The modulation by 5-HT of glutamatergic inputs from the raphe pallidus to rat hypoglossal motoneurones, in vitro

Abstract

Decreases in the activity of 5-HT-containing caudal raphe neurones during sleep are thought to be partially responsible for the resultant disfacilitation of hypoglossal motoneurones. Whilst 5-HT has a direct excitatory action on hypoglossal motoneurones as a result of activation of 5-HT2 receptors, microinjection of 5-HT2 antagonists into the hypoglossal nucleus reduces motor activity to a much lesser extent compared to the suppression observed during sleep suggesting other transmitters co-localised in caudal raphe neurones may also be involved. The aim of the present study was therefore to characterise raphe pallidus inputs to hypoglossal motoneurones. Whole cell recordings were made from hypoglossal motoneurones in vitro. 5-HT evoked a direct membrane depolarisation (8.45 +/- 3.8 mV, P < 0.001) and increase in cell input resistance (53 +/- 40 %, P < 0.001) which was blocked by the 5-HT2 antagonist, ritanserin (2.40 +/- 2.7 vs. 7.04 +/- 4.6 mV). Stimulation within the raphe pallidus evoked a monosynaptic EPSC that was significantly reduced by the AMPA/kainate antagonist, NBQX (22.8 +/- 16 % of control, P < 0.001). In contrast, the 5-HT2 antagonist, ritanserin, had no effect on the amplitude of these EPSCs (106 +/- 31 % of control, P = n.s.). 5-HT reduced these EPSCs to 50.0 +/- 13 % of control (P < 0.001), as did the 5-HT1A agonist, 8-OH-DPAT (52.5 +/- 17 %, P < 0.001) and the 5-HT1B agonist, CP 93129 (40.6 +/- 29 %, P < 0.01). 8-OH-DPAT and CP 93129 increased the paired pulse ratio (1.38 +/- 0.27 to 1.91 +/- 0.54, P < 0.05 & 1.27 +/- 0.08 to 1.44 +/- 0.13, P < 0.01 respectively) but had no effect on the postsynaptic glutamate response (99 +/- 4.4 % and 100 +/- 2.5 %, P = n.s.). They also increased the frequency (P < 0.001), but not the amplitude, of miniature glutamatergic EPSCs in hypoglossal motoneurones. These data demonstrate that raphe pallidus inputs to hypoglossal motoneurones are predominantly glutamatergic in nature, with 5-HT decreasing the release of glutamate from these projections as a result of activation of 5-HT1A and/or 5-HT1B receptors located on presynaptic terminals.

Figure 1. The postsynaptic actions of 5-hydroxytryptamine are mediated via 5-HT₂ receptors

A, current clamp records showing membrane reponses to hyperpolarising and depolarising current pulses in the absence and presence of 5-HT. 5-HT evokes a membrane depolarisation and increase in cell input resistance. B, current–voltage relationships generated by step depolarisations from a holding potential of −70 mV in the absence (○) and presence (•) of 10 μm 5-HT. The 5-HT-mediated increase in cell input resistance is abolished by concurrent administration of the 5-HT₂ antagonist, ritanserin (Rit, 10 μm, ▾). C, the 5-HT₂ agonist, α-Me-5-HT (α-Me, 20 μm), but not the 5-HT_1A agonist, 8-OH-DPAT (8-OH, 50 μm) or the 5-HT_1B agonist, CP 93129 (CP, 10 μm), also evoked a membrane depolarisation. ***P < 0.001; **P < 0.01. D, α-Me-5-HT and CP 93129, but not 8-OH-DPAT, evoked increases in cell input resistance. E, current clamp records showing a 5-HT (10 μm) -evoked depolarisation (upper trace) that was markedly reduced in the presence of ritanserin (10 μm, lower trace). Both traces were obtained in the same neurone.

Figure 2. Stimulation of the raphe pallidus evokes glutamatergic excitatory postsynaptic currents in hypoglossal motoneurones

A. EPSCs were evoked in hypoglossal motoneurones by stimulation within the raphe pallidus, these EPSCs were unaffected by the 5-HT₂ antagonist, ritanserin (10 μm). Superimposed averages of 50 consecutive EPSCs in the presence and absence of ritanserin, with the motoneurone being held at a holding potential of −70 mV. B, at this holding potential, the NMDA antagonist, APV (50 μm), had no effect on the EPSC. In contrast, it was markedly reduced by the AMPA/kainateantagonist, NBQX (20 μm). C, these EPSCs were the result of activation of the cell bodies of neurones within the raphe pallidus. Pressure ejection of glutamate (▾, 10 mm, 20 p.s.i. (138 kPa)), for a, 50 ms and b, 60 ms, within the confines of the raphe pallidus evoked a concentration-dependent increase in synaptic activity and the induction of neuronal firing in a hypoglossal motoneurone. Action potential amplitudes have been truncated. c, membrane potential before (1), during (2), and after (3) the glutamate-evoked response. Same response as b at increased gain and faster time base. D, EPSCs with the same electrophysiological and pharmacological characteristics could still be evoked in a reduced slice preparation. E, schematic showing cuts made to reduce the coronal slice to a triangle of tissue and location of recording (*) and stimulating (•) electrodes. Adapted from Paxinos & Watson, 1998.

Figure 3. Spontaneous excitatory postsynaptic currents in hypoglossal motoneurones are glutamatergic

Current records showing spontaneous EPSCs in a hypoglossal motoneurone and their abolition by the AMPA/kainateantagonist, NBQX (20 μm, A) but not by the 5-HT₂ antagonist, ritanserin (10 μm, B).

Figure 4. 5-HT decreased the amplitude of EPSCs following activation of presynaptic 5-HT_1A and 5-HT_1B receptors

A. 5-HT (20 μm) decreases, in a reversible manner, the amplitude of EPSCs evoked in hypoglossal motoneurones following stimulation within the raphe pallidus. B, the 5-HT-evoked decreases in EPSC amplitude were mimicked by the 5-HT uptake inhibitor, fluoxetine (Fluo, 50 μm), the 5-HT_1A agonist, 8-OH-DPAT (8-OH, 50 μm) and the 5-HT_1B agonist CP 93129 (CP, 10 μm), but not the 5-HT₂ agonist, α-Me-5-HT (α-Me, 50 μm). The amplitude of the average of 50 consecutive EPSCs obtained during the peak agonist response is expressed as a percentage of the averaged EPSC obtained in normal aCSF prior to agonist application. C and D, 5-HT and its selective agonists also evoked significant increases in the paired pulse ratio. C, twin pulse stimulation within the raphe pallidus (stimulus interval 50 ms) evoked pairs of EPSCs in hypoglossal motoneurones. The amplitude of the second EPSC was compared to the first to give the paired pulse ratio (PPR). In this neurone, whilst the PPR in normal aCSF was 1.1, it was increased to 2.1 in the presence of 8-OH-DPAT (8-OH, 50 μm) suggesting that the agonist was having its effects via an action at presynaptic terminals. Recovery of the PPR was observed on washout. D, mean data showing increases in the PPR in the presence of 5-HT (10 μm), 8-OH-DPAT (50 μm) and CP 93129 (10 μm). E, CP 93129 evokes progressively less inhibition of sequential eEPSCs in a train. Superimposed averages of EPSCs evoked by a train of four pulses (stimulus interval = 50 ms, train frequency = 0.02 Hz) in normal ACSF and in the presence of CP 93129. Data are averages from 64 trials. F, CP 93129 increases the amplitude ratio of successive EPSCs in a train. Mean data of amplitude ratio (Amplitude EPSC_n/EPSC₁) of each EPSC in train of four relative to the first in normal ACSF (○) and in the presence of CP 93129 (▪). Data are averages from 5 neurones of 64 trials in each neurone. * denotes significance of amplitude ratio for each EPSC in the train in the presence of CP 93129 compared to the corresponding ratio obtained in normal ACSF (*P < 0.05).

Figure 5. CP 93129 decreases the frequency, but not the amplitude, of miniature excitatory postsynaptic currents

A, voltage clamp records showing miniature EPSCs recorded in the absence and presence of the 5-HT_1B agonist, CP 93129 (10 μm). B, C, cumulative probability histograms of the data from A showing that CP 93129 application resulted in a significant reduction in the frequency of mESPCs (B, P < 0.001) but had no effect on their amplitude (C, P = n.s.)