Evidence for a hypothalamic oxytocin-sensitive pattern-generating network governing oxytocin neurons in vitro

Abstract

During lactation and parturition, magnocellular oxytocin (OT) neurons display a characteristic bursting electrical activity responsible for pulsatile OT release. We investigated this activity using hypothalamic organotypic slice cultures enriched in magnocellular OT neurons. As shown here, the neurons are functional and actively secrete amidated OT into the cultures. Intracellular recordings were made from 23 spontaneously bursting and 28 slow irregular neurons, all identified as oxytocinergic with biocytin and immunocytochemistry. The bursting electrical activity was similar to that described in vivo and was characterized by bursts of action potentials (20.1 +/- 4.3 Hz) lasting approximately 6 sec, over an irregular background activity. OT (0.1-1 microM), added to the medium, increased burst frequency, reducing interburst intervals by 70%. The peptide also triggered bursting in 27% of nonbursting neurons. These effects were mimicked by the oxytocin receptor (OTR) agonist [Thr4, Gly7]-OT and inhibited by the OTR antagonist desGly-NH2d(CH2)5[D-Tyr2,Thr4]OVT. Burst rhythmicity was independent of membrane potential. Hyperpolarization of the cells unmasked volleys of afferent EPSPs underlying the bursts, which were blocked by CNQX, an AMPA/kainate receptor antagonist. Our results reveal that OT neurons are part of a hypothalamic rhythmic network in which a glutamatergic input governs burst generation. OT neurons, in turn, exert a positive feedback on their afferent drive through the release of OT.

Fig. 1.

HPLC of OT immunoreactivity (OT-IR) in extracts of cultured cells, culture medium, adult SON, and neurohypophysis (NH). Note that OT in cultures coeluted in the same fraction as that in adult tissues.

Fig. 2.

A, Spontaneous release of OT in organotypic cultures. Correlation between the number of OT cells and OT immunoreactivity (OT-IR) in medium wasr² = 0.83, p < 0.001. Media were collected after 5 d of incubation (see Materials and Methods). B, K⁺-evoked release of OT in organotypic cultures. The total amount of OT released during 56 mm [K⁺] stimulation was closely correlated to OT concentration in the culture medium before the medium was changed (r² = 0.93,p < 0.001).

Fig. 3.

OT-IR in culture medium in relation to the number of days of incubation in the medium and to the number of cultures in which one bursting OT neuron was recorded or not. Note that the probability of recording a bursting neuron was higher in culture with high OT-IR. p < 0.05, Mann–WhitneyU test. Numbers in brackets indicate numbers of cultures per group.

Fig. 4.

Bursting activity in OT neurons. A, Sequential histogram of action potential discharge (number of spikes per second) over a 10 min period. Note the recurrence of bursts over an irregular background activity. B, Examples of action potential discharges during bursts. Action potential frequencies have been calculated every 0.5 sec to visualize peak frequencies. InB₁, the profile is very similar to that of milk ejection bursts in vivo. In B₂, firing is more sustained throughout the burst.

Fig. 5.

Effect of OT on the bursting activity of OT neurons. A, Example of a spontaneously bursting OT neuron in which bath application of OT (0.1 μm; 10 min) increased dramatically the frequency of bursts (★), with no effect on their duration or the intraburst frequency of discharge.B, Example of a nonbursting OT neuron in which bath application of OT (0.1 μm; 15 min) switched the pattern of activity from a continuous (top panel) to a bursting mode (bottom panel).

Fig. 6.

Effect of an OTR agonist on the bursting activity of OT neurons. The specific OTR agonist [Thr⁴, Gly⁷]-OT (0.1 μm; 10 min) mimicked the effect of OT, facilitating bursting in a spontaneously bursting neuron (A) and evoking bursting in a nonbursting OT-cell.

Fig. 7.

Effect of an OTR antagonist on the bursting activity of OT neurons. A, Example of a spontaneous bursting (★) OT neuron whose activity was inhibited by bath application of a specific OTR antagonist desGly-NH₂d(CH₂)₅[d-Tyr²,Thr⁴]OVT (50 μm; 10 min). B, Example of a spontaneously bursting OT neuron (top panel) in which addition of OT (0.1 μm; 10 min) resulted in an increased burst frequency (middle panel). Simultaneous application of the OTR antagonist (50 μm; 15 min) inhibited the facilitatory effect of OT (bottom panel).

Fig. 8.

A, Effect of membrane potential on bursting activity. Example of an OT cell in which bursting activity was induced by bath-application of OT. The burst persisted when the cell was held at three different membrane potentials. Neither interburst intervals (A₁) nor burst duration (A₂) was affected by this manipulation. Note that hyperpolarizing membrane potential unmasked an abundant excitatory synaptic activity underlying the bursts (A₂,bottom record). In A₂, action potentials have been clipped to adjust to the scale. B, Summary of the effect of membrane potential on burst and interburst durations. These two parameters were unaffected in six of six cells tested. Data were normalized to the value obtained at −60 mV and are expressed as percentage changes of the mean ± SD.C, Summary graph of the effect of membrane potential on action potential discharge observed within the burst. Action potential frequency within the burst increased at depolarized potentials and decreased at hyperpolarized potentials.

Fig. 9.

Effect of CNQX on bursting activity. Example of a cell in which the bursting activity was facilitated by exogenous application of OT (top panel). Addition of 15 μm CNQX to the perfusion solution (middle panel) fully inhibited the bursting behavior. This effect was reversible on washout of CNQX (bottom panel). Note that during CNQX application, depolarizing membrane potential above spike threshold (−50 mV) enhanced action potential discharge but caused no burst.