Oxytocin (OT) is a nonapeptide that is primarily produced in the paraventricular (PVN) and supraoptical (SON) nuclei in the hypothalamus (1). Oxytocinergic magnocellular neurons in the PVN and SON project to the neurohypophysis, where OT is released into circulation. In contrast, oxytocinergic parvocellular neurons in the PVN project to several areas within the brain such as the amygdala, locus coeruleus, raphe nuclei, the nucleus tractus solitarii (NTS), the dorsal motor nucleus of the vagus, and the rostral ventrolateral medulla (2, 3).

In both the periphery and the central nervous system, the OT signal is transduced by a single OT receptor (4). The OT receptor belongs to the G-protein-coupled receptor family and is coupled to phospholipase C through Galphaq11 (5).

OT is classically related to reproductive phenomena, such as parturition and milk ejection. Nevertheless, in the last few decades, it has been proposed that OT plays a role in autonomic functions (e.g., social behavior, yawning, memory and learning, tolerance and dependence mechanisms, feeding, grooming, thermoregulation, and cardiovascular regulation) (2, 6-12).

Oxytocin fibers project from the PVN to a number of regions in the CNS that are important for cardiovascular regulation, such as the NTS, the dorsal motor nucleus of the vagus, and the rostral ventrolateral medulla (2, 3, 13). Microinjections of OT into NTS elicited increases in blood pressure and heart rate (14, 15), and a hypertensive effect was also observed after injection of OT in the dorsal vagal complex (16). In contrast, OT administered to the dorsal motor nucleus of the vagus elicited a decrease in heart rate (HR) (17).

Moreover, it has been suggested that OT acts as a modulator of the baroreceptor reflex in the NTS. In conscious rats, OT administered intracisternally has been shown to attenuate arterial baroreflex sensitivity (18). In contrast, OT in the solitary vagal complex facilitates the baroreceptor bradycardic response (19).

Genetically modified mice lacking the OT gene showed a slight hypotension and enhanced baroreflex gain over a small blood pressure range (20), supporting the role of OT in cardiovascular regulation.

OT has also been implicated in the cardiovascular response to physical exercise and stress adjustments (21-24). Central administration of VP-OT antagonist (25) or OT antisense oligomers (26) prevented the increase in heart rate seen following acute stress. During exercise, the decreased baseline in HR (an important adjustment to training) is accompanied by increased OT mRNA expression in the NTS (21, 22).

OT modifies the cardiovascular responses produced by other substances. It has been demonstrated that OT significantly counteracts the vasodepressor and bradycardic actions of the 2-adrenoceptor agonist clonidine (14). Receptor autoradiographical experiments showed that OT significantly reduced the affinity of the 2-adrenoceptor agonist binding sites in the NTS compatible with an antagonistic interaction with the 2-adrenoceptors (14). In addition, OT neurons in the paraventricular nucleus mediate increases in blood pressure and heart rate induced by stimulation of substance P receptors (27). Furthermore, the increase in MAP and HR in response to VP administered intracerebroventricularly is larger if rats are preinjected with OT (28).

The aim of the current work was to clarify the role of endogenous OT in central cardiovascular control in the NTS by microinjecting OT receptor antagonist and specific OT antiserum. Moreover, we analyzed whether OT modulates the central cardiovascular responses mediated by L-glutamate (GLU), the neurotransmitter involved in the baroreceptor reflex in this nucleus.


MATERIAL ANE METHODS
Animals

Male pathogen-free Sprague-Dawley rats (body weight: 250-350 g) were used. They were housed under standardized lighting and temperature conditions and had free access to food pellets and tap water.

Surgical procedure

On the day of the experiment, the animals were anesthetized with a mixture of -cloralose (35 mg/ kg, Merck) and urethane (1 g/kg, Sigma, St Louise, MO, USA). The trachea was cannulated with a curved plastic tube in order to keep the airway unobstructed when the head was flexed and so the animals were able to breathe freely. A plastic catheter (PE-50, Clay-Adams, NY, USA) containing heparin (50 IU/ ml in 0.9% NaCl w/v solution) was inserted into the femoral artery to record blood pressure and heart rate.

Animals were then placed in a stereostatic frame (Kopf, USA) and their heads were flexed 45°. After a midline incision through the skin, the dorsal neck muscles were dissected with a knife. The atlanto-occipital membrane was opened and the brainstem surface was exposed.

Body temperature was monitored during the experiment and kept constant at 37.5±0.5°C by means of a thermostatic blanket.

Microinjections into NTS

Microinjections into the NTS were made via a glass micropipette (tip diameter 40-50 µm) connected to a Hamilton micro-syringe (0.5 µl). The coordinates were 0.5 mm rostral and 0.5 mm lateral to the obex, and 0.5 mm below the surface. The drugs were diluted in artificial cerebrospinal fluid (aCSF) (0.12 M NaCl, 0.02 M NaH2CO3, 2 mM KCl, 0.5 mM KH2PO4, 1.2 mM CaCl2, 1.8 mM MgCl2, 0.5 mM Na2SO4, 5.8 mM D-glucose, pH 7.4). Each animal received only one microinjection (in a volume of 50 nl; injected in a time of 10-12 seconds in all cases).

At the end of each experiment, the site of the micropipette tip was identified by injecting 50 nl of evans blue through the micropipette. Anesthetized rats were killed by decapitation and the brains were removed, immediately frozen, and sectioned at 40 µm in a cryostat (H-550, Microm Instruments). Sections were stained with cresyl violet for histological verification of the microinjection site and all of the animals that were found to have an injection site outside the NTS were omitted from the analysis. A schematic representation of microinjection sites is shown in Fig. 1.

Fig. 1. Schematic representation of microinjection sites (symbols) in the dorsomedial part of the nucleus tractus solitarius in 6-8 animals (), 10-12 animals ( and ). AP, area postrema; CC, central canal; Cu, cuneate nucleus; Gr, gracile nucleus; Sol, nucleus tractus solitarius; sol, solitary tract; SolC, commisural part of the solitary nucleus; 10, dorsal motor nucleus of the vagus. Triangles are not drawn on the right side.

Experimental groups

The experimental groups were divided into several series for the various aims of the study:

1. To study the potential role of endogenous OT in central cardiovascular control in the NTS. In a first set of experiments, groups of animals were microinjected with a specific antagonist of OT receptors [d(CH2)5,Tyr(Me)2,Orn8]-vasotocin (OTA) (Peninsula Lab., CA, USA) (10 pmol) alone or in combination with an effective dose (10 pmol) of OT (Peninsula Lab., CA, USA) (n=5-7 rats in each group), which was previously obtained by our laboratory (14). In a second set of experiments, groups of animals received microinjections of a specific antiserum (Rabbit Purified Antiserum Ig G, Peninsula Lab. CA, USA) (OT-Ab) diluted 1:10, with no cross-reactivity with structurally similar peptides (% Cross-reactivity: Lys8-vasopressin < 0,01%; Arg8-vasopressin 0). Control rats received OT-Ab that was inactivated by heat (submerged in a water bath at 60°C for 1 hour) (n=5-7 rats in each group).

2. To evaluate any modulatory effects between OT and GLU, a subthreshold dose of OT (1 pmol) (14) was coinjected with a subthreshold (30 pmol) or an effective dose (1.5 nmol) of glutamate (29) (n=5-7 rats in each group). These doses of OT and GLU have been shown to interact with other neurotransmitters at the cardiovascular level in the NTS (14, 29).

In all groups, control animals received aCSF alone.

Cardiovascular measurements

Mean arterial pressure (MAP) and heart rate (HR) were recorded by connecting the femoral catheter to a Statham-type transducer (Statham Co., Puerto Rico) that was connected to a computerized data acquisition system (MacLab, AD Instruments). When the cardiovascular parameters were stabilized, MAP and HR were recorded for 10 min (immediately before the microinjections) and these data were used as basal values. After injections, the changes in MAP and HR were recorded during a 30-min period.

Statistical analysis

For the purpose of analysis, the peak effect (maximal response during the first 15 min after injections) was calculated for each parameter and for each animal using an IBM-XT computer and a program developed by Guna Consult (Stockholm, Sweden) (14, 30). The peak effect was expressed as a percent change from the respective mean basal values and showed the maximal responses during the first 15 minutes. In addition, the area created under the curve, which mainly reflected the duration of the effect of the 30-min recording period, was calculated for each parameter and for each animal. The area values were expressed in arbitrary units.

To analyze the interactions between OT and GLU on MAP and HR, data were collected 10 seconds after the injection (time of maximal effect of glutamate). The values were expressed as a percent change of each parameter from the respective basal values.

A parametric one-way analysis of variance (ANOVA) for multiple comparisons was used to test for overall effects. If the ANOVA was significant, a Newman-Keuls test was used for comparisons between experimental groups in the experiments with the specific antagonist of OT (OTA) and in the GLU experiments. The significance levels are indicated in each case.

Data from Anti-OT antibody (OT-Ab) experiments were analyzed by Student’s unpaired t-test to compare the control group with the experimental group.


RESULTS
Cardiovascular effects of microinjections of OTA into the NTS

A 10 pmol microinjection of the oxytocin antagonist, [d(CH2)5,Tyr(Me)2,Orn8]-vasotocin (OTA), into the medial NTS did not produce a significant change in the peak effect, MAP, or HR (Fig. 2 and 3).

Fig. 2. Effects of microinjections in the NTS of OTA, OT, and OT+OTA on mean arterial pressure. The peak effect (A) is shown as a percent change from respective basal values, and the overall effects (area values) are shown (B) as absolute values in arbitrary units. Means±SEM are given (n=5-7 rats in each group). The basal MAP values were: CSF group 70±7 mmHg; OT group 68±3 mHg; OTA group 73±8 mmHg; OT+OTA group 65±7 mmHg. *** p<0.001 *p<0.05 vs CSF group and OTA group (A) and *** p<0.001 vs. the remaining groups (B) according to ANOVA followed by Newman-Keuls multiple comparison test.

Fig. 3. Effects of microinjections in the NTS of OTA, OT, and OT+OTA on heart rate (HR). The peak effect (A) is shown as a percent change from respective basal values, and the overall effect (area values) is shown (B) as absolute values in arbitrary units. Means±SEM are given (n=5-7 rats in each group). The basal HR values were: CSF group 350±9 beats/min; OT group 343±18 beats/ min; OXA group 360±9 beats/min; OT+OXA group 361±19 beats/min. ** p<0.01 vs. the remaining groups according to ANOVA followed by Newman-Keuls multiple comparison test.

The presence of OTA decreased the hypertensive response produced by an effective dose of OT (Fig. 2). This blockade was statistically significant (p<0.01) when the 30-minute recording period was analyzed (Fig. 2B).

OTA also significantly blocked (p<0.01) the tachycardic response induced by OT (Fig. 3). This blockade was observed beginning in the first 15 minutes of recording (peak effect) and was maintained throughout the entire recording period (Fig. 3A and 3B).

Cardiovascular effects of microinjections of Anti-OT antibody into the NTS

The control group obtained by inactivation of Anti-OT antibody (OT-Ab) in a water bath at 60°C for 1 hour did not show any differences comparing with animals treated with aCSF.

Microinjections of Anti-OT antibody (OT-Ab) did not modify the values of MAP or HR during the 30 min of recording (Table 1). With regard to MAP, a slight decrease was observed in the area values, but it did not reach statistical significance (Table 1).

Table 1. Effect on mean arterial pressure and heart rate of microinjections of specific oxytocin antiserum (1:10) (OT-Ab) and Control (OT-Ab inactivated by heat) in the Nucleus tractus solitarii. Means+SEM. are shown, n=7-8 rats in each group. The maximal peak effects are expressed as percent change from the respective mean basal value. The area values formed under the curves are expressed in arbitrary units. No effects were observed according to Student´s t-test.

Cardiovascular effects of coadministration of OT and GLU in the NTS

As previously demonstrated (29), a dose of 1.5 nmol of GLU elicited a significant decrease in MAP and HR compared with control animals (aCSF) (Fig 4A, B).

Fig. 4. Effects on mean arterial pressure(MAP) (A) and heart rate (HR) (B) of microinjections of an effective dose of glutamate (1.5 nmol) alone or in combination with a threshold dose of oxytocin (1 pmol) into the NTS. Means±SEM are given (n=5-7 rats in each group). The basal MAP values were: CSF group 73±2 mHg; GLU group 71±4 mHg; GLU+OT group 69±5 mmHg. The basal HR values were: GLU group 321±28 beats/min; GLU+OT group 440±50 beats/min. * p<0.05 vs. the CSF group and the OT group *** p<0.001 vs. the remaining groups according to ANOVA followed by Newman-Keuls multiple comparison test.

OT at 1 pmol dose, which did not induce a change at the cardiovascular level, significantly (p<0.001) counteracted the vasodepressor effect elicited by the effective dose of GLU (Fig. 4A). At the HR level, GLU induced a bradycardic response compared with the control group. This effect was not observed with the coinjection of OT and GLU (Fig. 4B).

Fig. 5 shows a representative recording of the blockade produced by OT in the decrease of MAP elicited by GLU.

Fig. 5. Representative tracings of the effect of GLU 1.5 nmol (A) and GLU 1.5 nmol + OT 1 pmol (B) on arterial blood pressure. The decrease in arterial blood pressure elicited by GLU is counteracted by OT.

The coinjection of subthreshold doses of OT and GLU did not produce a change in MAP or HR (Table 2).

Table 2. Effects on mean arterial pressure and heart rate of microinjections of a threshold dose of glutamate (GLU) alone or in combination with a threshold dose of oxytocin (OT) into the Nucleus tractus solitarii. Results are shown as percentage of change with respective basal values 10 seconds after the microinjection. Means±SEM (n= 5-6 rats in each group). No significant changes were observed according to one-way ANOVA. The basal MAP values were: CSF group 80±4; GLU group 75±5 mHg; GLU+OXT group 74±5 mmHg. The basal HR values were: CSF group 342±25 beats/min; GLU group 340±23 beats/min; GLU+OXT group 349±23 beats/min.

DISCUSSION
The results obtained in the present paper demonstrate that OT plays a role in the NTS at the cardiovascular level since the specific OT antagonist [d(CH2)5,Tyr(Me)2,Orn8]-vasotocin blocks the hypertension and tachycardia induced by OT in the NTS. The modulation by OT of the cardiovascular response elicited by GLU in the NTS confirms the importance of OT in the cardiovascular adjustments in this nucleus.

However, it seems as though OT does not exert a tonic effect on the mechanism that modulates blood pressure and heart rate in the present model since the Anti-OT antibody (OT-Ab) did not induce changes in mean arterial pressure or heart rate.

Experiments have shown that brain peptides regulate blood pressure and heart rate (31, 32). For OT, there are several effects described in MAP, HR, and baroreflex activity (14, 15, 19, 21). There are also differences in OT cardiovascular effects, which are dependent on the area involved and the experimental models. For example, OT administered in the dorsal motor nucleus of the vagus elicited a decrease in HR effect block by OT antagonist (17) whereas in the NTS, an increase in the HR has been described after microinjections of OT (14, 15).

Although the role of OT in the cardiovascular control in the NTS has been previously described (14, 15), the present work is the first to show the blockade of the cardiovascular effects of OT by OT antagonist in the NTS. The OT antagonist [d(CH2)5,Tyr(Me)2,Orn8]-vasotocin (OTA) significantly blocked the increase in blood pressure and heart rate induced by an effective dose of OT and this blockade was maintained throughout the entire recording period. This finding confirms that these effects are specifically mediated by OT receptors.

The OT antagonist [d(CH2)5,Tyr(Me)2,Orn8]-vasotocin (OTA) is a specific OT antagonist (33), with antagonist efficacy proven in several functions induced by OT including sexual behavior and feeding studies (34-37). The specificity of the OTA to OT receptors (33, 38) excludes the possibility that the cardiovascular effects of OT observed in the NTS are induced not only through OT receptors, but also vasopressin receptors of the V1a-subtype (8).

Previously, it has been proposed that the bradycardic caused by OT was due to the stimulation of vagal outflow (19, 21); however, the specific increase in MAP and HR induced by OT in the NTS could indicate that OT also affects the sympathetic outflow. In fact, microinjections of OT in the rostral ventrolateral medulla also induced an increase of MAP and HR, and these effects have been suggested to involve the sympathetic outflow (39).

The modulation by OT of the cardiovascular response elicited by GLU in the NTS confirms the importance of OT in the cardiovascular adjustments in this nucleus. Our findings show that a subthreshold dose of OT significantly blocked the vasodepressor effect of GLU. OT also induced a reduction in the bradycardic response induced by GLU.

Although there are numerous neurotransmitters in the NTS, GLU is the principal excitatory transmitter and it plays a pivotal role in regulating cardiovascular functions within the NTS (40-42). GLU exerts its effects through the binding and activation of two classes of specific receptors: ionotropic and metabotropic. Since both receptor classes have been involved in the GLU actions at the cardiovascular level in the NTS (43, 44), in the present work, we cannot exclude their participation in the interactions observed with OT. GLU acts through ionotropic receptors to excite the second-order baroreceptor reflex NTS neurons and GLU also activates metabotropic receptors to modulate the excitability of NTS neurons at pre- and postsynaptic loci (43, 44). The interaction of GLU with amino acid gamma amino butyric acid (GABA) transmission in the NTS is well known; in fact, stimulation of metabotropic receptor type II can decrease GABA release (44).

As an established neurotransmitter/neuromodulator in the brain (45), OT is likely to modulate other neurotransmitter systems such as amino acids. In fact, the interactions between OT and GLU have been described in several functions, such as nociception (46, 47) and stress responses (48, 49). Furthermore, other neurotransmitters, such as the GABA, selectively inhibit OT secretion (50).

In the present paper, an inhibition of OT on GLU action at the cardiovascular level was observed. These effects could be a consequence of a specific interaction between OT receptors and GLU receptors. A long list of transmitter heteromers and homomers show that this is a fundamental mechanism in the brain and in the periphery (51). In fact, OT receptors have been shown to form oligomeric complexes in vivo (52). Thus, it can be speculated that the blocking effect of OT on the cardiovascular actions mediated via GLU receptors shown in this paper is a consequence of the receptor interactions.

It is also possible that OT/GLU interactions take place at synaptic membrane levels. Since GLU receptors in the NTS have been described at both the pre- and postsynaptic membrane level (43, 44), OT may block GLU receptor transduction, leading to a reduction of the vasodepressor and bradychardic response induced by GLU. In fact, OT has been described to modulate glutamatergic transmission at pre- and postsynaptic levels in the olfactory bulb (53).

Previous studies suggested that OT plays a role in the regulation of baroreceptor reflex function. Injections of OT had divergent effects on reflex bradycardia, an enhancement in the solitary vagal complex (19), but also a depression in the baroreceptor reflex after intracisternal administration (18). The participation of OT in the regulation of baroreceptor reflex is also supported by the findings in oxytocin-deficient mice, showing that deletion of the OT gene blunts the bradycardic, but also facilitates the tachycardic, response to depressor challenges (20).

The inhibitory action of OT in GLU mediated cardiovascular action observed in this paper suggests that OT induces a decrease in the baroreceptor reflex response, which was also previously observed (18). The inhibition of the baroreceptor reflex of HR control will result in a hypertensive response, an effect observed with the microinjection of OT in the NTS likely with high sympathetic tone.

However, when we coinjected threshold doses of both GLU and OT, we did not find significant changes in cardiovascular function. These results suggest that the modulatory role of OT appears after a significant release of this peptide within the NTS, supporting the hypothesis of the absence of a tonic effect of endogenous OT on central cardiovascular regulation.

In fact, these results are in agreement with the Anti-OT antibody (OT-Ab) results observed in the present study. The OT-Ab did not produce a modification in the mean arterial pressure or arterial pressure, suggesting that OT is not involved in tonic mechanisms that modulate blood pressure and heart rate maintenance at the NTS (at least in the present model). The role of endogenous OT in the maintenance of tonic blood pressure has been suggested since oxytocin-deficient mice showed a small hypotension (20). However, the results observed in the current paper seem to indicate that OT does not play a tonic role in blood pressure regulation. This divergence could be explained in the shift of the autonomic balance observed in the oxytocin-deficient mice (20).

In conclusion, our results are the first to indicate that 1) the specific OT antagonist [d(CH2)5,Tyr(Me)2,Orn8]-vasotocin blocks the hypertension and tachycardia induced by OT in the NTS and 2) OT modulates the cardiovascular response elicited by GLU in the NTS. These results confirm the modulatory role of OT on central cardiovascular regulation within the NTS. However, it seems as though OT does not exert a tonic effect on MAP and HR in the present model. These results could aid the understanding of the general neurochemical mechanisms that generate the cardiovascular responses from the NTS. Moreover, these results may be of relevance for the autonomic and behavioral effects mediated by central OT.

Acknowledgments: This work was supported by the Spanish DGCYT BFU2005-02241, BFU2008-03369 and Junta de Andalucia SEJ01323.

Conflict of interests: None declared.


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