Target-specific modulation of the descending prefrontal cortex inputs to the dorsal raphe nucleus by cannabinoids

Abstract

Serotonin (5-HT) neurons located in the raphe nuclei modulate a wide range of behaviors by means of an expansive innervation pattern. In turn, the raphe receives a vast array of synaptic inputs, and a remaining challenge lies in understanding how these individual inputs are organized, processed, and modulated in this nucleus to contribute ultimately to the core coding features of 5-HT neurons. The details of the long-range, top-down control exerted by the medial prefrontal cortex (mPFC) in the dorsal raphe nucleus (DRN) are of particular interest, in part, because of its purported role in stress processing and mood regulation. Here, we found that the mPFC provides a direct monosynaptic, glutamatergic drive to both DRN 5-HT and GABA neurons and that this architecture was conducive to a robust feed-forward inhibition. Remarkably, activation of cannabinoid (CB) receptors differentially modulated the mPFC inputs onto these cell types in the DRN, in effect regulating the synaptic excitatory/inhibitory balance governing the excitability of 5-HT neurons. Thus, the CB system dynamically reconfigures the processing features of the DRN, a mood-related circuit believed to provide a concerted and distributed regulation of the excitability of large ensembles of brain networks.

Keywords: anxiety; depression; glutamate receptors; optogenetics; synapse.

Fig. S1.

mPFC sends direct monosynaptic inputs to the DRN. (A, 1) Schematic of Fluoro-Gold injection in the DRN and subsequent retrograde transport of Fluoro-Gold through the mPFC axon terminal. (A, 2) Confocal image of a coronal brain slice containing the mPFC. (Inset) Two-photon image of the Fluoro-Gold–expressing cells bodies in the mPFC. Note the dense labeling with Fluoro-Gold of deep-layer cells in the mPFC. (A, 3) Confocal image of Fluoro-Gold in the DRN 1 wk postinjection. Aq, aqueduct.

Fig. S2.

mPFC axon terminals in the DRN. (A) Immunostaining for TPH2 (Left) and ChR2(H134R)-mCherry (Middle). (Right) Merged image is shown. (B) Higher magnification of TPH2-expressing 5-HT neuron in the DRN (Left) and ChR2(H134R)-expressing terminals (Middle). (Right) Merged image is shown. (C, 1) Two-photon image of Alexa-594–filled DRN 5-HT neuron (red) and GFP-expressing mPFC axon terminals (green). (C, 2) Whole-cell voltage-clamp recording from the Alexa-594–filled 5-HT cell in C1 and response to bath application of the 5HT_1A receptor agonist 5-CT.

Fig. S3.

ChR2(H134R)-expressing layer V pyramidal neurons respond directly to optical stimulation. (A, 1) Schematic of AAV-ChR2(H134R) injection in the mPFC. (A, 2) DIC image of mPFC-containing coronal brain slice. (A, 3) Immunostaining for NeuN and ChR2(H134R)-mCherry in the same brain slice as in A, 2. (B, 1) Schematic of whole-cell recording from a ChR2(H134R)-expressing layer V pyramidal neuron. (B, 2) DIC image of whole-cell recording from a ChR2(H134R)-expressing layer V pyramidal neuron. (B, 3) Current-clamp recording from a ChR2(H134R)-expressing layer V pyramidal neuron. (C, 1) Schematic of whole-cell recording from a ChR2(H134R)-expressing layer V pyramidal neuron and optical stimulation. (C, 2) Input/output curve of ChR2(H134R)-mediated current in response to increasing intensity of optical stimulation. (C, 3) Responses of the same ChR2(H134R)-expressing layer V pyramidal neuron to 500 ms of optical stimulation. (Above) In a current-clamp recording, the pyramidal neuron responded with action potential firing. (Below) In a voltage-clamp recording with TTX (1 μM), the ChR2(H134R)-mediated current initially peaked and was desensitized within 100 ms. Im, membrane current; Vm, membrane voltage.

Fig. 1.

mPFC-DRN inputs onto 5-HT neurons are monosynaptic, glutamatergic, and sufficient to drive action potential firing. (A) Schematics of adeno-associated virus (AAV)-ChR2 injection (Left) and DRN neuron recording from a midbrain slice (Right). ChR2-PSCs are blocked by NBQX (10 μM) or D-APV (50 μM) (B, 1: n = 5; B, 2: n = 5). Presynaptic release is inhibited by cadmium (Cd²⁺; 200 μM) or TTX (1 μM) (B, 3: n = 3; B, 4: n = 10). (B, 5) Inhibition of presynaptic release with TTX was partially rescued by 4-AP (1 mM; n = 6). Traces of a ChR2-EPSC (magenta arrow) and spontaneous EPSCs (sEPSCs) (orange arrows) in the presence of 2.5 mM extracellular Ca²⁺ (C, 1) or 4 mM strontium chloride (SrCl₂) (D, 1) are shown. (C, 2, Top and D, 2, Top) Scatter plot of ChR2-EPSCs (magenta) followed by sEPSCs (orange). (C, 2, Bottom and D, 2, Bottom) Peristimulus time histogram of ChR2-EPSCs (magenta) and sEPSCs (orange). (D, 3) Histogram of asynchronous EPSC (asEPSC) amplitudes at mPFC-DRN synapses onto 5-HT neurons (n = 9). (E) Recordings of ChR2-EPSCs [Left, membrane voltage (Vm) = −70 mV] and action-potential firing in current-clamp recordings (Right, Vm ∼ −70 mV). Cumulative distribution plot depicts the latency distribution of mPFC, i.e., mPFC-driven action potentials in DRN 5-HT neurons (49.4 ± 14.5 ms, n = 11). Amp., amplitude; Cum. Prob., cumulative probability; Freq., frequency.

Fig. S4.

Electrophysiological and immunohistochemical identification of 5-HT neurons in the DRN. (A) Schematic of whole-cell recording from a DRN 5-HT neuron. (B, 1) Whole-cell voltage-clamp recording from a 5-HT neuron and change in holding current in response to bath application of the 5HT_1A receptor agonist 5-CT (100 nM). (B, 2) Whole-cell current-clamp recording from a DRN 5-HT neuron. The neuron was brought to spike consistently with the current injection. A 5-CT bath application induced a characteristic 5HT_1A-mediated hyperpolarization and inhibition of spiking. (C) Characteristic firing pattern of DRN 5-HT neurons. (D, 1) Post hoc immunolabeling for TPH2 in a 5-HT neuron filled with biocytin during whole-cell recordings. (D, 2) Two-photon image of Alexa-594–filled 5-HT neuron in the DRN.

Fig. S5.

Pharmacological isolation of direct monosynaptic optically activated EPSCs results in a rightward shift in the time of EPSC onset and peak. (A) Representative averaged EPSC traces before and after TTX/4-AP application. (Inset, Top) Rightward shift in the time of EPSC onset. (Inset, Bottom) Rightward shift in the time of EPSC peak. (B, 1) Averaged EPSC onset before and after TTX/4-AP application (baseline, 5.24 ± 0.3; TTX/4-AP, 9.28 ± 1.0; n = 5; P = 0.004). (B, 2) Averaged time to EPSC peak before and after TTX/4-AP application (baseline, 12.1 ± 1.1; TTX/4-AP, 19.9 ± 1.8; n = 5; P = 0.006). (B, 3) Averaged EPSC tau before and after TTX/4-AP application (baseline, 15.5 ± 3.1; TTX/4-AP, 18.2 ± 3.2; n = 5; P = 0.55).

Fig. 2.

mPFC-DRN inputs to 5-HT neurons provide disynaptic feed-forward inhibitory connections. (A, 1, Left) Traces of ChR2-PSCs. (A, 1, Right) ChR2-PSC traces before and after bicuculline (BIC) (20 μM; Vm = −10 mV). (A, 2) Scatter plot of PSCs in response to BIC. (A, 3) Average population response of inward ChR2-EPSCs (blue) and outward IPSCs (red) to BIC. (B, 1 and 4) Individual traces of ChR2-EPSCs (blue, inward) and ChR2-IPSCs (red, outward) (Vm = −15 mV). (B, 2) Scatter plot of PSC latencies (dotted line, mean latency; gray, SD). (B, 3) PSC latencies plotted as 1/CV² (n = 6; P = 0.004). (B, 5) Scatter plot of PSC amplitudes. (B, 6) PSC amplitudes plotted as 1/CV² (n = 6; P = 0.046). (C, 1 and 2) Traces and scatter plot of ChR2-PSCs in response to NBQX and D-APV. (C, 3) Normalized population data of PSC amplitude during baseline and following NBQX and APV (n = 3). (D, 1 and 2) Traces and scatter plot of PSCs in response to bath application of TTX, followed by 4-AP. (D, 3) Normalized population data of PSC amplitudes during baseline, TTX, and TTX and 4-AP (n = 6). Gray circles indicate individual cell averages. CTL, control; norm., normalized.

Fig. S6.

Electrophysiological and immunohistochemical identification of local GABAergic interneurons located in the lateral wings of the DRN. (A1) Schematic of whole-cell recording from a DRN GABAergic interneuron. (A2) Immunostaining for GAD67 and ChR2(H134R)-mCherry. (Right) Higher magnification image of a DRN GABAergic neuron surrounded by ChR2-expressing mPFC axons and terminals. (B) Whole-cell voltage-clamp recording from a DRN GABA neuron with no significant change in holding current in response to bath application 5-CT (Vm = −55 mV). (C) Characteristic firing properties of a DRN GABAergic interneuron in the DRN. (D) Post hoc immunolabeling for TPH2 in a GABAergic interneuron filled with biocytin during a whole-cell recording. TPH2 was not detectable in the GABAergic interneuron. (E) Two-photon image of Alexa-594–filled GABAergic interneuron in the DRN. Note the extent of the dendritic arborization of the DRN GABAergic interneuron.

Fig. 3.

mPFC-DRN inputs to GABA neurons provide monosynaptic excitatory and disynaptic feed-forward inhibitory connections. (A, 1, Left) Schematic of a recording from a DRN GABAergic neuron. (A, 1, Right) Differential interference contrast (DIC) image of the DRN. (Inset) Recording from a GABA neuron. (A, 2) ChR2-PSCs are abolished by NBQX (n = 4). (B, 1 and 2) Averaged traces and scatter plot of ChR2-EPSCs in response to bath application of TTX, followed by 4-AP. (B, 3) Normalized population data of ChR2-EPSC amplitudes during baseline, TTX, and TTX and 4-AP (n = 7). (C, 1, Left) Traces of ChR2-PSCs onto a GABA neuron. (C, 1, Right) ChR2-PSC traces before and after BIC (Vm = −10 mV). (C, 2) Scatter plot of PSCs in response to BIC. (C, 3) Average population response of inward ChR2-EPSCs (blue) and outward IPSCs (red) to BIC. (D, 1 and 4) Individual traces of mixed ChR2-PSCs onto a GABA neuron (Vm = −25 mV). (D, 2) Scatter plot of PSC latencies (dotted line, mean latency; gray, SD). (D, 3) CV of PSC latencies plotted as 1/CV² (n = 3; P = 0.0001). (D, 5) Scatter plot of PSC amplitudes. (D, 6) CV of PSC amplitudes plotted as 1/CV² (n = 3; P = 0.001). Gray circles indicate individual cell averages.

Fig. 4.

mPFC-DRN synapses onto 5-HT and GABA neurons exhibit target-specific modulation by CBs. (A, 1, Left) Traces of ChR2-EPSCs onto 5-HT (green) and GABA (orange) neurons before and after (gray) bath application of 5-HT (30 μM). (A, 1, Right) 5-HT decreases ChR2-EPSC amplitude onto 5-HT and GABA neurons (solid circles indicate individual recordings). (A, 2) Average sensitivity of ChR2-EPSCs to 5-HT (5-HT cells, n = 5; GABA cells, n = 5; P = 0.4). (B, 1, Left) Traces of ChR2-EPSCs onto 5-HT (green) and GABA (orange) neurons before and after (gray) bath application of WIN 55, 212-2 (WIN; 10 μM). (B, 1, Right) WIN decreases ChR2-EPSC amplitude onto 5-HT and GABA neurons. (B, 2) Average sensitivity of ChR2-EPSCs to WIN (5-HT cells, n = 5; GABA cells, n = 5; P = 0.01). (C, 1) Traces and plot depicting the effect of WIN on the mixed inward and outward ChR2-PSCs onto a 5-HT neuron. (C, 2) Summary plot of WIN sensitivity on direct EPSCs and indirect IPSCs onto DRN 5-HT neurons (n = 4; P = 0.01). Gray circles indicate individual cell averages.

Fig. S7.

ChR2(H134R)-mediated presynaptic release of glutamate onto a 5-HT neuron is reduced by decreasing the extracellular concentration of Ca²⁺ and activating presynaptic GABA_B receptors. (A) Representative traces and amplitude plot of optically activated synaptic EPSC and the response to decrease the extracellular concentration of Ca²⁺ from 2.5 to 0.5 mM. (B) Representative traces and amplitude plot of optically activated synaptic EPSC and the response to bath application of the GABA_B-selective agonist baclofen.

Fig. S8.

DSE can be induced and expressed by optical stimulation of mPFC inputs onto DRN 5-HT and GABA neurons. (A, 1, Above) Representative traces of stimulating electrode-evoked EPSCs in response to a 5-s depolarization DSE protocol. (A, 1, Below) Bath application of AM-251 for at least 25 min blocked the DSE effect onto 5-HT neurons. (A, 2) Summary plot of DSE effect of electrically evoked AMPAR-mediated EPSCs (eEPSCs) from a 5-HT neuron. DSE experiments in the presence of AM-251 are represented in gray (n = 3), and experiments in regular conditions are represented in green (n = 6). (B, 1, Above) Representative traces of stimulating electrode-evoked EPSCs in response to a 5-s depolarization DSE protocol. (B, 1, Below) Bath application of AM-251 for at least 25 min blocked the DSE effect onto 5-HT neurons. (B, 2) Summary plot of DSE effect of eEPSCs from GABA neurons. DSE experiments in the presence of AM-251 are represented in gray, and experiments in regular conditions are represented in orange (gray, n = 16; orange, n = 10). (C, 1) Representative traces of optically stimulated EPSCs in response to a 5-s depolarization DSE protocol recorded from a 5-HT neuron. (C, 2) Representative plot of DSE of optically stimulated EPSCs from a 5-HT neuron. (D, 1) Representative traces of optically stimulated EPSCs in response to a 5-s depolarization DSE protocol recorded from a GABA neuron. (D, 2) Representative plot of the DSE of optically stimulated EPSCs from a GABA neuron. norm., normalized.

Fig. S9.

Model of mPFC projections to the DRN and the shift toward excitatory drive onto 5-HT neurons in the presence of elevated concentrations of extracellular CBs. High levels of CBs in the DRN result in the activation of presynaptic CB₁Rs, including, but not exclusively, those CBs on mPFC terminals in the DRN. The sensitivity of mPFC terminals to CBs is target-specific, thereby having a more robust effect at mPFC synapses onto DRN GABAergic neurons. Thus, in a switch-like manner, activation of CB₁R in the DRN powerfully gates information flow from the PFC in the DRN by favoring the direct excitatory drive of 5-HT neurons at the expense of the local feed-forward inhibitory mechanisms.