synaptic inputs to parvocellular neurosecretory neurons of the hypothalamic paraventricular nucleus (PVN) by the glucocorticoids
dexamethasone and corticosterone. The effect was maintained with dexamethasone conjugated to bovine serum albumin and was not
was not blocked by the nitric oxide synthesis antagonist NG-nitro-L-arginine methyl ester hydrochloride or by hemoglobin but was
blocked completely by the CB1cannabinoid receptor antagonists AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-
3-carboxamide] and mimicked and occluded by the cannabinoid receptor agonist WIN55,212-2 [(?)-(?)-[2,3-dihydro-5-methyl-3-(4-
morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate], indicating that it was mediated by
PCR analysis, were subject to rapid inhibitory glucocorticoid regulation, including corticotropin-releasing hormone-, thyrotropin-
Glucocorticoids secreted by the adrenal cortex exert inhibitory
ing but not limited to the negative feedback regulation of the
hypothalamic–pituitary–adrenal (HPA) axis by suppression of
the secretion of corticotropin-releasing hormone (CRH) from
neurons of the hypothalamic paraventricular nucleus (PVN)
(Herman et al., 1996; de Kloet, 2000). The glucocorticoid nega-
tive feedback regulation of the HPA axis occurs acutely by a rel-
regulating CRH and vasopressin (VP) expression in PVN
neurons (Keller-Wood and Dallman, 1984). Classical glucocor-
by binding to intracellular receptors and regulation of gene tran-
scription (Falkenstein et al., 2000). Recent evidence in different
membrane receptors and activation of nongenomic signaling
ically to cellular membrane sites (Suyemitsu and Terayama, 1975;
Harrison et al., 1979; Orchinik et al., 1991) and to induce rapid
influences on electrolyte movement across cellular membranes
for corticosterone, which appears to meet all of the criteria for a
functional membrane-associated corticosteroid receptor, has been
partially purified and characterized in neuronal membranes from
amphibian brain (Evans et al., 2000). However, it remains contro-
versial where in the mammalian brain and through which mecha-
HPA axis. Here, we show that glucocorticoids exert a rapid inhibi-
tory effect on glutamate release onto identified parvocellular neu-
of an endocannabinoid and results in the suppression of synaptic
Slice preparation. Male Sprague Dawley rats (3–5 weeks of age; Charles
River Laboratories, Wilmington, MA) were used in these experiments
according to a protocol approved by the Tulane University Institutional
Animal Care and Use Committee and in accordance with United States
4850 • TheJournalofNeuroscience,June15,2003 • 23(12):4850–4857
tobarbital sodium (50 mg/kg body weight) and decapitated. The brain
and immersed in cooled (1–2°C), oxygenated artificial CSF (aCSF). The
composition of the aCSF was as follows (in mM): 140 NaCl, 3 KCl, 1.3
MgSO4, 1.4 NaH2PO4, 2.4 CaCl2, 11 glucose, and 5 HEPES, pH adjusted
to 7.2–7.3 with NaOH. The hypothalamus was blocked and the caudal
surface of the block was glued to the chuck of a vibrating microtome
slices (350 ?m) containing the PVN were sectioned, bisected along the
in oxygenated aCSF at room temperature, where they were allowed to
equilibrate for ?1.5 hr before being transferred to the recording
cate glass (1.65 mm outer diameter, 1.2 mm inner diameter; KG33; Gar-
ner Glass, Claremont, CA) with a Flaming/Brown P-97 micropipette
puller (Sutter Instruments, Novato, CA) to a resistance of 3–4 M?. The
pipette solution contained (in mM): 120 K-gluconate, 10 KCl, 1 NaCl, 1
with 20 mM D-sorbitol.
a submersion recording chamber, secured to the floor of the chamber
with AgCl2-coated wire, and allowed to equilibrate for at least 15 min
before recordings. Medial PVN neurons were visualized with a cooled
CCD camera using infrared illumination and differential interference
contrast optics and patch-clamped under visual control. After achieving
ings with unstable series resistance were discarded. Whole-cell record-
ings were performed in voltage-clamp mode using an Axopatch 1-D
amplifier (Axon Instruments, Foster City, CA) and monitored continu-
ously on a digital storage oscilloscope (Hitachi, Tokyo, Japan). All data
were low-pass-filtered at 2 kHz, converted to digital video format at 22
kHz using a Neuro-Corder (NeuroData Instruments, New York, NY),
and stored on videotape for off-line analysis. Selected data were subse-
quently digitized off-line at 4 kHz and recorded on a personal computer
rons were recorded in the presence of the voltage-gated sodium channel
blocker tetrodotoxin (TTX) (1 ?M) at a holding potential of ?60 mV at
22–24°C. To focus on the fast, nongenomic actions of glucocorticoids, 3
min epochs of mEPSCs were collected just before bath application of
glucocorticoids and after 7 min of glucocorticoid application. The fre-
point at which the mEPSC had decayed by 63%) of mEPSCs were ana-
lyzed using the Minianalysis 5.0 program (Synaptosoft, Decatur, GA).
Statistical analyses were performed using the Student’s paired t test for
comparisons, and the Kolmogorov–Smirnov nonparametric test for
within-cell comparisons of mEPSC distribution; p values of ?0.05 were
Type II, putative parvocellular PVN neurons were differentiated from
tion (Hoffman et al., 1991; Tasker and Dudek, 1991; Luther et al., 2000).
The presence of transient outward rectification was assessed with a
current-clamp protocol consisting of a series of progressively more de-
lular neurons, was identified as a pronounced dampening of the
depolarization-induced action potentials. Type II neurons were divided
rons on the basis of the expression or lack of expression of a calcium-
dependent low-threshold spike (Luther et al., 2002). Only putative par-
vocellular neurosecretory neurons, which lacked a low-threshold spike,
were included in this study.
Drug application. Water-soluble forms of the steroids dexamethasone
(DEX) (0.01–100 ?M), corticosterone (1 ?M), and cholesterol (5 ?M)
(Sigma-Aldrich, St. Louis, MO) were directly dissolved in aCSF to final
concentrations and applied in the bath perfusion. The DEX–bovine se-
rum albumin (BSA) conjugate (10 ?M) (purified to remove free steroids
concentration of DEX–BSA, (10 ?M) was selected to obtain an effective
reduces the effectiveness of receptor binding by a factor of ?10. TTX (1
?M) (Sigma-Aldrich), the nitric oxide (NO) synthase (NOS) inhibitor
NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) (50 ?M)
(Tocris Cookson, Ellisville, MO), and the carbon monoxide (CO)–NO
scavenger hemoglobin (20 ?M; Sigma-Aldrich) were dissolved in sterile
II corticosteroid receptor antagonists spironolactone (10 ?M) and mife-
pristone (RU486) (10 ?M), the protein kinase blockers staurosporine (1
?M) and bisindolylmaleimide (GF109203X) (0.5 ?M) (Sigma-Aldrich),
the cannabinoid receptor agonist (R)-(?)-[2,3-dihydro-5-methyl-3-
thalenylmethanone mesylate (WIN55,212-2) (1 ?M), and the cannabinoid
phenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) (1 ?M), 1-(2,4-
zole-3-carboxamide (AM281) (10 ?M), and 6-iodo-2-methyl-1-[2-
(4-morpholinyl)ethyl]-1H-indol-3-yl] (AM630) (1–10 ?M) (Tocris
Cookson) were stored as 10 mM stock solutions in DMSO at ?20°C
and dissolved to their final concentrations in aCSF before bath appli-
cation. The cannabinoid analogs were also dissolved with 25%
?-cyclodextrin. The DMSO and ?-cyclodextrin solutions without the
cannabinoids or glucocorticoids had no effect on mEPSCs at the con-
centrations used. The nonhydrolyzable guanylyl nucleotide GDP-?-S
(500 ?M) (Sigma-Aldrich) and, in some cases, dexamethasone (1 ?M)
were included in the patch solution for intracellular application. To
prevent drugs destined for intracellular application from leaking into
the extracellular space before a membrane seal was obtained, the tips
of the patch pipettes were filled first with regular patch solution and
then back-filled with the drug solution.
Single-cell reverse transcription-PCR. The single-cell reverse transcrip-
(1996), with minor modifications. The cell contents were aspirated into
the patch pipette under visual control, taking care to avoid aspiration of
the nucleus, and transferred into 5 ?l of a lysis buffer containing (in ?l)
2.9 of diethyl pyrocarbonate (DEPC)-treated water, 0.7 of BSA, 0.7 of
at ?80°C or used immediately for RT. The lysate-buffer mixture was
?l of final RT master mix, containing (in ?l) 8.0 of DEPC-treated water,
of mixed deoxy NTPs (dNTPs) (10 mM), was added. Single-stranded
cDNA was reverse transcribed from the cellular mRNA by adding 0.7 ?l
15 min. The RNA strand in the RNA–DNA hybrid was removed by
adding 0.5 ?l of RNase H (2 U/?l) for 20 min at 37°C. All reagents were
obtained from Invitrogen (Madison, WI).
Watertown, MA) using a fraction of the single-cell cDNA as a template.
Reaction mixtures contained (30 ?l final volume): 2.2 mM MgCl2, a 0.5
ers, 2.5 U of Taq DNA polymerase, 2.9 ?l of 10? buffer, and 4 ?l of the
cDNA template made from the single-cell RT reaction. The thermal cy-
min for 50 cycles.
Primers for CRH and thyrotropin-releasing hormone (TRH) were
designed in our laboratory using Primer Select 4 (DNAStar, Madison,
WI), and primer designs for VP, oxytocin (OT) and glyceraldehyde-3-
Dietal.•GlucocorticoidInhibitionviaEndocannabinoidRelease J.Neurosci.,June15,2003 • 23(12):4850–4857 • 4851
phosphate dehydrogenase (GAPDH) were taken from Glasgow et al.
(1999). All primers were synthesized by Integrated DNA Technologies
(Coralville, IA). The following primers were used: CRH, 5?-GCCCCGC
AGCCGTTGAA-3? and 5?-GACCGCCTCCCTCTCTCCAG-3? (326 bp
product); TRH, 5?-GGGGACCTCGGTGCTGCCTTAGAC-3? and 5?-
CTGCCGCTTGACTTGGGGGACATC-3? (276 bp); OT, 5?-GACGGT
ACAAA-3? (462 bp); VP, 5?-CCTCACCTCTGCCTGCTACTT-3? and
5?-GGGGGCGATGGCTCAGTAGAC-3? (440 bp); GAPDH, 5?-GGAC
GCCAAAG-3? (441 bp). Each of the primers was used for every cell
analyzed. Final PCR products were visualized by staining with ethidium
bromide after separation by electrophoresis in 1.5–2% agarose gels. The
detect genomic DNA in each cellular template. In addition to the single-
cell GAPDH RT-PCR, whole hypothalamic slices were run in parallel
with the single-cell reactions as positive controls for the primers. Nega-
the RT step from the reaction, respectively. None of the cells tested
showed a genomic DNA PCR product.
To characterize the fast effects of glucocorticoids on putative
neurosecretory parvocellular PVN neurons, we performed
dial parvocellular region of the PVN in acutely prepared hypo-
thalamic slices. A total of 131 putative neurosecretory parvocel-
lular PVN neurons, identified on the basis of their visualized
position within the PVN and on electrophysiological criteria de-
scribed in Materials and Methods, were recorded in acute hypo-
thalamic slices from 80 rats. mEPSCs were recorded at a holding
potential of ?60 mV, which was close to the calculated chloride
AP-5 (50 ?M) and DNQX (30 ?M) (n ? 4) but were not affected
by the GABAAreceptor antagonist bicuculline methiodide (30
?M) (n ? 6), indicating that they were mediated by synaptically
released glutamate. Data were collected after an initial 10 min
baseline period was established during which the amplitude and
frequency of mEPSCs were stable. The mEPSCs recorded under
control conditions ranged in amplitude from 8.2 to 36.9 pA, in
The glucocorticoids dexamethasone and corticosterone applied
in the bath perfusion had no effect on membrane-holding cur-
rent or input conductance in any of the neurons tested but sup-
putative parvocellular neurosecretory neurons of the PVN (i.e.,
?10% change in mEPSC frequency). Dexamethasone (1 ?M), a
selective synthetic glucocorticoid receptor agonist, caused a sig-
(24.77 ? 1.95 vs 24.10 ? 1.76 pA; 98.18 ? 1.5% of baseline; p ?
0.13) or decay time (3.52 ? 0.17 vs 3.68 ? 0.16 msec; 105.0 ?
effect of dexamethasone on mEPSC frequency had a rapid onset
(3–5 min) and was dose dependent, with a threshold concentra-
tion between 10 and 100 nM and a saturating concentration be-
inhibitory effect on mEPSCs, causing a decrease in the mEPSC
frequency (1.90 ? 0.57 to 1.39 ? 0.43 Hz; 72.4 ? 4.6% of base-
line; p ? 0.01; n ? 7) (Fig. 1e), without affecting either the
of baseline; p ? 0.13) or decay time (4.06 ? 0.85 vs 3.79 ? 0.76
ticoid did not reverse after 60 min of washout of the steroid. The
effect was steroid-specific, because the corticosteroid precursor
cholesterol (5 ?M) and the physiologically inactive steroid iso-
pregnanolone (5 ?M) had no effect on mEPSCs (Fig. 1e). Thus,
glucocorticoids exerted a fast inhibitory effect on glutamate re-
lease onto PVN parvocellular neurons.
The rapid onset of the effect of glucocorticoid (3–5 min) sug-
gested a nongenomic mechanism of steroid action. We con-
ducted a series of experiments to determine whether the effect of
glucocorticoid on glutamate release was mediated by activation
of the classical intracellular corticosteroid receptors. The de-
crease in mEPSC frequency induced by dexamethasone was not
type I or type II corticosteroid receptor antagonists spironolac-
tone (10 ?M) and mifepristone (10 ?M), respectively (Fig. 2a).
Additionally, dexamethasone (1 ?M) applied directly into the
cytoplasm of parvocellular neurons by including it in the patch
frequency plots of mEPSC interval and amplitude distributions from the same cell showed a
significant reduction in mEPSC frequency ( p ? 0.01) with no change in mEPSC amplitude
frequency. Numbers in parentheses represent numbers of cells analyzed in each condition in
Glucocorticoids inhibited glutamate release onto PVN parvocellular neurons. a,
4852 • J.Neurosci.,June15,2003 • 23(12):4850–4857 Dietal.•GlucocorticoidInhibitionviaEndocannabinoidRelease
of baseline after 10 min of recording; n ? 7) (Fig. 2a). Also, bath
application of a membrane-impermeant DEX–BSA conjugate
(10 ?M) retained the inhibitory effect of dexamethasone on
mEPSC frequency (2.18 ? 0.77 to 1.39 ? 0.54 Hz; 60.2 ? 7.3%;
glucocorticoids on glutamate release was not mediated by the
classical intracellular corticosteroid receptors, and suggested a
mechanism involving a membrane-associated receptor.
We investigated the molecular mechanisms of the rapid glu-
cocorticoid effect in parvocellular neurons by testing for
pipette to block postsynaptic G-protein activity in the PVN par-
vocellular neurons. Intracellular application of GDP-?-S (10
min) blocked the decrease in mEPSC frequency evoked by dexa-
methasone (94.7 ? 4.7% of baseline; p ? 0.11; n ? 9) (Fig. 2b).
Because GDP-?-S is not membrane-permeant, this indicated
that the effect of glucocorticoid on glutamate release was depen-
rons. To determine the protein-kinase dependence of the glu-
cocorticoid effect, staurosporine, a broad-spectrum protein
kinase inhibitor, and GF109203X, a protein kinase C (PKC)-
selective blocker, were applied in the bath 10 min before dexa-
methasone application. Staurosporine (0.5 ?M) attenuated the
significant levels (82.5 ? 6.9% of baseline; p ? 0.25; n ? 5), and
GF109203X (0.5 ?M) blocked the dexamethasone effect com-
pletely (102.7 ? 5.8% of baseline; n ? 6) (Fig. 2b). Thus, the
inhibitory effect of glucocorticoids on glutamate release in PVN
is not yet known whether the kinase dependence of the action of
glucocorticoid is presynaptic and/or postsynaptic.
mate release, indicating a presynaptic steroid effect, and on the
other hand, that the effect of glucocorticoid was dependent on
steroid action. This suggested that the actions of glucocorticoid
and has been proposed as an intranuclear messenger in the reg-
ulation of GABA inputs to PVN magnocellular neurons (Bains
and Ferguson, 1997). We tested whether NO or CO functions as
the retrograde messenger that modulates glutamate release in
response to glucocorticoids. Bath application of L-NAME (50
(1 ?M) on mEPSC frequency (decrease to 68.4 ? 6.7% of base-
line; p ? 0.05; n ? 5) (Fig. 3a–c). Similarly, bath application of
hemoglobin (20 ?M), the extracellular NO and CO scavenger,
also failed to block the decrease in mEPSC frequency induced by
dexamethasone (1 ?M) (to 72.3 ? 10.7% of baseline; p ? 0.05;
role in the response to glucocorticoids.
sengers that reduce synaptic glutamate and GABA release (Shen
et al., 1996; Auclair et al., 2000; Wilson and Nicoll, 2001; Wilson
the retrograde messenger in the rapid, glucocorticoid-mediated
inhibition of glutamate release. Bath application of the type I
cannabinoid receptor (CB1) antagonists AM251 (1 ?M) and
AM281 (10 ?M) blocked the dexamethasone-induced reduction
in mEPSC frequency (AM251: 94.4 ? 2.7% of AM251 value, or
AM281 value, or 82.9 ? 8.7% of baseline, p ? 0.29, n ? 7) (Fig.
failed to block the effect of dexamethasone on mEPSC frequency
blocked the dexamethasone effect at a higher, nonselective con-
high-affinity synthetic cannabinoid agonist WIN55,212-2 mim-
icked and occluded the inhibitory effect of glucocorticoids on
1 ?M) caused a significant decrease in mEPSC frequency (70.3 ?
6.9% of baseline; p ? 0.01; n ? 13) but had no effect on mEPSC
amplitude (101.1 ? 3.4%) or decay time (104.6 ? 2.8%) (Fig.
4d,e). Dexamethasone application (1 ?M) after 10 min of
WIN55,212-2 application (1 ?M) failed to elicit any additional
decrease in mEPSC frequency (96.2 ? 5.8% of the WIN55,212-2
retrograde endocannabinoid messenger and activation of pre-
synaptic CB1receptors. Application of the cannabinoid antago-
nists AM281 (10 ?M) and AM630 (10 ?M) subsequent to dexa-
methasone application did not reverse the inhibitory effect of
dexamethasone on glutamate release (data not shown), which
suggested that the nonreversibility of the effect of glucocorticoid
was caused by a mechanism downstream from the presynaptic
Parvocellular PVN neurons tested for their sensitivity to glu-
cocorticoids or cannabinoids were screened for CRH, TRH, OT,
and the intracellular mineralocorticoid receptor antagonist spironolactone (10 ?M) failed to
ers abolished the effect of glucocorticoid on mEPSCs. Postsynaptic G-protein blockade with
itory effect of dexamethasone on mEPSCs, suggesting a PKC-dependent mechanism that is
The glucocorticoid effect was mediated by a postsynaptic, G-protein-coupled
Dietal.•GlucocorticoidInhibitionviaEndocannabinoidRelease J.Neurosci.,June15,2003 • 23(12):4850–4857 • 4853
and VP mRNA expression using the single-cell RT-PCR tech-
nique after experiments (Surmeier et al., 1996; Glasgow et al.,
1999). Of 30 neurons responsive to glucocorticoids, five ex-
pressed CRH, two expressed TRH, four expressed OT, and three
expressed VP mRNAs. Of 16 neurons responsive to cannabinoid
analogs, one expressed CRH, four expressed OT, and two ex-
pressed VP mRNAs. Each of the identified cells expressed only
one of the four mRNAs (Fig. 5), except one cell in which expres-
sion of both TRH and OT was detected. These findings indicate
rosecretory parvocellular PVN neurons, and suggest a general-
ized feedback inhibitory role of glucocorticoids in the regulation
of hypophysiotropic hormone secretion.
Together, our data demonstrate a fast, membrane corticosteroid
receptor-mediated suppression of excitatory input to neurose-
cretory parvocellular neurons of the PVN, and support a model
of acute glucocorticoid actions (Fig. 6). In this model, glucocor-
ticoids activate a postsynaptic G-protein-coupled membrane re-
ceptor that leads to the release of an endocannabinoid. The en-
docannabinoid acts as a retrograde messenger at presynaptic
glutamate terminals to inhibit glutamate release onto the parvo-
to these cells.
Several findings presented here support the involvement of a
extracellular corticosteroids, (2) the lack of effect of intracellular
of antagonists of the classical intracellular corticosteroid recep-
tors to block the acute corticosteroid effect. Additionally, block-
ade of the corticosteroid effect by blocking G-protein activity
selectively in the postsynaptic neuron implicates a G-protein-
oftheNOSinhibitorL-NAME(10?M).b,CumulativefrequencyplotsshowedasignificantreductioninmEPSCfrequency( p?0.05)withnochangeinmEPSCamplitude( p?0.24)inthepresence
interval( p?0.01)butnochangeinmEPSCamplitude( p?0.42)inWIN55,212-2.f,MeanchangesintheaveragemEPSCfrequencyinthepresenceofglucocorticoidandthecannabinoidreceptor
The effect of glucocorticoid on glutamate release was mediated by an endocannabinoid. a, The dexamethasone-induced suppression of mEPSCs was prevented by previous bath
4854 • J.Neurosci.,June15,2003 • 23(12):4850–4857Dietal.•GlucocorticoidInhibitionviaEndocannabinoidRelease
roid receptor in PVN parvocellular neurons is a G-protein-
coupled receptor. Whether the kinase dependence of the
response is associated with the postsynaptic G-protein signaling
that leads to the release of endocannabinoid or with the presyn-
aptic cannabinoid receptor signaling remains to be determined.
These findings are consistent with reports of G-protein and/or
brain, including radiolabeled corticosterone binding in amphib-
ian neuronal membranes (Orchinik et al., 1992) and cortisol
modulation of calcium currents in guinea pig CA1 pyramidal
neurons (ffrench-Mullen, 1995).
We found that the activation of a postsynaptic, G-protein-
dependent signaling mechanism led to the suppression of gluta-
mate release, a presynaptic endpoint, thus implicating the in-
volvement of a retrograde messenger. Inhibiting the retrograde
messengers NO and CO failed to block the effect of glucocorti-
coid on mEPSC frequency or inhibition of cannabinoid recep-
tors. Conversely, saturation with exogenous agonist or antago-
nist, respectively, abolished the effect of glucocorticoid on
mEPSCs completely, suggesting that the retrograde messenger
involved is an endocannabinoid. Our findings indicate that
the glucocorticoid-induced endocannabinoid actions were me-
diated by presynaptic CB1receptors, because they were blocked
by CB1receptor antagonists as well as by a CB2receptor antago-
nist at a concentration effective at both CB2and CB1receptors
but not at a concentration selective for CB2receptors. This is
consistent with the finding that CB1receptors are the predomi-
nant receptors in the brain (Matsuda et al., 1990; Munro et al.,
1993). Retrograde regulation of the synaptic release of glutamate
and GABA by endocannabinoids is emerging as a widespread
modulatory mechanism at synapses throughout the brain, in-
cluding the hippocampus, cerebellum, striatum, and neocortex
(Huang et al., 2001; Ohno-Shosaku et al., 2001). It is worth not-
ing that the CB receptor antagonists we used are considered in-
verse agonists (Gifford et al., 1997; Ross et al., 1999), although
they may not play an inverse agonistic role at the presynaptic
receptors in the PVN at the concentrations used. Future studies
will clarify the CB receptor mechanisms in the PVN.
Introduction of GDP-?-S into a single parvocellular neuron
blocked the actions of glucocorticoids completely, suggesting
that there was little or no spillover of endocannabinoids released
by adjacent neurons onto the recorded neurons. This is consis-
tent with a restricted secretion domain and limited spread of the
endocannabinoids in the extracellular space. Unlike the gaseous
retrograde messenger NO, which has been shown to spread to
activate local GABA circuits in the PVN (Bains and Ferguson,
1997), retrogradely released endocannabinoids might be ex-
pected to have a restricted range of action because of their li-
pophilic nature (Kreitzer and Regehr, 2002).
It is interesting to note that the inhibitory actions on PVN
neurosecretory cell activity of glucocorticoids and cannabinoids
applied directly in the PVN in our experiments are opposite
to the activational effects of systemic anandamide or ?9-
tetrahydrocannabinol on HPA hormone release (Murphy et al.,
1998). However, the effects of systemic cannabinoids on HPA
hormone secretion are accompanied by changes in PVN cate-
cholamine levels (Murphy et al., 1990; Rodriguez de Fonseca
et al., 1995), and are blocked completely by hypothalamic deaf-
effects of cannabinoids occur upstream of the PVN, and that
this cannabinoid-stimulated afferent excitation either is not
glutamatergic or is glutamatergic and strong enough to override
mate (GLU) release onto the PVN neuron?leading to decreased PVN neuronal activity and
A model of the rapid glucocorticoid actions in PVN parvocellular neurons. The
Dietal.•GlucocorticoidInhibitionviaEndocannabinoidReleaseJ.Neurosci.,June15,2003 • 23(12):4850–4857 • 4855
mediated by steroid actions at different central targets, either
indirectly via actions in the hippocampus or directly at the levels
of the hypothalamus and pituitary (Dallman et al., 1987). Glu-
cocorticoids have been reported to have direct inhibitory effects
on the electrical activity of some parvocellular neurons of the
PVN (Kasai and Yamashita, 1988; Saphier and Feldman, 1988),
although the inhibitory mechanisms have not been determined
and the identification of specific glucocorticoid-responsive PVN
for the inhibition by glucocorticoids of glutamate release onto
parvocellular neurosecretory neurons of the PVN, including
CRH-expressing cells, which resulted in the suppression of exci-
tatory inputs to these cells. This is the first demonstration, there-
fore, of a rapid inhibition of identified CRH neurons by cortico-
steroids, and provides a mechanism for the fast feedback
inhibition of CRH release by glucocorticoids directly at the level
of the hypothalamic CRH neurons.
In addition to the effect on CRH neurons, we found that glu-
cocorticoids also exerted a similar inhibitory effect on glutamate
release onto parvocellular TRH-expressing, OT-expressing, and
VP-expressing neurons. These peptides, along with CRH, are
thought to play an anorexigenic role in the central control of
and endocannabinoids both have well established central orexi-
that the modulatory cross talk between glucocorticoids and en-
docannabinoids that leads to reduced excitation of PVN parvo-
the central control of feeding and energy balance.
of a G-protein-dependent signaling pathway represents a novel
mechanism of rapid steroid action in the brain. This endocan-
nabinoid link between steroid hormonal and neuronal signaling
rograde modulators of synaptic function. The glucocorticoid-
cellular neurons is likely to play an important role in the acute
negative feedback effect of glucocorticoids on HPA axis activity.
Because the PVN is considered to be the main neuroendocrine
and autonomic effector in the control of stress, energy balance,
and homeostasis (Swanson and Sawchenko, 1980; Laugero,
2001), the modulatory cross talk between glucocorticoids and
tral mechanism for the well established orexigenic and homeo-
static actions of both glucocorticoids and cannabinoids. This
back actions of glucocorticoids in the PVN provides new poten-
tial targets for the therapeutic treatment of stress and feeding
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