the afferent limb of the micturition reflex presents an attractive target for pharmacological intervention in the treatment of bladder dysfunction , but the animal models currently available for testing potential therapeutic agents have limitations ( andersson , 2002 ) .
two types of in vitro preparations have been used to study bladder sensory activity in response to mechanical and chemical stimulation : an en block bladder / pelvic nerve preparation ( sengupta and gebhart , 1994 ; rong et al . , 2002 ) and
studies employing these models have contributed significantly to defining the peripheral neuronal mechanisms involved in bladder sensation .
studies focusing on central neuroregulation of the micturition reflex during bladder filling and micturition require an intact micturition reflex pathway and can therefore only be conducted in vivo .
animal models used to date require the use of anesthesia , which exerts its effects by enhancing inhibitory and/or suppressing excitatory neural output , likely compromising nerve activity recordings ( habler et al . , 1990 ; hara and harris , 2002 ; malley and vizzard , 2002 ) .
to address these limitations , we developed a new method for recording sensory nerve activity in vivo in the conscious animal .
we have adapted the ex vivo technique of multifiber sensory nerve recording to in situ recordings from the postganglionic bladder nerve in a conscious mouse
. bladder - specific afferent neural discharge and corresponding changes in intravesical pressure during bladder filling were recorded .
the validity of this model was tested by recording extracellular bladder sensory nerve activity in healthy animals and after inducing bladder overactivity with 0.5% acetic acid .
the functional contribution of c - fibers to the bladder sensory discharge during noxious / chemically induced bladder irritation was assessed using the trpv1 receptor antagonist , capsazepine ( daly et al . , 2007 ) .
male 6- to 8-week - old balbc mice ( charles river laboratories , st constant , qc , canada ) were maintained under standard laboratory conditions with free access to food and water .
all animal use procedures were approved by the university of vermont institutional animal care and use committee , and appropriate measures were taken to minimize pain and discomfort of the animals .
after anesthetizing mice with isoflurane , a lower midline abdominal incision was made and pe-10 tubing ( bd ) with a heat - flared end was implanted into the dome of the urinary bladder .
postganglionic bladder nerves running on the lateral aspect of the prostate were identified ( figure 1a ) , and a single branch was bluntly dissected approximately midway between the major pelvic ganglion ( mpg ) and the bladder neck . a 0.125-mm diameter , bipolar teflon - coated silver electrode ( wpi ,
sarasota , fl , usa , catalog number agt0510 ) with hook - shaped poles was positioned under the single postganglionic nerve .
isolation from surrounding tissues was achieved by slightly lifting the nerve and the electrode of the prostatic surface using a micromanipulator narishige mx-1 ( narishige scientific instrument lab . , tokyo , japan ) . after achieving a stable recording ,
the nerve - electrode complex was then embedded in a biocompatible silicone adhesive compound ( kwik - sil ; wpi , sarasota , fl , usa ; figure 1b ) .
this non - toxic compound secures the contact between the electrode and the nerve , anchors the nerve - electrode complex to the surrounding tissues , thereby preventing electrode movement and damage to the nerve .
the recording electrode and the ground reference electrode wire which was positioned within the abdominal cavity , were then externalized at the animal 's back and secured to paravertebral muscles with 6 - 0 nylon sutures .
the bladder tubing and the electrode were coiled into two separate subcutaneous pouches , and the animal was allowed to recover for 48  h. postoperative analgesia was maintained with buprenorphine ( 0.05  mg / kg s.c . ) .
analgesia was stopped 24  h prior to sensory nerve recording to prevent its suppressive effects on sensory neurotransmission .
( a ) postganglionic bladder nerves ( two branches are marked by black arrows ) running between the mpg and the bladder neck alongside the bladder vein ( marked by white arrow ) on the lateral aspect of the prostate ( total magnification 40 ) .
( b ) depiction of the teflon - coated silver electrode with exposed hook - shaped poles lying beneath the postganglionic bladder nerve ( marked by white arrow ) , embedded in the clear transparent silicone adhesive compound .
black arrow marks the 10 - 0 nylon ligature tied between the mpg and the recording electrode ( total magnification 30 ) .
the activity of sensory bladder nerves was recorded in situ in conscious animals . under brief inhalation anesthesia with isoflurane , pe-10 tubing and the electrode and ground wire
the mouse was placed in a flat bottom mouse restrainer lined with an absorbent pad ; the tubing and electrode were then connected to a pressure transducer ( grass model pt300 , quincy , ma , usa ) and a neurolog head stage ( nl100 , digitimer ltd , uk ) , respectively .
intravesical pressure was recorded and analyzed using labview software ( texas instruments , dallas , tx , usa ) .
electrical activity was transmitted through the head stage , amplified ( nl104 ) , captured and analyzed via a power 1401 interface and spike 2 software ( version 6.03 , ced , uk ) .
nerve activity was sampled at a rate of 25,000  hz and stored on a pc .
off - line analysis was performed using the spike 2 wavemark function ; action potentials were discriminated based on their waveform .
the animal was allowed to acclimate 1530  min before initiating continuous intravesical infusion of isotonic 0.9% nacl at a rate of 0.75  ml / h .
after three reproducible micturition cycles , the nacl solution was replaced by 0.5% acetic acid ( sigma chemical co. , st .
louis , mo , usa ) diluted in 0.9% saline . in a subsequent set of experiments , 10  m capsazepine ( sigma ) was added to the infusate after initial exposure to acetic acid .
intravesical pressure and sensory nerve activity were recorded simultaneously . at the conclusion of the sensory nerve activity recording ,
neurostimulation , as square - wave pulses ( 1.0  ms , 12  hz , 4  v ) , was applied through the bipolar silver electrode using a grass stimulator ( grass technologies , ma , usa ) . a segment of the postganglionic bladder nerve overlying the bipolar electrode was subsequently removed and evaluated histologically .
artifacts in sensory recordings caused by movement were eliminated by analyzing only that data obtained during intervals when the animal was not moving .
whole - nerve multifiber afferent activity was expressed as impulses per second ( hz ) and is referred to as frequency of afferent nerve discharge .
data was quantified using a spike processor , which counts the number of action potential units per second that cross a preset threshold .
the threshold value was set to the level of the smallest identifiable spike ( approximately twice the baseline noise level ) .
nerve activity is expressed as a mean frequency of firing obtained over the course of 10  s. quantitative analyses compare data from three areas of interest within the micturition cycle before and after pharmacological intervention : ( 1 ) at the lowest pressure point during the filling phase ( filling pressure ) , ( 2 ) at the voiding contraction threshold ( threshold pressure ) , and ( 3 ) at peak micturition pressure .
quantitative values are presented as the mean    sem , with differences between means determined using a paired student 's t - test .
the animal was continuously monitored and its physical activity was recorded . artifacts in sensory recordings caused by movement were eliminated by analyzing only that data obtained during intervals when the animal was not moving .
whole - nerve multifiber afferent activity was expressed as impulses per second ( hz ) and is referred to as frequency of afferent nerve discharge .
data was quantified using a spike processor , which counts the number of action potential units per second that cross a preset threshold .
the threshold value was set to the level of the smallest identifiable spike ( approximately twice the baseline noise level ) .
nerve activity is expressed as a mean frequency of firing obtained over the course of 10  s. quantitative analyses compare data from three areas of interest within the micturition cycle before and after pharmacological intervention : ( 1 ) at the lowest pressure point during the filling phase ( filling pressure ) , ( 2 ) at the voiding contraction threshold ( threshold pressure ) , and ( 3 ) at peak micturition pressure .
quantitative values are presented as the mean    sem , with differences between means determined using a paired student 's t - test .
during continuous filling with 0.9% nacl , cystometrograms showed two distinct patterns : ( a ) a steady intravesical pressure with pressure fluctuations of less than 2  cm h2o ( figure 2a ) , and ( b ) a filling phase with pressure fluctuations ranging from 2 to 10  cm h2o ( figure 2b ) . two distinct patterns recorded with cystometry : ( a ) steady intravesical pressure during the filling phase with minimal pressure fluctuations ; ( b ) phasic components ( small fluctuations in pressure ) during the entire filling phase .
intravesical infusion of 0.5% acetic acid evoked a significant reduction in intermicturition intervals coupled with an increase in average filling and threshold pressures .
the same mouse utilized for the basal and acetic acid urodynamic comparison was further challenged with a mixture of 0.5% acetic acid and 10  m capsazepine ( figure 3 ) . in the presence of capsazepine
, the bladder response to acetic acid was reduced as evidenced by an increase in intermicturition interval and a reduction in filling and threshold pressures .
0.5% acetic acid led to higher micturition frequency , multiple non - voiding contractions along with elevated filling , threshold and micturition pressures .
addition of capsazepine to the acetic acid solution reduced the number of non - voiding contractions and nerve activity .
the rectangles mark the points in the experiment where the infusate was changed ( m , micturition event ) .
continuous filling with saline in vivo generated a reproducible pattern of afferent nerve discharge ( n  =  8) .
in all animals , baseline sensory activity oscillated between 20 and 60  hz , and repeated bursts of afferent discharge were observed throughout the entire filling phase ( figure 2 ) . in some animals , low amplitude non - voiding bladder contractions ( 210  cm h2o ) seen throughout the filling phase correlated with phasic afferent discharge ( figure 2b ) .
phasic afferent discharge was also evident in a subgroup of animals that displayed only minimal fluctuations in intravesical pressure ( amplitude < 2  cm h2o ; figure 2a ) .
the terminal portion of the filling phase was characterized by a rapid increase in the frequency of sensory discharge that peaked at 97.4    20.0  hz once the bladder reached the threshold pressure .
the mean sensory nerve firing rate during micturition was 135.1    26.0  hz . in 20% of cases the experiment was unsuccessful due to excessive movement that lasted during entire recording .
intravesical infusion of 0.5% acetic acid led to a 1.9-fold increase in voiding frequency and reduced intermicturition intervals from 361.7    86.7  s to 188.7    27.8  s ( n  =  8) .
acetic acid induced 2.1-fold , 2-fold , and 2-fold increases in filling , threshold , and micturition pressures , respectively , that were associated with 2.8-fold , 2.4-fold , and 1.9-fold increases in excitatory afferent nerve discharge ( figure 4a ) .
( a ) barr graphs summarizing the data in filling ( fp ) , threshold ( tp ) , and micturition pressures ( mp ) ( n  =  8) .
a significant increase in all three parameters was observed following the infusion of 0.5% acetic acid .
the ( * ) mark a significant difference between 0.9% nacl and 0.5% acetic acid , ( ) indicate significant difference between 0.5% acetic acid and capsazepine .
( b ) summary of changes in sensory activity during filling , threshold , and micturition phases .
a significant increase in all three parameters was observed following the infusion of 0.5% acetic acid .
capsazepine , infused with 0.5% acetic acid , significantly reduced sensory nerve activity recorded during filling and threshold phases .
the ( * ) mark a significant difference between 0.9% nacl and 0.5% acetic acid , ( ) indicate significant difference between 0.5% acetic acid and capsazepine .
capsazepine opposed acetic acid - induced increases in the frequency of micturition and reduced bladder filling , threshold and micturition pressures by 25 , 31.2 , and 16.5% respectively ( n  =  8) .
the magnitude of the multifiber afferent response to bladder irritation with 0.5% acetic acid was significantly reduced in the presence of 10  m capsazepine .
acetic acid - evoked afferent discharge was inhibited by 36 and 23.4% at filling and threshold pressures , respectively , with no apparent effect on bladder contraction - related nerve activity ( figure 4b ) . at the conclusion of sensory nerve recording
electrostimulation - induced detrusor contraction resulted in an increase in intravesical pressure that ranged from 63 to 100% of the micturition pressure recorded during continuous bladder filling ( figure 5 ) . at the conclusion of the experiment neurostimulation
was applied to the postganglionic bladder nerve to confirm the viability of the nerve and specificity of the recording .
square - wave pulses ( 0.3  ms , 12  hz , 2  v ) were delivered by a grass stimulator , resulting in detrusor contraction with a rise in the intravesical pressure . to address the potential impact of emg artifacts originating from the striated urethral sphincter we conducted multifiber sensory recording with the nerve tied both proximal and distal to the recording electrode . simply tying the nerve and elevating it of the surrounding tissues did not prevent the propagation the action potential . insulating the nerve / electrode complex with
silicone glue however resulted in complete signal cessation . with the exception of microscopic foci of eosinophilic degenerative changes , morphological evaluation of the segment of the postganglionic bladder nerve overlying
hematoxylin and eosin stain of postganglionic bladder nerves removed 48  h following implantation of the recording electrode .
the nerve was healthy except for two microscopic foci of eosinophilic degenerative changes ( arrows ) .
during continuous filling with 0.9% nacl , cystometrograms showed two distinct patterns : ( a ) a steady intravesical pressure with pressure fluctuations of less than 2  cm h2o ( figure 2a ) , and ( b ) a filling phase with pressure fluctuations ranging from 2 to 10  cm h2o ( figure 2b ) . two distinct patterns recorded with cystometry : ( a ) steady intravesical pressure during the filling phase with minimal pressure fluctuations ; ( b ) phasic components ( small fluctuations in pressure ) during the entire filling phase .
intravesical infusion of 0.5% acetic acid evoked a significant reduction in intermicturition intervals coupled with an increase in average filling and threshold pressures .
the same mouse utilized for the basal and acetic acid urodynamic comparison was further challenged with a mixture of 0.5% acetic acid and 10  m capsazepine ( figure 3 ) . in the presence of capsazepine
, the bladder response to acetic acid was reduced as evidenced by an increase in intermicturition interval and a reduction in filling and threshold pressures .
0.5% acetic acid led to higher micturition frequency , multiple non - voiding contractions along with elevated filling , threshold and micturition pressures .
addition of capsazepine to the acetic acid solution reduced the number of non - voiding contractions and nerve activity .
the rectangles mark the points in the experiment where the infusate was changed ( m , micturition event ) .
continuous filling with saline in vivo generated a reproducible pattern of afferent nerve discharge ( n  =  8) .
in all animals , baseline sensory activity oscillated between 20 and 60  hz , and repeated bursts of afferent discharge were observed throughout the entire filling phase ( figure 2 ) . in some animals , low amplitude non - voiding bladder contractions ( 210  cm h2o ) seen throughout the filling phase correlated with phasic afferent discharge ( figure 2b ) .
phasic afferent discharge was also evident in a subgroup of animals that displayed only minimal fluctuations in intravesical pressure ( amplitude < 2  cm h2o ; figure 2a ) .
the terminal portion of the filling phase was characterized by a rapid increase in the frequency of sensory discharge that peaked at 97.4    20.0  hz once the bladder reached the threshold pressure .
the mean sensory nerve firing rate during micturition was 135.1    26.0  hz . in 20% of cases the experiment was unsuccessful due to excessive movement that lasted during entire recording .
intravesical infusion of 0.5% acetic acid led to a 1.9-fold increase in voiding frequency and reduced intermicturition intervals from 361.7    86.7  s to 188.7    27.8  s ( n  =  8) .
acetic acid induced 2.1-fold , 2-fold , and 2-fold increases in filling , threshold , and micturition pressures , respectively , that were associated with 2.8-fold , 2.4-fold , and 1.9-fold increases in excitatory afferent nerve discharge ( figure 4a ) .
( a ) barr graphs summarizing the data in filling ( fp ) , threshold ( tp ) , and micturition pressures ( mp ) ( n  =  8) .
a significant increase in all three parameters was observed following the infusion of 0.5% acetic acid .
the ( * ) mark a significant difference between 0.9% nacl and 0.5% acetic acid , ( ) indicate significant difference between 0.5% acetic acid and capsazepine .
( b ) summary of changes in sensory activity during filling , threshold , and micturition phases .
a significant increase in all three parameters was observed following the infusion of 0.5% acetic acid .
capsazepine , infused with 0.5% acetic acid , significantly reduced sensory nerve activity recorded during filling and threshold phases .
the ( * ) mark a significant difference between 0.9% nacl and 0.5% acetic acid , ( ) indicate significant difference between 0.5% acetic acid and capsazepine .
capsazepine opposed acetic acid - induced increases in the frequency of micturition and reduced bladder filling , threshold and micturition pressures by 25 , 31.2 , and 16.5% respectively ( n  =  8) .
the magnitude of the multifiber afferent response to bladder irritation with 0.5% acetic acid was significantly reduced in the presence of 10  m capsazepine .
acetic acid - evoked afferent discharge was inhibited by 36 and 23.4% at filling and threshold pressures , respectively , with no apparent effect on bladder contraction - related nerve activity ( figure 4b ) . at the conclusion of sensory nerve recording
electrostimulation - induced detrusor contraction resulted in an increase in intravesical pressure that ranged from 63 to 100% of the micturition pressure recorded during continuous bladder filling ( figure 5 ) .
at the conclusion of the experiment neurostimulation was applied to the postganglionic bladder nerve to confirm the viability of the nerve and specificity of the recording .
square - wave pulses ( 0.3  ms , 12  hz , 2  v ) were delivered by a grass stimulator , resulting in detrusor contraction with a rise in the intravesical pressure . to address the potential impact of emg artifacts originating from the striated urethral
sphincter we conducted multifiber sensory recording with the nerve tied both proximal and distal to the recording electrode . simply tying the nerve and elevating it of the surrounding tissues did not prevent the propagation the action potential .
insulating the nerve / electrode complex with silicone glue however resulted in complete signal cessation . with the exception of microscopic foci of eosinophilic degenerative changes , morphological evaluation of the segment of the postganglionic bladder nerve overlying
hematoxylin and eosin stain of postganglionic bladder nerves removed 48  h following implantation of the recording electrode .
the nerve was healthy except for two microscopic foci of eosinophilic degenerative changes ( arrows ) . the total magnification 400 .
mechano- and chemosensitivity have been recorded at the level of dorsal root afferent fibers in rats ( sengupta and gebhart , 1994 ) ; pelvic nerves in mice ( rong et al . , 2002 ;
2007 ) , rats ( sengupta and gebhart , 1994 ; namasivayam et al . ,
1998 ) , and cats ( floyd et al . , 1976 ; habler et al . , 1990 , 1993 ) ; lumbar splanchnic nerves in mice ( xu and gebhart , 2008 ) ; and postganglionic bladder nerves in guinea pigs ( zagorodnyuk et al . , 2006 ) . in vivo
anesthetized animal models ( cats and rats ) have been used as well ( mallory et al . , 1989 ; hara and harris , 2002 ; qin et al . , 2003 ) . in the present study
, we introduce a new model for recording bladder - specific sensory nerve activity in vivo in a conscious mouse .
the purpose of this model is to provide a new tool for the study of bladder sensory nerve activity that preserves micturition reflex pathways and avoids the suppressive effects of anesthesia on the nerve activity .
this communication represents both a description and validation of the model . in this study ,
continuous intravesical filling resulted in micturition cycles that were coupled with reproducible neural activity . during filling ,
sensory nerve activity recorded ex vivo was characterized by a gradual increase in frequency of multifiber nerve activity , whereas in vivo recording in a conscious mouse showed a complex pattern . in our study , a low level of relatively constant afferent nerve activity was observed during the initial phase of bladder filling .
small fluctuations in intravesical pressure ( < 10  cm  h2o ) that did not cause micturition led to bursts of afferent discharge . we were also able to detect bursts of afferent activity associated with minimal ( < 2  cm  h2o ) changes in the intravesical pressure . in this latter case , the observed sensory activity might have originated from micromotions in the bladder wall .
similar pressure transients have been reported in bladders of different animal species , including humans ( gillespie , 2004 , 2005 ) . in an ex vivo model ,
such spontaneous contractions have been recently shown to evoke afferent nerve firing in mouse bladders with detrusor overactivity ( mccarthy et al . , 2009 ) .
our study provides further evidence that bursts of such sensory nerve activity likely play a role in the sensation of bladder filling in both health and disease . in vivo models using anesthetized animals
more faithfully mimic physiological conditions than do in vitro studies because they maintain the anatomical integrity of the micturition reflex .
urethane anesthesia , however , inhibits nociceptive neurotransmission through its effect on n - methyl - d - aspartate receptors ( hara and harris , 2002 ) .
mechanosensory and nociceptive responses to bladder filling in animal models of bladder overactivity may therefore be reduced by anesthetic suppression of signals transmitted by c- and/or a fibers .
urethane or chloral hydrate anesthesia also leads to less efficient voiding , as evidenced by the significant post - void residual volume and increased frequency of micturition ( streng et al . , 2006 ) . to provide further insight into the contribution of different nerve populations , we used continuous intravesical infusions of 0.5% acetic acid to induce bladder overactivity .
acetic acid solutions have been shown to evoke a statistically significant increase in voiding frequency and intravesical pressure in conscious rats ( mccafferty et al . , 2008 ; mitobe et al . , 2008
here we showed that acetic acid - induced changes are coupled with a higher frequency of sensory neural activity .
we further documented reduced afferent responses to acetic acid in the presence of the trpv1 receptor antagonist , capsazepine .
capsazepine reduced filling and threshold pressures by 36 and 23.4% , respectively , providing additional support for a trpv1-dependent mechanism in chemical bladder irritation .
( 2007 ) , which showed that the response to intravesical infusion of acetic acid was attenuated in ex vivo bladder preparations from anesthetized trpv1 knockout mice . in our study , the sensory response to acetic acid infusion in a conscious mouse in the presence of capsazepine was reduced but not eliminated , suggesting that additional mechanisms are involved .
mechanosensitive a delta fibers are low threshold fibers ; therefore , it is possible that the increase in afferent firing and enhanced bladder activity was not due to direct excitation of afferents by acetic acid , but rather resulted from increased wall tension . a delta fibers , however , are connected to the detrusor muscle fibers in series ( gabella , 1995 ) ; thus , if they were the only fibers that contributed to the recorded sensory activity , the relationship between the bladder pressure and sensory firing would be linear .
the correlation between the intravesical pressure and sensory nerve activity during bladder filling observed in our study was complex , and the increase in nerve discharge frequency during filling was non - linear .
as evident from the data summary ( figure 4 ) , changes in the frequency of afferent nerve discharge following intravesical infusion of acetic acid or capsazepine exceeded changes in the intravesical pressure .
this suggests that afferent activity changes not only as a result of increased wall tension but also in response to direct stimulation of chemoreceptors and/or nociceptors in the bladder wall . to eliminate the efferent neurotransmission , we ligated the postganglionic bladder nerve between the electrode and mpg .
the effect of ligation is likely similar to the process which takes place in the distal stump of the cut peripheral nerve .
it was first described by waller in 1850 and since then it has been confirmed by others ( stoll et al . , 2002 ) .
 wallerian degeneration  includes disintegration and degeneration of axoplasma and acrolemma . at the same time macrophage recruitment
takes place , leading to rapid clearance of myelin debris , facilitating inflammatory processes leading to regeneration of the injured nerve .
hematoxylin and eosin staining showed that the morphology of the nerve overlying the electrode was preserved .
although eosinophilic degradation was evident , it was only present in the form of microscopic foci in the superficial portion of the nerve ( figure 5 ) .
in addition there is a possibility that ligation of the postganglionic bladder nerve might compromise bladder function .
however , the fact that we have ligated only one of four to six terminal branches of this nerve entering each side of the bladder makes this unlikely .
we conducted multifiber sensory recording with the nerve tied both proximal and distal to the recording electrode .
simply tying the nerve and elevating it from the surrounding tissues did not prevent the propagation of nerve activity recording during bladder filling due to emg artifacts .
imbedding the nerve / electrode complex in the silicone glue however resulted in complete cessation of the signal , confirming that the activity recorded is solely attributed to the sensory nerve firing .
our data provide evidence that an in vivo approach in a conscious animal yields reproducible results .
once we solved the technical issues and established the microsurgical techniques , only less than 20% of experiments had to be excluded from the analysis due to excessive movement artifacts . preserved continuity of regulatory pathways and the absence of anesthesia allows for detailed study of afferent nerve function in health and disease .
this model could complement currently available experimental methods in preclinical testing of therapeutic interventions that target the sensory limb of the micturition reflex .
it is also applicable to genetically altered animal models , providing an opportunity to examine molecular targets relevant to bladder sensitivity and thereby adding a valuable tool for the study of fundamental bladder neurophysiology .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 the goal of this study was to develop an in vivo awake mouse model for extracellular bladder sensory nerve recording . 
 a bipolar 125-m silver electrode was positioned under a single postganglionic bladder nerve . 
 efferent nerve signals were eliminated by tying off the postganglionic bladder nerve between the major pelvic ganglion and the recording electrode . 
 sensory nerve activity was measured in the conscious animals 48  h after surgery during continuous intravesical infusion of 0.9% saline/0.5% acetic acid followed by 0.5% acetic acid with capsazepine ( 10  m ) at a rate of 0.75  ml / h . 
 continuous infusion of 0.9% nacl led to a gradual increase in the frequency of sensory nerve firing that peaked upon reaching threshold pressure . 
 non - micturition contractions were observed in some animals during filling and other animals exhibited only minimal pressure fluctuations ; both types of events were associated with a rise in sensory nerve activity . 
 intravesical infusion of 0.5% acetic acid reduced the intermicturition interval . 
 this was associated with a 2.1-fold increase in bladder pressure during filling and a two - fold increase at both threshold and micturition pressures . 
 concurrent with these changes , sensory activity increased 2.8-fold during filling and 2.4-fold at threshold pressure . 
 subsequent intravesical infusion of capsazepine in 0.5% acetic acid reduced filling and threshold pressures by 21 and 31.2% , respectively , and produced corresponding decreases of 36 and 23.4% in sensory nerve activity . 
 the current study shows that multifiber sensory nerve recordings can be reproducibly obtained from conscious mice . 
Introduction
Materials and Methods
Data analysis
Results
Intravesical pressure during bladder filling and micturition
Multifiber bladder sensory nerve activity during bladder filling
Discussion
Conflict of Interest Statement