Excitation of tuberoinfundibular dopamine neurons by oxytocin: crosstalk in the control of lactation

Abstract

Milk production in the nursing mother is induced by the hormone prolactin. Its release from the anterior pituitary is generally under tonic inhibition by neuroendocrine tuberoinfundibular dopamine (TIDA) neurons of the arcuate nucleus. Successful nursing, however, requires not only production but also ejection of breast milk. This function is supported by the hormone oxytocin. Here we explored the possibility that interaction between these functionally complementary hormones is mediated by TIDA neurons. First, whole-cell patch-clamp recordings were performed on prepubertal male rat hypothalamic slices, where TIDA neurons can be identified by a robust and rhythmic membrane potential oscillation. Oxytocin induced a switch of this rhythmic activity to tonic discharge through a depolarization involving direct actions on TIDA neurons. The depolarization is sensitive to blockade of the oxytocin receptor and is mediated by a voltage-dependent inward current. This inward current has two components: a canonical transient receptor potential-like conductance in the low-voltage range, and in the high-voltage range, a Ca(2+)-dependent component. Finally, whole-cell and loose-patch recordings were also performed on slices from virgin and lactating female rats to evaluate the relevance of these findings for nursing. In these preparations, oxytocin was found to excite TIDA neurons, identified by their expression of tyrosine hydroxylase. These findings suggest that oxytocin can modulate prolactin secretion by exciting TIDA neurons, and that this may serve as a feedforward inhibition of prolactin release.

Keywords: arcuate nucleus; oscillation; prolactin.

Figure 1.

OT directly excites TIDA neurons via the OT receptor. A, Current-clamp whole-cell recording of a TIDA neuron in slice from prepubertal male rat. Portions of trace indicated by boxes in Aa shown expanded in Ab and Ac. Note typical rhythmic membrane potential oscillation under control conditions (Ab). OT administration is followed by a switch of the activity from rhythmic to tonic (Ac). B, Current-clamp recordings of TIDA neurons in the presence of TTX. Application of OT at 1 μm (Ba) or 10 nm (Bb) induces a depolarization. C, Dose–response histogram illustrating the relationship between OT concentration and the resultant membrane potential depolarization. Cells included have been exposed to a single concentration. At least five cells are included for each concentration. The Wilcoxon test was used to perform the statistical analysis between each condition: *p < 0.01 compared with OT concentration of 0 nm; #p < 0.01 compared with OT concentration of 1 nm and §p < 0.05 compared with OT concentration of 10 nm. No statistical difference measured between 1 and 10 μm. Vm: membrane potential. D, Representative TIDA neuron current-clamp recording performed with application of OTA. In the presence of the antagonist, OT administration fails to induce a switch of discharge activity. E, Current-clamp recording of a TIDA neuron in the presence of TTX. Application of the selective receptor agonist, TGOT, induces a depolarization.

Figure 2.

OT activates a net inward current. A, Representative voltage-clamp whole-cell recording from a TIDA neuron at a holding potential of −60 mV and in the presence of TTX (Aa), and Gaussian fits of averaged holding current frequency distribution histograms (Ab; n = 8 cells). Application of OT induces a partially reversible inward current. Statistical analysis performed with Wilcoxon test, *p < 0.05. B, Averaged voltage-clamp ramps (n = 5 cells) recorded in the presence of TTX under control condition (black) and at the peak of the OT response (red). C, OT-induced current obtained by subtraction of the traces shown in B (n = 5 cells). Note two distinct inward current components: a LV and a HV component. D, Plot of I_OT obtained at a holding potential of −60 mV in control aCSF (using recordings as the one shown in A), in the presence of 2-APB (blue; 100 μm), in the presence of flufenamic acid (red; 100 μm), and with the control aCSF replaced by Na⁺-substituted aCSF (pink) or low-Ca²⁺ aCSF (green). Light dots represent individual values; dark dots correspond to the mean value ± SEM. The Wilcoxon test was used to perform the statistical analysis between each condition: **p < 0.01; n.s., Not significant.

Figure 3.

Low and high voltage components of the OT-induced current. A, I_OT as recorded in voltage-clamp ramps from TIDA neurons as described in Figure 2C, shown in black. Blue curve shows I_OT obtained in the presence of 2-APB (100 μm; n = 5 cells). Note loss of the LV component but no effect on HV component. B, Red curve shows I_OT obtained during recording in Na⁺-substituted aCSF (n = 5 cells). Black curve, same as in A. Note reduction of the LV component but no significant effect on the HV component by Na⁺ substitution. C, Green curve shows I_OT obtained during recording in low-Ca²⁺- aCSF (n = 5 cells). Black curve, same as in A. The LV component is not changed but the HV component is significantly reduced in low Ca². The Wilcoxon test was used to perform the statistical analysis: *p < 0.05; n.s., Not significant.

Figure 4.

OT excites arcuate nucleus neurons in virgin female rats. A, Pie charts illustrating the proportion of dmArc cells about OT responses for all cells (Aa; n = 59), only TH-positive cells (Ab; n = 7), and only TH-negative cells (Ac; n = 8) recorded in slices from virgin female rats. Ba, Ca, Examples of recording from neurons in the dmArc of virgin female rats that increased firing following application of OT (1 μm) in TIDA (Ba) and non-TIDA (Ca) neurons. The recorded cells were filled with neurobiotin (NB; green) and stained by immunofluorescence for TH (red), shown in confocal micrographs in Bb, Cb. Green and red channels shown separately in smaller panels; larger panels shows merged image. The cell in Ba exhibits TH-immunoreactivity (Bb), indicating its TIDA identity. Cell shown in Ca does not stain with the TH antiserum (Cb). Scale bars, 50 μm.

Figure 5.

OT excites arcuate nucleus neurons in lactating female dams. A, Pie charts illustrating the proportion of dmArc cells about OT responses for all cells (Aa; n = 86), only TH-positive cells (Ab; n = 19) and only TH-negative cells (Ac; n = 33) recorded in slices from lactating dams. Ba, Ca, Examples of recordings from neurons in the dmArc of lactating female rats that exhibit increased firing following application of OT (1 μm) in TIDA (Ba) and non-TIDA (Ca) neurons. The recorded cells were filled with neurobiotin (NB; green) and stained by immunofluorescence for TH (red), shown in confocal micrographs in Bb, Cb. Green and red channels shown separately in smaller panels; larger panels shows merged image. The cell in Ba exhibits TH-immunoreactivity (Bb), indicating its TIDA identity. Cell shown in Ca does not stain with the TH antiserum (Cb). Scale bars, 50 μm.

Figure 6.

OT innervation of TIDA neurons is sparse. A, C, E, Fluorescence micrographs from coronal sections of the male (A), virgin female (C), and lactating (E) rat hypothalamus labeled with immunofluorescence for TH (red) to identify TIDA neurons and OT (green). Note dense OT-immunoreactive terminals of the median eminence (ME) but only occasional OT-immunoreactive axons in the Arc. Scale bars, 200 μm. B, D, F, Confocal micrographs of male (B), virgin female (D), and lactating (F) rat hypothalamus sections stained for TH and OT immunofluorescence as in A. Only scattered OT-immunoreactive fibers (arrowheads) are seen and very few contact points with TIDA neurons can be observed. 3V, Third ventricle; scale bars, 10 μm.