W.C. de Groat

Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1

In the urinary bladder, the capsaicin-gated ion channel TRPV1 is expressed both within afferent nerve terminals and within the epithelial cells that line the bladder lumen. To determine the significance of this expression pattern, we analyzed bladder function in mice lacking TRPV1. Compared with wild-type littermates, trpv1−/− mice had a higher frequency of low-amplitude, non-voiding bladder contractions. This alteration was accompanied by reductions in both spinal cord signaling and reflex voiding during bladder filling (under anesthesia). In vitro, stretch-evoked ATP release and membrane capacitance changes were diminished in bladders excised from trpv1−/− mice, as was hypoosmolality-evoked ATP release from cultured trpv1−/− urothelial cells. These findings indicate that TRPV1 participates in normal bladder function and is essential for normal mechanically evoked purinergic signaling by the urothelium.

Mechanisms underlying the recovery of urinary bladder function following spinal cord injury

Micturition in cats and rats with an intact neuraxis is dependent upon a spinobulbospinal reflex activated by A delta bladder afferents. This report describes changes in micturition reflexes 2 h to 14 weeks following spinal cord transection at the lower thoracic level. In acute spinal cats micturition reflexes were blocked, however, several weeks after transection, a long latency (180-200 ms) spinal reflex could be activated by C-fiber bladder afferents. This reflex was blocked by capsaicin in doses (20-30 mg/kg, s.c.) that did not affect micturition reflexes in intact cats. Micturition reflexes were unmasked in acute spinal and facilitated in chronic spinal cats by naloxone, an opioid antagonist. Spinal neurons and axons containing opioid peptides were more prominent below the level of transection in chronic spinal cats. VIP, a putative neurotransmitter in C-fiber bladder afferents, inhibited micturition reflexes when injected intrathecally (2-10 micrograms) in intact cats but facilitated micturition reflexes in spinal cats (doses 0.1-1 micrograms, i.t.). VIP-containing C-fiber afferent projections to lamina I of the sacral spinal cord expanded in spinal cats. Thus VIP afferents may have an important role in the recovery of bladder reflexes after spinal injury. Paraplegic animals also exhibit bladder-sphincter dyssynergia, which causes functional outlet obstruction. Studies in rats have revealed that outlet obstruction induced by partial urethral ligation facilitates spinal micturition reflex pathways and causes an expansion of HRP-labelled bladder afferent projections in the spinal cord. These findings raise the possibility that the alterations in central reflex connections in paraplegic animals may be induced in part by changes in peripheral afferent input secondary to outlet obstruction.(ABSTRACT TRUNCATED AT 250 WORDS)

Reflexes to sacral parasympathetic neurones concerned with micturition in the cat

1. Reflexes to sacral parasympathetic neurones were studied by electrophysiological techniques in decerebrate, in chloralose‐anaesthetized, and in chronic spinal cats.

NEURAL CONTROL OF URETHRAL OUTLET ACTIVITY IN VIVO: ROLE OF NITRIC OXIDE

The present study investigated the role of nitric oxide (NO) in the reflex changes in urethral outlet activity during micturition. Isovolumetric bladder contractions, urethral pressure and external urethral sphincter electromyogram (EUS EMG) activity were recorded independently in urethane-anesthetized rats. During reflex bladder contractions, the urethra exhibited reflex responses characterized by an initial decrease in urethral pressure in conjunction with a rise in bladder pressure. This was followed by a period of high frequency oscillations (HFOs) associated with maximal urethral relaxation and burst type EUS EMG activity. Administration of N-nitro-L-arginine (L-NOARG) 10 mg./kg. intravenously, a nitric oxide synthase inhibitor, reversibly decreased the magnitude (62%, p < 0.05) and duration (40%, p < 0.05) of reflex urethral relaxation (N = 7). In 4 additional experiments, L-NOARG (10 to 15 mg./kg. intravenously) completely eliminated reflex urethral relaxation during micturition, and this effect was reversed in all animals by the administration of L-arginine (100 to 150 mg./kg. intravenously). Administration of N-nitro-D-arginine (D-NOARG) (10 to 30 mg./kg. intravenously) had no effect on reflex urethral relaxation. Neuromuscular blockade (vecuronium bromide 5 mg./kg. intravenously) reversibly decreased resting urethral pressure and eliminated the HFOs. The urethral smooth muscle relaxation that remained after neuromuscular blockade was eliminated following administration of L-NOARG (10 mg./kg. intravenously) in 2 of 3 animals. These results suggest that reflex urethral responses during micturition involve changes in both smooth and striated muscle activity, and that the predominant neurotransmitter mechanisms that mediate reflex urethral smooth muscle relaxation involve NO.

Pontine control of the urinary bladder and external urethral sphincter in the rat

Neurons in the rostral pontine tegmentum are known to have an important role in controlling micturition. The present experiments used urethane anesthetized rats to examine the effects of electrical stimulation at various sites in the pons on bladder and external urethral sphincter activity and on the volume threshold for inducing micturition. Stimulation with short trains of pulses (50 Hz, 1-3 s trains, 1-15 V) in the laterodorsal tegmental nucleus (LDT), the periaqueductal grey (PAG) or the lateral parabrachial nucleus (L-PBN) elicited contractions of a partially filled, quiescent bladder. However stimulation during a bladder contraction aborted the contraction indicating that these areas have inhibitory as well as excitatory effects. Continuous stimulation (50 Hz) in the PAG or L-PBN during a cystometrogram decreased bladder capacity (mean decrease 36%). Conversely, continuous stimulation in the pontine reticular formation (in or near the dorsal subcoeruleus nucleus and medial parabrachial nucleus) increased bladder capacity (mean increase 50%). Stimulation at pontine sites (LDT, PAG and L-PBN) which elicited bladder contractions also elicited an increase in external urethral sphincter activity. A similar increase in urethral sphincter activity occurred during reflex micturition induced by bladder distension. These data suggest that bladder capacity and the coordination of bladder and external urethral functions are controlled by various neuronal populations in the rostral pons of the rat.

Parasympathetic preganglionic neurons in the sacral spinal cord

Two types of preganglionic neurons have been identified in the sacral parasympathetic nucleus (SPN) of the cat. These neurons could be differentiated by various characteristics including axonal conduction velocities, morphology, location in the nucleus, organ of innervation and central reflex mechanisms controlling their activity. Neurons having myelinated axons (B-PGNs) with conduction velocities between 3.3 and 13 m/s were located in the lateral band of the SPN and innervated the urinary bladder. Neurons with unmyelinated axons (C-PGNs) with conduction velocities of 0.5-1.4 m/s were located in the dorsal band of the nucleus and innervated the large intestine. B-PGNs were excited by distention of the bladder and inhibited by distension or mechanical stimulation of the intestine, whereas C-PGNs exhibited the opposite responses to these stimuli. C-PGNs often exhibited a low level of spontaneous discharge in absence of stimulation but exhibited marked firing (3.5-10 spikes/s) during a defecation reflex elicited by mechanical stimulation of the rectum-anal canal. The excitatory responses were elicited by C-fiber afferents via a spinal reflex pathway. B-PGNs were inactive when intravesical pressure was below the threshold for inducing micturition (5 cm H2O) but raising the pressure above the threshold induced firing consisting of repetitive bursts of action potentials occurring at relatively high frequencies (15-60 spikes/s). These bursts coincided with rhythmic bladder contractions. The frequency of bladder contractions and associated bursts of PGN-firing and the mean PGN-firing rate (2-8 spikes/s) increased as intravesical pressure was increased in steps between 5 and 30 cm H2O. However, as indicated by interspike interval histograms, the frequency of firing within a burst of action potentials was unchanged. It is concluded that the micturition reflex pathway is organized as a simple on-off switching circuit and that B-PGNs receive a maximal synaptic input when intravesical pressure exceeds the micturition threshold. This circuit was triggered by vesical A delta afferents via a spinobulbospinal pathway. Transection of the spinal cord interrupted the reflex pathway and blocked micturition. However, in chronic spinal animals a spinal reflex mechanisms emerged which contributed to the recovery of bladder function. This mechanism, which was weak or non-existent in animals with an intact neuraxis, exhibited a number of important differences from the normal micturition reflex, most notably being activated by a C-fiber afferent rather than a A delta afferent limb. The mechanism underlying the emergence of C-fiber evoked bladder reflexes in spinal animals is uncertain.

Reflex activation of sympathetic pathways to vesical smooth muscle and parasympathetic ganglia by electrical stimulation of vesical afferents.

1. Activation of the sympathetic input to the urinary bladder by electrical stimulation of afferent fibres in the pelvic nerve evoked three responses: (1) an initial transient rise in intravesical pressure, (2) an inhibition of neurally evoked bladder contractions and (3) an inhibition of transmission in vesical parasympathetic ganglia. Similar responses were elicited by stimulation of the hypogastric nerve. 2. The reflex responses were observed in acute spinal preparations (T10‐T12) and in cats with intact spinal cords, but were abolished by bilateral transection of the hypogastric nerves. 3. The inhibition of bladder contractions was antagonized by the administration of propranolol (200‐400 mug, I.A.), a beta‐adrenergic blocking agent. The inhibition of ganglionic transmission was antagonized by dihydroergotamine (30‐75 mug, I.A.), an alpha‐adrenergic blocking agent. The initial rise in intravesical pressure was not antagonized by either agent. 4. Electrical stimulation of other afferents (carotid sinus nerve and sciatic nerve) did not consistently elicit responses in the urinary bladder. However, stimulation of these afferents as well as pelvic nerve afferents evoked reflex firing in nerve filaments on the surface of the urinary bladder. The firing was abolished by transection of the ipsilateral hypogastric nerve. 5. It is concluded that stimulation of vesical afferents activates a spinal sympathetic reflex which results in closing of the internal urethral sphincter and a depression of bladder activity. The latter occurs by a direct depression of detrusor smooth muscle as well as a block of the neural input to the bladder. This vesico‐sympathetic reflex represents a negative feed‐back mechanism which may have an important role in the maintenance of urinary continence.

Neural Control of the Urethra

Coordination between the urinary bladder and the urethra is mediated by multiple reflex pathways organized in the brain and spinal cord. Some reflexes promote urine storage; whereas other reflexes facilitate voiding. During bladder filling, activation of mechanoreceptor afferent nerves in the bladder wall triggers firing in the cholinergic efferent pathways to the external urethral sphincter (EUS) and in sympathetic adrenergic pathways to the urethral smooth muscle. These storage reflexes are dependent upon interneuronal circuitry in the spinal cord. During voiding the spinal storage reflexes are inhibited by supraspinal mechanisms which originate in the pontine micturition center. Glutamatergic, serotonergic and alpha 1 adrenergic excitatory transmission as well as GABAergic/glycinergic inhibitory transmission have been implicated in the central control of sphincter reflexes. During voiding, a parasympathetic nitrergic inhibitory input to the urethral smooth is activated. This reflex mechanism which is triggered by bladder afferents persists in paraplegic rats and therefore must be mediated at least in part by spinal interneuronal circuitry. In female rats, the parasympathetic nitrergic pathway is prominent; but in male rats it is obscured by a dominant parasympathetic cholinergic excitatory input to the urethral smooth muscle. The function of the cholinergic pathway in voiding is uncertain. Stimulation of urethral afferents can also influence bladder activity. Contraction of the external urethral sphincter activates afferents that inhibit reflex bladder contractions; whereas infusion of fluid through the urethra facilitates bladder contractions. These reflexes are also organized in the spinal cord and presumably play a role in urine storage and elimination. Alterations in primitive bladder-to-urethra and urethra-to-bladder reflex mechanisms may contribute to neurogenic bladder dysfunction.Coordination between the urinary bladder and the urethra is mediated by multiple reflex pathways organized in the brain and spinal cord. Some reflexes promote urine storage; whereas other reflexes facilitate voiding. During bladder filling, activation of mechanoreceptor afferent nerves in the bladder wall triggers firing in the cholinergic efferent pathways to the external urethral sphincter (EUS) and in sympathetic adrenergic pathways to the urethral smooth muscle. These storage reflexes are dependent upon interneuronal circuitry in the spinal cord. During voiding the spinal storage reflexes are inhibited by supraspinal mechanisms which originate in the pontine micturition center. Glutamatergic, serotonergic and alpha, adrenergic excitatory transmission as well as GABAergic/glycinergic inhibitory transmission have been implicated in the central control of sphincter reflexes. During voiding, a parasympathetic nitrergic inhibitory input to the urethral smooth is activated. This reflex mechanism which is triggered by bladder afferents persists in paraplegic rats and therefore must be mediated at least in part by spinal interneuronal circuitry. In female rats, the parasympathetic nitrergic pathway is prominent; but in male rats it is obscured by a dominant parasympathetic cholinergic excitatory input to the urethral smooth muscle. The function of the cholinergic pathway in voiding is uncertain. Stimulation of urethral afferents can also influence bladder activity. Contraction of the external urethral sphincter activates afferents that inhibit reflex bladder contractions; whereas infusion of fluid through the urethra facilitates bladder contractions. These reflexes are also organized in the spinal cord and presumably play a role in urine storage and elimination. Alterations in primitive bladder-to-urethra and urethra-to-bladder reflex mechanisms may contribute to neurogenic bladder dysfunction.

The role of neuropeptides in the sacral autonomic reflex pathways of the cat.

Immunohistochemical and pharmacological studies were conducted to examine the origin and function of peptidergic nerves in the sacral autonomic system of the cat. Leucine-enkephalin (L-Enk) immunoreactivity was identified in nerve terminals in peripheral ganglia on the surface of the urinary bladder and in the parasympathetic nucleus in the sacral spinal cord. In colchicine-treated animals L-Enk was also detected in sacral preganglionic neurons (sPGN) identified by retrograde transport of a fluorescent dye. L-Enk terminals in bladder ganglia are believed to arise from sPGN since the terminals were eliminated by transection of the sacral ventral roots. Pharmacological studies indicated that exogenous as well as endogenously released enkephalins have an inhibitory action at both ganglionic and spinal sites in the sacral outflow to the urinary bladder. Peptides were also associated with afferents nerves in the sacral autonomic system. The distribution of substance P, VIP and cholecystokinin in the sacral dorsal horn paralleled the distribution of visceral afferent projections as demonstrated with HRP techniques. Dye labeling combined with immunohistochemistry revealed that some dorsal root ganglion cells projecting to the pelvic viscera contain substance P or VIP.

Excitatory and inhibitory influences on bladder activity elicited by electrical stimulation in the pontine micturition center in the rat

Electrical stimulation at various sites in the dorsal pontine tegmentum in urethane anesthetized rats modulated activity of the urinary bladder as well as efferent firing on bladder postganglionic nerves. Electrical stimulation (0.2 ms 50 Hz, 5-20 V or 30-150 microA, 2-5 s train duration) using a microelectrode (tip diameter, 10-20 microns) in an excitatory area located rostral and medial to the locus coeruleus evoked short latency (less than 2 s) large amplitude (greater than 20 cm H2O) bladder contractions and increased firing on the bladder postganglionic nerves. Stimulation at sites adjacent to the excitatory area inhibited bladder postganglionic nerve firing and bladder activity. Inhibitory responses were evident as either a decrease in intravesical pressure, an increased interval between bladder contractions, or an interruption or elimination of bladder contractions. The threshold intensity for excitation using a large electrode (2-4 V) was slightly higher than that for inhibition (1.5-2 V). The optimum sites for evoking bladder contractions were located in and close to the laterodorsal tegmental nucleus (LDT) and in the periaqueductal gray just dorsal or dorsolateral to the LDT. The extent of the area that induced bladder contractions was 0.5-1.2 mm in diameter in each rat when a microelectrode was employed for electrical stimulation. Electrical stimulation in the optimum site for evoking bladder contractions induced relatively little striated muscle activity and produced no short-latency blood pressure changes. The longer latency blood pressure changes associated with a spontaneous bladder contraction were still present following a stimulation of the dorsolateral pons. These data are consistent with the view that neurons in the dorsal pontine tegmentum play an important role in the regulation of urine storage as well as urine release.