α‐Melanocyte‐stimulating hormone inhibition of oxytocin neurons switches to excitation in late pregnancy and lactation

Abstract

Oxytocin is secreted into the periphery by magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei (SON and PVN) to trigger uterine contraction during birth and milk ejection during suckling. Peripheral oxytocin secretion is triggered by action potential firing, which is regulated by afferent input activity and by feedback from oxytocin secreted into the extracellular space from magnocellular neuron somata and dendrites. A prominent input to oxytocin neurons arises from proopiomelanocortin neurons of the hypothalamic arcuate nucleus that secrete an alpha-melanocyte-stimulating hormone (α-MSH), which inhibits oxytocin neuron firing in non-pregnant rats by increasing somato-dendritic oxytocin secretion. However, α-MSH inhibition of oxytocin neuron firing is attenuated in mid-pregnancy and somato-dendritic oxytocin becomes auto-excitatory in late-pregnancy and lactation. Therefore, we hypothesized that attenuated α-MSH inhibition of oxytocin neuron firing marks the beginning of a transition from inhibition to excitation to facilitate peripheral oxytocin secretion for parturition and lactation. Intra-SON microdialysis administration of α-MSH inhibited oxytocin neuron firing rate by 33 ± 9% in non-pregnant rats but increased oxytocin neuron firing rate by 37 ± 12% in late-pregnant rats and by 28 ± 10% in lactating rats. α-MSH-induced somato-dendritic oxytocin secretion measured ex vivo with oxytocin receptor-expressing "sniffer" cells, was of similar amplitude in PVN slices from non-pregnant and lactating rats but longer-lasting in slices from lactating rats. Hence, α-MSH inhibition of oxytocin neuron activity switches to excitation over pregnancy while somato-dendritic oxytocin secretion is maintained, which might enhance oxytocin neuron excitability to facilitate the increased peripheral secretion that is required for normal parturition and milk ejection.

Full text

Physiological Reports. 2022;10:e15226.
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1 of 10
https://doi.org/10.14814/phy2.15226
wileyonlinelibrary.com/journal/phy2
Received: 9 February 2022
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Revised: 21 February 2022
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Accepted: 23 February 2022
DOI: 10.14814/phy2.15226
ORIGINAL ARTICLE
α- Melanocyte- stimulating hormone inhibition of oxytocin
neurons switches to excitation in late pregnancy and
lactation
Michael R.Perkinson1,2,3
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Matthew K.Kirchner4
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MengZhang4
|
Rachael A.Augustine1,2,3
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Javier E.Stern4
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Colin H.Brown1,2,3
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.
© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.
1Brain Health Research Centre,
University of Otago, Dunedin, Aotearoa
New Zealand
2Centre for Neuroendocrinology,
University of Otago, Dunedin, Aotearoa
New Zealand
3Department of Physiology, University
of Otago, Dunedin, Aotearoa New
Zealand
4Center for Neuroinflammation and
Cardiometabolic Diseases, Georgia
State University, Atlanta, Georgia, USA
Correspondence
Colin H. Brown, Department of
Physiology, School of Biomedical
Sciences, University of Otago, PO
Box 56, Dunedin 9054, Aotearoa New
Zealand.
Email: colin.brown@otago.ac.nz
Funding information
This work was supported by a Manatu
Hauora | Health Research Council of
New Zealand Grant (Colin H. Brown),
a University of Otago Postgraduate
Scholarship (Michael R. Perkinson),
National Heart, Lung, and Blood
Institute Grant NIH HL090948
(Javier E. Stern), National Institute
of Neurological Disorders and Stroke
Grant NIH NS094640 (Javier E. Stern)
and National Heart, Lung, and Blood
Institute Grant 1F32HL158172- 01
(Matthew K.Kirchner).
Abstract
Oxytocin is secreted into the periphery by magnocellular neurons of the hypo-
thalamic supraoptic and paraventricular nuclei (SON and PVN) to trigger uterine
contraction during birth and milk ejection during suckling. Peripheral oxytocin
secretion is triggered by action potential firing, which is regulated by afferent
input activity and by feedback from oxytocin secreted into the extracellular space
from magnocellular neuron somata and dendrites. A prominent input to oxy-
tocin neurons arises from proopiomelanocortin neurons of the hypothalamic ar-
cuate nucleus that secrete an alpha- melanocyte- stimulating hormone (α- MSH),
which inhibits oxytocin neuron firing in non- pregnant rats by increasing somato-
dendritic oxytocin secretion. However, α- MSH inhibition of oxytocin neuron
firing is attenuated in mid- pregnancy and somato- dendritic oxytocin becomes
auto- excitatory in late- pregnancy and lactation. Therefore, we hypothesized that
attenuated α- MSH inhibition of oxytocin neuron firing marks the beginning of
a transition from inhibition to excitation to facilitate peripheral oxytocin secre-
tion for parturition and lactation. Intra- SON microdialysis administration of α-
MSH inhibited oxytocin neuron firing rate by 33±9% in non- pregnant rats but
increased oxytocin neuron firing rate by 37±12% in late- pregnant rats and by
28± 10% in lactating rats. α- MSH- induced somato- dendritic oxytocin secretion
measured ex vivo with oxytocin receptor- expressing “sniffer” cells, was of similar
amplitude in PVN slices from non- pregnant and lactating rats but longer- lasting
in slices from lactating rats. Hence, α- MSH inhibition of oxytocin neuron activity
switches to excitation over pregnancy while somato- dendritic oxytocin secretion
is maintained, which might enhance oxytocin neuron excitability to facilitate the
increased peripheral secretion that is required for normal parturition and milk
ejection.
KEYWORDS
lactation, oxytocin, pregnancy, somato- dendritic secretion, vasopressin

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1
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INTRODUCTION
In mammals, delivery and feeding of the newborn re-
quires the nonapeptide, oxytocin, which is synthesized
and secreted by magnocellular neurons of the supraoptic
nucleus (SON) and paraventricular nucleus (PVN) in the
hypothalamus. Oxytocin secretion into the circulation is
triggered by action potential (spike) invasion of the axon
terminals of the posterior pituitary gland. Over the course
of pregnancy, the oxytocin system is modified to prepare
for increased secretion necessary for uterine contraction
during parturition and milk ejection during suckling.
While intrinsic changes occur in oxytocin neurons over
pregnancy to facilitate increased activity (Armstrong
et al., 2002; Perkinson et al., 2021), changes in neuronal
and hormonal inputs also contribute to increased oxytocin
secretion at parturition and in lactation (Augustine et al.,
2017; Brussaard et al., 1997; Israel & Poulain, 2000; Oliet
et al., 2001; Stern et al., 2000).
In addition to axon terminal secretion, oxytocin neu-
rons also secrete oxytocin from their somata and den-
drites (somato- dendritic secretion) to modulate their own
excitability. Oxytocin neurons express oxytocin receptor
mRNA (Meddle et al., 2007) and oxytocin receptor acti-
vation increases intracellular calcium (Lambert et al.,
1994) to decrease the firing rate of oxytocin neurons by
triggering endocannabinoid release for retrograde inhi-
bition of glutamatergic synaptic transmission (Hirasawa
et al., 2004; Kombian et al., 1997). However, the activa-
tion of oxytocin receptors switches to excitation of oxy-
tocin neurons in lactation (Freund- Mercier & Richard,
1984; Moos et al., 1989a). Furthermore, oxytocin recep-
tor mRNA expression increases in oxytocin neurons over
pregnancy (Meddle et al., 2007) and somato- dendritic oxy-
tocin release increases immediately before each burst of
action potential firing in oxytocin neurons during lacta-
tion (Moos et al., 1989a). Hence, it appears that regulation
of oxytocin neuron activity by somato- dendritic oxytocin
secretion changes over pregnancy to facilitate increased
axon terminal secretion of oxytocin into the circulation
for birth and lactation.
While both depend on intracellular calcium, axon ter-
minal secretion and somato- dendritic secretion can occur
independently from oxytocin neurons, suggesting that
different mechanisms control secretion from different
compartments of the neurons (Pitra et al., 2019), which
would allow for differential modulation of the two modes
of secretion by afferent inputs (Ludwig & Leng, 2006).
Alpha- melanocyte- stimulating hormone (α- MSH) is syn-
thesized by proopiomelanocortin (POMC) neurons of the
hypothalamic arcuate nucleus and has been shown to dif-
ferentially modulate axon terminal secretion and somato-
dendritic secretion of oxytocin. Oxytocin neurons express
receptors for α- MSH (MC4R) and receive inputs from
POMC neurons (Douglas et al., 2002). In non- pregnant
rats, MC4R activation inhibits peripheral oxytocin secre-
tion through reduced action potential firing and simul-
taneously increases somato- dendritic oxytocin secretion
via increased intracellular calcium (Sabatier et al., 2003).
However, α- MSH inhibition of oxytocin neuron activity is
lost by mid- pregnancy (Ladyman et al., 2016) and it is un-
known whether α- MSH stimulation of somato- dendritic
oxytocin is modulated by reproductive status.
Therefore, we hypothesized that attenuation of α- MSH
inhibition of oxytocin neuron activity in mid- pregnancy
represents the beginning of a transition from inhibition
to excitation to facilitate peripheral oxytocin secretion for
parturition and lactation and that α- MSH stimulation of
somato- dendritic oxytocin secretion would be maintained
to support enhanced peripheral oxytocin secretion for par-
turition and lactation. Consistent with our hypotheses, we
found that intra- SON administration of α- MSH inhibited
oxytocin neurons in non- pregnant rats but excited oxyto-
cin neurons in late- pregnant and lactating rats and that
α- MSH- induced somato- dendritic oxytocin secretion in-
duced similar responses in oxytocin receptor- expressing
“sniffer” (snifferOT) cells in PVN slices from non- pregnant
and lactating rats.
2
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METHODS
2.1
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Ethics approvals
Electrophysiology procedures were approved by the
University of Otago Animal Ethics Committee and were
carried out in accordance with the New Zealand Animal
Welfare Act and associated guidelines. Sniffer cell pro-
cedures were approved by Georgia State University
Institutional Animal Care and Use Committee and were
carried out in accordance with NIH guidelines.
2.2
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Animals
For electrophysiology, 10- week- old, female Sprague–
Dawley rats were purchased from the University of
Otago Animal Facility. Rats were group- housed until
mid- pregnancy (gestation day 14, G14) then housed
individually until the day of the experiment. Rats were
kept in controlled light conditions (12h– 12h; lights on
at 07.00h; 22±1°C) with ad libitum access to standard
rat chow and tap water. To generate late- pregnant and
lactating rats, daily vaginal smears were taken to moni-
tor the estrous cycle (Hubscher et al., 2005). Following
identification of a pro- estrous smear, rats were housed

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PERKINSON et al.
overnight with a male rat and sperm present in the next
morning's smear was determined as G0. Lactating rats
typically gave birth on days 21– 22 of gestation (post-
partum day 1; PP1).
For sniffer cells experiments, non- pregnant Sprague–
Dawley rats were group- housed under controlled con-
ditions (light- dark cycle [12 h– 12 h; 22– 24°C]), with
ad libitum access to standard rat chow and tap water.
Lactating rats were purchased from the Charles River
Laboratory and delivered for use on PP14.
In all experiments, non- pregnant rats were freely-
cycling to avoid any confounding effect of the estrous
cycle.
2.3
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In vivo electrophysiology
On the day of electrophysiology, non- pregnant, late-
pregnant (G18– G21) or lactating (PP7– PP17) rats were
anesthetized with 1.25gkg−1 I.P. urethane (ethyl carba-
mate, Sigma, St Louis, MO, USA). Once cessation of the
pedal withdrawal reflex was confirmed, the left femoral
vein was catheterized to allow systemic administration
of (Tyr[SO3H]27)cholecystokinin fragment 26– 33 amide
(CCK8S; Sigma). The pituitary stalk and right SON were
exposed by transpharyngeal surgery (Brown et al., 2014).
A U- shaped microdialysis probe (permeable to 10kDa; in-
house design (Horn & Engelmann, 2001)) was placed onto
the ventral surface of the brain.
Extracellular single- unit recordings were made with
a glass recording microelectrode (15– 40MΩ; filled with
0.9% saline) connected to a Neurolog system (Digitimer
Ltd, UK). A side- by- side SNEX- 200 bipolar stimulating
electrode (Science Products GmbH, Hofheim, Germany)
was placed on the pituitary stalk to depolarize the axons
and elicit antidromic action potentials in SON neurons.
Neuronal activity was recorded via a CED 1401 analog–
digital interface (Cambridge Electronic Design) using
Spike 2 software (Cambridge Electronic Design) and
analyzed offline. Neurons were characterized as oxy-
tocin neurons on the basis of a transient excitation of
greater than 0.5spikes s−1 averaged over 5min follow-
ing IV CCK8S injection (20µgkg−1, 0.5mlkg−1 in 0.9%
saline) (Brown et al., 1996), or as vasopressin neurons
by transient inhibition, or no effect, following CCK8S
injection (Scott et al., 2009). The SON was continu-
ously dialyzed with artificial cerebrospinal fluid (aCSF;
mmolL−1: NaCl 138, KCl 3.36, NaHCO3 9.52, Na2HPO4
0.49, urea 2.16, CaCl2 1.26, MgCl2 1.18; osmolality 295–
300 mosmol kg−1) and switched to α- MSH in aCSF
(Sigma; 1.5mM) at 3μlmin−1 for 30min. At the end of
the experiment, the rats were euthanized by IV injection
of 0.5mlKCl (3molL−1).
2.4
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Slice preparation for snifferOT cells
Hypothalamic brain slices were prepared as previously
described (Pitra et al., 2019; Son et al., 2013). Briefly, rats
were anesthetized with pentobarbital (50 mg kg−1 I.P.).
Once fully anesthetized, rats were transcardially perfused
with ice- cold sucrose aCSF containing (in mM): 2.5KCl,
1MgSO4, 26NaHCO3, 1.25NaH2PO4, 20 D- glucose, 0.4
ascorbic acid, 2 CaCl2, and 210sucrose; pH 7.3; 295mosm.
The brain was dissected out and coronal slices (240 μm)
of the hypothalamus containing the PVN were cut in
the same ice- cold sucrose aCSF constantly bubbled with
95%O2/5%CO2. Once the brain slices were cut, they were
transferred to a holding chamber containing standard
aCSF warmed at 32°C for 20min and then resting at room
temperature for at least 40 min before the start of the
experiment.
2.5
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SnifferOT cells
Full methods are as described in Pitra et al. (2019). Briefly,
snifferOT cells were generated by culturing Chinese ham-
ster ovary (CHO) cells in Dulbecco's Modified Eagle
Medium containing 10% w/v fetal bovine serum, 1% w/v
penicillin– streptomycin, 1% w/v Na- Pyruvate, and 1% w/v
NaCO3 filtered once through a Nalgene filtration system,
and transfecting with pcDNA3.1+ containing human
oxytocin receptors cloned in EcoRI (5’) and XhoI (3’)
(plasmid obtained from Missouri S&T cDNA Resource
Center, Rolla, MO, USA) using lipofectamine, and stable
overexpression was achieved by geneticin (500mg ml−1)
selection (Piñol et al., 2014). SnifferOT cells were then
plated and transiently transfected to express the red fluo-
rescent genetically encoded calcium indicator (R- GECO;
GenScript, Piscataway, NJ, USA) with Fugene HD reagent
(Promega, Madison, WI, USA).
SnifferOT cells were resuspended in standard aCSF
([in mmol L−1]: 119NaCl, 2.5KCl, 1MgSO4, 26NaHCO3,
1.25NaH2PO4, 20 D- glucose, 0.4 ascorbic acid, 2 CaCl2,
and 2 pyruvic acid; pH 7.3; 295 mosm) with trypsin
(0.05%). SnifferOT cells were transferred directly onto
the lateral magnocellular subdivision of the PVN in
brain slices after pausing aCSF superfusion. After at
least 5min, aCSF superfusion was resumed for 5min to
wash off any unattached snifferOT from the slice before
proceeding with the recording. SnifferOT cells adopted
a rounded appearance when transferred to aCSF and
no further overt morphological changes were observed
during the equilibration period on the brain slices.
Experiments were restricted to preparations that had at
least five fluorescently visible sniffer cells in the field
(~10 on average).

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2.6
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Imaging and analysis of the calcium
changes in snifferOT cells
To record the calcium- induced fluorescence changes
of snifferOT cells, images were taken with the Andor
Technology Revolution system (iXON EMCCD camera
with the Yokogawa CSU 10 [Tokyo, Japan], confocal
scanning unit; Belfast, UK), at a rate of 4Hz. The sniff-
erOT cell fluorescence was imaged under a 488nm exci-
tation light and the calcium response was measured at
>495nm (Fluo- 5F). To test for somato- dendritic secretion
of oxytocin in response to α- MSH, hypothalamic slices
were constantly superfused with aCSF at 32°C. Slices
were stimulated with a 1ml bolus of α- MSH (1µM; con-
centration on slice ~0.1µM) and then with 1ml oxytocin
(10 µM; concentration on slice ~1µM; at least 10 min
after α- MSH). Drugs were pumped into the aCSF line by
hand at ~0.1 mls−1 and fluorescent calcium responses
were monitored in surrounding snifferOT cells. Responses
to α- MSH are reported only from snifferOT cells that re-
sponded to exogenous oxytocin with an increase in fluo-
rescence of >20% from baseline.
Each slice was imaged separately. Imaging data were
analyzed using ImageJ software (NIH). For quantitative
measurements, fractional fluorescence (F/F0) was deter-
mined by dividing the fluorescence intensity (F) within
a region of interest by a baseline fluorescence value (F0)
determined from 30 frames/images before stimulation
(Stern & Potapenko, 2013). Peak calcium amplitude was
the maximum F/F0 achieved after α- MSH. Latency to
the response was determined as the time between the
start of the α- MSH bolus and the start of the calcium
response of each sniffer cell. Response duration was
the duration from the start of the response to the return
baseline. The area under the curve was calculated for the
duration of the response. Response rates are the number
of cells that responded to both a- MSH and oxytocin rela-
tive to all cells that responded to oxytocin. SnifferOT cells
that showed intrinsic oscillatory calcium activity were
excluded from the analysis. To better display changes in
fluorescence levels, images were pseudocolored using
ImageJ.
2.7
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Data analysis and statistics
Hazard function analysis was done on the 10min pre- α-
MSH and last 10min of α- MSH administration (Methods
and Results in Supplementary Figures S1 and S2). All
values are reported as mean ± standard error of the
mean (SEM). Statistical analyses were completed using
Sigma Plot version 12 for Windows (Systat Software,
San Jose, CA, USA) or Prism version 9.1.0 (GraphPad
Software, San Diego, CA, USA). One- way or two- way
ANOVA was used to compare multiple groups; where
the F- ratio was significant, ANOVA was followed by all-
pairwise Holm– Sidak post hoc tests. The Student's t- test
was used to compare sniffer cell data. p<0.05 was con-
sidered significant.
3
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RESULTS
3.1
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Spontaneous firing rate of
magnocellular neurons was similar in all
reproductive states
Spontaneous firing rate was analyzed from the 10min
prior to α- MSH for 33 oxytocin neurons and 24 vaso-
pressin neurons from 13 non- pregnant rats, ten late-
pregnant rats and 17 lactating rats. There was no
difference between the firing rate of oxytocin neu-
rons (F2,29 = 0.18, p = 0.84, one- way ANOVA) in
non- pregnant (5.23 ± 0.72 spikes s−1, n = 10), late-
pregnant (5.19 ± 1.40spikes s−1, n = 7) and lactating
rats (4.59±0.70 spikes s−1, n=15). Vasopressin neu-
ron firing rate was also similar (F2,21= 1.37, p=0.28)
between non- pregnant (6.11±0.60spikes s−1, n=10),
late- pregnant (6.92±0.80spikes s−1, n=6), and lactat-
ing rats (5.08±0.85spikes s−1, n=8).
3.2
|
Alpha- melanocyte- stimulating
hormone inhibits oxytocin neurons
in non- pregnant rats but excites
oxytocin neurons in late- pregnant and
lactating rats
To determine whether α- MSH induced changes in ox-
ytocin neuron firing rate is affected by reproductive
status, in vivo electrophysiological recordings were
maintained during α- MSH administration for 30 min
in ten oxytocin neurons from nine non- pregnant rats,
seven oxytocin neurons from seven late- pregnant rats,
and 15 oxytocin neurons from 14lactating rats. The ef-
fect of intra- SON α- MSH administration on oxytocin
neuron firing rate was dependent on reproductive status
(interaction between REPRODUCTIVE STATUS and
TIME F(6, 87)=11.25, p<0.001, two- way RM ANOVA;
Figure 1). Consistent with previous observations, α- MSH
administration progressively decreased oxytocin neuron
firing rate in non- pregnant rats (p<0.001, Holm– Sidak
test). In contrast to non- pregnant rats, α- MSH adminis-
tration progressively increased the firing rate of oxytocin
neurons in late- pregnant rats (p=0.014) and lactating
rats (p=0.002).

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3.3
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Alpha- melanocyte- stimulating
hormone has no effect on the firing rate of
vasopressin neurons in non- pregnant, late-
pregnant, or lactating rats
To test whether the α- MSH effects on firing rate were
specific to oxytocin neurons, we made in vivo electro-
physiological recordings during α- MSH administration
in ten vasopressin neurons from seven non- pregnant
rats, six vasopressin neurons from five late- pregnant rats,
and eight vasopressin neurons from seven lactating rats.
Vasopressin neuron firing rate was unaffected by α- MSH
administration (interaction between REPRODUCTIVE
STATUS and TIME: F6, 63=1.24, p=0.30, two- way RM
ANOVA, Figure 2).
3.4
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Alpha- melanocyte- stimulating
hormone- induced similar snifferOT cell
calcium responses in PVN slices from non-
pregnant and lactating rats
To test whether α- MSH stimulated somato- dendritic oxy-
tocin secretion is maintained during lactation, snifferOT
cells were plated onto hypothalamic slices containing the
PVN and the calcium response induced by exogenous α-
MSH was recorded. α- MSH was superfused onto slices
from three non- pregnant rats (12 slices) and two lac-
tating rats (12slices). The proportion of snifferOT cells
that responded to α- MSH was similar in slices from non-
pregnant rats (61.5%; 32/52) and lactating rats (72.7%;
24/33; Chi- square=0.45, p=0.50). There was no differ-
ence in the peak amplitude (p= 0.93, Student's t- test),
the area under the curve (p=0.17) or latency to response
after α- MSH between slices from non- pregnant rats and
lactating rats (p=0.16; Figure 3). However, there was
a longer α- MSH- induced response duration in snifferOT
cells on slices from lactating rats than in non- pregnant
rats (p=0.03).
FIGURE  The effect of α- MSH on the firing rate of oxytocin
neurons is dependent on reproductive status. (a– c) Example
ratemeter recordings (in 1min bins) of oxytocin neurons during
microdialysis alpha- melanocyte- stimulating hormone (α-
MSH; 1.5mM) administration for 30min in non- pregnant (a),
late- pregnant (b) and lactating (c) urethane- anesthetized rats.
Microdialysis α- MSH administration was maintained for 30min
in 11 oxytocin neurons from ten non- pregnant rats, seven oxytocin
neurons from seven late- pregnant rats (G18– G21) and 15 oxytocin
neurons from 13lactating rats (PP7– L17). (d) Percentage change
in firing rate±SEM (in 10min bins) of oxytocin neurons during
30min microdialysis α- MSH administration compared to the
initial firing rate prior to α- MSH administration. Administration of
α- MSH caused a change in firing rate of oxytocin neurons that was
dependent on the reproductive status of the rats (REPRODUCTIVE
STATUS [RS]: F2,29=0.34, p=0.71; TIME: F2,29=0.43, p=0.66;
interaction between RS and TIME: F6,87=11.249, p<0.001, two-
way repeated measures ANOVA). **p=0.002 and ***p<0.001
compared to pre- α- MSH in non- pregnant rats, ‡p=0.014 compared
to pre- α- MSH in late- pregnant rats, ††p=0.002 compared to pre- α-
MSH in lactating rats, Holm– Sidak post hoc tests. However, there
were no differences in the firing rate of oxytocin neurons between
non- pregnant, late- pregnant and lactating rats at any time point

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4
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DISCUSSION
Here, we found that α- MSH inhibition of oxytocin neurons
switches to excitation in late- pregnant rats and that this
switch to excitation is maintained into lactation. We also
found that α- MSH- induced somato- dendritic oxytocin se-
cretion is maintained in lactating rats, with a modest in-
crease in the duration of the calcium response in snifferOT
cells on slices from lactating rats. While the prolonged
snifferOT cell response could result from subtle differences
in washout between brain slice preparations and/or re-
duced breakdown of oxytocin in the extracellular space
in lactation, the tortuosity of the extracellular space is de-
creased in the SON during lactation (Piet et al., 2004) and
hypothalamic oxytocinase activity is consistently higher
in lactating rats than in non- pregnant rats (Tobin et al.,
2014). Hence, the prolonged snifferOT cell response likely
results from increased α- MSH- induced somato- dendritic
oxytocin secretion in lactation, rather than reduced oxy-
tocin breakdown.
While the somato- dendritic oxytocin secretion trig-
gered by exogenous α- MSH was only modestly higher in
slices from lactating rats than in slices from non- pregnant
rats, it is possible that the stimulation by endogenous α-
MSH in lactation is more prominent than the endogenous
inhibition before pregnancy because arcuate nucleus
POMC mRNA expression increases in over the course of
pregnancy (Douglas et al., 2002). Indeed, oxytocin lev-
els rise in the SON during suckling in rats (Moos et al.,
1989b). Hence, plasticity in α- MSH modulation of oxy-
tocin neurons appears to be part of the suite of physio-
logical adaptations required to prepare the system for the
increased oxytocin secretion necessary for successful par-
turition and lactation.
α- MSH inhibits oxytocin neuron firing rate in non-
pregnant rats by increasing somato- dendritic oxytocin
secretion to trigger retrograde inhibition of synaptic trans-
mission via endocannabinoid activation of cannabinoid
1 (CB1) receptors on excitatory axon terminals (Sabatier
& Leng, 2006; Sabatier et al., 2013). The mechanism that
underpins the switch to α- MSH stimulation of oxytocin
neuron firing rate in late- pregnant and lactating rats has
yet to be determined but cannot be simply explained by
receptor downregulation or desensitization. We used the
hazard function to investigate changes in post- spike ex-
citability (Brown et al., 2007) of oxytocin neurons over
FIGURE  α- MSH has no effect on the firing rate of
vasopressin neurons. (a– c) Example ratemeter recordings (in
1min bins) of vasopressin neurons during microdialysis alpha-
melanocyte- stimulating hormone (α- MSH; 1.5mM) administration
for 30min in non- pregnant (a), late- pregnant (b) and lactating (c)
urethane- anesthetized rats. Microdialysis α- MSH administration
was maintained for 30min in 10 vasopressin neurons from seven
non- pregnant rats, six vasopressin neurons from five late- pregnant
rats (G18– G20) and eight vasopressin neurons from seven lactating
rats (PP7– L16). (d) Percentage change in firing rate±SEM (in
10min bins) of vasopressin neurons during 30min microdialysis
α- MSH administration compared to the initial firing rate prior to
α- MSH administration. Administration of α- MSH had no effect
on the firing rate of vasopressin neurons recorded from non-
pregnant, late- pregnant or lactating rats (RS: F2,21=0.16, p=0.23;
TIME: F2,21=0.65, p=0.57; interaction between RS and TIME:
F6,63=1.24, p=0.30, two- way repeated measures ANOVA)

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PERKINSON et al.
reproduction during α- MSH adminstration. While we
found a change in the mean late hazard, which infers the
influence of baseline membrane potential and ongoing
synaptic input on spiking activity, it was not specific to any
group (Figure S1). Hence, there appears to be no precise
change in post- spike excitability that accounts for the
change in response of oxytocin neurons to α- MSH over re-
productive states. Rather, it presumably involves changes
in the coupling of α- MSH, oxytocin, and/or endocannabi-
noid signalling in the oxytocin system.
4.1
|
α- MSH stimulation of somato-
dendritic oxytocin secretion
MC4R couples to multiple signaling pathways, includ-
ing mobilization of intracellular calcium (Kumar et al.,
2021), which presumably mediates α- MSH stimulation of
somato- dendritic oxytocin secretion. Given that the sniff-
erOT cell response to α- MSH- induced somato- dendritic
secretion was similar in slices from non- pregnant and lac-
tating rats, it appears likely that the switch from α- MSH
inhibition to excitation of oxytocin neurons is mediated
by changes downstream of somato- dendritic oxytocin
secretion.
Oxytocin inhibits oxytocin neurons in brain slices
from non- pregnant rats (Kuriyama et al., 1993) but is
auto- excitatory during lactation, inducing depolariza-
tion (Kawarabayashi et al., 1993; Wang et al., 2006) and
attenuating GABA inhibition (Brussaard et al., 1996),
and intracerebroventricular oxytocin receptor antago-
nist administration inhibits peripheral oxytocin secretion
during suckling (Lambert et al., 1993; Richard et al., 1991).
Hence, direct oxytocin neuron excitation by α- MSH-
induced somato- dendritic oxytocin secretion might over-
ride ongoing retrograde inhibition of excitatory synaptic
transmission by somato- dendritic oxytocin- induced endo-
cannabinoids in lactation.
4.2
|
Retrograde endocannabinoid
signaling from oxytocin neurons
While α- MSH- induced somato- dendritic oxytocin secre-
tion might directly excite oxytocin neurons in late preg-
nancy and lactation, it is possible that endocannabinoid
signaling also switches from inhibitory to excitatory. In
addition to CB1 receptors, endocannabinoids can also
activate transient receptor potential vanilloid (TRPV) re-
ceptors (Branco & Staras, 2009) that allow non- specific
cation influx to induce depolarization (Nilius et al., 2007).
ΔN- truncated TRPV1 (ΔN- TRVP1) mRNA is expressed
in oxytocin neurons but these do not contribute to the
basal activity of oxytocin neurons in non- pregnant and
late- pregnant rats (Perkinson et al., 2021). However, in
the presence of a CB1 antagonist, α- MSH causes a small
excitation of oxytocin neurons in non- pregnant rats
(Sabatier & Leng, 2006). While CB1 receptors are still
FIGURE  Somato- dendritic oxytocin secretion in response
to α- MSH administration is similar in hypothalamic slices from
non- pregnant and lactating rats. (a). Example image of calcium
responses (F/F0) from snifferOT cells plated onto a hypothalamic
slice containing the paraventricular nucleus from a non- pregnant
rat. The circles indicate cells that had fluorescence measured
shown in (b). (b) The coloured lines reflect the change in calcium
response (F/F0) of the five individual cells from one recording
(these changes can be seen in the video; Figure S3). The arrow
indicates the start of the α- MSH (1µM) bolus. (c– f) Summary data
of the calcium responses from snifferOT cells displaying the peak
amplitude (c), integrated area under the Ca2+ response curve (d),
latency to start of response (e) and duration of the calcium response
(f) (*p=0.027, unpaired Student's t- test). Data contains 32 oxytocin
responsive snifferOT cells plated onto hypothalamic slices from
three non- pregnant rats and 24 oxytocin responsive snifferOT cells
plated onto hypothalamic slices from two lactating rats

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PERKINSON et al.
functional during lactation (Vilela & Giusti- Paiva, 2014),
if CB1 receptor expression on excitatory inputs to oxytocin
neurons decreases in lactation, the balance of endocan-
nabinoid effects might shift from inhibition to excitation
during lactation.
4.3
|
Somato- dendritic oxytocin
release and food intake in
pregnancy and lactation
In addition to feedback regulation of oxytocin neuron ac-
tivity, somato- dendritic oxytocin secretion has been impli-
cated in mediating α- MSH’s satiety effects via activation
of ventromedial hypothalamic neurons (Sabatier et al.,
2013). During pregnancy and lactation, changes in the cen-
tral control of satiety cause hyperphagia to cope with the
metabolic demands of pregnancy and lactation (Trujillo
et al., 2011). ICV oxytocin inhibits food intake in non-
pregnant rats but increases food intake in mid- pregnant
rats (Douglas et al., 2007). Hence, the maintenance of α-
MSH- induced somato- dendritic oxytocin secretion might
also help coordinate increased food intake while facilitat-
ing oxytocin secretion for parturition and lactation.
4.4
|
Alpha- melanocyte- stimulating
hormone does not affect vasopressin
neuron activity
Vasopressin is secreted by a distinct population of magno-
cellular neurons of the SON and PVN to trigger water re-
absorption by the kidney in response to increased plasma
osmolality. Vasopressin neurons also undergo adaptations
over the course of pregnancy to increase blood volume
to cope with the cardiovascular demands of pregnancy
and lactation (Koehler et al., 1993; Prager- Khoutorsky &
Bourque, 2010). In non- pregnant rats, vasopressin neu-
rons do not express MC4R (Mountjoy et al., 1994) or re-
spond to α- MSH (Sabatier et al., 2003). We found that
vasopressin neurons remain unresponsive to α- MSH in
late pregnancy and lactation, further supported by no
change in the post- spike excitability (Figure S2), suggest-
ing that α- MSH is not involved in the reproductive plastic-
ity of vasopressin neurons.
5
|
CONCLUSION
Oxytocin neurons show dramatic plasticity in morphol-
ogy, intrinsic properties, and responses to afferent inputs
over the course of pregnancy to prepare for increased ox-
ytocin secretion necessary for parturition and lactation.
The present study adds a switch in α- MSH inhibition to
excitation to the suite of changes that promote oxytocin
secretion for parturition and lactation. While α- MSH-
induced somato- dendritic oxytocin secretion was main-
tained in lactation to contribute to the excitation, the
mechanisms by which is does so remain to be determined.
TRANSLATIONAL PERSPECTIVE
Preterm birth is a major cause of infant mortality and
lifelong morbidity. Appropriate activation of oxytocin
neurons is critical for normal birth and early activation
of oxytocin neurons can lead to preterm delivery. Our re-
search shows that α- MSH inhibition of oxytocin neurons
switches to excitation in late pregnancy. Hence, antago-
nising α- MSH activation of oxytocin neurons might pro-
vide a novel therapeutic target to reduce the risk of the
preterm birth in at- risk pregnancies.
CONFLICT OF INTEREST
The authors have no competing financial interests.
AUTHOR CONTRIBUTION
All authors contributed to the design and interpretation
of the experiments. Michael R. Perkinson, Matthew K.
Kirchner, Rachael A. Augustine, and Colin H. Brown
performed the experiments. Michael R. Perkinson and
Matthew K. Kirchner analyzed data. Michael R. Perkinson
prepared the figures and the first draft of the manuscript.
All authors reviewed the manuscript for intellectual con-
tent. The final version of manuscript for publication was
explicitly approved by all authors.
ORCID
Colin H. Brown https://orcid.org/0000-0003-2305-846X
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SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of the article at the publisher’s website.
How to cite this article: Perkinson, M. R.,
Kirchner, M. K., Zhang, M., Augustine, R. A., Stern,
J. E., & Brown, C. H. (2022). α- Melanocyte-
stimulating hormone inhibition of oxytocin
neurons switches to excitation in late pregnancy
and lactation. Physiological Reports, 10, e15226.
https://doi.org/10.14814/ phy2.15226