Ketamine attenuates the glutamatergic neurotransmission in the ventral posteromedial nucleus slices of rats

Ketamine was purchased from Gutian Pharma. Corp. (Fujian, China). 6,7-dinitro-quinoxaline-2,3(1H,4H)-dion (DNQX, a AMPA receptor antagonist), 2-Amino-5-phosphonovalerate-pharmacology(APV, a NMDA receptor antagonist),bicuculline (BIC,a blockade of GABA_A receptors), tetrodotoxin (TTX, a blockade of Na + channels) and strychnine (Str, a glycine receptors antagonist) were purchased from Sigma-Aldrich. Recording pipettes (tip diameter < 1 μM) were made from borosilicate glass capillaries (Sutter Instruments, Novato, CA) using the P-97 micropipette puller (Sutter Instruments, Novato, CA), as previous described [14].

Thirty Sprague–Dawley rats were purchased from animal center of the third military medical university (Chongqing, China). Thalamocortical slices were prepared as described previously [8] with a slight modification. Rats (7–15 days old) were deeply anesthetized with isoflurane and decapitated. After decapitated,the whole brain mass was isolated. The brain was then submerged in an ice-cold (0 °C) artificial cerebrospinal fluid (ACSF) containing (mM) NaCl 126, KCl 2.5,CaC1₂ 2,MgSO₄·7H₂O₂, NaHCO₃ 25,NaH₂PO₄·2H₂O 1.5,Glucose·H₂O 10, (pH = 7.34–7.45 when bubbled with 95% O₂–5% CO₂). Then a block tissue containing VPM was detached from the brain using a sharp blade. The tissue was again immersed in an ice-cold ACSF and further sectioned with the HM 650 V Vibroslicer (Thermos Instruments, US) to manufacture brain slice with 250 μM thickness slices (Fig. 1b). Before whole-cell recording, the slices were incubated at 32 °C in ACSF for 1 h. ACSF was saturated with a mixture of 95% O₂ and 5% CO₂.

In whole-cell recording, VPM neurons were located by a BX51WI microscope (Olympus, Japan) with an infrared camera. The intracellular solution for recording EPSCs contained (mM) 140CsCl, 2 Na₂ATP, 10 EGTA, and 10 HEPES (pH 7.4). The membrane potential was held at −65 mV. sEPSCs were recorded in the presence of 20 μM bicuculline and 1 μM strychnine. While mEPSCs were recorded in the presence of 20 μM bicuculline,1 μM strychnine and 0.5 μM TTX. The sEPSCs and mEPSCs were fltered at 3 kHz, digitized at 20 kHz with an HEKA EPC10 amplifier and Patch Master Software (HEKA InstrμMents, Inc., Lambrecht, DE), as previous described [15].

In the present study, all drugs were perfused by a BPS-4 perfusion system (ALA, USA), as previous described [8]. Ketamine was dissolved into ACSF and the concentrations of ketamine in were 30 μmol/L、100 μmol/L、300 μmol/L、1000 μmol/L respectively. We started to record the current after a 10 min duration of drug application and the recording time lasted for 5 min.

All currents were detected and analyzed using Mini Analysis 6 (Synaptosoft, Inc., Decatur, GA). For events recording, noise analysis was conducted for each cell and basal noise values during voltage-clamp recordings were <10 pA. The template wasselected visually according to the rise and decay phases of events, and the amplitude threshold was set to15 pA. The amplitude and frequency of currents were normalized to their mean value during the control period (5 min). The effects of drugs were expressed as % change (mean ± SEM) from pre-drug baseline, due to the large variability observed from neuron to neuron. The cumulative probability plots of the incidence of various inter-event intervals and amplitude, recorded in control conditions and during drug applications to the same neuro, as previous studies reported [16, 17]. We firstly run a normality test and the results show that the changes are normally distributed. Therefore, we used repeated measures one-way ANOVAs with post-hoc Bonferroni’s correction to evaluate discrepancies among groups, as previous study reported [15]. P < 0.05 was considered statistically significant.

Figure 2a showed the typical currents of sEPSCs recorded in a VPM neuron. Ketamine (30 μM and 100 μM) did not significantly affect the frequency of sEPSCs (P > 0.05, n = 8), only higher concentration of ketamine (300 μM and 1000 μM) decreased the frequency of sEPSCs (Fig. 2b, P< 0.05, n = 8). However, ketamine (30 μM–1000 μM) decreased the amplitude, as shown in Fig. 2b (P < 0.05, n = 8). Figure 2c showed the cumulative probability plots of the incidence of various inter-event intervals and amplitude.

mEPSCs were recorded in the presence of TTX (0.5 μM) and bicuculline (BIC, 20 μM, Fig. 3a). Compared with control group, ketamine had no significant effect on the frequency (Fig. 3b, P>0.05, n = 8). However, ketamine (100 μM–1000 μM) decreased the amplitude of mEPSCs (Fig. 3b, P< 0.05, n = 8). The cumulative probability plots of the incidence of various inter-event intervals and amplitude were showed in Fig. 3c. After ketamine (1000 μM) perfusion, we started to perfuse with ACSF. The time of perfuse with ACSF was also 10 min and the recording time lasted for 5 min. However, we found the amplitude and frequency of sEPSCs did not recover to the baseline (control) after wash out, as shown in Additional file 1: Figure S1A and B. The amplitude of mEPSCs did not recover to the level of control, but the frequency recover to the recover to the level of control (Additional file 1: Figure S1A and B).