Pharmacological Discrimination of Effects of MK801 on Thalamocortical, Mesothalamic, and Mesocortical Transmissions

N-methyl-d-aspartate/glutamate receptor (NMDAR) is one of the major voltage-sensitive ligand-gated cation channel. Several noncompetitive NMDAR antagonists contribute to pathophysiology of schizophrenia and mood disorders; however, the effects of inhibition of NMDAR on several transmitter system have not been well clarified. Thus, this study determined the selective NMDAR antagonist, MK801 (dizocilpine), on thalamocortical, mesothalamic, and mesocortical transmissions associated with l-glutamate, GABA, serotonin, norepinephrine, and dopamine using multiprobe microdialysis. Perfusion with MK801 into the medial prefrontal cortex (mPFC) increased and decreased respective regional releases of monoamine and GABA without affecting l-glutamate. The mPFC MK801-induced monoamine release is generated by the regional GABAergic disinhibition. Perfusion with MK801 into the reticular thalamic nucleus (RTN) decreased GABA release in the mediodorsal thalamic nucleus (MDTN) but increased releases of l-glutamate and catecholamine without affecting serotonin in the mPFC. The RTN MK801-induced l-glutamate release in the mPFC was generated by GABAergic disinhibition in the MDTN, but RTN MK801-induced catecholamine release in the mPFC was generated by activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/glutamate receptor (AMPAR) which received l-glutamate release from thalamocortical glutamatergic terminals in the mPFC. Perfusion with MK801 into the dorsal raphe nucleus (DRN) decreased GABA release in the DRN but selectively increased serotonin release in the MDTN and mPFC. These DRN MK801-induced serotonin releases in the both mPFC and MDTN were also generated by GABAergic disinhibition in the DRN. These results indicate that the GABAergic disinhibition induced by NMDAR inhibition plays important roles in the MK801-induced releases of l-glutamate and monoamine in thalamic nuclei and cortex.

3.1.2. Interaction between NMDAR and GABAA-R in the mPFC on Regional Extracellular Transmitter Levels.
Perfusions with 1 μM muscimol into the mPFC did not affect regional extracellular levels of Lglutamate, GABA, serotonin, norepinephrine, or dopamine (  Figure 1E) without affecting MK801-induced reduction of GABA release ( Figure 1B). These results suggest that MK801 increases monoamine release in the mPFC (threshold level is 5 μM) via regional GABAergic disinhibition (threshold level is 1 μM).

Effects of NMDAR in the RTN on Transmitter Releases in the mPFC and MDTN (Study_2).
Several previous reports demonstrated that the activation of thalamocortical glutamatergic pathway is one of the major responsible glutamatergic pathways of systemic MK801-induced Lglutamate release in the mPFC (medial prefrontal cortex) [15,17,18,20,21]. Our previous studies demonstrated that activation of glutamatergic neurons in the MDTN (mediodorsal thalamic nucleus) increased the L-glutamate release in the mPFC, IsC (insular cortex) and OFC (orbitofrontal cortex) [15,17,18,20,21]. The threshold concentrations of local administration of MK801 into the MDTN on L- Ordinates: mean ± SD (n = 6) of extracellular transmitter levels (µM or nM); abscissa: time after MK801 administration (min). Light blue and gray bars indicate perfusion with muscimol (Mus) and MK801 into the mPFC, respectively. * p < 0.05, ** p < 0.01; relative to control or 5 µM MK801 by multivariate analysis of variance (MANOVA) with Tukey's post hoc test.
3.1.2. Interaction between NMDAR and GABA A -R in the mPFC on Regional Extracellular Transmitter Levels Perfusions with 1 µM muscimol into the mPFC did not affect regional extracellular levels of  Figure 1E) without affecting MK801-induced reduction of GABA release ( Figure 1B). These results suggest that MK801 increases monoamine release in the mPFC (threshold level is 5 µM) via regional GABAergic disinhibition (threshold level is 1 µM).

Effects of NMDAR in the RTN on Transmitter Releases in the mPFC and MDTN (Study_2)
Several previous reports demonstrated that the activation of thalamocortical glutamatergic pathway is one of the major responsible glutamatergic pathways of systemic MK801-induced l-glutamate release in the mPFC (medial prefrontal cortex) [15,17,18,20,21]. Our previous studies demonstrated that activation of glutamatergic neurons in the MDTN (mediodorsal thalamic nucleus) increased the l-glutamate release in the mPFC, IsC (insular cortex) and OFC (orbitofrontal cortex) [15,17,18,20,21]. The threshold concentrations of local administration of MK801 into the MDTN on l-glutamate release in the MDTN and mPFC were 50 µM [17,21]. Contrary to the MDTN, the threshold concentrations of local administration of MK801 into the RTN (reticular thalamic nucleus) on l-glutamate release in the MDTN and mPFC were 5 µM and 1 µM, respectively [17,21]. These finding suggest that the MDTN is responsible region for thalamocortical glutamatergic transmission, but a candidate generator region is the RTN. To explore the threshold concentration of local administration of MK801 into the RTN on thalamocortical glutamatergic transmission and its associated other transmitter release, Study_2 was designed to determine the effects of MK801 (1, 5 and 50 µM) into the RTN on the releases of l-glutamate, GABA, serotonin, norepinephrine, and dopamine in the mPFC and MDTN.  Figure 2E) without affecting those of GABA or serotonin in the mPFC ( Figure 2B,C). Extracellular levels of catecholamine (norepinephrine and dopamine) were increased by 5 µM MK801 but not by 1 µM MK801 ( Figure 2D,E); however, extracellular l-glutamate level was increased by both 1 µM and 5 µM MK801 (Figure 2A). Therefore, the threshold concentration of local administration of MK801 into the RTN on releases of l-glutamate and catecholamine in the mPFC are 1 µM and 5 µM, respectively. glutamate release in the MDTN and mPFC were 50 μM [17,21]. Contrary to the MDTN, the threshold concentrations of local administration of MK801 into the RTN (reticular thalamic nucleus) on Lglutamate release in the MDTN and mPFC were 5 μM and 1 μM, respectively [17,21]. These finding suggest that the MDTN is responsible region for thalamocortical glutamatergic transmission, but a candidate generator region is the RTN. To explore the threshold concentration of local administration of MK801 into the RTN on thalamocortical glutamatergic transmission and its associated other transmitter release, Study_2 was designed to determine the effects of MK801 (1, 5 and 50 μM) into the RTN on the releases of L-glutamate, GABA, serotonin, norepinephrine, and dopamine in the mPFC and MDTN.  Figure 2E) without affecting those of GABA or serotonin in the mPFC ( Figure 2B,C). Extracellular levels of catecholamine (norepinephrine and dopamine) were increased by 5 μM MK801 but not by 1 μM MK801 ( Figure  2D,E); however, extracellular L-glutamate level was increased by both 1 μM and 5 μM MK801 ( Figure  2A). Therefore, the threshold concentration of local administration of MK801 into the RTN on releases of L-glutamate and catecholamine in the mPFC are 1 μM and 5 μM, respectively. Extracellular serotonin level was increased by 50 μM MK801 but not by 1 μM or 5 μM MK801 ( Figure 3C). Extracellular GABA level was decreased by 1 μM MK801 ( Figure 3B). Extracellular Lglutamate level was increased by 5 μM MK801, but not by 1 μM MK801 ( Figure 3A). Therefore, the threshold concentrations of local administration of MK801 into the RTN on releases of L-glutamate, GABA and serotonin in the MDTN are 5 μM, 1 μM, and 50 μM, respectively. Extracellular serotonin level was increased by 50 µM MK801 but not by 1 µM or 5 µM MK801 ( Figure 3C). Extracellular GABA level was decreased by 1 µM MK801 ( Figure 3B). Extracellular l-glutamate level was increased by 5 µM MK801, but not by 1 µM MK801 ( Figure 3A). Therefore, the threshold concentrations of local administration of MK801 into the RTN on releases of l-glutamate, GABA and serotonin in the MDTN are 5 µM, 1 µM, and 50 µM, respectively.

Effects of GABA
These results suggest that RTN MK801-induced l-glutamate release in the mPFC is generated by the GABAergic disinhibition and relatively activation of AMPAR in the MDTN, but is not modulated by these receptors in the mPFC. Contrary to l-glutamate, RTN MK801-induced catecholamine release in the mPFC is regulated by the GABAergic disinhibition in the MDTN, and activation of AMPAR in the MDTN and mPFC, but is not modulated by GABA A -R in the mPFC. Therefore, RTN MK801-induced catecholamine release in the mPFC is activated by AMPAR in the mPFC via activation of thalamocortical glutamatergic transmission. These results suggest that RTN MK801-induced L-glutamate release in the mPFC is generated by the GABAergic disinhibition and relatively activation of AMPAR in the MDTN, but is not modulated by these receptors in the mPFC. Contrary to L-glutamate, RTN MK801-induced catecholamine release in the mPFC is regulated by the GABAergic disinhibition in the MDTN, and activation of AMPAR in the MDTN and mPFC, but is not modulated by GABAA-R in the mPFC. Therefore, RTN MK801-induced catecholamine release in the mPFC is activated by AMPAR in the mPFC via activation of thalamocortical glutamatergic transmission.  Figure 5A). Contrary to L-glutamate, neither 50 μM RTN MK801-induced serotonin release nor reduction of GABA in the MDTN were affected by perfusion with 1 μM muscimol and perampanel into the MDTN (Figure 5B,C).
These results suggest that RTN MK801-induced L-glutamate release in the MDTN is generated by the GABAergic disinhibition in the MDTN, but is not modulated by AMPAR in the MDTN. Contrary to L-glutamate, RTN MK801-induced serotonin release and reduction of GABA release in the MDTN are not affected by GABAA-R or AMPAR in the MDTN.   Figure 5A). Contrary to l-glutamate, neither 50 µM RTN MK801-induced serotonin release nor reduction of GABA in the MDTN were affected by perfusion with 1 µM muscimol and perampanel into the MDTN (Figure 5B,C).
These results suggest that RTN MK801-induced l-glutamate release in the MDTN is generated by the GABAergic disinhibition in the MDTN, but is not modulated by AMPAR in the MDTN. Contrary to l-glutamate, RTN MK801-induced serotonin release and reduction of GABA release in the MDTN are not affected by GABA A -R or AMPAR in the MDTN.
the MDTN did not affect ( Figure 5A). Contrary to L-glutamate, neither 50 μM RTN MK801-induced serotonin release nor reduction of GABA in the MDTN were affected by perfusion with 1 μM muscimol and perampanel into the MDTN (Figure 5B,C).
These results suggest that RTN MK801-induced L-glutamate release in the MDTN is generated by the GABAergic disinhibition in the MDTN, but is not modulated by AMPAR in the MDTN. Contrary to L-glutamate, RTN MK801-induced serotonin release and reduction of GABA release in the MDTN are not affected by GABAA-R or AMPAR in the MDTN.

Effects of NMDAR in the DRN on Transmitter Releases in the DRN, mPFC and MDTN (Study_4)
The results of stdies_1~3 indicated the serotonergic transmission in the mPFC (medial prefrontal cortex) was regulated by independent system compared with other catecholamine, since serotonin release in the mPFC is not affected by thalamocortical glutamatergic transmission, but conversely mesothalamic serotonergic transmission possibly affects thalamocortical glutamatergic pathway in the MDTN (mediodorsal thalamic nucleus) [16]. To clarify the effects of serotonergic transmission on mesocortical serotonergic transmission (DRN-mPFC) and mesothalamic serotonergic transmission (DRN-MDTN), study_4 was designed to determine the effects of local administration of MK801 into the DRN (dorsal raphe nucleus) on serotonin release in the mPFC, DRN and MDTN.  Figure 6C) without affecting those of l-glutamate, GABA, norepinephrine or dopamine ( Figure 6A,B,D,E). Therefore, DRN MK801-induced serotonin release in the mPFC is probably induced by GABAergic disinhibition in the DRN. and 50 μM MK801 ( Figure 6C). Therefore, the threshold concentration of local administration of MK801 into the DRN on serotonin release in the mPFC is lower than 1 μM.

Concentration-Dependent Effects of Local Administration of MK801 into the DRN on Extracellular Transmitter Levels in the MDTN
Perfusions with MK801 (1 and 50 μM) into the DRN concentration-dependently increased extracellular serotonin level in the MDTN [FMK801(2,15) = 11.6 (p < 0.05), FTime(6.9,103.6) = 26.0 (p < 0.05),  Figure 7A,B,D). Extracellular serotonin level was increased by 50 µM MK801, but not by 1 µM MK801 ( Figure 7C). Our previous study demonstrated that local administration of 5 µM MK801 into the DRN increased serotonin release in the MDTN [16]. Therefore, taken together with our previous demonstration, the threshold concentration of local administration of MK801 into the DRN on serotonin release in the DRN is 5 µM.

Discussion
The threshold concentration of local administration (perfusion) of MK801 into the mPFC, MDTN, RTN, and DRN on several transmission systems demonstrated by this study and previous reports [15][16][17]20] are summarized in the Table 1.

Discussion
The threshold concentration of local administration (perfusion) of MK801 into the mPFC, MDTN, RTN, and DRN on several transmission systems demonstrated by this study and previous reports [15][16][17]20] are summarized in the Table 1.

Catecholaminergic Transmission Regulation System Associated with NMDAR
Local administration of MK801 into the mPFC increased and decreased regional respective monoamine and GABA releases without affecting l-glutamate release [15,20,21,24,30]. These mPFC MK801-induced monoamine release was inhibited by activation of GABA A -R in the mPFC (perfusion with 1 µM muscimol into the mPFC). Therefore, inhibition of NMDAR in the mPFC increases monoamine release induced by presynaptic GABAergic disinhibition in the mPFC. Indeed, the threshold concentrations of local administration of MK801 into the mPFC on GABA release (1 µM) is more sensitive compared with that of monoamine (5 µM) ( Table 1).
Contrary to intra mPFC regulation system, local administration of MK801 into the RTN increased releases of l-glutamate, norepinephrine and dopamine in the mPFC without affecting those of GABA or serotonin. The threshold concentration of local administration of MK801 into the RTN on releases of l-glutamate and catecholamine (norepinephrine and dopamine) in the mPFC were 1 µM and 5 µM, respectively. The RTN MK801-induced l-glutamate release in the mPFC was inhibited by the activation of GABA A -R and inhibition of AMPAR in the MDTN, but was not affected by the activation of GABA A -R or inhibition of AMPAR in the mPFC. These results suggest that an activation of glutamatergic neuronal activity induced by GABAergic disinhibition in the MDTN contributes to RTN MK801-induced l-glutamate release in the mPFC.
Various thalamic nuclei, which receive GABAergic inhibition from RTN [39], project excitatory glutamatergic terminals to superficial layers of the mPFC [38,41,42]. Glutamatergic neurons in the MDTN also receive GABAergic inhibition of intra MDTN GABA interneurons [17,20,21], whereas the inhibitory regulation from the RTN on glutamatergic neurons in the MDTN is predominant compared with that from GABAergic interneurons in the MDTN [15,20]. Indeed, the threshold concentration of local administration of MK801 into the MDTN and RTN on GABA release in the MDTN were 50 µM and 1 µM, respectively [15,17,20,21]. Therefore, one of the major responsible pathways of MK801-induced l-glutamate release in the mPFC is thalamocortical (from MDTN to mPFC) glutamatergic pathway, but generating mechanisms is GABAergic disinhibition from RTN to MDTN through NMDAR inhibition in the RTN.
Contrary to l-glutamate release, the RTN MK801-induced catecholamine release in the mPFC was inhibited by the activation of GABA A -R in the MDTN, inhibition of AMPAR in the MDTN and mPFC, but was not affected by the activation of GABA A -R in the mPFC. Electrophysiological study demonstrated that electrical stimulation of the MDTN increased the releases of glutamate and catecholamine in the mPFC without affecting those of serotonin [23]. Other line studies demonstrated that the afferents from LC compromise two types: the projects to the superficial layer of the mPFC of co-releasing norepinephrine with dopamine, while the other projects to the deep layer of the mPFC as the selective norepinephrine-releasing terminal [23,24,40]. The co-releasing terminals norepinephrine with dopamine from the LC receive excitatory thalamocortical glutamatergic projection which activates AMPAR on the catecholamine co-releasing terminals [23,24]. Taken together with these previous findings, the present study suggests that the hyperactivated glutamatergic transmission in the thalamocortical pathway (from MDTN to mPFC) enhances catecholamine release in the mPFC via activation of AMPAR in the mPFC.

Serotonergic Transmission Regulation System Associated with NMDAR
This study indicates the several specific regulation systems of serotonergic transmission in the mPFC and MDTN which are independent upon catecholamine release regulation system. The first, intra mPFC regulation of serotonergic transmission associated with NMDAR is resembling to the regulation systems of catecholaminergic transmission, since the selective noradrenergic, dopaminergic and serotonergic terminals in the deeper layers of mPFC receive GABAergic inhibition. However, the serotonergic terminal in the mPFC from DRN does not contact with thalamocortical glutamatergic afferents, whereas mesothalamic serotonergic transmission activates thalamocortical glutamatergic transmission, since an activation of serotonergic neuronal activities in the DRN increases serotonin release in the MDTN. Recent multiprobe microdialysis studies demonstrated that an activation of serotonergic neuronal activity enhances MDTN glutamatergic neurons through activation of excitatory 5-HT7 receptor in the MDTN [15,16]. In the present study, the norepinephrine release in the MDTN could be detected, but that of dopamine could not be detected. The present study has not determined the functionally impact of norepinephrine release in the MDTN on thalamocortical glutamatergic transmission, whereas our previous study suggest that the noradrenergic transmission from LC probably attenuates thalamocortical glutamatergic transmission via activates α1 adrenoceptor on the RTN GABAergic neurons, but norepinephrine release in the MDTN does not affect thalamocortical glutamatergic transmission [18]. Therefore, the regulatory effects of norepinephrine and serotonin in the thalamus are independent, since thalamocortical glutamatergic transmission is inhibited and enhanced by activation of respective α1 adrenoceptor on the GABAergic neuron in the RTN and 5-HT7 receptor on the glutamatergic neurons in the MDTN [15,16,18].
In the DRN, serotonin release is regulated by inhibitory GABA A -R predominantly, as indicated by decreased regional serotonin release following local administration of muscimol (GABA A -R agonist) into the DRN; however, GABA release is regulated by excitatory 5-HT7 receptor, reflecting decreased regional GABA release following local perfusions with SB269970 (5-HT7R antagonist) into the DRN [16]. Previous electrophysiological studies show that inhibitory 5-HT1AR and excitatory 5-HT2A and 5-HT7R response in serotonergic neurons were more than 90% and lower than 20% in the DRN, respectively [43]. In contrast with serotonergic neurons, inhibitory and excitatory 5-HT responses in GABAergic neurons were 15% and 80% in the DRN, respectively, likely relating to 5-HT7R excitatory responses [43]. Taken with these reports, GABA A -R and 5-HT7R predominantly inhibit serotonergic neurons and enhance GABAergic neurons, respectively. In the present study, local administration of MK801 into the DRN, decreased and increased releases of GABA and serotonin in the DRN, respectively. The opposite effects of MK801 between releases of GABA and serotonin were inhibited by local administration of muscimol in the DRN. Therefore, NMDAR in the DRN regulates predominantly GABAergic neurons rather than serotonergic neuronal activity in the DRN.

Pharmacological Discrimination of Effects of MK801 on Thalamocortical Glutamatergic Associated Catecholamine Release and Mesocortical Serotonergic Transmission
The present study demonstrated that inhibition of NMDAR directly inhibited GABAergic transmission, but enhanced indirectly glutamatergic and monoaminergic transmissions induced by GABAergic disinhibition. In other words, during resting stage, MK801 selectively inhibits NMDAR on GABAergic neurons, but cannot affect NMDAR on the glutamatergic or monoaminergic neurons. In generally, GABAergic interneuron is more sensitive to NMDAR antagonist compared with other types of neurons, since the resting membrane potential of GABAergic interneurons are more positive (−50~−60 mV) rather than those of monoaminergic and glutamatergic neurons [43,44]. NMDAR is voltage-sensitive ligand gated cation channel, since the extracellular magnesium and zinc ions bind to specific sites of NMDAR and blocks cation channel pore during resting stage. However, depolarization (higher than −20 mV) repels magnesium and zinc ions from the channel pore, resulting in voltage-dependently inflow of sodium and calcium ions, and outflow potassium ions [45]. Taken together with these findings, NMDAR on GABAergic neurons can voltage-dependently be activated by small amount of depolarization compared with NMDAR on other glutamatergic and monoaminergic neurons.
In this study, the threshold concentrations of local administrations of MK801 into the mPFC, RTN and DRN on regional GABAergic transmission were almost equal to be 1 µM. Contrary to GABA, the threshold concentrations of local administration of MK801 into the mPFC and RTN on catecholamine release in the mPFC were 5 µM, whereas those into mPFC and DRN on serotonin release in the mPFC were 5 µM and 1 µM, respectively. Therefore, serotonergic transmission is more sensitive to NMDAR antagonist rather than other catecholaminergic transmissions in the mPFC.
Subanesthetic doses of ketamine produces transient dissociative and psychotomimetic effects that resemble the positive and negative symptoms of schizophrenia [46]; however, numerous placebo-controlled studies have demonstrated that sub-anesthetic-dose ketamine exerts rapid, robust, and relatively sustained antidepressant effects in patients with antidepressant-resistant major depressive disorder and bipolar depression [11,[47][48][49][50]. Ketamine has also been shown to have distinct and independent anti-suicidal and anti-anhedonic effects in patients with mood disorders [11,12].
Neither clinical mechanisms between psychotomimetic and antidepressant effects of ketamine have remained to be clarified. Local administration of ketamine into the infralimbic mPFC reproduced the antidepressant-like actions of systemic ketamine administration [51]. Local administration of (R)-ketamine into the infralimbic mPFC and hippocampus also produced antidepressant-like action [52], whereas local administration of (R)-ketamine into the prelimbic mPFC nucleus accumbens could not produce [52]. These findings suggest that infralimbic mPFC, but not prelimbic mPFC, is responsible region of the antidepressant-like action of ketamine and (R)-ketamine. Moreover, the binding affinity (Ki values) of (S)-ketamine and (R)-ketamine to NMDAR are 0.7 and 2.6 µM, respectively [53]. Indeed, the anesthetic effects of (S)-ketamine is potent than that of (R)-ketamine [54]. The MK801 exhibits rapid antidepressant-like effects, but cannot keep long-lasting its action [55]. Clinical studies have also demonstrated that the antidepressant actions of ketamine against patients with major depression are more potent than those of other NMDAR antagonists, [56,57]. These clinical and preclinical demonstrations suggest that NMDAR inhibition, at least partially, contributes to rapid-acting antidepressant and anti-suicidal actions. Therefore, the superior clinical effects of ketamine against antidepressant-resistant depression is mediated multimodal mechanisms, but NMDAR antagonism plays important roles in the rapid-acting and anti-suicidal action of ketamine. Exactly, a recent preclinical study reported that inhibition of NMDAR-dependent bursting activity in the lateral habenula is probably associated with rapid-acting antidepressant-like effects of ketamine [58].
The thalamocortical glutamatergic transmission plays important roles in the function of neuro-cognition, including learning, memory, and perceptual integration [47,59,60]. The mesocortical serotonergic transmission contributes to improvement of depressive mood [61][62][63], and mesothalamic serotonergic transmission plays important roles in emotional perception via regulation of thalamocortical glutamatergic transmission [15,16,64]. Based on these findings, the present demonstration, the higher sensitivity of mesocortical serotonergic transmission and equivalent sensitivity of mesothalamic serotonergic transmission to MK801 compared with thalamocortical transmission of l-glutamate and secondary catecholamine release in the mPFC, suggests that lower concentration of MK801 affects emotional rather than cognitive disturbances. In the present study, inhibition of AMPAR in the MDTN and mPFC attenuated the MK801-induced glutamatergic and monoaminergic transmissions without affecting GABAergic disinhibition. Several preclinical studies reported the possibility that NMDAR inhibition conversely activates glutamatergic transmission associated with AMPAR through GABAergic disinhibition [58,65]. In spite of suspending clinical trials, the rapid-acting antidepressant-like effects of AMPAR positive allosteric modulator, S47445 have been also demonstrated by preclinical study [66], whereas the other line preclinical study suggested that AMPAR activation may not be necessary for the antidepressant effects of ketamine [67]. The detailed discussion regarding the effects of AMPAR activation and NMDAR inhibition on rapid-acting antidepressant-like action should be needed the results of an FDA approved clinical study using perampanel and ketamine (NCT03367533).

Conclusions
The present study determined the effects of MK801 on the thalamocortical (RTN-MDTN-mPFC) glutamatergic, mesothalamic (DRN-MDTN), and mesocortical (DRN-mPFC) serotonergic transmissions using multiprobe microdialysis, to clarify the NMDAR associated regulation systems in these three pathways. Inhibition of NMDAR in the RTN activates thalamocortical glutamatergic transmission induced by GABAergic inhibition and secondarily activated AMPAR in the MDTN. The enhanced l-glutamate release in the mPFC activates AMPAR on the regional co-releasing terminals resulting in an increase in releases of norepinephrine and dopamine, but does not affect serotonin release. Inhibition of NMDAR in the DRN enhances serotonin release in the DRN, MDTN and mPFC via GABAergic disinhibition in the DRN. The sensitivities of serotonin release in the mPFC to local administration of MK801 into the DRN is more predominant rather than that of catecholamine release in the mPFC induced by local MK801 administration into the RTN.