Serotonin enhances central olfactory neuron responses to female sex pheromone in the male sphinx moth manduca sexta

Abstract

In the brain of the sphinx moth Manduca sexta, sex-pheromonal information is processed in a prominent male-specific area of the antennal lobe called the macroglomerular complex (MGC). Whole-cell patch-clamp recordings from identified projection (output) neurons in the MGC have shown that serotonin [5-hydroxytryptamine (5-HT)] increases both the excitability of MGC projection neurons and their responses to stimulation with pheromone. At least two types of voltage-activated potassium currents in these cells are modulated by 5-HT. 5-HT decreases the maximal conductance of a transient potassium current (I(A)) and shifts its voltage for half-maximal inactivation to more negative potentials without affecting the half-maximal voltage for activation. This reduces the "window current" between the voltage activation and inactivation curves, decreasing the tonically active I(A) near the resting potential and causing the cell to depolarize. 5-HT's effect in this case is to decrease both the transient and resting K(+) conductance by modulating the same channel (I(A)). 5-HT also decreases the maximal conductance of a sustained potassium current [I(K(V))] without affecting its voltage dependence. Using HPLC, we show also that levels of 5-HT in the antennal lobes fluctuate significantly over a 24 hr period. Interestingly, 5-HT levels are highest at times when the moths are most active. We suggest that by controlling the responsiveness of antennal-lobe projection neurons to olfactory stimuli, 5-HT will have significant impact on the performance of odor-dependent behaviors.

Fig. 1.

A, Bodian stain of a section through the antennal lobe showing the glomerular structure of the antennal-lobe neuropil and clusters of cell bodies at the periphery.B, The framed cell body cluster fromA shown in higher magnification. C,In situ preparation of the antennal lobe showing a cell body cluster similar to that shown in A and B. D, Patch pipette in recording position on one of the cell bodies. Scale bars: A, 100 μm;B, 50 μm; C, 25 μm; D, 12.5 μm.

Fig. 2.

A, Whole-cell patch-clamp recording of responses of an MGC projection neuron to antennal stimulation with pheromone before (control), during (2 and 6 min), and after (wash) application of 5-HT (10⁻⁴m). The resting membrane potential was held at the control value by tonic current injection. Arrows indicate the positions of the last spike from the control trace(top) and the wash trace(bottom). 5-HT increased the number of spikes, the length of the spike train, and the duration of the depolarization elicited by stimulation with pheromone. These effects of 5-HT were reversible. The horizontal bar beneath the two recordings marks the duration of pheromone stimulation.B, Arborizations of the projection neuron within the cumulus (marked with the dottedline) of the MGC revealed by intracellular staining. Scale bar, 50 μm.

Fig. 3.

Effects of 5-HT on several parameters of the response of MGC projection neurons to pheromone.A, Mean percentage change in the number of spikes and the length of the spike train. In the presence of 5-HT (gray vertical bars), the number of spikes and the length of the spike train were increased significantly, by 18 ± 2% (p < 0.0001) and 23 ± 2% (p < 0.0001), respectively. Both effects showed significant recovery after washing with saline (whiteverticalbars;p < 0.0001). B, Mean percentage change of the slow component of pheromone-evoked depolarization. 5-HT had no significant effect on the amplitude of the depolarization but increased significantly the length and the integral of the response, by 32 ± 2% (p < 0.0001) and 35 ± 4% (p < 0.0001), respectively (grayverticalbars). Both effects were reversible after washing with saline (whiteverticalbars; p < 0.0001). * indicates significantly different from control (p ≤ 0.025). AP, Action potential.

Fig. 4.

A, Action potentials recorded from the cell body of an MGC projection neuron using voltage-clamp recording in cell-attached configuration. B, 5-HT effect on the pheromone response of an MGC projection neuron recorded as described inA. The horizontalbarbeneath the recording indicates the duration of pheromone stimulation. 5-HT increased reversibly the number of action potentials and the length of the spike train elicited by the pheromone. Recordings were high-pass-filtered on-line to eliminate slow components of the signal. This type of recording was used to identify pheromone-sensitive neurons and to test whether pheromone-induced responses were modulated by 5-HT. A whole-cell recording configuration was then established to examine the effects of 5-HT on voltage-gated K⁺ currents in the same cells.

Fig. 5.

Voltage-clamp analysis of the effect of 5-HT on the transient potassium current (I_A) in MGC projection neurons. All cells responded to pheromone in the pretest, and the responses were enhanced by 5-HT. The whole-cell configuration was established after the 5-HT effect of the pretest had fully reversed. I_A was isolated pharmacologically and by digital subtraction (see Materials and Methods). A, Steady-state activation.Current traces ofI_A before, during, and after application of 5-HT are shown. The holding potential was −60 mV. After a prepulse to −90 mV (2 sec), voltage was stepped from −45 to +60 mV in 15 mV increments. B, Steady-state inactivation.Currenttraces ofI_A before, during, and after application of 5-HT are shown. The holding potential was −60 mV. Test pulses (to +20 mV) were preceded by 2 sec preconditioning pulses between −90 and −20 mV in 10 mV increments. C, D, Conductance/voltage curves for activation (C; m³) and inactivation (D; h) ofI_A under control conditions (solid squares) and in 10⁻⁴m 5-HT (solidtriangles). Theopentriangles (5-HT*) represent the conductance under 5-HT scaled to the control. Values are means ± SEM (n = 4 for activation;n = 5 for inactivation), calculated as a fraction of the calculated maximal conductance under control conditions in each experiment. C, Steady-state activation. Thecurves are fits to a third-order Boltzmann relation (Eq.1, Materials and Methods) with the following parameters. Control: V_act = −32.69 mV;s_act = −17.51; 5-HT: V_act = −29.85 mV;s_act = −15.99. D, Steady-state inactivation. The curves are fits to a first-order Boltzmann relation (Eq. 1, Materials and Methods) with the following parameters. Control: V_0.5inact = −53.3 mV; s_inact = 7.23; 5-HT: V_0.5inact = −60.4 mV;s_inact = 5.35. E, To demonstrate the decrease in tonically activeI_A near the resting potential, the product of the activation and inactivation curves(from C, D) plotted as follows: $g/g_{max}=(1/1+e^{-(V-V_{{0.5}_{\text{act}}}){/}_{S_{\text{act}}}}{)}^3\times (1/1+e^{-(V-V_{{0.5}_{\text{inact}}}){/}_{S_{\text{inact}}}}{)}^1$for the control condition and for 5-HT application. Thesecurves showed the fraction of tonically activeI_A as a function of the membrane potential. The area between the curve and the baseline is decreased by 88% during 5-HT application. F, Arborizations of the MGC projection neuron recorded in Arevealed by staining via the patch pipette. The cumulus of the MGC is marked by the dottedline. Scale bar, 50 μm.

Fig. 6.

Voltage-clamp analysis of the 5-HT effect on the sustained potassium current I_K(V) in MGC projection neurons. All cells responded to pheromone in the pretest, and the responses were enhanced by 5-HT. The whole-cell configuration was established after the 5-HT effect of the pretest had fully reversed. I_K(V) was isolated pharmacologically or by holding the neuron at −40 mV whereI_A is almost completely inactivated.A, Steady-state activation.Current traces ofI_K(V) before, during, and after application of 5-HT are shown. The holding potential was −40 mV. Voltage was stepped from −35 to +70 mV in 15 mV increments. B, Steady-state activation. Conductance voltage curves for activation under control conditions (filled squares) and during application of 5-HT (filled triangles) are shown. Theopen triangles (5-HT*) represent the conductance under 5-HT scaled to the control. Conductances were calculated assumingE_K = −91.6 mV (see Materials and Methods). Values are means ± SEM from four experiments, calculated as a fraction of the calculated maximal conductance under control conditions in each experiment. The curves are fits to a third-order Boltzmann relation (Eq. 1, Materials and Methods) with the following parameters. Control: V_act = −18.5 mV; s_act = −22.5; 5-HT: V_act = −16.8 mV; s_act= −24.4. C, Arborizations of the MGC projection neuron recorded in A revealed by staining via the patch pipette. The cumulus of the MGC is marked by the dotted line. Scale bar, 50 μm. D, Steady-state inactivation. Current traces ofI_K(V) were determined by applying preconditioning pulses (2 sec) between −90 and 0 mV in 15 mV increments before the test pulses to +20 mV.I_A was blocked with 4–5 × 10⁻³m 4-AP. Data were not plotted because I_K(V) did not show obvious inactivation.

Fig. 7.

5-HT levels (±SEM) in the antennal lobes ofM. sexta at different time points over two 24 hr periods. Male moths were maintained for at least 9 d (at least 7 d as pupae and 2 d as adults) under a long-day photoperiod regimen of 14:10 hr (light/dark). Periods during which the light was turned off (blackhorizontalbars) or on (whitehorizontalbar) are indicated along thex-axis. The n values for each group are provided above the data points. A, B 5-HT levels of the first and second run in which eight and seven time points were examined, respectively. C, 5-HT concentrations from the first and second runs combined. Contrast analysis performed on the pooled data confirmed that there is a significant difference between maximum and minimum levels of 5-HT recorded in the antennal lobes of the moth (p = 0.0097).