INTRODUCTION

Major depressive disorder (MDD) is the most predominant illness among mental, neurological, and substance-use disorders (Collins et al, 2011). Despite significant progress in development of antidepressant treatments, the response and remission rates in depressed patients remain suboptimal (Shelton et al, 2010). Lately, combination strategies in treatment of MDD, and especially its treatment-resistant form, find more and more empiric support (Papakostas, 2009; Stahl, 2010). The effectiveness of augmentation of antidepressants with low doses of atypical antipsychotics (AAPs) is now well documented (Shelton et al, 2010; Nelson and Papakostas, 2009; DeBattista and Hawkins, 2009). Moreover, extensive clinical studies resulted in an official approval of some of these drugs for use in MDD.

The group of AAPs comprises agents with a wide variety of pharmacological profiles, with the antagonism at serotonin (5-HT)2A and dopamine D2 receptors serving as a common denominator. Because the first generation antipsychotics, acting primarily at the D2 receptors, do not possess antidepressant properties, the blockade of the latter receptors therefore does not appear to be the mechanism explaining the antidepressant action of AAPs. Indeed, the doses of AAPs used in depression treatment are much lower than those prescribed in psychotic states and generally provide clinically insignificant occupancy of D2 receptors. It is thus likely that the 5-HT2 receptors may be the main determinants of the beneficial clinical action of the AAPs in depression treatment (Celada et al, 2004; Szabo and Blier, 2002; Blier and Szabo, 2005). As selective serotonin reuptake inhibitors (SSRIs) attenuate norepinephrine (NE) neuronal activity via activation of 5-HT2A receptors, their blockade by AAPs reverses this effect (Dremencov et al, 2007a; Seager et al, 2005). This mechanism potentially contributes to the additive efficacy of such augmentation treatment. Although the efficacy of AAPs as SSRI-augmenting agents may largely be explained by the reversal of tonic inhibition of catecholaminergic neurons by 5-HT, their action at other receptors may also contribute to their clinical benefits. The monoaminergic properties vary from one AAP to another due to their differential affinity for various receptors that regulate the activity of monoamine neurotransmitters.

To date, the effectiveness of extended-release quetiapine in unipolar and bipolar depression has been assessed in 12 controlled, randomized, double-blind clinical studies totaling 4485 patients (McElroy et al, 2010). It was shown to be effective in the treatment of hQuet is a 3:1 mixture of Quet and NQuet. MDD when used alone, combined with antidepressants, or cognitive behavior therapy (McIntyre et al, 2007; El-Khalili et al, 2010; Bauer et al, 2009; Bortnick et al, 2011; Chaput et al, 2008; Cutler et al, 2009; Katila et al, 2008; Weisler et al, 2009). Not only the remission rate from MDD was increased, but the relapse was found to be less likely in patients who after alleviation of depressive symptoms were maintained on quetiapine (Liebowitz et al, 2010). This data set resulted in approval of the drug for use in MDD as an augmenting agent in the United States and European Union and as a second-line monotherapy in Canada.

It is important to mention that in humans quetiapine is extensively metabolized leading to over 20 metabolites (Goldstein and Arvanitis, 1995; Lindsay DeVane, 2001). N-Desalkyl quetiapine (NQuet) is one of the main active metabolites. It largely shares the pharmacological profile of quetiapine but has additional pharmacological targets potentially important in the treatment of MDD (Jensen et al, 2008). Having significant structural similarity with tricyclic antidepressants, NQuet has one of their prominent properties, a moderate affinity to the NE transporter (NET; Jensen et al, 2008). Unlike humans, rodents do not metabolize quetiapine to NQuet. In order to mimic the therapeutic conditions, NQuet was thus added to quetiapine in a ratio present in humans. The mixture used for experiments was thus termed hQuetiapine (for human quetiapine; hQuet).

Despite the established efficacy of quetiapine in the treatment of MDD, its mechanism of action is not entirely understood. Though the extended-release quetiapine formulation is approved for monotherapy use in depression, in many cases it is used in combination with SSRIs. Thus, the current study was aimed at investigating the effects of short- and long-term use of quetiapine alone, and in combination with the SSRI escitalopram (ESC) on neurotransmission in the 5-HT and NE system, which are known to have an important role in pathophysiology and treatment of MDD.

MATERIALS AND METHODS

Animals

Male Sprague Dawley rats (Charles River, St Constant, QC, Canada) weighing 270–320 g at the time of recording were used for the experiments. They were kept under the standard laboratory conditions (12 : 12 h light:dark cycle with free access to food and water). All animal handling and procedures were approved by our local Animal Care Committee (Institute of Mental Health Research, University of Ottawa, Ottawa, ON, Canada). Data were obtained from three to five rats per experimental group.

Treatments

Quetiapine and ESC were delivered via subcutaneously implanted osmotic minipumps at a daily dose of 10 mg/kg and NQuet at a dose of 3.3 mg/kg. These drugs were administered for 2 or 14 days alone and in combination. Control rats received physiological saline through an osmotic minipump as well. The 2- and 14-day length of drug administration was chosen to determine the immediate (at steady-state levels) and the clinically relevant long-term effects of the studied drugs upon monoaminergic systems.

In vivo Electrophysiological Recordings

Rats were anesthetized with chloral hydrate (400 mg/kg; i.p.) and placed in a stereotaxic frame. To maintain a full anesthetic state, chloral hydrate supplements of 100 mg/kg, i.p., were given as needed. Extracellular recordings of the 5-HT and NE neurons in the RD and the LC, respectively, were obtained using single-barreled glass micropipettes. Their tips were of 1–3 μm in diameter and impedance ranged 4–7 MΩ. All glass micropipettes were filled with 2 M NaCl solution. Prior to electrophysiological experiments, a catheter was inserted in a lateral tail vein for systemic i.v. injection of appropriate pharmacological agents when applicable.

Recording of the LC NE neurons. Micropipettes were positioned in mm from lambda at: AP, −1.0 to −1.2; L, 1.0–1.3; V, 5–7. Spontaneously active NE neurons were identified using the following criteria: regular firing rate (0.5–5.0 Hz) and positive action potentials of long duration (0.8–1.2 ms) exhibiting a brisk excitation followed by period of silence in response to a nociceptive pinch of the contralateral hind paw (Aghajanian and Vandermaelen, 1982a). Dose-response curves were obtained using only the initial response to the first dose injected to a single neuron of each rat.

Recording of the RD 5-HT neurons. Single-barreled glass micropipettes were positioned in mm from lambda at: AP, +1.0 to 1.2; L, 0±0.1; V, 5–7. The presumed 5-HT neurons were then identified using the following criteria: a slow (0.5–2.5 Hz) and regular firing rate and long-duration (2–5 ms) bi- or triphasic extracellular waveform (Aghajanian and Vandermaelen, 1982b).

Dose-response Curves

Dose-response curves assessing the effect of a 2-day administration of hQuet on the responsiveness of 5-HT2A receptors and α2-adrenergic autoreceptors were constructed for systemic i.v. injections of the 5-HT2A agonist DOI and the α2-adrenergic agonist clonidine. Dose-response curves were plotted using GraphPad software.

Extracellular Recordings and Microiontophoresis of Pyramidal Neurons in CA3 Dorsal Hippocampus

Extracellular recordings and microiontophoresis of CA3 pyramidal neurons were carried out with five-barreled glass micropipettes. The central barrel used for the unitary recording was filled with 2M NaCl solution, the four-side barrels were filled with the following solutions: 5-HT creatinine sulfate (10 mM in 200 mM NaCl, pH 4), (±)-NE bitartrate (10 mM in 200 mM NaCl, pH 4), quisqualic acid (1.5 mM in 200 mM NaCl, pH 8), and the last barrel was filled with 2 M NaCl solution used for automatic current balancing. The micropipettes were descended into the dorsal CA3 region of the hippocampus using the following coordinates: 4 mm anterior and 4.2 mm lateral to lambda (Paxinos and Watson, 1998). Pyramidal neurons were found at a depth of 4.0±0.5 mm below the surface of the brain. Because the pyramidal neurons do not discharge spontaneously in chloral hydrate-anesthetized rats, a small current of quisqualate (+1 to –6 nA) was used to activate them to fire at their physiological rate (10–15 Hz; Ranck, 1975). Pyramidal neurons were identified by their large amplitude (0.5–1.2 mV) and long-duration (0.8–1.2 ms) simple action potentials, alternating with complex spike discharges (Kandel and Spencer, 1961). The duration of microiontophoretic application of 5-HT and NE was 50 s. The 50-s duration of microiontophoretic application of the pharmacological agents and the ejection currents (nA) were kept constant before and after each i.v. injection throughout the experiments. Neuronal responsiveness to the microiontophoretic application of 5-HT and NE, prior to and following i.v. injections, was assessed by determining the number of spikes suppressed per nA.

Assessment of the Tonic Activation of Postsynaptic α1 and α2 Adrenoceptors in CA3 Pyramidal Neurons

The degree of tonic activation of postsynaptic α-adrenergic receptors was assessed following 14-day hQuet administration. The assessment of the tonic activation of postsynaptic receptors is more accurate when the firing rate of the recorded neuron is low (Haddjeri et al, 1998b). Therefore, the firing rate of pyramidal neurons was reduced by lowering the ejection current of quisqualate. The degree of tonic activation of postsynaptic α2 and α1 adrenoceptors was assessed using the selective antagonists idazoxan and prazosin, respectively (Ghanbari et al, 2011). Upon obtaining a low steady firing baseline, idazoxan (1 mg/kg) and prazosin (100 μg/kg) were systemically administered to assess the changes in the firing activity in rats administered with saline or hQuet for 14 days. In order to avoid drug residual effects, only one neuron in each rat was tested.

Assessment of the Tonic Activation of Postsynaptic 5-HT1A Receptors

The degree of tonic activation of postsynaptic 5-HT1A receptors was assessed following 14-day hQuet administration. The assessment of the tonic activation of postsynaptic 5-HT1A receptor is more accurate when the firing rate of the recorded neuron is low. Therefore, the firing rate of pyramidal neurons was reduced by lowering the ejection current of quisqualate. After stable firing baseline is obtained, the selective 5-HT1A receptor antagonist WAY 100635 (100 μg/kg) was administered systemically in four incremental doses of 25 μg/kg each at time intervals of 2 min. Neuronal response at each dose point was obtained for construction of the dose-response curve. Such curves represent stable changes in the firing rate of pyramidal neurons as percentages of baseline firing following each systemic drug administration. In order to avoid drug residual effects, only one neuron in each rat was tested.

Assessment of NE Reuptake in vivo

To evaluate the effectiveness of hQuet on the blockade of NET reuptake, the recovery of the firing activity of pyramidal neurons following the microiontophoretic application of NE was assessed using the recovery time 50 (RT50) value. NE exerts an inhibitory action upon firing of pyramidal neurons. The time necessary for their firing to recover (RT50) is mainly dependent on the activity of the NET (De Montigny et al, 1980). The RT50 value was obtained by calculating the time in seconds required for the neuron to recover 50% of its initial firing rate at the end of the microiontophoretic application of NE onto CA3 pyramidal neurons (De Montigny et al, 1980).

Stimulation of the Ascending 5-HT Pathway

The ascending 5-HT pathway was electrically stimulated using a bipolar electrode (NE-100, David Kopf, Tujunga, CA). The electrode was implanted 1 mm anterior to lambda on the midline with a 10° backward angle in the ventromedial tegmentum and 8.0±0.2 mm below the surface of the brain. Two hundred square pulses of 0.5 ms in duration were delivered by a stimulator (S48, Grass Instruments, West Warwick, RI) at an intensity of 300 μA and a frequency of 1 Hz. The effects of 1 Hz stimulations of the ascending 5-HT fibers were assessed prior to and following i.v. injections of the α2-adrenoceptor agonist clonidine (10 and 400 μg/kg, respectively) while recording from the same neuron. The low and high doses of clonidine were used to assess the responsiveness of the α2-adrenergic auto- and heteroreceptors, respectively. Previous studies showed that clonidine is 10-fold more potent at the α2-adrenergic autoreceptors than at the α2-adrenergic heteroreceptors on 5-HT terminals (Frankhuyzen and Mulder, 1982; Maura et al, 1985). The low dose of clonidine (10 μg/kg) potentiates the effect of stimulation of the 5-HT pathway by stimulating the α2-adrenergic autoreceptors that are present on NE terminals, leading to inhibition of NE firing and disinhibition of 5-HT terminals (Lacroix et al, 1991). Indeed, the effect of the low, but not the high, dose of clonidine was abolished when the NE neurons were lesioned (Mongeau et al, 1993). On the other hand, the high dose of clonidine (400 μg/kg) inhibits the effect of 5-HT stimulation by acting on the α2-adrenergic heteroceptors, located on the 5-HT terminals, leading to inhibition of 5-HT release. Therefore, 1 Hz stimulations of 5-HT bundle result in a greater 5-HT release and increased SIL value after the i.v. injection of the low clonidine dose and a smaller 5-HT release resulting in a shorter inhibition of pyramidal firing (smaller SIL) following a high dose of clonidine.

The stimulation pulses and the firing activity were analyzed by computer using Spike 2 (Cambridge Electronic Design Limited, Cambridge, UK). Peristimulus time histograms of hippocampal pyramidal neurons were generated to determine the suppression of firing measured in absolute silence (SIL) value in ms. The SIL represents the duration of a total suppression of the hippocampal neuron.

Statistical Analysis

All results are expressed as mean±SEM. Statistical comparisons between differences in spontaneous firing of DR 5-HT and LC NE neurons in rats treated with saline, ESC, hQuet, and ESC+hQuet combination were carried out by one-way analysis of variance and multiple comparison procedures using Fisher's PLSD post-hoc test. Data were obtained from three to five rats per experimental group. Statistical significance was taken as P<0.05.

Drugs

Quetiapine fumarate and NQuet were provided by Astra Zeneca, ESC was provided by Lundbeck (Copenhagen, Denmark; the doses for the above drugs are expressed as salt), MDL100907 (Servier, Courbevoie, France), WAY 100635, clonidine hydrochloride, idazoxan hydrochloride, DOI, 5-HT creatinine sulfate, (±)-NE bitartrate, quisqualic acid, and desipramine were purchased from Sigma (St Louis, MO); WAY 100635 and ESC oxalate were dissolved in distilled water, NQuet and quetiapine fumarate were dissolved in physiological saline.

RESULTS

Assessment of the Effects of 2- and 14-day Administration of ESC, hQuet and their Combination on the Mean Firing Rate of NE Neurons

In line with previous data (Dremencov et al, 2007a), both short- and long-term ESC administration led to significant decreases in NE spontaneous firing when compared with controls (2 days: −47%, P<0.001; 14 days: −35%, P<0.01; Figure 1a and b). Administration of hQuet led to a significant increase in the NE neuronal firing after both 2 and 14 days (2 days: +40%, P<0.01; 14 days: +28%, P<0.001; Figure 1a and b). When the two drugs were coadministered for either 2 or 14 days, NE neuronal firing was not only fully restored compared with that of ESC-treated rats but also increased significantly compared with the control level (2 days: +27%, P<0.05; 14 days: +25%, P<0.001; Figure 1a and b).

Figure 1
figure 1

Effect of acute and sustained administration of hQuet, ESC, and their combination on NE-spontaneous firing rate. Firing rate of LC NE neurons after 2- (a) and 14-day (b) of drugs administration. The number of neurons recorded in each group is displayed in respective histograms. The data are expressed as mean firing rates±SE of the mean (SEM). *P<0.05, **P<0.01, and ***P<0.001.

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Assessment of the Effects of 14-day Administration of hQuet of the Tonic Activation of Postsynaptic α1 and α2 Adrenoceptors on the Dorsal Hippocampus CA3 Pyramidal Neurons

Pyramidal neurons in the CA3 layer of the dorsal hippocampus experience constant (tonic) activation by NE released from terminals. The effect of NE on pyramidal neurons is inhibitory and mediated by α1 and α2 adrenoceptors. Systemic application of the selective α2- and α1-adrenoceptor antagonists idazoxan and prazosin, respectively, did not modify the firing activity of pyramidal neurons in control rats (Figure 2a). However, in rats administered with hQuet for 14 days, consecutive i.v. injections of idazoxan significantly enhanced the firing activity of CA3 pyramidal neurons by 260±38% (P<0.001; Figure 2b). The blockade of α1-adrenergic receptors with prazosin did not alter the firing of pyramidal neurons in rats receiving hQuet for 14 days.

Figure 2
figure 2

Assessment of tonic activation of α adrenoceptors in dorsal hippocampus. Integrated firing-rate histograms of dorsal hippocampus CA3 pyramidal neurons illustrating the effects of systemic administration of idazoxan (1000 μg/kg), prazosin (100 μg/kg) in control (a) and 14-day hQuet- (b) treated rats. The arrows represent the consecutive injections of idazoxan and prazosin. The overall changes of the firing activity of pyramidal neurons after systemic injections of idazoxan and prazosin in controls and rats that received hQuet for 14 days (c). The number above each bar corresponds to the ejection current of NE in nA applied for 50 s. ***P<0.001.

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Assessment of NE Reuptake Potential of NQuet

NQuet, an active metabolite of quetiapine produced in humans but not in rats, appears to be a moderate blocker of NET (Ki=58 nM; Jensen et al, 2008). To assess the potential of NQuet to inhibit the reuptake of NE in vivo, the effect of direct microiontophoretic application of NE onto pyramidal neurons of the hippocampus was studied in anesthetized rats. It was found that NQuet administered i.v. at a dose of 0.5–1 mg/kg significantly increased the RT50 value compared with control rats (P<0.01; Figures 2 and 3). Furthermore, when rats were given hQuet (given as a 3 : 1 mixture of quetiapine and NQuet) for 14 days, RT50 values were increased more than two-fold (P<0.001; Figure 3). These observations indicate that hQuet exerts significant NE reuptake blockade in vivo.

Figure 3
figure 3

Assessment of the effect of NQuet at the NET. Histograms representing the recovery time (RT50) of dorsal hippocampus CA3 pyramidal neurons from microiontophoretic applications of NE in control rats, rats acutely injected with NQuet (0.5–1 mg/kg), and rats that received a long-term hQuet regimen. RT50 values (mean±SEM) represent the time (in seconds) required by the recorded neuron to recover 50% of its firing activity from the end of the microiontophoretic application of NE. The number of neurons tested is depicted in each histogram.

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Assessment of the Effects of hQuet on Locus Coeruleus NE Neurons: Role of 5-HT2A Receptors

The 5-HT system can inhibit NE neuronal activity via the activation of 5-HT2A receptors (Szabo and Blier, 2002). hQuet is known to have affinity for these receptors (Jensen et al, 2008). As expected, the dose of the selective 5-HT2A receptor agonist DOI required for the complete inhibition of NE neuronal firing rate was significantly higher in rats administered with hQuet for 2 days compared with controls (DOI ED50: control=20±8 μg/kg vs hQuet=55±16 μg/kg; Figure 4a and b). The blockade of 5-HT2A receptors by hQuet, documented by the present experiments, would thus prevent a potential 5-HT-mediated attenuation of the NE neuronal activity induced by ESC.

Figure 4
figure 4

Assessment of the hQuet 5-HT2A receptor antagonism. Representative integrated firing-rate histograms of LC NE neurons illustrating the effect of i.v. administration of 5-HT2A receptor agonist DOI in suppressing neuronal activity of rats administered with vehicle (a) or hQuet (10 mg/kg/day; b) for 14 days, and the relationship between the degree of suppression of LC NE firing activity and doses of DOI administered i.v. in vehicle and hQuet-administered rats. Outer lines (c) represent the SE of the regression line (DOI ED50: control=20±8 μg/kg, hQuet=55±16 μg/kg).

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Assessment of the Effects of hQuet on Locus Coeruleus NE Neurons: role of α2 Adrenoceptors

Adrenergic α2-autoreceptors regulate the firing rate and the release capacity of NE neurons in a negative feedback manner. Thus, in control rats, activation of these receptors by systemic administration of the selective α2-adrenergic agonist clonidine led to the complete cessation of the spontaneous discharge (ED50=2.1±0.5 μg/kg; Figure 5a). In rats exposed to hQuet for 2 days, the dose of clonidine required for the complete inhibition of neuronal discharging was significantly greater (ED50=5.4±1 μg/kg; Figure 5b). In line with its documented pharmacological properties (Jensen et al, 2008), this increase in the amount of clonidine necessary to inhibit NE neurons indicates that hQuet effectively blocks somatodendritic α2-adrenergic autoreceptors. This property is likely responsible for the increase in the discharge rate of NE neurons, following both 2 and 14 days of hQuet administration, respectively.

Figure 5
figure 5

Assessment of hQuet antagonism at presynaptic α2 adrenoceptors. Representative integrated firing rate histograms of LC NE neurons illustrating the effect of i.v. administration of the α2-adrenergic autoreceptor agonist clonidine in suppressing neuronal activity of rats administered with vehicle (a) and hQuet (10 mg/kg/day; b) for 14 days, and the relationship between the degree of suppression of LC NE firing activity and doses of clonidine administered i.v. in vehicle- and hQuet-administered rats. Outer lines (c) represent the SE of the regression line (clonidine ED50: control=2.1±0.5 μg/kg, hQuet=5.4±1 μg/kg).

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Effects of 14-day hQuet Administration on the Responsiveness of Terminal α2 Adrenoceptors

The ascending 5-HT pathway was stimulated to determine whether 14-day administration of hQuet had the ability to antagonize terminal α2 adrenoceptors and thus modulate the endogenous release of 5-HT and NE in the synaptic cleft. Systemic administration of the low dose of the α2-adrenoceptor agonist clonidine (10 μg/kg) significantly enhanced the suppression of the firing rate of hippocampus pyramidal neurons in the control rats, whereas high dose of clonidine (400 μg/kg) reversed this effect bringing the SIL below the pre-injection value (control, pre-clonidine: 43±2 ms; post-clonidine 10: 73±5 ms, P<0.001; post-clonidine 400: 29±1 ms; P<0.001; Figure 6a, c, e). The low dose of clonidine still significantly increased the suppression of CA3 pyramidal neurons in rats administered with hQuet for 14 days (hQuet 14 days: pre-clonidine 40±2 ms; post-clonidine 10: 55±3 ms, P<0.01; Figure 6b and c), although to a lesser extent than in the control rats (P< 0.01, compared with post-clonidine 10 in controls), thus suggesting a diminished function of α2-adrenergic autoreceptors on NE terminals. Following the 14-day administration of hQuet, the high dose of clonidine reversed the SIL-prolonging action of 10 μg/kg clonidine injection. The magnitude of the effect was blunted and the post-clonidine 400 value in rats receiving hQuet for 14 days was significantly higher than that in controls (hQuet 14 days: post-clonidine 400: 38±2 ms; control: post-clonidine 400: 29±1 ms; Figure 6e and f), indicating diminished functioning of α2-adrenergic receptors on 5-HT terminals.

Figure 6
figure 6

Assessment of hQuet antagonism at postsynaptic α2 adrenoceptors. Peristimulus time histograms illustrating effects of stimulation of the ascending 5-HT pathway on the firing activity of CA3 pyramidal neurons in control (a) and 14-day hQuet-exposed rats (b). The effect of the 5-HT-pathway stimulation prior to and following the systemic administration of clonidine at doses of 10 and 400 μg/kg, respectively (control: c and d, hQuet: e and f, respectively). The overall effect of clonidine administration in controls (g) and hQuet-treated rats (h). The numbers in the columns correspond to the number of recorded neurons. **P<0.01 and ***P<0.001, comparing the SIL value of the respective group with the basal level. ##P<0.01, comparing the SIL value following clonidine in hQuet group with the respective values in controls. SIL: absolute silence.

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Assessment of the Effects of 2- and 14-day Administration of ESC, hQuet and their Combination on the Firing Rate of 5-HT Neurons

Short-term ESC administration resulted in a 65% decrease in the spontaneous firing rate of 5-HT neurons (P<0.001; Figure 7a). hQuet administered for 2 days decreased the spontaneous firing rate of 5-HT neurons by 43% (P<0.001; Figure 7a). hQuet combined with ESC for 2 days led to the same decrease of the spontaneous firing of 5-HT neurons as that of rats treated with ESC alone (65% decrease, P<0.001; Figure 7a).

Figure 7
figure 7

Effect of acute and sustained administration of hQuet, ESC, and their combination on 5-HT-spontaneous firing rate. The firing rate of DR 5-HT neurons after 2- (a) and 14-day (b) drugs administration. The number of neurons recorded in each group is displayed in each histograms. The data expressed as mean firing rate±SE of the mean (SEM). ***P<0.001.

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As previously reported, 5-HT neuronal firing returned to the control level after ESC was administered for 14 days (El Mansari et al, 2005; Figure 7b). Sustained hQuet administration yielded a significantly dampened firing when compared with controls (46% decrease, P<0.001). hQuet given in combination with ESC also led to significant inhibition of spontaneous firing activity of 5-HT neurons (62% decrease, P<0.001; Figure 7b).

Assessment of the Effect of 14-day Administration of ESC, hQuet and their Combination on the tonic Activation of Postsynaptic 5-HT1A Receptors in the Dorsal Hippocampus CA3 Pyramidal Neurons

Pyramidal neurons in CA3 layer of the dorsal hippocampus receive its serotonin innervation from the dorsal and median raphe nuclei. The effect of 5-HT on pyramidal neurons is inhibitory and mainly mediated by 5-HT1A receptors. All antidepressant medications thus far tested, as well as electro-convulsive shocks and stimulation of the vagus nerve (undertaken to achieve antidepressant action), produce an increase in tonic activation of pyramidal neurons (Manta et al, 2009; Haddjeri et al, 1998b; indicated by the disinhibition of firing rate in response to the blockade of 5-HT1A receptors by highly potent and selective antagonist WAY 100635). Importantly, no significant disinhibition occurs in control rats (Figure 8a).

Figure 8
figure 8

Assessment of tonic activation of 5-HT1A receptors in dorsal hippocampus. A and B are integrated firing-rate histograms of dorsal hippocampus CA3 pyramidal neurons illustrating systemic administration of 5-HT1A-receptor antagonist WAY 100635 in four incremental doses of 25 μg/kg in vehicle (a) and 14-day hQuet- (10 mg/kg/day; b) treated rats. Each bar corresponds to 50 s application of 5-HT and the number above each bar corresponds to the ejection current in nA. Each arrow indicates a single injection of 25 μg/kg of WAY 100635. The overall effect of cumulative systemic administration of WAY 100635 on baseline firing of CA3 pyramidal neuron in vehicle and hQuet-administered rats (c, expressed as % of change in basal firing). *P<0.05 and **P<0.01.

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It was found that chronic administration of hQuet produced a significant increase in tonic activation of postsynaptic 5-HT1A receptors located on the dorsal hippocampus CA3 pyramidal neurons (230±28%; Figure 8b). ESC administered on its own for 14 days also produced a marked increase (511±87%; Figure 8d). When hQuet was coadministered with ESC, the increase in tonic activation was in the same range as that obtained with ESC alone (471±46%; Figure 8c).

Assessment of the Effects of hQuet on Dorsal Raphe Nucleus 5-HT Neurons: Role of α1 Adrenoceptors

Stimulation of α1 adrenoceptors located on the cell bodies of 5-HT neurons leads to the decrease of their spontaneous firing rate. hQuet has moderate affinity for α1 adrenoceptors (Ki for quetiapine=22 nM and for NQuet=144 nM; Jensen et al, 2008). It was found that acute i.v. injection of hQuet completely inhibited the firing of 5-HT neurons (ED50=0.5±0.2 mg/kg; Figure 9). This inhibition could be partially reversed by the administration of the potent NE reuptake blocker desipramine by displacing hQuet from α1 adrenoceptors through an additional enhancement of endogenous NE. As NQuet also has moderate affinity for 5-HT1A receptors (Ki=45 nM), the desipramine injection was followed by administration of the potent and selective 5-HT1A receptor antagonist WAY 100635, which expectedly led to the complete restoration of 5-HT neuronal firing. It is worth mentioning that the blockade of 5-HT1A receptors by WAY 100635 without desipramine administration could not reverse the inhibitory effect of hQuet at all, emphasizing the principal role of α1 adrenoceptors (data not shown). These results provide a possible explanation for the decrease of the 5-HT neuronal firing observed with both the 2- and 14-day regimens of hQuet.

Figure 9
figure 9

Effect of acute hQuet administration on 5-HT neuronal firing. Representative single-unit extracellular recording from the 5-HT neuron during the consecutive acute injections of hQuet, followed by the NET inhibitor desipramine and the selective 5-HT1A-receptor agonist WAY 100635. hQuet was given as a 3 : 1 mixture of quetiapine and NQuet.

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DISCUSSION

The present study put into evidence that hQuet, administered for both 2 and 14 days, increased the NE neuronal discharge rate and the overall NE neurotransmission after 14 days. hQuet was found to block cell body and terminal α2-adrenergic receptors but not the α2-adrenergic receptors located postsynaptically. In contrast, both pre- and postsynaptic α1 receptors were blocked by the hQuet. The documented antagonism of 5-HT2A receptors by hQuet was demonstrated in vivo. NQuet was shown to possess significant NET-blocking property, both when acutely administered on its own and when given on a long-term basis as a part of hQuet. The inhibitory influence of the SSRI ESC on NE-spontaneous neuronal discharge was reversed by hQuet, both after 2 and 14 days of concomitant drug administration. The firing rate of 5-HT neurons, however, was significantly decreased in rats receiving hQuet alone or in combination with ESC after both 2 and 14 days. Despite this dampening of firing, the overall 5-HT neuronal transmission was enhanced following long-term hQuet administration (see Figure 10 for schematic explanation of effects).

Figure 10
figure 10

Representative schema of 5-HT and NE neuronal interactions and changes evoked by the hQuet/NQuet administration upon neuronal elements. hQuet was found to block cell body and terminal α2-adrenergic auto- and heteroreceptors but not the α2-adrenergic receptors located on CA3 pyramidal neurons. In contrast, α1 receptors on 5-HT and pyramidal neurons were blocked by the hQuet. NQuet was shown to effectively block the NET. hQuet was shown to activate the cell body 5-HT1A autoreceptors and enhance the tonic activation of the postsynaptic 5-HT1A receptors in hippocampus. hQuet was found to block 5-HT2A postsynaptic receptors. Following the long-term hQuet administration, the spontaneous firing rate of NE neurons increased above the baseline, whereas that of 5-HT neurons decreased. (+), excitatory effect on firing activity or neurotransmitter release; (−), inhibitory effect on firing activity or neurotransmitter release.

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hQuet was found to produce very profound noradrenergic effects: both the spontaneous firing and the overall NE neuronal transmission were increased by sustained administration of hQuet. This effect is likely due to action of hQuet at several NE neuronal elements. Antagonism of α2-adrenergic cell-body autoreceptors that exert a negative feedback control over NE neuronal firing is known to increase the NE neuronal discharge. Both the optimal blockade of this receptor by the selective antagonist idazoxan and its sustained antagonism by mirtazapine, an effective antidepressant with prominent α2-adrenergic blocking properties, were previously documented to elevate the NE neuronal firing rate above the control level (Dremencov et al, 2007a; Freedman and Aghajanian, 1984; Haddjeri et al, 1998a). The α2 antagonistic potential of hQuet was assessed after 2 days of administration. The observed right shift of the dose-response curve of α2-adrenoceptor agonist clonidine clearly confirms that hQuet effectively blocks this receptor.

The potency of hQuet was nevertheless lower than that of idazoxan because the latter was still able to reverse the suppression action of clonidine. This is consistent with the much greater affinity of idazoxan for α2-adrenergic receptors than quetiapine and NQuet (Mallard et al, 1992; Jensen et al, 2008).

SSRIs administered for short-term or chronically are known to inhibit the spontaneous firing of NE neurons (Dremencov et al, 2007a; Szabo et al, 2000). This phenomenon was reproduced in the present study. The above effect takes place due to the SSRI-induced increase in endogenous stimulation of excitatory 5-HT2A receptors, located on the GABA neurons that inhibit the firing rate of the NE neurons (Aston-Jones et al, 1991; Szabo and Blier, 2001). The observed decrease in the discharge rate of NE neurons is likely counterproductive in treatment of MDD and may underlie the fatigue and asthenia observed in some patients chronically treated with SSRIs (Nutt, 2008; Kasper and Pail, 2010). Our results demonstrate that the addition of hQuet (exhibiting 5-HT2A receptor antagonism confirmed by the right shift of the 5-HT2A agonist DOI dose-response curve in rats subjected to 2-day hQuet administration) to the ESC regimen not only reversed the inhibitory influence of an SSRI upon NE neuronal firing but also increased it above the baseline level. This observation is in line with previous electrophysiological data, as well as notion that concomitant administration of SSRI with 5-HT2A-receptor blockers produces a significant increase in levels of the extracellular NE in rat frontal cortex (Szabo and Blier, 2002; Seager et al, 2005; Hatanaka et al, 2000). It is noteworthy that the addition of 5-HT2A receptor antagonist to the SSRI has been shown to result in an increased antidepressant effect in numerous animal and clinical studies (Nemeroff, 2005; Tohen et al, 2003; Papakostas, 2005). The potency of hQuet was nevertheless lower than that of MDL100907, which completely prevents the inhibitory effect of DOI on NE neuronal firing (Szabo and Blier, 2001), consistent with the higher affinity of MDL100907 than hQuet for the 5-HT2A receptors.

The present study also put into evidence that NQuet possessed NET-inhibiting properties and thus contributes to the NE-activating profile of the parent compound by preventing recycling and thus increasing the levels of synaptically available NE. Interestingly, the tricyclic antidepressant desipramine provides a similar degree of NE inhibition in rats when administered acutely in the same dose range as NQuet (Lacroix et al, 1991; Curet et al, 1992). Considering that low doses of desipramine lead to a 75% blockade of the NET in the 25–100 ng/ml range (Gilmor et al, 2002), it can be speculated that the plasma levels of NQuet in the 100 ng/ml range, forming as a result of 300 mg/day of Quet, also blocks NE reuptake to a clinically significant degree. The exact NET-inhibiting potency of NQuet remains, however, to be determined in humans.

When terminal α2 auto- and heteroreceptors that control the release of NE and 5-HT, respectively, are overstimulated by the reuptake-produced increased synaptic levels of NE, they gradually desensitize (Szabo and Blier, 2000). This decrease in sensitivity of terminal inhibitory α2 receptors leads to the increased release in NE and 5-HT. A similar functional change is produced by the α2-adrenergic antagonist mirtazapine (Haddjeri et al, 1998a).

The overall increase in the NE neuronal transmission can be attributed to the increased firing of NE neurons, the inhibition of NE reuptake by NQuet, and the attenuated function of terminal α2-adrenergic receptors on NE terminals. This was put into evidence by the observed enhancement in tonic activation of the postsynaptic adrenoceptors. Indeed, the degree of activation of postsynaptic α2 adrenoceptors was enhanced in rats receiving hQuet on a long-term basis. No such increase could be detected at postsynaptic α1-adrenergic receptors because of their effective blockade by the hQuet. The variability of the α2-antagonistic potential of hQuet between different receptor sites (ie ability to block auto- and terminal receptors but not the α2 adrenoceptors located on the cell body of pyramidal neurons in hippocampus) is not unusual. Similar changes were previously documented with the α2-adrenoceptor antagonist mirtazapine (Haddjeri et al, 1998a; Mongeau et al, 1994).

hQuet administered i.v. at a dose of 1 mg/kg abolished the discharge of 5-HT neurons. Indeed, all AAPs, but paliperidone, decrease the spontaneous firing rate of 5-HT neurons when administered acutely (Dremencov et al, 2007b; Gartside et al, 1997; Hertel et al, 1997; Sprouse et al, 1999; Stark et al, 2007). Two actions on the cell body of DR 5-HT neurons can mediate this decrease: the blockade of α1 adrenoceptors and/or the activation of 5-HT1A autoreceptors. As quetiapine and NQuet have significant affinities for both 5-HT1A and α1-adrenergic receptors (Jensen et al, 2008; Schotte et al, 1996), they likely suppress the 5-HT firing by acting on both receptors. This was confirmed by the observation that the quetiapine-induced suppression of 5-HT spontaneous firing could be completely reversed only when both these receptors were blocked (Figure 9). The same phenomenon was true for risperidone (Dremencov et al, 2007a, 2007b). The decrease in the firing rate of 5-HT neurons observed in rats treated with hQuet for both 2 and 14 days, respectively, likely took place due to the same inhibitory mechanisms. The firing rate of 5-HT neurons decreased with short-term ESC administration returns to control levels when the SSRI is given chronically (El Mansari et al, 2005). When hQuet and ESC are coadministered, however, this recovery did not take place (Figure 7). The latter is likely explained by the sustained blockade of the α1 adrenoreceptors.

Despite the observed decrease in 5-HT spontaneous firing in both the hQuet and hQuet+ESC groups, the overall 5-HT neurotransmission was found to be enhanced, as indicated by the increase in tonic activation of postsynaptic 5-HT1A receptors located on the CA3 hippocampal pyramidal neurons. The 5-HT neuronal tone thus appears to increase independently of the firing rate of 5-HT neurons in DR. The observed increase in 5-HT neuronal tone in rats administered with hQuet on a long-term basis likely stems from the direct activation of 5-HT1A receptors (Quetiapine Ki=717 nM, NQuet Ki=45 nM) combined with the augmented 5-HT release capacity, stemming from the blockade of release-inhibiting terminal α2 heteroreceptors. Interestingly, even though the firing rate of 5-HT neurons was the same in rats receiving hQuet alone and those administered hQuet in combination with ESC (Figure 7), the degree of tonic activation of postsynaptic 5-HT1A receptors was significantly higher in the latter group (Figure 8). This finding advocates for the additive benefit of combined administration of hQuet and SSRIs. The same is likely the case for other AAPs: long-term administration of risperidone, for instance, dampens the spontaneous activity of 5-HT neurons (Dremencov et al, 2007b), however, the concentration of 5-HT increases in both DR and prefrontal cortex (Hertel et al, 1999).

Limitations

Although the proposed NET-blocking properties of NQuet were confirmed in vivo by our study, its potency at human receptors in clinical conditions remains to be tested. Furthermore, though we attempted to mimic the pharmacokinetic balance of the parent compound and the active metabolite NQuet that naturally occurs in humans but not in rodents, it is not certain how close the attained blood levels of the studied compounds were to the absolute concentrations observed clinically. Although measurement of the blood levels of the tested drugs would give a definite answer, we believe that the fundamental findings obtained as a result of the present study are not undermined by the lack of this verification.

Conclusion

Both short- and long-term administration of hQuet enhanced the firing rate of NE neurons. Addition of hQuet to the SSRI regimen reversed the inhibitory action of the latter upon NE spontaneous firing (which is likely contributing to the limited benefit of SSRIs in some patients, as well as to some of their side-effects). The overall NE neuronal transmission was enhanced by long-term hQuet administration. Despite the inhibited spontaneous firing of 5-HT neurons after 2 and 14 days, respectively, of treatment with both hQuet and its combination with ESC, the overall 5-HT neurotransmission increased, as indicated by the enhancement of tonic activation of hippocampal 5-HT1A receptors. The effectiveness of hQuet and its combination with SSRIs in depression treatment can possibly be explained by its positive effect on NE and 5-HT neuronal tone.