Local Corticotropin-Releasing Factor Signaling in the Hypothalamic Paraventricular Nucleus

Abstract

Corticotropin-releasing factor (CRF) neurons in the hypothalamic paraventricular nucleus (PVN) initiate hypothalamic-pituitary-adrenal axis activity through the release of CRF into the portal system as part of a coordinated neuroendocrine, autonomic, and behavioral response to stress. The recent discovery of neurons expressing CRF receptor type 1 (CRFR1), the primary receptor for CRF, adjacent to CRF neurons within the PVN, suggests that CRF also signals within the hypothalamus to coordinate aspects of the stress response. Here, we characterize the electrophysiological and molecular properties of PVN-CRFR1 neurons and interrogate their monosynaptic connectivity using rabies virus-based tracing and optogenetic circuit mapping in male and female mice. We provide evidence that CRF neurons in the PVN form synapses on neighboring CRFR1 neurons and activate them by releasing CRF. CRFR1 neurons receive the majority of monosynaptic input from within the hypothalamus, mainly from the PVN itself. Locally, CRFR1 neurons make GABAergic synapses on parvocellular and magnocellular cells within the PVN. CRFR1 neurons resident in the PVN also make long-range glutamatergic synapses in autonomic nuclei such as the nucleus of the solitary tract. Selective ablation of PVN-CRFR1 neurons in male mice elevates corticosterone release during a stress response and slows the decrease in circulating corticosterone levels after the cessation of stress. Our experiments provide evidence for a novel intra-PVN neural circuit that is activated by local CRF release and coordinates autonomic and endocrine function during stress responses.SIGNIFICANCE STATEMENT The hypothalamic paraventricular nucleus (PVN) coordinates concomitant changes in autonomic and neuroendocrine function to organize the response to stress. This manuscript maps intra-PVN circuitry that signals via CRF, delineates CRF receptor type 1 neuron synaptic targets both within the PVN and at distal targets, and establishes the role of this microcircuit in regulating hypothalamic-pituitary-adrenal axis activity.

Keywords: CRF; CRFR1; CRH; CRHR1; HPA axis; stress.

Figure 1.

CRFR1-GFP neurons in the PVN receive contacts from CRF neurons and are activated by CRF. A–C, Validation of CRFR1-GFP transgenic mice. A, B, Expression of the CRFR1-GFP transgene (A) is highly correlated with CRFR1 receptor immunoreactivity (B). C, The merged image shows colocalization (white arrowheads). Scale bar, 100 μm. D–F, CRF-positive fibers (D) are closely apposed to CRFR1-GFP neurons (E), which express the LDCV marker CGA (F). G, Merged image shows a CRF⁺/CGA⁺ puncta apposed to a CRFR1-GFP fiber (yellow arrow). Scale bar, 10 μm. H, CRFR1-GFP neurons express functional CRFR1 receptors. Left, Representative traces of CRFR1-GFP neurons before and during CRF application in aCSF (top traces), in fast synaptic blockers (AP5, DNQX, and picrotoxin; middle traces), and in synaptic blockers plus the selective CRFR1 antagonist Antalarmin (bottom traces). Right, Summary histograms of exogenous CRF-induced changes in firing rate in aCSF (top), synaptic blockers (middle), and CRFR1 antagonist (bottom). *p < 0.05.

Figure 2.

PVN-CRFR1-GFP neurons are activated by locally released CRF. A, Example micrograph of the PVN from an animal with the genotype CRFR1-GFP; CRF-cre, injected in the PVN with AAV encoding Cre-dependent ChR2 with a 2A-Ruby reporter. CRFR1 neurons (green) are intercalated with CRF-cre neurons that have been infected with mChR2–2A-mRuby-expressing virus (Red). Scale bar, 100 μm. B, Representative traces of photocurrents recorded in CRF neurons. In CRF neurons infected with ChR2-Ruby virus, photostimulation (1 s pulse) induced typical ChR2-mediated currents (bottom trace), whereas photostimulation with the same intensity and duration failed to induce photocurrent in CRF neurons infected with control virus (middle trace). C, Sample traces of action potential firing in a CRF-ChR2 neuron with 5 and 50 Hz optical stimulation (laser duration, 1–5 ms), firing slows with 50 Hz stimulation. D, Entrainment of CRF-ChR2 neurons to direct optical stimulation: percentage change in spike fidelity and ChR2-mediated CRF neurons firing (n = 8). E, Schematic experimental setup for optogenetic activation of CRF release in the PVN. CRFR1-GFP; CRF-cre animals were injected in the PVN with AAVs encoding Cre-dependent ChR2 with a 2A-Ruby reporter. Recordings were performed on green fluorescent neurons (CRFR1-GFP) while activating CRF neurons with laser light. F, Activation of CRF neurons by optical stimulation causes an increase in action potential firing in a neighboring CRFR1-GFP neuron in the presence of synaptic blockers (top traces, 1 Hz; middle traces, 20 Hz). Note that action potentials are not correlated with laser pulses. The excitatory effect is blocked by Antalarmin preincubation, indicating that the increase in firing is mediated by CRFR1 receptors (bottom traces). Right panels, Quantification of the change in firing rate of CRFR1-GFP neurons with optical activation of CRF neurons in synaptic blockers and with Antalarmin preincubation. *p < 0.05.

Figure 3.

Electrophysiological and molecular properties of CRFR1-GFP neurons in the PVN. A, Representative responses of CRFR1-GFP neurons to a negative current injection step (−100 pA, 500 ms). B, Eighty percent of the CRFR1-GFP neurons fire at least one action potential in response to a −100 pA hyperpolarization current (type A), 11% express a small “hump” (type B), and 9% show no LTS (type C). C, Majority CRFR1-GFP neurons express a small sag in response to a hyperpolarization current. The arrow points out the “sag,” indicative of the presence of hyperpolarization-activated channels. D, A current–frequency plot showing high spike frequency adaptation of CRFR1-GFP in response to a series of depolarization currents. E, A representative image, and summary table of single-cell reverse-transcription PCR results. F, Summary and location information for 50 identified PVN CRFR1-GFP neurons.

Figure 4.

Mapping of monosynaptic inputs to PVN CRFR1 neurons. A, B, The expression pattern of tdTomato reporting Cre expression in CRFR1-GFP; CRFR1-Cre;Ai9 mice (A) is highly correlated with GFP expression from the validated CRFR1-GFP transgene (B). C, The merged image showing neurons expressing both reporters (arrowheads). Scale bar, 100 μm. D, Schematic experimental design for pseudotyped rabies virus assisted monosynaptic retrograde tracing. E, Quantification of monosynaptic inputs to CRFR1 neurons in the PVN. Note that CRFR1 neurons receive ∼90% of their synaptic inputs from within the hypothalamus, especially from the PVN itself. PM, Premammillary nucleus; PO, preoptic area; Pe, periventricular nucleus; LH, lateral hypothalamus. F, Selective expression of TVA-mCherry in CRFR1 neurons within the PVN. G, Retrograde labeled presynaptic neurons within the PVN. H, Merged image showing the presence of starter neurons, which initiate trans-synaptic infection to presynaptic neurons. Arrowheads indicate starter neurons, which express both TVA-mCherry and PTRV-eGFP. I–N, Representative images of presynaptic neurons found in septum (LS and MS; I), BST (J), POA (K), SON (L, bottom right,), DMH/VMH and ARC (M), and PB (N). Scale bar, 100 μm.

Figure 5.

PVN CRFR1 neurons receive intra-PVN inputs. A–C, In PTRV-assisted mapping of CRFR1 neuron synaptic inputs, many of the presynaptic neurons (green, left) are positive for CRF (middle panels). The merged image shows that many CRF neurons make monosynaptic connections with CRFR1 neurons. Arrowheads point to the neurons that express eGFP and are immunoreactive for CRF. Insets, Higher-power micrographs of CRF-positive PTRV traced neurons. D, Quantification of CRF⁺ input neurons. E–G, We also identified many presynaptic neurons (green, left) as positive for AVP (middle). The merged image shows the abundance of AVP⁺ magnocellular neurons that are presynaptic to CRFR1 PVN neurons. H, Quantification of AVP⁺ input neurons. I–K, Presynaptic OT⁺ neurons were also labeled by monosynaptic tracing, but were less frequent. L, Quantification of OT⁺ input neurons. Scale bars: 100 μm for C, G, K; 5 μm for inserts.

Figure 6.

PVN-CRFR1 neurons make local projections within the nucleus. A, Selective expression of ChR2-YFP in CRFR1-Cre neurons in the PVN. B, Representative traces of photocurrents recorded in CRFR1 neurons. In CRFR1 neurons expressing ChR2, 1 s photostimulation induced typical ChR2-mediated currents (bottom trace), whereas photostimulation with the same intensity and duration failed to induce photocurrents in the CRFR1 neurons infected with control virus (middle trace). C, Sample traces of action potential firing from a CRFR1-ChR2 neurons with 5 and 20 Hz optical stimulation (laser duration 1–5 ms), note the slowing of firing with 20 Hz stimulation. D, Entrainment of CRFR1-ChR2 neurons to direct optical stimulation: percentage change in spike fidelity and ChR2-mediated CRFR1 neurons firing (n = 10). E, Schematic experimental design. Laser-evoked postsynaptic responses were recorded in neighboring PVN neurons. (F) Representative traces from a neighboring neuron in the PVN, showing laser-evoked IPSCs, which are blocked by Bic and are restored after washout. Blue, Photostimulation; gray lines, individual traces; dark lines, averaged traces. G, Delay of laser-evoked eIPSCs from PVN neurons (n = 15). H, Summary histogram of eIPSCs in the PVN, which is blocked by selective GABA_A antagonist bicuculline (40 μm; n = 6; p < 0.05, paired Student's t test). I, Representative trace of a laser-evoked IPSC, which is blocked by TTX and rescued by 4-AP. Blue, Photostimulation; gray lines, individual traces; dark lines, averaged traces. J, Summary histogram of eIPSCs blocked by TTX and rescued by 4-AP (n = 4). *p < 0.05.

Figure 7.

Mapping of PVN-CRFR1 neuron projections. A, Schematic of experimental design for synaptophysin-assisted mapping of projections of PVN-CRFR1 neurons. B, Selective expression of synaptophysin-eYFP in CRFR1-cre neurons the PVN. C–H, Representative images of synaptophysin-eYFP fluorescence in the LS (C), BST (D), PAG (E), lateral parabrachial nucleus (LPB) and LC (F), VLM (G), and NTS (H). Scale bar, 100 μm.

Figure 8.

PVN-CRFR1 neurons make long-range projections to NTS. A, Schematic of the experimental design. B, A micrograph of the NTS from a CRFR1-Cre mouse injected with ChR2 into the PVN. Green, CRFR1-ChR2-YFP; blue, anti-tyrosine hydroxylase (TH) staining. C, Representative traces of laser-evoked EPSCs in a NTS neuron, which are blocked by AP5/DNQX and restored after washout. Gray lines, Individual traces; dark lines, averaged traces. D, Summary histogram of laser-evoked EPSCs in the NTS, which are blocked by AP5/DNQX (n = 6, p < 0.05, paired t test). E, Plot of the delay before an eEPSC from light onset in NTS neurons (n = 7). **p < 0.01.

Figure 9.

Selective ablation of PVN-CRFR1 neurons cause HPA axis hyperactivity. A, Selective expression of AAV-FLEX-DTR-YFP in the PVN. B, DT injection ablates CRFR1 neurons in the PVN (gray, nuclear counterstaining with ToPro). C, Ablation of CRFR1 neurons in adult mice increases peak corticosterone release during an immobilization stress and delays the fall in circulating corticosterone levels after release from immobilization (n = 9, and n = 12, for CRFR1-control and CRFR1-DTR, respectively; p < 0.01, two-way ANOVA). Controls are CRFR1-Cre littermates injected with control virus (AAV-DIO-eYFP), then injected with DT after 3 weeks of recovery. *p < 0.05; **p < 0.01.

Figure 10.

Proposed model for intra-PVN CRF signaling. CRFR1 neurons (green) receive monosynaptic inputs from both CRF neurons (red) and AVP neurons (blue) from within the PVN, together with other hypothalamic inputs, to coordinate PVN activity during stress. CRFR1 neurons make inhibitory synapses onto CRF neurons, functioning as a synaptic negative feedback on HPA axis activity. CRFR1 neurons also synapse on other resident PVN neurons to coordinate activity of CRF neurons with other endocrine axes. CRFR1 neurons also send excitatory long-range projections to potentiate autonomic tone during stress.