INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The nucleus locus coeruleus (LC) contains the major group of noradrenergic neurons and projects broadly throughout the CNS. LC neurons play an important role in many physiological functions such as autonomic control, arousal, sleep, cognition, memory, and emotion (Aston-Jones et al. 1991). All pontine noradrenergic groups, including the LC, the A5, and A7 cell groups, contribute to the noradrenergic innervation of the spinal cord (Westlund et al. 1983). In the rat, LC neurons project primarily to the dorsal horn and intermediate zone, whereas A5 and A7 neurons project to somatic motor neurons and the intermediolateral cell column (Fritschy and Grzanna 1990). There is considerable evidence that the bulbospinal noradrenergic system plays a significant role in pain modulation and analgesia (Basbaum and Fields 1984; Jones 1991; Jones and Gebhart 1986a, 1987). For example, stimulation of the LC causes an increase in norepinephrine release in the spinal cord, which inhibits the nociceptive transmission in the dorsal horn through ₂-adrenergic receptors (Crawley et al. 1979; Jones and Gebhart 1987; Pan et al. 2002; Westlund et al. 1983). Also, the antinociception induced by electrical and chemical stimulation in the LC is diminished by depletion of neuronal norepinephrine with 6-hydroxydopamine (Margalit and Segal 1979) or by intrathecal administration of ₂- but not ₁-adrenergic receptor antagonists (Jones and Gebhart 1986b). These studies suggest that stimulation of LC neurons activates the descending noradrenergic system, which inhibits nociceptive transmission through ₂-adrenergic receptors in the spinal cord.

There is substantial evidence indicating that the LC is one of the major targets of endogenous opioid neurons and is an important structure in mediating the analgesic effect of opioids. In this regard, the enkephalinergic neurons in the rostral medulla provide major afferent inputs to noradrenergic LC neurons (Drolet et al. 1992). Also, morphine microinjected into the LC produces analgesia (Bodnar et al. 1990; Jones and Gebhart 1988). Furthermore, destruction of the LC attenuates the antinociceptive efficacy of systemically administered morphine (Kostowski et al. 1978), while electrical stimulation in the LC enhances morphine-induced antinociception (Segal and Sandberg 1977). However, these behavioral data are not consistent with the electrophysiological studies showing that systemic or local administration of opioids generally inhibits the discharge activity of LC neurons (Bird and Kuhar 1977; Hirata and Aston-Jones 1996; Valentino and Wehby 1988). The reasons underlying this discrepancy are not clear. It has been shown that anesthetics may alter the effect of morphine on the neuronal activity in the LC (Valentino and Wehby 1988). It is important to note that these previous electrophysiological studies on the effect of opioids on LC neurons have largely ignored the functional diversity of the heterogenous output neurons in the LC.

The supraspinal -opioid receptors (OR) are also involved in the modulation of nociception. For instance, intracerebroventricular injection of OR agonists, such as [D-Pen², D-Pen⁵]-enkephalin (DPDPE), produces analgesia, which is blocked by OR antagonists (Calcagnetti et al. 1988; Ossipov et al. 1995). Furthermore, microinjection of DPDPE into the rostral ventromedial medulla produces antinociception (Kovelowski et al. 1999; Thorat and Hammond 1997). Additionally, both autoradiographic and immunocytochemistry studies have shown that the OR is located in the LC (Arvidsson et al. 1995; Mansour et al. 1995). An ultrastructural study has further demonstrated that the OR is primarily located on the GABAergic and glutamatergic nerve terminals in the LC (van Bockstaele et al. 1997). Since GABAergic terminals are juxtaposed to noradrenergic neurons in the LC, there is a likely potential functional interaction between GABAergic terminals and noradrenergic LC neurons (Berod et al. 1984). Thus the presynaptic OR in the LC may play an important role in the control of the activity of LC neurons through regulation of the inhibitory GABAergic input. However, the functional significance of OR in the control of the excitability of spinally projecting LC neurons has not been studied previously. In the current study, using both retrograde labeling and in vitro electrophysiological techniques, we tested the hypothesis that activation of presynaptic OR attenuates the GABAergic synaptic input, which contributes to excitation of spinally projecting LC neurons.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Retrograde labeling of spinally projecting LC neurons

The spinal cord of Sprague-Dawley rats (3-4 wk old; Harlan, Indianapolis, IN) was injected with a fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI; Molecular Probes, Eugene, OR) under halothane anesthesia. The tracer (10-15 mg dissolved in 200 µl of DMSO) was pressure-injected bilaterally into the thoracic (T₃-T₄) spinal cord in a volume of 100 nl using a glass micropipette (15- to 25-µm tip diam). The pipette was positioned stereotaxically in the dorsal horn of the spinal cord, and the DiI injection was performed using a microinjection system (Picospritzer II; General Valve, Fairfield, NJ) and monitored through an operating microscope, as described previously (Kangrga and Loewy 1994). DiI was chosen because this tracer has been used in a previous study and is devoid of toxicity to neurons (Kangrga and Loewy 1994). After injection, the muscles and skin were sutured and the wound was closed. Animals were returned to their cages for 3-7 days, which is sufficient time to permit retrograde tracer transport to the LC. The surgical preparations and experimental protocols were approved by the Animal Care and Use Committee of the Penn State University College of Medicine and conformed to the National Institutes of Health guidelines on the ethical use of animals. All efforts were made to minimize both the suffering and number of animals used.

Slice preparations

Three to 7 days after the DiI injection, the rats were rapidly decapitated under halothane anesthesia, and the brain was quickly removed and immersed into ice-cold, preoxygenated (95% O₂-5% CO₂) sucrose artificial cerebrospinal fluid (aCSF) solution for 1-2 min. A tissue block containing the LC was cut and glued onto the stage of the vibratome (Technical Product International, St. Louis, MO). Coronal slices containing the LC (250-300 µm in thickness) were cut from the tissue block in ice-cold sucrose aCSF. The slices were preincubated in the aCSF oxygenated with 95% O₂-5% CO₂ at 36°C for 1 h before being transferred into the recording chamber. The sucrose aCSF was composed of the following (in mM): 234 sucrose, 3.6 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 1.2 NaH₂PO₄, 12.0 glucose, and 26.0 NaHCO₃. The aCSF contained the following (in mM): 126.0 NaCl, 2.5 KCl, 1.3 MgSO₄, 2.4 CaCl₂, 1.2 NaH₂PO₄, 11.0 glucose, and 25.0 NaHCO₃ (pH 7.4; osmolarity, 295-300 mOsm).

Recordings of postsynaptic currents of LC neurons

Recordings of postsynaptic currents were performed using the whole cell voltage-clamp method, as we described previously (Li and Pan 2001; Li et al. 2001). The electrode for the whole cell recordings was triple-pulled with a puller (P-97; Sutter Instrument, Novato, CA) using borosillicate glass capillaries (OD 1.2 mm; ID 0.86 mm; World Precision Instruments, Sarasota, FL). The resistance of the pipette tip for the recordings of postsynaptic currents was 5-10 M when filled with the intracellular solution containing the following (in mM): 110.0 Cs₂SO₄, 0.5 CaCl₂, 2.0 MgCl₂, 5.0 EGTA, 5.0 HEPES, 5.0 ATP-Mg, 5 tetraethyl ammonium chloride (TEA), and 1 guanosine 5'-O-(2-thiodiphosphate) (GDP--S); adjusted to pH 7.2-7.3 with 1 M of CsOH and osmolarity 280-290 mOsm. GDP--S and K⁺-channel blockers (Cs⁺ and TEA) were used to inhibit a potential postsynaptic effect of DPDPE through the action of G proteins and to block the activation of K⁺ channels that may result from the postsynaptic effect, respectively. The slice was placed in a glass-bottomed chamber (Warner Instruments, Hamden, CT) and fixed with a grid of parallel nylon threads supported by a U-shaped stainless steel weight. The slice was perfused at 3.0 ml/min at 36°C maintained by an in-line solution heater and a temperature controller (TC-324; Warner Instruments). The solution in the recording chamber can be completely exchanged within 1 min.

DiI-labeled LC neurons were briefly identified in the slice with epifluorescence (rhodamine filter; Fig. 1) on a fixed stage microscope (BX50WI; Olympus, Tokyo, Japan). The neurons were then viewed with Nomarski optics through a water immersion objective. The tissue image was captured and enhanced through a CCD camera and displayed on a video monitor. After neurons were identified, positive pressure was applied to the pipette, which was then advanced toward the identified neuron (Fig. 1). Once the pipette touched the membrane of the neuron, the pressure was immediately released and slight negative pressure was applied to establish a high resistance seal. The cell membrane was then ruptured by further suction to record in the whole cell configuration. Recordings of postsynaptic currents began about 5 min after the whole cell access was established and the current reached a steady state. Miniature inhibitory postsynaptic currents (mIPSCs) and miniature excitatory postsynaptic currents (mEPSCs) were recorded at a holding potential of 0 and 70 mV, respectively (Li and Pan 2001; Li et al. 2001; Pan et al. 2002). All mIPSCs were recorded in the presence of tetrodotoxin (TTX, 1 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM), and mEPSCs were recorded in the presence of TTX (1 µM) and bicuculline (20 µM). Since miniature postsynaptic currents are Ca²⁺-independent, we did not test the effect of reducing the extracellular Ca²⁺ level or Ca²⁺ channel blockers on miniature postsynaptic currents in this study.

View larger version (37K): [in this window] [in a new window]   Fig. 1. Photomicrographs showing a 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI)-labeled locus coeruleus (LC) neuron in the brain slice. A: LC neuron labeled with DiI (red) viewed with fluorescence illumination. B: same DiI-labeled LC neuron (*) with an attached recording patch-clamp electrode () viewed with bright-field Nomarski optics.

To study the evoked IPSCs or EPSCs (eIPSCs/eEPSCs) in DiI-labeled LC neurons, synaptic currents were evoked by electrical stimulation (0.1 ms, 0.3-0.8 mA, and 0.2 Hz) through a bipolar tungsten electrode connected to a stimulator (World Precision Instruments). The tip of the stimulating electrode was placed 200-800 µm away from the recorded LC neuron. The internal pipette solution contained the following (in mM): 110.0 Cs₂SO₄, 0.5 CaCl₂, 2.4 MgCl₂, 5.0 BAPTA, 10.0 HEPES, 5 Na₂ATP, 0.33 GTP-tris salt, 10.0 lidocaine N-ethyl bromide (QX314), and 5.0 TEA-Cl (pH 7.3; osmolarity, 275-280 mOsm), as described in a previous study (Chiou and Huang 1999). TEA and QX314 were used to prevent K⁺ and Na⁺ channel activation, respectively, in these voltage-clamp experiments. Based on the optimal reversal potentials determined for CNQX-sensitive EPSCs and bicuculline-sensitive IPSCs using this pipette solution (Chiou and Huang 1999), the eEPSCs and eIPSCs were recorded at a holding potential of 70 and 10 mV, respectively.

Recordings of the discharge activity of LC neurons

Recordings of the discharge activity and membrane potential were performed using the whole cell current clamp method. The tissue processing, cell identification, and recording procedures were similar to those used for postsynaptic current recordings. The resistance of the recording pipettes was 5-10 M when filled with the following intracellular solution (in mM): 135.0 potassium gluconate, 5 KCl, 0.5 CaCl₂, 2.0 MgCl₂, 5.0 EGTA, 5.0 HEPES, 5.0 ATP-Mg, and 0.5 Na-GTP (pH 7.2-7.3; osmolarity, 280-290 mOsm). Recordings of the cell activity began about 5 min after the whole cell access was established and the membrane potential reached a steady state. Signals were filtered, recorded, stored, and analyzed as described above. A liquid junction potential of 15 mV (for the potassium gluconate pipette solution) was corrected during off-line analysis.

Dopamine--hydroxylase immunocytochemistry staining and intracellular labeling

To ensure that the DiI-labeled cells were noradrenergic neurons, some recorded LC neurons were labeled with the pipette solution containing 0.1% biocytin (Li and Pan 2001). Immunocytochemistry staining of dopamine--hydroxylase (DH), a specific marker for noradrenergic neurons, was performed. At the end of the experiment, the slice was fixed by submersion in 4% paraformaldehyde in PBS (pH:7.4) after recording and kept at 4°C for 2-10 days. The sections were cut to 35 µm in thickness and collected free floating in 0.1 M PBS. For DH staining, the sections were rinsed in 50 mM Tris buffered saline (TBS) and blocked in 4% normal goat serum in TBS for 1 h. Sections were then incubated with the mouse anti-DH monoclonal antibody (1:300; Chemicon International, Temecula, CA) for 2 h at room temperature and overnight at 4°C. Subsequently, sections were rinsed in TBS and incubated with the secondary antibody [goat anti-mouse IgG conjugated with Alexa Fluor-488 (5 µg/ml; Molecular Probe)] for 1.5 h. To visualize LC cells labeled by biocytin, the sections were rinsed for 20 min in TBS after the DH staining and incubated with streptavidin conjugated Alexa Fluor-594 (5 µg/ml, Molecular Probe) for 1.5 h at room temperature. The sections were rinsed in TBS for 40 min and mounted on slides, dried, and coverslipped. The sections were viewed using a confocal microscope (Carl Zeiss, Jena, Germany), and the areas of interest were photographed.

Experimental protocols

The resting membrane potential and the input resistance were continuously monitored throughout the recording period. Recordings were abandoned if the input resistance changed more than 15% (Li and Pan 2001; Li et al. 2001). After recording the mIPSCs or mEPSCs for 3 min as the baseline control, 1 µM DPDPE was perfused into the slice for 4-5 min. To determine the role of OR in the effect of DPDPE on mIPSCs and mEPSCs of DiI-labeled neurons in the LC, the specific OR antagonist, 1 µM naltrindole, was applied to the slice for 5 min followed by perfusion of 1 µM DPDPE plus 1 µM naltrindole. To further examine the differential effect of DPDPE on the GABAergic and glutamatergic synaptic inputs to DiI-labeled LC neurons, the effect of 1 µM DPDPE on eIPSCs and eEPSCs was tested using the same protocol as described above.

To study the effect of DPDPE and bicuculline on the excitability of DiI-labeled LC neurons, the effect of 1 µM DPDPE or 20 µM bicuculline on the discharge activity of LC neurons was tested using the whole cell current-clamp recordings. Finally, to examine the role of GABA release in the excitatory effect of DPDPE on the discharge activity of DiI-labeled LC neurons, 1 µM DPDPE was perfused into the slice in the presence of 20 µM bicuculline. TTX, CNQX, bicuculline methiodide, DPDPE, and naltrindole were obtained from Sigma-RBI (St. Louis, MO). Drugs were freshly dissolved in the aCSF and perfused into the slice chamber using syringe pumps. The effective concentrations of DPDPE, naltrindole, bicuculline, and CNQX have been determined in previous studies (Chiou and Huang 1999; Kohno et al. 1999; Li and Pan 2001; Li et al. 2001; Vaughan and Christie 1997).

Data analysis

Data are presented as means ± SE. The mIPSCs, mEPSCs, and the discharge frequency of DiI-labeled LC neurons were analyzed off-line with a peak detection program (Minianalysis; Synaptosoft, Leonia, NJ). The cumulative probability of the amplitude and inter-event interval of mIPSCs and mEPSCs was compared using the Komogorov-Smirnov test, which estimates the probability that two distributions are similar. At least 100 mIPSCs and mEPSCs were used in each analysis. The effects of drugs on the amplitude of eEPSCs and eIPSCs were analyzed using Clampfit (Axon Instruments), as we described previously (Pan et al. 2002). Statistical analyses of the effects of drugs on the amplitude and frequency of postsynaptic currents and the firing activity of LC neurons were determined by paired t-test or nonparametric ANOVA test followed by a post hoc test. P < 0.05 was considered to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Stable recordings were obtained from slices maintained in vitro for 4-6 h. Once the whole cell recording was established, the mIPSCs, mEPSCs, or spontaneous activity of DiI-labeled LC neurons typically can be recorded for 30 min without noticeable changes in the resting membrane potential and input resistance. In all the slices examined, the DiI-labeled LC neurons were DH positive (Fig. 2A). We also confirmed that all the recovered cells labeled with biocytin had DH immunoreactivity and were located in the LC proper (Fig. 2B).

View larger version (48K): [in this window] [in a new window]   Fig. 2. Photomicrographs showing DiI-labeled LC noradrenergic neurons. Images are in all cases single confocal optical sections. A: LC neurons labeled with DiI (red, a), dopamine--hydroxylase (DH) immunoreactivity (green, b), and digitally merged images (c) of DiI and DH staining viewed under a confocal microscope. Magnification, ×800. B: confocal images showing a recorded LC neuron labeled with biocytin (red, a), DH immunoreactivity (green, b), and digitally merged images (c) of biocytin and DH staining. Magnification, ×2,400.

Effect of DPDPE on mIPSCs and mEPSCs of spinally projecting LC neurons

The mIPSCs of DiI-labeled LC neurons were abolished by 20 µM bicuculline (n = 6, Fig. 3A), and the mEPSCs were eliminated by 10 µM CNQX (n = 6, Fig. 3B). In the presence of TTX and CNQX, application of 1 µM DPDPE significantly decreased the frequency of mIPSCs of nine DiI-labeled LC neurons from 2.1 ± 0.5 to 0.7 ± 0.2 Hz (P < 0.05, Fig. 4). The effect of DPDPE was observed within 2 min following DPDPE perfusion, and the frequency of mIPSCs generally returned to the control level 15-20 min after washout. However, DPDPE did not significantly alter the amplitude of mIPSCs of these neurons (24.5 ± 2.4 vs. 24.2 ± 2.3 pA, Fig. 4). The cumulative probability analysis of mIPSCs revealed that the distribution pattern of the inter-event interval of mIPSCs shifted toward right in response to DPDPE, but the distribution pattern of the amplitude was not affected by DPDPE (Fig. 4B). The effect of DPDPE on mIPSCs was further analyzed by measuring the decay time constant of the mIPSCs. The decay phase of mIPSCs was well-fitted by a single exponential fit. The decay time constant of mIPSCs before and during DPDPE application was identical (4.0 ± 0.3 ms). In the presence of 1 µM naltrindole, 1 µM DPDPE failed to significantly alter the frequency of mIPSCs of six DiI-labeled LC neurons (2.0 ± 0.7 vs. 2.0 ± 0.6 Hz, Fig. 4D).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and miniature excitatory postsynaptic currents (mEPSCs) of DiI-labeled neurons in the LC. A: original tracings of mIPSCs recorded at a holding potential of 0 mV from a DiI-labeled neuron in the LC during control and application of 20 µM bicuculline. B: representative tracings of mEPSCs recorded at a holding potential of 70 mV from a DiI-labeled LC neuron during control and application of 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Note that bicuculline completely blocked spontaneous mIPSCs, while CNQX completely blocked spontaneous mEPSCs.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Effect of 1 µM [D-Pen2, D-Pen5]-enkephalin (DPDPE) on mIPSCs of DiI-labeled neurons in the LC. A: representative traces from a DiI-labeled neuron in the LC showing spontaneous mIPSCs recorded during control and application of 1 µM DPDPE. B: cumulative probability plot showing the distribution of the inter-event interval and the peak amplitude of this neuron during control and DPDPE application. C: summary data showing the effect of 1 µM DPDPE on the frequency and the amplitude of mIPSCs (n = 9). D: summary data showing the effect of 1 µM naltrindole plus 1 µM DPDPE on the frequency and amplitude of mIPSCs (n = 6). Data presented as means ± SEM; *P < 0.05 compared with the control (paired t-test).

In the presence of TTX and bicuculline, application of 1 µM DPDPE did not significantly alter the frequency and amplitude of mEPSCs in nine DiI-labeled LC neurons (Fig. 5). The cumulative probability analysis of mEPSCs of these neurons demonstrated that the distribution patterns of the inter-event interval and the amplitude were not affected by 1 µM DPDPE (Fig. 5B).

View larger version (26K): [in this window] [in a new window]   Fig. 5. Effect of 1 µM DPDPE on mEPSCs of DiI-labeled neurons in the LC. A: representative tracings from a DiI-labeled neuron in the LC showing spontaneous mEPSCs recorded at a holding potential of 70 mV during control and application of 1 µM DPDPE. B: cumulative probability plot showing the distribution of the inter-event interval and the peak amplitude of this neuron during control and DPDPE application. C: summary data showing the effect of 1 µM DPDPE on the frequency and the amplitude of mEPSCs. Data presented as means ± SE (n = 9).

Effect of DPDPE on eIPSCs and eEPSCs of spinally projecting LC neurons

To determine the relative contributions of GABAergic and glutamatergic inputs to spinally projecting LC neurons and the effect of DPDPE, the eIPSCs and eEPSCs were recorded from a separate group of DiI-labeled LC neurons. In 16 of 20 neurons studied, both IPSCs and EPSCs were evoked at the same stimulating intensity. All the eIPSC and eEPSCs appeared to be monosynaptic since the latency was constant following electrical stimulation. Also, neither conduction failure nor an increase in latency occurred when stimulation frequency was increased to 20 Hz, consistent with the criteria used for identification of the monosynaptic input (Kohno et al. 1999). The peak amplitude of eIPSCs was 252.0 ± 43.3 pA, which was about three times larger than that of eEPSCs (78.9 ± 8.3 pA, P < 0.05, n = 16, Fig. 6).

View larger version (19K): [in this window] [in a new window]   Fig. 6. Differential effects of DPDPE on evoked IPSCs (eIPSCs) and evoked EPSCs (eEPSCs) of DiI-labeled neurons in the LC. A: original tracings of eIPSCs (a, holding potential = 10 mV) and eEPSCs (b, holding potential = 70 mV) of a DiI-labeled LC neuron during control and application of 1 µM DPDPE. Inhibitory effect of DPDPE was reversed by 1 µM naltrindole. Traces were averages of 8 consecutive responses. B: summary data showing the effect of DPDPE on the peak amplitude of eIPSCs and eEPSCs in 8 DiI-labeled LC neurons. Data presented as means ± SE; *P < 0.05 compared with the control (paired t-test).

The effect of DPDPE on eIPSCs and eEPSCs was further examined in 8 of 16 DiI-labeled neurons. DPDPE significantly inhibited the peak amplitude (~49.4%) of eIPSCs but not eEPSCs (Fig. 6). In three DiI-labeled LC neurons, 1 µM DPDPE failed to alter significantly the amplitude of eIPSCs in the presence of 1 µM naltrindole (Fig. 6A). In four DiI-labeled LC neurons tested, the eIPSCs were completely eliminated by perfusion of 20 µM bicuculline, while the eEPSCs were abolished by 20 µM CNQX (data not shown).

Effects of DPDPE and bicuculline on the discharge activity of DiI-labeled LC neurons

Under current-clamp conditions, the spontaneous discharge activity was recorded from 31 DiI-labeled LC neurons. A majority of DiI-labeled neurons (47/58, 81%) in the LC fired spontaneously (Fig. 7). The frequency of spontaneous activity ranged from 0.1 to 19.6 Hz (3.1 ± 0.7 Hz). Application of 1 µM DPDPE significantly increased the frequency of spontaneous discharge activity in 9 of 11 DiI-labeled LC neurons from 4.6 ± 0.7 to 6.2 ± 1.0 Hz (an increase of 34.8%, P < 0.05, Fig. 7). In these nine LC neurons, the membrane potential of five cells was depolarized from 58.5 ± 4.9 to 51.8 ± 3.7 mV (P < 0.05), one was slightly hyperpolarized (63.9 to 68.6 mV), and the remaining three were not changed by DPDPE. The firing frequency of 1 of 11 LC neurons was decreased from 7.9 to 6.1 Hz; the remaining one neuron was not affected by DPDPE.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Effect of 1 µM DPDPE on the spontaneous discharge activity of a DiI-labeled LC neuron. A: representative recordings showing spontaneous discharge activity of the same LC neuron during control and DPDPE application. B: summary data showing the effect of 1 µM DPDPE on the firing activity of 9 DiI-labeled LC neurons. Data presented as means ± SE; *P < 0.05 compared with the control (paired t-test).

The discharge activity of 20 DiI-labeled LC neurons was recorded before and after application of 20 µM bicuculline. While bicuculline did not significantly alter the firing activity of 7 DiI-labeled LC neurons, it significantly increased the discharge frequency of 13 neurons (from 1.8 ± 0.5 to 2.8 ± 0.6 Hz, P < 0.05). In these 13 neurons, the membrane potential of 7 neurons was depolarized from 55.8 ± 4.6 to 47.4 ± 3.6 mV (P < 0.05), and the membrane potential of the other 6 neurons was not altered by 20 µM bicuculline. In 6 of the above 13 DiI-labeled LC neurons, the effect of DPDPE plus bicuculline was further tested. In the presence of 20 µM bicuculline, application of 1 µM DPDPE failed to significantly alter the discharge frequency of these neurons (2.8 ± 0.6 vs. 3.0 ± 0.6 Hz, Fig. 8).

View larger version (28K): [in this window] [in a new window]   Fig. 8. Effect of 20 µM bicuculline plus 1 µM DPDPE on the discharge activity of DiI-labeled neurons in the LC. A: histogram of the firing frequency of a LC neuron during control, application of 20 µM bicuculline, and application of 20 µM bicuculline plus 1 µM DPDPE. The bin size is 1 s. B: representative tracings showing the firing activity of the same LC neuron during control, application of bicuculline, and bicuculline plus DPDPE. C: summary data showing a lack of effect of 1 µM DPDPE on the firing activity of LC neurons in the presence of 20 µM bicuculline. Data presented as means ± SE (n = 6). *P < 0.05 compared with the control (Kruskal-Wallis ANOVA test followed by Dunn's post hoc test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This is the first study determining the functional significance of OR in the regulation of inhibitory and excitatory synaptic inputs to spinally projecting LC neurons. In this study, we found that DPDPE significantly decreased the frequency of mIPSCs, but not mEPSCs, of spinally projecting LC neurons without altering their amplitude and the decay time constant. The inhibitory effect of DPDPE on GABA-mediated mIPSCs of neurons was abolished by a OR antagonist, naltrindole. Furthermore, DPDPE preferentially inhibited the peak amplitude of eIPSCs, but not eEPSCs, of LC neurons. Additionally, we examined the effect of activation of OR on the discharge activity of spinally projecting LC neurons. We found that DPDPE or bicuculline significantly increased the firing frequency of a majority of LC neurons. The excitatory effect of DPDPE on spinally projecting LC neurons was diminished in the presence of bicuculline. Therefore these data provide important functional evidence that activation of presynaptic OR excites a population of spinally projecting LC noradrenergic neurons through inhibition of GABAergic synaptic inputs. This mechanism may contribute to the analgesic action produced by -opioid agonists.

Since the LC contains heterogenous output neurons (Aston-Jones et al. 1991; Fritschy and Grzanna 1990), we used a combination of retrograde labeling and in vitro electrophysiological techniques to specifically examine the synaptic inputs to spinally projecting LC neurons. The approach used in this study has important advantages for studying heterogenous neuronal groups with spinal projections since retrogradely labeled bulbospinal neurons can be visualized in defined cytoarchitectonic regions in vitro. The LC neurons receive both GABAergic and glutamatergic inputs (Aston-Jones et al. 1991). Both tract-tracing and electrophysiological studies have revealed that the major GABAergic inputs to the LC are from the nucleus prepositus hypoglossi (Ennis and Aston-Jones 1989a,b), and the nucleus paragigantocellularis provides the major glutamatergic inputs to the LC (Ennis and Aston-Jones 1988; Ennis et al. 1992). In the present study, we find that both spontaneous mIPSCs and mEPSCs were recorded in spinally projecting LC neurons. Since mIPSCs of LC neurons were eliminated by bicuculline, the mIPSCs represent the quantal release of GABA from the presynaptic terminals. On the other hand, mEPSCs of LC neurons were completely blocked by CNQX, suggesting that the mEPSCs reflect the quantal release of glutamate from the presynaptic terminals. Our data are compatible with a previous study showing that the postsynaptic potential evoked by focal stimulation within the LC results from glutamate acting mainly at non-N-methyl-D-aspartate (NMDA) receptors and GABA acting at GABA_A receptors (Cherubini et al. 1988). These data suggest that these LC neurons are dually modulated by inhibitory GABAergic and excitatory glutamatergic inputs. Using the same stimulation intensity and by isolating EPSCs (70 mV) and IPSCs (10 mV) based on their reversal potentials, we determined the relative contributions of EPSCs and IPSCs to synaptic inputs to DiI-labeled LC neurons. We found that GABAergic inputs had a larger influence on the synaptic responses of LC neurons than glutamatergic inputs at the same driving force. This finding is similar to what reported for ventrolateral periaqueductal gray neurons (Chiou and Huang 1999). Therefore the inhibitory GABAergic synaptic input likely plays a dominant role in controlling the excitability of spinally projecting LC neurons.

The LC noradrenergic neurons receive extensive afferent inputs from enkephalinergic neurons in the rostral medulla (Drolet et al. 1992). In the LC, enkephalin is the most concentrated endogenous opioid peptide (Zamir et al. 1984), although enkephalin-containing neurons are not present in the LC (Drolet et al. 1992). Also, LC neurons are innervated directly and indirectly by the other enkephalin-rich areas, such as the periaqueductal gray (Ennis et al. 1991). In the present study, we observed that DPDPE significantly reduced the frequency without altering the amplitude and kinetics of mIPSCs of spinally projecting LC neurons. This finding is consistent with the ultrastructural evidence that the OR is located on the presynaptic GABAergic terminals in the LC (van Bockstaele et al. 1997). Furthermore, we have demonstrated that DPDPE significantly inhibited GABA-mediated eIPSCs of DiI-labeled LC neurons. The binding affinity of DPDPE for the -opioid receptor is 175 times greater than that for the µ-opioid receptor (Mosberg et al. 1983). Since the effect of DPDPE on eIPSCs/mIPSCs was completely abolished by a specific -opioid antagonist, naltrindole, it suggests that the effect of DPDPE on neurotransmission in the LC is through activation of -opioid receptors. Thus our study provides important physiological evidence that activation of presynaptic OR attenuates inhibitory GABAergic inputs to spinally projecting LC neurons. It has been reported that the antinociceptive effect of DPDPE is mediated, in part, by µ-opioid receptors (Fraser et al. 2000; He and Lee 1998). Because we did not determine if the effect of DPDPE is affected by µ-opioid antagonists in this study, we cannot exclude the possibility that the effect of DPDPE on LC neurons may be partially mediated by µ-opioid receptors. Previous studies have indicated that OR may play a role in regulating glutamate release in the LC. In this regard, stimulation of nucleus paragigantocellularis or local application of glutamate activates LC neurons (Ennis and Aston-Jones 1988; Ennis et al. 1992). Also, the OR is located presynaptically on the glutamatergic terminals in the LC (van Bockstaele et al. 1997). However, in the present study, we did not observe any effect of DPDPE on the mEPSCs and eEPSCs of DiI-labeled LC neurons, suggesting a lack of a functional role of presynaptic OR in the regulation of glutamatergic synaptic inputs to spinally projecting LC neurons. The reasons for the differential effect of DPDPE on the GABAergic and glutamatergic synaptic inputs to spinally projecting LC neurons are not fully known. It is possible that the lack of effect of DPDPE on mEPSCs in spinally projecting LC neurons may result, in part, in inadequate numbers of presynaptic OR or a lack of presynaptic K⁺ channels that couple to the OR on the glutamatergic nerve terminals (Vaughan et al. 1997).

GABA is an important inhibitory neurotransmitter regulating the activity of LC neurons since GABA inhibits spontaneous firing of LC neurons by increasing Cl conductance, which hyperpolarizes LC neurons (Shefner and Osmanovic 1991). It has been demonstrated that GABA_A receptors are present in the LC (Palacios et al. 1981), and almost one-half of the terminals in the LC take up GABA (Perez de la Mora et al. 1981). Also, the glutamic acid decarboxylase-positive nerve terminals are juxtaposed to cell bodies and dendrites of noradrenergic neurons in the LC (Berod et al. 1984). The major source of GABAergic inputs to the LC appears to originate from the nucleus prepositus hypoglossi (Ennis and Aston-Jones 1989a). This is because stimulation of the nucleus prepositus hypoglossi inhibits LC neurons, and such an effect is blocked by GABA_A antagonists (Ennis and Aston-Jones 1989a). In the present study, we observed that bicuculline significantly increased the discharge activity of a population of spinally projecting LC neurons with a decrease in the membrane potential (depolarization), suggesting that these LC neurons were tonically inhibited by synaptic GABA release. Furthermore, we found that DPDPE significantly increased firing activity and depolarized the membrane potential of most spinally projecting LC neurons. This effect of DPDPE is probably due to disinhibition of the GABAergic input to these neurons because the direct postsynaptic effect of DPDPE on the LC neurons is hyperpolarization by opening of G protein-coupled inwardly rectifying potassium channels (North et al. 1987). This interpretation is supported by our data showing that the excitatory effect of DPDPE on these LC neurons was completely eliminated in the presence of bicuculline. Thus activation of presynaptic OR could excite these LC neurons through disinhibition of GABAergic inputs. These data strongly suggest that DPDPE directly inhibits tonically active GABAergic synaptic inputs, thus disinhibiting the LC output neurons projecting to the spinal cord. Since the postsynaptic effect of DPDPE on LC neurons has been shown in a previous study (North et al. 1987), we focused on the functional significance of presynaptic OR in the regulation of excitatory and inhibitory synaptic inputs to LC neurons in this study. DPDPE increased the firing activity of most DiI-labeled cells, suggesting an important role of presynaptic OR is inhibition of GABAergic inputs to spinally projecting LC neurons. It should be acknowledged that young rats were used in this study, and it is uncertain if DPDPE and bicuculline have similar effects on LC neurons in adult animals. Since we did not examine the effect of DPDPE on nonspinally projecting LC cells in this study, it is not clear whether the DPDPE-mediated presynaptic disinhibition and its excitatory effect are selective for spinally projecting LC neurons.

We observed that DPDPE did not increase the excitability of all spinally projecting LC neurons. It is important to emphasize that the overall effect of DPDPE on spinally projecting LC neurons depends critically on the dynamic balance of its presynaptic (disinhibition) and postsynaptic inhibitory (hyperpolarization) actions. We found that, in most of the spinally projecting LC neurons, the major effect of DPDPE is the attenuation of the inhibitory drive of GABAergic inputs. It overcomes the DPDPE-induced hyperpolarization and, thus results in the excitation of these neurons. Collectively, our study suggests that despite postsynaptic inhibition of LC neurons produced by release of endogenous opiates or following systemically administered opioids, the OR mediated presynaptic disinhibition may lead to increased spinal norepinephrine release and analgesia. This presynaptic disinhibition produced by activation of OR may account for the analgesic actions of supraspinal OR activation or analgesia elicited by intra-LC injection of nonselective opioid agonists.

In summary, this study provides important new information that activation of presynaptic OR by DPDPE increases the excitability of a population of spinally projecting noradrenergic LC neurons by inhibition of GABAergic inputs. Thus the presynaptic OR likely plays an important role in the regulation of the excitability of spinally projecting LC neurons and the descending noradrenergic inhibitory system. These findings are important for our understanding of the physiological function of presynaptic OR in the LC and the mechanisms of the analgesic action produced by OR agonists.