INTRODUCTION

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The locus coeruleus (LC) is a compact noradrenergic nucleus in the pons that sends extensive projections throughout the CNS (Amaral and Sinnamon 1977; Dahlström and Fuxe 1964; Moore and Bloom 1979; Ungerstedt 1971). The LC has been considered to play an important role in such brain functions as vigilance, attention, and mediation of stress response (Aston-Jones et al. 1991; Foote et al. 1980, 1983; Hobson et al. 1975; Olpe et al. 1985; Tanaka et al. 1983; Tsuda et al. 1982). Tonic activity of rat LC neurons was found to vary in association with changes in behavioral state. The highest levels of neuronal activity in the LC are observed during wakefulness, while lower firing rates occur when animals are drowsy (AstonJones and Bloom 1981a,b; Foote et al. 1980). It has been proposed that recurrent collaterals of the LC projections release norepinephrine (NE) on to the LC neurons themselves (Aghajanian et al. 1977). Egan et al. (1983) suggested that released NE onto other LC neurons mediates the inhibitory postsynaptic potential (IPSP) through ₂-adrenoceptors. Firing rate of spontaneous action potentials, a pacemaker-like regulatory activity in the LC neurons, was reduced during the hyperpolarization induced by exogenously applied NE via ₂-adrenoceptors (Williams and North 1985).

Methylphenidate (MPH; Ritalin) is the most widely used drug for the treatment of children presenting the attention deficit hyperactivity disorder (ADHD) (Hunt et al. 1984). Biochemical studies have shown that MPH, like D-amphetamine, enhances the release and/or blocks the re-uptake of NE and dopamine (DA) in mammalian brain (Axelrod 1970; Carlsson et al. 1966; Ferris et al. 1972; Hendley et al. 1972; Raiteri et al. 1974; Ross 1978). Administration of MPH decreased the rate of spontaneous firing in rat LC neurons in vitro (Lacroix and Ferron 1988; Olpe et al. 1985). However, little is known about the mechanism underlying the inhibitory action of MPH on the neuronal activity in the LC. The purpose of the present study is to determine the effects of MPH on membrane potential and neuronal excitability in LC neurons and to examine whether these actions are mediated by intrinsic NE. Preliminary findings of this work have appeared in abstract form (Kidani et al. 2000).

	METHODS

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Brain slices containing the LC were obtained from rats in a manner described previously (Ishimatsu and Williams 1996; Williams et al. 1984). Male Wistar rats, 150-200 g, were killed by a heavy blow to the chest, and their brains were rapidly removed and immersed for 8-10 s in a cooled artificial cerebrospinal fluid (ACSF, 4-6°C) that was prebubbled with 95% O₂-5% CO₂. Horizontal brain slices (250-300 µm in thickness) were cut with a Vibroslice (Campden Instruments) in cooled ACSF and left to recover for 1 h in oxygenated ACSF at room temperature (22-24°C). A hemisected slice was then transferred to a recording chamber and submerged in the ACSF at 32-33°C. The composition of the ACSF was as follows (in mM): 126 NaCl, 2.5 KCl, 2.4 CaCl₂, 1.2 MgCl₂, 21 NaHCO₃, 1.2 NaHPO₄, and 11 D-glucose (pH: 7.4 and 295-305 mOsm). Intracellular recordings were made with glass microelectrodes filled with 2 M KCl (tip resistance: 26-40 M). Whole cell tight-seal recordings were made from LC neurons using the slice patch technique (Blanton et al. 1989; Coleman and Miller 1989). Patch pipettes were filled with the internal solution containing (mM): 130 KCl, 20 NaCl, 0.3 CaCl₂, 1 MgCl₂, 1 ethylene glycol-bis(-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA); 2 ATP (Mg-ATP); 0.25 GTP; 10 N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) (pH 7.3 adjusted by KOH, 280 mOsm). The tip resistance of a whole cell patch-pipette was 3-5 M. Voltage and current were recorded with an Axoclamp-2A amplifier and were monitored continuously with a memory oscilloscope (Nihon-Kohden, RTA-1100). During the whole cell voltage-clamping, sample frequencies were between 4.5 and 6 kHz and the amplifier gain was 0.8-2.5 nA/mV. A pClamp system (Axon Instruments) operating on a computer (PowerMac G4: Apple Computer) was used to analyze the membrane potential and current. The drugs used, GTP, EGTA, NE, yohimbine, prazosin, and tetraethylammonium (TEA) were purchased from Sigma-Aldrich Fine Chemicals (St. Louis, MO). Tetrodotoxin (TTX) was purchased from Wako Pure Chemical Industries (Osaka, Japan). MPH hydrochloride was a gift from Novartis Pharma. Drugs were directly dissolved in the ACSF. Each experimental value was presented as the mean ± SE and was analyzed by unpaired Student's t-test.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

MPH-induced hyperpolarization in LC neurons

LC neurons showed tonic firing of spontaneous action potentials with a frequency of 0.5-3 Hz, when impaled by a microelectrode. Bath application of MPH (30 µM) for 1-10 min caused a hyperpolarizing response (Fig. 1A) in 29 out of 32 neurons. In the remaining three neurons, MPH produced no changes in membrane potential. The firing of spontaneous action potentials was blocked during the hyperpolarization induced by MPH. The membrane potential and spontaneous action potentials recovered within 15-20 min after withdrawal of MPH from the superfusing solution. Properties of the MPH-induced hyperpolarization were analyzed at 60 mV, a membrane potential level at which the spontaneous action potentials were blocked. Electrotonic potentials produced by injection of hyperpolarizing current pulses with duration of 200 ms were depressed during the MPH-induced hyperpolarization (Fig. 1Ab). These results indicate that MPH decreases input resistance of LC neurons. When MPH (30 µM) was first applied to the ACSF, LC neurons showed hyperpolarization with amplitude of 12.0 ± 1.4 mV (n = 29) at 60 mV. The effect of MPH was use dependent: the MPH-induced hyperpolarization was diminished when the drug was repeatedly applied at an interval of 30-40 min. The amplitude of the hyperpolarization produced by the second application of MPH (30 µM) was 3 ± 2 mV (n = 12; Fig. 1Bb). Therefore the hyperpolarization produced by the first application of MPH was used for data analysis in the following study. Consequently, in the experiments testing the effect of drugs on the MPH response, the control and test neurons were two different sets. Figure 1C shows the voltage-current relationships (V-I curves) taken before and during the application of MPH (30 µM). In the presence of MPH, the V-I curve decreased in its slope and intersected the control curve at 107 mV. Pooled data showed that the reversal potential of the MPH-induced hyperpolarization was 91 ± 4 mV (n = 5).

View larger version (32K): [in this window] [in a new window]   Fig. 1. Effects of methylphenidate (MPH, 30 µM) on the membrane potential and input resistance of locus coeruleus (LC) neurons. A: an example of the MPH-induced hyperpolarization recorded at 55 mV. Note that spontaneous firing was blocked by MPH (a). Ab: the membrane potential was manually reset to 60 mV during MPH-induced hyperpolarization with current injection. B: 2 consecutive records of the hyperpolarization induced by repeated application of MPH (30 µM). The membrane potential was initially held at 60 mV under current-clamp condition. Records a and b were taken from the same cell at an interval of 20 min. C: effect of MPH (30 µM) on the V-I curve obtained by injection of rectangular current pulses with duration of 250 ms.  and , data obtained before and 10 min after application of MPH (30 µM), respectively. All data were obtained from A. The reversal potential of the MPH-induced hyperpolarization was 107 mV.

It has been shown that synaptic transmission in the LC was blocked in an ACSF containing 0 mM Ca²⁺ and 11 mM Mg²⁺ (Ca²⁺-free ACSF) (Egan et al. 1983). Figure 2A shows the effect of MPH (30 µM) on the membrane potential of LC neurons in the Ca²⁺-free ACSF. When the external solution was switched to the Ca²⁺-free ACSF, LC neurons exhibited a slow depolarization (10 ± 2 mV, n = 6) associated with an increase in the firing rate of spontaneous action potentials. Application of MPH (30 µM) to the Ca²⁺-free ACSF produced no obvious hyperpolarizing response. Pooled data showed that the MPH-induced hyperpolarization was almost completely depressed in the Ca²⁺-free ACSF (Fig. 2C). We next examined the effect of TTX (1 µM) on the hyperpolarization induced by MPH in LC neurons. Figure 2B shows an example of these experiments. When TTX (1 µM) was applied to the ACSF, spontaneous firing of the action potential was completely suppressed. TTX (1 µM) produced a depolarizing response with amplitude of 2.3 ± 1.1 mV (n = 7) in LC neurons (Fig. 2Ba). Addition of MPH (30 µM) to an ACSF containing TTX (1 µM) produced a hyperpolarizing response with amplitude of 5-10 mV in individual neurons (Fig. 2Bb). Averaged data show that the amplitude of the MPH (30 µM)-induced hyperpolarization was 8 ± 1 mV (n = 7) in the TTX (1 µM)-containing ACSF (Fig. 2C).

View larger version (34K): [in this window] [in a new window]   Fig. 2. Effects of Ca2+-free ACSF (A) and TTX (1 µM; B) on the MPH-induced hyperpolarization. A: effects of MPH (30 µM) on the membrane potential examined in Ca2+-free artificial cerebrospinal fluid (ACSF) in an LC neuron. Open horizontal bar indicates the period of bath-application of Ca2+-free ACSF. MPH (30 µM) was added to the Ca2+-free ACSF as indicated by the solid horizontal bar. Dashed line indicates the initial resting membrane potential of 52 mV. B: depression of the MPH-induced hyperpolarization by bath application of TTX (1 µM). a: a depolarization induced by bath-application of TTX (1 µM). b: MPH (30 µM) was added to ACSF containing TTX (1 µM). The resting membrane potential (55 mV) is indicated by the dashed line. C: pooled data for the effects of Ca2+-free solution and TTX (1 µM) on the MPH-induced hyperpolarization. Ordinate indicates the amplitude of the hyperpolarization. Vertical line on each column indicates SE of mean. Asterisk indicates the statistical significance obtained by unpaired Student's t-test (**P < 0.005, *P < 0.01). The number of experiments is shown in parentheses.

Contribution of NE to the MPH-induced hyperpolarization

MPH has been reported to enhance the release of NE and/or inhibit its re-uptake system in central neurons (Axelrod 1970; Carlsson et al. 1966; Ferris et al. 1972; Hendley et al. 1972; Raiteri et al. 1974; Ross 1978). We examined whether NE mediates the hyperpolarization induced by MPH in LC neurons. Bath application of yohimbine (1 µM), an ₂-adrenoceptor antagonist, produced a depolarizing response with amplitude of 6 ± 1 mV (n = 6) in LC neurons. Figure 3A shows the effect of yohimbine (1 µM) on the MPH-induced hyperpolarization in an LC neuron. Addition of MPH (30 µM) to yohimbine-containing ACSF for 10 min produced no visible hyperpolarization in this LC neuron. Pooled data showed that the amplitude of MPH-induced hyperpolarization was significantly depressed by yohimbine (1 µM; Table 1). It has been shown that prazosin (10 µM), an _2B/2C-adrenoceptor blocker, also antagonizes NE-induced outward current in dissociated LC neurons (Arima et al. 1998). In the present study, the effect of prazosin on the MPH-induced hyperpolarization was examined in LC neurons. Bath application of prazosin (10 µM) produced no obvious depolarization in LC neurons. Figure 3B shows an example of the MPH-induced hyperpolarization recorded in an ACSF containing prazosin (10 µM). MPH (30 µM) produced the hyperpolarization with amplitude of 6 mV in this particular LC neuron (Fig. 3B). Statistical data showed that prazosin (10 µM) significantly depressed the hyperpolarization induced by MPH (30 µM; Table 1).

View larger version (32K): [in this window] [in a new window]   Fig. 3. Effects of yohimbine (1 µM) and prazosin (10 µM) on the MPH-induced hyperpolarization in LC neurons. A: sample record of an effect of MPH (30 µM) in the presence of yohimbine (1 µM). B: an effect of MPH (30 µM) in the presence of prazosin (10 µM). A and B were taken from different neurons. The membrane potentials of these neurons were held at 58 mV (A and B). The period of bath-application of MPH is indicated by solid horizontal bars. Application of yohimbine and prazosin is indicated by open horizontal bars.

Effects of MPH on the membrane current in LC neurons

LC neurons were voltage-clamped at 60 mV with a whole cell configuration. Bath application of MPH (30-300 µM) caused an outward current (I_MPH) in 22 of 24 LC neurons (Fig. 4A). In the remaining two neurons, MPH did not change the holding current and membrane conductance. The I_MPH reached its maximum amplitude within 3 min after beginning of the application of MPH (30 µM) and recovered within 20 min after withdrawal of MPH. At a concentration of 0.1-1 µM, MPH produced no detectable response in LC neurons (n = 5). MPH (30 µM) produced the outward current with amplitudes of 110 ± 6 pA (n = 17). However, when the concentration of MPH was increased to 100-300 µM, the amplitude of I_MPH was almost the same as those produced by 30 µM MPH (Fig. 4B). Interestingly, the I_MPH appeared to be transient during a continuous application of MPH. Figure 4B shows the time course of the outward current during a prolonged application of MPH (300 µM) in an LC neuron. The I_MPH declined within 20 min even in the presence of MPH. To examine the sensitivity of ₂-adrenoceptor, NE (30 µM) was applied to a neuron where the I_MPH had been suppressed by a prolonged application of MPH (300 µM). NE (30 µM) produced an outward current with amplitude of 72 ± 5 pA (n = 8), even in the presence of MPH (300 µM). It has been shown that no detectable desensitization of ₂-adrenoceptors occurs during a continuous application of NE to LC neurons (Surprenant and Williams 1987; Williams et al. 1985). Supporting these reports, the amplitude of the I_NE was maintained as long as NE (30 µM) was present in the superfusing solution for 45 min (Fig. 4C). These results suggest that the decline of the I_MPH is due to a decrease in the release of NE from noradrenergic nerve terminals.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Whole cell patch-clamp recordings of the outward currents produced by MPH (30 and 300 µM) and norepinephrine (NE; 30 µM) in LC neurons. A: example of the outward current (IMPH) produced by MPH (30 µM). B: time course of the IMPH obtained by a continuous application of MPH (300 µM) for 45 min. NE (30 µM) was applied to the external solution containing MPH (300 µM). The periods of application of these drugs are indicated by solid horizontal bars. C: steady outward current induced by continuous application of NE (30 µM) for 45 min. B and C were taken from the same neuron.

Cocaine and desmethylimipramine, nonselective re-uptake inhibitors for catecholamines, have been shown to enhance the response to exogenous NE in LC neurons (Egan et al. 1983; Surprenant and Williams 1987). The effect of MPH on the outward current induced by exogenously applied NE was examined by whole cell patch-clamp techniques. In the control, NE (0.1-1 µM) produced no detectable outward current in the ACSF. When NE (10 µM) was added to the normal ACSF for 1-5 min, LC neurons showed outward current with amplitude of 51 ± 3 pA (n = 8). NE, at a concentration of 100 µM, produced an outward current with amplitude similar to that produced by 10 µM NE. Application of MPH (1 µM) did not produce any change in the membrane current and conductance of LC neurons. In the same cells, NE (1 µM) produced the outward currents with amplitude of 47 ± 4 pA (n = 8) in the presence of MPH (1 µM; Fig. 5). MPH (1 µM) also enhanced the outward current produced by NE (10-100 µM) in LC neurons (Fig. 5). The effect of MPH (1 µM) in enhancing the NE-induced response was reversible. The I_NE was restored, when LC neurons were superfused with the recovery solution for 20 min.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Effect of MPH (1 µM) on the outward current induced by NE (0.1-100 µM) in an LC neurons. Left and right: taken before and 15 min after application of MPH (1 µM), respectively. The holding membrane potential was 60 mV. Horizontal bars indicate the period of the application of NE.

MPH activates inward rectifier K+ channels in LC neurons

Current-voltage relationships (I-V curves) were constructed by step command potentials with duration of 200 ms (Fig. 6A). MPH (30 µM) increased the amplitude of currents induced by step command potentials, indicating that the membrane conductance of LC neurons was increased by MPH (Fig. 6Ab). The component of current activated by MPH (net I_MPH) was obtained by digital subtraction of the control I-V curve taken in the ACSF from that recorded in the presence of MPH (30 µM). The net I_MPH showed inward rectification in LC neurons (Fig. 6B). The I_MPH reversed polarity at 102 ± 6 mV (n = 8) in the ACSF (containing 2.5 mM K⁺; Fig. 6B). The reversal potential of I_MPH was shifted to 78 ± 2 mV (n = 6) and 66 ± 2 mV (n = 6) when it was recorded in external solutions containing 6.5 and 10.5 mM K⁺, respectively. Figure 6C shows the relationship between the reversal potential of I_MPH and the concentration of extracellular K⁺. The slope of this line was 54 mV for a 10-fold change in external K⁺ concentration. This is similar to the expected value of the equilibrium potential for K⁺ by the Nernst equation. It has been reported that Ba²⁺, at a micromolar concentration, selectively blocks the inward rectifier K⁺ current in various central neurons (North 1989). The effects of MPH (30 µM) on the membrane current of LC neurons were examined in the presence or absence of Ba²⁺ (100 µM). In neurons superfused with the ACSF, MPH produced an outward current of 113 ± 3 pA (n = 11). Bath application of Ba²⁺ (100 µM) produced an inward current associated with a decrease in membrane conductance. The amplitude of the outward current induced by MPH (30 µM) was 28 ± 2 pA (n = 4) in the presence of Ba²⁺ (100 µM). The I-V curve showed that Ba²⁺ (100 µM) almost completely suppressed the inward rectification of I_MPH (Fig. 6B). The present study showed that the MPH-induced hyperpolarization was blocked in the Ca²⁺-free ACSF (Fig. 2A). To confirm the Ca²⁺-dependent NE release by MPH, the effect of Co²⁺, a nonselective blocker for Ca²⁺ current, on I_MPH was examined in LC neurons. In the presence of Co²⁺ (1 mM), MPH (30 µM) produced 7 ± 3 pA (n = 5) outward current in LC neurons.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Electrophysiological properties of the IMPH in LC neurons. A: membrane conductance was measured by applying step command potentials with duration of 200 ms. Bottom records (a-c) were inward currents induced by step command potentials. They were recorded at the times indicated by the respective letters in the top record. B: graph shows I-V curves of the net IMPH obtained by subtraction of control I-V curve from that taken in the presence of MPH (30 µM).  and , obtained before and after application of Ba2+ (100 µM), respectively. Data were obtained from A. C: relationship between the reversal potential of the IMPH (ordinate) and the concentration of external K+. Bars indicate the SE of mean. Line was fitted by eye.

	DISCUSSION

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The present study showed that MPH (30 µM) produced a hyperpolarizing response associated with a block of the spontaneous firing of action potentials in LC neurons. Under whole cell patch-clamp condition, MPH (30-300 µM) caused an outward current in a great majority of LC neurons. It has been shown that synaptic transmission in the LC is blocked in the Ca²⁺-free ACSF (Egan et al. 1983). We examined whether or not MPH directly produced the hyperpolarization (and outward current) in rat LC neurons. The MPH-induced hyperpolarization was blocked, when LC neurons were superfused with the Ca²⁺-free ACSF. Furthermore, Co²⁺ (1 mM), a nonselective Ca²⁺ channel blocker, also strongly depressed the I_MPH in LC neurons. These results suggest that a neurotransmitter secondary mediates the hyperpolarization (and the outward current) induced by MPH in LC neurons. MPH, like D-amphetamine, has been reported to enhance the release and/or to block the re-uptake of NE and DA in mammalian brain (Axelrod 1970; Carlsson et al. 1966; Ferris et al. 1972; Hendley et al. 1972; Raiteri et al. 1974; Ross 1978). Electrophysiological studies have shown that NE produces a hyperpolarizing response mediated by ₂-adrenoceptors in LC neurons (Aghajanian and VanderMaelen 1982; Arima et al. 1998; Egan et al. 1983; Williams and Marshall 1987). The present study clearly showed that NE mediates the hyperpolarization induced by MPH because the MPH-induced hyperpolarization was strongly depressed by yohimbine, an antagonist for ₂-adrenoceptors (1 µM). Prazosin (10 µM), which blocks _2B/2C-adrenoceptors in cultured LC neurons (Arima et al. 1998), also depressed the MPH-induced hyperpolarization. The prazosin-induced depression of the I_MPH may not be mediated by ₁-adrenoceptors in rat LC neurons because the ₁-adrenoceptor has been shown to mediate a depolarizing response at early stages of development, but it is almost absent in adult rats (Williams and Marshall 1987). We suggest that the MPH-induced hyperpolarization is mediated by activation of _2B/2C-adrenoceptor subtypes in LC neurons of adult rats.

The ionic mechanism underlying the I_MPH was examined in LC neurons. The I_MPH was associated with an increase in the membrane conductance. The I_MPH reversed polarity at a membrane potential that was close to the equilibrium potential for K⁺. The reversal potential of the I_MPH changed by 54 mV per decade change in the external K⁺ concentration as predicted by the Nernst equation for K⁺. These results indicate that I_MPH is carried exclusively by K⁺. Current-voltage relationship showed that the I_MPH had a characteristic inward rectification in most LC neurons. Ba²⁺ (100 µM), a selective blocker for the inward rectifier K⁺ current (North 1989) reduced not only the amplitude but also the inward rectification of the I_MPH. These results suggest that MPH activates the inward rectifier K⁺ channels of LC neurons. These electrophysiological properties of the I_MPH are comparable to those of the I_NE in LC neurons (Arima et al. 1998; Egan et al. 1983).

It has been suggested that recurrent collaterals of the projections of noradrenergic neurons release NE on to the LC neurons themselves (Aghajanian et al. 1977). Spontaneous release of NE onto the somatodendritic membrane of other LC neurons produces inhibitory responses (Egan et al. 1983). Chemical studies have shown that tonic release of NE from rat LC neurons is increased when animals are exposed to continuous stressor (Ida et al. 1985; Tanaka et al. 1983; Tsuda et al. 1982). Tonic release of NE on LC neurons produces a hyperpolarization associated with increased potassium conductance via ₂-adrenoceptor. The present study showed that superfusion of LC neurons with the Ca²⁺-free ACSF resulted in a depolarization associated with an increase in the frequency of spontaneous action potentials. Yohimbine also produced a depolarizing response in LC neurons. Furthermore the inhibition of NE-re-uptake by MPH resulted in the hyperpolarization of LC neurons. These findings support the assumption that tonic release of NE from adrenergic nerve terminals regulates the membrane excitability of LC neurons. The present study showed, however, that TTX (1 µM) did not completely blocked the MPH-induced hyperpolarization, although it blocked the spontaneous firing of the action potential in LC neurons. A TTX-independent mechanism might underlie the MPH-induced NE release in the rat LC.

It has been demonstrated that cocaine and desmethylimipramine, irreversible NE re-uptake inhibitors, markedly enhance the response to exogenous NE in LC neurons (Surprenant and Williams 1987). The present study showed that MPH, at a concentration of 1 µM, produced no outward current but reversibly enhanced the I_NE in LC neurons. In addition, the present study showed that the MPH-induced hyperpolarization ran down, when a brief application of MPH (30 µM) was repeated in LC neurons. Furthermore, the I_MPH declined during a continuous application of MPH (300 µM) for 20 min. The decrease in the I_MPH amplitude may not be due to loss of the sensitivity of ₂-adrenoceptors at postsynaptic membrane because application of NE (30 µM) clearly produced an outward current in neurons in which the I_MPH had been suppressed by prolonged exposure to MPH. Furthermore detectable desensitization of ₂-adrenoceptors did not occur during a continuous application of NE to LC neurons (see also Surprenant and Williams 1987; Williams et al. 1985). These results suggest that the release of NE is depressed during a continuous exposure to MPH of LC neurons. Because MPH has been known to facilitate the release of NE in mammalian central neurons (Axelrod 1970; Carlsson et al. 1966; Ferris et al. 1972; Hendley et al. 1972; Raiteri et al. 1974; Ross 1978), the elevated levels of NE may inhibit the release of NE via presynaptic ₂-autoreceptors. Alternatively, excess release of NE by prolonged application of MPH may result in the depletion of NE at noradrenergic nerve terminals.

The LC has been known to correlate with the level of vigilance and attention in mammals (Aston-Jones and Bloom 1981b; Aston-Jones et al. 1991; Foote et al. 1980; Hobson et al. 1975). Extracellular recordings showed that intravenous or intraperitoneal administration of MPH decreased firing rate of spontaneous activity in rat LC neurons (Lacroix and Ferron 1988; Olpe et al. 1985). The present study has shown that the MPH-induced inhibition of the firing activity of LC neurons is due to the hyperpolarization of the postsynaptic membrane. Electrical stimulation of noradrenergic nerves or administration of NE increases the discrimination of incoming external stimuli by reducing the background neuronal activity, therefore augmenting the signal-to-noise ratio (Foote et al. 1975; Segel 1985; Waterhouse et al. 1980). We suggest that the therapeutic effect of MPH on children presenting the ADHD is correlated with the enhancement of the action of NE in depressing the firing activity of LC neurons.