Brain slice preparation
The use of animals conformed to the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences of the Physiological Society of Japan (1988) and was approved by the Animal Care Committee of the Jikei University School of Medicine, Tokyo, Japan. Wistar rats (7-21 days; weighing 17-50 g) of either sex were anesthetized by intraperitoneal ketamine (100-150 mg/kg) injection or brief isoflurane (5%) inhalation and decapitated immediately after the disappearance of the righting reflex. Using a vibration slice cutter (DTK-1000, Dosaka, Kyoto, Japan), two to three 400-μm thick horizontal brain slices through the Sp5c were made in ice-cold low-Ca2+ and high-Mg2+ artificial cerebrospinal fluid (ACSF) containing (in mM) NaCl 125, KCl 2.5, CaCl2 0.1, MgCl2 5.0, NaH2PO4 1.25, D-glucose 12.5, L-ascorbic acid 0.4, and NaHCO3 25 and saturated with 95% O2 + 5% CO2 (pH = 7.4). The slices were incubated in "normal" ACSF (CaCl2 2 mM and MgCl2 1.3 mM) for 30-40 min at 37°C and then kept at room temperature until the recordings.
Whole-cell recording
Two types of internal solutions were used [
1]. A "CsCl-based" internal solution contained (in mM) 140 CsCl, 1 CaCl
2, 2 MgATP, 1 EGTA, and 10 HEPES, pH 7.3, with CsOH. The estimated equilibrium potential of Cl
- with this internal solution was approximately 0 mV. This solution was used to record miniature inhibitory postsynaptic currents (mIPSCs) and miniature excitatory postsynaptic currents (mEPSCs), which appear independent of presynaptic action potentials. The frequencies of mIPSCs and mEPSCs reflect spontaneous and tonic transmitter release from inhibitory and excitatory presynaptic axon terminals, respectively. The mIPSCs were recorded in isolation at a holding potential of -70 mV in the presence of kynurenic acid (1 mM; an ionotropic glutamate receptor blocker; Sigma) and TTX (1 μM; a voltage-dependent Na
+ channel blocker; Alomone, Jerusalem, Israel), while mEPSCs were recorded in the presence of picrotoxin (100 μM; a GABA
A and GABA
C receptor blocker; Sigma), strychnine (1 μM; a glycine receptor blocker; Sigma), instead of kynurenic acid and TTX [
2]. A "low-Cl" internal solution contained (in mM) 135 gluconic acid potassium, 0.1 CaCl
2, 2 MgCl
2, 2 MgATP, 0.3 NaGTP, 1 EGTA, and 10 HEPES, pH 7.3, with KOH. The estimated equilibrium potential of Cl
- with this internal solution was -90 mV. This internal solution was used to simultaneously record mEPSCs and mIPSCs from SG neurons in the Sp5c. In these experiments, the membrane potential was held around -40 mV, a value in between the reversal potentials of EPSCs and IPSCs, enabling simultaneous but separate recordings of inward (excitatory) and outward (inhibitory) postsynaptic currents. The tip resistance of the electrode with these solutions was 3-7 MΩ.
The slices were secured in a recording chamber (~0.5 ml volume) and continuously perfused with ACSF at a flow rate of 2-3 ml/min. Using infrared differential interference contrast optics or oblique illuminating systems combined with videomicroscopy (BX51; Olympus, Tokyo), the SG of Sp5c was identified as a lucent, rostrocaudally extending structure adjacent to the dark and opaque rostrocaudally running bundles of the trigeminal nerve at the lateral edge of the brainstem. The neurons located in the SG were visually identified, and all recordings were made from healthy-appearing neurons. Immediately (within 10 s) after the membrane rupture that established the whole-cell recording mode, we confirmed that the resting membrane potential was more polarized than -45 mV without current injection and, by rapidly manipulating the amplifier controls, that action potentials in response to positive current injection were overshooting. The cells without these properties were rare in our experimental conditions and were discarded when found. In the recordings with low-Cl internal solution, the resting potential and input membrane resistance were measured 5-10 min after the establishment of the whole-cell configuration. These values for the neurons recorded with the CsCl internal solution were not measured after stabilization because the resting membrane potential was almost 0 mV due to K channel blockade with Cs. The slices were perfused with "normal ACSF" during the search for and establishment of whole-cell configuration, and the data used for the analyses were sampled after at least a 10-min perfusion with specific ACSFs containing drugs for pharmacological isolation of the components of interest in each experiment. Only one neuron in a slice was recorded for pharmacological analyses. The nominally "Ca2+ free" ACSF contained 3.3 mM MgCl2 and 0.2 mM EGTA (instead of 2 mM CaCl2 and 1.3 MgCl2) and was used to examine the role of extracellular Ca2+. The membrane current was recorded with an AxoPatch 200B (Axon Instruments). In a subset of the experiments, evoked IPSCs (eIPSCs), which were evoked by focal stimulation at a submaximal intensity (0.1 Hz; 0.08-0.5 mA; 100 μsec) with a bipolar concentric electrode placed within the Sp5C near the recording site (< 500 μm), were recorded together with spontaneous IPSCs (sIPSCs) in the absence of TTX and in the presence of kynurenic acid (1 mM).
In general, if a sole application of an antagonist exerts its effect by blocking a certain type of receptor, this effect should depend on how much these target receptors had been previously activated by endogenous ligands. Because esmolol markedly facilitated release at a much higher concentration than that at which it antagonizes β receptors, the β receptor agonist isoproterenol was pre-applied at 100 μM 10 min prior to esmolol in some of the experiments. Also, in a subset of cells, the effects of landiolol, which is another β blocker with a similar chemical structure to esmolol, were observed to examine whether landiolol also facilitates mIPSCs.
In general, an increase in the frequency of miniature postsynaptic events by a drug implies an effect on the presynaptic release mechanism [
12]. Accordingly, the increase in mIPSC frequency, but not that of mEPSC, as described in the Results, might indicate that esmolol selectively affected GABAergic and/or glycinergic presynaptic terminals in the Sp5c.
We also analyzed the effect of extracellular Ca
2+ deprivation on esmolol modulation of mIPSC frequency to examine whether the effect of esmolol depends on the presence of extracellular Ca
2+ and its entry into the presynaptic terminals, which is the most critical step of transmitter release [
13], to further identify the mechanism underlying the increase in mIPSC frequency with esmolol.
The signals were sampled with a PowerLab interface (AD Instruments) at 4 kHz. The series resistance was monitored but not compensated. The whole-cell capacitance was monitored and compensated. There were no apparent changes in the series resistance and whole-cell capacitance during the recordings for the neurons used in this study. The original traces in the figures and curve-fitting calculations were made with the Igor Pro graphic program (WaveMetrics). Postsynaptic currents were identified first automatically and then manually with visual identification of all events with IgorPro procedures written by F.K.