INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE PRESSOR REGION of the rostral ventrolateral medulla (RVLM) is critical in the generation and maintenance of sympathetic activity and is also an essential part of the central baroreflex pathways (8, 9, 15). The GABAergic synapse is believed to be a principal relay in the RVLM in transmission of baroreceptor information, because blockade of GABA receptors in this area abolished the vasomotor component of the reflex (8, 15). By contrast, the excitatory amino acids (EAAs) in the RVLM are thought to play a little role in mediating cardiovascular responses to baroreceptor stimulation. This assumption is supported by numerous findings that the blockade of ionotropic EAA receptors in the region with local administration of kynurenate did not affect sympathetic baroreflexes in rats (27, 40), rabbits (41), or cats (12). This contrasts with the essential role of EAAs in the RVLM in mediating many other cardiovascular reflexes. In particular, administration of kynurenate into the RVLM has been shown to attenuate the pressor and sympathoexcitatory reflex responses to hypothalamic stimulation (40), muscular contraction (4), noxious stimulation (34), and renal and vagal afferent stimulation (6, 41) in anesthetized animals. Furthermore, this also contrasts with the ability of glutamate, given into the RVLM, to enhance the sympathetic baroreflex, indicating that local glutamate-sensitive inputs may also play a role in modulating baroreflexes by endogenous EAAs (33). Importantly, all studies that have reported the lack of the effects of kynurenate or other EAA receptor antagonists in the RVLM on baroreflexes have been conducted using acute, anesthetized animal preparations. However, anesthesia may significantly alter the normal functioning of baroreflexes, typically attenuating the gain and/or operational range of the reflex (10, 19, 32, 37). It is possible that this attenuation is partly due to a decreased responsiveness of the RVLM neurons to EAAs because the pressor responses to local glutamate injections have been shown to be reduced in anesthetized rats (3). In this way, the functional significance of EAA-sensitive inputs in the RVLM in controlling baroreflexes may be diminished under anesthesia, which would explain the inability of kynurenate to affect these reflexes.

In the present study, we determined whether the blockade of EAA receptors in the RVLM by local microinjections of kynurenate alters the renal sympathetic and cardiac responses to baroreceptor stimulating and unloading without confounding action of anesthesia. We administered the drugs bilaterally using a recently developed microinjecting system, which enabled us to selectively modulate neuronal activity in highly circumscribed regions of the ventrolateral medulla in conscious rabbits (33). To test the influence of anesthesia on the role of EAA receptors in modulating baroreflexes, the effects of kynurenate microinjections into the RVLM have also been determined after induction of pentobarbital sodium anesthesia.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The experiments were performed in 15 conscious rabbits of either sex, weighing 2.7-3.5 kg, and bred and housed at the Baker Heart Research Institute in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and fully conform with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society (1).

Surgical procedures. Three weeks before the experiments, rabbits were implanted under halothane anesthesia with metal guide cannulas for bilaterally microinjecting into the ventrolateral medulla as described in detail previously (33). Anesthesia was induced using propofol (Diprivan, 10 mg/kg iv, Zeneca, UK) and maintained with halothane (Fluothane, Zeneca, UK). The animal was placed in a stereotaxic frame with lambda and bregma parallel to the horizontal stereotaxic arm, and the angle of the stereotaxic drill was set at 12° from the vertical with the tip pointing rostrally. Holes (1.5 mm in diameter) were then drilled 3.0 mm bilaterally from the central fissure and 4.2 mm posterior to lambda. A stainless steel pin (60-mm length, 0.2-mm diameter) held in a stereotaxic clamp was used to take bilateral measurements from lambda to the base of the skull via the holes. These measurements were used to position the end of the guide cannula 8.0 mm above the base of the skull, which conforms to 7.0 mm above the intended injection site. In addition, the difference between the left and right measurements was used to equal the distance between the guides and the midsagittal plane. The sterilized guide cannula (0.71-mm O.D., 22.5-mm long) was then mounted in a stereotaxic clamp, inserted into the brain, and fixed in position with dental cement. One week before the experiments, a bipolar renal nerve electrode for recording RSNA was implanted according to the method of Dorward and colleagues (11).

Microinjections into the ventrolateral medulla. The histological examination revealed that our guide cannula implantation approach provided the minimal bias between the positions of the left and right injection sites with respect to the midsagittal plane. However, there was a certain variability in the location of injection sites in the ventrolateral medulla in the rostrocaudal direction presumably due to some differences in the shape of the skull that were observed between rabbits. Therefore, in each animal, the location of injection sites in the pressor region of the RVLM was verified both functionally, using unilateral and bilateral microinjections of glutamate (5-10 nmol in 50-100 nl), and histologically. In eight rabbits, glutamate increased mean arterial pressure (MAP) by more than 20 mmHg and the injection sites were found in the ventrolateral medulla +2.0 to +3.0 mm from the obex, i.e., in the pressor region of the RVLM in the rabbit (9, 33). These animals were included into the RVLM group (Fig. 1). In five rabbits, glutamate produced little pressor responses and injection sites were found in the intermediate ventrolateral medulla (IVLM) at the level of 0 to +1.0 mm from the obex. These animals formed the IVLM group (Fig. 1). In two rabbits, glutamate did not change MAP, while the injection sites were found dorsorostral to the pressor region at the level of +3.2 to +3.5 mm from the obex. These rabbits were used for control microinjection experiments. The microinjections into the ventrolateral medulla were made through a stainless steel needle (315-µm O.D.) connected via polyethylene SP8 tubing to a 250-µl Hamilton syringe. The injection volume was controlled by measuring the displacement of a small air bubble in the polyethylene tubing.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Schematic representation of coronal view of the rabbit medulla with circles showing distribution of injection sites in the coronal plane in the rostral (+2 to +3 mm from the obex) ventrolateral medulla (RVLM) and intermediate (0 to +1 mm from the obex) ventrolateral medulla (IVLM). IO, inferior olive; NA, nucleus ambiguus.

Blood pressure and renal sympathetic nerve activity measurement. On the day of the experiment, the animal was placed in a standard rabbit box (15 × 40 × 18 cm, width × length × height). Under local anesthesia, the central ear artery and marginal ear vein were catheterized and the plug of the renal nerve electrode was retrieved from under the skin. The renal sympathetic nerve activity (RSNA) and pulsatile arterial pressure (measured with a Statham 23Dc pressure transducer) were continuously monitored throughout the experiment and were sampled at 500 Hz using an analog-to-digital data-acquisition card. The beat-to-beat MAP, heart rate (HR), and integrated RSNA were detected on-line using a LabVIEW program (28). Because voltages recorded from RSNA electrodes vary considerably between animals, in each experiment the values were normalized to the upper plateau of the first control baroreflex curve, which was taken to equal 100 normalized units (nu).

Experimental protocol. At the beginning of the experiment, each rabbit was subjected to microinjections of glutamate into the ventrolateral medulla as described above. A 30- to 40-min recovery period was allowed between the mapping injections and the remainder of the experiment. RSNA and HR baroreflexes were then assessed before and 15-20 min after bilateral injection of kynurenate (10 nmol in 200 nl) into the ventrolateral medulla. Microinjection of this dose of kynurenate into the RVLM has been shown to reduce the pressor response to electrical stimulation of sciatic (17) or abdominal vagus nerve (6) in the rabbit. To test the influence of anesthesia on the baroreflex responses to kynurenate, we determined the effects of the drug in some rabbits from the RVLM group after induction of the surgical level of pentobarbital sodium anesthesia (45 mg/kg iv, n = 4). In addition, in six rabbits, the cardiovascular responses to unilateral injection of glutamate into the RVLM were determined also 10 min after local injection of kynurenate.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Left: representative recording of changes in mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA), and heart rate (HR) produced by intravenous infusions of phenylephrine (PE) and sodium nitroprusside (NP) in a conscious rabbit. Dashed lines mark the beginning and conclusion of infusions. Right: relationship between MAP, RSNA, and HR obtained from the baroreflex testing shown left. Each dot represents value averaged over 2-s interval. Dashed curves represent sigmoid 5 parameter logistic function applied to raw data. bpm, Beats/min.

Statistical analysis. Values were expressed as means ± SE. A split plot (nested) repeated-measure ANOVA was used to compare the resting values and responses to kynurenate between the RVLM and IVLM groups of rabbits (26). A multifactor repeated-measure ANOVA was used to determine the effect of anesthesia, drug treatment, and their interaction. The between-animal sum of squares (SS) as well as the treatment SS were removed from the total SS to obtain a within-animal SS (38). Effects were considered significant and the null hypothesis was rejected when P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Resting values of MAP, HR, RSNA, and respiratory rate were not different between the RVLM (n = 8) and IVLM (n = 5) groups of rabbits (Table 1). Bilateral microinjections of glutamate (10 nmol) into the RVLM promptly increased RSNA and MAP by 38 ± 10 nu and by 34 ± 7 mmHg, respectively (P < 0.01). By contrast, microinjections of the same dose of glutamate into the IVLM caused a moderate pressor or depressor response, and as such, the pooled data did not show any overall effect on RSNA (+11 ± 10 nu) or MAP (+6 ± 7 mmHg).

Bilateral microinjection of kynurenate into the RVLM did not alter resting MAP, HR, or RSNA (Table 1). By contrast, administration of kynurenate into the IVLM increased RSNA by 27 ± 11 nu (P = 0.059) and MAP by 27 ± 2 mmHg (Table 1). The MAP and RSNA responses began within 1-2 min after the injection, gradually reaching a maximum 30-40 min later, and returned to pretreatment levels within 2 h after injection. The HR was not altered by microinjections of kynurenate into the IVLM (Table 1). The respiratory rate was not different before or after the injection of kynurenate either into the RVLM or IVLM (Table 1). Administration of kynurenate into the RVLM did not alter the MAP response to local unilateral injection of glutamate, which was +24 ± 4 and +25 ± 6 mmHg before and after kynurenate, respectively (n = 6; Fig. 3). However, the RSNA response to glutamate injections into the RVLM tended to decrease after kynurenate pretreatment from +23 ± 7 to +13 ± 6 nu (P > 0.05).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Original recordings of MAP and RSNA responses to injections of glutamate (Glu; 10 nmol) into the RVLM before and 10 min after local injection of kynurenate (Kyn; 10 nmol) from the same rabbit. Arrows mark injection of glutamate and kynurenate.

Microinjections of kynurenate into the RVLM decreased the gain of the RSNA baroreflex by 53 ± 10% but did not affect the lower or upper plateau of this reflex (Table 2 and Fig. 4). This change was mediated by a reduction in the average reflex curvature of 51 ± 9% (Table 2). This decrease was due to a similar attenuation in both the upper (59 ± 15%, P < 0.01) and lower (44 ± 12%, P < 0.01) curvatures, which reflected a decrease in the threshold pressure (8 ± 3 mmHg, P < 0.05) and increase in the saturation pressure (+12 ± 2 mmHg, P < 0.01) of the reflex, respectively (Table 2 and Fig. 4). Similarly, administration of kynurenate into the RVLM did not alter the lower or upper plateau of the HR baroreflex but decreased the reflex gain by 45 ± 10% (Table 3 and Fig. 4). This change was due to a reduction in the average curvature (43 ± 9%), which was, in turn, mediated by concurrent changes in the upper (49 ± 10%, P < 0.01) and lower (32 ± 9%, P < 0.01) curvatures of the HR baroreflex (Fig. 4). After administration of kynurenate, the threshold and saturation pressures of the HR baroreflex decreased (10 ± 2 mmHg) and increased (+17 ± 4 mmHg), respectively (P < 0.05; Table 3).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effects of kynurenate microinjections into the RVLM on RSNA and HR baroreflexes in conscious rabbits. Circles on curves represent resting values. Open dot and dashed line represent control, and black dot and solid line indicate kynurenate (10 nmol). Error bars are average SE calculated from ANOVA, indicating variation within animals (n = 8). Right: average gain before (open bar) and after (gray bar) treatment; dots represent the values obtained from all 8 rabbits before and after treatment. *P < 0.01 vs. control. nu, Normalized units.

Microinjections of kynurenate into the IVLM shifted the RSNA baroreflex curve to higher pressures but did not alter the upper plateau range or lower plateau of this reflex (Table 2 and Fig. 5). In contrast to the RVLM group, neither the gain (7 ± 7%) nor curvature (+1 ± 15%) of the RSNA baroreflex was changed by kynurenate. The administration of kynurenate into the IVLM shifted the HR baroreflex curve to higher pressures and increased the lower plateau by 38 ± 13 beats/min, without affecting the upper plateau of the reflex (Table 3 and Fig. 5). Microinjections of kynurenate just rostral to the RVLM in two other rabbits did not affect the RSNA or HR baroreflex parameters (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effects of kynurenate microinjections into the IVLM on RSNA and HR baroreflexes in conscious rabbits (n = 5). Symbols, abbreviations, and error bars as in Fig. 4.

Induction of pentobarbital sodium anesthesia increased resting MAP from 82 ± 1 to 100 ± 1 mmHg (P < 0.01, n = 4) and HR from 194 ± 12 to 310 ± 21 beats/min (P < 0.001) without altering RSNA (Fig. 6). The anesthesia reduced the gain and range of the RSNA baroreflex by 78 ± 13% (P < 0.001) and 40 ± 9% (P < 0.01), respectively. Similarly, the gain and range of the HR baroreflex were reduced by 82 ± 12 and 48 ± 12%, respectively (P < 0.001). The reduction in the range of the HR baroreflex was mediated by a marked increase in the lower plateau (+120 ± 19 beats/min), whereas the upper plateau remained unaffected by anesthesia (Fig. 6). Under these conditions, bilateral microinjection of kynurenate into the RVLM did not cause any further alteration in the gain and range of the RSNA or HR baroreflexes (Fig. 6).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effects of kynurenate microinjections into the RVLM on RSNA and HR baroreflexes in anesthetized rabbits. Gray line, dot (left) and bar (right) represent the control baroreflex parameters before anesthesia in the same rabbits. Other symbols, abbreviations, and error bars as in Fig. 4. *P < 0.001, between the conscious and anesthetized state (n = 4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The major finding of the present study is that bilateral microinjection of the broad-spectrum EAA receptor antagonist kynurenate into the pressor region of the RVLM markedly reduced the gain of RSNA and HR baroreflexes without altering resting cardiovascular parameters in conscious rabbits. This indicates that EAA-mediated inputs in the RVLM may subserve to sensitize the baroreflex and thus to increase the RSNA and HR responses to a given change in blood pressure. To our knowledge, this is the first direct evidence that tonic EAA-mediated inputs in the RVLM can modulate the renal sympathetic and cardiac responses to acute changes in blood pressure in conscious animals via enhancing the buffering capacity of the baroreflex.

This effect of kynurenate was likely to be specific to the pressor region of the RVLM since its microinjections into the IVLM or rostral to the RVLM did not affect the baroreflex sensitivity. By contrast, previous studies reported that microinjections of kynurenate as well as NMDA and non-NMDA receptor antagonists into the RVLM left sympathetic and cardiac baroreflexes intact in anesthetized animals (12, 27, 40, 41). The reason for such discrepancy may relate to the influence of anesthetics, because in our study kynurenate, given into the RVLM in the same rabbits after induction of pentobarbital sodium anesthesia, did not alter RSNA or HR baroreflexes. The lack of the effect of kynurenate appeared to be due to the pentobarbital sodium anesthesia severely compromising the baroreflex gain, which is likely to limit the ability of the premotor neurons to be further inhibited. The strong inhibiting action of barbiturate anesthesia on the baroreflex gain has also been reported previously (18, 31). Although other commonly used anesthetics, such as urethane and chloralose, generally inhibit baroreflexes to a lesser extent (19, 37), chloralose-urethane still reduces the RSNA baroreflex gain to ~60% of the value in conscious rabbits (10, 11). Considering that, in the present study, kynurenate decreased the baroreflex gain to a similar extent, it is plausible that its effect may still be masked under less suppressive chloralose and/or urethane regimens.

The attenuation in the baroreflex gain after the kynurenate injection was mediated by a decrease in the reflex curvature, which is likely to reflect a diminished responsiveness of the RVLM neurons to the afferent signals from baroreceptors (7). By contrast, we recently found that glutamate microinfusion into the RVLM increased the RSNA baroreflex gain solely via augmenting the reflex range in conscious rabbits, which rather indicates an increase in excitatory capacity of the motoneuron pool, possibly via the recruitment of previously silent units (33). The dissimilarity between the studies suggests that kynurenate and glutamate in the RVLM can alter baroreflexes acting via different EAA receptor subtypes. The effect of glutamate was mediated, at least in part, via blocking metabotrobic EAA receptors because microinjection of kynurenate into the RVLM did not alter the pressor response to local glutamate injection. The lack of effect of kynurenate on the glutamate-induced pressor responses is in agreement with previous findings that metabotrobic EAA receptors mediate cardiovascular responses evoked by glutamate injections into the RVLM in anesthetized rats and rabbits (13, 42). This does not imply, however, that ionotropic EAA receptors in the RVLM cannot mediate, under particular conditions, the glutamate-induced excitation, because kynurenate has been found to block the pressor effect of glutamate in conscious rats (2).

In the present study, microinjections of kynurenate into the RVLM did not alter resting MAP, HR, or RSNA in conscious or anesthetized rabbits. The lack of effect of kynurenate in the RVLM on arterial pressure and basal firing frequency of the local vasomotor neurons was also documented previously in a number of studies in normotensive anesthetized rats (21, 25, 27, 40) and rabbits (41) and also in conscious rats (2). One explanation of this phenomenon is that EAA-mediated input onto the vasomotor RVLM neurons is not tonically active, and resting activity of these cells is determined by their intrinsic pacemaker properties (16). Alternatively, a tonically active EAA-mediated input to the RVLM does contribute to the basal drive of the vasomotor RVLM neurons (21). However, kynurenate, given into the RVLM, does not change arterial pressure, because it simultaneously removes, possibly via a presynaptic action, local inhibitory inputs originating from the caudal ventrolateral medulla (CVLM). Pharmacological inhibition of neuronal activity in the CVLM eliminates EAA-sensitive inhibitory inputs and kynurenate decreases arterial pressure significantly below the initial resting levels (21). Thus, this hypothesis suggests that endogenous EAAs in the RVLM exert both direct excitatory and indirect inhibitory (the CVLM dependent) influences on the local vasomotor neurons and that these opposing influences are in balance at resting conditions.

Importantly, if the latter hypothesis is correct, then one may expect that naturally occurring alterations of neuronal activity in the CVLM, such as during baroreceptor loading or unloading (14, 22), would also change the balance and thus the net effect of kynurenate on the RVLM neurons. In particular, an attenuation of inhibitory activity of the CVLM with a fall in arterial pressure (14, 22) would decrease influence of EAA-sensitive inhibitory inputs (Fig. 7). This would shift the balance toward excitation and thus result in the overall inhibitory influence of kynurenate. This, in turn, would attenuate the sympathoexcitatory response to a given decrease in arterial pressure, reducing the upper baroreflex curvature. By contrast, an increase in activity of the CVLM with a rise in arterial pressure may also enhance EAA-mediated inhibitory influence, shifting the balance toward inhibition. This would result in the net excitatory influence of kynurenate and lead to an attenuated sympathoinhibitory response to a given rise in arterial pressure, thus decreasing the lower baroreflex curvature. This model should also assume the existence of EAA-independent barosensitive inhibitory inputs from the CVLM to RVLM (not shown on Fig. 7 for simplicity), which bring the baroreflex plateaus to pre-kynurenate levels when arterial pressure deviates further from resting values. Thus, current findings that kynurenate is equally effective in decreasing the upper and lower curvatures, while producing little effect on resting arterial pressure, are more consistent with the presence of both excitatory and inhibitory EAA-mediated inputs in the RVLM. On the contrary, the model that suggests the existence of only excitatory EAA-mediated inputs, which are not tonically active at resting conditions, is more likely to predict no effect of kynurenate on baroreflexes when blood pressure increases above resting levels.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Schematic model showing the effect of interaction of an excitatory amino acid (EAA) receptor antagonist kynurenate with the proposed EAA-mediated excitatory and inhibitory inputs to the RVLM neurons (21) during baroreceptor unloading (A) and activation (B). A: attenuation in inhibitory activity of the caudal ventrolateral medulla (CVLM) with a fall in arterial pressure decreases influence of EAA-sensitive inhibitory inputs, resulting in the net inhibitory action of kynurenate. This action attenuates the sympathoexcitatory response to a given decrease in arterial pressure, reducing the upper baroreflex curvature (right, gray arrows). B: increase in activity of the CVLM with a rise in arterial pressure enhances EAA-sensitive inhibitory influence, resulting in the net excitatory action of kynurenate. This blunts the sympathoinhibitory response to a given rise in arterial pressure, thus decreasing the lower baroreflex curvature (right, black arrows). For elaboration of this model, see DISCUSSION. , EAA receptor.

It is noteworthy that the balance between EAA-mediated excitation and inhibition of resting activity of the RVLM neurons is not always the case. In particular, it may chronically be shifted to excitation in hypertension (5, 20). In addition, this balance may depend on the experimental preparation and anesthetic used, since injection of kynurenate into the RVLM has recently been found to decrease arterial pressure in urethane-anesthetized rabbits (6).

In contrast to the RVLM injections, administration of kynurenate into the IVLM 0-1.0 mm rostral to the obex level increased resting arterial pressure and RSNA, indicating that EAA inputs were tonically active in this portion of the ventrolateral medulla in agreement with previous studies in anesthetized rabbits (29, 30). However, we also cannot exclude some leakage of kynurenate into the neighboring depressor region of the CVLM, where blockade of EAA inputs to sympathoinhibitory neurons increases arterial pressure and sympathetic activity as shown previously in anesthetized animals (27, 29).

In this study, injections of kynurenate into the IVLM did not alter the gain or range of the sympathetic baroreflex in conscious rabbits. These results might seem somewhat surprising, considering that EAAs play an important role in mediating baroreceptor reflexes at the level of the CVLM, which forms afferent input to the sympathoexcitatory neurons of the RVLM (14, 22, 27). However, it has been shown previously that, to affect the baroreflex in the rabbit, it was necessary to treat the rostrocaudal extent of the ventrolateral medulla from 1.3 mm rostral to 3 mm caudal to the obex using three to five consecutive injections (100 nl each) of kynurenic or kainic acids (29, 30). Thus, in the present study, the single injection of kynurenate was presumably not sufficient to alter the baroreflex transmission in the depressor region of the ventrolateral medulla.

In conclusion, the results of the present study suggest that endogenously released EAA neurotransmitters in the RVLM are important in modulating the renal sympathetic and cardiac baroreflexes in conscious rabbits. However, under resting conditions, blockade of EAAs in the RVLM has little effect on arterial pressure. The current results are consistent with existence of both excitatory and inhibitory EAA-mediated inputs to this region as it was suggested previously (21). Anesthesia can mask the functional significance of EAAs in the RVLM in controlling the baroreflexes, which may explain why previous studies in anesthetized animals found no effect of blocking EAA receptors in this region on sympathetic baroreflexes.

Perspectives