PMC

GnRH neurons are immunoreactive for GIRK2 (K_ir3.2). A, Single confocal plans showing GnRH neurons (red), colabeled with GIRK2 (green; scale bar, 10 μm) in 11-d-old explants (N = 3). Representative pictures showing all GnRH neurons examined were colabeled with the GIRK2 antibody. B, Single confocal plans showing GnRH neurons (red), with background (green) when GIRK2 antibody was omitted and used to set the camera at the level of nonspecific staining; the experiment was run simultaneously with the one shown in the second row in A.

A subset of ARC POMC neurons is colabeled with OFQ. A1–A3, Representative photographs showing ARC neurons immunoreactivity for POMC (red) and OFQ (green) in an adult male mouse. The box in A1 is enlarged in A2 and A3 to show a lateral subset of POMC neurons colabeled with OFQ (low magnification, A1; high magnification, A2, A3). Scale bars, 50 μm. The arrows in A2 and A3 point to the neuron indicated in the high-magnification images in B. White arrowheads in A2 show OFQ neurons colabeled with POMC in A3. Empty arrowheads in A3 show POMC neurons that are not colabeled with OFQ in A2. B, Representative photographs showing examples of ARC POMC neurons coexpressing OFQ. Scale bar, 10 μm.

POA GnRH neurons are apposed by OFQ-immunoreactive fibers. A, Representative stack of confocal images showing GnRH neurons labeled with GFP- (green) and OFQ- (red) immunoreactive fibers in the POA of an adult male mouse. Scale bar, 20 µm. Enlarged image from boxed area in B. Scale bar, 5 µm. C, Single confocal plan from the boxed area showing an OFQ-immunoreactive fiber apposed to a GnRH fiber. D, Representative stacks of confocal images showing GnRH neuron cell bodies contacted by an OFQ-immunoreactive fiber (arrows, merged yellow). Scale bar, 5 µm. The specificity of the OFQ antibody is shown in Figure 8-1.

OFQ-evoked inhibition of GnRH neuronal activity is mediated by the receptor ORL1. A, Left, Calcium imaging recordings showing OFQ-evoked largely reduced inhibition in the presence of the ORL1 antagonist UFP-101. Right, Summary data showing the average frequency of calcium oscillations in all GnRH neurons tested during the control (SFM), pretreatment (UFP-101), treatment (+OFQ), and the washout (SFM WO) periods (n = 113, N = 3). au, Arbitrary unit. *Statistical significance between two consecutive periods. B, Left, The ORL1 antagonist SB also reduced the OFQ-evoked inhibition. Right, Summary data showing the average frequency of calcium oscillations in all GnRH neurons tested during the control (SFM), pretreatment (SB 162111), treatment (+OFQ), and washout (SFM WO) periods (n = 244, N = 6).

GnRH neurons express the N/OFQ receptor ORL1. A, High-magnification image of GnRH neurons (arrows) recorded with calcium imaging. Initially identified by their bipolar morphology (left), GnRH neurons loaded with the calcium-sensitive dye Calcium Green-1 AM (middle) were imaged. The phenotype of cells was confirmed post hoc by immunocytochemistry (right). Scale bar, 50 µm. B, Embryonic GnRH neurons express Orl1. Transcripts for the receptor ORL1 were found in single GnRH neurons. Adult brain was used as positive control and water and reverse transcriptase were used as negative controls, respectively. The volume of template and number of cycles are as indicated.

The OFQ-evoked inhibition activates GIRK channels, insensitive to tertiapin-Q but inhibited by protein kinase C phosphorylation. A, Left, Calcium imaging recordings showing that a specific blocker of G-protein inwardly rectifying potassium channels, TPNQ, was ineffective on the OFQ-evoked inhibition. Right, Summary data showing the average frequency of calcium oscillations in all GnRH neurons tested during control (SFM), pretreatment (TPNQ), treatment (+OFQ), and washout (SFM WO) periods (n = 80, N = 3). au, arbitrary unit. *Statistical significance between two consecutive periods. B, Left, TPNQ was also ineffective on GABA_B-evoked inhibition with BAC. Right, Summary data showing the average frequency of calcium oscillations in all GnRH neurons tested during control (SFM), pretreatment (TPNQ), treatment (+BAC), and washout (SFM WO) periods (n = 20, N = 2). C, Left, The protein kinase C activator PMA prevented OFQ-evoked inhibition. Right, Summary data showing the average frequency of calcium oscillations in all GnRH neurons tested during control (SFM), pretreatment (PMA), treatment (+OFQ), and washout (SFM WO) periods (n = 118, N = 4). D, Left, PMA was also profoundly blunted the GABA_B-evoked inhibition. Right, Summary data showing the average frequency of calcium oscillations in all GnRH neurons tested during control (SFM), pretreatment (PMA), treatment (+BAC), and washout (SFM WO) periods (n = 92, N = 3).

POMC-immunoreactive fibers contact GnRH neurons and POMC fibers in the POA coexpress OFQ. A–C, Representative stack of confocal images showing GnRH neurons labeled with GFP- (green) and POMC- (red) immunoreactive fibers in an adult male mouse. Scale bar, 20 μm. Enlarged image from boxed area in B. Scale bar, 5 μm. C, Single confocal plan from boxed area showing a POMC-immunoreactive fiber apposed to this GnRH cell body in the POA. D, Representative confocal images showing fibers immunoreactive for OFQ and POMC in the POA. Scale bars, 10 μm. Note individual varicosities in the same fiber which are immunolabeled for only OFQ (single-labeled; open arrowheads, third panel) or for both OFQ and POMC (dual-labeled; filled arrowheads), suggesting that spatial localization of these peptides might occur in processes. E, F, Representative confocal image (Z projection, ∼10 μm) showing immunoreactivity for OFQ (green) and POMC (red) in fibers surrounding GnRH (blue) neurons in the POA. Scale bar, 50 μm. F1–F3, Boxed areas in E enlarged show contacts (arrowheads) between GnRH and POMC fibers (F1), between GnRH and OFQ fibers (F2), and near cell bodies (F3). Scale bars, 10 μm.

OFQ inhibits the GnRH neuron firing rate in acute brain slices, independent of GABAergic and glutamatergic inputs. A, Left, Electrophysiological recording of adult GFP-tagged GnRH neurons showing that OFQ (1 µm, 100 nm, and 10 nm) evoked a potent decrease in GnRH neuron firing rate. Right, Summary data showing quantification of firing rate (in Hz) in individual GnRH neurons tested at the different concentrations during the control (CTRL), treatment (OFQ), and washout (WO) periods. The values represent the average of 1 s bins for the last 1 min of each period identified on the traces. B, Left, OFQ (10 nm) in the presence of AABs (BIC, 20 µm; CNQX, 10 µm; d-AP5, 20 µm) still decreased GnRH neuron firing rate. Right, Summary data showing quantification of the firing rate (in Hz) in individual GnRH neurons tested during the control (CTRL), pretreatment (AAB), treatment (+OFQ), and washout (WO) periods. The values represent the average of 1 s bins for the last 1 min of each period identified on the traces.

Blockers are effective in GnRH neurons identified as OFQ responsive. A–D, Left, Calcium imaging recordings of explants showing that OFQ (100 pm) evoked a potent decrease in the frequency of calcium oscillations in GnRH neurons (first application, A–D). The inhibition is repeatable (second application, A) and blocked by UFP-101 (ORL1 antagonist), cesium (broad-spectrum blocker of K_ir channels), and PMA (PKC activator). A–D, Right, Heatmaps of changes in the levels of intracellular calcium in cells recorded simultaneously during different paradigms (left). Each row represents changes in a single cell. White lines indicate the time of drug application. Note the large number of GnRH cells inhibited by OFQ (first application, A–D), the repeatability of the inhibition (second application, A), and the effectiveness of UFP-101 (ORL1 antagonist), cesium (broad-spectrum blocker of K_ir channels), and PMA (PKC activator; B, C, and D, respectively, second application) to prevent OFQ inhibition. Note the homogeneity of the response throughout the cells within an explant.

The receptor ORL1 is G_i/o coupled and activates GIRK channels to inhibit GnRH neuronal activity. A, Left, Calcium imaging recordings, after a 4 h pretreatment with PTX (250 ng/ml), showing OFQ (100 pm) evoked only a mild inhibition of GnRH neuronal activity. Right, Summary data showing the average frequency of calcium oscillations in all GnRH neurons pretreated with PTX and tested during the control (PTX-SFM), treatment (+OFQ), and washout (SFM WO) periods (n = 89, N = 3). au, Arbitrary unit. *Statistical significance between two consecutive periods. B, Left, The OFQ-evoked inhibition is insensitive of IBMX and FSK. Right, Summary data showing the average frequency of calcium oscillations in all GnRH neurons tested during control (SFM), pretreatment (IBMX + FSK), treatment (+OFQ), and washout (SFM WO) periods (n = 161, N = 3). Note that the IBMX + FSK treatment is not showing a significant increase due to the calcium plateau evoked in some cells, which results in an underestimate of peak detection. C, D, Left, The broad-spectrum blocker of K_ir channels, Cs or Ba, reduced the OFQ-evoked inhibition. Right, Summary data showing the average frequency of calcium oscillations in all GnRH neurons pretreated with Cs or Ba and tested during the pretreatment (Cs or Ba) and treatment (+OFQ) periods (Cs: n = 169, N = 5; Ba: n = 36, N = 4).

OFQ inhibits GnRH neuron calcium oscillation, independent of GABAergic and glutamatergic inputs. A, Heatmaps of changes in levels of intracellular calcium in cells recorded simultaneously during the paradigm in B, independently repeated in five different explants. Each row represents changes in a single cell. White lines indicate the time of drug application. Note the consistency of the response to OFQ within each explant and across explants (). B, Left, Calcium-imaging recordings of explants showing OFQ (100 pm) evoked a potent decrease in the frequency of calcium oscillations in GnRH neurons (kymograph from the cell shown on the top trace). Right, Empty circles, Summary data showing the average frequency of calcium oscillations for each explant. To minimize the impact of the cell-to-cell heterogeneity, the values from all individual cells originating from the five explants in A were combined. Bars, Summary data showing the average frequency of calcium oscillations in all GnRH neurons tested during the control (SFM), treatment (OFQ), and the washout (SFM WO) periods (n = 144, N = 5). au, Arbitrary unit. *Statistical significance between two consecutive periods. C, Left, The OFQ-evoked inhibition persisted in the presence of AABs (BIC, 20 µm; CNQX, 10 µm; d-AP5, 20 µm). Right, Summary data showing the average frequency of calcium oscillations in all GnRH neurons tested during the control (SFM), pretreatment (AAB), treatment (+OFQ), and washout (SFM WO) periods (n = 113, N = 3).

OFQ as a new player in the ARC regulation of GnRH neurons. A, Left, Electrophysiological recording of an adult GFP-tagged GnRH neuron showing OFQ (10 nm) suppressed the firing rate of kisspeptin-10 (kp-10; 100 nm)-activated GnRH neurons. Right, Corresponding periodogram illustrating the changes in GnRH neuron firing rate (in Hz) over time (i.e., the increase in firing rate induced by kp-10 and its suppression by OFQ). The bars represent the instant frequency in 10 s bins. B, Electrophysiological recording of another adult GFP-tagged GnRH neuron showing OFQ (10 nm) suppressed the firing rate of kp-10 (100 nm)-activated GnRH neurons. The inhibition evoked by OFQ was partially antagonized by UFP-101 (500 nm) but was reestablished by a second application of OFQ after the washout of UFP-101. C, Electrophysiological recording of a third adult GFP-tagged GnRH neuron showing the partial antagonism of OFQ by UFP-101. Note that, without UFP-101, the OFQ evoked a pause in the tonic firing for ∼11 min (red arrow, on the left), while with UFP-101 the pause was ∼2.5 min (red arrow, on the right; i.e., an approximately four times faster recovery despite being further from the initial kp-10 application. D, Quantification of the OFQ inhibition, before (square) and after (circle) the application of UPF-101. The values represent the firing rate 4 min after OFQ or UFP-101 plus OFQ application, normalized to the firing rate in the last minute of aCSF application before application of OFQ (empty square) or UFP-101 before UFP-101 plus OFQ (empty circle) application, respectively. E, Diagram representing where OFQ stands within the already established connectivity around GnRH neurons. GnRH neuron (blue) receive excitatory inputs from RP3V kisspeptin neurons (red) at the soma. ARC kisspeptin neurons (purple), autoregulated via a neurokinin B and dynorphin loop, contact GnRH neuronal processes around the median eminence. Both kisspeptin neuronal populations are linked via ARC → RP3V connections. POMC neurons (green) contact GnRH neurons (; ). ARC kisspeptin neurons contact POMC neurons (; ). OFQ fibers (orange), possibly originating from a subpopulation of POMC neurons and/or another unidentified neuronal population, contact GnRH neurons, providing an inhibitory signal.