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Departments of Physiology and Pharmacology, University of Tennessee, Memphis, Tennessee 38163
Address all correspondence and requests for reprints to: Steven L. Bealer, Ph.D., Department of Physiology, University of Tennessee, 94 Union Avenue, Memphis, Tennessee 38163. E-mail: sbealer{at}physio1.utmem.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-adrenergic antagonist phentolamine. Dialysates
and plasma were collected, and OT concentrations were determined using
RIA. Dialysis of the PVN with ACSF containing histamine significantly
increased the release of OT systemically and centrally within the PVN.
Furthermore, the increases in OT concentration in dialysates and plasma
were prevented by simultaneous administration of chlorpheniramine (an
H1 receptor antagonist) or ranitidine (an H2 receptor antagonist) as
well as by the adrenergic antagonist phentolamine. These data
demonstrate that histamine acts within the PVN to increase both
systemic and intranuclear release of OT. Furthermore, the increased OT
release induced by histamine is dependent upon stimulation of both H1
and H2 histaminergic receptors and subsequent activation of
-noradrenergic receptors. These findings suggest that histamine
induces systemic and intranuclear OT release by stimulating the release
of norepinephrine. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several lines of evidence suggest that central histaminergic neurons
participate in the neuroendocrine regulation of OT secretion in
response to physiological stimuli. For example, intracerebroventricular
administration of a histamine synthesis inhibitor or histaminergic H1
or H2 receptor antagonists prevents suckling-induced OT release into
the systemic circulation during lactation (7). Further, depletion of
central histamine with the synthesis inhibitor
-fluoromethylhistidine also impairs OT release and OT messenger RNA
expression induced by dehydration (8). Conversely,
intracerebroventricular administration of histamine increases the
systemic secretion of OT (9), the expression of c-fos in
OT-containing cells (10), and the messenger RNA for OT (11) in
magnocellular neurons of the PVN and SON. Although histaminergic
neurons localized in the posterior hypothalamus provide a dense
innervation of the PVN and SON (12, 13), it has not been reported
whether histamine acts within these nuclei to stimulate systemic OT
secretion, and it is also unknown at present whether histamine might
play a role in activating the intranuclear release of the peptide.
Therefore, one goal of the present studies was to evaluate the effects
of selective histaminergic stimulation of the PVN on the systemic and
intranuclear release of OT.
In addition, a number of the cardiovascular and hormonal
responses elicited by central histamine are reduced or abolished by
interference with noradrenergic neurotransmission (14, 15, 16), and
centrally administered histamine has been shown to directly enhance the
release of norepinephrine in the PVN (17), suggesting the presence of
stimulatory histamine receptors on noradrenergic nerve terminals in
this region. Because central -adrenoreceptor stimulation increases
the systemic and central release of OT (3, 6), it is therefore possible
that the stimulatory effects of histamine on OT are similarly mediated
by the release of norepinephrine. Therefore, a second goal of these
studies was to determine whether noradrenergic mechanisms are involved
in the excitatory effects of locally applied histamine in the PVN on
systemic and/or central OT release.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surgery
On the day before the experiment, animals were anesthetized with
methohexital (brevital sodium, 60 mg/kg), and a loop style
microdialysis probe (18, 19) was stereotaxically positioned so that the
tip was placed at the dorsal and lateral extent of the PVN (-1.9 mm
posterior to bregma; +0.6 mm lateral to the midline; -8.2 ventral to
the surface of the skull) (6, 16, 17). Dialysis probes were secured
with small screws placed in the skull and dental acrylic. In addition,
polyethylene catheters (PE-50 anchored to PE-10 tubing, filled with
heparin, 50 U/ml) were implanted in a femoral artery and a femoral
vein. The catheters were led sc to exit the skin between the scapulae,
where they were secured. The rats were returned to their home cages and
recovered overnight.
The dialysis membrane used in these microdialysis probes was Qupra-ammonium-rayon with a molecular mass cut-off of 40,000 Da and a diameter of 250 µm (Asahi Medical, Tokyo, Japan). The dimensions of the membrane exchange area at the tip of the microdialysis probe were 1.5 x 0.7 x 0.3 mm (length x width x depth).
Protocol
On the day of the experiment, rats were tested in plastic cages.
The input port of the dialysis probe was connected to a remote 3-cc
syringe with polyethylene tubing, and the syringe was placed in a
syringe pump. Polyethylene tubing attached to the output port of the
dialysis probe was led to chilled plastic collection tubes.
All dialysis probes were perfused with artificial cerebrospinal fluid (ACSF) containing bacitracin (20 µM) at a flow rate of 1 µl/min. The dialysate obtained during the initial 45–60 min of probe perfusion was discarded. After equilibration, dialysate was collected during 100 min of probe perfusion with normal ACSF/bacitracin solution (control period; CONT-ACSF). At the end of this period, dialysate probe perfusion with normal ACSF was continued on one group of animals; in another group of animals, the perfusate was changed to ACSF containing histamine (HA; 50 mM; Sigma Chemical Co., St. Louis, MO). At the completion of this 100-min experimental perfusion period (EXP period), dialysate probes in all animals were perfused with normal ACSF for a final 100-min period for recovery (REC period). This concentration of histamine releases norepinephrine in the PVN without producing cardiovascular responses (17).
Additional groups of animals were used to examine the effects of H1 and H2 receptor blockade on central and peripheral OT release induced by histamine. In these animals, the 100-min perfusion with normal ACSF (CONT-ACSF) was followed by perfusion with ACSF containing the H1 antagonist chlorpheniramine (8 mM) or the H2 receptor antagonist ranitidine (6 mM; CONT-Antag). This period was followed by perfusion with ACSF containing the antagonist and histamine (50 mM; EXP period). This concentration of chlorpheniramine abolishes H1 receptor-mediated effects of histamine when perfused through microdialysis probes placed adjacent to the PVN (17). Ranitidine has not previously been administered through microdialysis membranes. The concentration of this H2 antagonist was selected for use in these studies based upon results obtained after the administration of similar concentrations of other H2 antagonists (17), H1 antagonists (17), and similar molecular mass catecholamine antagonists (6, 16, 20) through identically constructed microdialysis probes. These groups of animals were not observed during a REC period.
Finally, a separate group of animals received microdialysis probe
perfusion with normal ACSF during the CONT and REC periods and ACSF
containing histamine and phentolamine (HA/PHEN; 50 mM HA
and 6 mM PHEN) during the EXP period. This concentration of
phentolamine was used because it is effective in preventing
-adrenergically mediated responses when administered through
identically prepared microdialysis probes placed in the PVN (6, 16).
In these studies, dialysate was perfused at a rate of 1 µl/min and was collected for 100 min. This flow rate was selected to maximize the dialysate concentration of OT, as recovery of the peptide from brain extracellular fluid is typically between 1–6% (21, 22) and is inversely proportional to the dialysate flow rate (21). Preliminary experiments from our laboratory found that the dialysate concentration of OT obtained using faster flow rates was below the detection limits of the RIA. The 100-min collection periods were therefore necessary to obtain sufficient sample volume for reliable measurement of OT.
Blood samples (800 µl) were obtained from the arterial catheter for measurement of plasma concentrations of OT at 30 and 60 min after the start of the CONT, EXP, and REC periods. In initial studies, we also obtained blood samples after 5 and 10 min of the EXP period. However, there were no significant differences in plasma OT between animals perfused with ACSF and rats receiving ACSF containing histamine at these observation times. Therefore, blood was sampled in all other experimental groups only at 30 and 60 min of the EXP period. After each collection, the blood was centrifuged, the plasma was placed in chilled plastic tubes, and the red cells were suspended in saline and infused back into the animal through the venous catheter.
Histology
After the experiments, rats were anesthetized with pentobarbital
(Nembutal; 60 mg/kg), and the brains were removed after transcardial
perfusion with saline. The brains were placed in sucrose formalin
(30%) for a minimum of 3 days and subsequently blocked, frozen,
sectioned (40 µm), and stained with cresyl violet. These sections
were observed under the light microscope for determination of the
proper placement of the dialysis probe.
Assays
All dialysate and plasma samples were frozen and stored at -80
C for subsequent analysis of OT concentrations using a RIA with a
detection limit of 0.4 pg (23). In vitro analysis of OT
recovery with the dialysis probes and dialysate flow rate used in these
studies indicated a recovery between 8–11%. OT values were not
corrected for recovery.
Data analysis
Only animals that had detectable concentrations of OT in the
dialysate and histological verification of probe tip placement adjacent
to the PVN were included in the data analysis. If an animal had
detectable neurochemical levels during the CONT period but undetectable
levels during the EXP and/or REC periods, the concentrations were
recorded as zero for the EXP and/or the REC period.
Data were analyzed using a two-factor ANOVA with repeated measures. Significant differences among individual means were determined using a Newman-Keuls a posteriori test. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a schematic
representation of a coronal brain section taken at the level of the
PVN. Dialysates with detectable concentrations of OT were obtained from
animals with microdialysis probe tips located within the
cross-hatched area. The OT concentrations in dialysates from
probes placed farther from the PVN were undetectable. Furthermore,
animals were eliminated from data analysis if there was damage to the
PVN. Eighteen animals had dialysis probe placements that were more than
600 µm from the PVN or that substantially damaged the PVN. In all
such cases, OT was not detectable in the dialysates, and the animals
were eliminated from the data analysis.
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shows the effects on plasma OT
concentrations of dialysis probe perfusion of the PVN with ACSF
containing histamine alone and in combination with selective H1 and H2
receptor blockers. For clarity, values for plasma OT during CONT-ACSF
and CONT-Antag periods represent mean concentrations measured in the
30- and 60-min samples. Administration of ACSF containing histamine
through the dialysis probe significantly increased the plasma
concentration of OT. Neither chlorpheniramine nor ranitidine alone
affected the plasma OT concentration (CONT-Antag). However, the
increase in plasma OT produced by dialysis administration of histamine
was not evident in dialysates collected from animals perfused with
histamine containing either chlorpheniramine (H1 antagonist) or
ranitidine (H2 antagonist) during the EXP period.
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). Figure 3 also shows
that although administration of ACSF containing chlorpheniramine or
ranitidine alone via the dialysis probe did not alter baseline
concentrations of OT, each antagonist prevented the increase in
intranuclear OT in response to histamine. Although the dialysate OT
concentration tended to be higher when ACSF contained histamine and
either the H1 or H2 antagonist, neither value was significantly greater
than the OT level measured during administration of the antagonist
alone (CONT-Antag). Furthermore, the dialysate OT concentration during
administration of histamine alone was significantly greater than the
peptide concentration in groups administered histamine and
chlorpheniramine or histamine and ranitidine.
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shows plasma concentrations of
OT before (CONT 30 and 60), during (EXP 30 and 60), and after (REC 30
and 60) dialysate probe perfusion with ACSF vehicle, ACSF containing
histamine, or ACSF containing both histamine and phentolamine.
Concentrations of OT in plasma obtained during the CONT periods were
not different among any of the experimental groups. Plasma OT
concentrations were also similar between ACSF-treated animals and
histamine-treated animals at 5 min (ACSF, 10.6 ± 2.7 pg/ml; HA,
11.1 ± 2.6 pg/ml) or 10 min (ACSF, 13.3 ± 2.8 pg/ml; HA,
19.7 ± 2.4 pg/ml) of the EXP period (data not shown). However,
plasma OT concentrations were significantly increased by histamine
administration to the PVN at 30 and 60 min of the EXP period, whereas
microdialysis probe perfusion with normal ACSF did not alter the plasma
concentration of OT at any time. In addition, when the - adrenergic
antagonist phentolamine was included in the dialysate, the increase in
plasma OT induced by histamine was abolished. Plasma OT concentrations
were significantly greater in animals whose microdialysis probes were
perfused with ACSF and histamine during the EXP period than in those
receiving either ACSF alone or ACSF containing histamine and
phentolamine.
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. In animals perfused with ACSF
vehicle, dialysate concentrations of OT did not change significantly
over time and tended to decrease during the EXP and REC periods.
However, inclusion of histamine in the dialysate during the EXP period
resulted in a significant increase in OT, which returned toward control
values during the REC period. Similar to the increase in plasma OT
produced by histamine, the enhanced intranuclear OT release in response
to histamine was prevented by concomitant administration of
phentolamine. Further, OT concentrations in dialysates obtained from
animals whose dialysis probes were perfused with ACSF and histamine
were significantly higher than OT in dialysates from animals perfused
with either ACSF alone or ACSF containing histamine and phentolamine
during the EXP period.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-adrenergic receptors, the present studies suggest that
one mechanism underlying histaminergic activation of the OT system
involves mediation by stimulatory noradrenergic networks in the
PVN. The cell bodies of all central nervous system neurons using histamine as a neurotransmitter are located in the tuberomammillary nucleus of the posterior hypothalamus (12, 13), from which axons of the histamine-containing neurons project to many areas of the brain, including most major loci contributing to autonomic and endocrine regulation. The diencephalon receives the highest density of histamine neurons in the brain (13), and more specifically related to the present study, the PVN and SON both are innervated with numerous histaminergic nerve fibers (24, 25, 26). Dense concentrations of H1 receptors have been identified in the PVN and SON (27, 28). Although the distribution of H2 receptor in rat brain has not been established, H2 receptors have been identified in the hypothalamus of guinea pig (29), and functional evidence suggests that this receptor type is found in magnocellular nuclei (30).
The involvement of central histaminergic pathways in the neuroendocrine regulation of OT secretion has not been examined extensively, but several previous studies have implicated histamine in activation of systemic OT release. Perhaps most notably, OT secretion is increased after intracerebroventricular administration of histamine (9), whereas depletion of central neuronal histamine abolishes the increase in the plasma OT concentration in response to suckling (7) and dehydration (8). Furthermore, blockade of histamine receptors delays the delivery of pups during parturition (31) and prevents the increase in systemic OT release observed during suckling (7). The results of the present studies are consistent with the proposition that the physiological stimuli evoking OT secretion activate central histamine-containing networks and suggest that one site of action for histamine is the PVN. This is supported by results from the present studies demonstrating that histamine elicited significant and sustained increases in plasma OT upon administration to the PVN via the microdialysis probe, but was ineffective if probes were misplaced outside this region or if the PVN were damaged by the surgery.
The present findings also extend previous results by showing for the first time that histamine induces the release of OT within the PVN, concomitant with the systemic release of the peptide. There is now a considerable body of evidence that OT is released within the magnocellular nuclei during periods of systemic OT release, e.g. induced by hyperosmolality, during parturition and lactation (6, 21, 32, 33, 34, 35). The source of this intranuclear OT is most likely dendritic processes (see Ref. 3 for review). From results of studies in which OT or OT antagonists have been applied to the magnocellular nuclei, it has been proposed that the intranuclear released OT exerts a positive feedback action on its own systemic release; furthermore, this action appears to be obligatory for the synchronous recruitment and activation of the entire OT neurosecretory population to allow appropriate amounts of the peptide to be released into the systemic circulation (4, 5, 35). The present results, therefore, implicate histamine as an important neuromessenger in this critical neuroendocrine mechanism.
It is noteworthy that in the present studies the effect of histamine on both systemic and intranuclear OT release was prevented by simultaneous treatment with either an H1 or an H2 receptor antagonist. It is not clear at present why both receptor types are involved, but a similar phenomenon has been reported in earlier studies on histamine-induced OT secretion (9) and suckling-induced OT release (7), both of which are blocked by either an H1 or an H2 antagonist.
Further work is required to establish the precise mode of action of
histamine on OT neurons. It is possible that histamine fibers directly
contact OT neurons, as suggested by electrophysiological evidence (30).
However, in that study, histamine exerted a direct inhibitory effect on
OT neuronal firing, which is at variance with the present and previous
findings. Alternatively, the observation in our studies that the
-adrenergic antagonist phentolamine prevented histamine-induced
activation of systemic and central OT release suggests that
histamine’s actions on OT release may be indirect and involve an
interaction with stimulatory noradrenergic mechanisms.
There is now considerable evidence that norepinephrine exerts a
major stimulatory influence on OT secretion in a variety of
physiological contexts (see Ref. 3 for review). Noradrenergic systems
arising from the lower brain stem provide a dense innervation of the
magnocellular nuclei (36), and electron microscopic studies document
direct synaptic contacts between noradrenergic fibers and OT-positive
perikarya and processes (37, 38). Microinjection studies from this
laboratory (39, 40) indicate that activation of the
1-adrenergic receptors within the PVN and SON stimulates
OT release. In addition, pharmacological interference with
noradrenergic neurotransmission reduces OT secretion evoked by stress
(41), cholecystokinin treatment (42), and suckling (23). Furthermore,
1-adrenergic blockade increases the latency of the
OT-mediated milk ejection reflex (43) and prevents the increase in
plasma OT induced by suckling (44). Finally, suckling increases
norepinephrine turnover in the PVN and SON (23), and we have recently
demonstrated with microdialysis that the release of norepinephrine in
the PVN is increased during suckling (6). Furthermore, central
-adrenoreceptor blockade prevents the increase in intranuclear OT
release evoked by suckling in lactating rats (6), as it blocked
histamine-induced OT release in the present studies.
Thus, the present studies demonstrate that activation of -adrenergic
receptors is necessary for histamine-induced systemic and intranuclear
release of OT. It is possible that an intact noradrenergic stimulatory
drive to OT neurons is required for the excitatory effects of
histamine. Alternatively, noradrenergic nerve terminals in the PVN may
contain histamine receptors positively coupled to norepinephrine
release. Such a functional relationship between central histaminergic
and noradrenergic systems has been demonstrated in several experimental
conditions. For example, administration of histamine increases
norepinephrine release from brain slices containing the rat
hypothalamus (45) or from isolated synaptosomes (46). Furthermore,
in vivo studies demonstrated that local administration of
histamine increases norepinephrine release in the hypothalamus of the
cat (47) and the PVN of conscious rats (17). Functionally, several
responses evoked by central administration of histamine can be blocked
by the destruction of central noradrenergic nerve terminals or
pharmacological blockade of adrenergic receptors. For example, the
pressor response evoked by both ventricular injections and local
administration of histamine to the PVN is prevented by blockade of
-adrenergic receptors (15, 16). In addition, -adrenoreceptor
blockade decreased histamine-induced stimulation of corticosterone in
response to stress (14). The present experiments further support a
similar relationship between histamine and norepinephrine in control of
both systemic and central release of OT.
The present studies focused on mechanisms in the PVN controlling OT secretion, and further work will be required to examine whether similar pathways are operational in the SON, which also receives dense noradrenergic (36) and histaminergic innervation (24, 48). At present there is little information regarding differential effects of histamine stimulation of PVN vs. SON neurons and systemic or intranuclear release of OT. However, the functional studies described previously reporting the effects of central histamine blockade on suckling-induced systemic OT release (7) and on pup delivery (31) suggest that histamine is excitatory to neurons in both the PVN and SON under these conditions.
In these experiments, histamine perfusion of the dialysis probes in animals with simultaneous H1 or H2 receptor blockade occurred during the third collection period, whereas histamine perfusion in control animals was performed during the second collection period. This procedure was necessary to determine the effect of histamine antagonists alone on basal OT secretion. It is unlikely that changes in dialysis membrane characteristics between the second and third collection periods could account for differences in the OT content of the dialysate in these groups, as previous studies have demonstrated that membrane transit of biogenic amines (norepinephrine) across similar microdialysis probe membranes is constant for 5–6 h (18, 19).
In summary, these experiments have demonstrated that central
administration of histamine in the region of the PVN increases the
release of systemic OT and increases intranuclear release of OT in the
PVN. In addition, blockade of either H1 or H2 antagonists prevents OT
release both into the blood and centrally in the PVN. Finally,
stimulation of -adrenergic receptors is required for the
histamine-induced increases in central and peripheral OT release. In
conjunction with previous findings, these data suggest that central
histamine pathways may contribute to the stimulation of OT release in
specific physiological conditions via a presynaptic activation of
norepinephrine release.
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Acknowledgments |
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Footnotes |
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Received June 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-adrenoceptors
control cardiovascular responses to hyperosmolality. Am J Physiol
272:R1283–R1289
1-adrenergic and -amino-3-hydroxy-5-methylisoxazole-4-propionic
acid-sensitive excitatory amino acid mechanisms. Endocrinology 133:2855–2860[Abstract]
1-adrenergic mechanisms. Endocrinology 132:658–666[Abstract]
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