Download MSH-induced inhibition of oxytocin cells

Am J Physiol Regul Integr Comp Physiol 290: R577–R584, 2006.
First published November 3, 2005; doi:10.1152/ajpregu.00667.2005.
CALL FOR PAPERS
Neurohypophyseal Hormones: From Genomics and Physiology
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Presynaptic actions of endocannabinoids mediate ␣-MSH-induced inhibition
of oxytocin cells
Nancy Sabatier and Gareth Leng
Centre for Integrative Physiology, The University of Edinburgh, George Square Edinburgh, United Kingdom
Submitted 13 September 2005; accepted in final form 25 October 2005
(SON) contains magnocellular neurons
that project to the posterior pituitary where they secrete oxytocin and vasopressin into the circulation. Vasopressin and
oxytocin are also released centrally and are involved in several
behaviors (47). Central release originates, in part, from parvocellular neurons of the paraventricular nucleus (PVN), but both
peptides are also released from the dendrites of magnocellular
neurons of the PVN and SON (20). Dendritically released
oxytocin has autoregulatory actions on SON neurons; notably,
these include facilitating the synchronized bursting that accompanies reflex milk ejection in lactating rats (26).
Dendritic oxytocin release can be triggered by agents that
mobilize calcium from intracellular stores, including oxytocin
itself (16) and can occur without an increase in electrical
activity and without oxytocin secretion from nerve terminals
(23). Thus, in some physiological conditions, including suckling, osmotic challenge, and, in response to some specific
stressors (20), dendritic release occurs independently of secretion from the nerve terminals.
We recently studied the effect of ␣-melanocyte-stimulating
hormone (␣-MSH), a peptide produced by neurons in the
arcuate nucleus (43), on SON oxytocin neurons. Centrally
injected ␣-MSH and oxytocin have similar actions on many
behaviors in the rat, and it seems possible that centrally
released oxytocin mediates some of the behavioral effects of
centrally administered ␣-MSH. The melanocortin 4 (MC4)
receptor mRNA is expressed abundantly in the SON and PVN
(28), and MC-containing neurons of the arcuate nucleus project
directly to the SON (35).
We previously showed that ␣-MSH differentially regulates
dendritic and systemic release of oxytocin (34). Central injections of ␣-MSH or MC4 agonists decrease the electrical activity of oxytocin cells and, consequently, reduce peripheral
oxytocin secretion. However, ␣-MSH induces oxytocin release
from dendrites in isolated SON, probably as a consequence of
its stimulating effect on intracellular calcium stores. In the
present study, we investigated the mechanisms by which
␣-MSH inhibits oxytocin cells. Oxytocin, like ␣-MSH, mobilizes intracellular calcium stores in oxytocin cells (16) and
triggers presynaptic inhibition of afferent inputs that is mediated by cannabinoids (9). We therefore hypothesized that the
same mechanism might underlie the inhibitory effects of
␣-MSH. To test this, we recorded from oxytocin cells in the
SON in vivo, and showed that cannabinoid antagonists applied
to the SON by retrodialysis could block the response of
oxytocin cells to intracerebroventricular injection of ␣-MSH.
We then went on to compare the effects of cannabinoids and
␣-MSH applied to the SON on the responses of SON neurons
to electrical stimulation of an afferent pathway. The pathway
chosen for these studies was the projection from the organum
vasculosum of the lamina terminalis (OVLT). The OVLT
Address for reprint requests and other correspondence: N. Sabatier, Univ. of
Edinburgh Centre for Integrative Physiology, Hugh Robson Bldg, George
Square, Edinburgh EH8 9XD, UK (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
dendritic release; supraoptic nucleus; organum vasculosum of the
lamina terminalis; presynaptic inhibition; ␥-aminobutyric acid; melanocortin 4 receptor; ␣-melanocyte-stimulating hormone
THE SUPRAOPTIC NUCLEUS
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Sabatier, Nancy, and Gareth Leng. Presynaptic actions of endocannabinoids mediate ␣-MSH-induced inhibition of oxytocin cells.
Am J Physiol Regul Integr Comp Physiol 290: R577–R584, 2006. First
published November 3, 2005; doi:10.1152/ajpregu.00667.2005.—We
recently showed that central injections of ␣-melanocyte-stimulating
hormone (␣-MSH) inhibits oxytocin cells and reduces peripheral
release of oxytocin, but induces oxytocin release from dendrites.
Dendritic oxytocin release can be triggered by agents that mobilize
intracellular calcium. Oxytocin, like ␣-MSH, mobilizes intracellular
calcium stores in oxytocin cells and triggers presynaptic inhibition of
afferent inputs that is mediated by cannabinoids. We hypothesized
that this mechanism might underlie the inhibitory effects of ␣-MSH.
To test this, we recorded extracellularly from identified oxytocin and
vasopressin cells in the anesthetized rat supraoptic nucleus (SON).
Retrodialysis of a CB1 cannabinoid receptor antagonist to the SON
blocked the inhibitory effects of intracerebroventricular injections of
␣-MSH on the spontaneous activity of oxytocin cells. We then
monitored synaptically mediated responses of SON cells to stimulation of the organum vasculosum of the lamina terminalis (OVLT); this
evoked a mixed response comprising an inhibitory component mediated by GABA and an excitatory component mediated by glutamate,
as identified by the effects of bicuculline and 6-cyano-7-nitroquinoxaline-2,3-dione applied to the SON by retrodialysis. Application of
CB1 receptor agonists to the SON attenuated the excitatory effects of
OVLT stimulation in both oxytocin and vasopressin cells, whereas
␣-MSH attenuated the responses of oxytocin cells only. Thus ␣-MSH
can act as a “switch”; it triggers oxytocin release centrally, but at the
same time through initiating endocannabinoid production in oxytocin
cells inhibits their electrical activity and hence, peripheral secretion.
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ENDOCANNABINOIDS, ␣-MSH, AND OXYTOCIN CELLS
projects to the SON both directly and indirectly via the nucleus
medianus (18, 24); this projection has been well characterized
in previous studies, and stimulation of the OVLT evokes a
mixed response that has an inhibitory component mediated
by GABA and an excitatory component mediated by glutamate (4).
MATERIALS AND METHODS
RESULTS
Effect of cannabinoid antagonist on MSH-induced inhibition
of oxytocin cells. Although intracerebroventricular injection of
␣-MSH inhibits oxytocin cells, ␣-MSH induces dendritic reAJP-Regul Integr Comp Physiol • VOL
Fig. 1. Effect of the CB1 antagonist AM251 on ␣-melanocyte-stimulating
hormone (␣-MSH)-induced inhibition of oxytocin cells. A: inhibition of an
oxytocin cell by ␣-MSH (300 ng, icv). At the second ␣-MSH injection, the
inhibition is prevented by retrodialysis of the SON with 100 ␮M AM251. B:
mean change (⫾SE) in the firing rate of 7 oxytocin cells in response to
intracerebroventricular injection of 300 ng ␣-MSH before (closed symbols)
and during (open symbols) retrodialysis with AM251.
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Electrophysiology. Experiments were performed on adult female
Sprague-Dawley rats anesthetized with urethane (1.25 g/kg ip). A
femoral vein and trachea were cannulated, and for intracerebroventricular injections, an injection cannula was implanted into the lateral
ventricle (coordinates from bregma: 0.6 mm posterior; 1.6 mm lateral;
4 mm depth). The pituitary stalk, the left SON, and the OVLT were
exposed transpharyngeally, and a microdialysis probe loop was placed
onto the ventral surface of the SON (21). The extracellular activity of
single neurons was recorded by a glass micropipette placed through
the center of the probe loop. A bipolar side-by-side stimulating
electrode (model SNEX-200; Clarke Electromedical) was placed on
the pituitary stalk to deliver matched biphasic pulses (1 ms, ⬍1 mA)
to antidromically identify SON neurons. A bipolar concentric stimulating electrode (model SNEX-100, Clarke Electromedical) was
placed in the OVLT to deliver single shocks (1 Hz, matched biphasic
1-ms pulses, 0.1–1 mA peak to peak) or trains of shocks (20 Hz for
200 ms every 5 s, 0.1–1 mA). Oxytocin cells were distinguished from
vasopressin cells by their firing pattern and by their opposite response
to intravenous CCK [20 ␮g/kg, cholecystokinin-(26 –33)-sulfated;
Bachem; Refs. 31 and 32]. Artificial cerebrospinal fluid (aCSF; pH
7.2, composition in mM: 138 NaCl, 3.36 KCl, 9.52 NaHCO3, 0.49
Na2HPO4, 2.16 urea, 0.49 NaH2PO4, 1.26 CaCl2, 1.18 MgCl2) was
dialyzed at 3 ␮l/min, and changed to aCSF containing CNQX,
bicuculline, WIN 55,212–2 mesylate N-(cyclopropyl)-5Z, 8Z, 11Z,
14Z-eicosatetraenamide (ACPA) (in Tocrisolve 100), AM 251 (all
Tocris Cookson, Avonmouth, UK), or ␣-MSH (Calbiochem-Novabiochem, Nottingham, UK).
Data analysis. Responses evoked by OVLT stimulation were
initially analyzed using Spike2 software (CED, Cambridge, UK).
Stimulation-evoked spike activity was visualized with raster displays
and peristimulus time histograms (PSTH) were constructed to quantify the responses. For each cell, the responses to trains were analyzed
by constructing PSTHs for 1,000 s (200 trains, 250 bins of 20 ms) in
control conditions and during and after drug application by retrodialysis to the SON. The PSTHs were first normalized to the ongoing
spontaneous firing rate, measured as the average rate in the 2.5-s
periods before each stimulus train. For PSTHs in control conditions,
the stimulus-evoked response profile was then identified. Peristimulus
inhibition was recognized when five or more consecutive bins each
contained fewer spikes than expected from the prestimulation control,
and peristimulus excitation when five or more consecutive bins
contained more than the expected number of spikes. The response
amplitude was defined as the number of additional spikes per stimulus
train during the identified periods. To assess changes in responses, the
same poststimulus periods were used to quantify response amplitudes
as those identified in control PSTHs. For the experiment with intracerebroventricular injection of ␣-MSH, firing rate in 30-s bins in the
5 min before injection was compared with the firing rate between 10
and 15 min after injection. In all cases, the duration of the experimental procedures meant that only one or two cells could be fully
tested in any one experiment.
lease of oxytocin (34). In vitro, oxytocin presynaptically inhibits excitatory inputs via release of endocannabinoids (9), so
we investigated whether ␣-MSH inhibits oxytocin cells by a
similar mechanism. The effect of ␣-MSH (300 ng icv) was
compared before and during retrodialysis of the SON with the
CB1 antagonist AM251 (10 –1,000 ␮M). The response to
␣-MSH was measured as the difference in firing rates in the 5
min before and 10 –15 min after ␣-MSH injection. In control
conditions, ␣-MSH produced a mean change of ⫺0.7 ⫾ 0.2
spikes/s from an initial rate of 3.6 ⫾ 0.3 spikes/s (P ⬍ 0.01).
After 30- to 45-min retrodialysis with AM251, the inhibitory
response to ␣-MSH was absent in six of seven cells (Fig. 1),
including in all three during dialysis with 1 mM AM251, in
two of three during 100 ␮M AM251, and in one during 10 ␮M
AM251. For these seven cells, the response to ␣-MSH in the
presence of AM251 was an increase of 0.4 ⫾ 0.2 spikes/s
from an initial rate of 3.3 ⫾ 0.3 spikes/s (P ⬍ 0.01 vs. initial
response).
We also analyzed the effect of AM251 on the background
activity of 10 oxytocin cells. After 30 to 55 min of retrodialysis
with AM251, the mean background firing rate increased by
0.4 ⫾ 0.15 spikes/s from an initial rate of 4 ⫾ 0.6 spikes/s (P ⬍
0.05 Student’s paired t-test).
These experiments indicated that ␣-MSH-induced inhibition
of oxytocin cells is mediated by endogenous cannabinoids. At
many sites in the CNS, including in the SON, cannabinoids are
known to act presynaptically. We therefore went on to compare
the effects of ␣-MSH and cannabinoids on the responses of
SON neurons to activation of an afferent pathway, the OVLT.
Effects of OVLT stimulation on oxytocin and vasopressin
cells. Typically, SON neurons responded to single-shock stimulation of the OVLT with a brief inhibition followed by a more
ENDOCANNABINOIDS, ␣-MSH, AND OXYTOCIN CELLS
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Fig. 3. Characteristics of the responses of SON neurons to OVLT stimulation.
Aa: mean response (⫾SE) to OVLT stimulation of SON neurons that displayed
excitation (Excit) only (n ⫽ 27). Ab: mean amplitude (top, left), onset latency
(top, right), duration (bottom, left), and peak time (bottom, right) of the
responses of 16 vasopressin (VP) and 11 oxytocin (OT; closed and open bars,
respectively) cells included in Aa. Ba: mean response (⫾SE) to OVLT
stimulation of SON neurons that were inhibited then excited (n ⫽ 13). Bb:
mean amplitude (top, left), onset latency (top, right), duration (bottom, left),
and peak time (bottom, right) of the responses of 4 vasopressin and 9 oxytocin
cells. Inhib, inhibition.
Fig. 2. Effects of organum vasculosum of the lamina terminalis (OVLT)
stimulation on electrical activity of supraoptic nucleus (SON) neurons. Aa and
Ba: firing rate of a vasopressin cell (Aa) and an oxytocin cell (Ba) during
OVLT stimulation [200 trains of stimulation (1,000 s) at 20 Hz]. Ab and Bb:
rasters of the responses of the cells shown in Aa and Ba illustrating excitation
(Ab) and inhibition followed by excitation (Bb); the onset of stimulus trains is
indicated by the vertical line at t ⫽ 0. Ac and Bc: peristimulus time histograms
(PSTH) averaging the responses to the 200 trains of stimulation in the neurons
shown in Aa and Ba, illustrating excitation and inhibition followed by excitation. The period of train stimulation is indicated by the shaded bar.
AJP-Regul Integr Comp Physiol • VOL
9 oxytocin cells) that were inhibited and then excited, the mean
inhibition was ⫺0.6 ⫾ 0.09 spikes/train, maximal at 97 ⫾ 17
ms, and the mean excitation was 1.4 ⫾ 0.35 spikes/train,
maximal at 358 ⫾ 43 ms (Fig. 3B). In the two cells that were
inhibited only (both vasopressin cells), the mean response was
⫺0.6 ⫾ 0.2 spikes/train, maximal at 170 ⫾ 42 ms (not shown).
Thus inhibitory responses were slightly more prominent among
oxytocin cells than among vasopressin cells (9/20 vs. 6/22
cells). Otherwise, responses to OVLT stimulation were very
similar between oxytocin cells and vasopressin cells.
To test whether the responses to OVLT stimulation were
stable over time, we monitored responses during ⬎90 min of
microdialysis with aCSF (Fig. 4). For all seven cells tested
(vasopressin cells and oxytocin cells), the response to stimulation remained apparently unchanged; initially, the mean excitatory response of these cells was an additional 2.8 ⫾ 0.4
spikes/train, and after 90 min, the response was an additional
2.9 ⫾ 0.4 spikes/train (Fig. 4C; mean change 0.05 ⫾ 0.1
spikes/train, no significant difference).
Effects of glutamate and GABAA antagonists on responses to
OVLT stimulation. To characterize the neurochemical nature of
the responses to OVLT stimulation, we applied glutamate and
GABAA receptor antagonists to the SON by microdialysis
(retrodialysis). The concentrations quoted for all drugs are
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prolonged excitation, as reported by others (46). Short-latency
inhibitions (mean onset 15 ⫾ 3 ms) were apparent in all five
oxytocin cells tested and in four of six vasopressin cells. Two
cells showed short-latency excitation only. In subsequent experiments, we applied brief trains of stimuli to the OVLT (20
Hz for 200 ms every 5 s). In these conditions, the excitatory
effects summated, giving clearer overall excitation. We used
raster displays to visualize the responses and PSTHs to quantify them.
In response to these trains (Fig. 2), 27 cells were excited,
with an onset latency of 61 ⫾ 9 ms and duration of 468 ⫾ 28
ms. Two cells were inhibited only (not shown), and 13 were
inhibited (latency 12 ⫾ 9 ms, duration 183 ⫾ 16 ms) and then
excited (latency 230 ⫾ 26 and 397 ⫾ 40 ms). The mean onset
latency, duration, time to peak response, and peak response
amplitude of vasopressin cells were very similar to those of
oxytocin cells (Fig. 3).
In cells that were excited only, the mean response for the 27
cells (16 vasopressin and 11 oxytocin cells) was an additional
1.8 ⫾ 0.2 spikes/train, and the responses were maximal at
220 ⫾ 9 ms (Fig. 3A). Among the 13 cells (4 vasopressin and
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ENDOCANNABINOIDS, ␣-MSH, AND OXYTOCIN CELLS
those in the microdialysate. Resulting tissue concentrations in
the SON are three to four orders of magnitude lower, as
estimated previously (21). The glutamate antagonist 6-cyano7-nitroquinoxaline-2,3-dione (CNQX, 1 mM) was retrodialyzed for 15–25 min during recording from four cells excited
by OVLT stimulation. Within 10 min, both the background
electrical activity and the excitatory response to OVLT stimulation were abolished in all four cells (Fig. 5A); both background activity and excitatory response recovered within 30
min of returning to aCSF.
The GABAA antagonist bicuculline (2 mM) was applied
while recording from seven cells that, in response to OVLT
stimulation, were either inhibited only or were inhibited and
then excited. After 10 –25 min, the inhibition was abolished in
five of the seven cells (Fig. 5B) and markedly attenuated in the
other two; the mean response reversed from ⫺0.33 ⫾ 0.085
spikes/train to ⫹0.34 ⫾ 0.26 spikes/train (n ⫽ 7; mean change
0.67 ⫾ 0.25 spikes/train), apparently unmasking coincident
excitation. Despite this dramatic effect on stimulation-evoked
inhibition, bicuculline had little sustained effect on background
activity (mean change after 20 min: 0.3 ⫾ 0.2 spikes/s, from
3.4 ⫾ 1.1 to 3.7 ⫾ 1.1 spikes/s, n ⫽ 7), although an earlier
transient excitation was generally observed. Recovery of responses after changing back to dialysis with aCSF was slow,
(40 min or more), consistent with previous studies.
These results are consistent with the conclusion that the
excitatory and inhibitory responses reflect glutamate and
GABA inputs acting mainly at AMPA/kainate receptors and
GABAA receptors, respectively, in agreement with previous
studies (4).
Effects of cannabinoids on background firing rate. The
effects of the CB1 agonist WIN 55,212–2 on background firing
rate were tested on 10 vasopressin cells (3 continuously firing,
7 phasic) and 8 oxytocin cells. The firing rate 5 min before
retrodialysis with WIN 55,212 (100 ␮M for 35– 45 min) was
compared with the rate in the last 1,000 s of retrodialysis. WIN
55,212 produced a small (not significant) reduction in the firing
rate of oxytocin cells (mean change, ⫺0.45 ⫾ 0.25 spikes/s)
AJP-Regul Integr Comp Physiol • VOL
Fig. 5. Effect of glutamate and GABAA antagonists on responses of SON
neurons to OVLT stimulation. A, left: vasopressin cell recorded during retrodialysis with the glutamate antagonist CNQX (1 mM). The background firing
was abolished shortly after starting the antagonist application and recovered
after return to artificial cerebrospinal fluid (aCSF). A, right: raster display of
the same recording showing the loss of the excitatory response to OVLT
stimulation during retrodialysis with CNQX. B: firing rate of a vasopressin cell
during retrodialysis with 2 mM bicuculline, showing little effect on background firing rate (top); raster display of the same recording showing the
inhibition evoked by OVLT stimulation is abolished by bicuculline (middle);
PSTHs constructed before and during bicuculline application showing total
loss of the inhibitory response in the presence of bicuculline (bottom).
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Fig. 4. Stability of the response to OVLT stimulation in SON neurons. A:
example of a vasopressin cell recorded for 105 min during OVLT stimulation.
B: PSTHs constructed at the beginning of the recording (left) and 90 min later
(right). C: mean amplitude (⫾SE) of excitation in response to OVLT stimulation at t ⫽ 0 and t ⫽ 90 min (n ⫽ 7). Note that the response is very stable
over time.
and a small (not significant) increase in vasopressin cells (mean
change, 0.3 ⫾ 0.47 spikes/s) (Fig. 6A).
Effects of cannabinoid agonists on excitatory response of
oxytocin and vasopressin cells. We then investigated whether
cannabinoid agonists modulate the afferent inputs to the SON.
The CB1 agonists WIN 55,212–2 (250 ␮M, 12 cells) and
ACPA (1 mM, 5 cells) were applied to the SON by retrodialysis for 30 – 60 min during recordings of 12 vasopressin cells
and five oxytocin cells that were excited by OVLT stimulation.
Both agonists had similar effects on vasopressin and oxytocin
cells, so results from the two types of neurons and the two
agonists were pooled.
In 13 of 17 cells, the excitatory response to stimulation was
reduced by ⬎20% in the presence of a CB1 agonist; four cells
were not affected. Overall, the mean excitatory response was
reduced by 0.38 ⫾ 0.09 spikes/train (28 ⫾ 5%, n ⫽ 17; P ⬍
ENDOCANNABINOIDS, ␣-MSH, AND OXYTOCIN CELLS
1.2 ⫾ 0.7 spikes/train, and in the additional presence of CB1
agonist, this was reduced by 0.4 ⫾ 0.08 spikes/train.
Effects of ␣-MSH on the excitatory response of oxytocin
cells. We demonstrated (see Fig. 1) that ␣-MSH-induced inhibition of oxytocin cells is prevented by blocking endogenous
cannabinoid actions, indicating that the inhibitory effect of
␣-MSH might be mediated by endocannabinoids. If so, the
effects of ␣-MSH on the responses of oxytocin cells to OVLT
stimulation should be similar to those of CB1 agonists. To test
this, ␣-MSH (500 ␮M) was retrodialyzed onto the SON for
30 – 60 min. In the seven oxytocin cells tested, ␣-MSH attenuated the excitatory response to OVLT stimulation by 24 ⫾ 3%
(Fig. 8; initial response, 1.0 ⫾ 0.3 spikes/train; during ␣-MSH,
0.7 ⫾ 0.2 spikes/train; mean change ⫺0.3 ⫾ 0.01 spikes/train;
P ⬍ 0.05). By contrast, ␣-MSH had no significant effect on the
responses of four vasopressin cells tested (initial response
1.9 ⫾ 0.3 spikes/train, during ␣-MSH, 2.2 ⫾ 0.5 spikes/train;
mean change 0.3 ⫾ 0.3 spikes/s, significantly different from
oxytocin cells P ⬍ 0.05).
DISCUSSION
0.001, paired Student’s t-test, Fig. 6B). The 17 cells include
three that initially were inhibited and then excited in response
to OVLT stimulation. For these three cells, we tested the CB1
agonist in the presence of bicuculline. In this condition, the
inhibitory component of the response was abolished, so we
could ensure that the reduction of excitation by CB1 agonist
was not the result of an increase in inhibition (Fig. 7). In these
three cells, the response in the presence of bicuculline was
OVLT stimulation evoked mixed excitatory and inhibitory
responses in SON neurons that were sensitive to the AMPA/
kainate receptor antagonist CNQX and the GABAA receptor
antagonist bicuculline, respectively. The excitatory responses
of both oxytocin and vasopressin cells were attenuated by
direct application of CB1 agonists to the SON, whereas direct
application of ␣-MSH attenuated the responses of oxytocin
cells only. Moreover, the inhibitory effects of intracerebroventricular ␣-MSH on the spontaneous activity of oxytocin cells
were blocked by application of a CB1 antagonist to the SON.
Together, these results suggest that ␣-MSH inhibits oxytocin
cells by inducing the production of cannabinoids that presynaptically attenuate excitatory inputs to the SON.
Fig. 7. Effect of CB1 agonist on the response to OVLT stimulation in the
presence of bicuculline in a vasopressin cell. A: firing rate of a phasic
vasopressin cell during retrodialysis of bicuculline (2 ␮M) and CB1 agonist
WIN 55,212 (250 ␮M) in the presence of bicuculline. B: PSTHs constructed in
control condition, during bicuculline retrodialysis, during bicuculline combined with CB1 agonist, and after washout. The inhibition in response to
OVLT stimulation was abolished by bicuculline, unmasking an excitation. In
the continuing presence of bicuculline, the excitation was strongly reduced by
CB1 agonist application. After washout, the control response to OVLT stimulation partially recovered within 20 min.
Fig. 8. Effect of ␣-MSH on the excitatory response of oxytocin cells to OVLT
stimulation. A: recording of an oxytocin cell during retrodialysis with 500 ␮M
␣-MSH. B: PSTHs comparing the excitatory responses to OVLT stimulation of
the oxytocin cell shown in A before and during retrodialysis with ␣-MSH. The
excitation was reduced by 29% in this cell (mean 24% for all oxytocin cells).
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Fig. 6. Effect of CB1 agonists on background firing rate of SON neurons and
on their excitatory responses to OVLT stimulation. A: mean firing rate of 10
vasopressin and 8 oxytocin cells before and during retrodialysis with CB1
agonists WIN 55,212 and ACPA (250 ␮M-1 mM). The firing rates of neither
vasopressin nor oxytocin cells were significantly affected. B: firing rate of an
oxytocin cell during retrodialysis with CB1 agonist WIN 55,212 (1 mM) and
PSTHs comparing the excitatory responses to OVLT stimulation before and
during retrodialysis with WIN 55,212. The excitatory response to stimulation
was reduced by 29% in this cell (mean 28% for all cells).
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AJP-Regul Integr Comp Physiol • VOL
activity in vivo, and the first to identify actions on a characterized afferent projection. However, many studies on different
neurons have shown that presynaptic inhibitory effects of
cannabinoids are very widespread in the CNS (e.g., 15, 44).
␣-MSH had an effect similar to CB1 agonists, as it reduced
by 24% the excitatory response to OVLT stimulation. However, unlike CB1 agonists, its effects were selective for oxytocin cells; responses of vasopressin cells were unaffected.
Recently, we showed that intracerebroventricular injection of
␣-MSH inhibits oxytocin cells but not vasopressin cells in vivo
(34). In the present study, this ␣-MSH-induced inhibition was
prevented by application of CB1 antagonist AM251 onto the
SON. The simplest explanation is that ␣-MSH inhibits oxytocin cells by depressing glutamate afferents through the release
of endocannabinoids from oxytocin cells. The production of
endocannabinoids is regulated by calcium influx through voltage-dependent channels (19) and generally depends on elevation of intracellular Ca2⫹ concentration ([Ca2⫹]i) (8, 42). Like
oxytocin (16), ␣-MSH triggers an increase in [Ca2⫹]i in oxytocin cells (34), which could lead to production of endocannabinoids.
In SON neurons, dendritic peptide release can either precede
(26) or succeed systemic release (20), or it can occur without
systemic release, such as during forced-swimming (45). We
showed recently that ␣-MSH can also differentially regulate
dendritic and systemic release of oxytocin as ␣-MSH inhibits
the electrical activity of oxytocin cells in vivo and, as a
consequence, systemic release of oxytocin, but induces dendritic release of oxytocin (34). Oxytocin and ␣-MSH have
similar effects on several behaviors in rats; in particular, both
suppress feeding (2, 41), and both stimulate female and male
sexual behavior (1). There are many centrally projecting oxytocin neurons in the PVN, and these have generally been
assumed to govern behavioral effects of oxytocin. However,
although these neurons synthesize oxytocin, this is unlikely to
be their major neurotransmitter product; most centrally projecting peptidergic neurons also synthesize a conventional
neurotransmitter, which appears to be their main synaptic
output. Indeed, large dense-cored vesicles that contain peptides
are quite rare in synapses, but they can be released from all
parts of neurons. Morris and Pow (27) estimated that half of the
peptide release in the CNS is from extrasynaptic sites. The
centrally projecting oxytocin neurons project mainly outside
the hypothalamus, notably to the brainstem and spinal cord,
where there are dense terminal projections. Within the hypothalamus itself and in adjacent structures, the innervation is
more sparse, and mismatches have been noted between the
distribution of oxytocin receptors and oxytocin innervation.
Large quantities of oxytocin are released from the dendrites
of magnocellular neurons. The concentration in the extracellular fluid of the SON is 10 –100 times higher than in the
systemic circulation (20), and the basal release rate of oxytocin
from the isolated SON is 1 pg oxytocin/min per SON (26).
Oxytocin receptors expressed within the brain appear identical
to these expressed peripherally, with affinities for oxytocin in
the nanomolar range (3). Because the half-life of oxytocin in
the brain is estimated to be 19 min (25), it is difficult to see
how the magnocellular neuronal dendrites cannot be a major
contributor to modulation of central oxytocin receptors, assuming no barrier to diffusion of oxytocin within the brain. Thus
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The OVLT, adjacent to the anteroventral wall of the third
ventricle, is prominently involved in body fluid and electrolyte
homeostasis, and it projects to the SON both directly, and
indirectly via the nucleus medianus (24). Consistent with
previous reports (10, 17, 46), OVLT stimulation evoked two
main types of responses in SON neurons: 1) excitation or 2)
inhibition followed by excitation. The stimulation used here
(trains at 20 Hz for 200 ms every 5 s) elicited moderate
responses that were stable for long periods in control conditions. Both the background firing of SON neurons and excitatory responses to OVLT stimulation were abolished by the
AMPA/kainate antagonist CNQX, and the inhibitory responses
were abolished by the GABAA antagonist bicuculline. This is
consistent with in vitro electrophysiological experiments in
hypothalamic explants, showing that OVLT stimulation
evoked inhibitory postsynaptic potentials in SON neurons that
were sensitive to bicuculline and excitatory postsynaptic potentials (EPSPs) that were mainly sensitive to CNQX. The
residual component of EPSPs was blocked by the N-methylD-aspartate (NMDA) antagonist 2-amino-5-phosphonovalerate
(APV, also APS) APV (46). However, recent recordings in
hypothalamic explants showed that GABAB receptors also
modulate both excitatory and inhibitory postsynaptic currents
evoked by OVLT stimulation in SON neurons (13). In the
present experiments, although bicuculline blocked the inhibitory response of SON neurons to OVLT stimulation, it had
little effect on background firing rate. This is surprising,
because about half of the afferent input to SON neurons is
thought to be mediated by GABA (39), but we have reported
this relative lack of effect in previous experiments (22). It
seems that SON neurons compensate for a progressive diminution of GABA input with an decrease in intrinsic excitability. Similar perverse stability of spontaneous activity has been
described in other circumstances (e.g., 6), possibly indicating
that spontaneous firing rate is strongly buffered by multiple
negative feedback mechanisms, including feedbacks mediated
by nitric oxide (38), adenosine (29, 30), dynorphin (5), vasopressin (21, 33), oxytocin (14), and endocannabinoids (9), as
well as intrinsic activity-dependent hyperpolarization (12).
In the present study, CB1 agonists inhibited the excitatory
response to OVLT stimulation by 20 –30% in both oxytocin
cells and vasopressin cells, indicating that inputs to both cell
types are sensitive to presynaptic inhibition by cannabinoids.
The concentration of CB1 agonist used in these experiments
was chosen to produce an estimated extracellular concentration
of 0.25–1 ␮M, similar to the dose shown to be effective in the
experiments of Hirasawa et al. (9). CB1 receptors have been
localized by immunohistochemistry to both excitatory and
inhibitory axon terminals innervating dendrites in the SON (9),
and the cannabinoid agonist presynaptically inhibits spontaneous EPSCs and IPSCs in SON neurons recorded in slices (37).
As with bicuculline, CB1 agonists had surprisingly little effect
on spontaneous firing rate, but oxytocin cells were activated in
the presence of a cannabinoid antagonist, indicating tonic
activity of endogenous cannabinoids. Again, we suspect that
agonist-induced changes in excitability are buffered by negative feedback, whereas interfering with these mechanisms has
clearer consequences. Presynaptic actions of cannabinoids in
the SON have been reported recently from in vitro studies; the
present study is the first to characterize actions on identified
oxytocin and vasopressin cells, the first to show endogenous
ENDOCANNABINOIDS, ␣-MSH, AND OXYTOCIN CELLS
ACKNOWLEDGMENTS
This work was supported by research grants from Merck & Co, by the
Biotechnology and Biological Sciences Research Council, and by the European Commission FPG funding (contract no. LSHM-CT-2003-503041).
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