VIP, CRF, and PACAP act at distinct receptors to elicit different cAMP/PKA dynamics in the neocortex

Abstract

The functional significance of diverse neuropeptide coexpression and convergence onto common second messenger pathways remains unclear. To address this question, we characterized responses to corticotropin-releasing factor (CRF), pituitary adenylate cyclase-activating peptide (PACAP), and vasoactive intestinal peptide (VIP) in rat neocortical slices using optical recordings of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) sensors, patch-clamp, and single-cell reverse transcription-polymerase chain reaction. Responses of pyramidal neurons to the 3 neuropeptides markedly differed in time-course and amplitude. Effects of these neuropeptides on the PKA-sensitive slow afterhyperpolarization current were consistent with those observed with cAMP/PKA sensors. CRF-1 receptors, primarily expressed in pyramidal cells, reportedly mediate the neocortical effects of CRF. PACAP and VIP activated distinct PAC1 and VPAC1 receptors, respectively. Indeed, a selective VPAC1 antagonist prevented VIP responses but had a minor effect on PACAP responses, which were mimicked by a specific PAC1 agonist. While PAC1 and VPAC1 were coexpressed in pyramidal cells, PAC1 expression was also frequently detected in interneurons, suggesting that PACAP has widespread effects on the neuronal network. Our results suggest that VIP and CRF, originating from interneurons, and PACAP, expressed mainly by pyramidal cells, finely tune the excitability and gene expression in the neocortical network via distinct cAMP/PKA-mediated effects.

Figure 1.

VIP and CRF elicit different cAMP/PKA responses in pyramidal neurons. (A) PKA activation in pyramidal cells expressing the AKAR2 probe to VIP (250 nM), CRF (250 nM), and forskolin (FSK, 13 μM). Traces show average F535/F480 emission ratios measured at the soma of individual pyramidal neurons delineated on pseudocolor image a. Arrows indicate time points corresponding to pseudocolor images below. Pseudocolor images show the ratio value (coded by the pixel hue) and the intensity of the fluorescence (coded by the pixel intensity), on the scale indicated by the calibration square (20 μm wide). Grayscale image shows the 535 nm fluorescence emission. Inset: VIP and CRF dose–response histograms. Data are expressed in percent of the response to FSK. Numbers of recorded neurons are indicated in each bar (statistical significance: ***P < 0.0001; *P < 0.05). (B) cAMP elevation in pyramidal cells expressing Epac1-camps in response to VIP (250 nM), CRF (250 nM), and FSK (13 μM) in the presence of IBMX (200 μM). Traces show average F480/F535 emission ratios measured at the soma of individual neurons shown on the grayscale image. Arrows indicate time points corresponding to pseudocolor images. Note that cAMP/PKA responses to CRF declined during peptide application.

Figure 2.

Expression of VIP receptors in identified neocortical neurons. (A) Electrophysiological and scPCR analysis of a neuron visually selected under infrared video microscopy. This neuron exhibited a piriform soma with a prominent apical dendrite (upper left) and a reduction of action potential amplitude and frequency along the discharge (upper right) typical of pyramidal neurons. Expression of vGlut1, PAC1, VPAC1, CB, CCK, and SOM mRNAs was detected in this neuron upon agarose gel analysis of scPCR products (lower panel) run in parallel with 100 bp DNA ladder as a molecular weight marker. (B) This vertical fusiform interneuron fired short-duration action potentials with large afterhyperpolarizing potentials at a sustained high frequency, typical of fast-spiking interneurons. This neuron expressed GAD65, GAD67, PAC1, PV, CCK, and NPY mRNAs. (C) Expression of VIP receptors in pyramidal neurons (vGluT+/GAD−) and interneurons (GAD+).

Figure 3.

Pharmacology of VIP and CRF responses. (A) Upper: responses of individual pyramidal cells expressing AKAR2 to CRF (250 nM) were prevented by the CRF receptor antagonist Astressin (500 nM). Lower: Responses to VIP (10 nM) were prevented by the VPAC1 antagonist PG97-269 (200 nM). Application of forskolin (13 μM) provides control for responsiveness of AKAR2 and allowed for the quantification of peptidergic responses. (B) Responses to CRF (250 nM) were abolished by the CRF receptor antagonist Astressin (500 nM). Responses to VIP (10 nM) were largely reduced by the VPAC1 antagonist PG97-269 (200 nM). Antagonists were applied at least 5 min prior to agonist application. Numbers of tested cells and statistical significance are indicated (***P < 0.0001).

Figure 4.

PACAP elicits persistent cAMP/PKA responses in pyramidal neurons via PAC1 receptors. (A–C) Responses of pyramidal cells expressing AKAR2 (PKA) or EPAC1-camps (cAMP) probes to PACAP (250 nM), to the PAC1 agonist Maxadilan (250 nM), and to FSK (13 μM). Variations of cAMP levels were recorded in the presence IBMX (200 μM). Recorded neurons are indicated by arrowheads on grayscale images showing the 535 nm fluorescence emission (right panels). Note that PACAP and Maxadilan had similar effects and that responses to both agonists persisted after peptide washout. (D) Traces show mean (solid lines) ± standard error of the mean (color shades) onset of AKAR2 responses to 250 nM VIP, CRF, and PACAP. For each experiment, traces recorded for individual neurons were normalized to the maximal response and then averaged. The zero time point was defined as half-maximal activation, and the averaged traces obtained from individual experiments were averaged between different experiments. Note the slow onset of PACAP responses.

Figure 5.

Neuropeptide-specific inhibition of the IsAHP tonic I_K in pyramidal cells. (A) Effects of neuropeptides (250 nM) on the phasic IsAHP activated by a train of action potentials (left panels) and on the holding current (tonic I_K, plotted in right panels) were recorded in voltage-clamp mode at a holding potential of −67 mV. Neuropeptides concomitantly reduced the amplitude of IsAHP and tonic I_K as compared with control conditions (ACSF). Subsequent forskolin application (FSK, 13 μM) further reduced the amplitude of both IsAHP and tonic I_K. VIP elicited a smaller reduction of the slow AHP amplitude than CRF and PACAP. Conversely, the onset of PACAP effects was slower than those of VIP and CRF effects. (B) Traces show mean (solid lines) ± standard error of the mean (color shades) onset of tonic I_K inhibition in response to 250 nM of VIP, CRF, or PACAP. Individual traces were normalized to the maximal tonic I_K. Zero time point was defined as the point where the inhibition was 50%. Note the slow onset of the PACAP effect.