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The Journal of Physiology logoLink to The Journal of Physiology
. 2014 Nov 25;593(Pt 1):217–230. doi: 10.1113/jphysiol.2014.275701

GABAergic transmission and enhanced modulation by opioids and endocannabinoids in adult rat rostral ventromedial medulla

Ming-Hua Li 1,✉, Katherine L Suchland 1, Susan L Ingram 1
PMCID: PMC4293064  PMID: 25556797

Abstract

Neurons in the rostral ventromedial medulla (RVM) play critical and complex roles in pain modulation. Recent studies have shown that electrical stimulation of the RVM produces pain facilitation in young animals (postnatal (PN) day < 21) but predominantly inhibits pain behaviours in adults. The cellular mechanisms underlying these changes in RVM modulation of pain behaviours are not known. This is in part because whole-cell patch-clamp studies in RVM to date have been in young (PN day < 18) animals because the organization and abundance of myelinated fibres in this region make the RVM a challenging area for whole-cell patch-clamp recording in adults. Several neurotransmitter systems, including GABAergic neurotransmission, undergo developmental changes that mature by PN day 21. Thus, we focused on optimizing whole-cell patch-clamp recordings for RVM neurons in animals older than PN day 30 and compared the results to animals at PN day 10–21. Our results demonstrate that the probability of GABA release is lower and that opioid and endocannabinoid effects are more evident in adult rats (mature) compared to early postnatal (immature) rats. Differences in these properties of RVM neurons may contribute to the developmental changes in descending control of pain from the RVM to the spinal cord.

Key points.

  • Electrical stimulation of the rostral ventromedial medulla (RVM) facilitates pain behaviours in neonates but inhibits these behaviours in adults. The cellular mechanisms underlying these changes in RVM modulation of pain behaviours are not known.

  • We optimized whole-cell patch-clamp recordings for RVM neurons in animals older than postnatal day 30 and compared the results to postnatal day 10–21 animals.

  • Our results demonstrate that the γ-aminobutyric acid (GABA) release is lower and opioid effects are more evident in adult rats compared to early postnatal rats.

  • A cannabinoid receptor antagonist significantly increased GABA release in mature but not in immature RVM neurons suggesting the presence of local endocannabinoid tone in mature RVM.

Introduction

The rostral ventromedial medulla (RVM) is an important site of supraspinal pain modulation. This region exerts bidirectional control, sending both descending inhibitory and excitatory projections to dorsal horn spinal cord neurons (Urban & Gebhart, 1999; Heinricher & Ingram, 2008). Recently, studies comparing neonates with adults have observed striking differences in how the RVM controls nociception at these ages. For example, electrical stimulation of the RVM facilitates pain behaviours in the neonates but inhibits these behaviours in adults. Conversely, RVM lesions increase pain thresholds in neonates, but decrease thresholds in adults. Further, microinjection of opioids into the RVM in neonates facilitates nociception whereas this has long been known to support analgesia in adults (McGaraughty & Heinricher, 2002; Hathway et al. 2009, 2012).

In adults, the functionally opposing facilitation and inhibition of pain has been well characterized at both the single cell and circuit level and arises from different populations of RVM neurons, termed ‘ON-cells’ and ‘OFF-cells’ based on their responses to nociceptive input (Fields, 1985; Heinricher & Fields, 2013). OFF-cells display an abrupt pause in ongoing activity right before a nociceptive reflex and contribute inhibitory influences that descend from the RVM (Fields et al. 1983; Heinricher et al. 1994, 2009; Urban & Gebhart, 1999). ON-cells show a burst of activity immediately preceding nociceptive input, and facilitate nociceptive responses in the spinal cord (Heinricher et al. 1989; Fields et al. 1991; Heinricher & Neubert, 2004; Neubert et al. 2004; Kincaid et al. 2006; Xu et al. 2007). ON-cell activity is critical for hyperalgesia (Cleary & Heinricher, 2013) and both in vivo and in vitro studies show that ON-cells are directly inhibited by μ-opioid receptor (MOPr) agonists (Barbaro et al. 1986; Pan et al. 1990, 1997; Heinricher et al. 1994; Meng & Johansen, 2004). These studies form the basis of the current model of descending control of pain in adult rats.

In vitro studies have delineated subpopulations of RVM neurons based on their opioid sensitivity. ‘Secondary cells’ (presumed ON-cells) are directly hyperpolarized by MOPr agonists (Pan et al. 1990, 1997). ‘Primary cells’ (presumed OFF-cells) are not directly hyperpolarized by MOPr agonists, but are disinhibited by direct opioid receptor-mediated presynaptic inhibition of GABAergic input (Pan et al. 1990; Heinricher & Ingram, 2008). The majority of these in vitro studies, however, are from neonatal RVM neurons because the organization and abundance of myelinated fibres in this region make it a challenging area for whole-cell patch-clamp recording in adults. The goal of the present study was to test the hypothesis that GABAergic transmission in RVM neurons and responses to opioids are differentially regulated during development. Developmental changes in GABAergic and glutamatergic receptors are stable in many brain areas by PN day 21 (Wu et al. 1996; Ben-Ari, 2002; Ewald & Cline, 2009). We have optimized the RVM slice preparation from older animals (PN day 30–90) and taken advantage of a fluorescent opioid peptide, dermorphin-Alexa Fluor 594 (DERM-A594) to label opioid-sensitive ON-cells. DERM-A594 is internalized with the MOPr in MOPr-expressing neurons in RVM (Phillips et al. 2012) so ON-cells can be identified in the slice. GABAergic synaptic transmission and effects of opioids and endocannabinoids were examined in DERM-A594-labelled and unlabelled neurons and compared between immature (PN day 10–21) and mature (PN day 30–90) RVM neurons.

Methods

Ethical approval

All procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health and approved by the Institutional Animal Care and Use Committee of Oregon Health and Science University.

RVM slice preparation

Male Sprague–Dawley rats (immature PN day 10–21; mature PN day 30–90) (Harlan, Livermore, CA, USA) were deeply anaesthetized with isoflurane (4%) and the brains were rapidly removed and placed in ice-cold ‘cutting buffer’ containing (in mm): 75 NaCl, 2.5 KCl, 0.1 CaCl2, 6 MgSO4, 1.2 NaH2PO4, 25 NaHCO3, 2.5 d-dextrose, 50 sucrose. In some experiments, transcardial perfusion was performed before slice cutting (Ye et al. 2006). This process helps cell survival and improves cell visibility in adult RVM. We did not observe any difference in intrinsic membrane properties and GABAergic signalling in RVM neurons from rats with or without transcardial perfusion. Coronal slices (180–200 μm) were cut in 95% O2 and 5% CO2 oxygenated cutting buffer. Slices were incubated in warm (35˚C) 95% O2 and 5% CO2 oxygenated artificial cerebrospinal fluid (aCSF) for at least 40 min and maintained at room temperature afterward until transfer to a recording chamber. The aCSF contained (in mm): 126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 21.4 NaHCO3, 11.1 d-dextrose, pH 7.4, and the osmolarity was adjusted to 300–310 mosmol l–1.

Whole-cell patch-clamp recordings

RVM neurons were visualized in the triangular midline region dorsal to the pyramidal tracts, rostral to the inferior olive, including and immediately surrounding the raphe magnus using an OLY-150IR video camera (Olympus, Lake Success, NY, USA) coupled to a TV monitor (Sony Corporation of America). Whole-cell patch-clamp recordings were made from visually identified RVM ON-cells (DERM-A594 labelled) or unlabelled cells with an Olympus BX51W1 upright differential interference contrast (DIC) microscope (Olympus) equipped with infrared optics and epi-fluorescence filters. An Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) was used for voltage and current clamp recordings. Patch pipettes were pulled from borosilicate glass (1.5 mm diameter; WPI, Sarasota, FL, USA) on a two-stage puller (PP83, Narishige, Tokyo, Japan). Pipettes had a resistance of 2–4 MΩ when filled with the intracellular solution. Experiments looking at evoked (eIPSCs) and spontaneous miniature GABAA-mediated inhibitory postsynaptic currents (mIPSCs) used an intracellular pipette solution containing (mm): 140 CsCl, 10 Hepes, 4 MgATP, 3 NaGTP, 1 EGTA, 1 MgCl2, 0.3 CaCl2 (pH adjusted to 7.3 with CsOH, 290 mosmol l–1). With this internal solution, the reversal potential of Cl− (ECl) is ∼0 mV, so the GABAA receptor-mediated currents are inward at a holding potential of −70 mV. A junction potential of −5 mV was corrected during recording. GABAergic events were isolated in the presence of glutamate receptor antagonists 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX; 10 μm) and dl-2-amino-5-phos-phonopentanoic acid (APV, 50 μm). Events were low-pass filtered at 5 kHz and sampled at 10–20 kHz. Individual events were detected offline using Axograph X software (Sydney, Australia) and visually confirmed. Spontaneous mIPSCs were recorded in the presence of 500 nm TTX. eIPSCs were recorded in aCSF solution containing 10 μm NBQX and 50 μm APV with a bipolar stimulating electrode placed ∼200–300 μm distally from the recorded cell. Stimulation pulses (range 0.1–10 mA, 2 ms) were delivered at 0.05 Hz with a stimulation isolator (WPI). For paired-pulse ratio measurements, two stimuli were applied at an interval of 100 ms. The paired-pulse ratio was calculated by dividing the amplitude of the second evoked IPSC by the amplitude of the first IPSC (P2/P1). Current-clamp experiments used an intracellular pipette solution containing (in mm): 138 KMeSO4, 10 KCl, 10 Hepes, 4 MgATP, 3 NaGTP, 1 EGTA, 1 MgCl2, 0.3 CaCl2 (pH 7.3 with KOH, 290 mosmol l–1). Data from current-clamp recordings were only included for analyses if the action potential amplitude was at least 50 mV. Series resistance was compensated by ∼80% and was monitored regularly during recordings. During each experiment, a voltage step of  −10 mV from the holding potential was applied periodically to monitor cell capacitance and access resistance. Recordings in which access resistance or capacitance changed by >15% during the experiment were excluded from data analysis. In some experiments, the internal pipette solution also contained 0.3% biocytin for post hoc immunohistochemical staining. All recordings were made in aCSF at an ambient temperature of 30–31˚C. One neuron was recorded per slice and 4–5 slices were recorded per rat. Each set of experiments was repeated using at least 3–4 different rats and no more than two cells from a single rat were included in a specific dataset.

RVM microinjection

RVM ON-cells are differentiated from OFF-cells because they express MOPrs (Fields & Heinricher, 1985; Pan et al. 1990; Heinricher et al. 1994). In order to record from identified RVM ON-cells, some rats were microinjected with DERM-A594 before slice preparation. Microinjections of DERM-A594 selectively label MOPr-expressing neurons in the RVM (Phillips et al. 2012) (Fig. 1). Briefly, male rats were deeply anaesthetized with i.p. ketamine (37.5 mg/kg)/xylazine (7.5 mg/kg)/acepramozine (1.5 mg/kg) cocktail and a 23-gauge stainless steel guide cannula was lowered into the RVM using stereotaxic techniques (immature, AP: −8.5 mm; ML: 0.0 mm; DV: −6.9 mm from bregma; mature, AP: −2.1; ML: 0.0 mm; DV: −7.9 mm from lambda). A 31-gauge injection cannula that extended 2 mm beyond the tip of the guide cannula was inserted and DERM-A594 (300 ng (0.5 μl)–1 in 32% DMSO and saline) was administered over 100 s. The injection cannula remained in place for an additional 60 s post-injection to minimize backflow up the cannula tract. The injection and guide cannula were removed and the brain was extracted for electrophysiological recording. DERM-A594 binds to MOPrs and is rapidly internalized (Arttamangkul et al. 2000, 2006; Macey et al. 2010, 2014; Phillips et al. 2012). DERM-A594 labelling was observed as long as 5 h after injection.

Figure 1. DERM-A594 labels a subpopulation of RVM neurons.

Figure 1

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An in vivo marker for ON-cells, DERM-A594 (300 ng (0.5 μl)–1), was microinjected into the RVM prior to the rat being killed. Top panels: a DERM-A594-labelled cell (red) was whole-cell patch-clamped and filled with biocytin (green). Bottom panels: a biocytin-labelled cell that was not labelled with DERM-A594. Far right panel in the first row: DERM-A594-labelled neuron with epi-fluorescence with the recording electrode.

Control experiments using microinjection of the vehicle for DERM-A594 (DMSO + saline) were performed. We did not find any significant difference in mIPSC frequency between vehicle and DERM injections from mature rats (vehicle: 0.4 ± 0.1 Hz compared to DERM-A594: 0.4 ± 0.1 Hz, n = 5–11 Mann–Whitney U = 19, P > 0.05). In addition, we also determined that superfusion of 32% DMSO on RVM slices did not affect mIPSC frequency or amplitude (n = 3, data not shown).

Chemicals

2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX), dl-2-amino-5-phosphonopentanoic acid (APV), bicuculline, tetrodotoxin (TTX), muscimol and CGP 88594 were purchased from Abcam (Cambridge, MA, USA). Naloxone, met-enkephalin (ME) and [d-Ala2, NMe-Phe4, Gly5-ol]-enkephalin (DAMGO) were purchased from Sigma (St Louis, MO, USA). SR141716 was purchased from Tocris (Tocris Bioscience, Bristol, UK). All chemicals were dissolved according to the manufacturer's instructions and made in stock solution kept at −20˚C, then diluted to the final concentration into aCSF on the day of the experiment.

Data analyses

Weighted decay time constants were calculated from double exponential fits with the equation:

graphic file with name tjp0593-0217-m1.jpg

where If and Is are the amplitudes of the fast and slow decay components and tf and ts are the decay time constants (Rumbaugh & Vicini, 1999). All data are reported as mean ± SEM. Student's t test and two-way ANOVA were used where appropriate to determine statistical significance. The criterion for significance was set at P < 0.05.

Results

Whole-cell patch-clamp recordings were performed from a total of 167 cells from immature (PN day 10–21) and 124 cells from mature (PN day >30; range 30–50 days of age with some rats as old as 90 days) rats. Microinjections of DERM-A594 into the RVM labelled neurons expressing MOPrs (presumed ‘ON-cells’) which were compared to unlabelled RVM neurons. In some experiments, neurons were filled with biocytin to confirm co-labelling with DERM-A594 in recorded cells with post hoc immunohistochemistry (Fig. 1). If responses were not different in labelled and unlabelled neurons, the neurons were grouped and only differences between immature and mature RVM slices were compared.

The action potential firing rate of RVM neurons is altered with development

The resting membrane potentials of RVM labelled and unlabelled neurons were determined in a subset of recordings of RVM neurons from immature and mature rats. There was no significant difference in resting membrane potentials between immature (−50 ± 2 mV, n = 10) and mature (−51 ± 2 mV, n = 8) labelled RVM neurons (t(16) = 0.50, P > 0.05). Similarly, the resting membrane potentials were not different between immature (−52 ± 5 mV, n = 5) and mature (−53 ± 3 mV, n = 8) unlabelled RVM neurons (t(11) = 0.19, P > 0.05).

Spontaneous firing rates of immature and mature RVM neurons were different. Current-clamp recordings from immature slices showed that ∼65% (21 of 32) of RVM neurons were spontaneously active. By contrast, only 14% (2 of 14) of mature neurons were spontaneously active (Fig. 2A). Both regular and irregular firing patterns were observed in RVM slices (Fig. 2B). In cells that were not spontaneously active, current was injected with a step protocol consisting of a series of incremental current injections (50 mA, 500 ms duration), starting with a 50 mA hyperpolarizing current injection. Fewer action potentials were elicited with current injection in mature (n = 12) compared to immature RVM neurons (n = 17; two-way ANOVA, Interaction, F(4,108) = 14.57, P < 0.01; Fig. 2C). These data suggest that the excitability of RVM neurons decreases with development.

Figure 2. Excitability of RVM neurons is different with development.

Figure 2

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A, fewer cells (2 out of 14) exhibit spontaneous action potential (AP) firing in mature RVM neurons compared to immature RVM (21 out of 32). B, representative traces showing both regular and irregular firing patterns exist in RVM neurons. C, summary graph showing fewer APs are generated with varying stimulus currents in mature neurons (∗∗P < 0.01).

Opioid-induced G protein-coupled inwardly-rectifying potassium (GIRK) currents are larger in mature RVM ON-cells

Previous studies have demonstrated that activation of MOPrs selectively hyperpolarizes a subpopulation of RVM neurons through activation of GIRK channels in immature rats (Pan et al. 1990, 1997). Whole-cell voltage-clamp recordings were performed with KMeSO4-based internal solution from rat RVM slices. The selective MOPr agonist [d-Ala2, NMe-Phe4, Gly5-ol]-enkephalin (DAMGO, 1 μm) stimulated outward GIRK currents in a subpopulation of RVM neurons from slices without prior microinjection of DERM-A594. DAMGO elicited larger outward currents at −70 mV in mature (59 ± 16 pA, n = 5) compared to immature neurons (17 ± 3 pA, n = 10) RVM neurons (t(13) = 3.4, P < 0.01, Fig. 3A). The effects of DAMGO were also recorded in labelled RVM neurons from slices with prior microinjection of DERM-A594. Similarly, there were larger outward GIRK currents in mature labelled (60 ± 5 pA, n = 3) compared to immature labelled (24 ± 6 pA, n = 3, P = 0.01, t(4) = 4.5) RVM neurons. There were no significant differences in GIRK currents measured in DERM-A594-labelled neurons and those recorded without DERM-A594 microinjections at each age.

Figure 3. Effects of opioids in RVM neurons.

Figure 3

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A, summary data showing that DAMGO-mediated currents were significantly larger in mature (n = 5) compared to immature RVM neurons (n = 10, ∗∗P < 0.01). Inset: representative trace of DAMGO-mediated outward current in RVM neurons. Space represents I–V protocol steps. Scale bars: 10 pA, 1 min. B, summary data showing increased DAMGO inhibition of mIPSC frequency in mature (n = 9) compared to immature (n = 6) unlabelled RVM neurons (∗P < 0.05). C, Gaussian fits to mIPSC amplitude histograms were not different in the absence and presence of DAMGO in either immature (n = 6) or mature (n = 9) unlabelled RVM neurons.

Opioid inhibition of GABAergic mIPSCs is greater in mature RVM neurons

Opioid inhibition of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) was determined with the selective μ-opioid receptor agonist DAMGO (1 μm). GABAA-mediated mIPSCs were isolated in the presence of NBQX (10 μm), APV (50 μm) and TTX (500 nm). DAMGO was more effective in reducing GABAA-mediated mIPSC frequency in mature (52 ± 6%, n = 9) compared to immature (13 ± 7%, n = 6) unlabelled RVM neurons (t(13) = 4.35, P < 0.01; Fig. 3B). There was no difference in mIPSC amplitude in the presence or absence of DAMGO in immature unlabelled RVM neurons (81 ± 10 pA compared to baseline 88 ± 16 pA, n = 6, paired t test, t(5) = 0.70, P > 0.05). Similarly, the mean amplitude before and during DAMGO application to mature unlabelled RVM neurons was not different (108 ± 38 pA compared to 94 ± 34 pA after DAMGO application, n = 9, paired t test, t(8) = 1.10, P > 0.05; Fig. 3C). The non-selective μ/δ-opioid receptor agonist met-enkephalin (ME, 10 μm) also produced more effective inhibition of mIPSCs in mature RVM neurons (58 ± 7%, n = 9) than in immature RVM neurons (25 ± 11%, n = 8, t(15) = 2.63, P < 0.05). The 10–90% rise time and decay kinetics of the mIPSCs were not altered in the presence of DAMGO in immature and mature RVM (data not shown). Taken together, these results indicate that opioid receptors inhibit GABA neurotransmission to a greater extent in mature RVM slices than in immature slices.

The probability of GABA release is less in mature RVM neurons

To determine if GABA release probability from RVM GABA terminals is altered during development, we examined paired-pulse ratios (Melis et al. 2002). Both paired-pulse facilitation and depression were observed in DERM-A594-labelled and unlabelled neurons at each age without a noticeable preference, so labelled and unlabelled neurons were grouped at each age. Paired-pulse ratios (P2/P1) were significantly greater in mature (1.6 ± 0.2, n = 8) compared to immature RVM neurons (1.0 ± 0.1, n = 10, t(16) = 2.34, P < 0.05; Fig. 4A and B). Consistent with this finding, evoked IPSCS (eIPSCs) were typically smaller at the same stimulus intensity (0.5 mA constant current) in mature RVM neurons (154 ± 27 pA in mature, n = 9, compared to 387 ± 69 pA in immature, n = 10, t(17) = 3.00, P < 0.01; Fig. 4C and D). Together, these results indicate that the probability of GABA release in mature RVM is significantly decreased compared to that in immature RVM.

Figure 4. Evoked release probability is lower in mature RVM neurons.

Figure 4

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A, representative traces showing evoked GABAA IPSCs using a paired-pulse protocol in immature and mature RVM neurons. Each trace represents the average of 10 responses to stimuli at 0.03 Hz. B, bar graph showing a significant increase in paired pulse ratios (P2/P1) in mature (n = 10) compared to immature RVM neurons (n = 8) (∗P < 0.05). C, representative traces showing evoked GABAA IPSCs (eIPSCs) in immature and mature RVM neurons. Each trace represents the average of 10 responses to stimuli at 0.05 Hz. D, summary of data indicating that eIPSC amplitudes were significantly smaller in mature grouped RVM neurons at the same stimulus intensity (0.5 mA) (n = 9–10, ∗∗P < 0.01).

In many brain areas, GABAA receptors are excitatory until ∼PN 7 when expression of the potassium chloride cotransporter 2 (KCC2) shifts the reversal potential for chloride to more hyperpolarizing potentials. It is not known when this shift occurs in RVM neurons. The reversal potential for GABAA receptor-mediated chloride currents elicited by superfusion of the GABAA agonist muscimol was determined using a KMeSO4-based intracellular solution in immature and mature RVM neurons (Fig. 5A). Current–voltage (I–V) curves were plotted for the subtracted currents (muscimol minus control). There was no shift in the reversal potential of I–V curves between immature and mature RVM neurons (−70 ± 2 mV in immature, n = 16 compared to −67 ± 2 mV in mature, n = 7, t(21) = 1.06, P > 0.05; Fig. 5B and C). These results indicate that the chloride gradient in rat RVM neurons does not change between PN day 10 and 90.

Figure 5. The reversal potential for GABAA-mediated currents does not change with RVM development.

Figure 5

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A, representative traces of current–voltage (I–V) relations obtained in aCSF (left), 10 μm muscimol (middle) and subtracted (muscimol – control) (right). B, I–V curves were plotted for the subtracted traces. C, reversal potentials were similar for GABAA (EGABAA) for immature and mature RVM neurons.

There is less spontaneous GABA release in mature RVM

Whole-cell patch-clamp recordings of DERM-A594-labelled and unlabelled RVM neurons from immature and mature slices were used to monitor GABAergic mIPSCs isolated in the presence of NBQX (10 μm), APV (50 μm) and TTX (500 nm). The frequency of mIPSCs was significantly lower in mature unlabelled (0.6 ± 0.1 Hz, n = 13) compared to immature unlabelled neurons (1.3 ± 0.2 Hz, n = 12; Mann–Whitney U = 30.5, P < 0.05; Fig. 6A and B). In DERM-A594-labelled neurons, the mIPSC frequency was not different in mature RVM (0.8 ± 0.1 Hz, n = 20) compared to immature (1.1 ± 0.1 Hz, n = 15; Mann–Whitney U = 97, P > 0.05; Fig. 6B). The mIPSCs were completely blocked by bicuculline (20 μm) confirming that these currents were mediated by GABAA receptors (data not shown). There were no age-related changes in mean mIPSC amplitude in unlabelled RVM neurons from immature (71 ± 7 pA, n = 8) compared to mature rats (78 ± 16 pA; n = 9, t(15) = 0.38, P > 0.05; Fig. 6C). These results indicate that the decrease in frequency in unlabelled neurons was not a function of decreased mIPSC amplitude. There were also no differences in rise time or decay kinetics between labelled and unlabelled neurons in each age group so neurons were pooled. No significant differences were noted in 10–90% rise time from mature (1.0 ± 0.1 ms, n = 14) and immature (1.0 ± 0.1 ms, n = 11) or the weighted decay time constant (mature, 15.0 ± 1.5 ms versus immature, 16.3 ± 1.9 ms; Fig. 6D).

Figure 6. GABAergic synaptic transmission is less evident in mature RVM neurons.

Figure 6

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A, representative traces of GABAergic mIPSCs (in the presence of TTX) in immature and mature RVM neurons. B, summary bar graph showing mIPSC frequency measured from DERM-A594-labelled and unlabelled RVM neurons. A significant difference in GABAA mIPSC frequency was observed in mature unlabelled RVM neurons (∗P < 0.05). C: left, cumulative frequency plot of mIPSC amplitudes from all unlabelled RVM neurons showing no difference in the amplitude distribution from immature and mature RVM neurons; right, mean amplitude determined from Gaussian fits to mIPSC amplitude histograms were not different in unlabelled RVM neurons from the two ages (P > 0.05). D, summary of data showing that 10–90% rise time and decay kinetics of mIPSCs are not different in immature and mature grouped RVM neurons (P > 0.05).

Antagonism of GABAB receptors does not modulate mIPSC frequency in RVM

To determine whether activation of GABAB receptors reduces the probability of GABA release in mature RVM, we examined the effects of the GABAB antagonist CGP 88594. CGP 88594 (5 μm) did not change the frequency or amplitude of mIPSCs in DERM-A594-labelled or unlabelled neurons in either immature or mature RVM neurons. Therefore, labelled and unlabelled neurons were grouped at each age. In immature RVM neurons, mIPSC frequency was 0.9 ± 0.1 Hz in the presence of CGP 88594 compared to the baseline mIPSC frequency (1.2 ± 0.2 Hz; n = 8, Mann–Whitney U = 27, P > 0.05; Fig. 7A). In mature RVM neurons, mIPSC frequency was 1.0 ± 0.1 Hz in the presence of CGP 88594 compared to a basal frequency of 0.8 ± 0.1 Hz (n = 6, Mann–Whitney U = 11.5, P > 0.05; Fig. 7C). Similarly, there was no significant change in the Gaussian mean of mIPSC amplitudes in the absence and presence of CGP 88594 in immature (80 ± 6 pA, compared to 94 ± 13 pA; n = 8, paired t test, t(7) = 0.91, P > 0.05; Fig. 7B) and mature (85 ± 9 pA, n = 6 compared to 81 ± 10 pA; paired t test, t(5) = 0.54, P > 0.05; Fig. 7D) RVM recordings. These data indicate that GABAB receptors do not modulate presynaptic GABA release in the RVM.

Figure 7. Antagonism of GABAB receptors does not affect mIPSCs in either immature or mature RVM.

Figure 7

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A, cumulative frequency plot of mIPSC inter-event interval and grouped data showing no significant difference in the frequency distribution of mIPSCs in the absence and presence of the GABAB antagonist CGP 88594 from immature grouped RVM neurons. B, cumulative frequency plot of mIPSC amplitude and summary data showing that mean amplitudes determined from Gaussian fits to mIPSC amplitude histograms are not changed with CGP 88594 superfusion in immature grouped RVM neurons (P > 0.05). C, cumulative frequency plot of mIPSC inter-event interval and grouped data showing no significant difference in mIPSC frequency in the absence and presence of CGP 88594 from mature grouped RVM neurons. D, cumulative frequency plot of mIPSC amplitude and summary data showing that mean amplitudes determined from Gaussian fits to mIPSC amplitude histograms are not different with CGP 88594 superfusion in mature grouped RVM neurons (P > 0.05).

Opioid receptor antagonism does not modulate basal mIPSC frequency in the RVM

The RVM is a major brain site for opioid analgesia. It is possible that the lower probability of GABA release observed in mature RVM is due to an increase in endogenous opioids acting on presynaptic opioid receptors. To test this hypothesis, the effects of the non-selective opioid antagonist naloxone were determined on mIPSC frequency. As shown in Fig. 8A, mIPSC frequency did not change in the presence of 1 μm naloxone in immature grouped RVM neurons (control, 1.2 ± 0.2 Hz compared to naloxone, 1.0 ± 0.2 Hz, n = 15, Mann–Whitney U = 80, P > 0.05; Fig. 8A). The Gaussian means of mIPSC amplitudes were unaffected by naloxone in immature RVM neurons (control, 62 ± 4 pA versus naloxone, 61 ± 5 pA, n = 11, paired t test, t(10) = 0.22, P > 0.05; Fig. 8B). There were also no significant effects of naloxone on mIPSC frequency observed in mature RVM neurons (basal: 0.6 ± 0.1 Hz, n = 12; naloxone: 0.6 ± 0.1 Hz, n = 12, Mann–Whitney U = 67, P > 0.05; Fig. 8C). Mean amplitude of mIPSCs determined from Gaussian fits to mIPSC amplitude histograms did not change in mature RVM neurons (basal: 65 ± 9 pA, n = 11) in the presence of naloxone (68 ± 9 pA, n = 11, paired t test, t(10) = 0.20, P > 0.05; Fig. 8D). These data indicate that the reduced mIPSC frequency observed in mature RVM is not due to a change in endogenous opioid release during RVM maturation.

Figure 8. Opioid receptor antagonism does not change basal mIPSC frequency in either immature or mature RVM.

Figure 8

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A, cumulative frequency plots of mIPSC inter-event interval and summary data showing that mIPSC frequency is not altered by superfusion of the non-selective opioid receptor inhibitor naloxone (1 μm) in either immature or mature grouped RVM neurons (P > 0.05). B, cumulative frequency plots of mIPSC amplitude and summary data of mean amplitudes determined from Gaussian fits to mIPSC amplitude histograms are not changed in immature grouped RVM neurons by application of naloxone (P > 0.05). C, cumulative frequency plots of mIPSC inter-event interval and summary data showing that there was no significant difference in the presence of naloxone in mature grouped RVM neurons. D, cumulative frequency plots of mIPSC amplitude and summary data of mean amplitudes determined from Gaussian fits to mIPSC amplitude histograms are not changed in mature grouped RVM neurons by application of naloxone (P > 0.05).

Antagonism of cannabinoid receptors increases presynaptic GABA release in mature RVM

The cannabinoid antagonist SR141716 (rimonabant) was used to determine whether endogenous cannabinoids (endocannabinoids) inhibit GABA release in mature RVM. We observed no differences in the effects of SR141716 on mIPSC frequency or amplitude when recording from DERM-A594-labelled and unlabelled neurons, so neurons were grouped at each age. In the presence of SR141716, mIPSC frequency was increased by 77 ± 20% (n = 9) in mature, but was largely unaffected in immature RVM neurons (7 ± 3%, n = 8; t(15) = 3.28, P < 0.01; Fig. 9C). SR141716 did not affect mIPSC amplitude in either immature (basal: 95 ± 14 pA and SR141716: 91 ± 13 pA, n = 7; paired t test, t(6) = 0.25, P > 0.05; Fig. 9D) or mature (basal: 89 ± 13 pA compared to SR141716: 86 ± 12 pA, n = 7; t(6) = 0.11, P > 0.05; Fig. 9D) RVM neurons.

Figure 9. Cannabinoid antagonists increase mIPSC frequency in mature RVM.

Figure 9

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A and B, representative traces showing mIPSCs in the absence and presence of SR141716 (SR, 3 μm) from immature and mature RVM neurons. C, summary bar graph showing that SR141716 increases mIPSC frequency more in mature RVM (∗∗P < 0.01). D, mean amplitudes determined from Gaussian fits to mIPSC amplitude histograms were not different in the presence of SR141716 in either immature or mature RVM neurons (P > 0.05).

Discussion

This study is the first whole-cell patch-clamp analysis of RVM neurons that characterizes excitability, GABAergic neurotransmission and opioid/endocannabinoid actions in adult RVM neurons. The results provide evidence that RVM neurons from mature rats are less excitable than those recorded from immature rats. Opioid activation of GIRK currents are increased and inhibition of presynaptic GABA release in unlabelled RVM neurons are enhanced in mature RVM compared to immature RVM. Interestingly, the probability of GABA release is significantly lower in mature RVM than in immature RVM. Cannabinoid receptor antagonism increased mIPSC frequency in mature RVM but had no effect in immature RVM slices indicating that presynaptic inhibition by endogenous cannabinoid release is increased in adults.

Characteristics of RVM neurons in neonates compared to adults

An anatomical substrate for a descending pathway (dorsolateral funiculus) from the RVM to the spinal cord is detectable at birth (Leong et al. 1984) but is not functional in terms of descending inhibition until at least PN day 10–12, with adult-like responses to nociceptive stimuli to the skin appearing about PN day 21 (Fitzgerald & Koltzenburg, 1986). Electrical stimulation in the RVM facilitates pain behaviours in neonatal rats (PN day <21) but produces biphasic inhibition and facilitation in adult rats (Hathway et al. 2009). Further, opioid microinjections into the RVM result in increased nociception in neonates but suppress nociception in older animals (Hathway et al. 2012). Thus, it is clear that descending nociceptive pathways change with development (Fitzgerald, 2005). The cellular basis for these observations is not understood, primarily due to the lack of electrophysiological data from older rats. The transition to adult-like behaviours occurs around PN day 21, similar to the time course for development of GABA and glutamate systems in many brain areas (Lujan et al. 2005). In these studies, we focused on changes in GABAergic neurotransmission in immature and mature RVM.

Many studies over the past 20 years have characterized subpopulations of RVM neurons in terms of their neurotransmitter expression, spontaneous activity and opioid sensitivity (Ossipov, 2012). In these studies, opioid-sensitive (secondary, presumed ON-cells) and opioid-insensitive (primary, presumed OFF-cells) RVM neurons have been observed (Marinelli et al. 2002, 2005; Zhang et al. 2006; Zhang & Hammond, 2010; Zhang & Pan, 2010, 2012). Both classes of neurons include neurons that are spinally projecting and non-spinally projecting RVM output neurons (Marinelli et al. 2002; Zhang et al. 2006; Zhang & Hammond, 2010). In addition, multiple subpopulations have been identified based on firing patterns in studies from younger animals (PN day < 21) (Zhang et al. 2006; Zhang & Hammond, 2010). Our results in immature rats are consistent with these previous studies in that we observe both irregular and regular firing patterns in RVM neurons. In addition, in opioid-sensitive neurons from immature RVM, we observed similar DAMGO-mediated GIRK currents (Pan et al. 1990, 1997; Zhang & Hammond, 2010). In opioid-insensitive RVM neurons, similar opioid inhibition of GABAergic mIPSCs was observed to previous studies (Pan et al. 1990, 1997). Overall, our results in the immature RVM slices are very consistent with previous reports.

Recordings from RVM neurons in adult animals have been largely restricted to in vivo single-unit recordings and intracellular recordings from RVM slices. This is due to the organization and abundance of myelinated fibres in this region in adults that make it a challenging area for whole-cell patch-clamp recording. We have developed a procedure that allows us to routinely record from adult RVM slices. We find significant differences in excitability, GABA release and responses of mature RVM neurons to opioids and cannabinoids. First, the majority of neurons in mature slices did not exhibit the spontaneous firing activity readily observed in immature RVM. In addition, mature RVM neurons were less excitable than neurons from immature rats and significantly more injected current was necessary to drive the mature neurons to firing threshold. Previous intracellular recordings from adult RVM neurons observed differences in spontaneous activity of primary (opioid-insensitive) versus secondary (opioid-sensitive) RVM neurons. The secondary (opioid-sensitive) neurons were spontaneously active but primary neurons (opioid-insensitive) were not (Pan et al. 1990). We investigated the possibility that the reduced spontaneous activity in mature RVM neurons in our studies might reflect skewed sampling of unlabelled (opioid-insensitive) neurons. However, we found that some DERM-A594-labelled neurons in mature RVM (opioid-sensitive cells) were not spontaneously active. DERM-A594 did not suppress spontaneous activity in these cells because exogenous DERM-A594 had been washed from the slices for several hours prior to recordings and we observed no effects of naloxone in slices from mature RVM. The differences between our results and the earlier studies may be a function of the different recording procedures: intracellular versus whole-cell patch-clamp recordings.

Opioid agonists have larger pre- and postsynaptic effects in mature animals. We found that the MOPr-selective agonist DAMGO and the non-selective agonist ME induced larger GIRK currents in mature compared to immature RVM neurons. These results suggest that coupling of MOPrs to postsynaptic effectors is increased with age, which is strongly supported by a previous study (Fernandez-Alacid et al. 2011). We also observed greater opioid inhibition of presynaptic GABA release in mature RVM neurons.

Another major difference between mature and immature RVM is the amount of spontaneous GABA release observed under basal conditions (aCSF with NBQX and TTX). First, we observed a significant difference in paired pulse ratios in mature compared to immature RVM neurons, indicating a lower probability of release in mature RVM. Consistent with this finding, mIPSC frequencies in mature RVM were lower compared to immature RVM. Interestingly, the different mIPSC frequency was observed when recording from unlabelled cells (presumed OFF-cells) with no significant change in the DERM-A594-labelled neurons (presumed ON-cells). No changes were observed in mIPSC amplitude, rise time or decay time, further indicating that a developmental change occurs in presynaptic terminals. GABAergic neurotransmission plays an important role in RVM descending pain control circuits (Drower & Hammond, 1988; Heinricher et al. 1991; Heinricher & Kaplan, 1991; da Silva et al. 2010). Opioids activate descending anti-nociception by inhibiting GABA release onto RVM OFF-cells (Heinricher et al. 1994). Thus, the combined effects of lower release probability and stronger coupling of MOPrs to effectors in mature RVM may explain the shift toward inhibition of pain by the bulbospinal descending pathway in adults (Hathway et al. 2009, 2012).

Modulation of presynaptic GABA release by endogenous cannabiniods

A change in presynaptic release probability could be caused by endogenous neuropeptides or neurotransmitters that inhibit presynaptic release. GABAB receptors play important roles in synaptic processing within the brain and are present at both post- and presynaptic sites (Kulik et al. 2002; Lujan, 2007). Their activation can hyperpolarize neurons and inhibit neurotransmitter release from presynaptic terminals. However, we did not observe an effect of the GABAB antagonist CGP 88594 on mIPSCs from either immature or mature RVM neurons, indicating that GABAB receptors do not modulate the probability of GABA release in the RVM. We also observed no effect of the opioid antagonist naloxone on mIPSCs, implying that endogenous opioid release does not modulate GABA release in mature RVM.

Cannabinoid agonists produce analgesia when microinjected into the RVM (Martin et al. 1998; Meng et al. 1998). At a cellular level, cannabinoid receptor 1 (CB1) agonists inhibit GABA release onto a subpopulation of RVM neurons (Vaughan et al. 1999; Vaughan & Christie, 2005). Our present results show that the CB1 antagonist SR141716 significantly increases mIPSC frequency in mature RVM neurons. The increase in mIPSC frequency with SR141716 without a change in mIPSC amplitude suggests that endogenous cannabinoids inhibit presynaptic GABA release in adult RVM. Our results are also consistent with the findings of Vaughan and colleagues in that CB1 receptor antagonists had no effect on their own in young rat RVM slices (PN day 12–24) (Vaughan et al. 1999) because SR141716 did not affect mIPSC frequency in immature RVM (PN day 10–20) in our studies. Thus, we find that there is a significant developmental change in the modulation of GABAergic neurotransmission in the RVM by endocannabinoids.

Relevance to descending control of pain

The RVM is a major brain site for opioid analgesia (Heinricher & Ingram, 2008; Heinricher & Fields, 2013). Opioid disinhibition of GABAergic inhibition of OFF-cells is the well-established mechanism for opioid-induced analgesia in adult rats. Hathway and colleagues showed that behavioural responses to electrical stimulation and opioid microinjection into the RVM were different in neonatal compared to adult rats (Hathway et al. 2009, 2012). Our findings provide a cellular basis for the differences in mature RVM. We find a lower amount of spontaneous GABA release impinging onto unlabelled cells and greater opioid inhibition of presynaptic GABA release in mature RVM compared to immature RVM. Both of these results support the likelihood that electric stimulation of the RVM or opioid microinjection into the RVM produces antinociception. In addition, the RVM is also a necessary site for the anti-nociceptive actions of cannabinoids and several studies have observed synergistic analgesic interactions between cannabinoids and opioids (Meng et al. 1998; Vaughan et al. 1999; Cichewicz, 2004; Vaughan, 2014). Our finding that both opioids and endocannabinoids play an important role in mature animals suggests that the more effective actions of opioids and endocannabinoids in mature RVM may be the basis for the developmental switch observed in in vivo studies of RVM modulation of pain (Hathway et al. 2009, 2012).

Acknowledgments

The authors thank Dr Mary M. Heinricher for critical reading of the manuscript.

Glossary

aCSF

artificial cerebrospinal fluid

APV

dl-2-amino-5-phosphonopentanoic acid

DAMGO

[d-Ala2, NMe-Phe4, Gly5-ol]-enkephalin

DERM-A594

dermorphin-Alexa Fluor 594

eIPSC

evoked inhibitory postsynaptic current

GIRK current

G protein-coupled inwardly-rectifying potassium current

mIPSC

miniature inhibitory postsynaptic current

ME

met-enkephalin

MOPr

μ-opioid receptor

NBQX

2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione

PN

postnatal

RVM

rostral ventromedial medulla

Additional information

Competing interests

None of the authors have any conflicts of interests to declare.

Author contributions

M.-H.L. designed the project, conducted experiments, analysed and interpreted data, wrote the manuscript and provided partial funding. K.L.S. conducted RVM microinjections. S.L.I. helped to design the project, interpreted data, provided funding and revised the paper. All authors approved the final version of the manuscript.

Funding

These studies were supported by grants from NIH (DA027625, S.L.I.), and American Heart Association (13SDG14590005, M.-H.L.).

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