Raphé neurons stimulate respiratory circuit activity by multiple mechanisms via endogenously released serotonin and substance P

Abstract

Brainstem serotonin (5-HT) neurons modulate activity of many neural circuits in the mammalian brain, but in many cases endogenous mechanisms have not been resolved. Here, we analyzed actions of raphé 5-HT neurons on respiratory network activity including at the level of the pre-Bötzinger complex (pre-BötC) in neonatal rat medullary slices in vitro, and in the more intact nervous system of juvenile rats in arterially perfused brainstem-spinal cord preparations in situ. At basal levels of activity, excitation of the respiratory network via simultaneous release of 5-HT and substance P (SP), acting at 5-HT(2A/2C), 5-HT(4), and/or neurokinin-1 receptors, was required to maintain inspiratory motor output in both the neonatal and juvenile systems. The midline raphé obscurus contained spontaneously active 5-HT neurons, some of which projected to the pre-BötC and hypoglossal motoneurons, colocalized 5-HT and SP, and received reciprocal excitatory connections from the pre-BötC. Experimentally augmenting raphé obscurus activity increased motor output by simultaneously exciting pre-BötC and motor neurons. Biophysical analyses in vitro demonstrated that 5-HT and SP modulated background cation conductances in pre-BötC and motor neurons, including a nonselective cation leak current that contributed to the resting potential, which explains the neuronal depolarization that augmented motor output. Furthermore, we found that 5-HT, but not SP, can transform the electrophysiological phenotype of some pre-BötC neurons to intrinsic bursters, providing 5-HT with an additional role in promoting rhythm generation. We conclude that raphé 5-HT neurons excite key circuit components required for generation of respiratory motor output.

Figure 1.

Rhythmogenic medullary slices contain neurons immunoreactive for 5-HT and substance P. A, Low-power (5×) IR-DIC image (left) and schematic (right) of medullary slice preparation showing configuration with microinfusion pipettes in the n. raphé obscurus (RO) or bilaterally in the pre-BötC. Inspiratory activity was recorded from XII nerve (integrated signal, ∫XII) with suction electrodes. IO, Inferior olive; NA, semicompact division of nucleus ambiguus; RP, n. raphé pallidus; XII N., hypoglossal nerve tract. B, High-power (63×) IR-DIC image of RO neurons (see inset in A) with patch-clamp recording pipette within slice. C, Left, Immunoreactivity for TpOH (Cy5 fluorescence images) was found in three subpopulations: n. raphé obscurus (1), n. raphé pallidus (2), and the parapyramidal region (3). Right, Enlargement of box enclosing ventral part of n. raphé obscurus shown on left. D, Left, TpOH-immunoreactive neurons in n. raphé obscurus (Cy5 fluorescence). Middle, SP-immunoreactive neurons (Alexa Fluor 488 fluorescence). Right, Merged image showing colocalization of TpOH- and SP-immunoreactive neurons. Arrows indicate the same set of neurons and highlight examples of double-labeled neurons.

Figure 2.

Spontaneous firing of neurons in the n. raphé obscurus. A, Current-clamp recording from a representative raphé neuron firing at ∼1 Hz, in aCSF with [K⁺]_o of 3 mm (gray trace) or 9 mm (black trace). B, Typical action potential shape, with visible shoulder and prominent AHP as shown in inset on the right, which is an expanded view of the region of the action potential bounded by the box on the left. C, Relationship between firing frequency and injected current for the first (○) and last (●) interspike intervals (n = 22 neurons) obtained during step injection of bias current. Difference in spike frequencies at a given current reflects spike frequency adaptation during sustained steps in bias current. D, Example of a regularly, tonically spiking raphé obscurus neuron filled with biocytin (1%) (Texas Red fluorescence, left). The same slice was stained for TpOH-immunoreactive neurons (Alexa Fluor 488 fluorescence, middle). Merged image (right) shows that the tonically spiking neuron was serotonergic.

Figure 3.

The n. raphé obscurus is interconnected with the pre-BötC and the XII motor nucleus. A, Simultaneous recording from the XII nerve (∫XII, top) and from a neuron in the n. raphé obscurus (bottom), showing that some raphé neurons increase their spike rate during inspiratory network activity. B, Expanded view of augmented neuronal spiking during inspiratory activity (from A indicated by *). C, Injecting hyperpolarizing current (20 pA) into the raphé neuron to prevent spontaneous firing reveals excitatory depolarizing synaptic potentials phase locked to XII motor output. D, Expanded view of depolarizing potential during inspiratory activity (indicated by *). E, Composite reconstruction of two biocytin-filled raphé obscurus (RO) neurons and two pre-BötC inspiratory neurons illustrating patterns of axonal projections. A pre-BötC inspiratory neuron (green) with axonal projection toward RO and a RO neuron (blue) with projection into pre-BötC were both labeled in the same slice. Other neurons (RO red, pre-BötC black) were labeled and reconstructed in individual slices and rescaled so that all neurons could be composited to represent different spatial patterns of synaptic projections. This other RO neuron projected to hypoglossal (XII) motor nucleus with collateral projections toward pre-BötC. The other pre-BötC neuron (left, black) projected through RO with collateral branching and arborizations in this region. 5SP, Spinal trigeminal nucleus.

Figure 4.

The pre-BötC region is innervated by serotonergic and SP-containing fibers, and a subset of presynaptic boutons on inspiratory neurons colocalize 5-HT and SP. A, Confocal image showing simultaneous labeling of an inspiratory pre-BötC neuron (red, AMCA-Avidin D) surrounded by fibers immunoreactive for 5-HT (blue, Cy3) and SP (green, Alexa Fluor 488) (63× magnification, 2-dimensional projection from merged image stack). B–D, Magnification of area outlined by white box in A showing examples of SP (B), 5-HT (C), and colocalization of 5-HT and SP (D, merged images) in presynaptic boutons (arrows) on a dendrite of an electrophysiologically identified inspiratory neuron. Images are from a single scan plane (63×, 0.3 μm optical section thickness).

Figure 5.

Inspiratory motor output in the rhythmic slice in vitro depends on endogenous activation of 5-HT_2A and NK-1 receptors. Bath application of methysergide (MeSG, n = 9), ketanserin (KET, n = 8), MDL 11,939 (n = 6), RS 102221 (n = 7), GR 113808 (n = 3), and SR 140333 (n = 7) produced a concentration-dependent depression of baseline inspiratory activity (monitored by recordings of integrated XII motoneuron population activity), with a reversible reduction in the inspiratory XII burst frequency (left) and burst amplitude (right). Panels show steady-state dose–response relations normalized to control. Amplitudes are measured from the peak of the integrated XII population activity. Data points represent mean values ± SEM.

Figure 6.

Spontaneous inspiratory activity depends on activation of 5-HT₂ and NK-1 receptors within the pre-BötC in vitro. A–C, Inspiratory frequency, as monitored by continuous recording of XII motoneuron population activity, is reversibly reduced with bilateral microinfusion into the pre-BötC of MeSG (5 μm) (A), the 5-HT₂ receptor antagonist KET (7.5 μm) (B), or the NK-1 receptor antagonist SR 140333 (7.5 μm) (C). D, Recording of XII motor output (∫XII), showing the effects of bilateral microinfusion of 5-HT (30 μm) into the pre-BötC, which increased inspiratory frequency and decreased burst amplitude. E, F, Inspiratory frequency plotted as function of time, showing reversible augmentation of inspiratory frequency by microinfusion of 5-HT (30 μm) (E) or Sar-Met SP (1 μm) (F), a selective NK-1 receptor agonist, into the pre-BötC.

Figure 7.

Endogenous 5-HT release is critical for inspiratory pattern generation and motor output in situ through multiple 5-HT receptor subtypes. A, Steady-state inspiratory burst frequency versus concentration of 5-HT_2A (MDL 11,939), 5-HT_2C (RS 102221), and 5-HT₄ (GR 113808) receptor antagonists applied via the perfusate in situ. Only the 5-HT_2C antagonist significantly decreased burst frequency (shown normalized to control values, data points are mean values ± SEM) indicating a 5-HT_2C receptor component for modulation of inspiratory rhythm generation. B–D, All three antagonists decreased the amplitudes (normalized to control, mean values ± SEM) of integrated inspiratory bursts in motor nerves (PN in B, XII in C) and pre-BötC (D) population activity in a concentration-dependent manner, indicating involvement of multiple receptor subtypes in regulation of inspiratory activity amplitude. The larger antagonism of PN activity compared with XII output by GR 113808 suggests differential contributions of 5-HT₄ receptors in regulation of spinal and cranial motor outputs. E, Representative recordings of integrated XII, PN, and pre-BötC population activity recorded in the in situ preparation illustrating typical large reductions of inspiratory burst amplitudes with only small perturbation of burst frequency by antagonism of 5-HT_2A receptors with MDL 11,939.

Figure 8.

NK-1 receptor activation in situ contributes to maintenance of inspiratory burst amplitude. A, Blocking endogenous substance P activity through antagonism of NK-1 receptors with SR 140333 did not significantly alter inspiratory burst frequencies in the XII nerve and pre-BötC (data not shown) as indicated by the steady-state responses to SR shown. B, Attenuation of integrated inspiratory burst amplitudes in the pre-BötC and XII nerve by SR, indicating disproportionate reductions in pre-BötC population activity and XII motor output, where the steeper attenuation of XII activity suggests additional NK-1 modulation of XII inspiratory burst generation downstream from the pre-BötC. Data points represent mean values ± SEM.

Figure 9.

Serotonin and substance P in concert are required to generate and maintain the respiratory rhythm and motor output in situ. Antagonism of 5-HT_1,2 receptors with the nonselective receptor antagonist MeSG (20 μm) did not affect inspiratory rhythm generation, as shown by the lack of effect on inspiratory burst frequency (XII/PN), but strongly reduced PN and XII nerve burst amplitudes. Addition of the NK-1 antagonist SR (20 μm) nearly eliminated all inspiratory burst activity, as shown by the significant large decreases in burst frequency and amplitudes. This suggests that endogenous 5-HT and NK-1 receptor activation work in concert to generate and maintain inspiratory activity in situ. Error bars indicate SEM.

Figure 10.

Increased firing of raphé neurons in slices stimulates inspiratory output via activation of 5-HT₂ and NK-1 receptors. A, XII motor output (top, ∫XII) recorded simultaneously with extracellular potential of a raphé obscurus neuron (bottom trace). AMPA (5 μm) microinfused into the n. raphé obscurus progressively increased firing rate of the raphé neuron (6.5-fold) and inspiratory frequency (1.9-fold). There was also an increase in tonic activity in hypoglossal motoneurons, as seen by the shift in baseline of the integrated XII population activity. B, Relationship between inspiratory burst frequency (normalized to baseline) and average firing rate of raphé neurons (n = 12 slices). Data points are mean values ± SEM. C, Top, The excitatory effect of raphé stimulation on burst frequency [control (CTRL)] was partially blocked by bath-applied MeSG (5 μm), and nearly eliminated by coapplication of SR 140333 (MeSG + SR; 7.5 μm). Middle, The excitatory effect was also partially blocked by bath-applied KET (7.5 μm), and further attenuated by coapplying SR 140333 (KET + SR). Bottom, MDL 11,939 (10 μm) also partially blocked the excitation, which was also further attenuated by coapplying 7.5 μm SR (MDL + SR).

Figure 11.

The raphé obscurus in situ drives frequency modulation of the respiratory network through 5-HT and NK-1 receptors. A, Simultaneous recordings of population activity in the raphé obscurus (raw and integrated multiunit activity shown) and motor output nerves (PN, XII) show spontaneous activity of raphé neurons as well as respiratory modulation of raphé activity in situ. The respiratory modulation of raphé neuron activity indicates functional connections between the respiratory network and the raphé system. B, AMPA (5 μm) injection into raphé obscurus causes excitation of raphé population, which then drives an increase of inspiratory burst frequency. C, D, Blocking NK-1 receptors and then NK-1 plus 5-HT receptors progressively reduced the AMPA-raphé-driven excitation of the respiratory network. Plotted in C are the PN inspiratory burst frequencies before and during raphé stimulation, where frequency is normalized to baseline frequency before stimulation. In D, the peak frequencies (normalized to baseline, mean values ± SEM) during stimulation are plotted before and after systemic application of SR and then SR plus MeSG, showing strong attenuation of peak responses by the antagonists. These data indicate functional connectivity between the raphé obscurus and inspiratory rhythm-generating components of the respiratory circuit mediated by 5-HT and NK-1 receptors.

Figure 12.

Serotonin and SP released from n. raphé obscurus depolarize inspiratory XII motoneurons and pre-BötC neurons in slices. A, Whole-cell current-clamp recording of XII motoneuron that depolarized in response to focal microinfusion of aCSF with elevated K⁺ (15 mm) into the n. raphé obscurus to stimulate serotonin neurons. CNQX (20 μm) was present in the bath. This depolarization was partially blocked by bath-applied ketanserin (7.5 μm) and further attenuated by coapplying SR 140333 (7.5 μm). B1, Example of intrinsically bursting pre-BötC neuron that was depolarized by raphé stimulation. This first augmented the intrinsic bursting frequency and then reversibly transformed the firing pattern from bursting to tonic spiking. B2, In the same neuron as B1, bath-applied KET (7.5 μm) decreased the initial burst frequency (left) and attenuated the effect of raphé stimulation (middle). B3, In the same neuron, coapplication of SR 14033 (7.5 μm) further reduced the initial bursting frequency relative to KET alone (left, note the change in time scale relative to B2) and nearly abolished the excitation that occurred during raphé stimulation (middle).

Figure 13.

Serotonin depolarizes pre-BötC neurons in part by activating a nonselective cation current. A, XII inspiratory activity (top, ∫XII) recorded simultaneously with membrane potential of a pre-BötC neuron (bottom). At the network level, bath-applied 5-HT (30 μm) increased inspiratory frequency. There was also increased motoneuron tonic activity as seen by the upward shift in baseline of the integrated XII population activity. At the cellular level, 5-HT depolarized the pre-BötC neuron. The depolarization induced ectopic bursts (examples indicated by *) out of phase with XII motor output, indicative of an intrinsically bursting neuron. B1, Whole-cell I–V relations recorded in voltage clamp from an intrinsically bursting pre-BötC neuron during slow voltage ramps (30 mV/s) with (red trace) and without (black trace) bath-applied 5-HT (30 μm). Rhythmic excitatory synaptic transmission was blocked by CNQX (20 μm). Reversal potential for the total leak current (E_Leak) before 5-HT application is indicated, estimated by extrapolating the linear region of I–V relation after linear regression (dashed line). B2, 5-HT-induced current obtained by subtracting the I–V curves shown in B1. The reversal potential (E_R) in this example was near −35 mV. C1, I–V relations obtained as in B1 under control conditions (gray), in bath-applied TTX (1 μm; black), and in TTX with 5-HT (red). C2, 5-HT induced current in TTX (E_R ≈ −42 mV). D1, I–V relations obtained with bath-applied TTX, TEA, and Cd²⁺ and Cs⁺-based internal solution. D2, The 5-HT-induced current (difference current) had an E_R ≈ −7.5 mV, which was identical to total leak reversal potential obtained before 5-HT application.

Figure 14.

Serotonin converts some nonintrinsically bursting inspiratory pre-BötC neurons into intrinsically bursting cells. A, Current-clamp recording from an inspiratory pre-BötC neuron that burst in phase with inspiratory XII nerve output (data not shown). B, After bath-applied Cd²⁺ to block synaptic transmission, this neuron became silent. In response to applied depolarizing current, the neuron spiked tonically, but did not burst rhythmically. This response is characteristic of pre-BötC nonintrinsically bursting neurons. C, Subsequent bath application of 5-HT (30 μm) transformed the neuron into an intrinsic rhythmic burster. Note the increase in intrinsic bursting frequency at higher applied current, indicating voltage-dependent bursting properties. D, Washout of 5-HT caused transformation back to a nonbursting neuron.