14 day) also with a PR typically showing a higher amplitude, shorter duration AHP (left side, depolarized to 50 mV to induce a single action potential), and nonadapting firing frequency during depolarizing pulses (right side). This particular PR cell had a prominent "LTS" (burst of action potentials on a hump after the end of hyperpolarizing pulses, diagonal arrow after the rebound burst) currents. It should be noted, however, that both PR and NPR cells could also exhibit a prominent "A" current. Calibration bar for A and B, vertical 10 mV, horizontal 50 ms. C: graph of AHP amplitude vs. duration in PR (, n = 91) and NPR (, n = 49) PnC neurons recorded at all ages (12-21 days). There was no statistically significant difference between PR and NPR cells in terms of AHP duration (although numerical decreases across age were greater in NPR than PR cells, see text); however, AHP amplitude was significantly greater in PR cells (see text). D: example of a PR cell (12 day) that responded following PPN stimulation (1-s train of 0.5-ms pulses of 500-µA amplitude at 60 Hz) showing a depolarization from the prestimulation RMP (62 mV) and prolonged action potential train (top record). Example of a NPR cell (18 day) that was hyperpolarized from RMP (60 mV) following identical stimulation applied to the PPN (bottom record). Calibration bar vertical 10 mV, horizontal 1 s. Dashed lines added to shown prestimulation membrane potential.

PROLONGED RESPONSES (PRS). If trains of stimuli were used, the brief, single action potential responses described above became PRs in 91/140, or 65%, of PnC neurons (Fig. 2D, top). The PR (operationally described as the duration of the train of action potentials induced) was comparable to results previously described in PnC neurons in the decerebrate cat (Garcia-Rill et al. 2001). However, the duration of the depolarization underlying these action potentials varied considerably, typically lasting <30 s. A maximum stimulus train duration of 1 s was selected for testing because stimulation for longer durations is known to recruit locomotion in decerebrate animals (Garcia-Rill 1991; Garcia-Rill et al. 1987, 1988; Skinner and Garcia-Rill 1984), while 1-s trains elicit only brief spinal cord activation. This also allowed comparison with previous results on PnC neurons in decerebrate animals (Garcia-Rill et al. 2001). Figure 3A shows the responses of a PnC neuron following administration of trains of increasing duration. In general, the membrane potential was depolarized briefly (1-2 s) by short-duration (100-ms) trains at 60 Hz, but depolarized for longer durations (>5-10 s) by longer duration (1-s) trains (Fig. 3A). The mean ± SD amplitude of the depolarization induced by PPN stimulation (using trains of 1 s at 60 Hz) was 4.8 ± 1.3 mV (n = 24). Two types of predepolarization activity were evident in PnC neurons with PRs. In some PnC cells (39%), the membrane potential was depolarized during the stimulus train and the PR began during, at the end, or within 1 s of the termination of the train (Fig. 3B). In the other PnC neurons with PRs (43%), the membrane potential was hyperpolarized during or at the end of the stimulation train (Fig. 3, A and C). That is, the PR could be elicited following depolarization or hyperpolarization induced by the stimulation. The remaining 18% of cells showed a poststimulation membrane potential (preceding the PR) equal to the prestimulation membrane potential. As evident in the figures provided (e.g., Figs. 3 and 4), there was a delay between the end of the stimulus train and the beginning of the PR in many neurons. In a group of PR neurons (n = 20) tested with identical stimulation parameters, the latency of the PR was similar using trains of 10 Hz (0.3 ± 0.5 s), 30 Hz (0.6 ± 0.8 s), and 60 Hz (0.5 ± 0.6 s), but increased significantly (P < 0.05) when using trains of 90 Hz (1.3 ± 1.1 s). It should be noted that the increase in latency was not due to a difference in the membrane response preceding the PR, i.e., a higher amplitude hyperpolarization or other factor. There was no obvious difference in electrophysiological properties between neurons that responded within or immediately following the stimulation train (e.g., Fig. 2D), compared with those that responded after some delay (e.g., Fig. 3, A-C). Even in the same cell, the latency of the PR could differ somewhat across repeated trials (Fig. 3C).

View larger version (62K): [in this window] [in a new window]   Fig. 3. Effects of stimulus duration and frequency on PRs. A: trains of different durations of PPN stimulation using 0.5-ms pulses at the same frequency and amplitude (60 Hz, 400 µA) showed increasing duration PRs as train duration increased from 100 ms (top record) to 250 ms (middle record) to 1 s (bottom record). Note that the PR and underlying depolarization endured for many seconds following the 1-s train when the PnC neuron (17 day) was held at 50 mV (membrane depolarization above prestimulation membrane potential shown as dashed line). B: trains of different frequencies of stimulation using 0.5-ms pulses at the same duration and amplitude (1 s, 400 µA) were applied to the PPN during recording of a PnC neuron (14 day) held at 53 mV. Stimulation at 10 and 30 Hz was not as effective in inducing PRs, whereas 60-Hz stimulation elicited the longest duration PR superimposed on a long-lasting depolarization, and, in this PnC cell, 90-Hz stimulation induced a PR almost as long as when using 60-Hz stimulation (in other cells, 90-Hz stimulation was not as effective as 60-Hz stimulation, see text). Note, however, that the amplitude of the membrane depolarization underlying the PR of action potentials increased in amplitude from prestimulation membrane potential (dashed line) with frequency up to 60 Hz, then decreased at 90 Hz. C: PR of a PnC neuron (12 day) held at 53 mV before PPN stimulation (300 µA, 60 Hz, 1-s train; top record), which failed to reset to the MP following intracellular hyperpolarizing pulses that reset the membrane potential to 60 mV (middle record, 0.1 nA, 2 s) or to 80 mV (bottom record, 0.3 nA, 1 s). Note the lack of change in the average firing frequency after the DC current was turned off. Calibration bars vertical 10 mV, horizontal 1 s.

View larger version (43K): [in this window] [in a new window]   Fig. 4. Effects of stimulus amplitude and membrane potential on PRs. A: responses of a PnC neuron (14 day) held at 53 mV following stimulation of the PPN using a 1-s train of 0.5-ms pulses delivered at 60 Hz, but with increasing amplitude. The response following 200-µA stimulation produced a long depolarization but only a few action potentials (top record). Stimulation at 300 µA induced a PR with a long train of action potentials (middle record, partly expanded to show underlying depolarization from holding potential, not easily visible due to the sampling rate), which increased in duration and firing frequency after 400-µA stimulation (bottom record, also partly expanded to show increase in underlying depolarization at 300 and 400 µA compared with 200 µA). B: effect of membrane potential on PPN stimulation-induced responses in the same neuron shown in A. At 49 mV holding potential, PPN stimulation (300 µA, 60 Hz, 1-s train) induced a PR and underlying depolarization (top record), which decreased markedly in firing frequency and slightly in membrane depolarization amplitude as the membrane was hyperpolarized to 51, 56, and 68 mV (middle to bottom). Calibration bars for A and B, vertical 10 mV, horizontal 1 s.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Effects of stimulation frequency on the duration of the PR and on the firing rate of PnC cells during the PR. A: in the same PnC cells, the mean ± SE duration of the PR was statistically longer following 60- and 90-Hz trains than 10- or 30-Hz trains of the same duration (1 s) and amplitude (400 µA; ** P < 0.01). While the mean ± SE duration of the PR was statistically shorter using 10-Hz trains than 30- and 60-Hz trains, there was no further increase in PR duration using 90-Hz trains, i.e., there may have been a plateau to this effect. B: firing frequency of PnC neurons during the 2nd second of the PR (firing frequency during the 1st second of the PR was not consistent across neurons) at different frequencies of stimulation. Stimulation using 60-Hz trains induced statistically significant higher firing frequencies than 10- or 30-Hz trains, whereas 90-Hz trains induced lower firing frequencies than 60-Hz trains, but still higher than 10- or 30-Hz trains (** P < 0.01). That is, maximal firing frequencies (~10 Hz in PnC neurons) during PRs were induced by PPN stimulation at 60 Hz.

View larger version (33K): [in this window] [in a new window]   Fig. 6. Changes in input resistance during PRs. A: PPN stimulation (200-µA pulses at 60 Hz for 1 s) induced a PR in this PnC neuron (21 day) held at 55 mV (top record). Intracellular hyperpolarizing pulses (0.3 nA, 100 ms at 2 Hz) were delivered before stimulation and throughout the PR, including a period in which the membrane potential was manually restored to 55 mV (bottom record). This test revealed an increase in resistance (increased amplitude pulses during underlined period) compared with pre-PPN stimulation resistance. The dashed line indicates the peak response of the hyperpolarizing pulses applied before stimulation. Calibration bars, vertical 10 mV, horizontal 500 ms. B: graph of the input resistance (M) at the peak of the hyperpolarizing pulses applied to the PnC neurons (n = 12) tested before (Pre-STIM) PPN stimulation and during the PPN stimulation-induced PR but after the membrane potential had been restored (as in the underlined period in the bottom record in A). Note that input resistance increased significantly (** P < 0.01) in all 12 neurons tested during the PR with compensated membrane potential.

Pharmacological properties

We then studied the possible nature of the mechanism(s) involved in generating PRs. One potential mechanism that is known to produce responses greatly outlasting the activation of inputs is that of plateau potentials. Plateau potentials are intrinsic membrane properties such that prolonged depolarization can be induced by brief depolarizing steps, which can be reset by hyperpolarizing steps and are usually calcium channel dependent (Hultborn and Kiehn 1992). PPN stimulation-induced PRs in PnC neurons, however, could not be elicited by depolarizing steps (Fig. 2, A and B), or reset, once elicited, using hyperpolarizing steps (Fig. 3C, n = 91). Moreover, superfusion with the calcium channel blocker nifedipine (10 µM) failed to block PPN stimulation-induced PRs (n = 7, not shown). A final test for the presence of plateau potential properties was the determination of a negative slope on the I-V curve, characteristic of bistable properties (Kim and Chandler 1995). Figure 7A shows the PR of a PnC neuron following PPN stimulation using a 1-s long, 60-Hz train of pulses of 500 µA in amplitude. Ramp stimulation was induced, in this case between 75 and 20 mV, before, during, and after recovery from TTX superfusion (0.3 µM; Fig. 7B). The I-V curves during two cycles of ramp stimulation after TTX were linear and overlapped considerably (Fig. 7C), indicative of the absence of bistable properties (i.e., no negative slope in the I-V curve) in seven PnC cells with PRs tested in this manner. It should be noted that, since TTX superfusion blocked sodium channels and the generation of action potentials, PPN stimulation-induced depolarization and PRs in PnC neurons were absent, in keeping with their synaptic nature.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Effects of ramp stimulation and TTX on current-voltage (I-V) curve of PnC neurons with PRs. A: intracellular recording from a relatively depolarized (44 mV holding potential) PnC neuron (14 day) showing a 5-s PR following stimulation of the PPN (60 Hz, 500 µA, 1-s train). Calibration bars as noted for A and B. B: ramp stimulation from 75 to 20 mV at 6 mV/s before (top record), during (middle record), and after (bottom record) recovery from TTX superfusion (0.3 µM). C: I-V curve of the PnC cell shown in A and B showing a linear slope, i.e., there was no negative slope in the curve, therefore this neuron did not have bistable properties. The graph represents superimposed curves calculated over the 2 cycles of stimulation shown in B, i.e., during TTX superfusion. The RMP of this cell was 60 mV, as evident in the I-V curve.

Since many PPN neurons are cholinergic, we tested the effects of cholinergic agents on the manifestation of PRs in PnC neurons. Superfusion (30 µM) of the muscarinic cholinergic antagonist scopolamine (SCOP) reduced or blocked the underlying depolarization and PR induced by PPN stimulation (1-s train of 60 Hz at 400-µA amplitude) in 5/22 cells tested (Fig. 8A) but was ineffective in reducing the PR (tonic firing) or underlying depolarization in 17/22 cells. It is not clear why SCOP was ineffective in modulating PRs in some neurons, since the cells tested probably had access to the SCOP at the concentration used. One possibility is that some PRs are not mediated by cholinergic mechanisms. On the other hand, superfusion (5 µM) or micropressure application (30 µM), of the cholinergic agonist carbachol (CAR) on PR neurons was found to induce long-lasting depolarization in most PnC neurons. Figure 8B shows the responses of a PnC neuron following PPN stimulation (a PR) and after CAR superfusion (5 µM; depolarization and prolonged activation), along with blockade of the CAR-induced effect by superfusion (30 µM) of the muscarinic receptor blocker scopolamine (SCOP) 2 min before CAR superfusion. A total of 33 PnC neurons was tested using CAR, 24 PR type cells, and 9 NPR type cells. Interestingly, 20/24 PR and 5/9 NPR cells were depolarized by CAR (as in Fig. 8B), while two cells of each type were hyperpolarized by CAR and two cells of each type showed no response. Since some NPR cells were depolarized by CAR but showed no PRs, either the cholinergic receptors were incapable of inducing PRs or the effects of CAR on NPR neurons were indirect. It is not clear why CAR did not affect two of the PR cells tested, whether CAR had insufficient access to the cells being recorded, or whether some PRs were due to noncholinergic mechanisms.

View larger version (60K): [in this window] [in a new window]   Fig. 8. Effects of cholinergic agents on PR neurons. A: PPN stimulation induced a PR in this PnC neuron (17 day) with a 65-mV RMP (control, top record). However, the PR was blocked by superfusion of the muscarinic cholinergic antagonist scopolamine (SCOP; 30 µM) 3 min before this trial (middle record), an effect that recovered following wash out of SCOP (bottom record). Note that both the action potential train of the PR and the underlying depolarization were blocked or decreased by SCOP. Calibration bar for A and top record in B, vertical 10 mV, horizontal 1 s. B: PPN stimulation induced a PR in another PnC neuron (17 day) with a 65-mV RMP (control, top record). Micropressure application of the cholinergic agonist CAR (30 µM; arrow, middle record) induced a prolonged depolarization in this PnC neuron that recovered after wash out (not shown). The membrane potential was held at 70 mV because SCOP generally induced a hyperpolarization on most PnC neurons tested. Also note that the sampling rate is responsible for the variable action potential amplitude in this longer record. However, if carbachol (CAR; 30 µM) micropressure application (arrow) was preceded by SCOP (30 µM) 3 min prior to the start of the bottom record), the CAR effect was blocked by the SCOP pretreatment. Calibration bar for middle and bottom records in B, vertical 10 mV, horizontal 2 s.

Interestingly, some PnC neurons were hyperpolarized by CAR (2 of each type), in keeping with known cholinergic inhibition in this region (see following text). For example, Fig. 10 (middle) shows the hyperpolarization induced in a PnC neuron by PPN stimulation, an effect matched by CAR superfusion (5 µM). The two NPR neurons that were hyperpolarized by CAR had relatively low R_in (63 ± 11 M) compared with PR neurons that were depolarized by CAR (95 ± 41 M), or cells of both types that showed no response to CAR (105 ± 23 M).

Since a subpopulation of cholinergic PPN neurons also contain glutamate as a co-transmitter (Lai et al. 1993), we tested the possible roles of ionotropic and metabotropic glutamate receptors on PnC neurons showing PPN stimulation-induced PRs. The nonspecific ionotropic glutamate receptor antagonist kynurenic acid (300 µM) failed to block PPN stimulation-induced PRs in five cells tested, as did the metabotropic glutamate receptor antagonist MCPG (300 µM; n = 4; not shown).

To test the potential mechanism for the increased resistance/decreased conductance, we used BaCl₂ superfusion (0.5-2 mM) to test the possibility that it was due to closure of potassium channels. Figure 9A shows the effects of BaCl₂ superfusion (1 mM) on the manifestation of the PR in a PnC neuron. BaCl₂ superfusion reduced or completely blocked the depolarization induced by PPN stimulation without affecting baseline membrane potential. During BaCl₂ superfusion and wash out, the amplitude of the AHP was reduced and AHP duration increased, an effect that was completely reversed by 5 min after wash out. While it is problematic to measure AHP amplitude during high-frequency firing, since this would tend to yield a lower amplitude AHP than after spontaneous/individual spikes, the effects of BaCl₂ on AHP amplitude are all the more impressive. Representative recordings before, during, on wash out, and after recovery from BaCl₂ are shown in Fig. 9B. AHP amplitude during the control PR, which were reduced by the high firing frequency, was decreased during BaCl₂ superfusion even though isolated spikes were being measured. The amplitude of the AHP began to recover during wash out when individual spikes were measured. The reduction in mean ± SD AHP amplitude from control (19 ± 1 mV, artificially low due to repetitive firing), during BaCl₂ superfusion (15 ± 2 mV, P < 0.01) and during wash out (19 ± 2 mV, individual spikes) are shown in graphed form in Fig. 9C.

View larger version (37K): [in this window] [in a new window]   Fig. 9. Effects of barium on PRs. A: stimulation of the PPN (300 µA, 60 Hz, 1 s) induced a PR in this PnC neuron (14 day) held at 52 mV (top record), which was blocked during BaCl2 (1 mM) superfusion (Ba2+ superfusion, 2nd record) and remained partially blocked soon after barium wash out began (2 min after Ba2+, 3rd record), but recovered completely by 5 min (bottom record). B: the properties of the PnC neurons studied were altered by Ba2+ such that the amplitude of the AHP decreased and its duration was increased. Examples of action potentials before superfusion (control), in which AHP amplitude decreased and duration increased, during barium superfusion (Ba2+) and wash out, but recovered by 5 min after wash out (recovery). C: graph of the mean ± SD of the amplitude of the AHP in 5 PnC neurons with PRs that were tested with Ba2+. Note the decrease in amplitude of the AHP during Ba2+ superfusion (Ba2+) and wash out (wash) compared with before Ba2+ superfusion (cont) (** P < 0.01).

Anatomical characteristics

The present studies include only limited morphometric analysis of somatic size in well-injected neurons. These cells were measured as follows: dendrites were truncated at the base, the long and short axes of the cell body measured for each neuron, and long versus short axis ratio calculated. The area of each cell was also measured using an image analysis system with National Institutes of Health software. There were no statistically significant differences between PR and NPR cells in terms of long versus short axis ratio (35 PR cells 1.86 ± 0.51 vs. 20 NPR cells 1.98 ± 0.75), or in terms of cell area (35 PR cells 451 ± 190 µm² vs. 20 PR cells 393 ± 155 µm²). There was a large variability in cell area measures, suggesting the presence of different cell sizes within each population. Of course, recordings were carried out over an age range during which there is considerable growth, which may have contributed to the lack of anatomical differentiation of PnC meurons correlating with physiological responses. When the measured neurons were divided according to age, PR cells <16 days (n = 18) had a mean area of 420 ± 209 µm² compared with those 16 days, which had an area of 484 ± 165 µm² (n = 17; NS). The difference across age was greater for NPR neurons (<16 days, n = 10, 346 ± 129 µm² vs. 16 days, n = 10, 442 ± 173 µm²; NS). Although there was a numerical increase across age for both cell types, the difference was not statistically different. The variability in cell sizes suggests the presence of groups of cells of different mean areas. There were also no statistically significant differences between PR and NPR cell area across age. For illustrative purposes, we have included reproductions of injected PR and NPR cells in Fig. 10. In this case, the PR neuron (328 µm²) was typical of injected PR and most NPR neurons. The NPR neuron shown was hyperpolarized by PPN stimulation and was larger than most other NPR neurons (625 µm²).

View larger version (26K): [in this window] [in a new window]   Fig. 10. Hypothetical role of descending PPN projections to PnC and their ultimate effects on the spinal cord. The records in the top half of this figure (PnC) include an example (17 day) of the 65% of PnC neurons with a PR following PPN stimulation (PnC, top); and of 1 of the few NPR PnC neurons (18 day) found to be hyperpolarized following PPN stimulation (PnC, bottom) using identical stimulation parameters (0.5-ms pulses, 60 Hz, 300 µA, 1-s trains). Drawings of the 2 cells shown are on the left side (PnC, PR cell, NPR cell). Please note that PR and most NPR cells were similar in size and morphology to the PR cell shown here, while only a few NPR cells were as large as the one shown here. The relatively small (328 µm2) PR neuron, by definition, exhibited a prolonged response following PPN stimulation using a 1-s train of 300-µA pulses delivered at 60 Hz (PnC, top left). The same cell was depolarized following a 1-min application of CAR (5 µM; PnC, top right). The larger (625 µm2) NPR neuron (PnC, bottom) showed hyperpolarization following PPN stimulation using the same parameters (PnC, bottom left) and also was hyperpolarized following a 1-min application of CAR (5 µM; PnC, bottom right). The records on the bottom half of this figure (Spinal Cord) are electromyograms (EMG) of hindlimb antagonist muscles (extensor above, flexor below) showing alternating activity (locomotion) in response to PPN stimulation in the decerebrate animal using 60-Hz pulses slowly ramped up to threshold and then applied continuously (bottom underline labeled "60 Hz Cont."). The locomotion recruited was interrupted by a brief, high-frequency train (thick bar labeled "100 Hz, 300 ms") of stimuli to the PPN, leading to decreased extensor and increased flexor muscle tone. Ultimately, these transient effects were overcome by the continued 60-Hz stimulation. The hypothetical Push-Pull mechanism proposed suggests that PPN stimulation at the appropriate frequencies (~60 Hz) for recruiting locomotion activates large numbers of PnC neurons (presumably interneurons that ultimately activate reticulospinal elements) via cholinergic receptors to drive these cells into a new state (Push), while inhibiting other PnC neurons that normally inhibit muscle tone (Pull). Presumably (and less certain given the paucity of evidence), when a sudden, high-frequency stimulus is applied to the PPN (e.g., a startling stimulus), there is preferential activation of pathways that inhibit extension and activate flexion, such as during the SR. Much additional evidence is needed to support this suggestion, although the findings described herein lend some support to this hypothesis. Calibration bars, PnC, PR, and NPR cell bar 100 µm; top left records PPN stimulation 10 mV and 1 s; top right records CAR superfusion 10 mV and 20 s; Spinal Cord, bottom EMG records 500 µA, 1 s.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Implications of major results

The implications of the main findings described herein are as follows. 1) PPN stimulation using medium frequency stimulation (60 Hz) induced PRs with longer duration in a large population (65%) of small-medium PnC neurons compared with stimulation at lower frequencies. These results are similar to those previously described in the decerebrate cat and suggest the presence of a frequency-dependent effect of PPN projections to PnC neurons (Garcia-Rill et al. 2001). 2) PPN stimulation at about 60 Hz was the most effective frequency range for eliciting the maximum firing rates (about 10 Hz) in PR cells following PPN stimulation (compared with 10, 30, or 90 Hz). This effect indicates that the maximal firing frequency induced following PPN stimulation was at frequencies known best to induce locomotion, suggesting a possible explanation for the long-known, but little understood need to use such frequencies when stimulating the mesopontine region to induce locomotion (Garcia-Rill 1991; Shik et al. 1966). In addition, 10 Hz is in the range of frequencies (5-20 Hz) required for electrical stimulation of the medioventral medulla to induce locomotion (Kinjo et al. 1990). However, additional study is need to verify that activity in some PnC neurons at a, presumably maximal, rate of 10 Hz is correlated with recruiting locomotor, or other types of motor activty. Interestingly, this is the mean frequency of physiological tremor, the upper limit of individual movements, and is thought to originate as a descending command (reviewed in Llinas 2001). This command is thought to act as a cueing function for synchronizing motoneurons, to provide inertia for overcoming friction and viscosity in muscles, and as a control system for binding inputs and outputs in time (Llinas 2001). 3) The developmental decrease in AHP duration across age may be simply an indication of maturing mechanisms that will allow neurons to respond at faster firing rates. However, the selective decrease in AHP duration in NPR but not in PR cells suggests that PR cells have a stable, maximal firing rate early in development, which is only marginally changed with maturation. Given the range of the durations of the AHP (90-130 ms), firing rates would be expected at 8-12 Hz. 4) PRs were voltage dependent, being present at a range between 65 and 45 mV (see following text). 5) PR cells did not show a negative slope in the I-V curve, induction of the PR by depolarizing pulses or resetting of the PR by hyperpolarizing pulses. In addition, the calcium channel blocker nifedipine failed to alter the manifestation of PRs, so that these responses are unlikely to be plateau potentials (Hultborn and Kiehn 1992). 6) However, PRs could be reduced or blocked in some neurons by the muscarinic antagonist SCOP in neurons that were also depolarized by superfusion of the muscarinic agonist CAR, suggesting that a muscarinic receptor is involved in generating some PRs. However, the effects of SCOP were not evident in a number of PnC neurons tested, indicating that there may be multiple mechanisms, as yet unidentified, involved in the generation of PRs. 7) Some PnC neurons were hyperpolarized by CAR superfusion, suggesting that descending PPN projections have differential effects on certain populations of PnC neurons. 8) Although some PPN neurons also release glutamic acid, PRs induced in PnC cells by PPN stimulation were not reduced or blocked by ionotropic or metabotropic glutamate antagonists, suggesting that PRs are not glutamate dependent. 9) PRs may have multiple components, an early phase of higher amplitude followed by a late phase during which there was an increase in input resistance, suggesting that PPN stimulation may lead to the closure of some channels in PnC neurons with PRs. The finding that PRs were reduced by membrane hyperpolarization, and blocked by BaCl₂ superfusion, indicates that PRs may be mediated by the closure of potassium channels. These results are in keeping with findings indicating that muscarinic agonists can depolarize the membrane in certain neurons, leading to lowered spike frequency accommodation and loss of the slowly decaying portion of the AHP, changes that greatly enhance the level of neuronal excitability (Washburn and Moises 1992). Voltage-clamp analysis of these effects showed that the muscarinic-induced depolarization resulted from inhibition of voltage-activated and voltage-insensitive potassium leak currents (Womble and Moises 1992). These results suggest that PPN projections to PnC may activate muscarinic receptors that block potassium channels and lead to depolarization and higher firing frequencies. Additional testing is required to determine the potential contributions of voltage-activated versus leak K⁺ currents in the manifestation of PRs in PnC neurons. Moreover, we do not know whether the early and late phases of the PR are differentially controlled. For example, nicotinic and muscarinic activation of pontine neurons has been reported previously (Stevens et al. 1992). Some PnC neurons may resemble thalamic cells, which, in response to PPN stimulation, show a nicotinic receptor-induced decrease in R_in, followed by a muscarinic receptor-induced increase in R_in (Curro-Dossi et al. 1991). However, pharmacological studies need to be performed to determine whether early and late phases might be modulated differentially.

Functional implications

The PPN sends widespread projections throughout the pontomedullary reticular formation (Reese et al. 1995; Rye 1997; Scarnati and Florio 1997), including the anterior pontine region (PnO) (Mitani et al. 1988). Injections of cholinergic agonists into a region called the pontine inhibitory area induce a REM sleep-like state (Baghdoyan et al. 1984; Yamamoto et al. 1990), an effect thought to be mediated via muscarinic blockade of an outward, G protein-coupled, K⁺ current (Shuman et al. 1995). Lesion of this area can produce REM sleep without atonia (Henley and Morrison 1974; Jouvet 1975), but these lesions may also damage neurons/axons responsible for REM sleep initiation and maintenance (Sanford et al. 1994). Comprehensive electrophysiological studies on pontine reticular neurons have been performed by McCarley (reviewed in Steriade and McCarley 1990), who found LTS burst and nonbursting neurons, some with a transient outward A current (similar to those reported herein for the PnC). These neurons receive excitatory cholinergic input from the laterodorsal tegmental nucleus (LDT) (Imon et al. 1996) and are depolarized by nicotinic and muscarinic agonists (Gerber et al. 1991; Stevens et al. 1993), although some cells are hyperpolarized by muscarinic agonists (Greene et al. 1989).

Descending PPN projections to the posterior pontine region (PnC) may be involved in, for example, modulation of the startle response (SR) (see comprehensive reviews on the SR for more information: Davis 1984; Koch 1999; Swerdlow et al. 1992). The pathway for the SR, which includes giant neurons in the PnC, has been worked out (Davis 1984). These giant cells make up only about 1% of PnC neurons (Koch 1999), but when lesioned by excitotoxic agents injected into this region, the amplitude of the SR is reduced (Koch et al. 1992). PPN lesions have been reported to reduce prepulse