Performance, properties and plasticity of identified oxytocin and vasopressin neurones in vitro

Abstract

The neurohypophysial hormones oxytocin (OT) and vasopressin (VP) originate from hypothalamic neurosecretory cells in the paraventricular and supraoptic (SON) nuclei. The firing rate and pattern of action potentials arising from these neurones determine the timing and quantity of peripheral hormone release. We have used immunochemical identification of biocytin-filled SON neurones in hypothalamic slices in vitro to uncover differences between OT and VP neurones in membrane and synaptic properties, firing patterns, and plasticity during pregnancy and lactation. In this review, we summarise some recent findings from this approach: (i) VP neuronal excitability is influenced by slow (sDAP) and fast (fDAP) depolarising afterpotentials that underlie phasic bursting activity. The fDAP may relate to a transient receptor potential (TRP) channel, type melastatin (TRPM4 and/or TRPM5), both of which are immunochemically localised more to VP neurones, and especially, to their dendrites. Both TRPM4 and TRPM5 mRNAs are found in the SON, but single cell reverse transcriptase-polymerisation suggests that TRPM4 might be the more prominent channel. Phasic bursting in VP neurones is little influenced by spontaneous synaptic activity in slices, being shaped largely by intrinsic currents. (ii) The firing pattern of OT neurones ranges from irregular to continuous, with the coefficient of variation determined by randomly distributed, spontaneous GABAergic, inhibitory synaptic currents (sIPSCs). These sIPSCs are four- to five-fold more frequent in OT versus VP neurones, and much more frequent than spontaneous excitatory synaptic currents. (iii) Both cell types express Ca(2+)-dependent afterhyperpolarisations (AHPs), including an apamin-sensitive, medium duration AHP and a slower, apamin-insensitive AHP (sAHP). In OT neurones, both AHPs are enhanced during pregnancy and lactation. During pregnancy, the plasticity of the sAHP is blocked by antagonism of central OT receptors. AHP enhancement is mimicked by exposing slices from day 19 pregnant rats to OT and oestradiol, suggesting that central OT and sex steroids programme this plasticity during pregnancy by direct hypothalamic actions. In conclusion, the differences in VP and OT neuronal function are underlain by differences in both membrane and synaptic properties, and differentially modulated by reproductive state.

Figure 1. Phasic bursting VP neurone and DAP

A. Voltage record of phasic bursting VP neurone recorded with the whole cell patch technique in the SON of a hypothalamic slice made from an adult female rat. Bursts are underlain by a plateau potential (white dashed line), and separated by silent periods consisting of a slow depolarisation. The grey dashed line indicates the peak of the post-burst hyperpolarisation. B. Ratemeter histogram of the spike activity shown in A. Note the highest firing rate occurs in the first 2 sec of the burst (blackened line). C. DAP activated by one spike (left) summates after two spikes, instigating a burst.

Figure 2. DAPs and AHPs in MNCs

Afterpotentials in a VP neurone were generated by a train of action potentials evoked by intracellular current injections (20, 5 ms depolarising pulses, 100–250 pA, 20Hz). A. In artificial cerebrospinal fluid, the train was followed by the mAHP, which was subsequently followed by the sDAP. B. Bath application of apamin (100 nM) completely blocked the mAHP and unmasked the fDAP, which was followed by the sDAP. C. Additional application of Cs⁺ (5 mM) blocked the sDAP and revealed the sAHP. D. Superimposed traces of A–C illustrate the temporally overlapping, multiple afterpotentials. E: Expanded portion of the trace in C (indicated by underline) revealed that the onset of the fDAP occurred after the 1st action potential and its amplitude increased with each subsequent action potential until a plateau was reached after 12 spikes. F. Inward tail currents underlying the DAPs (I_DAPs) were generated by 50 ms steps to 0 mV from a holding potential of −60 mV. Tail currents with similar time courses as the fDAP and sDAP were obtained, and the application of 5 mM Cs⁺ blocked only I_sDAP. With permission from Teruyama and Armstrong (80).

Figure 3. Localization of TRM4 type channels in the SON

A. Left panel: immunochemical localisation of TRPM4 in a 40 μm coronal section of SON of adult male rat. Most neurones are lightly stained, as are thick processes characteristic of dendrites (arrows). Labelling from polyclonal antibody (1:7500) raised in rabbit against TRPM4 (Abcam, Cambridge, UK), and followed by goat-anti-rabbit antibody conjugated to Alexa 568 (Invitrogen, Carlsbad, CA). Right panel: immunochemical localization of VP-associated neurophysin (VP-NP) in the same tissue section shown in left panel. VP-NP was localised using a monoclonal antibody (1:500) relatively specific for VP-NP (PS41, provided by H. Gainer, NIH), followed by goat-anti-mouse antibody conjugated to Alexa 488 (Invitrogen). Images from 2 μm optical sections taken on a confocal microscope (BioRad 1024, Hercules, CA). Note the double labelling of the dendrites (arrows) and many neurones. B. mRNA coding for TRPM4 is expressed in the SON. Total RNA was extracted from SON tissue punched out from brain slices, and reverse transcribed. Amplified products of the expected size were obtained for TRPM4 (267 bp). Rat kidney, heart, and tongue epithelial tissues (TRC) known to express TRPM4 served as positive controls. C. Individual SON neurones were collected from dissociated SON tissue. Following total RNA extraction and reverse-transcription from these single cells, PCRs for VP and TRPM4 were performed to verify the collected cells are MNCs. The cDNA from each neurone was amplified by PCR for TRPM4 and TRPM5. Amplified products of expected size for TRPM4 were obtained from 5 out of 7 cDNA libraries derived from those single MNCs. Of these 5, 4 neurones also contained VP mRNA.

Figure 4. OT and E₂ enhance AHPs in OT neurones from pregnant rats

Slices from Day 19 pregnant rats were exposed either to a vehicle control or a combination of E₂ (500 pM) and OT (10 nM) for 2 hrs. After washing, whole cell recordings were made in the SON and tested for AHPs, then later identified as VP or OT types with immunocytochemistry using standard procedures (e.g., Teruyama and Armstrong, 2007). Left top panel: The AHPs following a 1 sec train of spikes at 20 Hz in treated (grey) and control (black) slices. Traces represent averages of 7 control and 6 treated OT neurones. Treated neurones had a much larger sAHP. Left bottom panel: Histogram showing the difference in the means (± s.e.m.) of treated and untreated OT neurones of sAHP area. Inset: the increase in the sAHP was also accompanied by a longer decay constant for the sAHP. Right top panel: AHPs in VP neurones were not affected. Note the presence of DAPs. Right bottom panel: Histogram showing the difference in the means (± s.e.m.) of treated and untreated VP neurones of sAHP area.

Figure 5. The decay of IPSCs was slower during early lactation in OT and VP neurones

A, superimposed traces of monoquantal, mIPSCs from OT neurones in slices taken from a virgin (left) and early-lactating rat (right). Inset: In the inset, traces from each set are averaged, and the averaged traces scaled to the peak and superimposed. Note the longer decay of mIPSCs in the OT neurone from the lactating rat (black trace, arrow). B, superimposed traces of monoquantal, mIPSCs from VP neurones in slices taken from a virgin (left) and early-lactating rat (right). Inset: In the inset, traces from each set are averaged, and the averaged traces scale to the peak and superimposed. Note the longer decay of mIPSCs in the VP neurone from the lactating rat (black trace, arrow). C, Left Panel: the decay tau of mIPSCs in OT neurones is significantly slower in early-lactating (Days 2–6) compared to virgin rats. Numbers represent the number of neurones for which averaged mIPSCs were taken (*p = 0.0045). Right Panel: mIPSC amplitudes in OT neurones were not significantly different in virgin compared to lactating rats. D, Left Panel: the decay tau of mIPSCs is significantly slower in VP neurones in early-lactating (Days 2–6) compared to virgin rats. Numbers represent the number of neurones for which averaged mIPSCs were taken (*p = 0.0041). Right panel: mIPSC amplitudes in VP neurones were not significantly different in virgin compared to lactating rats. Modified with permission from Li (115).