NMDA receptors trigger neurosecretion of 5-HT within dorsal raphe nucleus of the rat in the absence of action potential firing

Abstract

Activity and calcium-dependent release of neurotransmitters from the somatodendritic compartment is an important signalling mechanism between neurones throughout the brain. NMDA receptors and vesicles filled with neurotransmitters occur in close proximity in many brain areas. It is unknown whether calcium influx through these receptors can trigger the release of somatodendritic vesicles directly, or whether postsynaptic action potential firing is necessary for release of these vesicles. Here we addressed this question by studying local release of serotonin (5-HT) from dorsal raphé nucleus (DRN) neurones. We performed capacitance measurements to monitor the secretion of vesicles in giant soma patches, in response to short depolarizations and action potential waveforms. Amperometric measurements confirmed that secreted vesicles contained 5-HT. Surprisingly, two-photon imaging of DRN neurones in slices revealed that dendritic calcium concentration changes in response to somatic firing were restricted to proximal dendritic areas. This implied that alternative calcium entry pathways may dominate the induction of vesicle secretion from distal dendrites. In line with this, transient NMDA receptor activation, in the absence of action potential firing, was sufficient to induce capacitance changes. By monitoring GABAergic transmission onto DRN 5-HT neurones in slices, we show that endogenous NMDA receptor activation, in the absence of postsynaptic firing, induced release of 5-HT, which in turn increased the frequency of GABAergic inputs through activation of 5-HT(2) receptors. We propose here that calcium influx through NMDA receptors can directly induce postsynaptic 5-HT release from DRN neurones, which in turn may facilitate GABAergic input onto these cells.

Figure 1. 5-HT neurones can reliably be selected from acute DRN slices

A, current clamp profile of 5-HT neurone in acute brain slice of DRN from adult male animals (6–8 weeks, example trace). Long (400 ms) depolarizing and hyperpolarizing current pulses were injected into each neurone. B, current clamp profile of non-5-HT neurone in acute brain slice in DRN (example trace). Note the presence of H current, which is typical for non-5-HT neurones. C, confocal image of 5-HT neurone filled with Alexa 594. Note the magnocellular nature of the soma and dendrites. D, confocal image of a non-5-HT neurone.

Figure 2. Voltage-dependent calcium channel activation induces exocytosis of 5-HT containing vesicles from somatic nucleated outside-out patches

A, inward current, membrane capacitance and membrane conductance in nucleated patches from DRN from adult male animals (6–8 weeks, averaged trace, n = 7) during 10 ms depolarization to 0 mV. Calcium channels were preferably activated beyond −20 mV, which resulted in capacitance changes, indicative of exocytosis (averaged trace, N = 7). B, I–V relationship of inward current activated in nucleated patches from DRN neurones (n = 7). C, the integral of the calcium current was calculated to produce the absolute Ca²⁺ influx. D, capacitance changes in nucleated patches of DRN neurones are proportional to the amount of, and therefore most likely a consequence of, calcium influx (compare panel C and D). E, repetitive depolarization (0.1 Hz, 100 ms, 0 mV) of nucleated outside-out patch (see inset, carbon fibre approaching from right) of an identified 5-HT neurone induced amperometric spikes (overlay of 13 traces from one example recording, n = 10). F, individual amperometric spikes taken from same recording at an increased time resolution.

Figure 3. Somatic vesicle release evoked by a single action potential

A, inward current, membrane capacitance and membrane conductance during a single action potential in nucleated patches (averaged trace, n = 7). The voltage template is shown above the current trace (bracket line: voltage protocol was adjusted to start and end at −70 mV to perform capacitance recordings). B, analogous responses during a train of action potentials in nucleated patches (averaged trace, n = 7). The voltage template is shown above the current trace. C, pair wise comparison of capacitance changes between single and train of action potentials (paired t test, P < 0.05, paired data from individual experiments). D, capacitance changes are dependent on number of action potentials.

Figure 4. Dendritic calcium influx induced by single somatic action potentials is restricted to proximal regions

A, overview of a 5-HT neurone. B, magnified image of a dendritic region used for line-scans. Dashed line indicates location where line-scan was taken. C, line-scan image following dendritic fluorescence of Fluo4 over time. D, green fluorescence transients (Δgreen/red ratio) in response to either single somatic action potentials (left) or trains of 5 action potentials (right) at different dendritic locations. E, summary data (n = 8), showing that AP-induced changes in ΔG/R ratio decrease with distance from the soma. Note that at 150 μm distance from the soma, a single action potential induces almost no fluorescence change.

Figure 5. NMDAR activation induces exocytosis without action potential firing of postsynaptic compartment

A, experimental set-up, with the nucleated patch positioned in front of a double-barrelled electrode attached to a piezo-element. NMDA (100 μm) is rapidly applied (200 ms) by repositioning of the double barrelled electrode. B, average NMDA-induced current and corresponding membrane capacitance during NMDA application recorded from adult (6–8 weeks, n = 6) male animals voltage-clamped at −70 mV (pooled data, 6 nucleated patches, total of 61 applications, mean ±s.d.: 10 ± 3 applications per patch). Note that during the full protocol, nucleated patches were continuously voltage clamped at −70 mV to prevent activation of voltage dependent calcium channels. C, average of NMDA-induced currents, capacitance changes and membrane conductance traces from one particular nucleated patch, which lacked NMDA induced capacitance changes (n = 8). D, same as C, but in this example on average, the NMDA applications resulted in increased membrane capacitance, being indicative of vesicle release. E, cumulative all-point histograms of the recording in C, showing the lack of capacitance changes when comparing the blue and red regions. F, cumulative all-point capacitance histograms of the blue and red regions of the recording shown in D, indicating exocytosis. G, cumulative all-point histograms of capacitance changes of all pooled recordings and analysing the black, blue and red regions of the average as shown in B. H, probability histogram of capacitance changes when pooling all trials from all recordings. In five out of six recordings, on average 40% of the trials gave capacitance changes > 3 fF. In addition, an average of 40%‘failures’ (i.e. changes between −3 and +3 fF) occurred, where in a minority of the trials negative events occurred, which may imply that in addition endocytosis may occur. As indicated in text, in one recording (shown in C and E) only failures were observed. Moreover, three very negative events, referred to in text, shown in H (i.e. < −10 fF) were all from one recording also showing failures and exocytotic events.

Figure 6. Postsynaptic NMDA receptor activation facilitates GABAergic transmission

A, voltage protocol for NMDAR activation by endogenous glutamate. Experiments were performed upon preloading with tryptophan, and recorded in the presence of CNQX (1 μm), L-CGG-III (10 μm) and CPPG (30 nm) in addition fluoxetine (10 μm). B and C, example traces of IPSCs at start (B, shown by left box in A) and more toward the end (C, right box in A) at −30 mV (calibration 20 pA, 20 ms). D, at −30 mV the specific NMDA antagonist APV (50 μm) significantly suppressed the tonic increase in sIPSC frequency (n = 7, ANOVA followed by a post hoc Bonferroni multiple comparison test, *P < 0.05). E, at −30 mV the 5-HT₂ antagonist ketanserin (1 μm) also significantly suppressed the tonic increase sIPSC frequency (n = 7, ANOVA followed by a post hoc Bonferroni multiple comparison test, *P < 0.05).