Neural Control of Lower Urinary Tract Function
Summary and Keywords
Functions of the lower urinary tract to store and periodically eliminate urine are regulated by a complex neural control system in the brain and lumbosacral spinal cord that coordinates the activity of smooth and striated muscles of the bladder and urethral outlet via a combination of voluntary and reflex mechanisms. Many neural circuits controlling the lower urinary tract exhibit switch-like patterns of activity that turn on and off in an all-or-none manner. During urine storage, spinal sympathetic and somatic reflexes are active to maintain a quiescent bladder and a closed outlet. During micturition, these spinal storage reflexes are suppressed by input from the brain, while parasympathetic pathways in the brain are activated to produce a bladder contraction and relaxation of the urethra. The major component of the micturition switching circuit is a spinobulbospinal parasympathetic pathway that consists of essential relay circuitry in the periaqueductal gray and pontine micturition center. These circuits in the rostral brain stem are, in turn, regulated by inputs from the forebrain that mediate voluntary control of micturition. Thus neural control of micturition is organized as a hierarchical system in which spinal storage reflexes and supraspinal voiding reflexes are regulated voluntarily by higher centers in the brain. In young children the voluntary mechanisms are undeveloped and voiding is purely reflex. Voluntary control emerges during maturation of the nervous system and depends on learned behavior. Diseases or injuries of the nervous system in adults cause re-emergence of involuntary micturition, leading to urinary incontinence.
The storage and periodic elimination of urine depend on the coordinated activity of two functional units in the lower urinary tract (LUT): (1) a reservoir (the urinary bladder) and (2) an outlet consisting of the bladder neck, urethra, and urethral sphincter. Coordination between these organs is mediated by a complex neural control system in the brain and spinal cord (de Groat, Griffiths, & Yoshimura, 2015). Thus, urine storage and release are highly dependent on central nervous system pathways. This distinguishes the LUT from other viscera, such as the gastrointestinal tract and cardiovascular organs that maintain a certain level of function in the absence of extrinsic neural input.
The LUT is also unusual in its pattern of activity and organization of neural control mechanisms. For example, micturition is under voluntary control and depends on learned behavior that develops during maturation of the nervous system, whereas many other visceral functions are regulated involuntarily. The LUT has only two modes of operation: storage and elimination. Thus, many of the neural circuits have switch-like patterns of activity (de Groat, 1975; de Groat & Wickens, 2013; Fowler, Griffiths, & de Groat, 2008), unlike the tonic neural control of cardiovascular organs. Micturition also requires the integration of autonomic and somatic efferent mechanisms to coordinate the activity of visceral organ smooth muscle (in the bladder and urethra) with that of urethral striated muscles (de Groat et al., 2015).
This article reviews the innervation of the LUT and the organization of the central neural pathways controlling urine storage and elimination.
The innervation of the LUT is derived from three sets of peripheral nerves: sacral parasympathetic (pelvic nerves), thoracolumbar sympathetic (sympathetic chain and hypogastric nerves) and sacral somatic nerves (primarily the pudendal nerves) (Figure 1).
Parasympathetic Efferent Pathways
The sacral parasympathetic pathway provides the major excitatory input to the bladder. Cholinergic preganglionic neurons located in the intermediolateral region of the lumbosacral spinal cord send axons to cholinergic ganglion cells in the pelvic plexus and bladder wall. Transmission in bladder ganglia is mediated by a nicotinic cholinergic mechanism. The ganglion cells, in turn, excite bladder smooth muscle by releasing acetylcholine, which activates post-junctional M3 muscarinic receptors (Figure 1). These receptors induce muscle contractions by releasing calcium from intracellular stores and by initiating muscle action potentials and calcium influx through L-type Ca2+ channels (Hashitani, Bramich, & Hirst, 2000). Binding of intracellular calcium to calmodulin activates myosin light-chain kinase that phosphorylates myosin type II light chain allowing myosin to interact with actin, leading to force generation (Andersson & Arner, 2004).
In various animals, such as rodents or rabbits, stimulation of parasympathetic nerves also produces bladder contractions that are resistant to muscarinic receptor blocking agents. These contractions are mediated by adenosine triphosphate (ATP) (Andersson & Arner, 2004; Burnstock, 2001; Ralevic & Burnstock, 1998), which excites the smooth muscle by acting on ligand-gated P2X1 receptors to increase intracellular calcium. Purinergic transmission has an important physiological role in animal bladders but is not important in the normal human bladder. However, ATP appears to have an excitatory function in bladders from patients with pathological conditions such as detrusor overactivity, chronic urethral outlet obstruction, or interstitial cystitis (Burnstock, 2001).
Parasympathetic pathways to the urethra induce relaxation during voiding. Inhibitors of nitric oxide synthase (NOS) block this relaxation indicating that nitric oxide is the inhibitory transmitter (Andersson & Arner, 2004). Nitric oxide stimulates guanylyl cyclase to increase the levels of cyclic GMP that in turn activates protein kinase G, leading to activation of potassium channels and desensitization of the contractile machinery to Ca2+.
Sympathetic Efferent Pathways
Sympathetic preganglionic pathways that arise from the T11 to L2 spinal segments pass to the sympathetic chain ganglia and then to prevertebral ganglia in the superior hypogastric and pelvic plexuses. Norepinephrine, released from sympathetic postganglionic nerves, contracts smooth muscle of the urethra and bladder base by activating α1-adrenergic receptors, and relaxes smooth muscle of the bladder body by activating β3-adrenergic receptors. α1-adrenergic receptors elicit contractions in the urethra and bladder neck by releasing Ca2+ from intracellular stores.
β3-adrenergic receptors elicit relaxation by increasing cyclic AMP and activating protein kinase A that acts in part by inducing a hyperpolarization of the cells either by opening K+ channels or by stimulating an electrogenic ion pump (Andersson & Arner, 2004).
Somatic Efferent Pathways
Somatic efferent pathways to the external urethral sphincter (EUS) are carried in the pudendal nerve from motoneurons in the S3–S4 segments of the human spinal cord (Figure 1A) and from various caudal lumbosacral segments in animals. Striated muscles of the pelvic floor, including the levator ani, coccygeus, and puborectalis muscles, are innervated by the levator ani nerve which arises from the S3–S5 segments of the sacral spinal cord (Barber et al., 2002); although there are reports that the pudendal nerve may also provide an innervation to these muscles in humans (Wallner, Maas, Dabhoiwala, Lamers, & DeRuiter, 2006).
Afferent axons innervating the lower urinary tract arise in the lumbosacral dorsal root ganglia and are contained in the three sets of peripheral nerves. The most important afferents for inducing sensations of bladder fullness and initiating micturition are those that travel in the pelvic nerves to the sacral spinal cord. These afferents consist of small myelinated (A-δ) and unmyelinated (C) axons that convey impulses from mechanosensitive and chemosensitive receptors located at various sites in the bladder wall. Urethral afferents in the pudendal nerves that respond to urine flow trigger reflexes to promote efficient voiding; while EUS afferents inhibit bladder activity (de Groat & Yoshimura, 2009).
Non-neuronal cells may also participate in lower urinary tract sensory mechanisms (Figure 2) (Birder & Andersson, 2013). Urothelial cells on the luminal surface of the bladder have specialized sensory and signaling properties dependent upon: (1) expression of stretch (Piezo), nicotinic, muscarinic, tachykinin, adrenergic, and capsaicin (TRPV1) receptors, (2) sensitivity to transmitters released from nerves and (3) ability to release chemical mediators such as ATP, acetylcholine and nitric oxide (NO) that can regulate the activity of adjacent afferent nerves and thereby trigger bladder sensations, local vascular changes and/or reflex bladder contractions. The role of ATP in urothelial signaling has garnered particular attention in recent years, and it has been recently determined that the mechanism controlling ATP release from the urothelium involves pannexin channels; large-pore ion channels that may be modulated by stretch or intracellular calcium signaling (Beckel et al., 2015). In the urethra, serotonergic and cholinergic paraneurons (also termed neuroendocrine cells) may have a similar sensory function (Kullmann et al., 2018).
Activity of the Lower Urinary Tract During Storage and Voiding
Intravesical pressure measurements during bladder filling in both humans and animals reveal low and relatively constant bladder pressures when bladder volume is below the threshold for inducing voiding (Figure 3). The accommodation of the bladder to increasing volumes of urine is primarily a passive phenomenon dependent upon the intrinsic properties of the bladder smooth muscle and quiescence of the parasympathetic efferent pathway. Additionally, in some species urine storage is also facilitated by sympathetic reflexes that mediate an inhibition of bladder activity, closure of the bladder neck, and contraction of the proximal urethra (de Groat & Lalley, 1972). During bladder filling, activity of the sphincter electromyogram (EMG) also increases reflecting an increase in efferent firing in the pudendal nerve and an increase in outlet resistance that contributes to the maintenance of urinary continence (Figure 3). These storage reflexes are mediated by spinal reflex pathways (Figure 4).
The storage phase of the urinary bladder can be switched to the voiding phase either involuntarily or voluntarily (Figure 3). The former is readily demonstrated in the human infant (Figure 3A) when the volume of urine exceeds the micturition threshold. At this point, increased afferent firing from tension receptors in the bladder produces firing in the sacral parasympathetic pathways and inhibition of sympathetic and somatic pathways (Figure 4B). The elimination phase consists of an initial relaxation of the urethral sphincter followed by a contraction of the bladder, an increase in bladder pressure, and flow of urine. Relaxation of the urethral outlet is mediated by activation of a parasympathetic reflex pathway to the urethra that releases nitric oxide as well as by removal of sympathetic and somatic excitatory inputs to the urethra. Voiding reflexes are mediated by a supraspinal pathway (Figure 4B).
In animals, voiding is not only initiated in response to bladder distension but also to mark territory (i.e., scent marking) (Hou et al., 2016; Keller et al., 2018). In rats and mice both types of voiding are characterized by intermittent urine flow generated by rhythmic contractions and relaxations of the EUS evident in EUS EMG recordings as bursting activity occurring at a frequency of 5–7 Hz. This EUS EMG activity facilitates urine flow and increases voiding efficiency. EUS bursting is generated by reflex mechanisms in the L3–L4 segments of the lumbar spinal cord and is facilitated by supraspinal pathways (Chang, Cheng, Chen, & de Groat, 2007; Keller et al., 2018).
Overview of the Organization of the Central Nervous System Circuitry Controlling the Lower Urinary Tract
The neural pathways controlling LUT function are organized as reflex and voluntary on–off switching circuits that maintain a reciprocal relationship between the urinary bladder and urethral outlet. Pathways involved in urine storage and voiding are organized at different levels of the central nervous system (Figure 4). Studies by Barrington (Barrington, 1925, 1933) beginning in the early part of the 20th century and later by Langworthy (Langworthy, Kolb, & Lewis, 1940) and by Tang (Tang, 1955) revealed that an essential part of the circuitry underlying reflex micturition in cats is located in the rostral brain stem, while storage mechanisms are mediated by pathways in the forebrain and spinal cord. Their experiments showed that decerebration at the level of inferior colliculus or brain lesions at various sites above the inferior colliculus facilitated reflex voiding while damage to an area slightly more caudal in the rostral pontine tegmentum (termed the pontine micturition center, PMC, or Barrington’s nucleus) eliminated reflex voiding. Furthermore, transection of the neuraxis at any point caudal to the PMC produced a similar block of voiding but preserved storage reflexes. Thus, it was concluded that the PMC contains circuitry essential for the generation of reflex bladder contractions and that this circuitry receives tonic inhibitory input from sites in the forebrain.
Transneuronal pseudorabies virus (PRV) tracing and brain imaging in animals and humans provided further support for the role of the PMC in lower urinary tract function and identified other groups of neurons in the brain that are involved in the control of bladder (Nadelhaft & Vera, 2001; Nadelhaft, Vera, Card, & Miselis, 1992; Sugaya, Roppolo, Yoshimura, Card, & de Groat, 1997), urethra (Vizzard, Erickson, Card, Roppolo, & de Groat, 1995), and the external urethral sphincter (Marson, 1997; Nadelhaft & Vera, 1996, 2001). In the brain stem of the rat, PRV labelled neurons are located in the PMC, PAG, medullary raphe nuclei, which contain serotonergic neurons; the locus coeruleus, which contains noradrenergic neurons, and the A5 noradrenergic cell group (Figure 5). More rostral regions in the hypothalamus (lateral medial preoptic and paraventricular nucleus), dorsal thalamus, the primary and secondary motor cortices and entorhinal and piriform cortices also exhibit virus-infected cells. In the cat, PRV tracing from the urinary bladder or the EUS identified a cluster of neurons extending from an area adjacent to the locus coeruleus ventrolaterally into the pontine reticular formation (de Groat et al., 1998).
Although transection of the spinal cord rostral to the lumbosacral level abolishes voluntary voiding, reflex bladder contractions and involuntary voiding begin to recover within a few weeks after transection. However, voiding is inefficient due to loss of coordination between the bladder and urethral sphincter (Figure 3C). Thus, in the absence of supraspinal control, spinal pathways are capable of generating reflex bladder contractions; but the circuitry mediating these contractions is markedly different than the circuitry in spinal intact animals (de Groat, 1997; de Groat et al., 1981; de Groat & Yoshimura, 2012). The emergence of this circuitry several weeks after spinal cord injury may be due in part to a reorganization of spinal reflex pathways in response to degeneration of supraspinal inputs.
Anatomy of the Spinal Circuitry Controlling the Lower Urinary Tract
The reflex circuitry controlling the lower urinary tract consists of four basic components: primary afferent neurons, spinal efferent neurons, spinal interneurons, and neurons in the brain that modulate spinal reflex pathways.
Afferent Projections in the Spinal Cord
Afferent pathways from the bladder project through the pelvic nerves into Lissauer’s tract at the apex of the dorsal horn in the caudal lumbosacral spinal cord and then send collaterals laterally and medially around the dorsal horn into laminae V–VII and X at the base of the dorsal horn (Figure 6A) (Morgan, Nadelhaft, & de Groat, 1981). The lateral pathway terminates in the region of the sacral parasympathetic nucleus and also sends some axons to the dorsal commissure (Figure 6A). Afferent projections from the EUS are similar to those of bladder afferents (Thor, Morgan, Nadelhaft, Houston, & de Groat, 1989).
Parasympathetic preganglionic neurons innervating the lower urinary tract are located in the intermediolateral grey matter (laminae V–VII) in the caudal lumbosacral segments of the spinal cord (Figure 6D); whereas sympathetic preganglionic neurons are located in medial (lamina X) and lateral sites (laminae V–VII) in the rostral lumbar spinal cord. Motoneurons innervating the striated urethral sphincter muscles are located in lamina IX in the ventral horn.
Interneurons involved in spinal reflex circuits controlling LUT functions have been identified by retrograde transneuronal transport following injection of pseudorabies virus (PRV) into the LUT of the rat (Nadelhaft et al., 1992; Vizzard et al., 1995). Large populations of interneurons are located just dorsal and medial to the preganglionic neurons as well as in the dorsal commissure and lamina I after infecting the bladder (Figure 6C), urethra or external urethral sphincter. A population of interneurons has also been identified in the dorsal commissure in the L3–L4 spinal segments after injecting PRV into the EUS or bladder of the rat (Karnup, Kim, & de Groat, 2017). As mentioned earlier, circuitry in this region of the spinal cord is essential for generating the EUS bursting during voiding in the rat.
The spinal neurons involved in processing afferent input from the lower urinary tract have been identified by the expression of the immediate early gene, c-fos (Figure 6B). In the rat, chemical or mechanical stimulation of the bladder and urethra increases the levels of Fos protein primarily in the dorsal commissure, the superficial dorsal horn, and in the area of the sacral parasympathetic nucleus (Figure 6B). Some of these interneurons make local connections in the spinal cord and participate in segmental spinal reflexes; whereas others send ascending projections to regions in the brain that are involved in the supraspinal control of lower urinary tract function (Birder, Roppolo, Erickson, & de Groat, 1999).
Spinal Tracts Connecting the Spinal Cord and Brain
Sensory pathways carrying information from the bladder to the brain are located in the superficial part of the dorsolateral funiculus in cats (Barrington, 1925, 1933) and in the most lateral part of the spinal cord midway between the anterior and posterior horns in humans (Nathan & Smith, 1951). Patients with bilateral lesions of the spinothalamic tract lose the normal sensation of bladder filling and the desire to void as well as the sensations of pain and temperature from the bladder and urethra (Nathan, 1956; Nathan & Smith, 1951). However, patients with unilateral lesions usually do not exhibit a change in bladder function or a change in the normal sensation of a full bladder (Nathan, 1956). In humans and cats the ascending pathways are partially crossed within the spinal cord. In cats the spinal tract neurons which are located in lamina I on the lateral side of the dorsal horn and in lateral lamina V and VI of the sacral spinal cord segments project bilaterally to the periaqueductal gray but not to the thalamus (Klop, Mouton, Kuipers, & Holstege, 2005). Neurons in this region also project to the hypothalamus in rats (Birder et al., 1999). Following damage to the cauda equina or the spinal cord below mid-lumbar level, afferent axons passing through prevertebral sympathetic nerves and the sympathetic chain to the rostral lumbar or caudal thoracic segments can also initiate sensations from the lower urinary tract (Nathan, 1956).
A second spinal pathway ascending from the pelvic viscera that initiates painful sensations is located in the dorsal columns (Al-Chaer, Feng, & Willis, 1998; Kuru, 1965; Willis, Al-Chaer, Quast, & Westlund, 1999). This pathway originates in spinal neurons in the region of the dorsal commissure and projects along the midline to make synaptic connections with neurons in the nucleus gracilis, which then relay information to the ventral posterior lateral nucleus of the thalamus. The pathway has been identified in various species using anatomical and electrophysiological techniques. In humans, destruction of this pathway in the dorsal columns has been effective in reducing cancer pain in the pelvic organs (Nauta et al., 2000).
Because some patients with complete spinal cord injury exhibit a vague perception of bladder filling, it has been suggested that the extraspinal sensory pathways passing through the vagus nerves may carry information from the bladder to the brain (Krhut et al., 2017). Evidence for this pathway was obtained through axonal tracing studies in rats which identified neurons in the nodose ganglia labeled via lipophilic tracers injected into the bladder wall (Herrity, Rau, Petruska, Stirling, & Hubscher, 2014). Brain imaging studies in patients with complete spinal cord injury revealed significant activity in several brain regions including the nucleus of the solitary tract, insular lobe, anterior cingulate gyrus and prefrontal cortex (Krhut et al., 2017). It was concluded that extraspinal sensory pathways may develop following spinal cord injury and that the vagus nerve may play a role in sensory re-innervation of the bladder.
Descending projections to the sacral spinal cord have been identified by injection of anterograde tracers into various regions of the cat brain (Figure 7). Tracers injected into the PMC labels terminals in the sacral parasympathetic nucleus, some of which make excitatory synaptic connections with preganglionic neurons. Tracer is also located on the lateral edge of the dorsal horn and the dorsal commissure (Blok, de Weerd, & Holstege, 1997; Blok & Holstege, 1997), areas containing dendrites of preganglionic neurons, sphincter motoneurons and afferent inputs from the bladder (Figure 7). Conversely, projections from neurons in the ventrolateral pons in the cat, an area identified as the pontine urine storage center (PUSC) (Kuru, 1965), terminate rather selectively in the sphincter motor nucleus (Figure 7) (Holstege, Griffiths, de Wall, & Dalm, 1986). Thus, the sites of termination of descending projections from the pons are optimally located to regulate reflex mechanisms at the spinal level. Tracer injected into the paraventricular nucleus of the hypothalamus labeled terminals in the sacral parasympathetic nucleus as well as the sphincter motor nucleus (Figure 7) (Holstege & Mouton, 2003).
In humans the descending tracts subserving conscious control of micturition and coordination of bladder function are located within the lateral columns at the level of the central canal in close association with the ascending sensory pathway throughout the length of the spinal cord (Nathan & Smith, 1958).
Organization of Urine Storage Reflexes
Sympathetic Storage Reflex
Surgical interruption or pharmacological blockade of the sympathetic innervation can reduce urethral outflow resistance, reduce bladder capacity and increase the frequency and amplitude of bladder contractions recorded under constant volume conditions (de Groat, Booth, & Yoshimura, 1993). Sympathetic reflex activity is elicited by a sacrolumbar intersegmental spinal reflex pathway that is triggered by bladder afferent activity in the pelvic nerves (de Groat & Lalley, 1972) (Figure 4A). This reflex pathway is inhibited when bladder pressure is raised to the threshold for producing micturition. The inhibitory response is abolished by transection of the spinal cord at the lower thoracic level, indicating that it originates at a supraspinal site, possibly the PMC (Figure 4B). Thus, the vesico-sympathetic reflex represents a negative feedback mechanism that allows the bladder to accommodate larger volumes during bladder filling but is turned off during voiding to allow the bladder to empty completely.
Urethral Sphincter Storage Reflex
Motoneurons innervating the striated muscles of the external urethral sphincter (EUS) exhibit a tonic discharge that increases during bladder filling (Thor & de Groat, 2010). This activity is mediated in part by a spinal reflex pathway (the guarding reflex) activated by low-level afferent input from the bladder (Figure 4A) and is evident as an increase in EUS EMG activity during bladder filling (Figure 3B).
Contractions of the EUS also induce afferent firing in the pudendal nerve that in turn activates spinal inhibitory mechanisms (de Groat et al., 2001; McGuire, Morrissey, Zhang, & Horwinski, 1983) that suppress preganglionic neurons and interneurons on the micturition reflex pathway (de Groat, 1978; de Groat, Booth, Milne, & Roppolo, 1982). Thus, this bladder-to-EUS-to-bladder reflex pathway represents a second negative feedback mechanism that promotes urinary continence. Activation of afferents in the pudendal nerve, some of which innervate the EUS, also elicits reflex contractions of the EUS and contribute to continence (Thor & de Groat, 2010). During micturition the firing of sphincter motoneurons and the negative feedback are inhibited. This inhibition can also be produced by electrical stimulation of the PMC (Kruse, Mallory, Noto, Roppolo, & de Groat, 1991; Kruse, Noto, Roppolo, & de Groat, 1990) and is weaker in chronic spinal cord transected animals (Thor & de Groat, 2010) indicating that it is dependent, in part, on supraspinal mechanisms.
Brain Stem Storage Mechanisms
Studies in cats also suggest that neurons in the pontine urine storage center (PUSC) (Figure 4A) promote continence by generating a tonic excitatory input to the EUS motoneurons (Holstege et al., 1986; Holstege & Mouton, 2003). Electrical stimulation in this region (Kuru, 1965; Kuru & Iwanaga, 1966) induces contractions of the EUS (Holstege et al., 1986; Koyama, Ozaki, & Kuru, 1966; Kuru, 1965).
Electrical stimulation of the PUSC also inhibits reflex bladder activity, increases bladder capacity and inhibits the excitatory bladder effect of PMC stimulation (Sugaya, Nishijima, Miyazato, & Ogawa, 2005). It has been proposed that PUSC neurons activate descending inhibitory pathways to the sacral parasympathetic nucleus (Sugaya et al., 2005), because neurons in this region send inputs to the nucleus raphe magnus (NRM) in the medulla which contains serotonergic neurons that project to the lumbosacral spinal cord. Chemical (Chen et al., 1993) or electrical stimulation in the NRM (Athwal et al., 2001; de Groat, 2002a; McMahon & Spillane, 1982; Morrison & Spillane, 1986; Sugaya et al., 1998) induces serotonergic inhibition of reflex bladder activity.
Organization of Voiding Reflexes
Role of PMC and Spinobulbospinal Micturition Reflex
Recordings of electrical activity in bladder efferent nerves (Figure 8) support the concept that the micturition reflex is mediated by a pathway passing through a switching center in the rostral pons. Stimulation of bladder afferent nerves evokes long latency discharges (120–150 ms) on bladder postganglionic nerves (Figure 8) in cats with an intact neuraxis or after supracollicular decerebration, but not after transection of the spinal cord at the thoracic level (de Groat, 1975; de Groat & Ryall, 1969). The evoked reflexes are unmasked by partial filling of the bladder to elicit a basal level of afferent firing. They also exhibit an unusual temporal facilitation in which the first stimulus during a train (0.5–1 Hz frequency) does not evoke a response, while the next few stimuli evoke gradually increasing responses (wind-up), eventually producing a self-sustaining micturition reflex (Figure 8). These observations indicate that even under optimal conditions with tonic afferent input from bladder mechanoceptors, electrical stimulation of bladder afferents only activates the micturition switching circuit after a delay of several seconds.
Bladder afferent nerve stimulation also evokes neuronal firing in the PMC at latencies ranging from 30–40 ms; and electrical stimulation in the PMC evokes bladder contractions and postganglionic nerve firing at latencies of 60–75 ms (de Groat, 1975; Noto, Roppolo, Steers, & de Groat, 1991). The sum of the latencies of the putative ascending and descending limbs of the reflex approximates the latency of the entire reflex pathway (120 ms). Pharmacological experiments in rats have revealed that glutamate is an essential transmitter in the ascending, pontine, and descending limbs of the spinobulbospinal micturition reflex pathway, as well as in spinal reflex pathways controlling the bladder and external urethral sphincter (Chang, Cheng, Chen, & de Groat, 2006; Yoshiyama & de Groat, 2005). Glutamatergic synaptic mechanisms involving both N-methyl-D-aspartate (NMDA) and non-NMDA receptors appear to interact synergistically to mediate transmission in these pathways.
Anterograde axonal tracing studies in cats revealed that neurons in the PMC make excitatory synaptic connections with bladder preganglionic neurons in the sacral spinal cord (Blok & Holstege, 1997). However, electrophysiological experiments in cats in which EPSPs were evoked in bladder preganglionic neurons by stimulation of the PMC indicate that the descending pathway from the PMC to bladder preganglionic neurons is polysynaptic and strongly facilitated during the micturition reflex (Sasaki & Sato, 2013). The latter finding is consistent with other studies in cats indicating that the descending PMC-spinal cord limb of the micturition reflex requires afferent feedback from the bladder to induce large-amplitude bladder contractions and that it can be modulated at the spinal level possibly at an interneuronal site by segmental afferent inputs (Kruse et al., 1991; Kruse, Mallory, Noto, Roppolo, & de Groat, 1992).
Properties of Neurons in the PMC
Single unit recording in the PMC of the cat (Figure 9) (de Groat et al., 1998; Koshino, 1970; Sasaki, 2002, 2004, 2005; Sugaya et al., 2005; Sugaya et al., 2003; Tanaka, Kakizaki, Shibata, Ameda, & Koyanagi, 2003) and rat (Elam, Thoren, & Svensson, 1986; Willette, Morrison, Sapru, & Reis, 1988) with the bladder distended under isovolumetric conditions revealed several populations of neurons exhibiting firing correlated with reflex bladder contractions including: (1) neurons that are silent in the absence of bladder activity but fire prior to and during reflex bladder contractions (direct neurons, 21%) (Figure 9A), (2) neurons that are active during the period between bladder contractions and are inhibited during contractions (inverse neurons, 51%) (Figure 9B) and (3) neurons that fire transiently at the beginning of bladder contractions (on-off neurons, 4%). Tonic firing that was not correlated with bladder activity was also identified in a large percentage (25%) of PMC neurons (termed independent neurons) (Figure 9C). All of these neurons are localized primarily in the region of the locus coeruleus complex.
Subpopulations of direct and inverse neurons in the cat have also been identified based on slow changes in firing during and between bladder contractions (Sasaki, 2004). Approximately 50% of direct neurons exhibit tonic firing between bladder contractions; whereas the remainder are quiescent until 0.5–1.2 seconds prior to a bladder contraction. The majority of inverse neurons (84%) stop firing during a bladder contraction after a delay of 4–11 seconds; whereas a small number exhibit only a reduction in firing. A large percentage of direct neurons project to the lumbosacral spinal cord (Sasaki, 2002; Sugaya et al., 2003); whereas only a small percentage of inverse neurons send projections to the cord. Thus, it has been speculated that inverse neurons function as local inhibitory neurons in the PMC. Both direct and inverse neurons exhibit excitatory synaptic responses to electrical stimulation of afferent axons in the pelvic nerve (de Groat et al., 1998). Direct neurons fire at a mean latency of 62 ms after a stimulus; whereas inverse neurons fire at a shorter latency of 25–30 ms followed by an inhibition at a latency of 80 ms and then a late excitation at 250–300 ms.
In the rat 79% of neurons in the PMC (Barrington’s nucleus) are activated by bladder distension, consistent with its role as a pontine micturition center (Rouzade-Dominguez, Miselis, & Valentino, 2003). The majority of bladder-responsive neurons (73%) are also activated by colon distension, although no neurons were selectively activated by distension of the colon. These data support proposals based on PRV tracing experiments that neurons in the PMC may coordinate the functions of multiple pelvic organs and not only those of the lower urinary tract (Rouzade-Dominguez et al., 2003; Valentino, Kosboth, Colflesh, & Miselis, 2000; Vizzard, Brisson, & de Groat, 2000).
Approximately half of the neurons in the rat and mouse PMC express corticotrophin releasing hormone (CRH) (Rouzade-Dominguez et al., 2003; Valentino et al., 1994; Verstegen, Vanderhorst, Gray, Zeidel, & Geerling, 2017); and it is estimated that slightly more than half of PMC neurons projecting to the lumbosacral spinal cord in the mouse express CRH (Verstegen et al., 2017). CRH negative neurons express estrogen-type 1 receptor (Esr1) (Keller et al., 2018). Both types of neurons express type 2 vesicular glutamate transporter (VGLUT2), a marker for glutamatergic excitatory neurons, indicating that two excitatory pathways project from the PMC to the sacral parasympathetic nucleus. Optogenetic stimulation of CRH positive neurons in the PMC causes a bladder contraction in anesthetized mice (Hou et al., 2016), but in awake mice produces weak voiding and inconsistent urethral sphincter bursting (Keller et al., 2018). On the other hand, optogenetic stimulation of the Esr-1 positive neurons in awake male mice produces bladder contractions, sphincter bursting and efficient voiding. Tracing experiments revealed that CRH and Esr1 neurons in the mouse PMC project to the SPN and DCM in the L6–S1 spinal cord but only Esr1 neurons project to the DCM in the L3–L4 spinal cord, which has been identified as the location of the EUS bursting center in the rat (Chang et al., 2007). Thus, it has been proposed that activation of Esr1 neurons in the PMC triggers micturition, while the function of CRH neurons is uncertain (Keller et al., 2018).
Axonal tracing studies revealed that the rat PMC receives inputs from the ventrolateral PAG, lateral hypothalamic and the preoptic area (Valentino et al., 1994); however, more recent studies in the mouse that identified extensive dendritic bundles of CRH positive neurons extending beyond the borders of the PMC raise the possibility that these neurons receive additional inputs from other areas of the brain (Verstegen et al., 2017).
Role of the PAG
Early studies in cats (Gjone, 1966; Kabat, Magoun, & Ranson, 1936; Koyama, Makuya, & Kuru, 1962; Langworthy & Kolb, 1935; Skultety, 1959) revealed that stimulation at sites in the PAG could either excite or inhibit bladder activity. The effects of stimulation were dependent on the state of the bladder. For example, when stimulation was applied with the bladder partially full and relatively inactive, excitatory effects were commonly elicited; however when the bladder was full and exhibiting large amplitude reflex contractions, stimulation at the same site produced inhibition. Reflex bladder activity was also enhanced by elimination of parts of the PAG by focal lesions or serial transections through the mesencephalon (Langworthy & Kolb, 1933; Ruch & Tang, 1956; Tang, 1955). This finding raised the possibility that a mesencephalic bladder inhibitory center tonically controlled micturition. An inhibitory region seems to be located in the dorsolateral margin of the rostral PAG (Numata et al., 2008) because chemical or electrical stimulation at this site inhibits reflex bladder contractions as well as the contractions induced by electrical stimulation of the PMC. Injection of bicuculline, a GABAA receptor antagonist, into the PMC blocks the PAG induced inhibition of PMC stimulation, indicating that GABA is the transmitter in the inhibitory pathway (Numata et al., 2008).
Other sites in the PAG seem to have a facilitatory role in micturition. Electrical stimulation in the ventrolateral region of the PAG evokes bladder contractions (Matsuura, Downie, & Allen, 2000; Noto, Roppolo, Steers, & de Groat, 1989; Taniguchi et al., 2002) and firing of bladder postganglionic nerves (Noto et al., 1991); while injections of cobalt chloride, a synaptic inhibitory agent, or an opioid receptor agonist (Matsumoto, Levendusky, Longhurst, Levin, & Millington, 2004) into this region suppresses reflex micturition. These data raise the possibility that the ventrolateral PAG is an essential component of the micturition reflex.
Electrical recordings in the PAG indicate that it may serve as a relay and coordinating center on the ascending limb of the micturition reflex pathway. In the rat electrical stimulation of bladder afferents in the pelvic nerve elicits negative field potentials in the dorsal PAG at a mean latency of 13 ms, which is considerably shorter than the mean latency of field potentials in the region of the PMC (42 ms) (Noto et al., 1989). In the cat a similar difference between latencies of pelvic afferent evoked field potentials in the PAG (11 ms) (Duong, Downie, & Du, 1999) and PMC in the (30–40 ms) (de Groat, 1975) has also been noted.
Subsequent studies in the cat and rat provided further support for the idea that bladder afferent information is relayed through the PAG. Axonal tracing studies in the cat revealed that spinal tract neurons located in lamina I on the lateral edge of the sacral dorsal horn, a region receiving primary afferent input from the bladder (Morgan et al., 1981), send a prominent direct axonal input through the lateral funiculus to the PAG (Blok, Deweerd, & Holstege, 1995; Holstege & Mouton, 2003) (Figure 4B). Injection of retrograde tracers into the lateral funiculus at the lumbar level labels the same group of sacral spinal tract neurons (de Groat et al., 1981). The PMC, on the other hand, receives a weaker input directly from the spinal cord and this input does not terminate on the PMC output neurons that send information back to the sacral parasympathetic nucleus. Axonal tracing in the cat also identified projections from the PAG to the PMC (Blok & Holstege, 1994; Kuipers, Mouton, & Holstege, 2006), raising the possibility that ascending afferent information from the bladder is relayed through synapses in the PAG to the PMC. Thus it has been proposed that the PAG has an essential role in the spinobulbospinal micturition reflex pathway (Holstege & Mouton, 2003; Noto et al., 1989).
Experiments in cats (Takasaki, Hui, & Sasaki, 2011), however, have raised questions about the importance of the PAG in reflex micturition. When the mesencephalon was serially transected at various levels that interrupt the connections between the PAG and the PMC, reflex bladder contractions persisted after transections at rostral levels that eliminated connections with the dorsal half of the PAG. Reflex micturition also persisted after more caudal transections that eliminated connections with both the dorsal and ventral half of the PAG or eliminated the most rostral part of the PMC. On the other hand, transections caudal to the PMC abolished reflex micturition. It was concluded that the PAG does not have an essential role in reflex micturition but rather is involved in transmitting bladder filling information to higher brain centers. Subsequently, the techniques used in the transection experiments were questioned by other investigators (Stone, Coote, Allard, & Lovick, 2011) who noted that the transections were often incomplete and a part of the caudal ventrolateral PAG was preserved in some experiments.
In the rat, the role of the PAG is even less clear because prominent ascending projections from the lumbosacral spinal cord have been detected in the PMC as well as the PAG (Blok & Holstege, 2000; Ding et al., 1997). Thus the organization of the ascending limb of the micturition reflex is uncertain and may vary in different species. Brain imaging studies (Tai et al., 2009) in the rat revealed that neuronal activity in the PAG increases during slow bladder filing (Figure 10) indicating that afferent activity from the bladder is received and processed in the PAG prior to micturition; however a similar signal was not detected in the PMC during filling (Figure 10). On the other hand during micturition, signals were detected in the PAG and the PMC. Similar results have been reported during brain imaging in humans (Figure 11). These results suggest that the PAG in the rat serves as a relay station for transmitting afferent information from the bladder to the PMC but that the switch from urine storage to voiding occurs in the PMC.
Properties of Neurons in the PAG
Single unit recordings in the PAG and adjacent mesencephalic reticular formation in decerebrate unanesthetized cats during rhythmic reflex bladder contractions under isovolumetric conditions revealed firing patterns similar to those recorded in the PMC including: (1) tonic storage neurons that are partially inhibited during bladder contractions (43%), (2) phasic storage neurons that are completely inhibited during bladder contractions (15%), similar to inverse neurons in the PMC, (3) phasic micturition neurons that are only active during micturition (13%) (Liu et al., 2004), similar to the activity of direct neurons in the PMC. A fourth type of neuron (29%), classified as tonic micturition neurons that are active throughout storage and micturition but increase their firing during bladder contractions, may be similar to the transient neurons identified in the PMC. Among the 84 neurons recorded in this study, 16 were located in the PAG and the remainder were located just ventral to the PAG. Storage neurons in the PAG seem to be located in the middle part of the PAG, whereas micturition neurons are distributed in a broader area.
The PMC–PAG switch
Pharmacological studies indicate that circuitry in the PMC and PAG allows the spinobulbospinal micturition reflex pathway to function as a switch that is either in a completely “off” mode (storage) or maximally “on” mode (voiding). Injections of excitatory amino acids into the PMC (Mallory, Roppolo, & de Groat, 1991) or PAG (Taniguchi et al., 2002) evoke bladder contractions in cat and rat. On the other hand, microinjections of low doses of inhibitory agents such as GABAA receptor agonists (muscimol) or opioid peptides at these sites increases the bladder volume threshold for inducing micturition without altering the magnitude of the micturition reflex measured as the amplitude of voiding contractions (Mallory et al., 1991; Matsumoto et al., 2004; Noto et al., 1991; Stone et al., 2011). Conversely, injections of GABAA receptor (bicuculline) or opioid receptor (naloxone) antagonists reduce the bladder volume threshold, indicating that tonic activation of inhibitory receptors in these centers can alter the set point of the micturition switch (Mallory et al., 1991; Noto et al., 1991; Stone et al., 2011). Because pharmacologic modulation of the PAG circuitry clearly alters the bladder volume threshold, it seems reasonable to conclude that PAG input to the PMC switching circuit also regulates the set-point for the micturition switch. A model of the PAG–PMC circuitry that generates an all-or-none micturition reflex response and efficient voiding resulting in complete bladder emptying has been published (de Groat & Wickens, 2013).
Forebrain Control of Micturition
Lesioning and electrical stimulation studies indicate that voluntary control of micturition depends on connections between the frontal cortex and other forebrain structures, including the anterior cingulate gyrus, insula, amygdala, bed nucleus of the stria terminalis and septal nuclei, where electrical stimulation elicits excitatory bladder responses (Andersson & Pehrson, 2003; de Groat et al., 1993). Damage to the cerebral cortex, due to tumors, aneurysms or cerebrovascular disease, appears to remove inhibitory control of the pontine micturition center, resulting in bladder overactivity. Pharmacological studies in rats indicate that decerebration or brain damage induced by occlusion of the middle cerebral artery induces bladder overactivity in part by eliminating tonic glutamatergic and dopaminergic inhibitory mechanisms and unmasking a dopaminergic excitatory mechanism (Yokoyama, Yoshiyama, Namiki, & de Groat, 2002).
Human brain and animal imaging studies using various methods have examined the areas of the brain involved in the control of micturition (Athwal et al., 2001; Blok, Sturms, & Holstege, 1998; Fowler, 2006; Fowler & Griffiths, 2010; Griffiths, Tadic, Schaefer, & Resnick, 2007; Griffiths & Fowler, 2013; Kavia, Dasgupta, & Fowler, 2005). Some studies evaluated the brain areas responsible for the perception of bladder fullness and the sensation of the desire to void during bladder filling (Athwal et al., 2001; Tadic, Tannenbaum, Resnick, & Griffiths, 2013). Others have examined brain activity during micturition (Blok et al., 1998; Blok, Willemsen, & Holstege, 1997; Nour, Svarer, Kristensen, Paulson, & Law, 2000) or voluntary contractions of the pelvic floor during urine withholding (Blok, Willemsen, et al., 1997). During urine storage, activation occurs in the PAG, thalamus, insula, prefrontal cortex, anterior cingulate, pons, medulla and supplementary motor area (Figure 11). These results are consistent with the notion that the PAG receives information about bladder fullness and then relays this information (possibly through the thalamus) to other brain areas involved in the control of urine storage. The insula, where normal visceral sensations such as desire to void are thought to be mapped, is regarded as a key center for processing bladder afferent input. During voiding activation occurs in the prefrontal cortex, insula, hypothalamus, PAG and in a region of the dorsal pons (Figure 11), comparable to the location of the PMC in rats (Figure 10) and cats.
The importance of the prefrontal cortex in bladder control was established in clinical studies (Andrew & Nathan, 1964) showing that lesions located in white matter tracts in the medial prefrontal regions between the superior frontal gyrus and the cingulate gyrus clinically were associated with long-term bladder dysfunctions including urgency, frequency and sometimes incontinence. Andrew and Nathan (Andrew & Nathan, 1964, 1965) speculated that cortical control of micturition might be mediated by pathways passing through septal and hypothalamic nuclei. However more recent anatomical studies in rats (Valentino, Miselis, & Pavcovich, 1999; Valentino et al., 1994) revealed direct pathways to the PMC from the motor, insular and infralimbic cortices, the bed nucleus of the stria terminalis, a region near the medial amygdaloid nucleus as well as from the hypothalamus and PAG. Thus, multiple forebrain circuits may interact at the level of the PMC to control micturition.
Spinal Micturition Reflex Pathways
Micturition can be induced by two spinal reflex mechanisms. In neonatal animals of many species the adult form of reflex voiding induced by bladder distension is not functional and voiding is elicited by stimulation of cutaneous afferent input when the mother licks the perineal region of the young animal during grooming. This micturition reflex is organized in the lumbosacral spinal cord and is downregulated during postnatal development but reemerges in adults within one to weeks after supralumbar spinal cord injuries (de Groat, 2002b) which eliminates voluntary and supraspinal control of voiding.
Spinal cord injury initially produces an areflexic bladder and complete urinary retention followed by a slow development of automatic micturition and bladder hyperactivity (Figure 3C) mediated by spinal reflex pathways. However, voiding is commonly inefficient due to simultaneous contractions of the bladder and urethral sphincter (bladder–sphincter dyssynergia) (Figure 3C). Electrophysiological studies in animals have shown that after spinal cord injury the micturition reflex pathway has a different afferent limb and occurs with a shorter central delay (15–30 ms versus 60–75 ms). The afferent limb of the spinal micturition reflex in cats with chronic spinal transection consists of unmyelinated (C-fiber) axons (Cheng, Liu, Chang, Ma, & de Groat, 1999; de Groat et al., 1990; de Groat et al., 1981) (Figure 12); whereas in cats with an intact spinal cord, myelinated (A-δ) afferents activate the micturition reflex (de Groat et al., 1981; de Groat & Ryall, 1969) (Figure 12). In normal cats capsaicin, a neurotoxin known to disrupt the function of C-fiber afferents, does not block the A-δ-afferent fiber evoked reflex. However, in cats with chronic spinal injury capsaicin completely blocks the C-fiber afferent-evoked reflex (Cheng et al., 1999). The emergence of C-fiber evoked bladder reflexes seems to be mediated by several mechanisms including changes in central synaptic connections and alterations in the properties of the peripheral afferent receptors leading to sensitization of the C-fiber afferents and the unmasking of mechano-sensitivity (de Groat & Yoshimura, 2006, 2012). In acute spinal cord transected cats, bladder reflexes can also be unmasked by sensitizing the C-fiber afferents with intravesical infusion of acetic acid (Xiao et al., 2014).
While much is known about the physiology of the lower urinary tract and its neural control, less is known about pathophysiology and the mechanisms underlying many types of lower urinary tract dysfunctions including: (1) urgency, urinary frequency and urge incontinence termed collectively overactive bladder; (2) underactive bladder characterized by failure to completely empty the bladder; (3) interstitial cystitis/bladder pain syndrome, which involves chronic inflammation of the bladder and painful urges to void; (4) neurogenic disorders that arise from damage or dysfunction of the central neural pathways after spinal cord injury, stroke, Parkinson disease or multiple sclerosis. Current pharmacological treatments for these disorders that primarily target symptoms rather the pathophysiological mechanisms exhibit limited efficacy. Thus, development of new therapies to treat lower urinary tract dysfunctions is a high priority.
Animal models of lower urinary tract disorders are used to test new therapies but clinical relevance of existing models is questionable due to the uncertainty about the underlying mechanisms of lower urinary tract symptoms and differences in voiding function in animals and humans. For example, animal models of overactive bladder commonly involve intravesical instillation of irritating substances such as acetic acid or capsaicin that activate sensory nerves. However, these models assume that the cause of overactive bladder is related to increased excitability of peripheral afferent nerves, a condition that has not yet been confirmed in patients. The recent development of large scale genomic and proteomic characterization of patient samples may help with the creation of better animal models. Paired with recent advances in genomic editing, such as CRISPR/Cas9, it should soon be possible to specifically induce in animals the genomic changes that underlie the lower urinary tract dysfunctions occurring in patients and thereby allow identification of new therapies in animal models that more accurately predict clinical efficacy.
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