Presynaptic α4β2 nicotinic acetylcholine receptors increase glutamate release and serotonin neuron excitability in the dorsal raphe nucleus

Abstract

Several behavioral effects of nicotine are mediated by changes in serotonin (5-HT) release in brain areas that receive serotonergic afferents from the dorsal raphe nucleus (DRN). In vitro experiments have demonstrated that nicotine increases the firing activity in the majority of DRN 5-HT neurons and that DRN contains nicotinic acetylcholine receptors (nAChRs) located at both somata and presynaptic elements. One of the most common presynaptic effects of nicotine is to increase glutamate release. Although DRN receives profuse glutamatergic afferents, the effect of nicotine on glutamate release in the DRN has not been studied in detail. Using whole-cell recording techniques, we investigated the effects of nicotine on the glutamatergic input to 5-HT DRN neurons in rat midbrain slices. Low nicotine concentrations, in the presence of bicuculline and tetrodotoxin (TTX), increased the frequency but did not change the amplitude of glutamate-induced EPSCs, recorded from identified 5-HT neurons. Nicotine-induced increase of glutamatergic EPSC frequency persisted 10-20 min after drug withdrawal. This nicotinic effect was mimicked by exogenous administration of acetylcholine (ACh) or inhibition of ACh metabolism. In addition, the nicotine-induced increase in EPSC frequency was abolished by blockade of α4β2 nAChRs, voltage-gated calcium channels, or intracellular calcium signaling but not by α7 nAChR antagonists. These data suggest that both nicotine and endogenous ACh can increase glutamate release through activation of presynaptic α4β2 but not α7 nAChRs in the DRN. The effect involves long-term changes in synaptic function, and it is dependent on voltage-gated calcium channels and presynaptic calcium stores.

Figure 1.

Electrophysiological characteristics of the DRN serotonergic neurons. A, Current traces in response to hyperpolarizing and depolarizing current steps recorded from a 5-HT neuron. Inset, An expanded AP from the same neuron. B, Plots of discharge frequency as a function of injected current obtained from 13 identified 5-HT neurons are shown. Filled circles represent the instantaneous frequency obtained with the reciprocal of the first interspike interval. Open circles represent the frequency obtained from the last interspike interval. C, Microphotographs illustrating the recorded cell in A, after biocytin staining (top), 5-HT immunoreactivity (middle), and merge (bottom).

Figure 2.

Low concentrations of nicotine enhance glutamatergic synaptic transmission. A, sEPSCs recorded by using whole-cell patch-clamp technique (voltage-clamp mode) in the presence of bicuculline (10 μm). The traces show 10 s taken in control conditions (left), after nicotine (300 nm) added to the bath solution (middle), and after 20 min wash (right). A region of the middle trace (dashed box) is shown below at a slower sweep time. Two of the synaptic events are indicated by asterisks. B, Time–frequency histogram (10 s bin) for the same cell in A, showing that nicotine increased the frequency but did not change the amplitude of sEPSCs (inset). C, Summary of the results from five neurons showing the effect on sEPSCs frequency in the presence of nicotine (*p < 0.05). D, Microphotographs illustrating the recorded cell in A after biocytin staining (left), 5-HT immunoreactivity (middle), and merge (right).

Figure 3.

Nicotinic effects are mimicked by endogenous ACh. A, Traces showing sEPSCs recorded from a 5-HT-positive neuron in control, in the presence of nicotine, 10 min after nicotine washout, and 10 min after adding CNQX (10 μm). B, C, Cumulative probability distributions of frequencies and amplitudes for the same neuron in A, in control (dark gray traces), nicotine (black traces), and 10 min wash (light gray traces). D, Average normalized time–frequency histogram from 16 5-HT neurons tested with nicotine (1 μm). Inset, Normalized time–frequency histogram from five 5-HT neurons tested with eserine (10 μm). E, Summary of the results showing the effect on sEPSC frequency with nicotine alone, nicotine in the presence of TTX, ACh, and eserine. All experimental groups were compared against control (before drug application). The last three bars represents the wash time after nicotine alone. All the experiments were done in the presence of bicuculline (10 μm). ACh experiments were done in the presence of bicuculline and atropine (10 μm) (*p < 0.05, **p < 0.01).

Figure 4.

Nicotinic effects are performed through β2-containing nAChRs. A, Traces showing sEPSCs recorded from a 5-HT-positive neuron in the presence of MLA (100 nm, top), MLA plus nicotine (1 μm, middle), and nicotine washout (bottom). B, Time–frequency histograms from 10 identified 5-HT neurons tested with nicotine (1 μm) in the presence of MLA. The inset shows the normalized sEPSC frequency. C, Traces showing sEPSCs recorded from a 5-HT-positive neuron in the presence of DHβE (100 nm, top), DHβE plus nicotine (1 μm, middle), and nicotine washout (bottom). D, Time–frequency histogram from 12 identified 5-HT neurons tested with nicotine (1 μm) in the presence of DHβE. The inset shows the normalized sEPSC frequency. In all the experiments, nicotine was applied after 10 min pretreatment with DHβE or MLA.

Figure 5.

Nicotinic effects are mimicked by the α4β2 nAChR-selective agonist RJR-2403. A, Traces showing sEPSCs recorded from a 5-HT-positive neuron in control (top), in the presence of RJR-2403 (100 nm, middle), and RJR-2403 washout (bottom). B, Time–frequency histogram from five identified 5-HT neurons tested with RJR-2403. The inset shows the normalized sEPSC frequency. C, Traces showing sEPSCs recorded from another 5-HT-positive neuron in control (top), in the presence of the selective α7 nAChR agonist PNU-282987 (100 nm, middle), and PNU-282987 washout (bottom). D, Time–frequency histograms from five identified 5-HT neurons tested with PNU-282987. The inset shows the normalized sEPSC frequency.

Figure 6.

Nicotinic action mechanism is presynaptic. Aa, Traces showing sEPSCs recorded from a 5-HT-positive neuron with BAPTA (10 mm) in the recording pipette (top) and after adding nicotine in the bath solution (bottom). Ab, Traces showing sEPSCs recorded from a 5-HT-positive neuron with BAPTA-AM (10 mm) applied in the bath solution (top) and after adding nicotine (bottom). Ac, Bar histogram shows normalized sEPSC frequency. B, Paired-pulse protocol is illustrated in Ba. A concentric stimulation electrode (S) with a 10 μm tip diameter was placed in the proximities of the DRN, ventral to the aqueduct. Excitatory evoked currents were recorded from 5-HT neurons within the DRN in the presence of bicuculline (10 μm). Evoked current traces of a single neuron in response to paired stimuli (S1 and S2) are shown in Bb (top). Gray traces are the responses in control conditions. Black traces are the responses in the presence of nicotine (1 μm). Temporal course of the first stimulus amplitude (S1) from the cell in Bb (top) is shown in Bb (bottom). Dark gray circles represent the control, black circles represent nicotine, and light gray circles represent nicotine washout. The circles at zero current represent synaptic failures. Bc, Bar histograms show the change in the normalized S1 amplitude induced by nicotine (top; ***p < 0.001, n = 6) and the change in the paired-pulse ratio induced by nicotine (bottom; ***p < 0.001, n = 6).

Figure 7.

Nicotinic effects depend on VGCCs and intracellular CICR. A, Time–frequency histogram shows the effect of nicotine on the sEPSC frequency in the presence of CdCl₂ (gray bar). B, Time–frequency histogram shows the lack of effect of nicotine on the sEPSC frequency in the presence of a mixture containing the Ca²⁺ channel blockers ω-agatoxin-TK, ω-conotoxin-GVIA, and nitrendipine (gray bar). C, Time–frequency histogram shows the lack of effect of nicotine on the sEPSC frequency in the presence of the SERCA blocker thapsigargin (gray bar). D, Bar graph shows the effect of nicotine on the sEPSC frequency in slices pretreated with CdCl₂, Ca²⁺ channel blockers, thapsigargin, CPA, or ryanodine (**p < 0.01). Gray bar represent the control (before nicotine application).

Figure 8.

Model summarizing nicotinic effects on glutamate terminals in the DRN. A, In physiological conditions, the cholinergic tone is regulating the excitatory glutamatergic input to the DRN 5-HT neurons through the activation of α4β2 nAChRs, located at glutamate terminals. Released ACh is quickly metabolized by the enzyme acetylcholinesterase. As a result, the Ca²⁺ influx into the glutamate terminals and glutamate release will be maintained at low levels. B, When nicotine is present (for example, in smokers), more α4β2 nAChRs will be activated, because nicotine cannot be degraded in the synaptic cleft. This will enhance Ca²⁺ entry, followed by depolarization of glutamate terminals and activation of VGCCs. This, in turn, will increase even more intracellular calcium and produce CICR from the endoplasmic reticulum (ER) through the activation of ryanodine receptors (RyR). This last event generates a long-term potentiation of glutamate release.