Lactation induces increased IPSC bursting in oxytocinergic neurons

Abstract

Hypothalamic magnocellular neurosecretory cells (MNCs) undergo dramatic structural reorganization during lactation in female rats that is thought to contribute to the pulsatile secretion of oxytocin critical for milk ejection. MNCs from male rats generate robust bursts of GABAergic synaptic currents, a subset of which are onset-synchronized between MNC pairs, but the functional role of the IPSC bursts is not known. To determine the physiological relevance of IPSC bursts, we compared MNCs from lactating and non-lactating female rats using whole-cell recordings in brain slices. We recorded a sixfold increase in the incidence of IPSC bursts in oxytocin (OT)-MNCs from lactating rats compared to non-lactating rats, whereas there was no change in IPSC bursts in vasopressin (VP)-MNCs. Synchronized bursts of IPSCs were observed in pairs of MNCs in slices from lactating rats. Our data indicate, therefore, that IPSC bursts are upregulated specifically in OT-MNCs during lactation, and may, therefore, contribute via rebound depolarization to the spike trains in OT neurons that lead to reflex milk ejection.

Keywords: GABA; IPSC; burst; lactation; oxytocin; synchronization.

Figure 1

Effect of lactation on spontaneous IPSC bursts in MNCs. (A) Representative recording of bursts of IPSCs in an unidentified MNC from a lactating rat before and after the addition of TTX. (B) Time histogram of the instantaneous IPSC frequency recorded in representative MNCs. IPSC bursts are indicated by a sharp rise in instantaneous IPSC frequency; they occur with a higher incidence in the MNC from a lactating rat (arrows, lower) than the MNC from a non–lactating rat (arrows, upper). (C) Average incidence of bursts of IPSCs (bursts/h) in MNCs from virgin and lactating rats. (D) Percent of MNCs displaying IPSC bursts (% cells_burst) in virgin and lactating rats. (E) Average incidence of bursts of IPSCs (bursts/h) in MNCs from non–lactating and lactating rats in the presence of TTX to block spiking. (F) Percent of MNCs displaying IPSC bursts in virgin and lactating rats in the presence of TTX. The numbers in the columns in all the figures represent the numbers of recorded cells in the averages. *, P < 0.05.

Figure 2

Upregulation of IPSC bursts in OT MNCs during lactation. (A) OT MNCs viewed in a brain slice under IR‐DIC (left) and epifluorescence (right). *, RFP(+) neurons. (B) Representative IPSC burst recorded in an OT RFP(+) MNC from a non–lactating rat. (C) Representative IPSC bursts recorded in an OT RFP(+) MNC from a lactating rat. (D) Average incidence of bursts of IPSCs (bursts/h) in OT‐RFP(+) MNCs from virgin and lactating rats. (E) Percent of OT‐RFP(+) MNCs displaying IPSC bursts (% cells_burst) in virgin and lactating rats. (F) Mean IPSC burst duration in OT‐RFP (+) MNCs from virgin and lactating rats. (G) Mean IPSC frequency in IPSC bursts in OT‐RFP(+) MNCs from non–lactating and lactating rats. **, P < 0.01.

Figure 3

IPSC bursts in VP and putative OT MNCs during lactation. (A) Pair of MNCs viewed under IR‐DIC (left) and epifluorescence illumination (right). White arrow denotes a GFP(‐), putative OT neuron; black arrow denotes a GFP (+) VP neuron. (B) Average IPSC burst incidence (bursts/h) and percent of cells displaying bursts (% cells_burst) in GFP(+) VP MNCs from virgin and lactating rats. (C) Average IPSC burst incidence (bursts/h) and percent of cells (% cells_burst) displaying bursts in GFP(‐), putative OT MNCs from virgin and lactating rats. (D) Mean IPSC burst duration and intra‐burst IPSC frequency (IPSC f_burst) in GFP(‐), putative OT MNCs from virgin and lactating rats. (E) Average IPSC burst incidence (burst/h) and percent of cells displaying bursts (% cells_burst) in VP‐GFP(‐), putative OT‐MNCs from virgin and lactating rats in TTX. **, P < 0.01.

Figure 4

Onset–synchronized bursts of IPSCs in pairs of MNCs from lactating rats. (A) Paired recordings of onset–synchronized IPSC bursts (open and filled arrows) in two MNCs from a lactating rat. Some large IPSCs in cell 2 are truncated. (B, C) Expanded traces from A showing the onset synchronization of the IPSC bursts in each of the MNCs. Open and filled arrows correspond to the bursts in A. (D) Time histograms of the instantaneous frequency of IPSCs from the paired recording in A, illustrating the incidence (arrows) of the two synchronized bursts (white and black arrows below).

Figure 5

The effect of simulated synchronized IPSC bursts on action potential frequency in pairs of putative OT neurons. (A) Onset‐synchronized IPSC bursts recorded in a pair of VP‐GFP(‐), putative OT neurons from a lactating rat. (B) The onsets of the two bursts in A shown at an expanded time scale. (C) The IPSC bursts in A were used as templates for simultaneous intracellular current injections into two VP‐GFP(‐), putative OT neurons from a lactating rat recorded in current clamp. Shown are the membrane potential (V) and injection current (I) traces taken from two cells (cell 1 and cell 2) that received the IPSC burst current injections. The two cells responded to the simulated IPSC bursts with a hyperpolarization followed by a rebound increase in spiking frequency. Membrane potentials were held near threshold with current injection (Vm = −46 mV with + 30 pA of holding current in cell 1; Vm = ‐54 mV with + 10 pA of holding current in cell 2). (D) Time histograms of the mean spiking frequencies in MNC pairs (n = 5) before and after the synchronized IPSC burst current injections (arrows). The simulated IPSC bursts were followed by an increase in frequency in the cell pairs. *P < 0.05, # P < 0.01, repeated measures ANOVA and Dunnett's post hoc test.

Figure 6

Nitric oxide dependence of lactation–induced increase in IPSC burst incidence. (A) Representative traces of IPSC bursts recorded in the NOS inhibitor L‐NAME (100 μmol/L) from a virgin rat (upper) and a lactating rat (lower). (B, C) Average incidence of IPSC bursts (burst/h) and percent of MNCs displaying IPSC bursts (% cells_burst) recorded in the presence of 100‐μmol/L L‐NAME in slices from virgin and lactating rats. NOS inhibition abolished the increase in IPSC burst incidence in OT MNCs and the increased percentage of OT MNCs that generated IPSC bursts in slices from lactating rats.