Introduction

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The norepinephrine (NE) and the serotonin (5-hydroxytryptamine; 5-HT) systems have both been implicated in anxiety and affective disorders. Although the etiopathology of these two disorders remains enigmatic, greater knowledge exists pertaining to the interactions/alterations of these monoaminergic systems during antidepressant drug treatment. It is well established that locus coeruleus (LC) NE neurons modulate the 5-HT system, and evidence is accumulating for a major influence of 5-HT on the NE system (see Haddjeri et al., 1997; Kaehler et al., 1999). The LC receives dense 5-HT projections coming from dorsal raphe and pericoerulear 5-HT neurons (Aston-Jones et al., 1991; Kaehler et al., 1999), which exert an inhibitory role (Léger and Descarries, 1978; Segal, 1979). This is supported by the observation that lesioning 5-HT neurons with a 5-HT neurotoxin produced a marked elevation of firing rate of NE neurons (Haddjeri et al., 1997). Long-term, but not acute or short-term (2-day) administration of SSRIs decreases the spontaneous firing activity of LC NE neurons in the rat (Béïque et al.,1998; Szabo et al., 2000; Szabo and Blier, 2001a; Grant and Weiss, 2001). This delayed reduction of NE neuron firing activity parallels the lag in onset of therapeutic action of antidepressant drugs in affective and anxiety disorders. Consequently, the therapeutic effect of drugs selective for the 5-HT system, like SSRIs, could thus be dependent on a modification of the efficacy of 5-HT transmission in the LC.

YM992 [(S)-2-[[(7-fluoro-4-indanyl)oxy]methyl]morpholine monohydrochloride] is a novel SSRI agent (K_i = 21 nM) that enhances 5-HT neurotransmission after long-term (21-day) administration and possesses 5-HT_2A-antagonistic properties (K_i = 86 nM; Hatanaka et al., 1996; Dong et al., 1999). In addition to blocking 5-HT reuptake, YM992 may contribute to superior antidepressant and antipanic activities by immediately blocking the 5-HT_2A receptors. Indeed, 5-HT is believed to exert a tonic inhibitory action on the firing activity of NE neurons via a 5-HT_2A receptor (Haddjeri et al., 1997; Szabo and Blier, 2001a). Changes in NE function in various brain areas by antidepressant drugs may play a crucial role in controlling 5-HT output, and NE/5-HT interactions may thus be ultimately relevant to antidepressant efficacy, as well to their side effect profile.

The present studies were designed to characterize the effects of acute administration of YM992 on the spontaneous firing activity of LC NE neurons and to ascertain its 5-HT_2A receptor-antagonistic potential in this brain region. Sustained treatment regimens with YM992 were administered to rats to assess whether their effects on the spontaneous firing activity of LC NE neurons differed from that previously obtained with SSRIs without 5-HT_2A receptor antagonism.

	Materials and Methods

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Animals and Sustained Treatments. The experiments were all carried out in male Sprague-Dawley rats (Charles River, St. Constant, QC, Canada) except for those assessing the effect of idazoxan (Charles River, Raleigh, NC). The rats weighed between 300 and 325 g and were kept under standard laboratory conditions (12/12-h light/dark cycle with access to food and water ad libitum). Rats were anesthetized with chloral hydrate (400 mg kg¹ i.p.) and mounted in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). Supplemental doses (100 mg kg¹ i.p.) were given to prevent any nociceptive reaction to pinching of the hind paw. Body temperature was maintained at 37°C throughout the experiments utilizing a thermistor-controlled heating pad (Gaymar Industries Inc., Orchard Park, NY). Prior to electrophysiological recording, a catheter was inserted in a lateral tail vein for systemic i.v. injection of drugs.

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Electrophysiological Experiments. Extracellular unitary recording of NE neurons were conducted with single-barreled glass micropipettes preloaded with fiberglass filaments (to facilitate filling) being pulled in a conventional manner, with the tips broken back to 1 to 3 µm and filled with a 2 M NaCl solution. Their impedance range was between 2 and 4 M. A burr hole was drilled 1 mm posterior to lambda and 1 mm lateral to midline for NE neuron recordings. NE neurons were recorded with micropipettes lowered at 0.7 mm interaural and 1.1 to 1.4 mm lateral. Spontaneously active NE neurons of the LC were identified using the following criteria: regular firing rate (1-5 Hz) and positive action potential of long duration (0.8-1.2 ms) exhibiting a characteristic burst discharge in response to nociceptive pinch of the contralateral hind paw. NE neurons were recorded for at least 1 min to establish basal firing rate. To determine possible changes of spontaneous firing activity of LC NE neurons in the treated animals, four to five electrode descents were carried out through this nucleus in control and YM992-treated rats.

Drugs. The following drugs were used: YM992 from Yamanouchi Pharmaceutical Co. (Ibaraki, Japan); MDL 100,907 from Marion Merrell Dow Inc. (Cincinnati, OH); 5,7-dihydroxytryptamine and creatinine sulfate from Sigma-Aldrich (St. Louis, MO); and 8-OH-DPAT, DOI, clonidine, ritanserin, and idazoxan from Sigma/RBI (Natick, MA). The concentrations and the doses used for these compounds were chosen on the basis of previous successful experiments carried out in our laboratory and others. Drugs administered i.v. were all dissolved in distilled water, whereas ritanserin was dissolved using some acetic acid and then titrated with distilled water to the appropriate concentration.

Statistical Comparisons. All results were expressed as mean (±S.E.M.) of single neuron values. Statistical comparisons of the number of NE neurons recorded per descent into the LC and spontaneous firing activity obtained in treated and control rats were carried out using Kruskal-Wallis one-way analysis of variance on ranks. Dunn's multiple comparison test was used to assess the difference between controls and treated groups. Analysis of the effects of idazoxan on NE neuron firing activity before and after injection in control and YM992-treated rats was performed with the above-mentioned statistical test. Correlational coefficients (r values) for the dose-response relationship observed in the LC were calculated. The S.E.M. for the ED₅₀ values were calculated by regression analysis, with the Y value of 50 used as the regressor. Differences between the two regressions were assessed by comparing their ED₅₀ values using the confidence intervals method when indicated. The 95% confidence limit was determined from Student's t distribution.

	Results

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Effect of Acute YM992 Administration on the Firing Activity of NE Neurons and Its Ability to Block the Inhibitory Action of DOI. Acute administration of the SSRI paroxetine or fluoxetine does not alter the firing activity of LC neurons (Béïque et al., 1999). However, due to the unique capacity that YM992 possesses in selectively blocking both 5-HT reuptake and 5-HT_2A receptors, this drug was injected while recording NE neurons. Acute systemic administration of YM992 at a dose of 4 mg kg¹, previously reported to completely inhibit dorsal raphe 5-HT neuron activity (Dong et al., 1999), decreased the firing rate of NE neurons by 29% (range 8-63%; n = 5; Fig. 1).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Integrated firing rate histogram of single LC NE neurons illustrating the effects of intravenous administration of various compounds on spontaneous discharge frequency. The preferential 5-HT2A agonist DOI attenuated the firing activity of a LC NE neuron in a naive rat, whereas a subsequent injection of the 5-HT2A/2C antagonist of ritanserin reversed that effect (A). In B, the intravenous administration of the SSRI/5-HT2A antagonist YM992 decreased, whereas a subsequent injection of DOI failed to produce an alteration in neuron firing rate (n = 5). Note that a final injection of the 2-adrenoceptor agonist clonidine produced the expected effect normally observed in naive animals.

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View larger version (21K): [in this window] [in a new window]   Fig. 2.   Relationship between the degree of suppression of LC NE firing activity and doses of the 5-HT2 agonist DOI administered intravenously in controls and YM992-preinjected rats. Only the initial response of a single NE neuron to the first dose of DOI in one rat was used to construct the curves. The arrows pointing to the data points on the dose-response curves correspond to the experiments illustrated in Fig. 1. Outer lines represent the standard error of the regression line. The DOI dose-response curve obtained in control rats was previously reported in Szabo et al. (2000) and is used herein because the two experimental series were performed concurrently.

Effect of Sustained YM992 Administration on the Firing Activity of LC NE Neurons. Given that acute injections of YM992 decreased NE neuron firing activity, as opposed to SSRIs and 5-HT_2A receptor antagonism alone, which leave it unaltered, rats were treated chronically with this compound. Five systematic electrode descents into the LC nucleus were carried out in rats treated with YM992 (40 mg kg¹ day¹), as well as in their respective controls, for 2, 7, or 21 days. An example of each is provided in Fig. 3. Since control groups treated with saline for varying durations did not differ from each other with respect to LC spontaneous firing activity, these data were merged to make up a single control group (range of firing, 1.2-4.1 Hz). Short-term (2-day) administration of YM992 resulted in a significant 66% decrease (range of firing, 0.2-1.9 Hz) in the spontaneous firing activity of NE neurons when compared with controls. There was a partial recovery of LC firing activity after a 7-day YM992 treatment, and when compared with control values, the decrease was then of 43% (range of firing, 0.3-2.8 Hz). Long-term (21-day) YM992 administration lead to a complete recovery (range of firing, 0.3-5.3 Hz) in the spontaneous firing activity of LC neurons, as indicated by a nonsignificant difference compared with the control rat values (Fig. 4). Analysis of the number of spontaneously active neurons in YM992-treated rats and control rats did not reveal significant differences (Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Integrated firing rate histograms of LC NE neurons, recorded in a single electrode descent through the LC showing their spontaneous firing activity in control rats (A) and in rats treated with 40 mg kg1 day1 of YM992 for 2 (B), 7 (C), and 21 days (D). The dashed lines in between neurons indicate an approximately 5-min time lapse. The number above each neuron indicates the depth from the floor of the fourth ventricle at which it was recorded. The time base applies to all four traces.

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Effects of 2-, 7-, and 21-day YM992 treatments (40 mg kg1 day1; n = 3 rats for each treatment group) on the firing activity of LC neurons. The horizontal rectangle represents the range (S.E.M. × 2) of the mean firing activity of neurons recorded in control rats. , P < 0.05 (Dunn's method) when compared with the control value. The number of neurons recorded is displayed in each box.

Assessment of the Function of ₂-Adrenoceptors Controlling NE Neuron Firing Activity in YM992-Treated Rats. The ₂-adrenoceptor located on the cell body of LC NE neurons is important in the negative feedback regulation of the firing activity of these neurons (Freedman and Aghajanian, 1984; Mateo et al., 1998). To determine whether the suppression of firing of NE neurons in rats treated with YM992 for 2 days was due to an increased activation of ₂-adrenoceptors, systemic injections of the selective ₂-adrenoceptor antagonist idazoxan (1 mg kg¹ i.v.) were carried out in controls and treated rats. The hypothesis tested here was that if YM992 produced a suppression of firing of NE neurons after 2 days of treatment by enhancing NE levels, the ₂-adrenoceptor antagonist should bring the discharge rate to the same level in both controls and YM992-treated rats. Examples of the effect of idazoxan on the same NE neurons are provided in Fig. 5. Idazoxan in fact did equalize the attenuated firing activity of NE neurons in rats treated with YM992 for 2 days and in the control group (Fig. 6).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Integrated firing rate histograms of LC NE neurons illustrating the effects of intravenous administration of the selective 2-adrenoceptor antagonist idazoxan augmenting the firing activity in a control rat (A) and a YM992-treated rat (B).

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Effect of the selective 2-adrenoceptor antagonist idazoxan on LC NE neuron firing activity in control and YM992-treated (40 mg kg1 day1) rats. The number of neurons recorded in two electrode descents before and after a single injection of idazoxan (1 mg kg1 i.v.) in control (n = 4) and 2-day YM992-treated rats (n = 5) is displayed in each box. Note that the firing activity of the NE neurons prior to idazoxan administration in control and YM992-treated rats is significantly different.  indicates a significant enhancement of NE neuron firing by idazoxan.  indicates a reduced NE neuron firing activity in treated compared with control rats before idazoxan injection. N.S., nonsignificant difference of NE neuron firing activity following idazoxan in control and YM992-treated rats.

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View larger version (18K): [in this window] [in a new window]   Fig. 7.   Integrated firing rate histograms of LC NE neurons illustrating the effect of intravenous administration of the selective 2-adrenoceptor agonist clonidine in suppressing firing activity. In A, subsequent injections of the selective 5-HT1A agonist 8-OH-DPAT partly restored the firing activity. In B, idazoxan completely reversed the inhibitory effect of clonidine (n = 7). Note that the effect of clonidine is blunted in the YM992-treated rat when compared with the control.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Relationship between the degree of suppression of LC NE firing activity and doses of clonidine administered intravenously in controls and YM992-treated rats. Only the initial response of a single NE neuron to the first dose of clonidine in every rat was used to construct the curves. Outer lines represent the standard error of the regression line. The arrows pointing to the data points on the dose-response curves correspond to the doses used in Fig. 7.

Effect of Intravenous Administration of 8-OH-DPAT on the Firing Activity of NE Neurons in YM992-Treated Rats. The prototypical 5-HT_1A agonist 8-OH-DPAT augments the spontaneous firing activity of NE neurons (Piercey et al., 1993; Szabo et al., 2000). However, a 21-day treatment with citalopram abolishes the excitatory effect of 8-OH-DPAT on NE neuron firing activity, thus indicating that drugs that selectively block 5-HT reuptake desensitize various populations of 5-HT_1A receptors. Systemic injection of 8-OH-DPAT produced a dose-dependent increase in the firing activity of NE neurons (Fig. 9A) and yielded an ED₅₀ of 15 µg kg¹ (Fig. 10). As expected, rats treated with 40 mg kg¹ day¹ of YM992 for 21 days did not present any incremental effect to 8-OH-DPAT injection (Fig. 9B), but a subsequent injection of the selective ₂-adrenoceptor agonist idazoxan (1 mg kg¹; n = 3) was able to increase the firing activity of LC neurons. The enhancing action of 8-OH-DPAT (30-120 µg kg¹ i.v.) on NE neuronal firing activity in 21-day YM992-treated rats was abolished, even at extremely high doses (Fig. 10).

View larger version (26K): [in this window] [in a new window]   Fig. 9.   Integrated firing rate histogram of LC NE neurons illustrating the enhancing effect of intravenous administration of the 5-HT1A agonist 8-OH-DPAT on firing activity in a control rat (A) and in a rat treated with YM992 (40 mg kg1 day1; n = 6) (B). The identity of the NE neuron recorded in the YM992-treated rat was assessed by showing the enhancing effect on firing activity of a subsequent intravenous administration of the 2-adrenoceptor agonist idazoxan on NE firing activity (n = 3).

View larger version (21K): [in this window] [in a new window]   Fig. 10.   Relationship between the degree of augmentation of LC NE firing activity and doses of 8-OH-DPAT administered intravenously in controls and YM992-treated rats. Only the initial response of a single NE neuron to the first dose of 8-OH-DPAT in every rat was used to construct the curves. The arrows pointing to the data points on the dose-response curves correspond to the doses used in Fig. 9. Outer lines represent the standard error of the regression line. The 8-OH-DPAT dose-response curve obtained in control rats was previously reported in Szabo et al. (2000) and is used herein because the two experimental series were performed concurrently.

	Discussion

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The present study revealed that the SSRI/5-HT_2A antagonist YM992 decreases the firing activity of NE neurons when administered in an acute and subacute fashion. Furthermore, YM992 abolished the suppressant effect of a subsequent injection of the 5-HT₂ receptor agonist DOI. Although DOI has affinity for 5-HT_2A and 5-HT_2C receptors, its action on NE neurons is likely mediated via the 5-HT_2A receptor subtype because the selective 5-HT_2A antagonist MDL 100,907 antagonized it as well (Szabo and Blier, 2001a). Based on these results, YM992 (4 mg kg¹ i.v.) appears to be an effective 5-HT_2A receptor antagonist able to block the inhibitory effects of DOI in the LC, even at doses up to 120 µg kg¹ (Fig. 2). In contrast, this dose of YM992 does not alter the responsiveness of medial prefrontal cortex neurons to microiontophoretic application of DOI, whereas the 5-HT_2A/2C receptor antagonist ritanserin was previously reported to be effective (Ashby et al., 1990; Dong et al., 1999). This apparent discrepancy in the 5-HT_2A receptor-antagonistic property of YM992 may be explained by the heterogeneous pharmacological profile of 5-HT₂ receptors in various brain structures (Bergqvist et al., 1999). For instance, ritanserin and YM992 both reverse the inhibitory effects of DOI on LC neuron firing (Rasmussen and Aghajanian, 1986; Chiang and Aston-Jones, 1993; Szabo et al., 2000), whereas the former drug only produces a small (16%) increase in NE neuron firing activity (VanderMaelen and Braselton, 1992). The effects of ritanserin may thus be mediated by 5-HT_2C receptors because YM992 did not block the effects of DOI in the medial prefrontal cortex. However, the possibility of edited forms of 5-HT₂ receptors has been proposed and cannot at present be ruled out (Fitzgerald et al., 1999; Rueter et al., 2000). Thus, the 5-HT₂ receptors that mediate the effect(s) of DOI vary considerably from one brain region to another.

The decrease of NE neuron firing activity due to an acute injection of YM992 (Fig. 1) stands in contrast to the lack of effects of SSRIs (Béïque et al., 1998). Also, when rats were treated with YM992 (40 mg kg¹ day¹) for a period of 2 days, the firing activity of NE neurons was markedly decreased (Figs. 3, 4, and 6). This effect of YM992 on LC firing activity was unexpected as it differed from that observed in rats treated for 2 days with paroxetine and citalopram, antidepressants that selectively block 5-HT reuptake and leave LC firing activity unaltered (Szabo et al., 2000). Interestingly, this decrease in LC firing activity after a 2-day YM992 administration (66%) was in the range of that reported with NE reuptake-blocking agents desipramine (10 mg/kg/day; ca. 70%) and reboxetine (2.5 mg/kg/day; ca. 68%; Szabo et al., 2000; Szabo and Blier 2001b). In light of this, controls and 2-day YM992-treated rats were challenged with the ₂-adrenoceptor antagonist idazoxan (1 mg kg¹ i.v.), and the mean spontaneous firing activities of NE neurons were equalized in these two groups. It thus appears that a 2-day YM992 administration increases NE synaptic availability, which consequently over-activates ₂-autoreceptors located on LC neurons to produce a suppression of NE neuronal firing activity. Recently, Hatanaka et al. (2000) reported an increase of NE in dialysates collected from the rat frontal cortex with YM992 or citalopram plus MDL 100,907, but not with citalopram or MDL 100,907 alone. These findings are in accord with electrophysiological data demonstrating that 5-HT agents such as MDL 100,907 and SSRIs, when given alone, fail to alter NE firing activity upon acute administration (Béïque et al., 1999; Szabo and Blier 2000a). Also, the increase in extracellular NE produced with YM992 is consistent with the effects on firing activity observed with desipramine (Thomas and Holman., 1991; Perry and Fuller, 1997; Mateo et al., 1998) and reboxetine (Sacchetti et al., 1999), although it is certainly not achieved by blocking NE reuptake because YM992 does not block this process (IC₅₀, 3100 nM; Hatanaka et al., 1996). This striking difference in the effect of 2-day YM992 and SSRI treatments on LC firing activity seems to be solely attributed to 5-HT_2A receptor antagonism in the presence of 5-HT reuptake blockade. The exact neurobiological basis for this peculiar action of YM992 remains to be elucidated.

When treatment duration with YM992 was further increased to 7 and 21 days, the firing rate of NE neurons increased and reached the control range (Table 1 and Fig. 4). In 21-day YM992-treated rats, the slope of the dose-response curve for the ₂-adrenoceptor agonist clonidine was tilted to the right with an ED₁₀₀ 4 times greater than that obtained in control animals (Fig. 8). Prolongation of YM992 treatment from 2 to 21 days therefore desensitizes ₂-adrenoceptors, presumably due to a sustained increase in NE concentration, and ultimately results in normalization of LC firing activity. This adaptive change cannot be attributed to ₂-adrenergic agonism of YM992 because it is devoid of affinity for these sites (K_i >10,000 nM; Hatanaka et al., 1996). This effect is different from that observed in desipramine- or reboxetine-treated animals where the firing rate of NE neurons was still attenuated and the inhibitory response to clonidine was either normal or slightly decreased after a 14- to 21-day treatment (Lacroix et al., 1990; Szabo and Blier, 2001b). Presumably, the firing rate of the NE neurons recovered in the presence of YM992 because this drug does not block NE reuptake (Hatanaka et al., 1996), obviously a major mechanism by which NE is removed from the extracellular milieu, particularly in the LC that contains a high density of NE transporters. It remains puzzling why this adaptive desensitization does not occur to a significant extent in the presence of NE reuptake and monoamine oxidase inhibition (Lacroix et al., 1990; Blier and de Montigny, 1994; Szabo and Blier, 2001b).

In addition to its indirect NE effects, YM992 abolished the incremental action of the selective 5-HT_1A receptor agonist 8-OH-DPAT on LC firing activity, similar to antidepressants selective for 5-HT or NE reuptake blockade (Szabo et al., 2000; Szabo and Blier, 2001b). The desensitization of somatodendritic 5-HT_1A autoreceptors has been a proposed mechanism of action of SSRIs (Blier and de Montigny, 1994). This phenomenon is well documented for the desensitization of 5-HT_1A autoreceptors, which impart a negative feedback influence on 5-HT neuron firing activity following long-term SSRI treatment including YM992, but not for drugs that block NE reuptake such as desipramine (Blier and de Montigny, 1994; Dong et al., 1999) and reboxetine (Szabo and Blier, 2001b). As with other SSRIs, YM992 induces an attenuation and normalization in 5-HT neuron firing activity after 2- and 21-day treatments, respectively, which is attributed to the initial over-activation of somatodendritic 5-HT_1A receptors and subsequent desensitization of these receptors in the latter treatment group (Dong et al., 1999). In addition, it appears that drugs that block 5-HT or NE reuptake produce a desensitization of 5-HT_1A receptors that control LC firing activity. This phenomenon is common to all major classes of antidepressant drugs tested thus far and may reflect an important finding with respect to the treatment of anxiety and affective disorders. Furthermore, these 5-HT_1A receptors are probably not the 5-HT_1A autoreceptor controlling 5-HT neuron firing activity (Szabo and Blier, 2001a,c). For instance, the selective 5-HT_1A antagonist WAY-100,635 at a dose of 0.1 mg kg¹ i.v. does not alter the firing rate of 5-HT neurons, but shuts off that of LC neurons. In contrast, a 5 µg/kg i.v. dose of 8-OH-DPAT completely suppresses 5-HT neuronal firing but does not alter NE neuronal firing (Szabo et al., 2000). In 5-HT-lesioned rats, the inhibitory and excitatory effects of WAY 100,635 and 8-OH-DPAT, respectively, on the firing rate of NE neurons are abolished (Haddjeri et al., 1997; Szabo and Blier, 2001a). It therefore appears that an intact 5-HT system is also necessary to produce alterations of LC firing activity by 5-HT_1A receptor ligands (Szabo and Blier, 2001a). Further research will be needed to determine the exact location of the 5-HT_1A receptors controlling LC activity. One possibility that has been put forth is their presence on excitatory neurons feeding onto 5-HT terminals in the LC (Szabo and Blier 2001a,c).

YM992 behaves as an SSRI on dorsal raphe 5-HT neurons during acute and sustained treatments (Dong et al., 1999). Unlike SSRIs, YM992 decreases NE neuron firing activity upon an acute and sustained 2-day administration that is normalized after 21 days. This rapid decrease in NE neuron firing activity may aid in alleviating the initial exacerbation of panic disorder symptoms generally observed in panic disorder patients with usual starting doses of SSRIs for major depression (Westenberg, 1996). Indeed, the enhancement of NE neuron firing and release achieved with the ₂-adrenoceptor antagonist yohimbine can produce anxiety in healthy volunteers and trigger panic attacks in patients with panic disorder (Charney et al., 1984). In contrast to SSRIs, which can decrease the spontaneous firing activity of LC neurons by as much as 50% upon long-term administration (Szabo et al., 2000), YM992 may guard against a lethargic effect sometimes produced by SSRIs (Montgomery et al., 1993), by maintaining a normal NE neuron firing rate (Blier, 2000). Consequently, this normalization of NE neuron firing activity, together with 5-HT_2A receptor antagonism, may also prove beneficial in attenuating sexual dysfunctions which commonly plague SSRI users. The normal firing activity of LC and dorsal raphe neurons in the presence of increased 5-HT and NE synaptic availability may contribute to a decreased side effect profile and an increase in efficacy as reported with drugs that increase both 5-HT and NE synaptic availability (Danish University Antidepressant Group, 1990; Einarson et al., 1999; Poirier and Boyer, 1999; Silverstone and Ravindran, 1999).