Department of Neuroscience, University of California, Riverside, California 92521
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Li, Zhenhui and
Glenn I. Hatton.
Histamine Suppresses Non-NMDA Excitatory Synaptic Currents in Rat
Supraoptic Nucleus Neurons.
J. Neurophysiol. 83: 2616-2625, 2000.
Whole cell patch-clamp
recordings were obtained from supraoptic neurons to investigate the
effects of histamine on excitatory postsynaptic currents evoked by
electrical stimulation of areas around the posterior supraoptic
nucleus. When cells were voltage-clamped at 70 mV, evoked excitatory
postsynaptic currents had amplitudes of 88.4 ± 9.6 pA and
durations of 41.1 ± 3.0 ms (mean ± SE;
n = 43). With twin stimulus pulses (20 Hz) used,
paired-pulse facilitation ratios were 1.93 ± 0.12. Bath
application of 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX) abolished
synaptic currents. Histamine at concentrations ~0.1-10 µM
reversibly suppressed excitatory postsynaptic currents in all
supraoptic neurons tested. Within 2 min after application of (10 µM)
histamine, current amplitudes and durations decreased by 61.5 and
31.0%, respectively, with little change in the paired-pulse facilitation ratio. Dimaprit or imetit (H2 or
H3 receptor agonists) did not reduce synaptic currents,
whereas pyrilamine (H1 receptor antagonist) blocked
histamine-induced suppression of synaptic currents. When patch
electrodes containing guanosine 5'-O-(2-thiodiphosphate) (GDP-
-S) were used to record cells, histamine still suppressed current amplitudes by 49.1% and durations by 41.9%. Similarly, intracellular diffusion of
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) and H7 did not abolish histamine-induced
suppression of synaptic currents, either. Bath perifusion of
8-bromo-quanosine 3',5'-cyclic monophosphate reduced current amplitudes
by 32.3% and durations by 27.9%. After bath perfusion of slices with
N
-nitro-L-arginine methyl ester (L-NAME),
histamine injection decreased current amplitudes only by 31.9%, much
less than the inhibition rate in control (P < 0.01). In addition, histamine induced little change in current
durations and paired-pulse facilitation ratios, representing a partial
blockade of histamine effects on synaptic currents by L-NAME. In
supraoptic neurons recorded using electrodes containing BAPTA and
perifused with L-NAME, the effects of histamine on synaptic currents
were completely abolished. Norepinephrine injection reversibly
decreased current amplitudes by 39.1% and duration by 64.5%, with a
drop in the paired-pulse facilitation ratio of 47.9%. Bath perifusion
of L-NAME, as well as intracellular diffusion of GDP-
-S, , 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine , or BAPTA, failed
to block norepinephrine-induced suppression of evoked
synaptic currents. The present results suggest that histamine
suppresses non-N-methyl-D-aspartate synaptic
currents in supraoptic neurons through activation of H1
receptors. It is possible that histamine first acts at supraoptic cells
(perhaps both neuronal and nonneuronal) and induces the production of
nitric oxide, which then diffuses to nearby neurons and modulates
synaptic transmission by a postsynaptic mechanism.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histamine (HA) has been proposed to function both as a transmitter
and a modulator in the brain (Hatton and Li 1998a;
Prell and Green 1986
). Released from presynaptic
terminals in response to electrical stimulation of the tuberomammillary
nucleus (TM) or to dehydration (Akins and Bealer 1993
;
Kjaer et al. 1994
), HA not only elicits excitatory or
inhibitory postsynaptic potentials (Weiss et al. 1989
;
Yang and Hatton 1994
) but also regulates gap junctional
communication (Hatton and Yang 1996
). The latter, as well as HA-induced prolonged depolarization and enhancement of depolarizing after potentials (DAPs) (Smith and Armstrong
1993
; Yang and Hatton 1989
), is mediated by
activation of various intracellular messenger systems (Leurs et
al. 1995
). Our recent experiments demonstrated that prevention
of G proteins, phospholipase C (PLC) or protein kinase C (PKC) from
activation blocks HA-induced depolarization and enhancement of currents
underlying depolarizing after potentials (IDAP)
(Li and Hatton 1996
; Li et al. 1999
).
Reduced nitric oxide (NO) production with inhibitors of NO synthase
(NOS) cancels HA-induced increases in dye coupling among supraoptic
nucleus (SON) neurons (Yang and Hatton 1999
).
The SON receives rich glutamatergic inputs that are critical in the
regulation of hypothalamic neuroendocrine activity (Armstrong 1995; Hatton 1990
; van den Pol et al.
1990
). Glutamate can bind to postsynaptic
non-N-methyl-D-aspartate (NMDA), mainly AMPA, and NMDA receptors to accomplish neurotransmission (Mayer and Westbrook 1987
; Ozawa et al. 1998
). Activation
of ionotropic glutamate receptors usually causes membrane
depolarization and burst activities in SON neurons, resulting in
increased release of both vasopressin and oxytocin (Gribkoff and
Dudek 1990
; Hu and Bourque 1992
; Moos et
al. 1997
; Wuarin and Dudek 1993
; Yang et
al. 1995
). Because HA is known to modulate excitatory
postsynaptic currents (EPSCs) in hippocampal neurons (Bekkers
1993
; Brown et al. 1995
; Nikmanesh et al.
1996
; Saybasili et al. 1995
), here we examined
whether HA influences non-NMDA glutamate receptor-mediated synaptic
currents in SON neurons and explored possible intracellular mechanisms involved. For comparison, we also tested the effects of norepinephrine (NE) on synaptic currents. NE is another neurotransmitter that is
reported to activate intracellular signal transduction systems similar
to those used by HA (Armstrong 1995
; Minneman and
Esbenshade 1994
). The results showed that HA reversibly
suppresses non-NMDA EPSCs via activating H1
receptors, and NO is partially responsible for these actions of HA. A
preliminary report of this study has appeared in abstract form
(Hatton and Li 1998b
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation
Male rats, 50-70 days, were decapitated using a rodent guillotine. Their brains were removed, and horizontal slices of the hypothalamus (250- to 300-µm thick) were cut using a vibratome. Oxygenated (95% O2-5% CO2) artificial cerebrospinal fluid (ACSF) contained (mM) 126 NaCl , 5 KCl, 1.3 NaHPO4, 2.4 CaCl2, 3.0, MgSO4, 26 NaHCO3, 10 glucose, and 5 3(N-morpholino)propanesulfonic acid buffer (310 mOsmol/l, pH 7.4). After incubation at room temperature (23°C) for >2 h, the slices were transferred to a submerging recording chamber and perifused with warmed ACSF (36°C) at 2 ml/min. To abolish GABAergic inhibitory synaptic inputs, bicuculline (10-15 µM) was added to the ACSF.
Electrophysiological recordings
Patch electrodes were pulled from borosilicate capillary tubing
and filled with the following solution (mM): 140 K+ gluconate, 2 MgCl2,10
HEPES, 2 K2ATP, and 0.4 Na2GTP (pH 7.25). Electrodes usually had outer
tip diameters of ~2.7 µm and DC resistances of 4-5 M. Whole
cell patch-clamp recordings were obtained from SON neurons using an
Axoclamp-2B amplifier (Axon Instruments, Foster City, CA). Patch
electrodes approached the SON under visual guidance using a dissecting
microscope. Whole cell recordings were obtained using current-clamp
techniques. Hyperpolarizing currents were repeatedly passed to induce
voltage responses that increased in amplitude when electrode tips
touched neuronal membranes. Gigaohm seals between electrodes and
cell membranes then were obtained by applying gentle suction. When a
seal was achieved, further brief suction was applied to break through
the membrane. Whole cell recordings were indicated by observation of
negative membrane potentials and action potentials. KCl/agar bridges
were used as reference electrodes and correction of liquid junction potential (
7 mV) was applied to recorded membrane voltages.
Continuous single-electrode voltage-clamp experiments were performed to
investigate the EPSCs. Cells were clamped around 70 mV, and amplifier
feedback gain was set at 40-90 nA/mV and low-pass filter at 1.0 kHz.
Series resistance was evaluated by measuring instantaneous currents
evoked by a hyperpolarizing pulse (10 mV, 40 ms) and ranged from 4 to
10 M
. Membrane conductance was monitored during the experiment using
hyperpolarizing pulses (
10 mV, 75 ms). Electrical stimulation was
delivered through a concentric electrode positioned in an area
immediately lateral to the posterior SON (Fig.
1A). Current intensity was
increased gradually until EPSCs were evoked by each stimulus (0.2 ms,
50-300 µA). Once per minute, four EPSCs at an interval of 5 s
were acquired consecutively and averaged. EPSC duration is the period
from the beginning of stimulating pulses to the return of the inward
current to baseline, whereas EPSC amplitude refers to the maximal value
of the inward current compared with baseline. The change in EPSC was
determined by comparing ion currents obtained before and 1, 3, 5, and
10 min after perifusion of slices with test agents. Data acquisition and analyses were performed using a computer operating AXOTAPE and
pCLAMP (Axon Instruments). All data are presented as means ± SE
and repeated measures ANOVA was applied for statistical analyses.
|
Chemicals
HA, NE, pyrilamine (H1 receptor
antagonist), dimaprit (H2 receptor agonist),
imetit (H3 receptor agonist, Tocris, Ballwin, MO), and N-nitro-L-arginine methyl
ester (L-NAME) were stored in concentrated solutions
(10
4-10
2 M) and diluted
into ACSF before experiments. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, RBI, Natick, MA), 2-amino-5-phosphonovaleric acid (APV), and
8-bromo-quanosine 3',5'-cyclic monophosphate (8-bromo-cGMP) were
directly dissolved in ACSF and applied. When needed, guanosine 5'-O-(2-thiodiphosphate) (GDP-
-S),
1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H7), and
1,2-bis(2-aminophenophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) were added to the electrode filling solution and applied
intracellularly after membrane rupture. Except where indicated, test
agents were usually bolus-injected into perifusion pipeline. It took
~30 s before test agents reached the recording chamber, after which
the effects of agents were observed. Because of prolonged effects on
SON neurons of activating intracellular second-messenger systems
(Armstrong and Sladek 1985
; Li and Hatton
1996
; Li et al. 1999
), such brief exposure of
SON cells to HA and NE, provided by bolus injections, achieves a much
better recovery rate than bath application. The final concentrations of
HA and NE in the recording chamber were ~10 µM (50 times dilution).
Except as otherwise stated, all chemicals used were purchased from
Sigma, St. Louis, MO.
Animal care and use during this study were in accordance with National Institutes of Health and institutional guidelines and policy on the use of animals in research. All efforts were made to minimize animal suffering and to reduce the number of animals used.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Evoked non-NMDA EPSCs in supraoptic nucleus neurons
Whole cell patch-clamp recordings were obtained from 68 SON
neurons. The resting membrane potentials ranged from 55 to
75 mV,
amplitudes of action potentials from 70 to 100 mV, and membrane input
resistances from 0.2 to 0.8 G
. In bridge mode without DC current
injection, 38% of these cells displayed phasic patterns of firing,
with DAPs following spontaneous single spikes. The remaining
recorded cells displayed continuous, irregular or burst patterns of
firing or were silent. Abundant spontaneous excitatory postsynaptic
potentials (EPSPs) were observed routinely, especially when the
membrane was hyperpolarized (Fig. 1B). Electrical
stimulation of an area lateral to the posterior SON evoked large
(10-30 mV) EPSPs that often triggered action potentials. These results
are consistent with previous reports (for review, see Hatton and
Li 1998a
).
In SON neurons showing evoked EPSPs, voltage-clamp experiments were
carried out and inward postsynaptic currents, i.e., EPSCs, were induced
after brief electrical stimulation (Fig.
2A). When cells were
voltage-clamped at 70 mV, evoked EPSCs had amplitudes of 88.4 ± 9.6 pA (n = 43). Their rise times ranged from 2-7 ms and durations were 41.1 ± 3.0 ms. With twin stimulus pulses used (20 Hz repeated every 5 s), EPSCs evoked by the second pulse
(171.2 ± 11.5 pA, 63.2 ± 4.8 ms; n = 43)
were always larger than those by the first pulse (for the amplitude and
duration, P < 0.001 and <0.05, respectively). Mean
paired-pulse facilitation (PPF) ratios were 1.93 ± 0.12. Bath
application of 10-20 µM CNQX, a non-NMDA receptor antagonist, for 2 min abolished EPSCs (Fig. 2B). CNQX did not change the PPF
significantly (from 1.91 ± 0.10 in control to 1.64 ± 0.26, P > 0.05). After bath perifusion of slices with 50 µM APV for 10 min, however, EPSCs decreased by ~10%
(n = 3). These data suggest that EPSCs recorded in the
current experiments are predominately gated by non-NMDA (AMPA/kainate) glutamate receptors.
|
Suppression of EPSCs by histamine
HA effectively suppressed EPSCs in all 15 SON neurons tested (Fig.
3). These cells displayed phasic or
nonphasic patterns of firing, with or without DAPs. Within 2 min after
HA application, EPSC amplitudes and durations decreased by 61.5 and
31.0%, respectively (from 67.5 ± 12.7 pA and 44.9 ± 4.8 ms
in control to 26.0 ± 6.4 pA to 31.0 ± 5.4 ms,
n = 15; P < 0.001 for both).
Similarly, HA suppressed EPSCs evoked by the second pulses. The
amplitudes and durations decreased by 59.4 and 27.5%, resulting in no
change in PPF (from 1.87 ± 0.13 in control to 1.95 ± 0.14, n = 15; P > 0.05). Recovery of EPSCs
from HA suppression was readily observed. EPSC amplitudes were 66.8, 86.7, and 95.6% of the control at 3, 5, and 10 min after HA
application, whereas EPSC durations were 83.7, 95.6, and 108.5%
(n = 15), respectively. Because EPSCs evoked by the
second pulses recovered faster, the PPF rose 118.2 and 126.2% of the
control (1.87 ± 0.13, n = 15) at 3 and 5 min
after HA application, although these differences were not statistically significant (P > 0.05). We examined the dose-response
relationship in another five cells, and bath perifusion of HA at
concentrations of 0.1, 1, and 10 µM suppressed EPSC amplitudes by
21.2, 47.6, and 70.2%, respectively (data not shown).
|
In contrast to HA, neither dimaprit nor imetit attenuated EPSCs (Fig. 4). Within 2 min after bolus injection of dimaprit, both EPSC amplitudes durations changed little (from 95.6 ± 12.9 pA and 41.1 ± 6.6 ms in control to 103.7 ± 22.1 pA and 40.5 ± 7.9 ms, n = 5; P > 0.05 for both). There was also no significant difference in PPF (1.65 ± 0.17 in control to 1.53 ± 0.22; P > 0.05). EPSC amplitudes and durations were 69.9 ± 8.3 pA and 44.2 ± 4.9 ms in control, and 77.7 ± 17.2 pA and 41.6 ± 5.8 ms within 2 min after imetit injection (n = 7), which are not significantly different (P > 0.05). The PPF was 1.95 ± 0.17 in control and 2.06 ± 0.23 within 2 min after imetit application (P > 0.05).
|
After bath application of 10-20 µM pyrilamine for 5 min, HA induced little change in EPSCs (Fig. 4A). EPSC amplitudes were 76.6 ± 9.7 pA in control and 70.6 ± 8.2 pA within 2 min after HA injection (n = 8; P > 0.05), and durations were 38.4 ± 5.6 ms and 42.3 ± 5.4 ms (P > 0.05, respectively). Similarly, the difference in PPF was also not significant (from 1.56 ± 0.11 to 1.62 ± 0.10; P > 0.05). These data suggest that HA suppress EPSCs in SON neurons by activating membrane receptors of the H1 subtype. NE also attenuated EPSCs; these results are described further in a later section.
Effects of histamine after the blockade of intracellular signal transduction in neurons
Histaminergic fibers and H1 receptors are
abundant in the SON, and their distribution patterns suggest that
locally released HA can affect not only SON neurons but also other
elements such as glia and presynaptic terminals (Inagaki et al.
1988; Kjaer et al. 1994
). The suppression of
EPSCs might be mediated by a direct action of HA at SON neurons from
which the EPSCs were recorded, by neuromodulators released from
neighboring cells after HA application, or by a decrease in transmitter
release resulting from the effects of HA and/or neuromodulators on the
presynapses. Although the PPF did not increase so greatly as to reach a
statistically significant level during HA application, there is still a
possibility that presynaptic modulation is partially responsible for
EPSC suppression induced by HA. We performed further experiments in the
current study, therefore to explore the mechanisms underlying EPSCs suppression.
When patch electrodes containing 0.5 mM GDP--S, a nonhydrolysable
GDP analogue, were used to record cells, EPSCs recorded were 145.0 ± 31.1 pA and 68.3 ± 4.1 ms (n = 4), which are
not significantly different from EPSCs in control (without GDP-
-S; P > 0.05; Fig. 5).
Within 2 min after HA injection, EPSCs were 73.8 ± 23.0 pA and
39.7 ± 3.3 ms, i.e., reductions of 49.1 and 41.9% in amplitude
and duration (P < 0.05 for both). The PPF went from
1.37 ± 0.17 in control to 1.76 ± 0.62 (P > 0.05). Intracellular diffusion of (10 mM) BAPTA to chelate
intracellular-free Ca2+, and (0.5 mM)
H7 to antagonize PKC activity, did not abolish HA-induced suppression of EPSCs, either (Fig.
6). In 11 SON neurons recorded using
electrodes containing BAPTA, EPSCs were 132.9 ± 41.2 pA and
38.6 ± 5.2 ms in control and 67.4 ± 24.9 pA and 18.6 ± 4.7 ms 2 min after HA injection, representing suppression by 49.3 and 51.8% in EPSC amplitude and duration, respectively. The change in
PPF from 1.79 ± 0.16 in control to 2.05 ± 0.26 was
nonsignificant (14.5%; P > 0.05). When both BAPTA and
H7 were diffused into the cells, HA reduced EPSC
amplitudes by 43.9% (from 129.7 ± 10.2 pA to 72.8 ± 23.1 pA; P < 0.01) and durations by 38.4% (from 59.4 ± 2.6 ms to 36.6 ± 10.4 ms; P < 0.05). The PPF
did not change significantly: 1.72 ± 0.13 in control to 2.19 ± 0.46 2 min after HA application (P > 0.05). Again,
EPSCs recorded using electrodes containing BAPTA and
H7 were not statistically different from those in
control (without BAPTA and H7; P > 0.05). These results suggest that a direct action of HA at SON
neurons recorded using patch electrodes is not the primary mechanism
underlying EPSC suppression. Rather it seems likely that this effect is
indirect and that HA first acts at SON cells (including neurons and
nonneurons), resulting in the production of NO, which then induces
postsynaptic changes in the SON neurons recorded.
|
|
Involvement of NO
Our previous studies have demonstrated that binding of HA to
H1 receptors activates an intracellular signal
transduction pathway including G-protein, PLC, inositol
1,4,5-trisphosphate, Ca2+ release from internal
stores, and PKC (Li and Hatton 1996; Li et al.
1999
). Ca2+-calmodulin complex and PKC
can further stimulate NOS and increase production of NO and cGMP
(Schuman and Madison 1994
). As a result, HA induces
prolonged membrane depolarization, enhances DAPs, and facilitates dye
transfer among SON neurons (Armstrong and Sladek 1985
;
Hatton and Yang 1996
; Smith and Armstrong
1993
). To investigate whether NO diffusion from neighboring
neurons is a potential mechanism for EPSC suppression, we carried out
further experiments using cGMP and L-NAME.
Bath perifusion of 1-2 mM 8-bromo-cGMP for 5 min reduced EPSC amplitudes by 32.3% (85.7 ± 17.8 pA in control to 58.0 ± 16.1 pA; n = 4; P < 0.01) and durations by 27.9% (from 46.3 ± 7.1 to 33.4 ± 9.5 ms; P < 0.05; Fig. 7). There was no change in PPF (from 1.63 ± 0.24 to 2.15 ± 0.65; P > 0.05). A 90% recovery in EPSC amplitude and duration was observed after washout for 10 min. After bath perifusion of slices with 20-25 µM L-NAME, a NOS inhibitor, for 5 min, HA injection decreased EPSC amplitudes only by 31.9% (from 65.0 ± 3.8 pA in control to 34.3 ± 5.0 pA 2 min after HA application, n = 9; P < 0.05; Fig. 8A). This attenuation was much less than that in control (61.5%; P < 0.01, Student's t-test). In addition, no change in EPSC durations (from 43.6 ± 4.4 to 36.1 ± 5.2 ms; P > 0.05) or in PPF (1.83 ± 0.13 to 1.90 ± 0.28; P > 0.05) were observed, suggesting a partial blockade of HA's effect by L-NAME. In SON neurons recorded using electrodes containing 10 mM BAPTA and perifused with L-NAME, the effects of HA on the EPSCs were abolished completely (Fig. 8B). The EPSC amplitudes were 119.8 ± 21.8 pA in control and 101.3 ± 27.8 pA within 2 min after HA injection (n = 4; P > 0.05), whereas durations were 43.3 ± 9.3 and 43.3 ± 6.0 ms, respectively (P > 0.05). The PPF was 1.61 ± 0.25 and 1.48 ± 0.21 before and 2 min after HA application (P > 0.05).
|
|
Comparison with NE
The SON receives dense noradrenergic innervation from the brain
stem nuclei (Armstrong 1995; Hatton
1990
). Binding to corresponding receptors in the membrane, NE
activates intracellular signal pathways, many of which also are
involved in mediating the effects of HA on SON neurons (Leurs et
al. 1995
). A recent report has demonstrated that NE can
suppress inhibitory postsynaptic currents (IPSCs) through a presynaptic
mechanism (Wang et al. 1998
). Therefore we examined
whether NE affects EPSCs in SON neurons and compared the effects of NE
with those of HA.
The EPSCs, with amplitudes of 72.3 ± 9.4 pA and durations of
34.6 ± 7.1 ms, were obtained from nine SON neurons. Within 1 and
3 min after NE bolus injection, EPSC amplitudes decreased to 46.6 ± 11.0 pA (by 35.6%; P < 0.01) and 44.0 ± 11.4 pA (by 39.1%; P < 0.001), respectively (Fig. 4),
whereas durations decreased to 17.1 ± 4.5 ms (by 50.6%;
P < 0.01) and 12.3 ± 4.1 ms (by 64.5%; P < 0.001). The PPF went from 1.67 ± 0.15 in
control to 0.87 ± 0.24 (47.9%; P < 0.01) within
3 min after NE injection (Figs. 5-7). The effects of NE on EPSCs were
reversible, and 95% recovery from inhibition of EPSC amplitudes and
durations was observed 10 min after NE injection. Intracellular
diffusion of GDP--S (0.5 mM), H7 (0.5 mM) and
BAPTA (10 mM) did not block NE-induced suppression of EPSCs. EPSC
amplitudes were 102.6 ± 9.3 pA in control (n = 20), and decreased to 62.9 ± 8.3 pA (by 38.7%; P < 0.001) and 45.0 ± 9.7 (by 56.1%; P < 0.001)
within 1 and 3 min after NE injection, respectively. Similar
attenuations in EPSC durations and PPF also were observed. In five SON
neurons bathed with a medium containing 20-25 µM L-NAME, EPSC
amplitudes were reduced from 59.3 ± 10.3 pA in control to
31.4 ± 8.0 pA (by 47.1%; P < 0.001) and
29.9 ± 9.3 pA (by 49.6%; P < 0.001) within 1 and 3 min after NE injection, respectively (Fig. 8A). EPSC
durations decreased from 44.0 ± 5.9 ms in control to 22.3 ± 8.4 ms (49.3%; P < 0.05), whereas the PPF was
1.83 ± 0.10 in control, but decreased to 1.13 ± 0.33 (by
38.3%; P < 0.05) within 3 min after NE treatment. The
combination of perifused L-NAME with intracellular diffusion of BAPTA
did not abolish the effects of NE on the EPSCs either (n = 2). These results suggest that NE suppresses EPSCs
in SON neurons through presynaptic mechanisms without involving NO production.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Neuroendocrine functions in the CNS are subject to continuous
control by extrinsic and intrinsic mechanisms to insure proper and
prompt responses to environmental stimuli (Hatton 1990;
Hatton and Li 1998a
). In this regard, the SON, the
magnocellular neurons of which synthesize and store either vasopressin
or oxytocin, receive massive neural terminal inputs from other CNS
nuclei (Armstrong 1995
; Leng et al.
1999
). Transmitters and/or modulators located at presynaptic
terminals in the SON include glutamate, GABA, NE, HA, acetylcholine,
ATP, oxytocin, vasopressin, angiotensin, opioid and natriuretic
peptides, and interleukins. Many of these substances not only evoke
postsynaptic potentials but also activate membrane metabotropic
receptors and influence intracellular biochemical processes
(Nicoll et al. 1990
). Besides those direct effects on SON neurons, released substances, i.e., modulators, can influence functions of other synapses. For example, bath application of oxytocin,
or inhibitors of aminopeptidase, an enzyme that degrades oxytocin,
reduced AMPA currents by ~35%, suggesting that endogenous oxytocin
can modulate excitatory inputs in the SON (Kombian et al.
1997
). Oxytocin also is known to suppress GABAergic synaptic transmission through postsynaptic mechanisms (Brussaard et al. 1997
). Activation of GABAB receptors
presynaptically inhibits AMPA and GABAA currents
(Kabashima et al. 1997
; Kombian et al. 1996
). In the present study, HA was found to suppress non-NMDA EPSCs in SON neurons by activating H1 receptors.
The suppression was accompanied by little change in the PPF. The
effects of HA remained after blockade of G-protein or PKC activation or
chelation of intracellular Ca2+ in cells where
EPSCs were recorded. EPSC suppression, however, was mimicked by an
increase in cytosolic cGMP but partially eliminated by NOS inhibition.
When combined with intracellular Ca2+ chelation,
NOS inhibition could completely eliminate EPSC suppression induced by
HA. These results support the hypothesis that HA induces NO production
in SON cells, and then NO postsynaptically suppresses the EPSCs in the
same and/or nearby neurons. Bekkers (1993)
reported that
in isolated or cultured hippocampal pyramidal neurons, HA enhances
NMDA, but not AMPA, synaptic currents via activating H3 receptors. Other studies also showed that HA
enhances NMDA currents in hippocampal neurons without involvement of
any HA receptor type known (Saybilisi et al. 1995
;
Vorobjev et al. 1993
). To the best of our knowledge,
therefore this is the first report regarding inhibitory effects of HA
on non-NMDA synaptic currents via mediation by H1 receptors.
Constituting more than one-quarter of the nerve terminals on SON
neurons (Meeker et al. 1993; van den Pol et al.
1990
), glutamatergic inputs are critical in the regulation of
hypothalamic neuroendocrine activity (Armstrong 1995
;
Hatton 1990
). Released from presynapses, glutamate can
bind to postsynaptic non-NMDA (mainly AMPA) and NMDA receptors to
accomplish neurotransmission (Mayer and Westbrook 1987
;
Ozawa et al. 1998
). SON neurons possess both non-NMDA
and NMDA receptors (Meeker et al. 1994a
), the activation
of which usually causes membrane depolarization and burst activities
(Gribkoff and Dudek 1990
; Hu and Bourque
1991
; Moos et al. 1997
; Wuarin and Dudek
1993
; Yang et al. 1995
). In the present study,
EPSCs were obtained from SON cells perifused with a medium containing 3 mM Mg2+ and voltage-clamped around
70 mV,
procedures that separate non-NMDA currents from NMDA ones
(Bekkers and Stevens 1993
; Mayer and Westbrook 1987
). Current isolation also was confirmed pharmacologically by application of CNQX and APV (Young and Fagg 1990
),
with the former greatly reducing or eliminating the EPSCs and the
latter having little effect. These results are consistent with the
previous reports showing that in in vitro preparations most excitatory synaptic inputs to SON neurons are mediated by non-NMDA receptors (Gribkoff and Dudek 1990
; Kabashima et al.
1997
; Kombian et al. 1997
; Wuarin and
Dudek 1993
).
Being evoked in cells displaying phasic or nonphasic patterns of
firing, EPSCs are likely to have been obtained from both vasopressin
and oxytocin neurons. There is an increasing body of evidence, however,
suggesting that oxytocin and vasopressin neurons are controlled
differently by glutamatergic inputs. Immunocytochemical experiments
using an antibody against glutamate receptor subunit 3 (GluR3) in the
rat hypothalamus reveal that these AMPA receptors are located in the
anterodorsal parts of the SON, and 45.8% of oxytocin neurons contain
GluR3 immunoreactivity (Ginsberg et al. 1995). In
contrast, only 1.1% of vasopressin neurons were found to be GluR3
immunopositive. Spontaneous miniature AMPA currents have been shown to
have larger amplitudes and longer durations in oxytocin, than in
vasopressin, neurons (Stern et al. 1999
). Neuronal
activities of putative oxytocin neurons, as well as oxytocin release
during lactation are preferentially increased by AMPA receptor
activation (Parker and Crowley 1993
;
Richardson and Wakerley 1997
), whereas NMDA receptors
are involved in the regulation of rhythmic firing pattern and mediate
EPSPs in vasopressin neurons (Hu and Bourque 1992
;
Moos et al. 1997
; Yang et al. 1994
).
Water deprivation, a strong stimulus for vasopressin secretion due to an increased plasma osmolality and a decreased blood volume,
significantly raises NMDA, but not non-NMDA, receptor densities in the
SON (Meeker et al. 1994b
). Therefore suppression by HA
of non-NMDA EPSCs observed in the present experiments might mainly
represent one source of inhibition to oxytocin neurons and thus to
oxytocin release.
HA, injected into the brain ventricles or SON, is known to elevate
plasma vasopressin concentration and induce antidiuresis (Bennett and Pert 1974; Dogterom et al.
1976
; Tuomisto and Eriksson 1979
;
Tuomisto et al. 1984
). HAergic projections from the TM, and HA receptors have been located in the SON (Inagaki et al. 1988
; Palacios et al. 1981
; Weiss et al.
1989
). An increase in HA release in the hypothalamus is
detected in response to electrical stimulation of the TM (Akins
and Bealer 1993
), dehydration, or injection of hypertonic
saline (Akins and Bealer 1990
; Kjaer et al.
1994
). HA predominantly excites putative vasopressin SON
neurons, enhances DAPs and promotes phasic firing (Armstrong and
Sladek 1985
; Haas et al. 1975
; Li and
Hatton 1996
; Smith and Armstrong 1993
). In
contrast, HA has no effect on, or even hyperpolarizes, the membrane in
most oxytocin neurons (Smith and Armstrong 1993
). TM
stimulation induces fast IPSPs in oxytocin neurons, and these are
blocked by H2-antagonists (Yang and Hatton
1994
). There is no direct evidence so far suggesting that HA
can increase oxytocin secretion either (Hatton 1990
;
Onodera et al. 1994
). Taken together, the present
results support the hypothesis that HA-induced suppression of EPSCs
functions as a supplementary mechanism to coordinate neuroendocrine
activities in the SON. Under conditions of increased demand for
vasopressin release, HA reduces the AMPA receptor-mediated glutamatergic drive to oxytocin neurons while exciting vasopressin neurons and promoting phasic firing, in such a way as to preferentially restore, e.g., water balance with little change in other neuroendocrine activities. It is also possible that this mechanism participates in
preventing premature oxytocin release before parturition, as suggested
in one study (Luckman and Larsen 1997
).
Our results imply that HA-induced suppression of EPSCs is mediated by
H1 receptor activation. It is unlikely that
H2 and H3 receptors are
involved because agonists of these receptors failed to mimic the
effects of HA. The finding that EPSC suppression was abolished by
H1 receptor antagonism also excludes a direct action of HA at AMPA receptors. H1 receptors in
the membrane usually are coupled to G proteins, and activation of these
receptors can increase the activities of PLC, PKC, and NOS, eventually
resulting in NO production and raised
[Ca2+]i (Hatton
and Yang 1996; Leurs et al. 1995
; Li and
Hatton 1996
; Li et al. 1999
). Because
intracellular diffusion of GDP-
-S, H7, and
BAPTA did not interrupt EPSC suppression, it is possible that HA's
effects are primarily indirect, i.e., acting at neighboring cells
and/or presynaptic terminals first. EPSC suppression could have been a
result of local release of neuromodulators after the HA stimulus. A
reduction in transmitter release from the presynapses also would cause
a similar EPSC suppression. Although many neuromodulators are supposed
to be released in response to HA treatment, in the present study we
only examined whether NO, a common intercellular modulator
(Garthwaite 1991
; Zhang and Snyder 1995
),
plays any role in HA-induced suppression of EPSCs. NO is known to
influence synaptic transmission, often through its actions at
postsynaptic NMDA receptors and transmitter secretion (Schuman
and Madison 1994
). In the SON, NOS is abundant (Bredt et
al. 1990
; Calka and Block 1993
; Miyagawa
et al. 1994
; Sanchez et al. 1994
), and NO can
increase dye transfer among neurons through gap junctions (Yang
and Hatton 1999
) and can modulate NMDA currents (Cui et al. 1994
). The present results showed that NOS inhibition
prevented EPSC suppression induced by HA, and cGMP reduced the EPSCs,
implying that NO at least partially participates in HA-induced
suppression of EPSCs. Our findings are consistent with previous
neuroendocrine studies showing that NO controls the release of both
vasopressin and oxytocin (Summy-Long et al. 1993
;
Yasin et al. 1993
). The effects of NO donors on EPSCs
were not examined here because NO itself can enhance the release of
glutamate, dopamine, serotonin, and acetylcholine from the presynapses
(Prast et al. 1996
; Schuman and Madison
1994
).
Further experiments are necessary to identify other potential factors
or mechanisms underlying EPSC suppression by HA. Indeed, bath
application of L-NAME only partially blocked HA-induced suppression of
EPSCs. HA is known to raise
[Ca2+]i by increasing
Ca2+ influx through membrane channels and
Ca2+ release from internal stores (Leurs
et al. 1995; Li et al. 1999
). Because NOS
activity is dependent on
[Ca2+]i, chelation of
internal Ca2+ with BAPTA can reduce NO production
(Kishi et al. 1996
). On the other hand, BAPTA diffusion
also can eliminate activation of other intracellular signal pathways
due to raised [Ca2+]i.
These actions of BAPTA might explain why intracellular diffusion of
BAPTA, simultaneously with L-NAME treatment, was needed for complete
abolition of EPSC suppression by HA.
Noradrenergic inputs play important roles in the regulation of
neuroendocrine secretion in the hypothalamus. The SON receives dense
noradrenergic innervation from brain stem nuclei and contains a high
density of 1 receptors, the activation of
which can increase activities of G protein, PLC, and PKC and elicits
postsynaptic membrane responses in SON neurons (Armstrong
1995
; Hatton 1990
; Michaloudi et al.
1997
; Minneman and Esbenshade 1994
). Like HA, NE
can depolarize SON neurons, enhance DAPs, and increase neuronal firing
rates (Li et al. 1999
; Renaud and Bourque
1991
; Yamashita et al. 1987
). NE also has been
demonstrated to suppress spontaneous miniature IPSCs in SON neurons by
activating
2 adrenergic receptors in
presynaptic terminals (Wang et al. 1998
). In the present
experiments, NE and HA reduced non-NMDA currents equally. However,
several lines of evidence suggest that NE differs from HA in the
mechanisms underlying EPSC modulation. First, NE was found to reduce
the EPSCs with a remarkable reduction in the PPF, effects that could not be abolished by intracellular treatments of recorded neurons with
GDP-
-S, H7, and BAPTA. Second, the maximal
responses to NE were observed usually 3-4 min after NE injection, when
EPSC suppression by HA already had started to recover. Third, NOS
inhibition abolished HA-, but not NE-, induced suppression of EPSCs.
These results suggest that NE suppresses non-NMDA currents via a direct action at the presynapses. In contrast, HA attenuation of EPSCs is
unlikely to be via presynaptic mechanisms because there were no
significant PPF changes after HA treatment. Although both HA and NE are
proposed to function as neuromodulators using common intracellular
signal transduction pathways (Nicoll et al. 1990
), our
observations in SON neurons suggest that they can target different sites to bring about a similar effect. As shown in a recent study, both
HA and NE enhance the currents underlying DAPs, but only HA's effects
are mediated by Ca2+ release from internal stores
(Li et al. 1999
).
In conclusion, the present results suggest that HA suppresses non-NMDA
synaptic currents in SON neurons through activation of
H1 receptors and NOS. It is possible that locally
released HA, like oxytocin and GABA (Brussaard et al.
1997; Kabashima et al. 1997
; Kombian et
al. 1996
, 1997
), can gate neural signals from other CNS neurons
by modulating non-NMDA currents to optimize neuroendocrine activities,
particularly during dehydration and the late term of pregnancy. Further
studies are needed to probe all intracellular signal transduction
pathways involved in mediating the effects of HA and NE on EPSCs.
![]() |
ACKNOWLEDGMENTS |
---|
This work was supported by National Institute on Neurological Disorders and Stroke Research Grants NS-16942 and NS-09140.
![]() |
FOOTNOTES |
---|
Address reprint requests to: G. I. Hatton.
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.
Received 26 August 1999; accepted in final form 13 January 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|