All experimental protocols in this work were reviewed and approved by the University of California Institutional Animal Care and Use Committee in compliance with the Animal Welfare Act and in accordance with Public Health Service Policy on Humane Care and Use of Laboratory Animals.
Sixteen male 11-week-old Sprague-Dawley rats (320 – 370 gm, Charles River Laboratories, Inc. Wilmington, MA, USA) were anesthetized with a combination of ketamine (50 mg/kg, im, Vedco Inc. St. Joseph, MO, USA) and xylazine (8 mg/kg, im, Vedco Inc. St. Joseph, MO, USA). A 4 – 5 mm segment of the aortic depressor nerve between the superior laryngeal nerve and vagus nerve/sympathetic trunk was carefully isolated and placed on a section of parafilm. The fluorescent dye crystals, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (FAST
DiI™ solid; DiI[
(3), Molecular Probes, Eugene, OR, USA), were placed on the aortic depressor nerve and the area was imbedded with Polyvinylsiloxane gel (Charlisle laboratories Inc. Rockville Centre, NY, USA). To allow for transport of the dye to the terminal boutons, the rats were allowed to recover for two weeks before the experimental protocols as previously reported (Chen et al., 2009
; Chen et al., 2005
). Pre-emptive analgesia (ketoprofen, 2 mg/kg sc, Fort Dodge Animal Health, Fort Dodge, Iowa, USA) and antibiotics (Baytril 5 mg/kg, im, Bayer HealthCare LLC, Shawnee Mission, Kansas, USA) were given immediately before surgery to prevent post-operative pain and infection. During surgery, the following procedure was used to assess adequacy of anesthesia and supplemental anesthesia (25% of initial dose) was given if 1) the eye blink reflex was present; 2) there was whisker movement; 3) paw withdrawal occurred upon pinch; or 4) irregular or sudden changes in breathing frequency were observed. Fluids (0.9% saline, 2 ml/100 g BW, sc) were administered post-operatively to prevent dehydration. Animals were kept on heating pads during recovery from anesthesia and were checked daily for signs of pain and infection.
Brainstem slice preparation
The rats were anesthetized with a combination of ketamine (50 mg/kg, im) and xylazine (8 mg/kg, im) and decapitated. The brain was rapidly exposed and submerged in ice-cold (< 4°C) high-sucrose artificial cerebrospinal fluid that contained (mM): 3 KCl, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 220 sucrose and 2 CaCl2, with a pH of 7.4 when continuously bubbled with 95% O2 / 5% CO2. Brainstem transverse slices (250 μm thick) were cut with Leica VT1000 S vibratome (Leica Microsystems, Inc. Bannockburn, IL, USA). After incubating for 45 min at 37°C in high-sucrose artificial cerebrospinal fluid, the slices were placed in normal artificial cerebrospinal fluid that contained (mM): 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 25 glucose and 2 CaCl2, with a pH of 7.4 when continuously bubbled with 95% O2 / 5% CO2. During the experiments a single slice was transferred to the recording chamber, held in place with a silk mesh, and continuously perfused with oxygenated artificial cerebrospinal fluid at a rate of approximately 3 ml/min. The perfusion line consists of an inner tube for artificial cerebrospinal fluid and an outer tube that was connected to a circulating water bath for temperature control. All experiments were performed at 33°-34°C.
All whole-cell voltage-clamp recordings were performed on second-order baroreceptive NTS neurons with attached fluorescent aortic depressor nerve boutons. The neurons were visualized with infrared differential interference contrast. The fluorescent boutons were visualized with an optical filter set for DiI (XF108, Omega Optical Inc., Brattleboro, VT, USA) and an image integrating system (InvestiGater, Dage-MTI, Michigan City, IN, USA). Borosilicate glass electrodes were filled with a KCl solution containing (mM): 130 KCl, 5 NaCl, 1 MgCl2
, 3 Mg-ATP, 0.2 Na-GTP, 10 EGTA, 10 HEPES. The pH was adjusted to 7.3 with KOH. Once the whole-cell configuration was established, the neuron was voltage-clamped at −60 mV. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded in the presence of the ionotropic glutamate receptor antagonists, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX, 10 μM) and (±)-2-amino-5-phosphonovaleric acid (AP-5, 50 μM) and a sodium channel antagonist, tetrodotoxin (1 μM). We and others previously showed that these inhibitory synaptic currents are GABAergic: having a reversal potential for chloride ions and being blocked by GABAA
receptor antagonists (Chen et al., 2005
; Glatzer et al., 2005
; McDougall et al., 2008
; Zhang et al., 2007
To determine whether activation of CB1Rs in the NTS decreases presynaptic GABA release, mIPSCs were recorded for six min during the control period and five min during perfusion with a cannabinoid receptor agonist, WIN 55212-2 (0.3 – 30 μM). For the WIN concentration-response curve, the doses of WIN were applied in a random order with a recovery time of 7-25 minutes between doses. Since WIN 55212-2 has some nonspecific effects, the response to WIN 55212-2 at concentration at 3 μM, which was found to be an effective functional mid-range dose, was again tested in the presence of the CB1R antagonist, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM 251, 5 μM) to verify that the inhibition of GABA release was mediated by activation of CB1Rs. In addition, a more selective CB1R agonist, methanandamide (3 μM), was also tested in the absence and presence of the CB1R antagonist. Finally, to demonstrate that activation of second-order baroreceptive NTS neurons evokes endogenous release of ECBs, leading to presynaptic inhibition of GABA release, the effects of depolarization of the postsynaptic neuron on mIPSC frequency and amplitude were examined in the absence and presence of the CB1R antagonist. The postsynaptic neuron was directly depolarized (−60 mV to +60 mV at 20 mV increments) for 20 s every 60 s with mIPSCs recorded for 40s following the neuronal depolarization (). The responses in mIPSC frequency and amplitude induced by the depolarizing steps were measured at the return of the membrane potential to −60 mV in order to control for the change in driving force for the chloride ions. The decrease in mIPSC frequency was greatest during the first 10 s upon return of the membrane potential to −60mV. The mIPSC frequency then gradually returned to baseline over the next 30 s. Thus the changes in mIPSC frequency reflected the full effect of depolarization. In a separate group of neurons this protocol was performed in the presence of AM 251.
Figure 5 Effects of postsynaptic membrane depolarization on presynaptic GABA release. A. Depolarization-induced disinhibition protocol. Brief (20 s) depolarizing steps were applied to the postsynaptic neurons every minute. Two minutes of mIPSC recordings before (more ...)
Data are expressed as means ± SE unless otherwise indicated. Differences were considered significant at p < 0.05. The statistical analyses were performed with SigmaStat software using a student’s t-test, one-way ANOVA, or two-way ANOVA, as described below.
To examine the effect of WIN 55212-2, the frequencies and amplitudes of mIPSCs recorded during the last three min of the control period were averaged as the control value. The same variables for mIPSCs recorded during the last three min of WIN 55212-2 perfusion at each concentration were averaged and expressed as a percent of the control value. The agonist concentration–response curve was analyzed with one-way ANOVA. To determine the effect of the CB1R antagonist on the agonist induced depression, mIPSC frequency during agonist (3 μM) perfusion was expressed as a percent of the control value. The data from neurons used to generate the concentration-response curve for WIN 55212-2 at the 3 μM dose were used to establish the agonist-induced effect in the absence of AM 251. In a separate group of neurons, the effects of the agonist at 3 μM were determined in the presence of AM 251 only. Thus, an unpaired t-test was used. For methanandamine, only one dose (3 μM) was tested and the effects of the agonist were tested both in the absence and presence of AM 251. Thus, the data were compared with a paired t-test.
To determine the effect of depolarization-induced ECB release on presynaptic GABA transmission, mIPSC frequency and amplitude recorded after the 20 s of depolarization were averaged for each depolarizing step. The data were compared with a two-way ANOVA with the postsynaptic membrane potential as one factor and the absence and presence of the CB1R antagonist as the other factor.
WIN 55212-2, methanandamide, and AM 251 were obtained from Tocris (Ballwin, MO, USA). NBQX, AP5, TTX, Mg-ATP, Na-GTP, EGTA, and HEPES were obtained from Sigma (St. Louis, MO, USA). All other chemicals were obtained from Fisher (Fairlawn, NJ, USA).