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The Journal of Neuroscience, June 1, 1999, 19(11):4270-4279
Nitric Oxide via cGMP-Dependent Mechanisms Increases Dye Coupling
and Excitability of Rat Supraoptic Nucleus Neurons
Qin Z.
Yang and
Glenn I.
Hatton
Department of Neuroscience, University of California, Riverside,
California 92521
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ABSTRACT |
Unlike many neuron populations, supraoptic nucleus (SON) neurons
are rich in both nitric oxide synthase (NOS) and the NO
receptor-soluble guanylyl cyclase (GC), the activation of which leads
to cGMP accumulation. Elevations in cGMP result in increased coupling
among SON neurons. We investigated the effect of NO on dye coupling in
SONs from male, proestrus virgin female, and lactating rats. In 167 slices 263 SON neurons were recorded; 210 of these neurons were
injected intracellularly (one neuron per SON) with Lucifer
yellow (LY). The typically minimal coupling seen in virgin females was
increased nearly fourfold by the NO precursor, L-arginine,
or the NO donor, sodium nitroprusside (SNP).
L-Arginine-induced coupling was abolished by a NOS
inhibitor. In slices from male and lactating rats who have a higher
basal incidence of coupling, SNP increased coupling by approximately
twofold over control (p < 0.03). SNP
effects were prevented by the NO scavenger hemoglobin (20 µM) and by the selective blocker of NO-activated GC, ODQ
(10 µM). These results suggest that NO released from
cells within the SON can expand the coupled network of neurons and that
this action occurs via cGMP-dependent processes. Because increased
coupling is associated with elevated SON neuronal excitability, we also
studied the effects of 8-bromo-cGMP on excitability. In both phasically
and continuously firing neurons 8-bromo-cGMP (1-2 mM), but
not cGMP, produced membrane depolarizations accompanied by membrane
conductance increases. Conductance increases remained when
depolarizations were eliminated by current-clamping the membrane
potential. Thus, NO-induced cGMP increases SON neuronal coupling and excitability.
Key words:
gap junctions; guanylyl cyclase inhibition; hemoglobin; L-NAME; ODQ; sodium nitroprusside; 8-bromo-cGMP
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INTRODUCTION |
Studies of the distribution of
neuronal nitric oxide synthase (NOS) have shown this enzyme to be
abundant in the dendrites, somata, and axon terminals of the neurons
constituting the magnocellular hypothalamo-neurohypophysial system
(Bredt et al., 1990 ; Dawson et al., 1991). Neurons of this system
synthesize, transport, and release either oxytocin or vasopressin, and
NOS has been found to be colocalized with both of these peptides
(Sanchez et al., 1994). NO is a membrane-permeant neuronal messenger
that is produced from L-arginine by the activation of NOS.
Typically, NO release from one cell type finds its way to its receptor,
soluble guanylyl cyclase (sGC), in nearby (within 200 µM)
target cells of another type, activating this enzyme and elevating
intracellular cGMP levels (Southam and Garthwaite, 1993; Wood and
Garthwaite, 1994). Magnocellular neurons of the paraventricular and
supraoptic (SON) nuclei are atypical in that they are rich not only in
NOS but also in both the  1 and  1 subunits of sGC (Furuyama et
al., 1993). Because this situation allows for NO activation of sGC
within the same neuron as well as between any nearby magnocellular
neurons, it is possible that NO plays an autoregulatory role in at
least some of the cGMP-dependent processes in these neurons.
Recently, it has been shown that the incidence of interneuronal dye
coupling (the number of coupled cells) and the extent of the coupled
network (the numbers of neurons coupled to each neuron) in the SON were
increased either via receptor-mediated activation of GC or by directly
elevating intracellular cGMP levels (Hatton and Yang, 1996).
Conversely, elevating cAMP levels drastically reduced coupling.
Although the hypothesis is straightforward that NO, either from
intrinsic sources or generated by cells in close proximity to SON
neurons, should enhance coupling, the effects of NO on coupling in
other neuronal systems have not been entirely consistent. NO donors
applied to striatal slices increased the incidence of coupling among
medium spiny neurons (O'Donnell and Grace, 1997), for instance,
whereas the same treatment applied to the retina has been found to
uncouple the heterologous gap junctions between AII amacrine cells and
bipolar cells (Mills and Massey, 1995). Horizontal cells also are
uncoupled from each other by the administration of the NO precursor,
L-arginine (Miyachi and Nishikawa, 1994). Here we present
evidence that coupling among SON neurons is enhanced markedly by either
exogenous (sodium nitroprusside, SNP) or endogenous
(L-arginine) sources of NO generation. Further, these
NO-mediated increases in coupling are blocked by the inhibition of
various enzymes in the pathway, e.g., NOS and sGC. In addition, elevated intracellular cGMP was found to increase membrane conductance and depolarize both putative oxytocin and vasopressin cells, suggesting that endogenously generated NO increases both SON neuronal coupling and excitability.
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MATERIALS AND METHODS |
Animals and procedure. Experiments were
performed on 167 slices prepared from as many young adult Sprague
Dawley rats of both sexes (50-65 d old, except for the lactating rats,
who were age 80-90 d). Of these, 46 rats were proestrus virgin
females, 105 were males, and 16 were lactating, actively
nursing mothers taken in approximately equal numbers on postpartum days
2, 4, 7, and 10. As in our previous studies (Yang and Hatton, 1987;
Hatton and Yang, 1989, 1994) the animals were introduced gently and
casually to a guillotine and decapitated, rapidly and nonstressfully,
without anesthesia. Then, 400-µm-thick slices, prepared on a
vibratome, were cut horizontally to avoid damage to the SON dendritic
zone. Cut into oxygenated medium at room temperature, the slices then were transferred to either a static bath chamber (Hatton et al., 1980)
or a perfusion chamber (Hatton et al., 1983) and maintained at
34-36°C. Transillumination of the slice permitted visual guidance of
the recording/injecting electrode into the SON.
As is standard in our coupling experiments, only one neuron per SON was
injected intracellularly with Lucifer yellow (LY). LY was the dye of
choice for several reasons. First, we wished to compare the present
results with those of our previous coupling studies; second, LY
transfer requires relatively large junctional conductances (Dermietzel
and Spray, 1993), permitting assessments of either increases or
decreases in response to experimental treatments; and third, the
determination of coupling incidence is rapid and direct, not requiring
complex histochemical processing that also reduces the recovery rates
of recorded and injected cells. After equivalent postinjection delays,
the cells injected in control medium or medium containing other
compounds were fixed in 4% buffered paraformaldehyde for 2 hr and then
transferred into Tris-buffered saline overnight. The slices were
ethanol-dehydrated, cleared, and mounted on glass slides in methyl
salicylate. LY-filled cells were located, counted, and photographed
under epifluorescence, special attention being paid to dendrodendritic
contacts, the sites of coupling between SON neurons (for review, see
Hatton, 1997). Conventional intracellular recording procedures were
used as in our previous coupling studies (Yang and Hatton, 1987, 1988; Hatton and Yang, 1994, 1996). Intracellular electrodes were filled with
either 5% LY (Stewart, 1978) in 0.25 M lithium acetate for coupling analyses or 1 M K-acetate in studies of membrane
conductance and excitability (resistances 80-180 M ). Measurements
of input conductance were made by passing brief hyperpolarizing current pulses (100 pA, 100 msec) through the recording electrode at rates from
0.6 to 1 Hz. Recordings were made with the bridge circuit of a
Neurodata Dual Intracellular Amplifier (New York, NY). Resting potentials were determined at the time of both entering and exiting the cell.
Solutions and drugs. The standard incubation medium was
composed of (in mM): 126 NaCl, 5.0 KCl, 26 NaHCO3, 1.25 NaH2PO4,
1.3 MgSO4, 2.4 CaCl2, 5 organic
buffer 3-(N-morpholino) propanesulfonic acid, and 10 glucose, pH 7.4, 310 mOsm/kg. All test compounds were dissolved in
standard medium and delivered to the recording chamber. Drugs used were
SNP (NO donor); hemoglobin or hydroxocobalamin (NO scavengers);
L-arginine (NO precursor); the NOS inhibitor N -nitro-L-arginine
methylester (L-NAME) and its inactive enantiomer, N-nitro-D-arginine
methylester (D-NAME); cGMP; 8-bromo-cGMP; the GC inhibitors
1H-[1,2.4] oxadiazolo [4,3a] quinoxalin-1-one
(ODQ), a selective blocker of NO-activated GC (Tocris Cookson, Ballwin, MO), or LY83583 (Calbiochem, La Jolla, CA). All other drugs were obtained from Sigma (St. Louis, MO).
Data analysis. Statistical analyses were performed by using
Fisher's Exact Test on the numbers of single versus coupled cells observed for the different treatments. The ratios of the numbers of
dye-coupled cells divided by the total number of dye-filled cells were
calculated and used in this analysis. This measure is the well
documented Dye Coupling Index and involves the same data analysis
procedure used in our previous studies (Hatton and Yang, 1996). For
purposes of graphic presentation the incidence of coupling is expressed
as the number of coupled cells per injection. Thus, if each cell were
coupled to one and only one other, this ratio would be 2.00. When the
incidence of coupling is low, the ratio approaches zero. In this study
this index of coupling varied from a minimum of 0.20 to a maximum of
1.50.
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RESULTS |
Membrane characteristics and firing patterns
The data that were analyzed were from 263 recorded SON neurons,
210 of which were LY-injected. The remaining 53 cells were studied for
their responsiveness to the membrane-impermeant cGMP and to the
membrane-permeant cGMP analog, 8-bromo-cGMP. These 263 neurons were
from 46 virgin female, 105 male, and 16 lactating rats. The membrane
characteristics, which were similar for all groups, were as follows
(means ± SEM for each measure): resting membrane potentials,
 57.5 (± 1.3 mV); action potential amplitudes, 62.4 (± 2.1 mV);
input resistances, 132 (± 6.4 M). Of the 210 recorded and injected
neurons, 17 cells displayed slow-irregular firing rates of <1 Hz, 26 cells exhibited phasic bursting activity (typical of vasopressin
neurons), and 113 cells displayed continuous firing, with rates ranging
from 1 to 36 Hz. Many of these were probably oxytocin cells (Poulain
and Wakerley, 1982). The remaining 54 cells were not spontaneously
active. Dye-coupling incidence was not associated selectively with any
of these activity patterns. Typical examples of LY fills of SON neurons
and of dye coupling observed in this study are shown in Figure
1. Note that all coupling involves the
dendrites, as has been found consistently among SON cells.
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Figure 1.
Fluorescence photomicrographs of LY-filled coupled
neurons. In each case only one cell was injected. A,
Coupled pair from SON of a virgin female brain slice treated with 500 µM L-arginine. B, Coupled
triplet from SON of a male brain slice treated with 100 µM sodium nitroprusside. Note that all coupling is via
the dendrites. Scale bar, 30 µm.
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Effects of the NO precursor, L-arginine
In our previous studies of coupling among SON neurons, we have
found that virgin female rats consistently display low basal rates of
coupling (Hatton et al., 1987; Yang and Hatton, 1987; Hatton and Yang,
1990). As seen in Figure 2, in slices
taken from virgin females and incubated in standard control medium,
there were 0.27 coupled neurons per injection. These data were based on
15 injections that yielded 13 single and two coupled pairs of
cells. Providing an abundance of the NOS substrate
L-arginine (500 µM) in the medium increased
this index of coupling to 1.00. This was based on 10 injections that
yielded six single and 10 coupled cells, the latter consisting of two
coupled pairs and two triplets. When similar slices were incubated with
both 500 µM L-arginine and a 50 µM concentration of the NOS inhibitor, L-NAME, the precursor-induced coupling increase was
abolished, because 10 injections yielded nine single and one coupled
pair of cells. A similar result was obtained from 10 injections in slices bathed in 50 µM L-NAME without
L-arginine (data not shown). The inactive enantiomer,
D-NAME, was ineffective in preventing the coupling
increase: 13 injections produced eight single and 12 coupled cells, the
latter consisting of three pairs and two triplets.
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Figure 2.
Effects of the NO precursor,
L-arginine, and blockade of NO synthase on coupling. The
number of coupled neurons per injection is given in four treatment
groups of slices taken from proestrus virgin female rats. Shown are
injections in slices incubated in standard medium (Control
Med), n = 15; in 500 µM
L-arginine, n = 10; in 50 µM L-NAME + 500 µM
L-arginine, n = 10; and in 50 µM D-NAME + 500 µM
L-arginine (n = 13). *Significantly
different from Control Med at p < 0.03.
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Effect of the NO donor, SNP
Figure 3 presents the data obtained
when SNP (100 µM) was added to the standard medium
bathing slices from virgin female rats. Control data are presented for
comparison. For the SNP treatment, 13 injections yielded seven single
and 13 coupled cells, five pairs, and one triplet, or 1.00 coupled
cells per injection. Incubating similar slices in medium containing 100 µM SNP plus the NO scavenger hydroxocobalamin (30 µM), after a 15 min preincubation in the medium
containing the 30 µM hydroxocobalamin alone, prevented this SNP-induced increase. Thus, 13 injections into cells in slices bathed in this medium yielded only four coupled cells, i.e., 11 single
and two coupled pairs, similar to the control condition. To obviate the
possible nonspecific effects of hydroxocobalamin, we repeated
this experiment on slices from male rats, using hemoglobin as the NO
scavenger (Fig. 4).
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Figure 3.
Effects of the NO donor, sodium nitroprusside
(SNP), and SNP plus the NO scavenger hydroxocobalamin
(HCA) on coupling. The number of coupled neurons per
injection is given in three treatment groups of slices taken from
proestrus virgin female rats. The Control Med group is
the same as in Figure 2. Shown are injections in slices
incubated in standard medium containing 100 µM SNP
(n = 13) and in 30 µM HCA + 100 µM SNP (n = 13). *Significantly
different from both groups at p < 0.02.
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Figure 4.
Effects on coupling of SNP administered along with
either hemoglobin (Hb) or the guanylyl cyclase
inhibitors LY83583 (LY) or ODQ. The number of
coupled neurons per injection is given in five treatment groups of
slices taken from male rats. Shown are injections in slices maintained
in Control Med (n = 22), in 100 µM SNP (n = 17), in 20 µm Hb+SNP (n = 17), in 10 µM LY+SNP (n = 8), or
in 10 µm ODQ+SNP (n = 9).
*Significantly different from Control Med and
HB+SNP at p < 0.05.  Significantly different from Hb+SNP,
LY+SNP, and ODQ+SNP at p < 0.01.
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The incidence of LY coupling among SON neurons under basal conditions
is significantly higher for males than for virgin females (Hatton and
Yang, 1990). We sought to determine whether NO was capable of further
increasing this already high coupling incidence in males. These data
are shown in Figure 4. In standard control medium, 22 injections
yielded 15 single and seven coupled pairs of cells (0.64 coupled cells
per injection). In medium containing 100 µM SNP, 17 injections yielded five pairs, two triplets, and two quadruplets,
or 24 coupled cells (1.41 coupled cells per injection). As in the
females, SNP-induced coupling was prevented in males by a NO scavenger.
Seventeen injections produced 12 single cells and five coupled pairs in
slices bathed in medium containing 20 µm of hemoglobin plus 100 µm
of SNP. To confirm the hypothesis that NO has its action on coupling in
these cells via the activation of GC, we incubated similar slices from
males in medium containing 100 µM SNP plus the GC
inhibitor LY83583 (10 µM). Nineteen LY injections yielded
17 single and two coupled pairs of cells (0.21 coupled cells per
injection). Similar results were obtained by using ODQ (10 µm), a
selective blocker of the NO-activated sGC. Twenty injections yielded 18 single fills, one pair and one triplet (0.25 coupled cells per
injection). As seen in Figure 4, these treatments reduced the coupling
to significantly below the male control levels. This suggests that
there is baseline NO release that activates a basal level of sGC and
that this activity has a tonic role in basal levels of interneuronal
coupling in male SONs.
Lactating rats also display incidences of coupling that are elevated
over virgin females (Hatton et al., 1987; Yang and Hatton, 1987; Hatton
and Yang, 1990, 1994). Coupling among SON cells from lactating rats
also was enhanced by SNP (Fig. 5, right).
In control medium, 19 injections produced 13 single and six coupled
pairs (0.63 coupled cells per injection), whereas in SNP-containing medium (100 µM) 12 injections resulted in four single and
18 coupled cells, consisting of three pairs and four triplets (1.50 coupled cells per injection). There were no discernible differences in coupling attributable to the number of days in lactation in either the
control or SNP groups.
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Figure 5.
Comparison of basal levels of coupling in control
medium (open bars) and in medium containing 100 µM SNP (filled bars) on coupling in
slices taken from virgin female, male, and lactating rats. For
lactating rats the injections are shown in control medium
(n = 19) and in SNP-containing medium
(n = 12); the other groups are the same as in
Figures 3 and 4. *Significantly different from own control at
p < 0.02-0.03. The relatively small number of
injections in control medium in this study resulted in nonsignificant
differences between the control groups.
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Summarized in Figure 5 are the comparisons of control and SNP
conditions for the three types of animals in this study. Clearly the
largest increase induced by the NO donor is in the virgin females, who
had the lowest basal incidence of coupling.
Effects of 8-bromo-cGMP on neuronal excitability
Responses to cGMP and 8-bromo-cGMP were studied in a separate set
of 38 phasically firing and 15 continuously firing neurons, all from
male rats. Twenty-one of the phasically firing cells were recorded
before and during a change in the bath concentration of 8-bromo-cGMP
from 0 to 2 mM. For the other 14 cells, responses to
nanodrop applications of the cGMP analog (1 mM) on the
slice surface were studied. Of the 35 neurons tested, two showed no response (one each for the bath-applied and nanodrop applications). The
remaining 33 cells showed membrane depolarizations in response to the
8-bromo-cGMP. Fifteen neurons studied at normal resting potential of
approximately 58 mV were depolarized by 6-10 mV, with some apparent
dependence on the duration and/or concentration of the nucleotide
application (Fig.
6A-C). In each case
the durations of the phasic bursts were increased also. Figure
6C shows that the membrane-impermeant cGMP had no effect on
either the membrane potential or the phasic bursts.
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Figure 6.
Effects of 8-bromo-cGMP on membrane potential and
burst duration of phasically firing, putative vasopressin neurons.
A, The addition of 8-bromo-cGMP to the medium resulted
in depolarization from normal membrane potential (dashed
line) and increased burst duration. The end burst is expanded
to show burst structure and apparent plateau potential.
B, These effects were repeatable 10 min later after a
return to baseline activity. C, Increasing the
concentration of 8-bromo-cGMP from 1 to 2 mM induced
apparently larger effects on membrane potential and burst duration
(compare A and C). Note in
C that 2 mM cGMP had no effect.
A and B are the same cell;
C is a different cell.
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Membrane conductance changes were measured in 26 neurons. Conductance
increases in response to bath applications of 8-bromo-cGMP, but not to
cGMP, were observed in all 20 of these cells. These increases in
conductance are seen in Figure 7,
A and C, in which a steady negative current moved
the pre-8-bromo-cGMP membrane potential to below spike threshold. In
response to 8-bromo-cGMP the cells depolarized and fired bursts of
action potentials. Previous incubation with the GC blocker LY83583 also
had no effect on the ability of 8-bromo-cGMP to depolarize and evoke
phasic bursting. The twelve continuously firing SON neurons were
depolarized, increased their firing rates, and showed increased
membrane conductances in response to these same concentrations of
8-bromo-cGMP (Fig. 8). To determine
whether the increased conductance seen in response to 8-bromo-cGMP was
attributable simply to membrane depolarization, we current-clamped
slices pretreated with LY83583 just below resting potential and
bath-applied the 8-bromo-cGMP. Figure 9
shows that the conductance change occurred without membrane
depolarization in six of six cells tested.
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Figure 7.
Effects of 1 mM 8-bromo-cGMP on
membrane conductances of two different phasically firing cells
(A, C) current-clamped at or slightly
below spike threshold by steady hyperpolarizing current (0.05 nA).
Pulses of hyperpolarizing current (detailed in B) were
administered at 1 Hz to monitor conductance changes. In each case the
bath application of 8-bromo-cGMP produced an increase in conductance
that was accompanied by a burst of action potentials. No effect on
conductance was seen in response to the application of cGMP
(C). Both cells were pretreated with LY83583 to
block endogenous GC activity.
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Figure 8.
Effects of 8-bromo-cGMP on membrane potential and
firing frequency (A) and on conductance
(B, C) of two continuously firing
neurons. Both cells were pretreated with LY83583 to block endogenous GC
activity.
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Figure 9.
Effects of bath-applied 8-bromo-cGMP on membrane
conductance of two cells, one phasically (A) and
one continuously (B) firing, for which the
membrane potentials were current-clamped at 1-2 mV below resting
potential. Both slices in which these cells were recorded were
pretreated with LY83583 to block endogenous guanylyl cyclase
activity.
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DISCUSSION |
Evidence presented here indicates that either exogenously supplied
or endogenously generated NO increases dye coupling among neurons in
the SON. Furthermore, increases are mediated by cGMP-dependent mechanisms, possibly involving protein kinase G and the phosphorylation of gap junction-associated proteins. Elucidation of these latter steps
in the process awaits further research. NO has been shown to have
similar enhancing effects on coupling among medium spiny neurons in the
striatum (O'Donnell and Grace, 1997) but has been shown to be an
uncoupler of neurons in the retina (Miyachi and Nishikawa, 1994; Mills
and Massey, 1995). Such cell-, connexin-, and messenger-specific
regulation of junctional permeability now is becoming recognized as the
rule rather than the exception (Bennett et al., 1991; Bennett, 1997;
Hatton, 1999). In the present study both the numbers of coupled pairs
of neurons and the numbers of neurons coupled to two or three others
were increased by NO, indicating that the coupled network was expanded
by this messenger. Similar effects on the incidence and extent of
coupling were obtained in earlier studies in which coupling increases
were induced by physiologically activating conditions or by stimulation
of excitatory afferents to the SON (Hatton et al., 1987; Yang and
Hatton, 1987; Hatton and Yang, 1990, 1996). Although cell type (i.e.,
oxytocin or vasopressin) was not determined immunocytochemically in the present study, the observed coupling increases were represented in
cells across all of the identified firing patterns and, thus, NO most
likely had similar effects on coupling among both oxytocin and
vasopressin neurons. The actual incidence and extent of coupling revealed by LY dye transfer in this study is an underestimate of the
actual coupling that exists among these neurons, because these numbers
are seen to be many-fold larger when a less highly charged and smaller
tracer molecule (e.g., neurobiotin) is used to estimate coupling
incidence (Hatton and Yang, 1994). Similar, but even more dramatic,
differences between LY and neurobiotin coupling have been reported in
the neocortex and retina (Vaney, 1991; Peinado et al., 1994).
Relation to hormone demand
Increased coupling among SON neurons is associated consistently
with physiological conditions of increased demand for peptide synthesis
and release, e.g., dehydration (Cobbett and Hatton, 1984) or lactation
(Hatton et al., 1987; Yang and Hatton, 1987). Similarly, the
physiological activation of this system during dehydration or lactation
has been found to result in an upregulation of NOS (Villar et al.,
1994; Blazquez et al., 1995; Wang and Morris, 1996) and/or its mRNA
(Ceccatelli and Eriksson, 1993; Kadowaki et al., 1994; Ueta et al.,
1995; Ceccatelli, 1997). SNP, IL-2, and acetylcholine also have been
shown to release vasopressin from hypothalamic slices. The effects of
the latter two substances were blocked by inhibition of NOS, indicating
that their actions were via this pathway (Raber and Bloom, 1994).
Unfortunately, no attempt was made in that study to determine whether
the peptide release was dependent on NO activation of sGC, although
this is probable. Finally, L-arginine or NO donors
administered intracerebroventricularly are capable of evoking
vasopressin release in vivo (Ota et al., 1993). Curiously,
however, intracerebroventricular administration of NOS inhibitors has
been found to facilitate the dehydration-induced release of oxytocin
(Summy-Long et al., 1993). It is difficult to discern the locus of
action of drugs placed into the ventricle, and effects at the level of
the hypothalamic neurons may differ from those at the neurohypophysis.
Nonetheless, it appears that there is a close positive relationship
among physiological activation of this system, peptide release,
upregulation of NOS, and increased coupling among the neurons of at
least the SON portion of the system.
Synaptic modulation of coupling
Two studies have shown that the brief activation of two different
monosynaptic afferent pathways to SON neurons was effective in
modulating coupling. The first of these studies (Hatton and Yang, 1990)
involved the stimulation of the lateral olfactory tract for 10 min at
10 Hz, which resulted in a significant increase in coupling among the
neurons of the SONs from lactating rats, but not from males or virgin
females. This experimental outcome was puzzling because, at that time,
little was known about NO either as an interneuronal or intraneuronal
messenger, the long-lasting intracellular consequences of ionotropic
glutamate receptor activation, or the plasticity of glutamate receptor
expression. Subsequent work, including the present study, indicates
that all of these factors appear to have played roles in the effects
leading to those earlier results. Thus, lateral olfactory tract
stimulation activates both NMDA and non-NMDA receptors (Yang et al.,
1995), primarily on SON dendrites (Smithson et al., 1989, 1992) where the coupling occurs (see Fig. 2). NMDA receptors (Meeker et al., 1994),
as well as NOS (noted above), are upregulated by the physiological stimulation of the magnocellular system. This may account for the
differential response of lactating versus virgin female or males rats
to the extent that lactation, like dehydration, is a general activating
stimulus in this system (see Hatton, 1997). NMDA receptor-mediated
increases in intracellular Ca2+ result in
Ca2+/calmodulin activation of NOS, with the
subsequent production of NO, activation of sGC, and the
accumulation of cGMP.
Investigation of a second monosynaptic pathway identified the
cGMP-dependent mechanisms as important enhancers of SON neuronal coupling (Hatton and Yang, 1996). Stimulation of the tuberomamillary nucleus resulted in prolonged enhancement of excitability in SON vasopressin neurons via H1-histamine receptors that are
linked via G-proteins to the activation of GC. Receptor activation by both the stimulation and incubation of the slices in 8-bromo-cGMP resulted in dramatic increases in coupling. Conversely, 8-bromo-cAMP sharply reduced coupling. One pathway involved in this
G-protein-mediated response to histamine involves a cascade of
phospholipase C-diacyl glycerol-protein kinase C activation of NOS.
Therefore, it is likely that these two excitatory inputs (i.e.,
glutamatergic and histaminergic) converge via the NOS-NO steps
investigated in the present study to use the same cGMP-dependent
mechanisms in the modulation of coupling.
Sex differences
One clue to the consistently observed sex differences in the basal
incidence of coupling emerged from the present findings. Inhibition of
either GC by LY83583 or sGC by ODQ reduced coupling in slices from
males to the same level as that found in the control virgin females
(~0.25 coupled cell per injection). This suggests that the sex
difference may be attributable to differential GC or adenylyl cyclase
activity levels in males and virgin females. Sex steroids may play an
influencing role in regulating the levels of these enzymes, because the
presence of testosterone has been found to be necessary to maintain the
high coupling incidence typical of males (Cobbett et al., 1987).
Similarly, the presence of estrogen maintains the relatively low
incidence of coupling observed in virgin females (Hatton et al.,
1992), and the more frequent coupling always found in lactating rats is
accompanied by plasma estrogen at its nadir (Taya and Greenwald,
1982).
Possible functional roles
NO release from magnocellular neurons may have a great many
functional consequences. Increased coupling among oxytocin neurons in
lactation is hypothesized to play a role in the synchronization of
bursting among these cells that occurs just before each milk ejection
during suckling of the pups (Hatton, 1997). The weak coupling (Yang and
Hatton, 1988) among vasopressin neurons should not produce synchrony,
but it appears to contribute importantly to their phasic bursting
patterns of firing, which are eliminated by uncoupling agents (Li et
al., 1998), patterns without which vasopressin release is inefficient
or lacking (Dutton and Dyball, 1979). NO may influence vasopressin
release via its effect on expanding the network of metabolically
coupled neurons that fire phasically, although asynchronously.
It is clear from the results presented here that the NO-induced
activation of sGC and the consequent accumulation of cGMP that normally
ensues not only affect coupling but are also capable of inducing or
enhancing SON neuronal excitability. The membrane depolarization
and conductance increases observed in response to elevated
intracellular cGMP in both putative oxytocin and vasopressin cells
suggest that the resultant action of endogenously released NO would be
to increase hormone release. Such release may be selective for one or
the other of the two peptides, depending on the spatial extent of NO
penetration within the SON, because its effective sphere of influence
is only ~200 µm (Wood and Garthwaite, 1994). It should be noted
that, although we observed substantial cGMP-dependent membrane
depolarizations and prolongation of phasic bursts, two earlier studies
of a few SON cells each found either no effect or slight
hyperpolarizations in response to 0.5 mM 8-bromo-cGMP (Akamatsu et al., 1993; Cui et al., 1994). No obvious explanation for
this apparent discrepancy presents itself.
Another functional consequence of NO released from magnocellular
neurons may be the dilation of local blood vessels, including those
that are found in extremely high density and close proximity to the
neurons in the SON. This would have the beneficial effect of increasing
blood flow, the delivery of nutrients, and the removal of metabolites
(for review, see Iadecola, 1993).
| |
FOOTNOTES |
Received Sept. 17, 1998; revised March 15, 1999; accepted March 18, 1999.
This research was supported by National Institutes of Health Research
Grants R01 NS09140 and R01 NS16942 from National Institute of
Neurological Disorders and Stroke. We thank Dr. Z.-H. Li for helpful
comments on an earlier draft of this paper and J. Kitasako for
technical assistance.
Correspondence should be addressed to Dr. Glenn I. Hatton, Department
of Neuroscience, University of California, Riverside, CA 92521.
| |
REFERENCES |
-
Akamatsu N,
Inenaga K,
Yamashita H
(1993)
Inhibitory effects of natriuretic peptides on vasopressin neuron mediated through cGMP and cGMP-dependent protein kinase in vitro.
J Neuroendocrinol
5:517-522[Web of Science][Medline].
-
Bennett MVL
(1997)
Gap junctions as electrical synapses.
J Neurocytol
26:349-366[Web of Science][Medline].
-
Bennett MVL,
Barrio LC,
Bargiello TA,
Spray DC,
Hertzberg E,
Saez JC
(1991)
Gap junctions: new tools, new answers, new questions.
Neuron
6:305-320[Web of Science][Medline].
-
Blazquez JL,
Pelaez B,
Pastor FE,
Lopez RM,
Amat P
(1995)
NADPH-diaphorase activity in the rat hypothalamo-neurohypophysial system after salt loading and rehydration.
Biomed Res
16:405-410.[Web of Science]
-
Bredt DS,
Hwang PM,
Snyder SH
(1990)
Localization of nitric oxide synthase indicating a neural role for nitric oxide.
Nature
347:768-770[Medline].
-
Ceccatelli S
(1997)
Expression and plasticity of NO synthase in the neuroendocrine system.
Brain Res Bull
44:533-538[Web of Science][Medline].
-
Ceccatelli S,
Eriksson M
(1993)
The effect of lactation on nitric oxide synthase gene expression.
Brain Res
625:177-179[Web of Science][Medline].
-
Cobbett P,
Hatton GI
(1984)
Dye coupling in hypothalamic slices: dependence on in vivo hydration state and osmolality of incubation medium.
J Neurosci
4:3034-3038[Abstract].
-
Cobbett P,
Yang QZ,
Hatton GI
(1987)
Incidence of dye coupling among magnocellular paraventricular neurons in male rats is testosterone-dependent.
Brain Res Bull
18:365-370[Web of Science][Medline].
-
Cui L-N,
Inenaga K,
Nagatomo T,
Yamashita H
(1994)
Sodium nitroprusside modulates NMDA response in rat supraoptic neurons in vitro.
Brain Res Bull
35:253-260[Web of Science][Medline].
-
Dawson TM,
Bredt DS,
Fotuhi M,
Hwang PM,
Snyder SH
(1991)
Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues.
Proc Natl Acad Sci USA
88:7797-7801[Abstract/Free Full Text].
-
Dermietzel R,
Spray DC
(1993)
Gap junctions in the brain: where, what type, how many, and why?
Trends Neurosci
16:186-192[Web of Science][Medline].
-
Dutton A,
Dyball REJ
(1979)
Phasic firing enhances vasopressin release from the rat neurohypophysis.
J Physiol (Lond)
290:433-440[Abstract/Free Full Text].
-
Furuyama T,
Inagaki S,
Takagi H
(1993)
Localizations of 1 and 1 subunits of soluble guanylate cyclase in the rat brain.
Mol Brain Res
20:335-344[Medline].
-
Hatton GI
(1997)
Function-related plasticity in hypothalamus.
Annu Rev Neurosci
20:375-397[Web of Science][Medline].
-
Hatton GI (1999) Synaptic modulation of neuronal coupling.
Cell Biol Int, in press.
-
Hatton GI,
Yang QZ
(1989)
Supraoptic nucleus afferents from the main olfactory bulb. II. Intracellularly recorded responses to lateral olfactory tract stimulation in rat brain slices.
Neuroscience
31:289-297[Web of Science][Medline].
-
Hatton GI,
Yang QZ
(1990)
Activation of excitatory amino acid inputs to supraoptic neurons. I. Induced increases in dye-coupling in lactating, but not virgin or male, rats.
Brain Res
513:264-269[Web of Science][Medline].
-
Hatton GI,
Yang QZ
(1994)
Incidence of neuronal coupling in supraoptic nuclei of virgin and lactating rats: estimation by neurobiotin and Lucifer yellow.
Brain Res
650:63-69[Web of Science][Medline].
-
Hatton GI,
Yang QZ
(1996)
Synaptically released histamine increases dye coupling among vasopressinergic neurons of the supraoptic nucleus: mediation by H1 receptors and cyclic nucleotides.
J Neurosci
16:123-129[Abstract/Free Full Text].
-
Hatton GI,
Doran AD,
Salm AK,
Tweedle CD
(1980)
Brain slice preparation: hypothalamus.
Brain Res Bull
5:405-414[Web of Science][Medline].
-
Hatton GI,
Ho YW,
Mason WT
(1983)
Synaptic activation of phasic bursting in rat supraoptic nucleus neurones recorded in hypothalamic slices.
J Physiol (Lond)
345:297-317[Abstract/Free Full Text].
-
Hatton GI,
Yang QZ,
Cobbett P
(1987)
Dye coupling among immunocytochemically identified neurons in the supraoptic nucleus: increased incidence in lactating rat.
Neuroscience
21:923-930[Web of Science][Medline].
-
Hatton GI,
Yang QZ,
Koran LE
(1992)
Effects of ovariectomy and estrogen replacement on dye coupling among rat supraoptic nucleus neurons.
Brain Res
572:291-295[Web of Science][Medline].
-
Iadecola C
(1993)
Regulation of the cerebral microcirculation during neural activity: is nitric oxide the missing link?
Trends Neurosci
16:206-214[Web of Science][Medline].
-
Kadowaki K,
Kishimoto J,
Leng G,
Emson PC
(1994)
Up-regulation of nitric oxide synthase (NOS) gene expression together with NOS activity in the rat hypothalamo-hypophysial system after chronic salt loading: evidence of a neuromodulatory role of nitric oxide in arginine vasopressin and oxytocin secretion.
Endocrinology
134:1011-1017[Abstract/Free Full Text].
-
Li Z-H,
Kumamoto K,
Mercier F,
Hatton GI
(1998)
Gap junctional communication is closely associated with expression of slow depolarizations in supraoptic nucleus neurons.
Soc Neurosci Abstr
24:1850.
-
Meeker RB,
McGinnis S,
Greenwood RS,
Hayward JN
(1994)
Increased hypothalamic glutamate receptors induced by water deprivation.
Neuroendocrinology
60:477-485[Web of Science][Medline].
-
Mills SL,
Massey SC
(1995)
Differential properties of two gap junctional pathways made by AII amacrine cells.
Nature
377:734-737[Medline].
-
Miyachi EI,
Nishikawa C
(1994)
Blocking effect of L-arginine on retinal gap junctions by activating guanylate cyclase via generation of nitric oxide.
Biogenic Amines
10:459-464[Web of Science].
-
O'Donnell P,
Grace AA
(1997)
Cortical afferents modulate striatal gap junction permeability via nitric oxide.
Neuroscience
76:1-5[Web of Science][Medline].
-
Ota M,
Crofton JT,
Festavan GT,
Share L
(1993)
Evidence that nitric oxide can act centrally to stimulate vasopressin release.
Neuroendocrinology
57:955-959[Web of Science][Medline].
-
Peinado A,
Yuste R,
Katz LC
(1994)
Extensive dye coupling between rat neocortical neurons during the period of circuit formation.
Neuron
10:103-114[Web of Science].
-
Poulain DA,
Wakerley JB
(1982)
Electrophysiology of hypothalamic magnocellular neurones secreting oxytocin and vasopressin.
Neuroscience
7:773-808[Web of Science][Medline].
-
Raber J,
Bloom FE
(1994)
IL-2 induces vasopressin release from the hypothalamus and the amygdala: role of nitric oxide-mediated signaling.
J Neurosci
14:6187-6195[Abstract].
-
Sanchez F,
Alonso JR,
Arevalo R,
Blanco E,
Aijon J,
Vazquez R
(1994)
Coexistence of NADPH-diaphorase with vasopressin and oxytocin in the hypothalamic magnocellular neurosecretory nuclei of the rat.
Cell Tissue Res
276:31-34[Web of Science][Medline].
-
Smithson KG,
Weiss ML,
Hatton GI
(1989)
Supraoptic nucleus afferents from the main olfactory bulb. I. Anatomical evidence from anterograde and retrograde tracers in rat.
Neuroscience
31:277-287[Web of Science][Medline].
-
Smithson KG,
Weiss ML,
Hatton GI
(1992)
Supraoptic nucleus afferents from the accessory olfactory bulb: evidence from anterograde and retrograde tract tracing in the rat.
Brain Res Bull
29:209-220[Web of Science][Medline].
-
Southam E,
Garthwaite J
(1993)
The nitric oxide-cyclic GMP signaling pathway in rat brain.
Neuropharmacology
32:1267-1277[Web of Science][Medline].
-
Stewart WW
(1978)
Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer.
Cell
14:741-759[Web of Science][Medline].
-
Summy-Long JY,
Bui V,
Mantz S,
Koehler E,
Weisz J,
Kadekaro M
(1993)
Central inhibition of nitric oxide synthase preferentially augments release of oxytocin during dehydration.
Neurosci Lett
152:190-193[Web of Science][Medline].
-
Taya K,
Greenwald GS
(1982)
Mechanisms of suppression of ovarian follicular development during lactation in the rat.
Biol Reprod
27:1090-1101[Abstract].
-
Ueta Y,
Yamashita H,
Kawata M,
Koizumi K
(1995)
Water deprivation induces regional expression of c-fos protein in the brain of inbred polydipsic mice.
Brain Res
677:221-228[Web of Science][Medline].
-
Vaney DI
(1991)
Many diverse types of retinal neurons show tracer coupling when injected with biocytin or neurobiotin.
Neurosci Lett
125:187-190[Web of Science][Medline].
-
Villar MJ,
Ceccatelli S,
Ronnqvist M,
Hokfelt T
(1994)
Nitric oxide synthase increases in hypothalamic magnocellular neurons after salt loading in the rat. An immunohistochemical and in situ hybridization study.
Brain Res
644:273-281[Web of Science][Medline].
-
Wang H,
Morris JF
(1996)
Constitutive nitric oxide synthase in hypothalami of normal and hereditary diabetes insipidus rats and mice: role of nitric oxide in osmotic regulation and its mechanism.
Endocrinology
137:1745-1751[Abstract].
-
Wood J,
Garthwaite J
(1994)
Models of the diffusional spread of nitric oxide: implications for neural nitric oxide signaling and its pharmacological properties.
Neuropharmacology
33:1235-1244[Web of Science][Medline].
-
Yang QZ,
Hatton GI
(1987)
Dye coupling among supraoptic nucleus neurons without dendritic damage: differential incidence in nursing mother and virgin rats.
Brain Res Bull
19:559-565[Web of Science][Medline].
-
Yang QZ,
Hatton GI
(1988)
Direct evidence for electrical coupling among rat supraoptic nucleus neurons.
Brain Res
463:47-56[Web of Science][Medline].
-
Yang QZ,
Smithson KG,
Hatton GI
(1995)
NMDA and non-NMDA receptors on rat supraoptic nucleus neurons activated monosynaptically by olfactory afferents.
Brain Res
680:207-216[Web of Science][Medline].
Copyright © 1999 Society for Neuroscience 0270-6474/99/19114270-10$05.00/0
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TOP
ABSTRACT
INTRODUCTION
