Bladder afferent pathway and spinal cord injury: possible mechanisms inducing hyperreflexia of the urinary bladder

Progress in Neurobiology Vol. 57, pp. 583 to 606, 1999 # 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0301-0082/99/$ - see front matter

PII: S0301-0082(98)00070-7

BLADDER AFFERENT PATHWAY AND SPINAL CORD INJURY: POSSIBLE MECHANISMS INDUCING HYPERREFLEXIA OF THE URINARY BLADDER NAOKI YOSHIMURA* Department of Pharmacology, University of Pittsburgh School of Medicine, W1353 Biomedical Science Tower, Pittsburgh, PA 15261, USA (Received 24 June 1998) AbstractÐLower urinary tract dysfunction is a common problem in patients with spinal cord injury (SCI). Since the coordination of the urinary bladder and urethra is controlled by the complex mechanisms in spinal and supraspinal neural pathways, SCI rostral to the lumbosacral level disrupts voluntary and supraspinal control of voiding and induces a considerable reorganization of the micturition re¯ex pathway. Following SCI, the urinary bladder is initially are¯exic, but then becomes hyperre¯exic because of the emergence of a spinal micturition re¯ex pathway. Recent electrophysiologic and histologic studies in rats have revealed that chronic SCI induces various phenotypic changes in bladder a€erent neurons such as: (1) somal hypertrophy along with increased expression of neuro®lament protein; and (2) increased excitability due to the plasticity of Na+ and K+ ion channels. These results have now provided detailed information to support the previous notion that capsaicin-sensitive, unmyelinated C-®ber a€erents innervating the urinary bladder change their properties after SCI and are responsible for inducing bladder hyperre¯exia in both humans and animals. It is also suggested that the changes in bladder re¯ex pathways following SCI are in¯uenced by neural-target organ interactions probably mediated by neurotrophic signals originating in the hypertrophied bladder. Thus, increased knowledge of the plasticity in bladder a€erent pathways may help to explain the pathogenesis of lower urinary tract dysfunctions after SCI and may provide valuable insights into new therapeutic strategies for urinary symptoms in spinal cord-injured patients. # 1999 Elsevier Science Ltd. All rights reserved.

CONTENTS 1. Introduction 2. Anatomy and innervation of the lower urinary tract 2.1. E€erent pathways 2.2. A€erent pathways 3. Re¯ex mechanisms for controlling micturition 3.1. Storage re¯exes 3.2. Voiding re¯exes 3.3. Supraspinal and spinal neurotransmitters controlling micturition 4. Functional properties of bladder a€erent pathways 4.1. In vivo function of bladder a€erent pathways 4.2. Electrophysiological properties of bladder a€erent neurons 4.2.1. Methodological summary for patch clamp recordings of bladder a€erent neurons 4.2.2. Passive membrane properties and action potentials of bladder a€erent neurons 4.2.3. Na+ currents in bladder a€erent neurons 4.2.4. Transient A-type K+ currents (IA) in bladder a€erent neurons 4.3. Histological and chemical properties of bladder a€erent neurons 5. E€ect of spinal cord injury on the micturition re¯ex 5.1. Changes in the micturition re¯ex following spinal cord injury 5.2. Autonomic dysre¯exia 6. E€ect of spinal cord injury on bladder a€erent pathways 6.1. Changes in in vivo functions of bladder a€erent pathways after spinal cord injury 6.2. Changes in electrical properties of bladder a€erent neurons following spinal cord injury 6.2.1. Methodological summary for spinal cord injury 6.2.2. E€ect of spinal cord injury on action potential characteristics 6.2.3. Plasticity in Na+ and K+ channels 6.3. Changes in histological and chemical properties of bladder a€erent neurons 7. Possible mechanisms inducing phenotypic changes in bladder a€erent pathways following spinal cord injury Acknowledgements References * Corresponding author. Tel.: +1 (412) 383-7368; Fax: +1 (412) 648-1945; E-mail: nyos+ @pitt.edu 583

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ABBREVIATIONS ACh AMPA 4-AP ATP BDNF bFGF CGRP CRF DA DRG EMG ENK EUS GABA Glu 5-HT

Acethylcholine a-Amino-3-hydroxy-5-methylisoxazole-4propionate 4-Aminopyridine Adenosine triphosphate Brain-derived neurotrophic factor Basic ®broblast growth factor Calcitonin gene-related peptide Corticotropin-releasing factor Dopamine Dorsal root ganglia Electromyography Enkephalin External urethral sphincter g-Aminobutylic acid Glutamic acid 5-Hydroxytryptamine

1. INTRODUCTION The lower urinary tract has two main functions: storage and periodic elimination of urine. These functions are regulated by a complex neural control system located in the brain and spinal cord. This control system functions like a switching circuit to maintain a reciprocal relation between the reservoir (bladder) and outlet (urethra and urethral sphincter) components of lower urinary tract (de Groat et al., 1993; Chai and Steers, 1996; Yoshimura and de Groat, 1997b). Spinal cord injury (SCI) rostral to the lumbosacral level impairs this voluntary and supraspinal control of voiding, leading to a marked reorganization of the micturition re¯ex pathways that coordinate bladder and sphincter function. SCI initially induces an are¯exic bladder and urinary retention followed by the emergence of automatic micturition mediated by spinal re¯ex pathways (Fam et al., 1988; de Groat, 1995; Chancellor and Blaivas, 1996; de Groat et al., 1997). The urinary bladder becomes hypere¯exic along with tonic activity of urethral sphincter (bladder-sphincter dyssynergia). These lower urinary tract dysfunctions then produce various urological problems such as urinary incontinence, recurrent urinary tact infection and vesicoureteral re¯ux with or without upper urinary tract deterioration in spinal cord-injured patients (Fam et al., 1988; Chancellor and Blaivas, 1996). Both clinical and experimental studies demonstrated that bladder hyperre¯exia after SCI is induced at least in part by phenotypic changes in unmyelinated C-®ber bladder a€erent pathways (de Groat et al., 1990; Geirsson et al., 1994; Cruz et al., 1997a). Electrophysiological studies also indicated that following SCI in the rat, capsaicin-sensitive bladder a€erent neurons exhibited somal hypertrophy and increased excitability due to the plasticity in Na+ and K+ channels, which might be responsible for the emergence of the C-®ber-mediated spinal micturition re¯ex following SCI (Yoshimura and de Groat, 1997a). In addition, it has been suggested that changes in bladder a€erent pathways are due to target organ±neural interactions mediated by neurotrophic factors released in the hypertro-

IA INa K MPTP NA NGF NMDA NO NOS NTF PAG SCI SP TTX VIP Vh

A-type K+ currents Na+ currents Slope factor 1-Methyl-4-phenyl-2, 3, 6-tetrahydropyridine Norepinephrine Nerve growth factor N-methyl-D-aspartate Nitric oxide Nitric oxide synthase Neurotrophic factor Periaqueductal gray Spinal cord injury Substance P Tetrodotoxin Vasoactive intestinal polypeptide Half-maximal potential

phied bladder muscle (de Groat, 1995; de Groat et al., 1997; Yoshimura and de Groat, 1997a; Yoshimura et al., 1998a). This review will describe: 1. the central and peripheral nervous control of lower urinary tract; 2. alterations in lower urinary tract function after SCI; and 3. plasticity in bladder a€erent pathways that might underlie the emergence of bladder hyperre¯exia after SCI, especially focusing on changes in functional properties of a€erent neurons innervating the urinary bladder. 2. ANATOMY AND INNERVATION OF THE LOWER URINARY TRACT The storage and periodical elimination of urine are dependent on the reciprocal activity of two functional units in the lower urinary tract: 1. a reservoir (the bladder); and 2. an outlet (bladder neck, and smooth and striated sphincter muscles of the urethra). During urine storage, the bladder outlet is closed and the bladder smooth muscle is quiescent allowing intravesical pressure to remain low over a wide range of bladder volumes [Fig. 1(A)]. During voluntary voiding, the initial event is a relaxation of the pelvic ¯oor and striated urethral sphincter muscles, followed by a detrusor muscle contraction and opening of the bladder neck. Re¯ex inhibition of the smooth and striated urethral sphincter muscles also occurs during micturition [Fig. 1(A)]. This activity is mediated by three sets of peripheral nerves: parasympathetic (pelvic), sympathetic (hypogastric) and somatic (pudendal) nerves (Fig. 2) (de Groat et al., 1993; Chai and Steers, 1996; Yoshimura and de Groat, 1997b). These nerves also contain a€erent axons innervating the lower urinary tract and the most important a€erents for initiating micturition are those carried in the pelvic nerve (Fig. 2) (JaÈnig and Morrison, 1986; HaÈbler et al., 1990; de Groat et al., 1993).

Bladder A€erent Pathway and SCI

Fig. 1. Combined cystometry and external urethral sphincter electromyography (EUS±EMG) recordings comparing re¯ex voiding responses in a normal adult (A, Intact) and in a SCI patient (B, SCI). The abscissa in all records represent bladder volume and the ordinate in cystometrograms represent bladder pressure. In panel A, a slow infusion of ¯uid into the bladder induces a gradual increase of EMG activity, but no apparent changes in bladder pressure. When a voluntary voiding starts, an increase of bladder pressure (voluntary bladder contraction) is associated with a cessation of EUS±EMG activity (synergic sphincter relaxation). On the other hand, in a SCI patient (B), the reciprocal relationship between bladder and sphincter is abolished. During bladder ®lling, uninhibited bladder contraction occurs in association with a increase in sphincter activity (dyssynergic sphincter contraction). Loss of the reciprocal relationship between bladder and sphincter in SCI patients interferes with bladder emptying (For further details and references, see text).

2.1. E€erent Pathways The parasympathetic e€erent pathway represents the major excitatory input to the bladder. Parasympathetic preganglionic axons originate in the intermediolateral column of the S2±S4 spinal cord and terminate on postganglionic neurons in the bladder wall and in the pelvic plexus which is a neural network located on lateral surface of the rectum in humans (Fig. 2). The parasympathetic preganglionic axons release acetylcholine (ACh) which activates postsynaptic nicotinic receptors (JaÈnig and McLachlan, 1987; Lundberg, 1996). Nicotinic transmission at ganglionic synapses can be regulated by various modulatory synaptic mechanisms which involve muscarinic, adrenergic and enkephalinergic

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receptors (Table 1) (de Groat and Booth, 1993). Parasympathetic postganglionic neurons in turn provide an excitatory input to the bladder smooth muscle. Parasympathetic postganglionic nerve terminals release ACh, which can excite di€erent types of muscarinic receptors (M2 and M3) which are present in the detrusor muscle (Table 1) (Kondo et al., 1995; Wang et al., 1995; Eglen et al., 1996; Yamaguchi et al., 1996). Muscarinic receptors are also involved in a presynaptic inhibition (M2) and facilitation (M1) of ACh release from postganglionic nerve terminals in the bladder (Table 1) (Somogyi and de Groat, 1992; Somogyi et al., 1994). Adenosine triphosphate (ATP) which is a cotransmitter also released from parasympathetic postganglionic terminals induces a rapid onset, transient contraction of the bladder (Table 1) (Hoyle and Burnstock, 1993; Lundberg, 1996). Due to the presence of ATP-mediated neural transmission, antimuscarinic agents do not completely abolish neurally-evoked bladder contractions in animals or humans, although the contribution of the purinergic pathway in human seems to be small (Luheshi and Zar, 1990; Andersson, 1993; Igawa et al., 1993b). The parasympathetic input to the urethra elicits inhibitory e€ects mediated at least in part via the release of nitric oxide (NO) which directly relaxes the urethral smooth muscle (Thornbury et al., 1992; Andersson, 1993; Bennett et al., 1995; Takeda and Lepor, 1995; Lundberg, 1996). In contrast to other transmitters which are stored and released from synaptic vesicles by exocytosis, NO is not stored, but is synthesized immediately prior to release by the enzyme nitric oxide synthase (NOS). NOS containing nerve terminals are found more densely in the bladder base and urethra than in the detrusor (Persson et al., 1993). Thus, it seems reasonable to assume that the excitation of sacral parasympathetic e€erent pathways induces a bladder contraction via ACh/ATP release and urethral relaxation via NO release (Table 1). Sympathetic preganglionic neurons located within the intermediolateral cell column of the Th11±L2 spinal cord make synaptic connections with postganglionic neurons in the inferior mesenteric ganglion as well as with postganglionic neurons in the paravertebral ganglia and pelvic ganglia (Fig. 2) (JaÈnig and McLachlan, 1987; de Groat et al., 1993; de Groat and Booth, 1993). Ganglionic transmission in sympathetic pathways is also mediated by ACh acting on nicotinic receptors. Sympathetic postganglionic terminals which release norepinephrine elicit contractions of the bladder base and urethral smooth muscle and relaxation of the bladder body mediated though adrenoceptors (Table 1) (Andersson, 1993; de Groat and Booth, 1993). In addition, postganglionic sympathetic input to bladder parasympathetic ganglia can facilitate and inhibit parasympathetic ganglionic transmission (de Groat and Booth, 1993). Somatic e€erent pathways which originate from the motoneurons in Onuf's nucleus of the anterior horn of the S2±S4 spinal cord innervate the external striated urethral sphincter muscle and the pelvic ¯oor musculature (Fig. 2). Somatic nerve terminals release ACh which acts on nicotinic receptors to

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Fig. 2. Diagram showing the sympathetic, parasympathetic and somatic innervation of the lower urinary tract. Sympathetic preganglionic pathways emerge from the thoracolumbar cord (Th11±L2) and pass to the inferior mesenteric ganglia. Preganglionic and postganglionic sympathetic axons then travel in the hypogastric nerve to the pelvic ganglia and lower urinary tract. Parasympathetic preganglionic axons which originate in the sacral cord (S2±S4) pass in the pelvic nerve to ganglion cells in the pelvic ganglia and postganglionic axons innervate the bladder and urethral smooth muscle. Sacral somatic pathways are contained in the pudendal nerve, which provides an innervation to the external urethral sphincter striated muscles. A€erent axons from the lower urinary tract are carried in these three nerve [Reproduced from Yoshimura and de Groat (1997b) with permission].

induce a muscle contraction (Table 1). Combined activation of sympathetic and somatic pathways elevates bladder outlet resistance and contribute urinary continence.

Several other nonadrenergic±noncholinergic transmitters such as Leucine enkephalin (ENK), vasoactive intestinal polypeptide (VIP) and neuropeptide Y have been identi®ed as modulators of

Table 1. Receptors in peripheral nervous pathways regulating lower urinary tract function E€erent Parasympathetic

Sympathetic

Ganglia

N(+), M(+) ENKd(ÿ)* VIP (+)

N(+), M(+) a1(+)$, a2 (ÿ)$ b (+)$

Bladder

M2 (+), M3 (+) P2X (+) M1 (+)*, M2 (ÿ)* NPY (ÿ)*

b1 (ÿ), b2 (ÿ) NPY (ÿ)*

M2 (+), M3 (+) NO (ÿ)

a1 (+), a2 (+)

Bladder neck and urethra Striated urethral sphincter

A€erent Somatic

NK1 (+), NK2 (+) CGRP (+), VIP (ÿ) H1 (+), B2 (+) Vanilloid (capsaicin) (+)

N(+)

Facilitatory and inhibitory responses are indicated by plus and minus in parentheses, respectively.*Presynaptic receptors.$Heterosynaptic inputs onto parasympathetic ganglion cells.Abbreviations: ENK, enkephalin; VIP, vasoactive intestinal peptide; NO, nitric oxide; NPY, neuropeptide Y; N, nicotinic receptor; M1, M2 and M3, muscarinic receptors; a1, a2, b1 and b2, adrenergic receptors; P2X, purinergic receptor; CGRP, calcitonin gene-relating peptide receptors; NK1 and NK2, tachykinin receptors; d, opioid receptors; H1, histamine receptor; B2, bradykinin receptor.

Bladder A€erent Pathway and SCI

e€erent inputs to the lower urinary tract (Table 1) (de Groat and Booth, 1993; Tran et al., 1994). 2.2. A€erent Pathways Sensory information including the feeling of bladder fullness or bladder pain is conveyed to the spinal cord via a€erent axons in the pelvic and hypogastric nerves (JaÈnig and Morrison, 1986; HaÈbler et al., 1990). Neuronal somata of these a€erent nerves are located in the dorsal root ganglia (DRG) at S2±S4 and T11±L2 spinal segmental levels (Fig. 2). The a€erent ®bers carry impulses from tension receptors and nociceptors in the bladder wall to neurons in the dorsal horn of the spinal cord. A€erent ®bers passing in the pelvic nerve to the sacral cord are responsible for initiating the micturition re¯ex. These bladder a€erents have myelinated (Ad-®ber) or unmyelinated (C-®bers) axons (de Groat et al., 1981; Mallory et al., 1989; Vera and Nadelhaft, 1990). Immunohistochemical studies indicate that bladder a€erent neurons contain various neuropeptides such as substance P (SP), calcitonin gene-related peptide (CGRP), VIP and ENK (Table 1) (Maggi, 1991; Keast and de Groat, 1992). The distribution of these peptidergic a€erent terminals in the spinal cord is similar to that of central projections of bladder a€erent neurons (de Groat, 1986; Steers et al., 1991a). SP and CGRP are present in a large percentage of C-®ber a€erent neurons (Maggi, 1993; Lawson et al., 1993). The release of these peptides in the bladder wall is known to trigger in¯ammatory responses, including plasma extravasation or vasodilation (Lundberg, 1996).

3. REFLEX MECHANISMS FOR CONTROLLING MICTURITION 3.1. Storage Re¯exes The bladder functions as a low pressure reservoir during urine storage. In both humans and animals, bladder pressures remain low and relatively constant when bladder volume is below the threshold for inducing voiding [Fig. 1(A)]. This is mainly due to the combined e€ect of: 1. a passive phenomenon depending on viscoelastic properties of the bladder wall; and 2. quiescence of the parasympathetic pathway to the bladder (de Groat et al., 1993; Yoshimura and de Groat, 1997b). In addition, during bladder ®lling, a€erent activity derived from the bladder activates a sacral to thoracolumbar intersegmental spinal re¯ex pathway which triggers ®ring in sympathetic pathways to the bladder (de Groat and Lalley, 1972). Activation of sympathetic e€erents then mediates an inhibition of bladder activity and contraction of the bladder neck and proximal urethra (de Groat and Theobald, 1976). Pudendal motoneurons are also activated by vesical a€erent input as the bladder ®lls, thereby inducing a contraction of the striated sphincter muscle which contributes to urinary continence (Shimoda et al., 1992; Fedirchuk and Shefchyk, 1993). Thus urine sto-

587

rage is mainly controlled by re¯exes integrated in spinal cord [Fig. 3(A)]. However, it is also reported that a supraspinal urine storage center is located in the dorsolateral pons. Descending inputs from this region activate the pudendal motoneurons to increase urethral resistance [Fig. 3(A)] (Holstege et al., 1986; Kohama, 1992). 3.2. Voiding Re¯exes When bladder volume reaches the micturition threshold, a€erent activity originating in bladder mechanoceptors triggers micturition re¯exes which consist of ®ring in the sacral parasympathetic pathways and inhibition of sympathetic and somatic pathways [Fig. 3(B)]. This leads to a contraction of the bladder and a concomitant relaxation of the urethra [Fig. 1(A)]. The a€erent ®bers which trigger micturition in the rat and cat are myelinated Ad®bers (de Groat et al., 1981; Mallory et al., 1989). These bladder a€erents in the pelvic nerve synapse on neurons in the sacral spinal cord, which then send their axons rostrally to a micturition center in the dorsolateral pons. This center contains neurons which are essential for inducing voiding re¯exes (Nishizawa et al., 1988; Kruse et al., 1990; Noto et al., 1991b; Mallory et al., 1991). Bilateral lesions in the region of the locus coeruleus in the cat or the Barrington's nucleus in the rat abolish micturition, while electrical or chemical stimulation of this region induces a bladder contraction and a reciprocal relaxation of the urethra, leading to bladder emptying (Sugaya et al., 1987; Noto et al., 1989; Yoshimura et al., 1990a; de Groat et al., 1993). It has been demonstrated that activity ascending from the spinal cord may pass through a relay center in the periaqueductal gray before reaching the pontine micturition center (Blok and Holstege, 1994; Blok et al., 1995). Thus voiding re¯exes depend on a spinobulbospinal pathway which passes through an integrative center in the brain [Fig. 3(B)]. This center functions as an `on±o€' switch activated by a€erent activity derived from bladder mechanoceptors and also receives inhibitory and excitatory inputs from the brain regions rostral to the pons. Re¯ex voiding is also facilitated by a€erent inputs from the urethra. This urethrovesical re¯ex triggered by urine ¯ow into the urethra enhances bladder contractions (Chai and Steers, 1996). During voiding re¯exes, activity in the pudendal e€erent pathway to the striated urethral sphincter is suppressed to reduce outlet resistance (Kruse et al., 1990; de Groat et al., 1993). This mechanism is mainly due to an inhibition of the pudendal motoneurons by the descending inputs from the dorsolateral pons (Mallory et al., 1989; Shimoda et al., 1992; Fedirchuk and Shefchyk, 1993). As mentioned in Section 2.1, an excitation of the sacral parasympathetic pathway also directly induces a relaxation of urethral smooth muscle mediated by the release of NO. 3.3. Supraspinal and Spinal Neurotransmitters Controlling Micturition Various neurotransmitters at the spinal and supraspinal level are involved in regulation of mic-

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Fig. 3. Diagrams showing neural circuits controlling continence and micturition. (A) Storage re¯exes. During urine storage, bladder distention produces low level ®ring in bladder a€erent pathways, which in turn stimulates: (1) the sympathetic out¯ow to the bladder outlet (bladder base and urethra); and (2) pudendal out¯ow to the external sphincter muscle. These responses are elicited by spinal re¯ex pathways. Sympathetic ®ring also inhibits detrusor muscle and transmission in bladder ganglia. A region in the rostral pons (pontine storage center) increases external urethral sphincter activity. (B) Voiding re¯exes. During elimination of urine, intense bladder a€erent ®ring activates spinobulbospinal re¯ex pathways passing through the pontine micturition center, which stimulate the parasympathetic out¯ow to the bladder and internal sphincter smooth muscle and inhibit the sympathetic and pudendal out¯ow to the bladder outlet. Ascending a€erent input from the spinal cord may pass through relay neurons in the periaqueductal gray (PAG) before reaching the pontine micturition center [Reproduced from Yoshimura and de Groat (1997b) with permission].

turition and continence (Fig. 4). Glutamic acid which is the major excitatory transmitter in the central nervous system has an important role in the control of the micturition re¯ex. Experiments in rats indicate that glutamatergic transmission in the spinal cord is essential for bladder and urethral re¯exes and for the spinal processing of a€erent input from the bladder. Both NMDA and AMPA/ kainate receptors are involved in glutamatergic transmission in the micturition re¯ex pathway (Yoshiyama et al., 1993, 1995, 1997; Sugaya and de Groat, 1994; Matsumoto et al., 1995a,b). Recent studies using spinal slice preparations from neonatal rats also revealed that the sacral preganglionic neurons directly receive glutamatergic excitatory inputs

through NMDA and AMPA/kainate receptors from spinal interneurons in the region of the sacral parasympathetic nucleus (Araki and de Groat, 1996). Thus, it is likely that glutamatergic transmission controlling the micturition re¯ex functions at various sites. A spinobulbospinal micturition re¯ex pathway controlling bladder and urethral re¯exes is also modulated by various neurotransmitters in the spinal cord such as norepinephrine (Yoshimura et al., 1990a,b; Ishizuka et al., 1996a,b) and 5-hydroxytryptamine (serotonin) (Thor et al., 1990; Steers et al., 1992b; Espey and Dawnie, 1995; Danuser and Thor, 1996) through di€erent receptor subtypes.

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589

Fig. 4. Diagram of neurotransmitters in spinal and supraspinal sites. Glutamate is the major excitatory transmitter in the control of micturition re¯ex. Modulation of the micturition re¯ex in the spinal cord occurs by segmental interneuronal mechanisms (ENK, GABA) or by descending input from the brain (5HT, NA, CRF). Modulation in the pontine micturition center can be activated in part by input from cortical±diencephalic areas. Facilitatory and inhibitory responses are indicated by plus and minus in parentheses, respectively. Abbreviations: acetylcholine (ACh); dopamine (DA); enkephalin (ENK); glutamate (Glu); 5-hydroxytryptamine (5HT); norepinephrine (NA); corticotropin-releasing factor (CRF); dopmine receptors (D1 and D2); opioid receptors (m and d); GABA receptors (A and B) [Reproduced from Yoshimura and de Groat (1997b) with permission].

The brain rostral to the pons (diencephalon and cerebral cortex) is also involved in excitatory and inhibitory regulation of the micturition re¯ex (Fig. 4). Experiments in monkeys with parkinsonism induced by a neurotoxin, MPTP, revealed that dopaminergic neurons originating in the substantia nigra tonically inhibit the micturition re¯ex through dopamine D1 receptors. Evidence for this was obtained in studies of MPTP-treated monkeys that exhibited hyperre¯exic bladders as reported in patients with Parkinson's disease; this hyperactivity was suppressed by activation of dopamine D1 receptors (Yoshimura et al., 1993, 1998b). GABA, opioids and ACh are also implicated as putative transmitters in spinal and supraspinal mechanisms modulating the micturition volume thresholds (Sugaya et al., 1987; Noto et al., 1991a,b; Steers et al., 1992a; Igawa et al., 1993a; de Groat et al., 1993; Araki, 1995). 4. FUNCTIONAL PROPERTIES OF BLADDER AFFERENT PATHWAYS 4.1. In Vivo Function of Bladder A€erent Pathways Electrophysiological studies in cats and rats have revealed that the normal micturition re¯ex is mediated by small myelinated Ad-®ber a€erents which respond to bladder distention (de Groat et al., 1981; Mallory et al., 1989; HaÈbler et al., 1990). In cats, C-®ber a€erents usually have highthresholds and are unresponsive to mechanical stimuli such as bladder distention and therefore have been termed `silent C-®bers'. However, many of these ®bers do respond to chemical, noxious or cold stimuli (HaÈbler et al., 1990; Fall et al., 1990). In

rats, Sengupta and Gebhart (1994) ®rst reported that mechanosensitive bladder a€erents which responded to bladder distension were detected in both Ad- and C-®ber groups. They also found that 30% of bladder a€erents were not responsive to any mechanical stimuli, and these unresponsive bladder a€erents included both Ad- and C-®bers. However, Dmitrieva and McMahon (1996) have recently demonstrated using rats that most myelinated Ad®ber bladder a€erents were mechanosensitive, while about one-half of unmyelinated C-®ber bladder a€erents had no clear mechanosensitivity (i.e. silent C-®bers), but responded chemical stimuli. They have also reported that nerve discharges induced by bladder distention were much lower in mechanosensitive C-®ber bladder a€erent ®bers than myelinated Ad®bers, suggesting that C-®ber bladder a€erents are less excitable than Ad-®ber a€erents in rats, as shown in cats. Moreover, since capsaicin, a neurotoxin of C-®bers, does not block normal micturition re¯exes in both cats and rats, C-®ber a€erents are not essential for normal voiding (de Groat et al., 1990; Cheng et al., 1993; Maggi, 1993). 4.2. Electrophysiological Properties of Bladder A€erent Neurons 4.2.1. Methodological Summary for Patch Clamp Recordings of Bladder A€erent Neurons The populations of DRG neurons which innervate the urinary bladder were labeled by retrograde axonal transport of a ¯uorescent dye, Fast Blue, injected into the wall of the bladder in rats. (Yoshimura et al., 1994, 1996; Yoshimura and de Groat, 1997a). Bladder a€erent neurons were dissociated from L6 and S1 DRG (Yoshimura et al.,

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N. Yoshimura

1994, 1996). Dye-labeled primary a€erent neurons that innervate the urinary bladder were readily identi®ed using a ¯uorescent microscope. Gigaohmseal whole-cell recordings were performed at room temperature on each freshly dissociated dye-labeled neuron. With these techniques, dye loading into the neurons by retrograde axonal transport did not change electrophysiological properties of the neurons (Yoshimura et al., 1994). 4.2.2. Passive Membrane Properties and Action Potentials of Bladder A€erent Neurons Based on current clamp recordings, bladder a€erent neurons were divided into two populations according to the electrical characteristics of their action potentials (Yoshimura et al., 1996). The most common population of bladder a€erent neurons (>70%) exhibited high-threshold, long-duration action potentials with an in¯ection on the repolarization phase (Fig. 5 and Table 2). These neurons were small in size, and had action potentials that were resistant to an application of tetrodotoxin (TTX), a Na+ channel blocker. The other population of bladder a€erent neurons which were larger in size than the neurons with TTX-resistant spikes

exhibited low-threshold, short-duration action potentials which were reversibly blocked by TTX (Fig. 5 and Table 2). Since pretreatment with systemic application of capsaicin, a C-®ber neurotoxin, reduced the number of bladder a€erent neurons with TTX-resistant spikes, TTX-resistant neurons are likely to be the origin of C-®ber a€erents (Yoshimura et al., 1996). It is also reported that small-diameter DRG cells with TTX-resistant Na+ currents are usually sensitive to capsaicin (Arbuckle and Docherty, 1995). The correlation of spike characteristics with other electrical and morphological properties of the neuron such as somal size, and action potential thresholds and duration was also reported by other investigators in unspeci®ed DRG neurons (Waddell and Lawson, 1990; Gold et al., 1996b; Cardenas et al., 1997). Thus, it is reasonable to assume that bladder a€erent neurons with TTXresistant humped spikes mainly represent C-®ber a€erent neurons. These neurons are less excitable due to higher thresholds for spike activation than the neurons with TTX-sensitive neurons (i.e. Ad®ber neurons). Another distinctive characteristic of bladder a€erent neurons with TTX-resistant action potentials

Fig. 5. Action potentials in bladder a€erent neurons from spinal intact rats. In current-clamp conditions, action potentials were evoked by depolarizing current pulses injected through the patch pipette. (A and B) Small-sized (24 mm) neuron. (C and D) Large-sized (35 mm) neuron. The small neuron exhibited a long-duration, high-threshold (ÿ18 mV) action potential with an in¯ection on repolarization phase (A), whereas the large neuron had a short-duration low-threshold (ÿ35 mV) spike with no in¯ection (C). The action potential of the small neuron, in which the spike in¯ection was supresssed by the removal of extracellular Ca2+ ions, was una€ected by the application of TTX (6 mM) (B), while the action potential in the large neuron was inhibited by TTX (2 mM) (D). Note voltage responses in the small neuron exhibited membrane potential relaxation during depolarizing current injections. The pulse protocols are shown in the insets [Reproduced from Yoshimura et al. (1996) with permission].

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591

Table 2. Membrane characteristics of bladder a€erent neurons with tetrodotoxin (TTX)-resistant and TTX-sensitive action potentials in spinal intact rats

No. of cells Diameter (mm) (range) Resting membrane potential (mV) Spike threshold (mV) Spike duration (msec)

TTX-resistant

TTX-sensitive

n = 38 24.22 1.8 (18±30) ÿ54.822.1 ÿ21.220.9 9.420.5

n = 11 32.4 21.2** (29±35) ÿ57.922.4 ÿ34.423.5** 5.620.8*

Values are mean 2SEM.*P < 0.05 and **P < 0.01 indicate di€erences between values obtained in neurons with TTX-resistant and TTX-sensitive action potentials.

was noted in regard to the e€ect on 4-aminopyridine (4-AP), a A-type K+ (IA) channel blocker, on the spike threshold (Yoshimura et al., 1996). When depolarizing currents were injected into the cell, voltage responses in bladder a€erent neurons with TTX-resistant spike usually exhibited relaxation phenomena at membrane potentials over ÿ45 to ÿ40 mV prior to spike activation (Fig. 6). Since an application of 4-AP (1 mM) suppressed this membrane potential relaxation and lowered the threshold for spike activation by ca 6 mV (Fig. 6), IA currents activated from resting membrane potentials may

contribute to high thresholds for spike activation in these TTX-resistant neurons (see details in Section 4.2.4). 4.2.3. Na+ Currents in Bladder A€erent Neurons Voltage clamp recordings of Na+ currents in bladder a€erent neurons revealed a similar correlation between cell size and sensitivity to TTX (Yoshimura et al., 1996). Both TTX-resistant and TTX-sensitive Na+ currents could be observed in single neurons, but usually one of the two currents predominated. TTX-resistant currents were promi-

Fig. 6. E€ect of 4-AP, an IA channel blocker, on voltage responses and action potentials in bladder a€erent neurons from spinal intact rats. In current-clamp conditions, voltage responses were evoked by 20 msec depolarizing current pulses injected through the patch electrode. The amplitude of the pulses was increased from ÿ20 pA in 10 pA steps. (A) Neuron with TTX-resistant spikes. (B) Neuron with TTX-sensitive spikes. Left traces, control. Right traces, 4-AP (1 mM) application. Action potentials in a neuron with TTX-resistant spike (A) were evoked at thresholds (dashed line) of ÿ22 and ÿ27 mV in the absence and presence of 4-AP, respectively, whereas no remarkable changes in spike con®guration were observed in (B). (C and D) Changes of membrane potentials at the end of command pulses (arrows of A and B) plotted against hyperpolarizing and depolarizing currents injected through patch pipette, before (q) and after 4-AP application (Q) in the same neurons as in (A) and (B), respectively. Note that the relaxation of membrane potentials in the depolarizing range over ÿ45 mV was suppressed by 4-AP application (C) [Reproduced from Yoshimura et al. (1996) with permission].

592

N. Yoshimura

nent (>85% of total Na+ currents) in small-sized bladder neurons whereas larger bladder a€erent neurons had TTX-sensitive currents comprising 60± 100% of the total Na+ currents (Fig. 7). These two di€erent Na+ currents exhibited di€erent voltagedependence. The threshold for activation of TTX-resistant Na+ currents was shifted by ca ÿ15 mV in the depolarizing direction when compared with TTX-sensitive Na+ currents (Fig. 7). Steady-state activation and inactivation of TTX-resistant Na+ currents were also displaced to more depolarized levels by 10 and 30 mV, respectively, in comparison with the TTX-sensitive Na+ currents (Fig. 8). Similar values in the voltage-dependence of Na+ currents were obtained by other investigators in unidenti®ed DRG neurons (Roy and Narahashi, 1992; Ogata and Tatebayashi, 1993; Elliott and Elliott, 1993). Thus these di€erent properties in voltage dependence of Na+ currents likely contribute to the higher spike thresholds in C-®ber bladder a€erent neurons with TTX-resistant action potentials than in those with TTX-sensitive action potentials. In addition, the TTX-resistant Na+ channel (named PN3 or SNS) has recently been cloned and con®rmed to be expressed in small-sized DRG neurons using in

situ hybridization or immunohistochemical methods (Akopian et al., 1996; Sangameswaran et al., 1996; Novakovic et al., 1998). 4.2.4. Transient A-Type K+ Currents (IA) in Bladder A€erent Neurons It has been documented that at least two di€erent types of transient IA currents are expressed in sensory neurons such as nodose ganglia and DRG cells (McFarlane and Cooper, 1991; Akins and McCleskey, 1993; Gold et al., 1996a). One of these IA currents exhibited slowly inactivating decay kinetics that is quite di€erent from the other typical fast inactivating IA currents. This slowly inactivating IA has a inactivation time constant between 150 and 300 msec and the voltage of half-maximal inactivation is reportedly displaced to a more positive membrane potential when compared with the fast inactivating IA. In addition, Gold et al. (1996a) identi®ed a third transient IA current which exhibited activation and inactivation kinetics similar to the fast inactivating IA, but had higher thresholds for activation. They have also reported that the slowly inactivating IA was selectively expressed in DRG neurons that had action potentials with in¯ec-

Fig. 7. Na+ currents in bladder a€erent neurons from spinal intact rats. (A) Superimposed traces of Na+ currents in a neuron with TTX-resistant spikes evoked by depolarizing voltage steps to +5 mV from a holding potential of ÿ60 mV in the absence (control) and presence (TTX) of TTX, and after the removal of Na+ ions in extenal solution (TTX + 0 mM Na+). (B) Superimposed traces of Na+ currents in a neuron with TTX-resistant spikes evoked by depolarizing voltage steps to ÿ10 mV from a holding potential of ÿ60 mV. (C and D) I±V curves for Na+ currents evoked by voltage steps (5 mV increments) ranging ÿ80 to +40 mV from a holding potential of ÿ60 mV in the absence (q) and presence (Q) of TTX, and after the removal of Na+ ions in external solution (r) in the same neurons as in (A and B), respectively. Note that the neurons in (A) exhibited TTX-resistant Na+ currents, while the Na+ current in B was mainly TTX-sensitive. [Reproduced from Yoshimura et al. (1996) with permission.].

Bladder A€erent Pathway and SCI

593

Fig. 8. Voltage-dependence of Na+ currents in bladder a€erent neurons from spinal intact rats. (A) Activation and inactivation characteristics of TTX-resistant Na+ currents (n = 8). The inactivation curve (q) is plotted as the normalized peak currents (I/Imax) vs the prepulse potential. The activation curve (r) is plotted as the relative Na+ conductance (G/Gmax) vs the command potential. The solid lines represent nonlinear least squares ®t to the Boltzmann equation. The Vh and K values for activation of TTX-resistant currents were ÿ11.4 and 8.5 mV, respectively. The inactivation curve for TTX-resistant currents had Vh of ÿ24.2 mV with K of 5.6 mV. (B) Activation and inactivation characteristics of TTXsensitive Na+ currents. The Vh and K values for activation of TTX-sensitive currents were ÿ20.6 and 6.1 mV, respectively (r). The inactivation curve for TTX-sensitive currents (q) had Vh of ÿ59.0 mV with K of 11.5 mV [Modi®ed from Yoshimura and de Groat (1997a) with permission].

tions and responded to capsaicin, whereas the fast inactivating IA was observed in large diameter DRG neurons without action potential in¯ections. Bladder a€erent neurons exhibited a similar distribution of two types of IA current; that is, small-sized neurons with TTX-resistant humped spike expressing slowinactivating IA and large-sized neurons with TTXsensitive spikes exhibiting fast-inactivating IA currents (Fig. 9) (Yoshimura et al., 1996). It was also observed in bladder a€erent neurons that steadystate inactivation of slowly inactivating IA currents was displaced by ca 20 mV in a more depolarizing direction than fast inactivating IA currents, and that 20% of the slow IA currents are still available at the resting membrane potential level between ÿ50 and ÿ60 mV while fast IA currents were almost completely inactivated at the membrane potential level (Fig. 10) (Yoshimura et al., 1996). This is in accordance with the ®ndings in current clamp recordings that bladder a€erent neurons exhibited membrane potential relaxation during depolarization, which was blocked by an application of 4-AP (see Section 4.2.2). Thus, in small sized bladder a€erent neurons, TTX-resistant high-threshold Na+ currents and slow IA currents contribute to the high thresholds for spike activation. Since the neurons with TTX-resistant humped spike are likely to be capsaicin sensitive C-®ber a€erent cells (Arbuckle and Docherty, 1995; Yoshimura et al., 1996), these functional properties of bladder a€erent neurons could explain inexcitability of C-®ber a€erent pathways. 4.3. Histological and Chemical Properties of Bladder A€erent Neurons A-®ber and C-®ber a€erent neurons are also distinguished by immunohistochemical staining for

neuro®lament protein in their somata. Neuro®lament is a cytoskeletal protein that is synthesized in cell bodies and delivered to axons by axoplasmic transport. The level of neuro®lament expression is known to correlated with axonal caliber and myelination (Ho€man et al., 1984, 1987). It has also been demonstrated that neuro®lament, especially 200 kDa subunit, is exclusively expressed in myelinated A-®ber DRG neurons, but not in unmyelinated in C-®ber neurons (Lawson et al., 1993). In bladder a€erent neurons from neurogenically normal rats, approximately two-thirds of the cells were neuro®lament-poor (i.e. C-®ber neurons) while the remaining one-third of cells exhibited intense immunoreactivity for neuro®lament (Ad®ber neurons) (Yoshimura et al., 1998a). It was also shown that neuro®lament immunoreactivity in bladder a€erent neurons negatively correlated with the sensitivity to capsaicin. A study using the cobalt uptake assay in DRG cell cultures revealed that ca 80% of neuro®lament-poor C-®ber bladder a€erent neurons were sensitive to capsaicin (Table 3) (Yoshimura et al., 1998a), which is similar to the previous ®ndings that the majority of nociceptive C®ber DRG neurons were sensitive to capsaicin (Lundberg, 1996). The predominance of neuro®lament-poor, C-®ber a€erent cells in the bladder a€erent population is in line with the ®nding in other studies such as conduction velocity measurement or histological analysis of the pelvic nerve that unmyelinated C-®ber bladder a€erents are more numerous than myelinated Ad-®ber a€erents in bladder a€erent pathways (Hulsebosch and Coggeshall, 1982; Vera and Nadelhaft, 1990).

594

N. Yoshimura

Fig. 9. Characteristics of IA current in bladder a€erent neurons from spinal intact rats. Inward currents were suppressed by equimolar substitution of choline for Na+ ions and reduction of Ca2+ ions in the external solution. (A and D) Outward K+ current evoked by voltage steps to +10 mV from holding potentials (Hp) of ÿ100, ÿ60 and ÿ40 mV in the neurons with TTX-resistant spikes and TTX-sensitive spikes, respectively. (B, C and E, F) Time-dependence of rise and decay phase of IA current in the neurons as in (A and D), respectively. The current was obtained by subtraction of K+ current evoked from the holding potential of ÿ40 mV from that at ÿ100 mV. The IA current obtained from the neuron in (A) was exponentially activated with time constant of 2.4 msec (B) and was not totally inactivated during 1 sec depolarizing pulse (time constant for inactivation; 284.3 msec) (C). The IA current of (D) was exponentially activated with time constant of 1.05 msec (E) and was inactivated more rapidly (time constant for inactivation; 17.8 msec) (F) than that of (C) [Reproduced from Yoshimura et al. (1996) with permission].

Fig. 10. Steady-state activation and inactivation characteristics of IA current in bladder a€erent neurons from spinal intact rats. (A) Activation characteristics of IA current obtained in the neurons with TTX-resistant-spikes (n = 8) (Q) and TTX-sensitive spikes (n = 7) (q). Relative IA conductances normalized to the maximal IA conductance (G/GMAX) were plotted against membrane potentials. (B) Inactivation characteristics of IA current obtained in the same neurons with TTX-resistant-spikes (n = 8) (R) and TTX-sensitive spike (n = 7) (r). Relative peak amplitude of IA current normalized to the maximal amplitude of IA current (I/IMAX) were plotted against membrane potentials. Vh and K were obtained by ®tting curves using the modi®ed Boltzmann equation. Note that Vh for IA inactivation in TTX-resistant neurons (ÿ77.5 mV) was displaced to more depolarized levels than that in TTX-sensitive neurons (ÿ90.5 mV) [Reproduced from Yoshimura et al. (1996) with permission].

Bladder A€erent Pathway and SCI

595

Fig. 11. Rhythmic bladder activity in normal cats (A) was unchanged following s.c. administration of capsaicin 30 mg kgÿ1 (C); but in chronic spinalized cats (B) capsaicin (20 mg kgÿ1, s.c.) abolished bladder activity (D). Vertical calibration represents 20 cmH2O, horizontal calibration represents 1 min. [Reproduced from de Groat et al. (1990) with permission].

5. EFFECT OF SPINAL CORD INJURY ON THE MICTURITION REFLEX 5.1. Changes in the Micturition Re¯ex Following Spinal Cord Injury The e€ect of SCI on lower urinary tract depends on the level, duration, and completeness of the cord lesion. However, the ®nal features of bladder dysfunction are usually characterized as upper motoneuron and lower motoneuron diseases, which correspond respectively with damage to the spinal cord rostral to the sacral cord, or damage to the sacral cord and/or cauda equina that give rise to the parasympathetic and somatic pathway to the bladder and urethral sphincter (Fam et al., 1988; Chancellor and Blaivas, 1996). Among patients with

SCI, upper motoneuron disease such as cervical and thoracic vertebral injuries forms the major group (Fam et al., 1988). Upper motoneuron type of SCI initially leads to a phase of spinal shock, which is followed by a recovery phase during which neurologic changes emerge. During the period of spinal shock immediately after SCI, there is a ¯accid paralysis and absence of re¯ex activity below the level of lesion; thus the urinary bladder become are¯exic. However, activity of the internal and external sphincter persists or rapidly recovers after suprasacral injuries. Therefore, because sphincter tone is present, urinary retention develops and patients have to be treated with intermittent or continuous catheterization to eliminate urine from the urinary bladder. Following the spinal shock phase, re¯ex detrusor activity reappears after 2±12 weeks in most

Table 3. Neuro®lament immunoreactivity and capsaicin sensitivity in dissociated bladder a€erent neurons from spinal intact and SCI rats Capsaicin sensitive

Capsaicin insensitive

Total

14.9% 30.5%

67.4 23.1% (26.820.4 mm) 32.6 23.1% (34.120.8 mm)

54.624.0% (27.220.7 mm)

45.424.0% (31.421.0 mm)

100% (29.22 1.2 mm)

B. Neurons (n = 126) from four SCI rats Neuro®lament poor 28.4% Neuro®lament rich 9.6% Total 38.021.2%* (29.120.8 mm)

9.7% 52.3% 62.021.2%* (36.12 0.8 mm)

38.1 20.6%** (27.020.7 mm) 61.9 20.6%** (37.720.9 mm) 100% (34.22 1.1 mm**)

A. Neurons (n = 149) from four spinal intact rats Neuro®lament poor 52.5% Neuro®lament rich 2.1% Total

Data are expressed as percentage of total cell population averaged in four rats of each group. Average cell diameters are indicated in parentheses. Statistically signi®cant di€erences between spinal intact and spinal transected animals in regard to neuro®lament content, capsaicin sensitivity and cell diameter are indicated by *P < 0.05 and **P < 0.01.

596

N. Yoshimura

cases (Fam et al., 1988; Chancellor and Blaivas, 1996). During the recovery phase, the detrusor develops involuntary re¯ex contraction responding to visceral stimuli such as bladder ®lling or suprapubic manual compression. During the ®rst phase of the recovery period, re¯ex bladder contractions are not sustained and generate low intravesical pressure, but over time the bladder contractions become more powerful and produce involuntary voiding. However, the bladder is usually only emptied partially because the striated urethral sphincter becomes dyssynergic (i.e. simultaneous contractions of the bladder and the striated urethral sphincter) [Fig. 1(B)]. Inecient voiding may also be due to unsustained bladder contractions. In normal micturition, activation of the pontine micturition center simultaneously induces bladder contractions and a suppression of sphincter activity [Fig. 1(A)] (de Groat et al., 1993; Yoshimura and de Groat, 1997b). A complete suprasacral lesion interrupts this coordination between the bladder and the striated sphincter. Thus, patients with chronic upper motoneurons lesions exhibit: 1. detrusor hyperre¯exia; 2. urethral smooth muscle synergia; and 3. striated urethral sphincter dyssynergia [Fig. 1(B)]. All patterns of detrusor hyperre¯exia associated with striated sphincter dyssynergia lead to high intravesical pressure and/or severe bladder trabeculation with the formation of diverticula, which often induce vesicoureteral re¯ux and the deterioration of upper urinary tract (Fam et al., 1988; Chancellor and Blaivas, 1996). In the lower motoneuron type of SCI, complete lesions of the sacral cord or the cauda equina usually result in ¯accidity of the bladder and its outlet. The bladder becomes are¯exic; thereby, bladder compliance and bladder capacity are increased. Pressures in the striated urethral sphincter are decreased. When the lesion is extended to thoracolumber spinal cord, the sympathetic out¯ow to internal smooth muscle sphincter of urethra is also damaged, and bladder neck becomes incompetent in association with hypoactive detrusor and striated sphincter (Fam et al., 1988; Chancellor and Blaivas, 1996; de Groat et al., 1997).

5.2. Autonomic Dysre¯exia Patients with suprasacral cord injuries at T6 or higher, often exhibit autonomic dysre¯exia, which is characterized by arterial pressor responses induced by stimuli such as bladder distension, fecal impaction or visceral in¯ammation below the level of spinal cord lesion (Fam et al., 1988). These stimuli cause excitation of the sympathetic pathway and induce arteriolar vasoconstriction and high blood pressure, as well as piloerection and sweating below the level of injury. Baroreceptor re¯exes stimulated by the high blood pressure activate vagus nerve activity and cause bradycardia. Since high blood pressure may cause a seizure or cerebral hemorrhage, autonomic dysre¯exia should be treated promptly with a-adrenergic receptor blocking agents and/or

removal of causative stimuli (e.g. emptying bladder by catheterization).

6. EFFECT OF SPINAL CORD INJURY ON BLADDER AFFERENT PATHWAYS 6.1. Changes in In Vivo Functions of Bladder A€erent Pathways After Spinal Cord Injury Reorganization of lower urinary tract function following SCI is also similarly observed in animals such as cats and rats (de Groat et al., 1990; Kruse et al., 1993). Both species with complete transection of thoracic spinal cord initially exhibit detrusor are¯exia, followed by the emergence of bladder hyperre¯exia with increased urethral sphincter activity. Electrophysiological and pharmacological studies have shown that the re¯ex pathways controlling the lower urinary tract are markedly di€erent between spinal intact and spinal injured animals. In spinal intact cats and rats, the micturition re¯ex is mediated by a long latency supraspinal re¯ex pathway passing through the pons which is activated by myelinated Ad-®ber bladder a€erents (de Groat et al., 1981; Mallory et al., 1989; de Groat et al., 1993). For example, in cats, ®ring of sacral parasympathetic neurons following stimulation of Ad®ber bladder a€erents in the pelvic nerve showed that the re¯ex occurs after a long latency (65±100 msec), which is the sum of latencies for a€erent spinobulbar (30±40 msec) and e€erent bulbospinal (45± 60 msec) components of the micturition re¯ex. However, electrophysiologic recordings in spinalized cats revealed that the central delay is considerably shorter (15±40 msec) than spinal intact animals, and that the a€erent limb of the micturition re¯ex consists of unmyelinated C-®ber a€erents (de Groat et al., 1990). The latter is also demonstrated in pharmacologic studies using capsaicin, a C-®ber neurotoxin. In chronic spinal cord-injured cats (3±6 weeks after SCI), subcutaneously administered capsaicin (20±30 mg kgÿ1) completely blocked bladder contractions induced by bladder distention whereas capsaicin had no e€ects on re¯ex bladder contractions in spinal intact cats (Fig. 11) (de Groat et al., 1990). Thus it is plausible that the interruption of pathways between spinal cord and the brain produces a considerable reorganization of the micturition re¯ex pathway, and that C-®ber bladder a€erents which usually do not respond to bladder distention (i.e. silent C-®ber) (HaÈbler et al., 1990) become mechano-sensitive and initiate automatic micturition after SCI. There is also evidence in the rat that C-®ber a€erents increase their excitability to induce bladder hyperre¯exia following the SCI. In chronic spinal injured rats, central delay of the micturition re¯ex becomes much shorter (<5 msec) than spinal intact rats (57±68 msec) (Mallory et al., 1989), and subcutaneous application of capsaicin suppressed hyperre¯exic nonvoiding bladder contractions which occur prematurely prior to voiding bladder contractions although voiding contractions are still triggered by Ad-®ber a€erents and not blocked by capsaicin (Fig. 12) (Cheng et al., 1995).

Fig. 12. Cystometrograms in anesthetized spinal intact and unanesthetized chronic SCI rats recorded under isotonic conditions with the bladder outlet open and the animals able to void. (A) Cystometric pattern in an anesthetized spinal intact rat. (B) Cystometric pattern in a chronic SCI rat before capsaicin treatment. Note relatively small nonvoiding contractions occurring during bladder ®lling and their amplitude progressively increasing with ®lling. (C) The same chronic SCI rat after capsaicin (125 mg kgÿ1, s.c.) pretreatment 4 days before the cystometric study. Note the nonvoiding contractions were eliminated but the voiding contraction was not altered. Vertical calibrations: intravesical pressure in cm H2O and horizontal calibrations: time in min. Arrows indicate beginning of saline infusion and asterisks (*) indicate voiding [Reproduced from Cheng et al. (1995) with permission].

Bladder A€erent Pathway and SCI 597

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N. Yoshimura

Clinical studies have also demonstrated that C®ber a€erents innervating the urinary bladder are involved in bladder hyperactivity and automatic bladder contractions in spinal cord-injured patients (Geirsson et al., 1995; Cruz et al., 1997a). In these studies, intravesical instillation of capsaicin suppressed bladder hyperactivity in patients with SCI. When administered intravesically in concentrations up to 2 mM, capsaicin increased bladder capacity and reduced the number of incontinence episodes in those patients. Resiniferatoxin, an agent with similar C-®ber desensitizing properties has been reported recently to have similar e€ects on neurogenic bladder hyperactivity (Cruz et al., 1997b). Taken together, it is clear that the functional properties of C-®ber a€erents in the bladder are altered following SCI, thereby inducing hyperre¯exic bladder activity in both humans and animals (Fig. 13). Other examples of a reorganization of C-®bermediated re¯ex pathways were obtained in studies of autonomic dysre¯exia or cold stimulation-evoked voiding re¯ex. Autonomic dysre¯exia (i.e. re¯ex hypertension) induced by bladder distention after SCI is triggered by capsaicin-sensitive bladder a€erents, since capsaicin treatment suppresses autonomic dysre¯exia in humans with SCI (Igawa et al., 1996) and similar arterial pressure responses induced by

bladder distension in rats (Cheng et al., 1993, 1995). It is also known that instillation of cold water into the bladder in patients with suprasacral cord lesions induces re¯ex voiding (the Bors Ice Water Test) (Bors and Comarr, 1971; Geirsson et al., 1993). This re¯ex does not occur in normal subjects, except for infants (Geirsson et al., 1994). It has been shown in the cat that C-®ber bladder a€erents are responsible for cold-induced bladder re¯exes (Fall et al., 1990). Intravesical administration of capsaicin to paraplegic patients also blocks the cold-induced bladder re¯exes, indicating that they are mediated by C-®ber a€erents in humans as well (Geirsson et al., 1995). Thus, it is suggested that cold-evoked bladder re¯exes mediated by C-®ber bladder a€erents are enhanced or unmasked after the elimination of supraspinal controls by SCI (Fig. 13). 6.2. Changes in Electrical Properties of Bladder A€erent Neurons Following Spinal Cord Injury 6.2.1. Methodological Summary for Spinal Cord Injury To prepare chronic spinal transected rats, the spinal cord was cut at the level of T8±T9 under halothane anesthesia (Kruse et al., 1993; Yoshimura

Fig. 13. Diagram of the central re¯ex pathways which regulate micturition in the cat and rat. With intact neuraxis, micturition is initiated by supraspinal re¯ex pathway passing through the pontine micturition center. The pathway is triggered by myelinated a€erents (Ad-®bers) connected to tension receptors in the bladder wall. In spinalized animals, connection the brainstem and the sacral spinal cord are interrupted () and micturition is initially blocked. In chronic SCI animals, a spinal re¯ex mechanism emerges which triggered by unmyelinated (C-®ber) bladder a€erents. The C-®ber re¯ex pathway is usually weak or undetectable in animals with an intact nervous system. Capsaicin blocks the C-®ber re¯ex in chronic SCI animals. Cold stimulation also activates the C-®ber mediated micturition re¯ex. However, following SCI voiding re¯ex in the rat is still triggered by myelinated Ad-a€erents connecting to spinal e€erent mechanisms (dotted arrow) while voiding re¯ex in the cat is totally abolished by capsaicin treatment.

Bladder A€erent Pathway and SCI

and de Groat, 1997a). During the ®rst 7±10 days after the transection when the bladder was are¯exic and the animals were in complete urinary retention, urine was eliminated from the bladder by manual compression two to three times a day; after this period the bladder was emptied once a day. Four weeks after spinal cord transection, bladder a€erent neurons were isolated from L6±S1 DRG for patch clamp recordings (see Section 4.2.1). 6.2.2. E€ect of Spinal Cord Injury on Action Potential Characteristics The underlying mechanism for inducing hyperexcitability of C-®ber bladder a€erents was investigated by whole-cell patch clamp recording in DRG neurons innervating the urinary bladder (Yoshimura and de Groat, 1997a). Chronic SCI in rats produced hypertrophy of bladder a€erent neurons as re¯ected by an increase in cell diameter and cell input capacitance (Table 4). This is in line with previous ®ndings that a€erent neurons in the L6±S1 DRG innervating the urinary bladder undergo somal hypertrophy (45±50% increase in cross-sectional area) in spinalized rats (Kruse et al., 1995). In addition to neuronal hypertrophy, bladder a€erent neurons in chronically spinalized rats increased their excitability. As described in Section 4.2.2, the majority (ca 70%) of bladder a€erent neurons exhibited TTX-resistant humped action potentials with higher threshold for activation than TTX-sensitive action potentials in the remaining bladder a€erent neurons (30%) (Yoshimura et al., 1996). However, in chronic spinal transected rats, 60% of bladder a€erent neurons exhibited TTX-sensitive low-threshold action potentials. The mean threshold for spike activation in spinalized rats (ÿ25.5 2 0.9 mV) was lower (21%) than measurements in intact animals (Table 4). 6.2.3. Plasticity in Na+ and K+ Channels The alteration of electrophysiological properties in bladder a€erent neurons after SCI was also re¯ected in changes in Na+ current distribution (Yoshimura and de Groat, 1997a). Consistent with the increment in the proportion of neurons with Table 4. Membrane characteristics of bladder a€erent neurons in spinal intact and SCI rats Intact

SCI

No. of cells Diameter (mm) Input capacitance (pF)

n = 90 25.1 22.8 27.2 23.8

n = 88 31.62 2.8** 39.62 2.4**

No. of cells Spike threshold (mV) Spike duration (msec)

n = 30 ÿ21.1 21.6 7.7 21.1

n = 30 ÿ25.52 0.9** 5.32 0.7*

No. of cells n = 60 Na+ current threshold (mV) ÿ26.5 21.4 Na+ current density (pA/pF) TTX-resistant 60.5 25.5 TTX-sensitive 32.1 29.5

n = 58 ÿ38.92 1.1**

599

TTX-sensitive spikes, the number of bladder a€erent neurons which predominantly expressed TTX-sensitive Na+ currents (60±100% of total Na+ currents) also increased (Fig. 14). The density of TTX-sensitive Na+ currents in bladder a€erent neurons signi®cantly increased from 32.1 to 80.6 pA pFÿ1, while TTX-resistant current density decreased from 60.5 to 17.9 pA pFÿ1 following SCI (Table 4). In addition, an increase in TTX-sensitive Na+ currents was detected in some bladder a€erent neurons that still retained a predominance of TTX-resistant currents (>50% of total Na+ currents) after SCI (Fig. 14). These data indicate that SCI induces a switch in expression of Na+ channels from TTX-resistant type to TTX-sensitive type. Since TTX-sensitive Na+ currents have a lower threshold for activation than TTX-resistant currents (Fig. 7), it is reasonable to assume that these changes in expression of Na+ channels in bladder a€erent neurons after SCI contribute to a low threshold for spike activation in these neurons. In addition, bladder a€erent neurons with TTXsensitive spikes in chronic spinalized rats exhibited no apparent membrane relaxation when membrane potentials were gradually depolarized by injecting currents into the cells. In these neurons, voltage responses induced by current injections was not altered by 4-AP application, although the neurons with TTX-resistant spike still had 4-AP-sensitive membrane potential relaxation during depolarization as found in spinal intact rats (Fig. 15). As described in Sections 4.2.2 and 4.2.4, the phenomenon of membrane potential relaxation is due to the activation of slowly inactivating IA currents which can be elicited by depolarization from the resting membrane potential (Yoshimura et al., 1996). Therefore it is likely that following SCI A-type potassium channels are suppressed in parallel with an increased expression of TTX-sensitive Na+ currents, thereby increasing excitability of bladder a€erent neurons. Since TTX-resistant Na+ currents and IA currents are preferentially expressed in small-sized C-®ber a€erent neurons in neurogenically intact rats (Gold et al., 1996a; Yoshimura et al., 1996), the changes in these channels after SCI mainly occur in C-®ber bladder a€erents neurons to increase cell excitability of these neurons. Since an immunohistochemical study using antibodies to TTX-resistant Na+ channel proteins revealed that TTX-resistant Na+ channels are located in small-sized DRG cell bodies and in super®cial laminae of the dorsal horn of the spinal cord (Novakovic et al., 1998), it appears that DRG cell bodies and terminals may share common properties in expression of ion channels. Therefore, if changes occurring in a€erent cell bodies also occur at peripheral receptors in the bladder or the spinal cord, these changes could contribute to the emergence of the C-®ber-mediated spinal micturition re¯ex following SCI.

17.92 9.2* 80.62 10.1**

6.3. Changes in Histological and Chemical Properties of Bladder A€erent Neurons

Values are mean 2SEM. *P < 0.05 and **P < 0.01 indicate di€erences between values obtained in neurons from spinal intact and SCI groups of animals.

Another type of plasticity in C-®ber bladder a€erent neurons following SCI was found in neuro®lament expression (Yoshimura et al., 1998a). Thirty-

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N. Yoshimura

Fig. 14. The classi®cation of bladder a€erent neurons from spinal intact and SCI rats based on the magnitude of TTX-sensitive Na+ currents. (A) Neurons from spinal intact animals (n = 60). (B) Neurons from spinal transected animals (n = 58). Abscissa, percentage of the total Na+ current that was TTXsensitive. Abscissa divided into 10 bins (10% for each bin). Ordinate, the fraction of the total population of cells exhibiting the corresponding percentage of TTX-sensitive Na+ currents. Note that bladder neurons from spinal intact animals (A) exhibited a bimodal pattern (either >60% or <20% of the total Na+ current) while bladder neurons from spinal transected animals (B) exhibited no obvious pattern in the expression of TTX-sensitive Na+ currents [Modi®ed from Yoshimura and de Groat (1997a) with permission].

two percent of bladder a€erent neurons from spinal intact rats exhibited positive neuro®lament immunoreactivity, which is a marker of myelinated A®ber a€erents, however, SCI signi®cantly increased the number of bladder a€erent neurons with prominent neuro®lament immunoreactivity to ca 60% of

total neurons (Table 3). The mean cross sectional area of bladder a€erent neurons in spinal cordinjured rats was signi®cantly larger than in spinal intact rats. Histograms of cross sectional somal area of bladder a€erent neurons clearly shows a shift in neuronal population from neuro®lament-poor cells

Fig. 15. E€ects of 4-AP, an IA channel blocker, on voltage responses in bladder a€erent neurons after SCI. In current-clamp conditions, voltage responses are evoked by 20 msec depolarizing current pulses injected through the patch electrode. Figures show changes in membrane potentials during an application of depolarizing pulses prior to spike activation. The duration of depolarizing pulses was indicated by arrows beneath voltage traces. The amplitude of the pulses was increased from 0 pA in 10 pA steps. (A) Neuron with TTX-resistant spikes. (B) Neuron with TTX-sensitive spikes. Left traces, control. Right traces, 4-AP (1 mM) application. In (A) the neurons exhibited membrane potential relaxation during depolarization, which was blocked by 4-AP (1 mM). In contrast, the neurons with TTX-sensitive spike exhibited no apparent relaxation during membrane depolarization and 4-AP had no e€ects on voltage responses induced by current injections (B).

Bladder A€erent Pathway and SCI

to neuro®lament-rich cells along with cell size increase following SCI (Fig. 16). Thus, it is plausible that 30±40% of small, neuro®lament-poor C-®ber bladder a€erent neurons increase in size and change to neuro®lament-rich neurons after SCI. In addition, a combination histochemical experiment in which neuro®lament immunoreactivity and capsaicin sensitivity were analyzed in cultured DRG preparations revealed that bladder a€erent neurons with de novo neuro®lament immunoreactivity lost their sensitivity to capsaicin, since the proportion of capsaicin-sensitive cells among neuro®lament-poor bladder a€erent neurons was similar (75±78%) in intact and spinal cord-injured animals although bladder a€erent neurons sensitive to capsaicin signi®cantly decreased from 55% to 38% of total population after SCI (Table 3) (Yoshimura et al., 1998a). Thus it is evident that bladder a€erent neurons exhibit various types of functional and morphologi-

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cal plasticity after SCI. Bladder a€erent neurons from chronic spinal cord-injured rats were: 1. larger in size; 2. more excitable due to increased expression of TTX-sensitive Na+ channels and suppression of IA channels; 3. more likely to exhibit neuro®lament immunoreactivity; and 4. less likely to respond to capsaicin (Fig. 17). Based on these observations, it appears that C-®ber bladder a€erent neurons which increase in size may change their phenotype to one resembling Ad-®ber bladder neurons after SCI. In addition, the reduction in capsaicin sensitivity in this species may explain why capsaicin treatment suppressed nonvoiding re¯ex bladder contractions during storage phase, but not voiding re¯exes in spinal cord-injured rats (Fig. 12) (Cheng et al., 1995). Since the voiding re¯ex in chronic paraplegic cats were completely

Fig. 16. Histograms of cross-sectional somal areas of L6 and S1 DRG cell pro®les innervating the urinary bladder in DRG sections. Abscissa, size distribution (cross-sectional somal area) with a bin width of 50 mm2. Ordinate, fraction of total cell population. Open and ®lled bars represent the fraction of neuro®lament-poor and neuro®lament-rich cell pro®les, respectively. (A) Neurons (n = 460) from two spinal intact rats. (B) Neurons (n = 423) from two spinal transected rats [Reproduced from Yoshimura et al. (1998a) with permission].

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Fig. 17. Diagrams of properties of bladder a€erent neurons in spinal intact rats (A) and rats with SCI (B). In (A), Ad-®ber bladder a€erent neurons which are large in size with positive expression of neuro®lament (NF) exhibit high excitability since the thresholds for spike activation is lower due to expression of TTX-sensitive Na+ currents (TTX-S INa) and fast A-type K+ currents (IA) than in small-sized C®ber bladder a€erent neurons whose thresholds for spike activation is relatively high because of the presence of TTX-resistant Na+ currents (TTX-R INa) and slow IA current. However, in (B), C-®ber bladder a€erent neurons after SCI increase their size along with increased NF expression, and become more excitable due to increased expression of TTX-S INa currents and reduced expression of slow IA currents.

blocked by capsaicin treatment (Fig. 11) (de Groat et al., 1990), the reduction in capsaicin sensitivity in bladder a€erent pathway after SCI might be less remarkable in cats when compared with rats.

7. POSSIBLE MECHANISMS INDUCING PHENOTYPIC CHANGES IN BLADDER AFFERENT PATHWAYS FOLLOWING SPINAL CORD INJURY How does SCI change the properties of bladder a€erent neurons? Fig. 18 depicts hypothetical mechanisms inducing hyperactivity of bladder a€erent pathways and the urinary bladder after SCI. As described in Section 5.1, SCI induces uncoordinated bladder and urethral sphincter activity, which increases urethral resistance and bladder work. Previous studies in chronic spinal injured rats revealed that bladder hyperre¯exia and uncoordinated urethral sphincter contractions induced inecient voiding (30-fold increase in residual volume), and eventually produced bladder and a€erent neuron hypertrophy (Kruse et al., 1993, 1995). Since bladder and neuronal hypertrophy were also observed in rats with urethral obstruction induced by partial urethral ligation (Steers et al., 1991a), it appears that changes in the target organ can in¯uence the properties of neurons innervating that organ (i.e. bladder). In spinal cord-injured rats,

urinary diversion, in which urine was diverted by anastomoses of ureters into colon or uterus, eliminated the bladder overdistention and increase in bladder work and also prevents the bladder and a€erent neuron hypertrophy (Kruse et al., 1995). Thus it is likely that phenotypic changes in bladder a€erent neurons are more likely due to the interaction with target organ, rather than the interaction with the injured spinal cord (Fig. 18). This conclusion is supported by the ®ndings that SCIinduced changes in ion channels, neuro®lament expression and capsaicin sensitivity were speci®cally observed in bladder a€erent neurons, but not in randomly selected population of L6±S1 DRG neurons or in neurons innervating the colon (Yoshimura and de Groat, 1997a; Yoshimura et al., 1998a). Target organ-neural interaction in bladder a€erent pathways following SCI might be mediated by an increase of neurotrophic factors produced in target organ tissues and transported retrogradely back to the neuronal cell bodies, since trophic factors such as nerve growth factor (NGF) increase in hypertrophied bladders in rats with partial urethral ligation (Buttyan et al., 1992; Steers et al., 1991a). In addition, hypertrophy of a€erent and e€erent neurons innervating the hypertrophic bladder is antagonized in part by autoimmunization against NGF (Steers et al., 1991b, 1996). Recent studies also revealed that local administration of NGF can sensitize a€erent terminals in the bladder to induce

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Fig. 18. Diagram of hypothetical mechanisms inducing hyperexcitability of the urinary bladder and bladder a€erent pathways following SCI. Injury to the spinal cord (1) abolished the reciprocal coordination between the bladder and sphincter (striated sphincter dyssynergia) (2), leading to functional obstruction of urethra. Increased urethral resistance by functional obstruction of urethra then induces bladder hypertrophy (3), resulting in increased excretion of neurotrophic factors (NTF) in the hypertrophied bladder smooth muscle (4). NTF taken up by a€erent terminals is transported through bladder a€erent ®bers (5) and induces phenotypic changes in bladder a€erent neurons (6). These changes which may occur in a€erent terminals then contribute hyperexcitability of the bladder a€erent pathways and the urinary bladder (7).

hyperactivity (Dmitrieva and McMahon, 1996), and that the majority of small-sized nociceptive bladder a€erent neurons have high-anity NGF receptors, trkA (Bennett et al., 1996). Several studies in other systems have provided evidence that functional and morphological properties of primary a€erent neurons could be altered by neurotrophic factors. Chronic administration of NGF reportedly increased cell size of small-diameter rat DRG neurons (Kornblum and Johnson, 1982). It has also recently demonstrated that overexpression of NGF in skin of transgenic mice induces hyperalgesia, an increase in the number of sensory ®bers and somal hypertrophy of a€erent neurons in trigeminal ganglia (Goodness et al., 1997). Previous studies also revealed that nerve injury which disrupt connections with target organs reduced neuro®lament expression in DRG neurons and that this e€ect is reversed in part by NGF application (Verge et al., 1990). In addition, capsaicin sensitivity (Winter et al., 1988; Aguayo and White, 1992; Bevan and Winter, 1995) and expression of TTX-resistant Na+ channels (Aguayo and White, 1992; Black et al., 1997; Oyelese et al., 1997) are at least in part regulated by NGF in DRG neurons innervating skin or muscle. However, NGF reportedly increases the expression of TTX-resistant Na+ channels (Aguayo and White, 1992; Black et al., 1997; Oyelese et al., 1997) and sensitivity to capsaicin (Winter et al., 1988; Aguayo and White, 1992; Bevan and Winter, 1995) in somatic a€erent neurons, which is opposite

to the changes in bladder a€erent neurons from chronic spinalized rats (Yoshimura and de Groat, 1997a; Yoshimura et al., 1998a). Therefore, there might be the involvement of other trophic factors such as brain-derived neurotrophic factor (BDNF) or basic ®broblast growth factor (bFGF), which are reportedly increased in hypertrophied bladder muscle following partial urethral obstruction (Buttyan et al., 1992). BDNF has been reported to regulate functional properties in somatic DRG neurons (Oyelese et al., 1997). It is also documented that bFGF increases Na+ currents in other systems such as carotid body chemoreceptor cells (Zhong and Nurse, 1995). Taken together, it is assumed that trophic factors released in target organs such as the urinary bladder could alter properties of their a€erent pathways (Fig. 18). The continued investigation of target organ±neural interaction in bladder a€erent pathways will provide further valuable insights into the mechanism inducing lower urinary tract dysfunction following SCI and may lead to the development of new treatments for urinary symptoms in spinal cord-injured patients.

AcknowledgementsÐI am indebted to Professor W. C. de Groat for his continuous support of my work and critical reading of the manuscript. The work from our laboratory has been supported by grants from the National Institute of Health, USA (DK 49430 and DK 51420). I also received support from the Ministry of Education, Science and

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Culture, Japan (Nos 07671720 and 08671812) and the American Paralysis Association (YA1-9801-2).

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