Peripheral insulin administration enhances the electrical activity of oxytocin and vasopressin neurones in vivo

Luis Paiva Image | Gareth Leng Image

Oxytocin neurones are involved in the regulation of energy balance through diverse central and peripheral actions and, in rats, they are potently activated by gavage of sweet substances. Here, we test the hypothesis that this activation is mediated by the central actions of insulin. We show that, in urethane-anaesthetised rats, oxytocin cells in the supraoptic nucleus show prolonged activation after i.v. injections of insu- lin, and that this response is greater in fasted rats than in non-fasted rats. Vasopressin cells are also activated, although less consistently. We also show that this activation of oxytocin cells is independent of changes in plasma glucose concentration, and is completely blocked by central (i.c.v.) administration of an insulin receptor antagonist. Finally, we replicate the previously published finding that oxytocin cells are activated by gavage of sweetened condensed milk, and show that this response too is com- pletely blocked by central administration of an insulin receptor antagonist. We con- clude that the response of oxytocin cells to gavage of sweetened condensed milk is mediated by the central actions of insulin.

electrophysiology, gavage, S961, supraoptic nucleus
Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK

Gareth Leng, Centre for Discovery Brain Sciences, The University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK.
Email: [email protected]

Funding information
Biotechnology and Biological Sciences Research Council, Grant/Award Number: BB/S000224/1


Insulin is widely known for its role in glucose homeostasis on pe- ripheral tissues, although its central effects are not yet fully elu- cidated. Once secreted into the circulation, insulin is transported into the brain by a saturable transport mechanism.1,2 Both exoge- nous insulin administration and glucose-stimulated insulin secre- tion result in a progressive increase of insulin in the cerebrospinal fluid (CSF) in several species, including humans.3-6 Accordingly, insulin concentrations in the CSF correlate with levels in plasma, although they are approximately 15-fold lower than plasma con- centrations in fasted rats.3 In the brain, regions sensitive to insulin include the hypothala- mus,7,8 which contains insulin-responsive neurones in several nu- clei.9-11 Amongst these, the insulin receptor (InsR) is abundantly expressed in the supraoptic nucleus (SON),12-14 which exclusively contains magnocellular oxytocin and vasopressin cells, and i.p. ad- ministration of insulin induces the expression of Fos protein in parvo- and magnocellular oxytocin cells of the paraventricular nu- cleus in rats.15 Explants of the hypothalamo-neurohypophysial sys- tem, including the SON and its projections to the posterior pituitary, release oxytocin and vasopressin in response to direct application of insulin,16 and central administration of insulin increases peripheral secretion of oxytocin in mice by a direct action on oxytocin cells.17 The peer review history for this article is available at https://publons.com/publon/10.1111/jne.12841. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
2020 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of British Society for Neuroendocrinology

Received: 8 October 2019 | Revised: 25 February 2020 | Accepted: 25 February 2020
DOI: 10.1111/jne.12841
Journal of Neuroendocrinology. 2020;00:e12841. https://doi.org/10.1111/jne.12841

In addition to their classical roles in reproduction,18,19 stress20 and water balance,21 oxytocin and vasopressin have roles in energy homeostasis.22 Both central and peripheral oxytocin administration exert anorexigenic effects, increase energy expenditure and induce lipolysis.23-26 Peripheral administration of both oxytocin and va- sopressin can induce the release of insulin from the pancreas27-30 and systemically administered oxytocin in humans (administered in- tranasally) has been reported to curb the meal-related increase in plasma glucose,31 as well as to improve β-cell responsivity and glu- cose tolerance in healthy men.32 Studies using well-validated radio- immunoassays in extracted plasma samples33 indicate that patients with metabolic syndrome exhibit higher circulating oxytocin concen- trations than normal individuals,34 and patients with diabetes have higher concentrations of vasopressin, as well as copeptin (which is co-secreted with vasopressin).35,36
In the present study, we examine whether peripheral (i.v.) ad- ministration of insulin affects the electrical activity of oxytocin and vasopressin cells in the SON of urethane-anaesthetised rats. The effect of different feeding states, and consequently different blood glucose concentrations, on these responses was also investigated. We investigated the role of brain InsR in these responses by blocking these receptors using an InsR antagonist. Finally, we tested whether the previously reported enhanced electrical activity of oxytocin cells in response to sweet food gavage37 was mediated by endogenous insulin release acting on brain InsRs.


2.1 | Animals

We used adult male Sprague–Dawley rats weighing 300-350 g. The rats had ad lib. access to food and water and were maintained under a 12:12 hour light/dark cycle (lights on 7.00 AM) at a room tempera- ture of 20-21°C. In most experiments, we used fasted rats to reduce the variability of blood glucose and gastric signals (induced by prior food consumption) that could affect neural activity and so, in these experiments, the food was removed overnight (~15 hours). All pro- cedures were conducted on rats under deep terminal anaesthesia in accordance with the UK Home Office Animals Scientific Procedures Act 1986 and a project licence approved by the Ethical Committee of the University of Edinburgh.

2.2 | Drugs

Human recombinant insulin solution (catalogue no. I9278; Sigma- Aldrich, St Louis, MO, USA) was diluted in 0.9% saline (B. Braun) at 0.25 U 100 µL-1. Glucose solution was prepared by dissolving 5% glucose (catalogue no. G5767; Sigma-Aldrich) in sterile distilled water. The InsR antagonist S961 (catalogue no. 051-86; Phoenix Europe GmbH, Karlsruhe, Germany) was dissolved at 0.33 nmol µL-
1 in artificial CSF (NaCl 138 mmol L-1, KCl 3.36 mmol L-1, NaHCO3 9.52 mmol L-1, Na2HPO4 2H2O 0.49 mmol L-1, urea 2.16 mmol L-1, CaCl2 1.26 mmol L-1, MgCl2 6H2O 1.18 mmol L-1; pH 7.5).

The responses of SON neurones to systemic insulin were tested by giving an i.v. bolus of 0.75 U kg-1. To determine the effect of re- storing circulating glucose content in insulin-responsive neurones, 400 µL of 5% glucose solution was given i.v. at 200 µL min-1. Glucose concentrations were then checked 5 and 20 minutes later and, if lower than basal values, were corrected by infusing an additional 300 and 100 µL of 5% glucose solution, respectively. To investigate the role of central InsRs on insulin responses, the InsR antagonist S96138 was given into the third ventricle using a 31-gauge needle inserted through the median eminence; 1 nmol (4.8 µg) of S961 was injected at 1 µL min-1. We chose a dose expected to be sufficient to block insulin receptors throughout the brain when given i.c.v., al- though lower than that needed to antagonise insulin actions if given peripherally. The affinity of S961 for both isoforms of the insulin receptor is close to that of insulin itself.38 Previous studies have re- ported that bilateral injections of 100 ng of S961 into the arcuate nucleus block the effects of insulin microinjected into the arcuate nucleus on lumbar sympathetic nerve activity in late pregnant rats.39 Studies using the closely related antagonist S661, which has proper- ties indistinguishable from those of S961, indicated that peripheral doses of 30 nmol kg-1 or more are needed to block the effects of
i.v. administration of 30 mmol kg-1 insulin on blood glucose levels.38 As detailed below, the i.c.v. application of S961 in our hands had no significant effect on plasma glucose concentrations.

2.3 | Sweet condensed milk gavage

In fasted rats, a gavage tube was inserted orally into the stomach to deliver a total volume of 5 mL of sweetened condensed milk (SCM; Nestle, Vevey, Switzerland) diluted 50% v/v in distilled water (40.8 kJ, 1.68 g sugar, 0.24 g fat) at 0.16 mL min-1.

2.4 | In vivo electrophysiology

Rats were briefly anaesthetised with isoflurane inhalation anaes- thesia, and then urethane (ethyl carbamate 25% solution) was in- jected i.p. at 1.25 g kg-1. A femoral vein was cannulated for drug administration and an endotracheal tube was inserted to maintain the airway open, and the SON and the pituitary stalk were ex- posed by transpharyngeal surgery.40 A bipolar stimulating elec- trode (SNEX-200X; Clark Electromedical Instruments, Reading, UK) was placed on the pituitary stalk, and a glass microelectrode (~1 µm tip; filled with 0.9% NaCl) was lowered into the SON under visual control for extracellular recording. The signal was ampli- fied using an Axonpatch 200B (Molecular Devices, Sunnyvale, CA, USA) connected to a HumBug 50 Hz noise eliminator (Quest Scientific Instruments Inc., Secaucus, NJ, USA) and was digital- ised with a CED-1401 laboratory interface (Cambridge Electronic Design, Cambridge, UK) connected to a PC running SPIKE2, version 7.20 (Cambridge Electronic Design). Most recordings were made from single neurones; in some experiments, the spike activity of two cells was recorded simultaneously; in these cases, the spikes were discriminated and analysed offline using the waveform function of SPIKE2. Recordings were made between 12.00 PM and 5.00 PM (lights on 7.00 AM to 7.00 PM). Rats were tested only once with insulin.

Supraoptic nucleus neurones were antidromically identified through stimulation of the pituitary stalk by matched biphasic pulses (1 ms, <1 mA peak to peak), which produce an antidromic spike at a constant latency (~10 ms) (Figure 1A). Oxytocin cells were discrim- inated from continuous-firing vasopressin cells (Figure 1B) by the shape of the interspike interval (ISI) distribution (Figure 1C,D) and by their opposite response to i.v. cholecystokinin (CCK) (CCK-8 sul- phated; catalogue no. H-2080; Bachem AG, Bubendorf, Switzerland) given at 20 μg kg-1, comprising a transient excitation of oxytocin cells, and no effect or short inhibition of vasopressin cells (Figure 1E, F).40,41 CCK was given at the end of the experiments to identify con- tinuously-firing cells.

2.5 | Recording and blood sampling protocols

2.5.1 | Effect of i.v. insulin

The spontaneous spiking activity of SON neurones was recorded for 20 minutes (basal activity) and for at least 60 minutes after i.v. insulin. Blood samples (50 μL) were taken to measure glucose imme- diately before administration of insulin or vehicle, as well as 15, 30, 60, 90 and 120 minutes later.

2.5.2 | Effect of restoring circulating glucose content in insulin-responsive neurones

The basal activity of SON neurones was recorded for 20 minutes, and for another 30 minutes after i.v. insulin. Then, glucose was given i.v. and the spike activity recorded for further 30 minutes. Blood glucose concentrations were measured before insulin, 30 minutes later (ie, before i.v. glucose) and 5 and 20 minutes after the first glucose injection. Only rats exhibiting in the last sample a blood glucose concentration within 15% of the value in the basal sample were used.

2.5.3 | Blockade of central InsRs

The basal spike activity of SON neurones in fasted rats was re- corded for 20 minutes. Then, S961 was given i.c.v. and spike activity recorded for 15 minutes. After this, insulin was given i.v. and the spike activity recorded for another 30 minutes. Blood glucose con- centrations were measured using an Accu-Chek Aviva meter (Roche Diagnostics GmbH, Mannheim, Germany) immediately before S961
injection, 15 minutes later (ie, before i.v. insulin) and 30 minutes after
i.v. insulin.

2.5.4 | Effect of central InsR blockade on SCM- stimulated activity of oxytocin cells

The basal spike activity of SON neurones was recorded for 20 minutes. Then, rats were injected i.c.v. with either vehicle or S961 and activity recorded for 10 minutes. After this, SCM was gavaged (over 30 minutes) and spike activity recorded for 1 hour. Blood samples (300 μL) were taken immediately before the i.c.v. injection, 10 minutes later (ie, before SCM gavage) and at 30 and 60 minutes after the start of gavage. Blood glucose concentra- tions were measured immediately after sampling; then, samples were centrifuged in EDTA-coated tubes, and plasma collected and stored at −80°C for insulin measurements using a rat/mouse insu- lin ELISA kit (catalogue no. EZRMI-13K; EMD Millipore, Burlington, MA, USA).

2.6 | Hazard functions

Hazard function displays how the excitability of a neurone changes with the time subsequent to the last spike, indicating the probability of a neurone firing a spike in a given period. For responsive neu- rones in fasted rats, we constructed ISI histograms in 10-ms bins of the 20-minute basal period and the last 30-minute after insulin administration. From these, hazard functions (in 10-ms bins) were constructed using the formula: [hazard in bin (t, t + 10)] = [number of ISIs in bin (t, t + 10)]/(number of ISIs of length > t) as described by Sabatier et al.42 Hazard functions plot the incidence of spikes as a proportion of the size of the residual tail of the ISI distribution. When plotted this way, a negative exponential distribution (the dis- tribution characteristic of random events) becomes a constant ‘haz- ard’ proportional to the average firing rate. Deviation from this then become interpretable as periods of decreased or increased excitabil- ity. Consensus hazard functions were calculated from the means of hazard functions.

2.7 | Statistical analysis

Data were analysed using Prism, version 6 (GraphPad Software Inc., San Diego, CA, USA). Responses to insulin were analysed by com- paring the mean firing rate in the 60-minute after insulin with the (basal) firing rate over the 20-minute control period. The changes were compared using a two-tailed Wilcoxon signed-rank test. The activity of phasic cells was analysed in SPIKE2; detection of a burst of activity was defined by spike activity lasting at least 5 seconds and containing >20 spikes followed by >5 seconds of spike silence between bursts. The mean burst duration, interburst interval and activity quotient (percentage of active time over the total time) over

Identification of supraoptic nucleus (SON) neurones. A, Voltage trace showing the electrical stimulation (red stimulus artefact; 0 ms) of the pituitary stalk that evokes an antidromic spike travelling to the soma of SON neurones at a constant latency (~10 ms) in each of two neurones (red spikes), and spike collision occurring when an spontaneous orthodromically traveling spike (black spike) encounters an antidromic spike (centre and right) extinguishing it. B, Raw voltage trace of a double recording showing the spike activity of a continuous- (black large spikes) and a phasic-firing (red short spikes exhibiting intermittent periods of activity) vasopressin cell. C, D, Typical interspike interval (ISI) histogram (frequency of time distributions between [two] occurring spikes) over 10 minutes and corresponding hazard function of the basal spontaneous activity of an (C) oxytocin cell, and (D) vasopressin cell. E, F, Pharmacological identification of SON neurones. Raw voltage trace (upper) and firing rate (spikes in 10-second bins; lower) of an (E) oxytocin, and (F) vasopressin cell exhibiting transient excitation and inhibition respectively in response to i.v. cholecystokinin (CCK) (20 µg kg-1) the 20-minute basal and 60 minutes after insulin were compared using Wilcoxon matched-pairs signed-rank test.
The effect of glucose on insulin-responsive cells was analysed by comparing the mean change in firing rate (spikes s-1 in 10-minute bins) before and after glucose (ie, 0-30 minutes vs 30-60 minutes) using Wilcoxon matched-pairs signed-rank test.

The effect of blockade of central InsRs was analysed by test- ing whether the mean change in firing rate in the 15-minute after S961 injection was significantly different from 0 (ie, from the basal rate) using a two-tailed Wilcoxon signed-rank test. Then, the mean change in firing rate over 30-minute after insulin was compared with the firing rate in the 15 minutes after S961 using a two-tailed Wilcoxon signed-rank test. One-way ANOVA fol- lowed by a post-hoc Bonferroni test was used to compare glucose profiles.

The mean change in firing rate over 60 minutes and the glucose profiles between fasted and non-fasted rats were compared using two-tailed Mann-Whitney test and two-way ANOVA followed by post-hoc Bonferroni multiple comparison tests, respectively. We also compared the change in firing rate to determine whether different treatments affect the responses of SON neurones to in- sulin using two-way ANOVA followed by a post-hoc Bonferroni test.
The effect of prior blockade of central InsRs on SCM-induced activity was analysed using a two-tailed Mann-Whitney test com- paring the mean change in firing rate over 60 minutes between i.c.v. control- and S961-treated rats. The change in firing rate (in 10-min- ute bins), blood glucose concentrations and plasma insulin content between the two groups were compared using two-way ANOVA, followed by a post-hoc Bonferroni test.
All data are reported as the mean ± SEM. P < 0.05 was consid- ered statistically significant, unless otherwise stated.


3.1 | Oxytocin cells

In both fasted and non-fasted rats, i.v. injections of insulin induced a prolonged increase in the firing rate of oxytocin cells, reaching

a plateau between 30 and 60 minutes later. All cells in fasted rats and all but one in non-fasted rats increased their activity after i.v insulin.
Recordings were made from 10 oxytocin cells in 10 fasted rats and from 10 cells in nine non-fasted rats (including one double recording). In non-fasted rats, the mean ± SEM (range) basal firing rate of 2.5 ± 0.4 (0.7-4.1) spikes s-1 increased by 0.9 ± 0.3 (0.1-2.5) spikes s-1 (averaged over the 60 minutes after i.v. insulin; P = 0.002, Wilcoxon signed-rank test) (Figure 2A,B). In fasted rats, oxytocin cells responded more strongly (Figure 2C): the basal firing rate of 2.4 ± 0.5 (0.6-4.8) spikes s-1 increased by 1.6 ± 0.3 (0.4-2.7) spikes s-1 (P = 0.002, Wilcoxon signed-rank test; P = 0.045 for comparison of fasted and non-fasted rats, Mann-Whitney U test).

3.2 | Vasopressin cells

In six fasted rats, recordings were made from 10 vasopressin cells (three phasic- and seven continuously firing) with a mean ± SEM (range) basal firing rate of 5.0 ± 0.8 (0.2-8.1) spikes s-1. After in- sulin, the rate increased by 1.0 ± 0.3 (−0.2 to 3.5) spikes s-1 over 60 minutes (P = 0.006, Wilcoxon signed-rank test) (Figure 2D). In the three phasic cells, insulin increased the burst duration from 32 ± 11 seconds to 82 ± 29 seconds, and decreased the interburst duration from 32 ± 15 seconds to 28 ± 15 seconds; the result-ing activity quotient increased from 0.5 ± 0.2 to 0.7 ± 0.2. The intraburst frequency was unchanged (basal: 3.6 ± 0.7 spikes s-1, insulin: 3.6 ± 0.9 spikes s-1). These changes were not statistically significant.
In 14 non-fasted rats, recordings were made from 16 vasopres- sin cells (eight phasic, eight continuous) with a basal firing rate of
5.1 ± 0.7 (1.4-10.1) spikes s-1. After insulin, the rate increased by 0.7 ± 0.3 (−0.7 to 2.8) spikes s-1 over 60 minutes (P = 0.028, Wilcoxon signed-rank test) (Figure 2D). In the eight phasic cells, insulin in- creased the burst duration (from 73 ± 17 seconds to 328 ± 137 sec- onds). In these cells, the interburst period was reduced (from 64 ± 26 to 61 ± 18 seconds); the activity quotient was increased from 0.6 ± 0.1 to 0.7 ± 0.1, and the intraburst frequency was increased from 6.6 ± 0.8 to 7.2 ± 0.7 spikes s-1. Eight of 10 vasopressin cells in fasted rats and nine of sixteen vasopressin cells in non-fasted rats increased their activity by more than 10% after i.v. insulin, and the mean response of all vasopressin cells tested was greater in fasted rats than in non-fasted rats, al- though this did not reach statistical significance (Mann-Whitney U test, P = 0.63).

3 | Hazard functions

In oxytocin cells, the hazard functions conformed to the profile previously reported as typical of oxytocin cells, reflecting a pro- longed post-spike refractoriness of 30-50 ms followed by a stable plateau of excitability.42 Insulin did not affect the duration of the post-spike refractoriness but elevated the plateau level of excitabil- ity (Figure 2E). In vasopressin cells, the hazard functions also conformed to the profile previously reported as typical of vasopressin cells, re- flecting a post-spike refractoriness of 20-50 ms followed by a pe- riod of hyperexcitability (reflecting a depolarising afterpotential) before reaching a stable plateau of excitability.42 Insulin did not affect the duration of the post-spike refractoriness or the plateau level of excitability but enhanced the post-spike hyperexcitability (Figure 2F).

3 | Effect of i.v. insulin on blood glucose and spike activity of SON neurones

At the time of recording from SON neurones (~3 hours after i.p. an- aesthesia), blood glucose concentrations were 12.7 ± 0.8 mmol L-1 in fasted rats and 19.9 ± 2.0 mmol L-1 in non-fasted rats. In both groups, plasma glucose levels were unchanged after injections of vehicle (0.9% saline) but fell after i.v. insulin, reaching a nadir after 60 minutes (two-way ANOVA; interaction F(18, 162) = 6.69; time, F(6, 162) = 20.11; treatment, F3,27 = 10.8; subject, F27,162 = 12.9, all P < 0.001) (Figure 3A). To test whether the activation of SON neurones by i.v insulin re- flected the reduction in plasma glucose concentrations, we injected insulin to activate SON neurones in 10 fasted rats and then gave i.v. glucose to restore basal glucose concentrations (one-way ANOVA, F1.936, 17.42 = 9.275 P = 0.002) (Figure 3B).

We tested five oxytocin cells in four of these rats. The mean ± SEM (range) basal firing rate (1.9 ± 0.7 [0.3-4.4] spikes s-1) in- creased by 1.1 ± 0.2 (0.4-1.7) spikes s-1 after insulin. After i.v. glucose, the firing rate continued to increase reaching a final mean change of
1.6 ± 0.4 (1.0-3.0) spikes s-1 (Wilcoxon matched-pairs signed-rank test, P = 0.06) (Figure 3C). We tested six vasopressin cells (of which one fired phasically) in six of the rats (one of the vasopressin cells was recorded simulta- neously with an oxytocin cell). The mean basal firing rate (5.2 ± 0.6 (3.4-6.9) spikes s-1) increased by 1.2 ± 0.3 (0.6-2.4) spikes s-1 after in- sulin. After glucose, the rate did not change significantly (final mean change 1.3 ± 0.3 [0.6-2.3] spikes s-1; Wilcoxon matched-pairs signed- rank test, P = 0.8) (Figure 3D). Thus, in the case of both oxytocin cells and vasopressin cells, responses to insulin were unaffected by i.v injections of glucose.

3.5 | Blockade of central InsRs before i.v. insulin

To test whether the activation of SON neurones by insulin involves brain InsRs, we studied the effect of central administration of S961 on the responses (Figure 4). At 30 minutes after insulin injection, the blood glucose concentration in rats pretreated with i.c.v. S961 had fallen by 7.4 ± 1.0 mmol L-1, similar to the fall in fasted rats injected

Effect of i.v. insulin on supraoptic nucleus neurones in fasted rats. A, Representative example of the firing rate of an identified oxytocin cell (in 30-second bins; upper; instantaneous frequency plot shown below) in response to an i.v. injection of insulin. The dashed line indicates the mean basal firing rate. B, Interspike interval distribution and average waveform (lower) over 10-minute period during baseline (left, corresponding to the solid line in A) and maximal neuronal response (right, corresponding to the dotted line in A) of the neurone shown in A. C, Average responses (mean ± SEM changes from baseline) of 10 oxytocin cells in fasted rats (black) and 10 in non-fasted rats (white) to
i.v. insulin. D, Average responses of 16 vasopressin cells in fasted rats (black) and 16 in non-fasted rats (white) to i.v. insulin. E, Mean ± SEM hazard functions of the 10 oxytocin cells in fasted rats before (closed symbols) and after insulin (open symbols). F, Mean ± SEM hazard functions of eight vasopressin cells in fasted rats before (closed symbols) and after insulin (open symbols; two cells excluded because the basal firing rates were too low to construct hazard functions)

However, i.v. insulin had no effect in rats pretreated with S961 (change −0.01 ± 0.1 [−0.4 to 0.3) spikes s-1 after i.v. insulin; Wilcoxon signed-rank test, P = 1.0) (Figure 4A). Similarly, the firing rate of eleven vasopressin cells (5.7 ± 0.3 [3.7- 7.7) spikes s-1; six continuous- and five phasic cells in 10 rats) was not significantly affected by S961 (change 0.16 ± 0.3 [−2.3 to 1.7) spikes s-1; Wilcoxon signed-rank test, P = 0.37). After i.v. insulin, their firing rate increased by 0.5 ± 0.4 [−1.5 to 3.3] spikes s-1, although this was not significant (Wilcoxon signed-rank test, P = 0.24) (Figure 4B). Thus, central administration of S961 blocked the responsiveness of oxytocin cells to systemic administration of insulin but had no sig- nificant effect on vasopressin cells.

Effect of i.v. glucose infusion in insulin-responsive neurones in fasted rats. A, Blood glucose concentrations were lowered after i.v. insulin, but not i.v. vehicle, in fasted (F) and non-fasted (NF) rats (*P < 0.05, Two-way ANOVA followed by a Bonferroni post-hoc test). B, Blood glucose concentrations after i.v. insulin and after i.v. insulin, and 5% glucose solution injections (arrows: as required) of all 10 rats where neuronal activity was recorded. After 30-minutes of i.v. insulin, the glucose concentration was significantly lower compared to all other blood samples (one-way ANOVA for repeated measures; ***P < 0.001, Bonferroni post-hoc test) with no significant differences between other samples. B, Blood glucose concentrations after i.v. insulin, and 5% glucose solution injections (arrows: 400 µL, *300 µL, *100 µL; * if required) of all animals (n = 10) where neural activity was recorded. C, D, After insulin, no significant differences in firing rate of (C) oxytocin and (D) vasopressin cells in glucose-treated rats were detected compared to non-glucose-treated fasted rats. Data are the mean ± SEM Effect of central insulin receptor (InsR) blockade on neuronal responses following i.v. insulin in fasted rats. A, B, Administration of S961 (1 nmol i.c.v.) blocked the increase in firing rate (in 10-minutes bins) induced by i.v. insulin in (A) oxytocin, but not (B) vasopressin cells, compared to fasted rats injected with i.v. insulin alone (Figure 3). C, Blood glucose concentration tended (P = 0.13) to increase 15 minutes after of i.c.v. InsR antagonist administration, insulin i.v. significantly lowered the blood glucose concentration after 30 minutes compared to basal, and InsR antagonist blood samples (***P < 0.001, one-way ANOVA for repeated measurements, followed by a Bonferroni post-hoc test; n = 15), in brackets, times after basal. Data are the mean ± SEM

3.6 | Effect of blockade of central InsRs on oxytocin spike activity induced by SCM gavage

Gavage of food rich in sugars, but not fat, results in a rise of blood glucose and insulin plasma concentration and a progressive increase in the electrical activity of oxytocin cells.37 Here, we tested whether this involves brain InsRs. Both vehicle- and S961-injected rats exhibited a significant in- crease in both blood glucose concentration and plasma insulin con- centration following SCM gavage (Figure 5A) with no significant differences between groups (glucose: two-way ANOVA for repeated measures: interaction, F3,24 = 0.7769, P = 0.52; time, F3,24 = 44.41,P < 0.0001; treatment, F1,8 = 0.4899, P = 0.5038; subjects, F8,24 = 29.9, P < 0.0001; insulin: interaction, F3,18 = 0.083, P = 0.97; interaction, F3,18 = 10.17, P = 0.0004; treatment, F1,6 = 0.18, P = 0.7; subjects, F6,18 = 2.1, P = 0.1). In vehicle-injected rats, as expected,37 SON oxytocin cells were progressively activated during SCM gavage. The firing rate of five oxytocin cells (from four rats) increased from 2.5 ± 0.3 [1.3-3.1] spikes s-1 by 1.1 ± 0.1 [0.7-1.5] spikes s-1 over during 60 minutes of gavage (Figure 5B). By contrast, in rats injected with S961, oxytocin cells did not respond to SCM gavage (Figure 5C). The firing rate of five oxytocin cells (from five rats) increased from 3.2 ± 0.8 (1.4-5.4) spikes s-1 by −0.1 ± 0.3 [−1.0 to 0.7] spikes s-1 during 60 minutes of gavage. This response was significantly different to the control group (*P = 0.016 Mann-Whitney test) (Figure 5D,E).


Recently, the role of oxytocin cells in metabolic regulation has at- tracted increasing attention. Most attention has been given to the oxytocin cells of the PVN because these include a small population of parvocellular neurones that project to the dorsal vagal complex and to the spinal cord, where their actions include effects on gastric reflexes, energy intake and thermogenesis.23,43,44 Until relatively recently, the magnocellular oxytocin cells, which comprise most of the oxytocin cells in the PVN and all those in the SON, were considered to have few central projections. However, these neurones, which all project axons Effect of central insulin receptor (InsR) blockade on sweet condensed milk (SCM)-induced increase in firing rate in oxytocin cells. A, Gavage of 5 mL of SCM significantly increased blood glucose concentrations (left; P < 0.05, Two-way ANOVA followed by a Bonferroni post-hoc test compared to baseline) and plasma insulin (right) in both i.c.v. vehicle- and S961-treated rats with no significant differences between groups. B, Representative examples showing the increase in firing rate (in 30-second bins) induced by SCM gavage (5 mL) in a vehicle-injected rat (upper) and blockade of SCM-induced response in an. S961-injected rat. C, Mean ± SEM change in firing rate over 60 minutes from all oxytocin cells recorded in i.c.v. vehicle- and S961-injected rats gavaged with SCM (*P < 0.05 Mann-Whitney test). D, The change in firing rate (in 10-min bins) was significantly different between groups after 30 minutes after the beginning of SCM gavage. Data are the mean ± SEM to the posterior pituitary, also release large amounts of oxytocin within the brain from their dendrites. This dendritic release is likely to have important effects at relatively local sites, including the amygdala and the ventromedial nucleus of the hypothalamus where abundant oxy- tocin receptors are expressed but which contain only sparse oxytocin fibres.23,45 In addition, it has recently become apparent that many mag- nocellular neurones have extensive axonal projections to diverse brain regions, including notably to the nucleus accumbens.

In the present study, systemic administration of insulin increased the electrical activity of both oxytocin and vasopressin SON cells, consistent with previous reports in humans and rats that insulin in- creases secretion of oxytocin and vasopressin.47-49 As originally conceived in the design of the present experiments, the dose and route of insulin administration followed the conventional design of insulin tolerance tests50 to produce an acute maintained hypoglycaemia. This bolus injection raises peripheral insulin concen- trations above the normal physiological range, which are then rapidly cleared. The evolution of oxytocin cell activity after insulin injections thus mirrored neither the changes in plasma glucose, nor the expected changes in peripheral insulin concentration. Insulin crosses the blood- brain barrier by an active transport mechanism that is saturated: at least 50% of maximal transport capacity is reached at euglycemic lev- els of plasma insulin; thus, supraphysiological levels of insulin in the plasma have little additional effect on insulin penetration into the brain beyond that seen at high physiological levels.1,51 Thus, the expected evolution of CNS insulin following i.v. bolus injection is a progressive rise when peripheral levels are elevated above normal levels, possibly explaining the progressive rise in oxytocin cell activity.

Brain InsRs play an important role in the control of energy balance as shown by selective genetically-induced decreased expression of brain InsRs which is linked to a peripheral metabolic alterations, includ- ing increased food intake, fat and body weight, as well as increased glu- cose and insulin resistance in rodents.52,53 Moreover, injection of the InsR antagonist S961 into the ventromedial nucleus increases blood glucose concentration in rats.54 In the present study, central adminis- tration of S916 prevented the insulin-induced responses in all oxytocin cells, indicating that systemic insulin penetrates into the brain to acti- vate SON neurones by actions on central InsRs. The central adminis- tration of S916 produced a non-significant increase in plasma glucose concentrations consistent with its reported effects in the ventromedial nucleus,54 and did not affect the effect of systemically applied insulin on plasma glucose levels, indicating that, at this dose and by this route, it did not block systemic effects of exogenous insulin.

In humans, glucose, but not fructose, infusion has been shown to prevent oxytocin and vasopressin release by insulin-induced hy- poglycaemia.55 In the present study, we infused bolus injections of glucose solution to approximately clamp circulating glucose concen- trations after i.v. insulin. Once the firing response was triggered, nei- ther oxytocin, nor vasopressin cells reduced their spike activity after glucose injections.
In non-fasted rats, which exhibited a more pronounced hyper- glycaemia than fasted rats, the responses of oxytocin cells were less prominent than in fasted rats. This may reflect InsR desensitisation in oxytocin cells, similarly to that shown in skeletal muscle in vivo56 and fibroblasts in vitro,57 where acute exposition to high glucose con- centration reduced insulin-stimulated glucose uptake and impaired InsR intracellular signalling, respectively. Alternatively, because, in fasted animals, blood glucose concentrations fell following insulin administration to concentrations lower than immediately after an- aesthesia, this might stimulate the hypothalamic-pituitary-adrenal as occurs in the insulin tolerance test,55 potentiating the release of oxytocin (and vasopressin).

A recent study17 raised a question about the capacity of SON neurones to respond to insulin administration because insulin given
i.c.v. induced an increase in Fos expression after 90-minutes in 13% of the PVN, but not SON, oxytocin cells compared to control mice. Nevertheless, SON neurones appear to be intrinsically sensitive to insulin and glucose because they express InsR12-14 and the enzyme glucokinase,58 a marker for glucose sensing. Moreover, vasopressin and oxytocin are released from SON explants in the presence of medium containing glucose and insulin.16 Although Fos protein has been widely used as a marker for neuronal activation, its lack of expression does not necessarily exclude changes in neural activity as observed in some conditions, and increased spike activity is not invariably linked to Fos expression.59,60 It appears that insulin might not induce the expected rapid expression of Fos (ie, 60-90 minutes) because Griffond et al15 reported that, at 1 hour after insulin i.p. (20 mg kg-1), there was little expression of Fos in PVN oxytocin cells. A limitation of the present study is that it involved urethane-anes- thetised rats. Urethane has long been the anaesthetic of choice for SON electrophysiological recordings because it provides a deep long-lasting stable anaesthesia compatible with transpharyngeal surgery without affecting the physiological responses of SON neurones.40 However, urethane raises blood glucose concentrations61,62 by increasing sym- pathetic tone63 and consequently increasing gluconeogenesis. Thus, blood glucose concentrations in both non-fasted and fasted anaes- thetised rats were higher than in conscious Sprague-Dawley rats.64 However, they were lower in fasted rats than in non-fasted rats, and
changed in the expected manner in response to i.v. insulin.

This work was supported by the BBSRC (BB/S000224/1).

The authors declare that they have no conflicts of interest.

The study was designed by GL and performed by LP. LP and GL ana- lysed the data and wrote the paper together. GL had full access to all the data and analyses, and takes responsibility for the integrity of the data and the accuracy of the analyses.

The datasets generated during and/or analysed during the present study are available from the corresponding author upon reasonable request.

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How to cite this article: Paiva L, Leng G. Peripheral insulin administration enhances the electrical activity of oxytocin and vasopressin neurones in vivo. J Neuroendocrinol.
2020;00:e12841. https://doi.org/10.1111/jne.12841