Are L-glutamate and ATP cotransmitters of the peripheral chemoreflex in the rat nucleus tractus solitarius

Experimental Physiology/2008/043653-R1

Are L-glutamate and ATP cotransmitters of the peripheral chemoreflex in the nucleus tractus solitarius?

Daniela Accorsi-Mendonça, Leni G.H. Bonagamba, Ricardo M. Leão and Benedito H. Machado*

Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, 14049-900, Ribeirão Preto, SP, Brazil

Running title: Glutamatergic and purinergic transmission in the NTS output neurons. Key words: glutamatergic, purinergic, NTS, sympathoexcitation, chemoreflex, RVLM * Corresponding author: Department of Physiology School of Medicine of Ribeirão Preto University of São Paulo 14049-900, Ribeirão Preto, SP, Brazil e-mail: [email protected] Phone: 55-16-3602-3015 FAX: 55-16-3633-0017

Physiology in Press; published online on October 22, 2008 as 10.1113/expphysiol.2008.043653

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ABSTRACT

Peripheral chemoreflex activation in awake rats or in the working heart brainstem

preparation (WHBP) produces sympathoexcitation, bradycardia and increase in the

frequency of phrenic nerve activity (PNA). Our focus is the neurotransmission of the

sympathoexcitatory component of the chemoreflex within the nucleus of tractus

solitarius (NTS) and recently we verified that the simultaneous antagonism of

ionotropic glutamate and purinergic P2 receptors in the NTS blocked pressor response

and increased thoracic sympathetic activity in awake rats and WHBP, respectively, in

response to peripheral chemoreflex activation. These previous data suggested the

involvement of ATP and L-glutamate in the NTS in the processing of the

sympathoexcitatory component of the chemoreflex by unknown mechanisms. For a

better understanding of these mechanisms, here we used a patch-clamp approach in

brainstem slices to evaluate the characteristics of the synaptic transmission of NTS

neurons sending projections to the ventral medulla, which include the pre-motor

neurons involved in the generation of the sympathetic outflow. The NTS neurons

sending projections to ventral medulla were identified by previous microinjection of the

membrane DiI tracer in the ventral medulla and the spontaneous (sEPSCs) and tractus

solitarius (TS)-evoked excitatory post-synaptic current (TS-eEPSCs) were recorded

using patch clamp. With this approach we verified that in NTS neurons projecting to the

ventral medulla: i) the sEPSCs and TS-eEPSCs of DiI-labeled NTS neurons were

completely abolished by DNQX, an antagonist of ionotropic non-NMDA glutamatergic

receptors, showing that they are mediated by L-glutamate; ii) application of ATP

increases the frequency of appearance of spontaneous glutamatergic currents reflecting

an increased exocytosis of glutamatergic vesicles; and iii) ATP decreases the peak of

TS-evoked glutamatergic currents. We conclude that L-glutamate is the main

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neurotransmitter of spontaneous and TS-evoked synaptic activities in the NTS neurons

projecting to ventral medulla and that ATP has a dual modulatory role on this excitatory

transmission facilitating the spontaneous glutamatergic transmission and inhibiting the

TS-evoked glutamatergic transmission. These data also suggest that ATP is not acting

as a cotransmitter with L-glutamate, at least at the level of this sub-population of NTS

neurons studied.

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The NTS is the site of integration of the viscerosensory information in the

brainstem including cardiovascular and respiratory afferents (Mifflin, 1992; Ciriello et

al., 1994; Chitravanshi & Sapru, 1995; Machado, 2001, 2004) and there is anatomical

and functional evidence that the first synapse of the peripheral chemoreceptor afferents

is located in the commissural NTS (Donoghue et al. 1984; Finley & Katz, 1992; Mifflin,

1992; Ciriello et al. 1994; Chitravanshi & Sapru, 1995; Paton et al. 2001). Activation of

the peripheral chemoreceptors by intravenous injection of potassium cyanide (KCN) in

awake rats or the working heart brainstem preparation (WHBP) produces bradycardia,

tachypnoea and increases in the sympathetic outflow and arterial pressure, (Franchini &

Krieger, 1992; Haibara et al. 1995; Boscan & Paton, 2001; Paton et al. 2002; Machado

& Bonagamba, 2005; Antunes et al. 2005b; Braga & Machado, 2006; Braga et al., 2007;

Zoccal et al., 2008). There is also evidence that the sympathoexcitatory component of

chemoreflex involves NTS neurons sending projections to the rostral ventrolateral

medulla [RVLM, (Ross, 1985; Urbanski & Sapru, 1988; Seller et al, 1990; Koshiya et

al., 1993; Granata, 1994; Aicher, et al., 1996)].

The increase in sympathetic activity during hypoxia condition is a physiological

response to increase the peripheral vascular resistance and the arterial perfusion

pressure. These autonomic and circulatory events occur in order to keep the cerebral

blood flow in the autoregulatory range. However, abnormal and frequent increase in the

sympathetic activity due to chemoreflex activation may contribute to the development

of hypertension, as observed in pathophysiological conditions (Machado, 2004). Our

laboratories are deeply involved with studies on the neurotransmission of the

sympathoexcitatory component of the chemoreflex at the NTS level, with special

attention to the role of L-glutamate and ATP as putative neurotransmitters or

neuromodulators (Haibara et al., 1995, 1999; de Paula et al., 2004; Machado &

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Bonagamba, 2005; Antunes et al., 2005; Braga & Machado, 2006; Braga et al., 2007).

In a series of recent studies we verified that the antagonism of L-glutamate ionotropic

and metabotropic receptors in the NTS was not effective in blocking the pressor

response in awake rats to chemoreflex activation and produced no effect on the

sympathoexcitatory response in the WHBP to chemoreflex activation (Haibara et al.,

1999; Machado & Bonagamba, 2005; Braga & Machado, 2006). The antagonism of P2

receptors in the NTS of awake rats was also not effective in blocking the pressor

response in awake rats or the sympathoexcitatory response of the chemoreflex in the

WHBP (Braga et al., 2007). Therefore, the possibility that both L-glutamate and ATP

might be acting as cotransmitters in the processing of the sympathoexcitatory

component in the NTS was considered and simultaneous blockade of glutamatergic and

purinergic receptors were performed. We verified that the simultaneous antagonism of

these receptors was effective in blocking the pressor response in awake rats and the

sympathoexcitatory response to chemoreflex activation in the WHBP (Braga et al.,

2007).

These findings, indicating that both L-glutamate and ATP are part of the

complex neurotransmission system of the sympathoexcitatory component of the

chemoreflex in the NTS, opened interesting possibilities to explore a possible

cotransmission of both neurotransmitter in the NTS. In order to test this hypothesis, we

combined in vivo retrograde labeling and in vitro whole cell patch clamp recordings in

brainstem slices containing the NTS to perform a characterization of glutamatergic and

purinergic mechanisms on the spontaneous and evoked excitatory neurotransmission of

NTS neurons sending projections to the ventral medulla, which includes the pre-motor

neurons involved in the generation of the sympathetic activity.

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Application of DiI into the ventral medulla and brainstem slices preparation

Wistar male rats (270-290 g) under tribromoethanol anesthesia (250 mg kg–1, ip,

Aldrich Chemical, St Louis, USA) were placed in a stereotaxic apparatus (David Kopf,

Tujunga, USA) for unilateral microinjection of the membrane tracer dye 1,1'-

dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiI, 0.5% in ethanol,

Invitrogen, Carlsbad, USA) into the ventral medulla. The stereotaxic coordinates

correspondent to the site of microinjections in the ventral medulla of DiI were 1.8 mm

lateral to the midline, 4.1 mm caudal to the bregma and 10.2 mm below the skull

surface.

Brainstem slices (250 µm thick) were sectioned at least 24 hours after DiI

microinjection in modified ice-cold (4C) artificial cerebrospinal fluid (aCSF)

containing the following (in mM): 75 sucrose, 87 NaCl, 2.5 KCl, 7 MgCl2, 1.25

NaH2PO4, 25 NaHCO3, 25 glucose and 0.2 CaCl2, (330-350 mOsm/Kg.H2O and pH

7.4) using an oscillating slicer (Vibratome 1000 plus, Vibratome, St. Louis, USA) and

then the slices were kept at 37C for 45 min. The site of DiI microinjection was

confirmed in fresh brainstem slices in a microscope equipped with fluorescence

(Olympus BX51WI, Olympus, Tokyo, Japan) using the nucleus ambiguus (NA) as an

anatomical reference. Thereafter, the slices were kept at room temperature (RT, 23-

25C) in normal aCSF containing (mM): 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4,

25 NaHCO3, 25 glucose and 2 CaCl2, with osmolality of 310-330 mOsm/Kg.H2O, a rate

flow of approximately 3 ml/min and continuously bubbled with 95% O2 and 5% CO2.

Neurons in brainstem slice were visualized by infrared differential interference contrast

(IR-DIC; Figure 1, panel A) and fluorescence microscopy using a 40X water immersion

objective (LUMPlain F1-IR, Olympus, Tokyo, Japan) and a CCD camera (C7500-50,

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Hamamatsu, Iwata-City, Japan) to identify DiI-labeled cell in the NTS slice (Figure 1,

panel B).

Whole cell patch-clamp electrophysiological recordings

Whole cell recordings (voltage clamp configuration) were made with patch

pipettes pulled from thick-walled borosilicate glass capillaries (Sutter Instruments,

Novato, USA) filled with an internal solution containing (mM): 150 KCl, 5 ethylene

glycol-bis (-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) and 10 N-2-

hydroxyethyl-piperazine-N’-2-ethanesulfonic acid (HEPES)(320-330 mOsm/Kg.H2O,

pH 7.4, final resistance: 4 to 8 M). The signals were acquired using an Axopatch 200B

amplifier (Axon Instruments, Sunnyvalley, USA) connected to a microcomputer via a

Digidata 1440A board (Axon Instruments, Sunnyvalley, USA). Data was low-pass

filtered at 2 kHz and acquired at 10 kHz. The holding potential was kept at -70 mV.

eEPSCs were generated by electrical stimulation of the TS using a bipolar

tungsten electrode (FHC, Bowdoin, USA) connected to an isolated stimulator (S48

Stimulator, Grass Instrument, West Warwick, USA). sEPSCs were recorded in 1-2

minutes periods and the data acquisition was performed with Clampex software

(pClamp 10, Axon Instruments, Sunnyvalley, USA). Some experiments were recorded

in the presence of bicuculline (20 µM) in the bath perfusion, in order to block

GABAergic currents or DPCPX (1 µM), an A1 receptor antagonist.

The peak amplitude of TS-eEPSC was measured using Clampfit (pClamp 10,

Axon Instruments, Sunnyvalley, USA) and the frequency, amplitude and half-width of

sEPSCs were analyzed using Minianalysis Program (version 5.0, Synaptosoft, Decatur,

USA). The data are expressed as mean ± standard error (SEM) and statistical

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significance (p<0.05) was determined by paired Student t test using the GraphPad Prism

4 software (GraphPad Prism, San Diego, USA).

TS-eEPSCs and sEPSCs in the NTS neurons projecting to ventral medulla

Electrical stimulation of the TS often evoked EPSCs in DiI-labeled NTS neurons.

These currents were completely blocked after the addition of 6,7-dinitroquinoxaline-

2,3(1H,4H)-dione (DNQX, 10 µM), a non-NMDA receptor antagonist, and bicuculline

(20 µM), a GABAA receptor antagonist (-104±14 vs -8±1.2 pA, n=8, p<0.05, Figure 2,

panels A and B) in the bath perfusion. sEPSCs in DiI-labeled NTS cells were also

recorded and DNQX and bicuculline were also effective in blocking these events (1.36

± 0.26 vs 0.05 ± 0.03 Hz, n=23, p<0.05, Figure 2, panels C and D).

Effect of ATP application on TS-eEPSCs and sEPSCs

Considering our previous data suggesting an interaction between glutamatergic

and purinergic neurotransmission in the NTS (Braga et al., 2007), in the present study

we analyzed the effects of ATP (500 µM) in the bath perfusion on the TS-eEPSCs and

sEPSCs in DiI-labeled NTS neurons. During the experiments the excitatory currents

were isolated by adding bicuculline and 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX,

1 µM), an A1 receptor antagonist, was also added in order to avoid a possible effect of

adenosine generated by ATP break-down.

ATP in the bath perfusion decreased the peak amplitude of TS-eEPSCs in all

recorded Di-labeled NTS neurons (-115.3 ± 24 vs -50 ± 10 pA, n=10, p<0.05, Figure 3,

panel A), suggesting that P2 receptors affect the glutamatergic neurotransmission in

NTS neurons. To analyze whether or not the mechanism by which ATP exerts its effect

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on evoked synaptic transmission involves a decrease in pre-synaptic glutamate release

and/or changes in post-synaptic neuron, we used a paired-pulse protocol. For this

purpose, we analyzed the paired-pulse ratio (PPR) of the amplitudes of two consecutive

TS-eEPSCs (P1 and P2 respectively), with inter-stimulus interval of 100 ms. A change

in the paired-pulse ratio (P2/P1) accompanying a decrease in the first TS-eEPSC peak

amplitude was taken as an indicator for inhibition at a pre-synaptic site (Kline et al.,

2002; Sekizawa et al., 2003; Ohi et al., 2007). ATP increased the PPR in DiI-labeled

neurons (0.6 ± 0.09 vs 0.86 ± 0.17 pA, n=10, p<0.05, Figure 3, panel B), suggesting that

P2 receptors are affecting the excitatory transmission by a pre-synaptic mechanism.

ATP in the bath perfusion increased the frequency of spontaneous synaptic

currents in ~70% of DiI-labeled NTS neurons. In responsive neurons we observed a

significant increase in the frequency of sEPSCs (0.55 ± 0.13 vs 4.4 ± 1.3 Hz, n=8,

p<0.05, Figure 3, panels C and D) and no changes in the amplitude (20 ± 3.4 vs 23

± 1.7 pA) or half-width (2.11 ± 0.37 vs 2.1 ± 0.23 ms) of these currents, suggesting that

activation of P2 receptors increases vesicular glutamate release from pre-synaptic

neurons.

Importance of electrophysiological data for understanding the chemoreflex

neurotransmission in the NTS

Here we evaluated aspects of the neurotransmission in the NTS neurons sending

projections to the ventral medulla, a group of neurons participating in generation of

sympathetic outflow (Seller et al., 1990, Koshiya et al., 1993). We employed

microinjections of the plasma membrane DiI tracer in the ventral aspect of the medulla

for a retrograde labeling of the NTS neurons projecting to this area. Although we aimed

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to reach the pre-motor sympathetic neurons in the RVLM, we assume that the DiI

microinjection was not restricted to the synaptic contact with these neurons and it may

have reached neurons other than those involved in the generation of the sympathetic

activity in the ventral medulla. Therefore, we considered the DiI-labeled NTS cells

generically as ventral medulla-projecting NTS neurons, which include the neurons

sending projections to the RVLM.

Recently, we demonstrated that combined microinjections of glutamatergic and

purinergic antagonists into the commissural NTS blocked the

sympathoexcitatory/pressor response to peripheral chemoreflex activation (Braga et al.,

2007). Therefore, we hypothesized that neurotransmission of the sympathoexcitatory

component of the chemoreflex involves an interaction between L-glutamate and ATP in

NTS and we evaluated this interaction in NTS neurons sending projections to ventral

medulla using electrophysiological approach.

The data of the present study support the concept that L-glutamate is the main

neurotransmitter involved in the synaptic activation of all studied NTS neurons sending

projections to the ventral medulla, since sEPSCs and TS-eEPSCs were abolished after

addition of DNQx, as previously described for NTS neurons (Andresen & Yang, 1990;

Kline et al., 2002; Jin et al., 2004; Accorsi-Mendonça et al., 2007; Kline et al., 2007).

Additionally, after DNQX application we did not observe any residual synaptic event

suggestive of a purinergic synaptic current, as observed in other brain areas, such as

cortex and habenula (Edwards et al., 1992; Pankratov et al., 2007).

The experiments involving ATP were performed in the presence of bicuculine

to block GABAergic currents and DPCPX to block possible effects of adenosine

receptors activation. ATP increased the frequency of the sEPSCs, suggesting that ATP

increases the probability of spontaneous L-glutamate release. The activation of somatic

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and/or pre-synaptic P2X receptors could depolarize the neuron/terminal leading to an

increased firing of the pre-synaptic neurons or an increased probability of release of

glutamatergic synaptic vesicles (Shigetomi & Kato, 2004). On the other hand ATP

inhibited the TS-eEPSC amplitude in Dil-labeled NTS neurons, probably acting pre-

synaptically reducing the release probability of these vesicles due to the observed

increase in the paired-pulse ratio. Therefore, the data of these experimental protocols

shows that in Dil-labeled NTS neurons, ATP has a dual effect on the glutamatergic

neurotransmission in the NTS, being both inhibitory or excitatory, probably by

differential activation of ionotropic (P2X) and metabotropic (P2Y) receptors (Heinrich

et al., 2008). Alternatively we suggest that the increased spontaneous glutamate released

by ATP application can activate pre-synaptic glutamatergic metabotropic receptors

causing the inhibition in the TS-evoked EPSC. A depletion of readily-available vesicles

with consequent reduction in the evoked release cannot be rule out. The findings of the

present study suggest that L-glutamate and ATP are not acting as cotransmitters in the

synaptic transmission in the NTS neurons sending projections to ventral medulla. This

possibility is supported by the fact that in the present study ATP presented only a pre-

synaptic effect, while a cotransmitter, in accordance with the concept described by

Burnstock (2005; 2007), should also activate its post synaptic receptors.

Taking into account a) that only the double antagonism of glutamatergic and

purinergic receptors in the NTS was effective in the blockade of the

sympathoexcitatory/pressor response to chemoreflex activation in awake rats and

WHBP, b) the electrophysiological data showing that in the NTS neurons sending

projections to the ventral medulla, L-glutamate is the main excitatory neurotransmitter

while ATP acts as an neuromodulator, we must considerer several other possibilities in

order to fit together all these findings. First, it is important to note that the

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sympathoexcitatory component of the chemoreflex is not restricted to the NTS-ventral

medulla projections (Figure 4), since there is experimental evidence that other important

areas in the brainstem such as the retrotrapezoid nucleus (Moreira et al, 2006; Takakura

et al., 2006) also participate in the generation of sympathetic and respiratory responses

to chemoreflex activation; second, as illustrated in Figure 4, there is also evidence that

the parabrachial nucleus (Haibara et al., 2002) and A5 area (Koshiya & Guyenet, 1994)

in the pons and the paraventricular nucleus of the hypothalamus (Olivan et al., 2001;

Reddy et al., 2005) participate in the sympathoexcitatory response of the chemoreflex,

but these alternative NTS projections were not yet evaluated using the approach

described in the present study. Therefore, at this time it is not possible to give a

definitive answer to the proposed question of this study about the overall glutamatergic

and purinergic cotransmission at the NTS level. However, the electrophysiological

findings of the present study clearly demonstrated that L-glutamate and ATP are not

acting as cotransmitters in the synaptic transmission in the NTS neurons sending

projections to the ventral medulla.

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Acknowledgements:

This work was supported by FAPESP (2004/03285-7) and CNPQ

(471184/2007-1). We thank Rubens F. Melo and Adriana Murashima for their technical

support.

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LEGENDS:

Figure 1. Identification of DiI-labeled NTS neurons

(A): DiI-labeled NTS neuron (arrow) viewed with infrared differential interference

contrast optic (IR-DIC) in the brainstem slice; (B): the same neuron under fluorescence

with an arrow indicating the DiI labeling.

Figure 2. Effect of bicuculine and DNQX on sPSCs and TS-ePSCs in DiI-labeled

NTS neurons

(A): Representative traces of TS- evoked post synaptic currents (ePSCs) from a DiI-

labeled NTS neuron in a control condition and after DNQX (10 µM) and bicuculline (20

µM) in the bath perfusion; (B): Mean peak amplitude of TS-ePSCs from DiI-labeled

NTS neurons (n=8) before and after DNQX and bicuculline in bath perfusion; (C)

Representative traces of spontaneous post synaptic currents (sPSCs) from a DiI-labeled

NTS neuron in a control situation and after DNQX and bicuculline in the bath

perfusion; (D) Mean frequency of sPSCs in DiI-labeled NTS neurons (n=23) before and

after DNQX and bicuculline in bath perfusion. Bic = bicuculline. Holding potential =

-70 mV. * p < 0.05.

Figure 3. Effect of ATP on sEPSCs and TS-eEPSCs in DiI-labeled NTS neurons

(A): Mean peak amplitude of TS-evoked excitatory post-synaptic currents (eEPSCs) in

DiI-labeled NTS neurons (n=10) before and after ATP (500 µM) in bath perfusion.

Inset: Representative traces of TS-eEPSCs before and after ATP application in the bath

perfusion; DPCPX (1 µM), an A1 receptor antagonist used to avoid the effect of

adenosine produced by ATP break-down; (B): Mean PPR (paired pulse ratio) of two

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consecutive eEPSCs (P1 and P2 respectively; time interval: 100 ms) in DiI-labeled NTS

neurons (n=10) before and after ATP in the bath perfusion. Inset: Representative traces

of two TS-eEPSCs before and after ATP application in the bath perfusion, P1 = Peak 1,

P2 = Peak 2; (C): Representative traces of sEPSCs from a DiI-labeled NTS neuron

before and after ATP in the bath perfusion; (D): Frequency of sEPSCs in DiI-labeled

NTS neurons (n=8) before and after ATP in the bath perfusion. Bic = bicuculline.

Holding potential = -70 mV. * p<0.05.

Figure 4. Schematic drawing of sympathoexcitatory pathways of the chemoreflex

in the brainstem, pons and hypothalamus

Lower pO2 in the arterial blood activates peripheral chemoreceptors located in the

carotid body. Experimentally this activation can be induced by intravenous injection of

potassium cyanide (KCN). The afferents of this reflex make the first synaptic contact in

the NTS, via tractus solitarius. NTS neurons sending projections to several other brain

areas are involved in the processing of the chemoreflex, such as paraventricular nucleus

of the hypothalamus (PVN, Olivan et al., 2001; Reddy et al., 2005), parabrachial

nucleus (PBN, Haibara et al., 2002), A5 area (Koshiya & Guyenet, 1994), pre-Bötzinger

nucleus (pre-BötC) and Bötzinger nucleus and retrotrapezoid nucleus (nRT, Moreira et

al., 2006; Takakura et al., 2006). Moreover, there is anatomical and functional evidence

of an important projection from NTS to the rostral ventrolateral medulla (RVLM,

Urbanski & Sapru, 1988; Aicher et al., 1996), in which are located the pre-motor

neurons involved in the generation of the sympathetic outflow (Sun et al., 1988).

Considering the importance of this NTS-RVLM projection in the context of the

sympatoexcitatory response of the chemoreflex, it was explored by using DiI tracer and

an electrophysiological approach. RVLM neurons send direct projections to the

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intermediolateral column of spinal cord (IML), which has connections with the

sympathetic ganglion. Sympathetic post-ganglionic fibers send projections to the end-

organs and release noradrenaline, especially to the resistance vessels, which increases

the vascular resistance and arterial blood pressure. Therefore, the chemoreflex

activation may produce sympathoexcitation by activation of one and/or several neuronal

pathways from the NTS illustrated in this panel. The electrophysiological data on a

possible cotransmission of L-glutamate and ATP described in the present study is

restricted to the sub-population of NTS neurons sending projections to the ventral

medulla.

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