Respiratory Effects of Kynurenic Acid Microinjected Into the Ventromedullary Surface of the Rat

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Brazilian Journal of Medical and Biological Research (1998) 31: 1339-1343 Tonic respiratory activity in the ventrolateral medulla 1339 ISSN 0100-879X Short Communication

Respiratory effects of kynurenic acid microinjected into the ventromedullary surface of the rat

F.P. Tolentino-Silva, Departamento de Fisiologia, Escola Paulista de Medicina, A.K. Russo, Universidade Federal de São Paulo, São Paulo, SP, Brasil S.L. Cravo and O.U. Lopes

Abstract

Correspondence Several studies demonstrate that, within the ventral medullary surface Key words F.P. Tolentino-Silva (VMS), excitatory amino acids are necessary components of the neural • Ventral surface of the Departamento de Fisiologia circuits involved in the tonic and reflex control of respiration and medulla EPM, UNIFESP circulation. In the present study we investigated the cardiorespiratory • Breathing pattern Rua Botucatu, 862 effects of unilateral microinjections of the broad spectrum glutamate • Cardiorespiratory control 04023-060 São Paulo, SP • antagonist kynurenic acid (2 nmol/200 nl) along the VMS of urethane- Kynurenic acid Brasil • Rat Fax: +55-11-573-7820 anesthetized rats. Within the VMS only one region was responsive to E-mail: [email protected] this drug. This area includes most of the intermediate respiratory area, partially overlapping the rostral ventrolateral medulla (IA/RVL). When Research supported by FAPESP. microinjected into the IA/RVL, kynurenic acid produced a respiratory depression, without changes in mean arterial pressure or heart rate. The respiratory depression observed was characterized by a decrease

Received April 8, 1997 in ventilation, tidal volume and mean inspiratory flow and an increase Accepted July 6, 1998 in respiratory frequency. Therefore, the observed respiratory depression was entirely due to a reduction in the inspiratory drive. Microin- jections of vehicle (200 nl of saline) into this area produced no significant changes in breathing pattern, blood pressure or heart rate. Respiratory depression in response to the blockade of glutamatergic receptors inside the rostral VMS suggests that neurons at this site have an endogenous glutamatergic input controlling the respiratory cycle duration and the inspiratory drive transmission.

The ventral medullary surface (VMS) is rostral ventrolateral medulla (RVL), caudal widely considered to be an important site for ventrolateral medulla (CVL) (4,5) and the the integration of many autonomic and so- caudal pressor area (CPA) (6-8). The RVL matic functions including the respiratory and overlaps most of the intermediate area, cardiovascular control (1,2). Within the VMS, whereas CVL overlaps the caudal respira- three areas essential for the respiratory activ- tory area. Although CPA stimulation pro- ity (rostral, intermediate and caudal) (3) co- duces a well-defined respiratory activation exist with sites responsible for the genera- (9), so far this response has not been related tion and maintenance of the vasomotor tone to any particular respiratory area. and arterial blood pressure regulation: the Although the localization of these respi-

Braz J Med Biol Res 31(10) 1998 1340 F.P. Tolentino-Silva et al.

ratory and cardiovascular areas in the VMS was computer-processed to quantify the pa- is well established, the neurotransmitters in- rameters of the respiratory pattern (ventila- . volved in these circuits remain largely un- tion, VE; tidal volume, VT; inspiratory time, known. Several studies indicate that excita- TI; expiratory time, TE; breathing frequency, tory amino acids are significant components f; mean inspiratory flow, VT/TI). Mean arte- of the neural circuits involved in the genera- rial pressure (MAP) and heart rate (HR) tion and maintenance of the respiratory were monitored continually throughout the rhythm and vasomotor tone (10,11). Topical experiment using a Statham transducer (P23 application to (12) or microinjections (9,13) Db) connected to the femoral artery, coupled of L-glutamate into the ventrolateral medul- to a Beckman R-611 polygraph. The animals lary surface elicit pronounced changes in were placed prone in a stereotaxic frame respiration and arterial blood pressure. In a (Stoelting) and temperature was maintained previous study (9) we demonstrated that, in at 37oC. Kynurenic acid (KA, 2 nmol/200 nl) the rat, three different breathing patterns was unilaterally microinjected into the VMS could be elicited from four distinct sites according to a method previously described within the VMS. When applied to the RVL (8). This method consistently allowed injec- or the CPA, L-glutamate produced increases tions to be placed inside the ventral surface. in ventilation, inspiratory drive and hyper- Each animal received one to three microin- tension. Within the CVL two respiratory jections, at least 2 mm apart, distributed at patterns were observed: microinjection into random along the VMS. A new site was the rostral half of the CVL produced respira- tested only after the effect of a previous tory depression, while in the caudal half microinjection had completely disappeared. increases in minute ventilation and inspira- At the end of the experiments the last injec- tory drive were associated with bradypnea. tion site was marked with 200 nl of 2% Whether all of these areas represent sites of Evan’s blue dye. The animals were sacri- tonic respiratory activity remains undeter- ficed with an overdose of urethane, and the mined. brainstem was removed and fixed in 10% To further characterize the role of gluta- formaldehyde. Injection sites were identi- matergic afferents present in the VMS in fied and plotted on a schematic diagram. respiratory control, in the present study we Other sites tested in the experiment were investigated the effects of microinjections of plotted according to their relative position the broad spectrum glutamate antagonist with respect to the site marked with the dye. kynurenic acid on the breathing pattern, ar- To further identify injection sites, the brain- terial blood pressure and heart rate of anes- stem was also cut coronally into 40-µm thick thetized rats. sections and stained with 1% neutral red. Experiments were performed on male Photomicrographs and schematic drawings Wistar rats (N = 25) weighing 300 ± 6 g of typical injection sites obtained with this anesthetized with urethane (1.2 g/kg, ip), method have been presented elsewhere (9). tracheotomized and placed in a closed cylin- The breath-by-breath respiratory pattern data der-shaped body plethysmograph (80 mm for each animal were interpolated at 15-s ID, 300 mm long) with openings at both ends intervals. All data are reported as means ± to allow the externalization of the head, ther- SEM. Friedman’s analysis of variance was mal probe, and arterial and venous catheters. used to test whether or not values changed Respiratory flow was recorded with a pneu- with time after glutamate microinjection, with motachometer (Fleisch No. 0000) connected subsequent post hoc comparisons by to the plethysmograph. After amplification Dunnett’s test. The criterion for statistical and analog to digital conversion, the signal significance was P<0.05.

Braz J Med Biol Res 31(10) 1998 Tonic respiratory activity in the ventrolateral medulla 1341

The distribution of responsive sites was RVL), unilateral microinjections of KA pro- systematically explored within the VMS by duced a reduction in ventilation without examining respiratory responses to unilat- changing blood pressure or heart rate. Taken eral microinjections of KA (2 nmol/200 nl) together, these data indicate that only in the at 74 points. The area explored extended IA/RVL are tonically active glutamatergic from 6 mm rostral to 2.5 mm caudal to the inputs required to control inspiratory drive calamus scriptorius (CS) and up to 4 mm and respiratory cycle duration. lateral to the midline. The area surveyed is Previous studies (10) reported that in illustrated in Figure 1. Active respiratory Figure 1 - The diagram repre- sites, i.e., sites where KA produced signifi- sents the ventral medullary sur- cant changes in respiration, were found in face of the rat summarizing re- the VMS in an area lying 2.3 to 3.8 mm sults obtained from 74 microinjections in 25 animals. Filled rostral and 1.5 to 2.5 mm lateral to the CS circles indicate sites where uni- (Figure 1). The time course and percentile lateral microinjections of kynu- changes of respiratory parameters are pre- VI renic acid produced decreases in ventilation, tidal volume and sented in Figure 2. Before KA, ventilation inspiratory drive concurrently was 113 ± 9.6 ml/min, tidal volume was 1.5 with tachypnea, whereas the ± 0.1 ml and VT/TI was 4.9 ± 0.6 ml/s (N = 9). small open dots indicate sites where no significant respiratory Sixty seconds after KA administration, ven- XII changes were obtained. For tilation was decreased by 27 ± 4.2%, tidal clarity all microinjections are volume by 38 ± 4.1% and VT/TI by 32 ± C1 represented on the left side re- 4.0%. Respiratory depression was accompa- gardless of the actual side of application. C1 = First cervical nied by a 19 ± 3.7% increase in respiratory nerve, VI and XII = 6th and 12th frequency (control = 81 ± 7.2 breaths/min). 2 mm cranial nerve rootlets, respec- Therefore, the observed respiratory depres- tively. sion was entirely due to reduction in inspiratory drive. Although a progressive reduction in both inspiratory and expiratory times was observed, these reductions did not reach lev- Table 1 - Cardiorespiratory responses produced by unilateral microinjections of kynurenic acid into the ventrolateral medulla. els of statistical significance (Table 1, Figure . 2). No significant changes in MAP or HR VE, Ventilation; VT, tidal volume; TI, inspiratory time; TE, expiratory time; f, breathing frequency; were observed (basal: 101.3 ± 7.6 and 389 ± VT/TI, mean inspiratory flow; MAP, mean arterial pressure; HR, heart rate; N, number of injection points. All values are reported as means ± SEM. Responses to kynurenic acid are 0.1; 60 s after KA: 106.9 ± 2.3 mmHg and reported as percent change in relation to the control value. N = 11. *P<0.05 compared to control 390 ± 0.1 beats/min, respectively). Microin- (Friedman's analysis of variance). jection of vehicle (200 nl of saline) into this area produced no significant changes in any Time Control % Change of the parameters analyzed. 15 s 30 s 45 s 60 s Previously (9), using unilateral microinjections of L-glutamate, we have demon- MAP (mmHg) 101.3 ± 7.6 +5.1 ± 2.3 +5.6 ± 2.3 +5.6 ± 2.3 +5.6 ± 2.3 strated the presence of cardiorespiratory ac- HR (beats/min) 389 ± 0.1 +0.1 ± 0.1 +0.1 ± 0.1 +0.1 ± 0.1 +0.1 ± 0.1 tive sites along a column in the VMS extend- f (breaths/min) 81 ± 7.2 +3.7 ± 1.2 +8.5 ± 1.4* +8.1 ± 2.0* +18.6 ± 3.7* ing from the VI cranial nerve to the first VT (ml) 1.5 ± 0.1 -15.5 ± 2.5* -23.1 ± 3.1* -31.0 ± 3.2* -37.8 ± 4.1* . cervical nerves in the caudal medulla. The VE (ml/min) 113.4 ± 9.6 -11.9 ± 2.0* -17.2 ± 3.4* -24.9 ± 3.4* -26.7 ± 4.2* results obtained in the present study demon- TI (s) 0.32 ± 0.02 -5.6 ± 6.6 -13.1 ± 6.2 -10.1 ± 4.1 -10.1 ± 4.1 strate that within the VMS only in the region TE (s) 0.50 ± 0.06 -1.6 ± 4.8 -1.5 ± 5.7 -11.9 ± 4.9 -16.2 ± 5.8 of the intermediate respiratory area overlap- VT/TI (ml/s) 4.9 ± 0.6 -6.8 ± 4.9 -13.9 ± 3.9* -25.9 ± 4.8* -32.2 ± 4.0* ping the rostral ventrolateral medulla (IA/

Braz J Med Biol Res 31(10) 1998 1342 F.P. Tolentino-Silva et al.

Figure 2 - Time course of 20 . sulted in apnea and decreased CO2 sensitiv- changes (mean ± SEM, % of V E VT . 10 ity in cats (16) or rabbits (17). control) in ventilation (VE), tidal 0 * * * All of these studies were restricted to the volume (VT), respiratory fre- -10 * * * quency (f), inspiratory time (TI), -20 * -30 * IA/RVL area. Our study demonstrated that expiratory time (TE) and mean in- % of control -40 in this area even unilateral blockade of gluta- spiratory flow (VT/TI) elicited by -50 unilateral microinjection of kynu- matergic afferents is capable of producing renic acid (2 nmol/200 nl) into significant reductions in ventilation. Con- 20 responsive sites of the ventro- TI TE trarily, in caudal portions of the VMS (in- lateral medulla. *P<0.05 com- 10 pared to control (Friedman's a- 0 cluding the caudal respiratory area overlap- nalysis of variance). -10 ping the caudal ventrolateral medulla and -20 % of control the caudal pressor area) unilateral glutama- -30 tergic blockade was unable to modify respi- * ration, even though these areas are respon- 20 VT/TI f * * 10 sive to glutamate stimulation (9). 0 * The local circuitry and the synaptic -10 * -20 * mechanisms underlying the IA/RVL control -30

% of control -40 of respiratory rithmogenesis remain largely -50 -5 0 15 30 45 60 -5 0 15 30 45 60 unknown. Neurons in this area are exten- Time (s) Time (s) sively connected to both the dorsal and ventral respiratory groups (18) and represent the anesthetized vagotomized rats, bilateral mi- link between the generators of respiratory croinjection of KA (45 mM) into an area rhythm and vasomotor tone (10). Recently equivalent to the IA/RVL here described Smith et al. (19) suggested that neurons in produced a 42% reduction in phrenic nerve the pre-Böotzinger complex, an area par- activity and a 27% increase in frequency, tially overlapping the IA/RVL region, re- simultaneously with a 15-mmHg hypoten- quire glutamatergic inputs to maintain the sion. Similar results were also obtained in membrane potential in the range for the in- other species. In cats, bilateral microinjec- trinsic oscillations adequate for the sponta- tions of KA into the IA/RVL resulted in neous activity and the mutual excitatory in- reduction of VT and a rise in respiratory teractions present in these cells. The origin frequency (14). This respiratory depression of these glutamatergic afferents remains un- was associated with a reduction in phrenic known. nerve activity with a decrease in the respira- In conclusion, although several sites in- tory cycle timing (15). Similarly to the re- volved in the regulation of inspiratory drive sults obtained here, in these studies KA pro- and timing mechanisms can be stimulated by duced minor or no alteration in mean arterial L-glutamate within the VMS, glutamatergic pressure or heart rate. Chemical or electro- inputs have a relevant tonic activity only in lytic inactivation of neurons in this area re- the IA/RVL region.

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