Brain serotonin (5-hydroxytryptamine, 5-HT) system is involved in a wide spectrum of physiological processes including sleep, cognition, sensory perception, motor activity, temperature regulation, appetite, hormone secretion, nociception and sexual behavior (1). Among at least 15 subpopulations of 5-HT receptors the 5-HT7 receptor is the latest subtype to be identified (2-4). A high density of 5-HT7 receptors has been identified in the thalamus, cortex, hippocampus, hypothalamus and raphe nuclei (5-7). Many antidepressant drugs influence the turnover and metabolism of 5-HT in the brain and dysfunctions of the serotonergic system are thought to be involved in the pathomechanism of depressive disorders (8). The presence of the 5-HT7 receptor in limbic structures suggests its involvement in the pathophysiology of mood disorders. This receptor has also been implicated in mood regulation, circadian rhytmicity and sleep, the disturbances of which are evident in the course of depressive disorders (9, 10). Our earlier study documented an attenuation of the effects of the activation of rat hippocampal 5-HT7 receptors after treatment with the tricyclic antidepressant, imipramine, and the selective 5-HT reuptake inhibitor, citalopram (11). In animal models, inactivation or blockade of the 5-HT7 receptor has been shown to induce antidepressant-like behavior (12-14). Recent studies have demonstrated a synergistic interaction between the specific 5-HT7 receptor antagonist SB 269970 and several antidepressant drugs (15, 16). Moreover, blockade of the 5-HT7 receptor may produce faster antidepressant effects than do contemporary antidepressant drugs (17). We have also demonstrated that stress-induced modifications of basal glutamatergic transmission and long-term potentiation in the frontal cortex were prevented by the 5-HT7 receptor antagonist (18). These findings support the hypothesis that the 5-HT7 receptor can be considered as a potential target for the action of antidepressant drugs.

The delayed onset of the therapeutic effects of antidepressants and the similar therapeutic efficacies of drugs with different primary pharmacological profiles suggest that adaptive mechanisms beyond monoaminergic modulation at synapses may be involved in the antidepressant actions. Growing evidence indicates that abnormalities in the excitatory amino acid transmission play not only an important role in the pathophysiology of mood disorders but also that a common mechanism of various antidepressant therapies may involve modifications in the function of brain glutamatergic system (19-22).

Abnormalities of several behavioral processes during depressive disorders suggest an involvement of the frontal cortex in the pathophysiology of depression and antidepressive drug action (23-25). We have previously shown that repeated administration of imipramine or citalopram, lasting for 2 weeks, resulted in a decrease in the amplitude of glutamate-mediated field potentials evoked in layer II/III of the rat frontal cortex by stimulation of underlying sites as well as in a reduction in the amplitude ratio of pharmacologically isolated NMDA to AMPA/kainate receptor-mediated components of the field potential (26). We have also demonstrated imipramine treatment-related decrease of spontaneous glutamate release from terminals synapsing onto layer II/III pyramidal cells in the frontal cortex (27). These results suggest that chronic treatment with antidepressants may attenuate glutamatergic transmission in rat cerebral cortex.

Because our previous study showed that serotonin, acting through 5-HT7 receptors, exerts a complex modulatory influence on glutamate- and GABA-mediated synaptic transmission in the rat hippocampus (28), it is conceivable that the antidepressant-like effects of the 5-HT7 receptor antagonist (12-14) might be related to its modulatory action on glutamatergic transmission, also in other brain regions. Therefore, in the present study we aimed at finding the effects of single and repeated administration 5-HT7 antagonist SB269970 on glutamatergic neurons and excitatory synaptic transmission in the rat frontal cortex.


MATERIALS AND METHODS
Treatment of animals

Experimental procedures were approved by the Animal Care and Use Committee at the Institute of Pharmacology, Polish Academy of Sciences, and were carried out in accordance with the European Community guidelines and national law. Male Wistar rats, weighing approx. 140 g at the beginning of the experiment, were housed in groups on a controlled light/dark cycle (light: 7.00-19.00). Standard food and tap water were available ad libitum. The rats received SB 269970 i.p. (1.25 mg/kg, dissolved in 0.9% NaCl, volume: 1 ml/kg; purchased from Tocris) either as a single injection or repeatedly for 14 days. In the former group the administration of SB 269970 was preceded by 13 daily injections of a 0.9% NaCl. The dose of SB 269970 was chosen on the basis of preliminary microdialysis experiments demonstrating 5-HT efflux in the frontal cortex (unpublished). The animals of the control group received 0.9% NaCl (1 mg/kg) once daily for 14 days.

Slice preparation

Brain slices were prepared 2 days after the last substance administration. Rats were anesthetized with isoflurane, decapitated, their brains were quickly removed and placed in ice-cold artificial cerebrospinal fluid (aCSF) containing (in mM): 130 NaCl, 5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1.25 KH2PO4, 26 NaHCO3, 10 D-glucose, bubbled with a mixture of 95% O2 and 5% CO2. Frontal cortical slices (420 µm thick) were cut in the coronal plane using a vibrating microtome (VT1000; Leica Microsystems). Slices were stored submerged in aCSF at 32±0.5°C.

Whole-cell recording

A slice was placed in the recording chamber where it was submerged and superfused at 3 ml/min with warm (32±0.5°C), modified aCSF containing (in mM): 132 NaCl, 2 KCl, 2.5 CaCl2, 1.3 MgSO4, 1.25 KH2PO4, 26 NaHCO3, 10 D-glucose, bubbled with 95% O2 - 5% CO2. Neurons were visualized using Zeiss Axioskop 2 upright microscope using Nomarski optics, a 40× water immersion lens and an infrared camera (29). Patch pipettes were pulled from borosilicate glass capillaries (Clark Electromedical Instruments) using Sutter Instrument P97 puller. The pipette solution contained (in mM): 130 K-gluconate, 5 NaCl, 0.3 CaCl2, 2 MgCl2, 10 HEPES, 5 Na2-ATP, 0.4 Na-GTP and 1 EGTA. Osmolarity and pH were adjusted to 290 mOsm and 7.2, respectively. Pipettes had open tip resistance of approx. 6 M. Signals were recorded using the SEC 05LX amplifier (NPI), filtered at 2 kHz and digitized at 20 kHz using Digidata 1322A interface and Clampex 9.2 software (Molecular Devices). Layer II/III cells were sampled from sites located approx. 2 mm lateral to the midline and approx. 0.3 mm below the pial surface. Pyramidal neurons were identified by morphological criteria (27) and by the characteristics of the response to intracellular current pulses (Fig. 1A).

Analysis of intrinsic excitability and spontaneous excitatory postsynaptic currents (sEPSCs)

Response characteristics of the recorded neurons were evaluated in the current clamp mode with intracellular injections of rectangular current pulses (500 ms). To determine the relationship between the current and firing rate, the number of spikes evoked by current steps of increasing amplitude (increments: 20 pA) were determined (30). The gain (slope) and firing threshold (measured as a current extrapolated at zero firing rate) parameters were determined from the straight lines fitted to raw data (Fig. 1D), and averaged (Fig. 1E, 1F).

Next, cells were voltage-clamped at -76 mV and sEPSCs were recorded for 8 min. Spontaneous EPSCs were detected off-line and analyzed using Mini Analysis software (Synaptosoft, USA). Data were accepted for analysis when the access resistance ranged between 15 and 18 M and it was stable (<25% change) during recording. Since the noise level of recordings was in a range of 4-5 pA, the amplitude and the area thresholds for the detection of an event were set to 7 pA and 25 fC, respectively, which maximized the correct identification of sEPSCs. Recorded traces were visually inspected following automated analysis to prevent false positive identification and false negative rejection of events. In part of the experiments, 2 mM kynurenic acid (Sigma Aldrich) was added to aCSF.

Statistical analysis was carried out using one-way ANOVA followed by the Dunnett's post hoc test (SigmaPlot 12, Systat Software Inc.).


RESULTS
The cells included in the analysis exhibited a regular spiking firing pattern in response to a depolarizing current pulse (Fig. 1A). Basic membrane properties did not differ significantly between the three experimental groups (number of control neurons: n=37; number of cells in slices from rats receiving single and repeated administration of SB 269970: n=38 and n=34, respectively; Fig. 1B, 1C). None of the recorded neurons demonstrated spontaneous spiking activity. Analysis of the relationship between injected current and firing rate (Fig. 1D, 1E, 1F) demonstrated that neither single nor repeated administration of SB 269970 modified the intrinsic excitability of frontal cortex pyramidal neurons.

Fig. 1. Treatment with SB 269970 does not influence the intrinsic excitability of pyramidal neurons. (A) A representative example of the response (upper trace) to a depolarizing current pulse (lower trace), typical of the layer II/III pyramidal cell. (B) Mean resting membrane potential (±S.E.M.). In this and in the following panels: con, control neurons (n=37); 1 x SB, cells (n=38) in slices from rats receiving single administration of SB 269970, 14 x SB, cells (n=34) in slices from rats receiving repetitive administration of SB 269970. (C) Mean input resistance (±S.E.M.). (D) A representative example of the response characteristics of a regular-spiking, pyramidal neuron. (E) Mean firing threshold (±S.E.M.). (F) Mean gain (slope of spiking rate vs. injected current relationship, ±S.E.M.).

At the holding potential of -76 mV sEPSCs were recorded as inward currents (Fig. 2A1, 2A2). These sEPSCs could be blocked by 2 mM kunyrenic acid, a non-selective antagonist at NMDA and AMPA/kainate receptors (Fig. 2A3). As illustrated in Fig. 2B, in cells prepared from rats treated with SB 269970 for 14 days the mean frequency of sEPSCs was significantly lower (P<0.05) in comparison to that in cells originating from control animals. Repeated treatment with SB 269970 also resulted in a reduction in the mean amplitude of sEPSCs (P<0.05). In contrast, single administration of SB269970 affected neither the mean frequency nor the mean amplitude of sEPSCs (Fig. 2B, 2C).

Fig. 2. Repeated administration of SB 269970 decreases the mean frequency and the mean amplitude of sEPSCs. (A1) A representative example of sEPSCs (downward deflections) recorded from a control neuron. (A2) A representative example of sEPSCs recorded from a neuron in a slice after repeated administration of SB 269970. (A3) A representative example of a recording in the presence of 2 mM kynurenic acid. (B) Mean (±S.E.M.) frequency of sEPSCs in control neurons (con) and in neurons originating from rats after single (1 x SB) and repeated (14 x SB) administration of SB269970. * P<0.05 vs. control. (C) Mean (± S.E.M.) amplitude of sEPSCs. Labels as in (B).

DISCUSSION
The major finding of the present study is that repetitive administration of SB269970 induces a strong reduction in the mean frequency and a smaller decrease in the mean amplitude of spontaneous EPSCs recorded from cortical pyramidal neurons 2 days after the end of the treatment with this selective 5-HT7 receptor antagonist.

As it was shown in our previous paper (27) most of the spontaneous EPSCs recorded from frontal cortical pyramidal neurons correspond to miniature EPSCs. Thus, the observed reduction in the frequency of sEPSCs is most likely related to presynaptic modifications of the mechanism of glutamate release (28). On the other hand, a decrease in the mean amplitude of sEPSCs may in principle result from pre- and/or postsynaptic modifications, involving a reduced glutamate content of synaptic vesicles within presynaptic terminals and/or alterations in postsynaptic reactivity to glutamate.

Some data show that serotonin regulates cortical function through modulation of glutamatergic signaling. Frontal cortex receives a dense serotonergic input from the dorsal raphe nucleus (DRN) (31). Serotonin, released from axon terminals of the raphe neurons, has been shown to affect the action of several neurotransmitters through heterosynaptic activation of 5-HT receptors located on neighboring axon terminals (32). Heterosynaptic activation of 5-HT receptors on glutamatergic neurons has been shown to inhibit or stimulate glutamate release in the cortex via 5-HT1A, 5-HT1B or 5-HT2A receptors (33-35). Thus it is conceivable that the decrease of glutamatergic transmission after chronic blockade of 5-HT7 receptors, seen in the present study, occurs as a result of the activation of other classes of 5-HT receptors by a prolonged, elevated level of 5-HT in the cortex.

It has been shown that the blockade of 5-HT7 receptors increases 5-HT raphe-hippocampus transmission (15). Enhancement of 5-HT transmission in such projection areas as the dorsal hippocampus after the administration of SB 269970 lasting 1 week has been reported by other researchers (17). So far only a few studies have examined the effect of SB-269970, administered alone or together with antidepressant drugs, on 5-HT release in the cortex. Wesolowska and Kowalska (36) have shown that SB-269970 increased the efflux of DA, NA, and 5-HT in the rat prefrontal cortex. On the other hand, some authors have found that SB-269970 significantly inhibits 5-HT efflux (37) and other investigators reported that inhibition of 5-HT release is likely to be mediated by 5-HT7 receptor agonists (38). There is a possibility that the SB-269970-induced increase in 5-HT release could be mediated by the inhibition of 5-HT7 receptors localized on GABAergic interneurons in the dorsal raphe nucleus (39). In a series of functional studies Roberts et al. (37) and Glass et al. (40) have proposed that 5-HT7 receptors in the DRN are localized, among others, on GABAergic cells. We have demonstrated that the 5-HT7 receptor-mediated increase in the sIPSCs frequency in hippocampal CA1 pyramidal cells is partially related to the activation of 5-HT7 receptors located on GABAergic interneurons (28).

Another possible mechanism of the observed reduction of glutamatergic transmission after 14 days of SB269970 injections may potentially be related to the direct effects of the interaction between SB 269970 molecules and the 5-HT7 receptor. It has been demonstrated that the activation of 5-HT7 receptors increases neurite outgrowth in hippocampal neurons in the cell culture (41). Both Galpha(s) and Galpha(12) proteins can regulate cellular morphology by activating intracellular signaling cascades. These effects are produced either by modulation of cAMP concentration (42) or direct binding of Galpha(s) protein to the cytoskeleton (43). The downstream effectors of Galpha(12) protein, which mediate changes in the actin cytoskeleton, are members of the Rho family of small GTPases, including RhoA, Rac1 and Cdc42 (44). The major functional effects of this pathway, including actin reorganization and the formation of neurite-like protrusions, are mediated by the activation of Cdc42 (41). Since the activation of the 5-HT7 receptor by Galpha(s) and Galpha(12) proteins stimulates glutamatergic synaptic transmission by a positive influence on a number of functional synapses, the prolonged blockade of the 5-HT7 receptor might account for the observed reduction of glutamatergic transmission. However, it has recently been shown that 5-HT7 receptor/Galpha(12) protein signaling pathway is functional only during early postnatal stages but not in adult animals (45), making this possibility less likely.

In conclusion, the results of the present study indicate for the first time that repeated administration of the 5-HT7 receptor antagonist SB269970 results in a decrease of glutamatergic synaptic transmission in the rat frontal cortex. This phenomenon may have consequences for the development of new antidepressant therapies based on 5-HT7 receptor antagonism.

Acknowledgements: Supported by the Ministry of Science and Higher Education (Warsaw, Poland) grant no. 0259/BP01/2010/38 and by the statutory funds from the Institute of Pharmacology, Polish Academy of Sciences, Cracow, Poland.

Conflict of interests: None declared.


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