Effects of MK-801 and CNQX, glutamate receptor antagonists, on bladder activity in neonatal rats

BRAIN RESEARCH ELSEVIER

Brain Research 640 (1994) l-t0

Research Report

Effects of MK-801 and CNQX, glutamate receptor antagonists, on bladder activity in neonatal rats Kimio Sugaya, William C. de Groat * Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

(Accepted 2 November 1993)

Abstract

This study was undertaken to examine the role of N-methyl-o-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamatergic receptors in the regulation of urinary bladder activity in the neonatal rat. Experiments were conducted using an in vitro spinal cord-bladder (SB) preparation from 1- to 5-day-old rats or awake neonatal rats 6 and 7 days old, SB preparations were isolated under hypothermic anesthesia. Isovolumetric bladder contractions occurred spontaneously, were induced by electrical stimulation (ES) of the bladder wall or were evoked reflexly by perineal tactile stimulation (PS). MK-801 (3-30 /zM), an NMDA receptor antagonist, enhanced the amplitude of spontaneous, ES- and PS-evoked contractions. Removal of the spinal cord after MK-801 abolished PS-evoked reflex contractions but did not change the amplitude of spontaneous and ES-evoked contractions. Removal of the spinal cord in the absence of MK-801 increased the amplitude of spontaneous and ES-evoked contractions, indicating that the bladder is subject to a tonic inhibitory control originating in the spinal cord. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 3-30 /~M), an AMPA receptor antagonist, decreased the amplitude of PS-evoked contractions and the frequency of spontaneous contractions in the SB preparation. Removal of the spinal cord after CNQX enhanced the amplitude of spontaneous and ES-evoked contractions but abolished PS-evoked contractions. The frequency of spontaneous contractions which decreased after CNQX increased to near control levels after removal of the spinal cord. In awake neonatal rats, intraperitoneal injection of MK-801 (3 mg/kg) induced spontaneous micturition. A large dose of CNQX (30 mg/kg) decreased PS-evoked micturition volume. These results suggest that NMDA glutamatergic receptors are involved in a lumbosacral spinal inhibitory mechanism controlling bladder activity; whereas AMPA glutamatergic receptors are involved in the perineal-to-bladder reflex pathway in neonatal rats. Key words: Glutamate; NMDA; AMPA; Micturition reflex; Urinary bladder

1. Introduction

Glutamic acid which functions as an excitatory neurotransmitter at various sites in the central nervous system [7,9,10,20,33] acts on at least three subtypes of ionotropic receptors [12,21]: N-methyl-o-aspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and 2-amino-4-phosphonobutyrate (AP4). N M D A and A M P A receptors have been implicated in the central control of micturition in the rat [16,19,26,30,31,34-38]. MK-801 (dizocilpine) [16,26,30, 31,34-37], a non-competitive N M D A receptor antago-

* Corresponding author. 13th Floor Biomedical Science Tower, Department of Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA. Fax: (1) (412) 648-1945. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(93)E1512-2

nist, and LY-274614 [38], a competitive N M D A receptor antagonist, have various actions on the micturition reflex in different preparations in the adult rat. For example, intravenous or intrathecal injections of MK801 suppressed reflex bladder contractions in urethane anesthetized rats [16,34-37]; whereas in decerebrate unanesthetized rats, intravenous administration of MK-801 did not affect bladder contractions [34]. However, in awake rats, systemic administration or intrathecal injection of MK-801 increased the amplitude or frequency of bladder contractions [30,31]. The effect of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an A M P A receptor antagonist, on the micturition reflex in the adult rat is uncertain [19,26,35]. Previous studies in this laboratory [26,35] revealed that C N Q X did not affect the micturition reflex in urethane anesthetized or decerebrate rats; whereas Matsumoto et al. [19]

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K Sugaya, I¥.C. de Groat/Brain Research 640 (1994) 1-10

r e p o r t e d that i n t r a v e n o u s or intrathecal injections of C N Q X i n h i b i t e d or abolished isovolumetric b l a d d e r c o n t r a c t i o n s in u r e t h a n c a n e s t h e t i z e d rats. O n the o t h e r hand, reflex m i c t u r i t i o n in chronic spinal rats was not affected by either MK-801 [26,34] or C N Q X [19,26]. This study was u n d e r t a k e n to e x a m i n e the role of glutamic acid in reflex m i c t u r i t i o n in the n e o n a t a l rat. In n e o n a t e s of m a n y species, the n e u r a l control of m i c t u r i t i o n u n d e r g o e s m a r k e d changes d u r i n g early postnatal d e v e l o p m e n t [4-6,28,29]. F o r example, micturition in n e o n a t a l rats a n d cats is m e d i a t e d by a spinal reflex pathway consisting of a somatic afferent limb in the p u d e n d a l nerve a n d a p a r a s y m p a t h e t i c efferent limb in the pelvic nerve [4,29]. This somatovesical reflex is activated w h e n the m o t h e r licks the p e r i n e a l region of the n e o n a t a l a n i m a l [28]. As the central n e r v o u s system m a t u r e s d u r i n g the postnatal period, the spinal m i c t u r i t i o n reflex is gradually replaced by a s p i n o b u l b o s p i n a l reflex pathway which is primarily responsible for m i c t u r i t i o n in adult animals [1,4,5,29]. This reflex pathway has an integrative c e n t e r in the rostral pons a n d has p e r i p h e r a l afferent a n d efferent limbs in the pelvic nerve [1,4,14,29]. T h e reflex is triggered by t e n s i o n receptor afferents which res p o n d to b l a d d e r distension [4,14]. I n adult a n i m a l s t r a n s e c t i o n of the spinal cord rostral to the lumbosacral level blocks the s p i n o b u l b o s p i n a l reflex a n d causes the r e a p p e a r a n c e of n e o n a t a l p e r i n e a l - i n d u c e d m i c t u r i t i o n reflex [29]. This indicates that the micturition reflex pathway can exhibit a c o n s i d e r a b l e degree of plasticity a n d that m a n y of the central m e c h a n i s m s controlling micturition in n e o n a t e s may be similar to those m e d i a t i n g a u t o m a t i c m i c t u r i t i o n in chronic spinal animals. T h e p r e s e n t e x p e r i m e n t s e x a m i n e d the role of N M D A a n d A M P A receptors in the lumbosacral spinal pathway regulating b l a d d e r activity in the awake n e o n a t a l rat a n d in an in vitro p r e p a r a t i o n of the n e o n a t a l rat spinal cord which also i n c l u d e d the lower urinary tract, the p e r i n e u m a n d the p e r i p h e r a l n e u r a l pathways to a n d from the lumbosacral spinal cord [22-25]. T h e effects of MK-801 a n d C N Q X in these p r e p a r a t i o n s indicate that both N M D A a n d A M P A receptors are involved in n e o n a t a l m i c t u r i t i o n reflexes. P r e l i m i n a r y data were p u b l i s h e d in abstracts [23,25].

2. Materials and methods 2.1. The in vitro neonatal rat spinal cord-bladder preparation

The urinary bladder and the spinal cord caudal to L1 were isolated together from 1- to 5-day-old Sprague-Dawley rats under hypothermic anesthesia. The spinal roots caudal to L 4 and the peripheral nerves to the urinary bladder, urethra and perineal skin were intact (Fig. 1). Sympathetic pathways to the lower urinary tract originating from the rostral lumbar spinal cord were interrupted. The

Needle forrecording bladder pressure

cord for ES

bladder

region Fig. 1. Diagram of the spinal cord-urinary bladder preparation showing position of electrodes for direct stimulation of the bladder and the needle for recording bladder pressure.

spinal cord-bladder (SB) preparation was pinned down on a layer of Sylgard resin (Dow Corning) in a recording chamber with ventral surface upwards. Perineal skin around the urethral orifice was stretched and pinned down bilaterally in order to obstruct the urethra and allow recording of isovolumetric bladder contractions. Removal and setup of the preparation was usually completed in less than 10 min. The preparation was continuously superfused with a Krebs solution. A 21-gauge needle was inserted into the bladder through the dome. The needle was connected via polyethylene tubes to a salineinfusion pump and a pressure transducer through a three-way stopcock. A chart recorder was used to display bladder pressure. The bladder was slowlyfilled (0.052 ml/min) with physiologicalsaline to a volume (0.1-0.2 ml) sufficient to elicit spontaneous bladder contractions. The physiological saline contained 2% blue food color (McCormick) to check for leakage of fluid around the needle and elimination of fluid through the urethral orifice. Bipolar silver electrodes (interelectrode distance, 1 mm) were positioned on the serosal surface of the bladder for electrical stimulation. Electrical stimulation (ES) of the bladder wall was elicited by trains of pulses (50 V, 1 ms pulse duration, 50 Hz intratrain frequency, 5 s train duration). Isovolumetric bladder contractions occurring spontaneously or induced by ES were recorded on a rectilinear paper recorder. Perineal stimulation (PS, 30 s) was performed with a fast, light rubbing of the perineal area with blunt forceps. This stimulation evoked reflex bladder contractions even when perineal skin was stretched and pinned down to obstruct the urethra. Changes in bladder activity were recorded following additions to the perfusion fluid of MK-801 (0.3-30/zM, Research Biochemical Inc.) or CNQX (0.3-30 /xM, Research Biochemical Inc.). MK-801 was dissolved in physiological saline; and CNQX was dissolved in physiologicalsaline at pH 11. The drug concentrations were calculated taking into account the total bath volume. Effects of these agents on decentralized lower urinary tract function were also studied using the preparation after removal of the spinal cord. Since CNQX was dissolved in

1<2Sugaya, W.C. de Groat/Brain Research 640 (1994) 1-10

3

basic saline (1 mg/ml, pH 11), the influence of this vehicle on SB preparation was also studied. Addition of basic saline to the perfusion fluid in the same volume (0.7 ml) used to administer 30 ~ M CNQX did not influence spontaneous, ES- and PS-evoked contractions (n = 2). The pH of the perfusion fluid in the recording chamber which had a volume of 100 ml changed slightly from pH 7.4 to 7.5 after addition of basic saline. The recording chamber consisted of a central compartment serving as the in vitro bath, surrounded by an outer, sealed compartment serving as a water jacket for regulating the bath temperature. The capacity of the recording chamber was 100 ml. Heated water was circulated through the jacket chamber from an external, temperature-regulated fluid reservoir. A gravity perfusion system was used to superfuse the preparation with a Krebs solution containing (mM): NaC1 113, KCI 4.7, CaC12 2.5, MgSO 4 1.2, NaHCO 3 25, KH2PO 4 1.2 and glucose 11.5. The solution was buffered to pH 7.4 and equibrated with 95% 0 2 and 5% CO 2 prior to infusion. The solution in the recording chamber was also bubbled with 95% 0 2 and 5% CO 2. The solution was preheated by passage through a heat exchanger before entering the recording chamber and was maintained at a constant temperature + 0.2°C which in different experiments ranged between 26°C and 27°C. The bath was drained by gravity or suction with the drainage flow rates adjusted to match fluid inflow rates.

plete, the bladder was exposed by an abdominal incision under hypothermic anesthesia and urine in the bladder was collected to measure the residual volume.

2.2. Awake neonatal rats

Spontaneous bladder contractions and contractions e v o k e d b y E S o r P S w e r e u s u a l l y e v i d e n t w i t h i n 20 m i n after beginning the recording. The amplitude of these contractions became stable within 40 min. Mean amplit u d e o f E S - (11.7 _+ 1.0 c m H 2 0 , n = 2 4 ) a n d P S - e v o k e d c o n t r a c t i o n s (9.5 _+ 1.2 c m H 2 0 , n = 2 4 ) w e r e n o t significantly different but both were significantly greater ( P < 0.0001 a n d P < 0.01, r e s p e c t i v e l y ) t h a n t h a t o f s p o n t a n e o u s c o n t r a c t i o n s ( 5 . 0 _ 0.6 c m H 2 0 , n - - 2 4 ) ( F i g . 2). M e a n f r e q u e n c y o f s p o n t a n e o u s c o n t r a c t i o n s w a s 1.3 __ 0 . 1 / m i n , a l t h o u g h i n s o m e p r e p a r a t i o n s , spontaneous bladder contractions were very infrequent and of low amplitude (< 1 cm H 20). Addition of MK-801 to the perfusion fluid in various concentrations (3-30 /zM) significantly enhanced the amplitudes of spontaneous, ES- and PS-evoked cont r a c t i o n s ( F i g s . 2 A a n d 3). T h e o n s e t o f t h e e f f e c t

2.3. Statistical analysis When bladder activity stabilized following setup of the preparation in the recording bath, amplitude a n d / o r frequency of spontaneous bladder contractions were measured for 10 min intervals to obtain an estimate of control bladder activity and the effects of drug treatment. Changes in bladder contractions are presented as percent change from control value. Results are reported as means+ S.E.M. Student's t-test for paired or non-paired data was used for statistical data analysis. A level of P < 0.05 was considered statistically significant.

3. Results 3.1. The in vitro neonatal rat spinal cord-bladder preparation

Six- and 7-day-old awake rats (n = 60) isolated from their mother were pretreated with physiological saline (5% body wt, subcutaneously [13]) four hours prior to study to increase urine formation. These rats were injected intraperitoneally with MK-801 (3 mg/kg, n = 1 0 ) or CNQX (3, 10 and 30 mg/kg, n = 1 0 for each dose). MK-801 was dissolved in physiological saline, and CNQX was dissolved in basic physiological saline pH 11. Therefore, two control groups were prepared. One group of control rats (n = 10) was injected intraperitoneally with physiological saline to compare with MK-801 injected group and the other control group (n = 10) was injected intraperitoneally with basic saline (pH 11) to compare with CNQX injected groups. These injected volumes were 30 ml/kg in all groups. Pups were held individually for 15 to 20 min after the injections; and voided urine was collected with a lcc syringe. The pups were held to prevent inadvertent stimulation of the perineum. After this period, continuous perineal stimulation (PS) using the tip of a lcc syringe was performed to induce micturition, and voided urine was collected again. After PS-induced micturition was com-

A

CONTROL

A F T E R MK-801

A F T E R C O R D F.,XTIRPATION

A PS

B

CONTROL

ES

PS

ES

AFTER CNQX

PS

A F T E R C O R D EXTIRPATION

A ES

PS

ES

PS

2~ emH,O

ES

20

~H,O

PS

Fig. 2. Effects of glutamate antagonists, MK-801 (dizocilpine, 30 p,M) and CNOX (6-cyano-7-nitroquinoxaline-2,3-dione, 30 ~M) and removal of the spinal cord on spontaneous, electrical stimulation (ES)-evoked and perineal stimulation (PS)-evoked bladder contractions. ES-evoked contractions were elicited by electrical stimulation of the bladder with trains (5 s duration) of pulses (50 V, 0.2 ms duration at 50 Hz) delivered by bipolar electrodes. PS consisted of tactile stimulation of the perineum for a period of 30 s. A: MK-801 enhanced the amplitude of spontaneous ES- and PS-evokcd contractions. Removal of the spinal cord did not alter the amplitude of spontaneous and ES-cvoked contractions but abolished PS-cvokcd contractions. B: CNOX decreased the amplitude of PS-cvoked contractions but did not alter the amplitude of spontaneous or ES-evoked contractions. Removal of the spinal cord enhanced the amplitude of spontaneous and ES-cvoked contractions but abolished PS-evoked contractions. Vertical calibration represents bladder pressure in cm H20. Horizontal calibration represents 1 min.

IC Sugaya, W.C. de Groat/Brain Research 640 (1994) 1-10

4

appeared within 3 min and was concentration dependent. After the highest concentration of MK-801 (30 /~M), there were marked increases in the mean amplitude of spontaneous (control = 4.7 + 1.0 cm H 2 0 , 111.6 + 30.0% increase, n = 9), ES-(control = 11.8 + 1.3 cm H 2 0 , 31.3 + 6.5% increase, n = 10) and PSevoked contractions (control = 6.6 + 1.3 cm H 2 0 , 124.9 + 25.1% increase, n = 10). Removal of the spinal cord after MK-801 did not alter the amplitude of spontaneous contractions (21.1 + 19.6% increase in comparison to contractions occurring after addition of 30 /.~M MK-801, n = 9) and ES-evoked contractions (11.6 + 9.9% increase, n = 10) but abolished PS-evoked contractions. Removal of the spinal cord in the absence of MK-801 also abolished PS-evoked contractions, but increased the amplitude of spontaneous (46.9 + 13.8% increase, n = 10) and ES-evoked contractions (46.1 + 17.2% increase, n = 8). This effect has been described previously [22,24] and is attributed to the elimination of a tonic inhibitory input from the spinal cord to the bladder. The persistence of the spontaneous contractions after elimination of neural input to the bladder indicates that they are mediated by the intrinsic properties of the bladder smooth muscle. The frequency of spontaneous contractions was not affected by MK-801 (control = 1.2 + 0 . 1 / m i n , 30 ~ M MK-801; 1.6 + 7.1% decrease, n = 9) or removal of the spinal cord (4.1 + 11.3% increase in comparison to contractions occurring after addition of 30/~M MK-801, n = 9). Addition of C N Q X (3-30 p.M) to the perfusion fluid significantly decreased the amplitude of PSevoked contractions, but did not affect the amplitude of spontaneous and ES-evoked contractions (Figs. 2B and 4). However, the frequency of spontaneous contractions significantly decreased after addition of 3-30 /xM C N Q X (control = 1.5 _+ 0 . 3 / m i n , 30 /xM CNQX;

I

f

250

~,

II ~ ~

200

Spontaneous ES-evoke d

~

***

,

PS-evoked

~'~ 150 100

0.3/tM

1/tM

3 #M

I0/~I

30/~M

Removal of

the ~

CNQX

Cord

Fig~ 4. Effects of C N Q X ( 0 . 3 - 3 0 / x M ) and removal of the spinal cord in the presence of C N Q X on the amplitude of spontaneous, ES- and PS-evoked contractions in 1- to 5-day-old rat SB preparations. Each value is mean:l: S.E.M. of at least 7 animals. Significant difference from controls or between 30 ~ M CNQX-treatment and after removal of the spinal cord (bars): *P < 0.05, * *P < 0.01 and * * * P < 0.001. Control = 100%.

26.6 + 5.1% decrease, n = 7) (Fig. 5). The onset of the effect appeared within 3 min. Addition of 3 / x M C N Q X abolished PS-evoked contractions in 2 of 8 preparations; whereas 3 0 / ~ M C N Q X abolished these contractions in 5 of 8 SB preparations. In 1 of 3 preparations in which the PS-evoked contractions were not abolished by 30 ~ M CNQX, the amplitude of PS-evoked contractions was not changed even by 100/xM CNQX. In the presence of 30/~M CNQX, the mean amplitude of the three types of bladder activity was: spontaneous (control = 4.0 5:1.1 cm H 2 0 , 10.3 5: 20.8% increase, n = 7), ES- (control = 10.9 + 2.5 cm H 2 0 , 1.3 + 9.0 % decrease, n = 8) and PS-evoked contractions (control = 12.8 + 2.2 cm HzO, 80.8 + 10.3% decrease, n = 8). Removal of the spinal cord after 30 ~ M C N Q X significantly increased the amplitude of spontaneous contractions (75.6 + 21.7% increase in comparison to contractions occurring after addition of 30 ~ M CNQX, n = 7,

400 350

300

mR Spontaneous ES-evoked

-T-

J

pS-evoked

250

~ 200 -

*

I

**

150-

~

100-

140 o 120 "~ ~r~ 100 80

'°

,<~ 5 0 0.3/~M

I #M

3/~M MX-801

I0/~M

30 #M

Removal of the Spinal Cord

Fig. 3. Effects of MK-801 (0.3-30 /zM) and removal of the spinal cord in the presence of MK-801 on the amplitude of spontaneous, ES- and PS-evoked contractions in 1- to 5-day-old rat SB preparations. Each value is m e a n + S.E.M. of at least 9 animals. Significant difference from controls: * P < 0.05, * * P < 0.01 and * * * P < 0.001. T h e amplitudes of spontaneous and ES-evoked contractions after 30 /zM MK-801 and after removal of the spinal cord were not significantly different. Control = 100%.

0

0.3

pM

1 pM

3 ¢M CNQX

10/~

30 pM

Removal of the Spinal Cord

Fig. 5. Effects of C N Q X ( 0 . 3 - 3 0 / z M ) and removal of the spinal cord in the presence of C N Q X on the frequency of spontaneous contractions in 1- to 5-day-old rat SB preparations. Each value is mean_+ S.E.M. of seven animals. Significant difference from controls: * P < 0.05 and * * P < 0.01. T h e frequencies of spontaneous contractions after 30 /.~M C N Q X and after removal of the spinal cord were not significantly different. Control = 100%.

K~ Sugaya, W.C. de Groat/Brain Research 640 (1994) 1-10

P < 0.05) and ES-evoked contractions (42.0 + 10.5% increase, n = 8, P < 0.01), but abolished PS-evoked contractions. The frequency of spontaneous contractions which decreased after C N Q X increased to near control levels (52.4 + 30.6% increase, n = 7) after removal of the spinal cord (Fig. 5). The combined effect of MK-801 and C N Q X on PS-evoked contraction was also studied. In 3 preparations, 10 or 30 /zM C N Q X decreased (51.3 + 12.2% decrease) the amplitude of PS-evoked contractions and the subsequent administration of MK-801 (10 or 30 /zM) reversed the inhibition (149.3 + 35.1% increase) (Fig. 6A). In different experiments (n = 3) illustrated in Fig. 6B, addition of 10 or 30 ~ M MK-801 enhanced (90.7 + 55.3% increase) the amplitude of PS-evoked contraction and the subsequent administration of C N Q X (10 or 30 /zM) decreased (62.0 + 11.5% decrease) the amplitude of PS-evoked contractions. In preparations in which the spinal cord was already removed, addition of MK-801 (10 /zM, n = 6) did not influence the amplitude of spontaneous (3.3 + 6.4% increase) and ES-evoked contractions (0.5 + 3.9% increase), nor the frequency of spontaneous contractions (8.0 + 5.3% decrease). Addition of C N Q X (10 /zM, n = 6) in this type of preparation also did not influence the amplitude of spontaneous (1.8 + 3.8% increase) and ES-evoked contractions (1.8 + 3.6% increase), and the frequency of spontaneous contractions (2.0 + 4.5% increase). 3.2. A w a k e neonatal rats

Prior to drug administration, all rat pups which were isolated from their mother for 4 hours had distended abdomens because their bladders were filled with urine. When drugs were injected intraperitoneally, about half of the pups in each group transiently voided small amounts of urine ( < 0.04 ml) probably in response to mechanical or nociceptive stimulation associated with

A

CNQX

5

Table 1 Effects of MK-801 (3 mg/kg, i.p.) on micturition and residual volumes in 6- and 7-day-old awake neonatal rats Urine volume Control MK-801 (saline) (3 mg/kg) Spontaneous micturition (ml) 0 0.25 + 0.02 PS-evoked micturition (ml) 0.37 + 0.03 0,12-t-0.03 * Residual urine (ml) 0.01 + 0.01 0.01 + 0.01 Total bladder volume (ml)

0.38 + 0.03

0,38 -1-0.01

Each value is the mean + S.E.M. (ml) of 10 animals. * Significant difference from controls (P < 0.0001).

the needle penetrating the abdominal wall. This initial voided urine was not included in the calculations. For 15-20 min after intraperitoneal injection, micturition did not occur in pups of two control groups receiving vehicle or the three groups receiving CNQX. Whereas in all MK-801 (3 mg/kg)-injected pups, spontaneous intermittent micturition responses began within 10 min and finished within 20 min after drug injection. Mean spontaneous voided volume was 0.25 + 0.02 ml in MK801 group (Table 1). This spontaneous voided volume was 66% (range 3 8 - 9 4 % ) of total bladder capacity. MK-801 injected pups showed walking motions during and after spontaneous micturition. PS-induced micturition was initiated 15-20 min after drug injection in all pups. PS was continued for 5 - 3 0 min until micturition was complete. PS-evoked micturition volume in the MK-801 treated group (0.12 + 0.03 ml) was significantly less than that in the control group (0.37 + 0.03 ml), but there was no difference between total micturition volume (i.e., spontaneous voided volume plus PSevoked micturition volume, 0.37 + 0.01 ml) in two groups. Residual volumes (0.01 + 0.01 ml) were same in both control and MK-801 groups. PS-evoked micturition volume and residual volume were not altered by treating the animals with either 3 m g / k g or 10 m g / k g CNQX. However, 30 m g / k g

MK-801 imin

B

MK-801

CNQX ~

j

~o~,o

Fig. 6. The combined effects of CNQX and MK-801 on PS-evokedcontractions in 2-day-old rat SB preparations. A: addition of CNQX (30/zM) decreased the amplitude of PS-evoked contractions and the subsequent administration of MK-801 (30 tzM) enhanced the amplitude of PS-evoked contraction to the level near the control. B: in another preparation, addition of MK-801 (10 /.LM) enhanced the amplitude of PS-evoked contraction and the subsequent administration of CNQX (10/~M) decreased the amplitude of PS-evoked contraction.

6

K. Sugaya, W.C. de Groat/Brain Research 640 (1994) 1-10

I

I

PMC

(2)i i

!(1)

? ES

/ /

..... <+)~O~-~--4~.2 \ (oExcitatory~Bladder

Lumbosacral spinal cord

/J

(oInhibitory~/~ ~ Pelvic nerve Perineum PS

Fig. 7. Diagram summarizing the putative central and peripheral neural pathways controlling bladder function in the neonatal rat. Excitatory ( + ) and inhibitory ( - ) pathways project from the lumbosacral spinal cord to the bladder. Primary afferent input from the perineum activates the excitatory outflow via a monosynaptic or polysynaptic pathway (dashed line) which depends on glutamatergic transmission and AMPA receptors. The excitatory outflow is also activated by afferent input from bladder tension receptors. The central component of the bladder-to-bladder reflex involves a spinobulbospinal pathway consisting of ascending (1) and descending (2) limbs to and from the pontine micturition center (PMC). A spinal bladder-to-bladder reflex pathway may also facilitate bladder activity under certain conditions. The perineal-to-bladder reflex is modulated by GABAergic and NMDA receptor-dependent mechanisms which may be in series. The tonically active inhibitory outflow to the bladder is also controlled by an NMDA receptor-dependent mechanism.

CNQX decreased (24%) PS-evoked micturition volume (0.26 + 0.02 ml) and increased the residual volume (0.09 + 0.02 ml) (Table 2). Total bladder volume (i.e., spontaneous voided plus PS-evoked micturition and residual volumes) were not altered by any doses of CNQX. PS-evoked micturition volumes and residual volumes were not changed in the control animals treated with vehicle solutions.

4. Discussion

This study revealed that bladder function in the neonatal rat is regulated by neural mechanisms which utilize glutamate as a transmitter. In the in vitro spinal

cord-bladder (SB) preparation intrinsic bladder activity evoked by bladder distension as well as reflex bladder contractions evoked by tactile perineal stimulation or by electrical stimulation of the bladder wall were altered by the administration of glutamate receptor antagonists. These agents also had effects on micturition in the unanesthetized rat pups. The results from both types of experiments indicate that glutamate is involved in inhibitory as well as excitatory pathways that control micturition in the neonatal animal (Fig. 7). In the intact unanesthetized rat pups, glutamate receptor antagonists could alter the micturition reflex by acting at various sites, including the brain, spinal cord and peripheral nervous system. However, the experiments conducted on the in vitro spinal cord-bladder preparation clearly identified the spinal cord as a principal site of action. MK-801 or CNQX did not alter intrinsic or electrically evoked bladder activity after removal of the spinal cord indicating that the drugs did not have direct effects on the bladder smooth muscle or the peripheral nervous system in absence of efferent outflow from the spinal cord. In addition the effects of the glutamate receptor antagonists on bladder contractions in the in vitro preparation were similar to those in intact rat pups suggesting that the actions of these agents on spinal pathways could account for all of the effects on bladder activity in the intact animals.

4.1. The role of NMDA receptors in the control of bladder activity The effects of MK 801 in adult [16,19,26,30,31,34-37] and neonatal animals [22-25] indicate that N M D A glutamatergic receptors are important for both inhibitory and excitatory neural control of bladder activity. Although MK-801, in high concentrations can also affect non-glutamtergic mechanisms, it seems reasonable to conclude that the effects on bladder activity are mediated by block of N M D A receptors since they occurred in low concentrations (threshold, 1 p.M) and since the effects of MK-801 on bladder reflexes in adult animals are mimicked by a competitive N M D A antagonist [38]. In the neonatal rat, administration of MK-801 either in vitro or in situ facilitated reflex

Table 2 Effects of CNQX (3, 10 and 30 mg/kg, i.p.) on micturition and residual volumes in 6- and 7-day-old awake neonatal rats Urine volume Spontaneous micturition (ml) PS-evoked micturition (ml) Residual urine (ml) Total bladder volume (ml)

Control (basic saline)

CNQX (3 mg/kg)

(10 mg/kg)

(30 mg/kg)

0 0.34 + 0.03 0.02 + 0.01 0.36 ± 0.03

0 0.39 _+0.02 0.01 _+0.01 0.40 _+ 0.02

0 0.34 + 0.02 0.03 _+0.01 0.37 ± 0.02

0 0.26 + 0.02 * 0.09 _+0.02 * * 0.35 ± 0.02

Each value is the mean _+S.E.M. (ml) of 10 animals. *'** Significant differences from controls: * P < 0.05 and ** P < 0.01.

K. Sugaya, W.C. de Groat/Brain Research 640 (1994) 1-10

bladder contractions, increased the amplitude of spontaneous contractions and induced voiding. These observations indicate NMDA receptors are necessary for the generation of a tonic inhibitory control of bladder activity. It seems likely that this inhibition consists of at least two components: (1) a peripheral component that suppresses the intrinsic contractile activity of the bladder smooth muscle and which is dependent on efferent outflow from the spinal cord [22,24] and (2) a central component that tonically inhibits the bladder-tobladder and the perineal-to-bladder reflex pathway. In a previous study [22,24] we showed that interruption of the efferent autonomic outflow from the lumbosacral spinal cord by: (1) transection of the spinal roots, (2) administration of a ganglionic blocking agent or tetrodotoxin or (3) by removing the spinal cord increased the amplitude of spontaneous and electrically evoked bladder contractions in the in vitro brainstem-spinal cord-bladder preparation. Since MK-801 induced similar effects, it is tempting to speculate that the proposed inhibitory outflow is dependent upon the activation of NMDA receptors (Fig. 7). Removing the spinal cord after treatment with MK-801 did not produce a further enhancement of bladder activity, indicating that peripheral inhibition was completely blocked by the drug. Thus the spontaneous contractions are myogenic in origin and induced by bladder distension, but are modulated by neural mechanisms. The peripheral inhibition seems to be complemented by a MK-801 sensitive central inhibitory mechanism which regulates the bladder-to-bladder reflex pathway. It is well established that awake neonatal rats at ages day 0 to day 10 do not exhibit a bladder-tobladder reflex or significant spontaneous voiding in the absence of perineal stimulation [13,17]. Thus, when separated from their mothers neonatal rats develop urinary retention and distended bladders. This was also noted in the present experiments. However, following the administration of MK-801, the animals exhibited a series of spontaneous voiding responses that released 66% of the bladder capacity. This suggests that the bladder-to-bladder reflex pathway is potentially functional in neonatal animals but tonically inhibited by a mechanism dependent upon activation of NMDA glutamatergic receptors. A similar situation exists in neonatal kittens ages day 1 to 10 which under normal conditions do not exhibit a bladder-to-bladder reflex or spontaneous voiding, but do exhibit bladder-to-bladder reflexes following chloralose or ketamine anesthesia [28]. In the kittens it has been speculated that anesthesia turns off an inhibitory mechanism that tonically controls the bladder-to-bladder reflex. It is possible that glutamatergic NMDA receptors are involved in this inhibitory mechanism in kittens, since ketamine which unmasks the bladder reflex is known to block NMDA receptors [27].

7

In contrast to neonatal rats with an intact neuraxis, in which the bladder reflex appears to be inhibited, decerebrate unanesthetized rat pups 6-11 days old do exhibit reflex bladder contractions in response to bladder distension [13]. These contractions produce partial but not complete bladder emptying and seem to be mediated by a spinobulbospinal reflex pathway. This finding suggests that bladder reflexes in 6-7 day neonatal rats may be inhibited by centers in the brain rostral to the pontine micturition center and that removal of this inhibition is sufficient to unmask voiding reflexes in response to bladder distension. Thus, MK801 could induce voiding in awake neonatal rat pups by blocking supraspinal as well as spinal inhibitory mechanisms. Previous studies [22,24] showed that reflex bladder contractions in the in vitro brainstem-spinal cordbladder preparation were tonically inhibited by a GABAergic mechanism that was blocked by bicuculline methiodide (BCMI), a GABA A receptor antagonist. BCMI produced changes in bladder activity similar but not identical to those induced by MK-801. For example, BCMI increased the amplitude of PS-evoked bladder contractions and increased the frequency as well as amplitude of spontaneous bladder contractions [22,24]. Thus, it is possible that GABAergic inhibitory interneurons are tonically activated by glutamatergic neurons via NMDA receptors (Fig. 7). The effect of BCMI has also been noted in the SB preparation (unpublished observation), indicating that the GABAergic inhibition is mediated by spinal circuitry and is not dependent on descending pathways from the brain. Intravenous or intrathecal administration of MK-801 also facilitates bladder activity in unanesthetized adult rats [30,31]. Thus, glutamatergic NMDA receptor-dependent inhibitory mechanisms appear to be important in neonatal and mature animals. On the other hand, in urethane anesthetized rats, NMDA antagonists suppress reflex bladder activity [16,34-38] and block the bulbospinal limb of the micturition reflex pathway [26]. The antagonists also inhibit the expression of immediate early genes (c-fos) induced in spinal neurons by activation of bladder afferents [2]. The depressant effect of the antagonists on c-fos expression is markedly reduced by spinal transection, indicating that the depression is dependent on the integrity of bulbospinal pathways [2]. Actions of NMDA antagonists at supraspinal sites are also likely since microinjection of glutamate agonists into the pontine micturition center and at other sites in the brainstem of the cat and rat facilitates bladder activity or can induce voiding [3,15,18,32]. These data suggest that NMDA receptors are likely to be involved in spinal and supraspinal excitatory mechanisms in the micturition reflex pathway. This function of NMDA receptors was not detected in the present experiments

8

K~ Sugaya, W.C. de Groat/Brain Research 640 (1994) 1-10

in neonatal animals, since none of the experiments were conducted in the presence of anesthesia which appears to be necessary to unmask the depressant effects of MK-801.

4.2. The role of AMPA receptors in the control of bladder activity This study revealed that the administration of CNQX to the in vitro SB preparation inhibited or abolished bladder contractions evoked reflexly by perineal stimulation but did not alter the contractions elicited by direct electrical stimulation of the bladder wall. Spontaneous bladder contractions were also influenced by CNQX, although only the frequency of the contractions was affected and this was only a modest decrease (27%). In intact animals, CNQX in a large dose had a weak depressant effect on the perineal-to-bladder reflex, presumably due to its low efficiency in penetrating the blood-brain barrier [8] or its rapid metabolism in vivo. Since the perineal-to-bladder reflex is likely to be mediated by a multisynaptic pathway [4], CNQX could block the reflex by acting on AMPA receptors on the preganglionic neurons or on spinal interneurons. Studies by other investigators have shown that AMPA receptors are involved in primary afferent evoked firing of dorsal horn interneurons [9,33] and in the activation of sympathetic preganglionic neurons in the cat spinal cord slices by focal stimulation of the intermediolateral nucleus [11] or in the rat by stimulation of axons in the lateral funiculus [7]. However, activation of sympathetic preganglionic neurons by dorsal root stimulation in neonatal rat spinal cord slices was not blocked by AMPA receptor antagonists, but was reduced by NMDA receptor antagonists [7]. NMDA receptors are also involved in the bulbospinal excitatory pathways to sympathetic preganglionic neurons in adult rats [10]. Thus, the role of NMDA and AMPA receptors seems to be different in sympathetic and parasympathetic reflex pathways. In the sympathetic system in the rat NMDA receptors are involved in segmental and bulbospinal excitatory pathways, whereas in the lumbosacral parasympathetic pathway to the bladder AMPA receptors mediate an excitatory segmental reflex and NMDA receptors are involved in an inhibitory mechanism. The small reduction in the frequency of spontaneous bladder contractions following CNQX administration is difficult to interpret. This effect did not occur after removal of the spinal cord; therefore it must be dependent on the integrity of neural pathways from the spinal cord to the bladder. Spontaneous contractions are generated by the intrinsic properties of bladder smooth muscle. However, various pharmacological and lesion experiments [22,24] mentioned above indicate

that in the in vitro SB preparation the bladder receives a tonic inhibitory outflow from the spinal cord. In the absence of drugs removal of this inhibitory pathway increases the amplitude, but not the frequency of spontaneous contractions. Thus, an effect of CNQX on the inhibitory pathway would not explain the effect of the drug on the frequency of contractions. On the other hand, we have shown previously that treatment of the in vitro preparations with bicuculline methiodide (BCMI), a GABA A receptor antagonist, does increase the frequency of spontaneous contractions [24]. This effect has been attributed to an increase in parasympathetic excitatory outflow to the bladder due to blockade of GABAergic inhibition in the spinal cord. A tonic excitatory input to the bladder in untreated preparations could occur in parallel with the inhibitory input (Fig. 7) and could arise from a pacemaker circuit within the spinal cord or be driven by afferent input from the distended bladder or from tonic afferent input from the stretched perineum. Thus, it is possible that CNQX blocks a tonic excitatory outflow and thereby reduces the frequency of contractions. It would be interesting in future experiments to determine the role of afferent input in activating a tonic excitatory pathway to the bladder by examining the effect of CNQX after transection of the dorsal roots. Also it would be important to evaluate whether CNQX also blocks the stimulatory effect of BCMI. A major role for bladder afferents in the activation of the tonic excitatory pathway seems unlikely since CNQX did not affect the amplitude of the bladder contractions evoked by direct stimulation of the bladder wall (i.e., ES-evoked contractions) which would be expected to strongly activate bladder afferent pathways and trigger bladder-to-bladder reflexes. Thus, these reflexes must be weak or inactive in the in vitro SB preparation or must be insensitive to CNQX. It has been reported that intravenous administration of CNQX does not influence: (1) distension-induced bladder contractions in anesthetized rats [35], (2) the spinobulbospinal and spinal reflexes elicited by electrical stimulation of afferent fibers in the pelvic nerve in anesthetized spinal intact and spinal transected rats [26], and (3) the increased expression of c-fos in spinal neurons induced by chemical irritation of the bladder in anesthetized spinal intact and transected rats [2]. However, other investigators [19] have reported that intravenous or intrathecal injections of CNQX reduce the frequency of isovolumetric bladder contractions in anesthetized rats, but do not affect isovolumetric bladder contractions in anesthetized chronic spinal rats. The reason for these different results with CNQX in adult rats is not known. Nevertheless, since CNQX affected bladder contractions in neonatal SB preparation but not in adult spinal rats, it seems reasonable to speculate that the functions of AMPA receptors in the

K Sugaya, W.C. de Groat~Brain Research 640 (1994) 1-10

neural control of micturition are different in neonatal and adult animals. In summary, these experiments together with results from previous studies suggest that glutamic acid is an important neurotransmitter in both the excitatory and inhibitory neural pathways regulating bladder activity in neonatal rats. Glutamatergic transmission involving AMPA receptors is important for excitatory reflex mechanisms, whereas NMDA receptors are involved in the generation of a tonic inhibitory control of bladder activity. These findings in neonatal rats demonstrate some similarities and some differences between neonates and adults. For example, in awake adult rats the major role of NMDA-glutamatergic transmission seems to be in the mediation of a tonic inhibitory control of bladder reflexes [30,31], as in the neonatal animal. However, in the anesthetized adult rat glutamatergic synapses involving NMDA [16,34-38] and possibly AMPA receptors [19] are important mediators of excitation in the spinobulbospinal micturition reflex pathway. Thus, some spinal glutamatergic controls of bladder function appear early in development, whereas others appear later in the postnatal period in concert with the maturation of bulbospinal pathways. Acknowledgements. This study was supported by NIH Grant DK37241 and NSF Grant BNS-8908934. The authors would like to thank Dr. M.N. Kruse for help in the initial experiments.

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[33] Yoshimura, M. and Jessell, T., Amino acid-mediated EPSPs at primary afferent synapses with substantia gelatinosa neurones in the rat spinal cord, J. Physiol., 430 (1990) 315-335. [34] Yoshiyama, M., Roppolo, J.R., Erickson, V. and de Groat, W.C., The effects of MK-801 on the micturition reflex in the rat - possible sites of action, Soc. Neurosci. Abstr., 16 (1990) 1064. [35] Yoshiyama, M., Roppolo, J.R. and de Groat, W.C., The effects of glutamate receptor antagonists on the micturition reflex in the rat, Soc. Neurosci. Abstr., 17 (1991) 1001. [36] Yoshiyama, M., Roppolo, J.R. and de Groat, W.C., Effects of MK-801 on the micturition reflex in the rat - possible sites of action, J. Pharmacol. Exp. Ther., 265 (1993) 844-850. [37] Yoshiyama, M., Roppolo, J.R., Rihmland, J., Blastos, B. and de Groat, W.C., The effects of MK-801, an NMDA receptor antagonist, on the micturition reflex in the rat, Neurosci. Lett., 126 (1991) 141-144. [38] Yoshiyama, M., Roppolo, J.R., Thor, K.B. and de Groat, W.C., Effects of LY-274614, a competitive NMDA receptor antagonist, on the micturition reflex in the urethane-anesthetized rat, Br. J. Pharmacol., 110 (1993) 77-86.