Oxytocin-induced antinociception in the spinal cord is mediated by a subpopulation of glutamatergic neurons in lamina I-II which amplify GABAergic inhibition

Characterization of fast spontaneous synaptic transmission in lamina II interneurons

In all lamina II neurons recorded in the whole-cell configuration of the patch-clamp technique (n = 39), spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs, respectively) were observed (Figure 1). Under our experimental conditions, the chloride equilibrium potential was set at -60 mV, and IPSCs were recorded as transient outward currents when lamina II neurons were held at 0 mV (Figure 1A). Both glycine- and GABA_A receptor-mediated sIPSCs (GABAA-R sIPSCs) were present and were blocked by 1 μM strychnine (not shown) and 10 μM bicuculline respectively (Figure 1A). We found that pharmacologically-isolated GABAA-R sIPSCs occurred at a mean frequency of 0.12 ± 0.02 Hz and exhibited a mean amplitude of 11.1 ± 1.5 pA (n = 6). For comparison, fast glutamatergic sEPSCs were detected as transient inward currents at a holding potential of -60 mV (Figure 1B). They occurred at a mean frequency of 1.26 ± 0.47 Hz and had a mean amplitude of -16.3 ± 0.8 pA (n = 16). These currents were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM) (Figure 1B), indicating that they were mediated by the AMPA subtype of glutamate receptors. The detailed properties of the kinetics of each type of current are given in Table 1 for spontaneous and miniature (i.e. recorded in the presence of 0.5 μM of TTX) synaptic events.

Consequences of oxytocin agonist application (TGOT) on spontaneous synaptic transmission recorded in lamina II neurons

Spontaneous AMPA-receptor-mediated EPSCs (AMPA-R sEPSCs) were recorded in the presence of 1 μM strychnine and 10 μM bicuculline (Figure 2A). Bath application of the selective oxytocin receptor agonist (TGOT 1 μM) increased the frequency of occurrence (but not the amplitude) of spontaneous AMPA-R sEPSCs (Figure 2A, histogram) in 5 out of 14 lamina II neurons (35.7%). This frequency of sEPSCs was of 1.04 ± 0.38 Hz under control conditions and increased to 1.79 ± 0.55 Hz in the presence of TGOT (n = 5). This change was reversible and found to be statistically significant (p < 0.05) and represented an average increase of 103 ± 27%. By contrast to its relatively modest effect on sEPSCs, TGOT (1 μM) induced a significant increase in the amplitude (Control: 14.4 ± 1.8 pA; TGOT: 25.5 ± 2.6 pA, n = 6; p < 0.05) and frequency of GABAA-R sIPSCs (Control: 0.12 ± 0.02 Hz; TGOT: 1.69 ± 0.61 Hz, n = 6; p < 0.05) in all lamina II neurons from which we recorded (n = 6 out of 6). The effect of TGOT was fully reversible after 10–15 minutes of washout and was never observed in the presence of OTA (d(CH₂)⁵ - [Tyr(Me)², Thr⁴-Tyr-NH₂⁹] ornithine vasotocin, 1 μM), a selective OT receptor antagonists (n = 5, not shown). This result indicated that the increase in frequency of sIPSCs was mediated by the activation of OT receptors.

Presynaptic OT receptors on glutamatergic synaptic terminals

In the presence of TTX (0.5 μM), TGOT also stimulated the frequency of AMPA-R mEPSCs (Figure 2A, left panel and histogram) in a subpopulation (50%, 6 out of 12) of lamina II neurons. The mean frequency of mEPSCs increased from 1.27 ± 0.30 Hz under control conditions to 2.31 ± 0.65 Hz in the presence of TGOT (1 μM). This change in frequency was statistically significant (p < 0.05) and represented an average increase of +96.8 ± 31.0% (n = 6; Figure 2A, right histogram) which was similar to that observed for sEPSCs. In sharp contrast with this result, TGOT failed to change the frequency of occurrence of GABAA-R mIPSCs (Figure 2B, right histogram) since, in all lamina II neurons recorded, the mean frequency remained stable (Control: 0.13 ± 0.03 Hz; TGOT: 0.12 ± 0.04 Hz, n = 6; p > 0.05; average change: -1.2 ± 9.1%, n = 6). TGOT had no apparent effect on the frequency of glycine receptor-mediated spontaneous and miniature synaptic transmission (not shown).

TGOT reveals a novel population of AMPA-R mEPSCs having slow rise and decay kinetics

Detailed analysis of mEPSC kinetics revealed that in the presence of TGOT a subpopulation of mEPSCs remained unchanged whereas an additional population of mEPSCs seemed to appear. As illustrated in Figure 3B–C, this "novel" population of miniature AMPA-R EPSCs had larger time to peak values (τ_R: +20.9 ± 4.6%, n = 6, p < 0.01), slower deactivation time constants (τ_D: +52.7 ± 7.9%; n = 6, p < 0.001) and smaller mean amplitudes (Amp.: -20.9 ± 2.0%; n = 6, p < 0.001). The effect of TGOT became particularly clear when the distribution of the monoexponential decay time constants of AMPA-R mEPSCs was represented under the form of a binned histogram (Figure 3A). Under control conditions (Figure 3A, top histogram), the distribution of the decay time constants could be adjusted by a Gaussian distribution centered at 3.21 ms, while in the presence of TGOT this distribution was strongly asymmetric because of the presence of the "newly" detected mEPSCs with slow decay time constants (Figure 3A, bottom histogram). These TGOT-sensitive mEPSCs were fully blocked by CNQX (10 μM) and disappeared upon washout of TGOT.

These results suggest that presynaptic OT receptors facilitate glutamatergic synaptic transmission by revealing a novel or previously silent or undetected population of EPSCs.

Immunohistochemical identification of PVN-activated (OT-sensitive) dorsal horn neurons

In order to localize and to tentatively identify the phenotype of the lamina II neurons activated upon OT release, we used an immunohistochemical approach based on c-Fos nuclear expression following PVN electrical stimulation in anaesthetized rats. As shown in Figure 4A and 4C, the c-Fos positive nuclei of PVN-activated cells in the spinal cord were rare and systematically localized in the most superficial layers of the dorsal horn (lamina I and outer part of lamina II: I/IIo) as well as in the sympathetic intermedio-lateral column (not shown). Lamina I/IIo cells immunopositive for c-Fos were mainly found in the ipsilateral dorsal horn, with a slight preference for the medial half of superficial laminae versus lateral half. This labeling matched the distribution of OT-positive fibers and OT-binding sites (see additional file 1). In good agreement, OT-containing fibers (brown staining in Figure 4B, arrowheads) could be found close to the c-Fos positive nuclei (blue staining in Figure 4B, arrows). PVN-activated cells were identified as neurons because c-Fos-positive nuclei were always surrounded by a thin (perinuclear) cytoplasmic compartment which was immunopositive for the neuronal marker MAP2 and negative for the astroglial marker GFAP (not shown). In a double immunofluorescence protocol against c-Fos and glutamic acid decarboxylase (GAD), the enzyme synthesizing GABA, c-Fos positive nuclei of PVN-activated cells were found in laminae with poor GAD-immunoreactivity (I/IIo) while lamina IIi showed a strong GAD-immunoreactivity (Figure 4C). Among 720 c-Fos positive nuclei observed in lumbar segments of two animals (80% in lamina I, 20% in lamina IIo), none were found in GAD positive neurons (Figure 4D–F), thus suggesting that PVN-activated neurons represented a population of non-GABAergic, i.e. glutamatergic neurons. In addition, we performed patch-clamp recordings of lamina II neurons in the current clamp mode to characterize the effect of TGOT application on the membrane potential at rest (i.e. no current injection: i = 0). As illustrated in figure 5 (top trace), TGOT superfusion (1 μM) did not modify the membrane potential (control: -63.6 ± 1.7 mV; TGOT: -60.8 ± 2.0 mV; n = 19; p > 0.05) in the vast majority of the recorded lamina II neurons (n = 19 neurons out of 21; 90.5%). Only a small fraction of neurons were depolarized transiently by TGOT (Figure 5, bottom trace; n = 2 neurons out of 21; 9.5%), a result which was in good agreement with the small number of c-Fos positive lamina neurons II following PVN stimulation in vivo. It should be noted that although TGOT was able to increase the frequency of occurrence of sEPSPs (spontaneous excitatory postsynaptic potentials) in some recorded neurons, these events did not reach an amplitude or a frequency sufficient to trigger action potentials.

Presynaptic effect of OT on glutamatergic lamina II neurons

As a first approach, we compared the effect of OT on spontaneous and miniature EPSCs/IPSCs. Whereas miniature currents (i.e. recorded in the presence of TTX) reflect only the activity of individual release sites, spontaneous synaptic currents allow action potentials and network activity-driven neurotransmitter release. TGOT did not change the frequency of occurrence of GABA_A-R-mediated miniature IPSCs (Figure 2B) but significantly increased AMPA-R-mediated synaptic transmission in 50% of the recorded lamina II neurons (Figure 2A). This suggested the presence of functional OT receptors on the presynaptic terminals of a subpopulation of glutamatergic neurons and the absence of such receptors on the synaptic endings of GABAergic dorsal horn interneurons. Although c-Fos expression was not observed in GAD positive neurons following PVN stimulation (Figure 4), we can not fully exclude that OT receptors are expressed by GABAergic interneurones. Should this be the case, we can however conclude that their activation is certainly not sufficient to directly increase the release probability of GABA (i.e. as seen in miniature transmission experiments; Figure 2) or to induce a significant action potential discharge in these neurons (see Figure 5 and related results in the text). These results are in agreement with those reported previously on cultured laminae I-III dorsal horn neurons [25]. A major difference observed in slices compared to dorsal horn primary cultures concerned the recruitment by OT of a population of AMPA-R mEPSCs with slow rise and decay kinetics (Figure 3). These mEPSCs were rare under control conditions (indicated by the histogram skewness in figure 3A) but were clearly seen after OT receptor stimulation. Their slow kinetics might indicate that they originate at synapses distant from the neuronal cell body, possibly on distal dendrites. In culture, dorsal horn neurons possess simpler dendritic trees and synapses normally impinging on distal dendrites might be established on the cell body and/or proximal dendrites. The existence of silent synapses due to the absence of functional postsynaptic AMPA-R has been previously shown in the spinal cord, in normal and pathological situations [26, 27]. Our results indicate for the first time that a pool of "presynaptically silent" synapses, which can be recruited or turned on functionally by a neuromodulator such as OT (Figure 3), might exist in the dorsal horn of the spinal cord. Although we cannot completely exclude a postsynaptic locus for the expression of this phenomenon [28, 29], the fast onset and reversibility TGOT effect on glutamatergic transmission argue rather in favor of the unmasking (i.e. by increasing the release probability) of presynaptically silent or whispering synapses at distal dendrites [29].

A subpopulation of OT-sensitive glutamatergic neurons recruits GABAergic interneurons in lamina II

In contrast with the effect on miniature IPSCs, OT receptor activation induced a massive and generalized increase in spontaneous GABAergic transmission, since it was observed in all lamina II neurons from which we recorded (Figure 2). This result is a key finding our study because it allows us to postulate that OT sensitive neurons, which are unlikely to be GABAergic (i.e. no effect of OT on the frequency of GABAA mIPSCs), are functionally connected (directly or indirectly) to all GABAergic interneurons in lamina II and are responsible for the facilitation of GABA release. Under the same conditions, however, the increase in spontaneous AMPA-R-mediated EPSCs concerned only a subpopulation of neurons (37%) comparable to that in which OT-receptor activation increased the frequency of mEPSCs. Our double immunofluorescence analysis indicates that c-Fos expressing neurons after PVN stimulation did not contain GAD, suggesting that the c-Fos positive neurons were likely to be glutamatergic. An interesting finding of the recent literature is the existence of a class of interneurons displaying no action potential discharge under resting conditions and receiving no apparent input from peripheral sensory fibers even when the latter were stimulated at C fiber strength [24]. These neurons are excited by PVN stimulation and the time-course of their excitation matched that of PVN-induced inhibition of Aδ/C afferent evoked electrical response. As indicated by the results from our current clamp experiments (Figure 5), the neurons directly depolarized by TGOT, represent a very small population of lamina II neurons and this finding is in good agreement with the c-Fos labeling seen in this lamina after PVN stimulation. This result strongly supports the existence of an organized local spinal circuit mediating the inhibitory effects of the descending hypothalamic pathway at the spinal level. Immunohistochemical experiments based on the revelation of c-Fos positive nuclei following PVN stimulation (Figure 4) were in line with our electrophysiological results. Only a small number of neurons was labeled after PVN stimulation and these neurons were located in lamina I and in the outer part of lamina II. It is interesting to note that this area is the main target in the dorsal horn for OT-positive hypothalamo-spinal fibers [8]. Recent work on rat or OT-knock-out mice spinal cord has shown that OT reduced glutamatergic monosynaptic EPSCs evoked by electrical stimulation of primary afferents in the superficial layers of neonatal spinal cord slices [18]. These results are not contradictory to our data since, as discussed in the work of Robinson [18], the presynaptic effect on primary afferent transmission might have been due to the activation of GABAergic neurons exerting an inhibitory effect on synaptic glutamate release from primary afferents.

Taken together, our results are therefore consistent with the hypothesis that the spinal excitation resulting from peripheral C and Aδ sensory neurons is reduced by a local network involving a subpopulation of glutamatergic neuron activated by OT which excites GABAergic interneurons in lamina II (Figure 6). This synaptic organization might also explain earlier observations made in vivo and showing that application of OT induced either excitation or inhibition of dorsal horn neurons [23].

Spinal cord slice preparation

In this study we used 15- to 30-day-old Wistar rats anesthetized with a single i.p injection of ketamine (150 mg/kg). A detailed description of the dissection procedure can be found elsewhere [30, 31]. Briefly, the spinal cord was removed by hydraulic extrusion and placed into ice-cold sucrose artificial cerebrospinal fluid (s-ACSF) containing (in mM): 248 sucrose, 11 glucose, 23 NaHCO₃, 2 KCl, 1.25 KH₂PO₄, 2 CaCl₂, and 1.3 MgSO₄ and was continuously bubbled with 95% O₂-5% CO₂. Transverse slices (600 μm thick) were cut from the lumbar segment of the spinal cord using a vibratome and stored at room temperature in an incubation chamber filled with oxygenated (95% O₂-5% CO₂) regular ACSF containing (in mM) 126 NaCl, 26 NaHCO₃, 1.25 NaH₂PO₄, 2.5 KCl, 2 CaCl₂, 2 MgCl₂, and 10 Glucose. Spinal cord slices were stored for at least 1 hour before being placed into the recording chamber. The slices were continuously superfused with oxygenated ACSF at a flow rate of approximately 3 ml per minute.

Patch clamp recordings, data acquisition, and analysis

All electrophysiological experiments were performed at room temperature. Lamina II neurons were recorded blindly using the whole-cell configuration of the patch-clamp technique and systematically dialyzed with biocytin in order to localize and determine the morphological characteristics of the recorded interneurons. Synaptic currents were recorded under voltage-clamp using an Axopatch 200B amplifier. The patch pipettes were filled with a solution containing (in mM): 80 Cs₂SO₄, 8 CsCl, 2 MgCl₂, 10 HEPES, 10 Biocytin, and adjusted to pH 7.3 with CsOH. In voltage-clamp mode, lamina II neurons were held at potentials of -60 mV and 0 mV to record glutamatergic and GABAergic synaptic currents, respectively. In a subset of experiments, we have used the current clamp mode to measure changes in the membrane potential (Em). Em was initially adjusted at -60 mV (i.e. with a small current injection if necessary), a value often corresponding to the resting membrane potential in our condition (no current injection: i = 0). For these experiments, cesium was replaced by potassium in the pipette solution described above. Synaptic currents were detected and analyzed individually using the WinEDR and WinWCP softwares (courtesy Dr J. Dempster, University of Strathclyde, Glasgow, UK) to extract their frequency of occurrence, amplitude, monoexponential rise time (τ_R), and decay time constants (τ_D). Results are expressed as mean value ± S.E.M. Statistical analysis was performed using parametric one-way ANOVA with Student's t-tests to compare means; sets of data were considered as different when p < 0.05.

Immunocytochemistry following in vivo stimulation of PVN

In anesthetized rats (alpha-chloralose 2% and urethane 20%) a bipolar electrode was implanted in the left paraventricular nucleus to perform 90 minutes stimulation (1 msec, 100 nA, 20 Hz). Immediately after the stimulation, tissues were fixed by an intracardiac perfusion of paraformaldehyde 4%. After a few hours of post fixation in the same fixative, spinal cords were removed, embedded in 5% agar and sectioned with a vibratome in 25 or 30 μm thick sections for direct observations or in 40 μm thick sections for preparation of semithin (1.5 μm) sections. Detailed procedures and antibodies used are indicated in additional file 1.

Drug application and perfusion solution

To activate oxytocin receptors, we used the selective agonist [Thr⁴, Gly⁷]-oxytocin (TGOT; Bachem, Germany) [32]. OT receptors were selectively inhibited with d(CH₂)⁵- [Tyr(Me)², Thr⁴-Tyr-NH₂⁹] ornithine vasotocin [33], a selective OT receptor antagonist (OTA; Sigma). Glutamate-receptor-mediated synaptic transmission was recorded in the continuous presence of the GABA_A-receptor antagonist bicuculline (BIC: bicuculline methiodide) and of the glycine-receptor antagonist strychnine (strychnine hydrochloride; Sigma). GABAergic synaptic currents were isolated in the presence of the general ionotropic glutamate receptor blocker kynurenic acid (2 mM) and of strychnine (1 μM) to block glycine receptors. To record miniature synaptic transmission, tetrodotoxin (0.5 μM) was present in the bath perfusion at a steady-state.