Pfizer Global Research & Development Sandwich Kent CT13 9NJ Evidence for the involvement of the medial prefrontal cortex and thalamus in the control of micturition in the female rat Yvonne Mbaki, Victoria Chapman, Rachel Conley 1, Rob Mason School of Biomedical Sciences, University of Nottingham, UK; 1 Discovery Biology, Pfizer Global R&D, Sandwich, UK Introduction Anatomical and imaging studies have demonstrated the involvement of multiple brain regions in the control of bladder function. Both the medial prefrontal cortex (mPFC) and thalamus have been shown to be involved in micturition although it remains unclear to what extent these two structures contribute towards efficient bladder voiding or continence. The mPFC is known to play a role in the conscious and social control of bladder function, in particular executive decision making of whether or not micturition should occur at a particular place or time 1. The thalamus, which has connections with the prefrontal cortex 2, has been implicated as a relay of sensory information arising from the bladder up to the cortex. The present study was conducted to examine the contribution of both the mPFC and thalamus in the control of micturition. In vivo electrophysiology under urethane anaesthesia examined neuronal activity recorded simultaneously in the mPFC and thalamus in response to normal bladder contractions in the female rat. References 1) KAVIA et al., (2005). J. Comp. Neurol., 493(1): ) VERTES, R.P. (2002). J. Comp. Neurol., 442: Discussion The present study identified neurones in the mPFC (anterior cingulate gyrus) and thalamus that were responsive to bladder contractions. Although both structures displayed similar bladder-evoked responses, the duration of single unit-suppression and burst firing pattern was different. Neurones in both regions displayed a lag in the onset of evoked-response (~ ms) after start of bladder contraction, suggesting both structures may not be directly involved in initiating micturition. This is surprising as the mPFC is known to be involved in making the decision when/where to void 4. An alternative view of mPFC control of micturition may include sensory processing of bladder information, e.g. perception of changes in bladder volumes detected after the onset of micturition. Additionally, it is possible that the mPFC may be involved in initiating the termination of micturition. The interconnectivity between thalamus and mPFC, coupled with the similarity of bladder-evoked responses, may imply the thalamus may mirror the role to that of the mPFC in regulation of micturition. From the literature, the mPFC is known to influence timing of micturition whilst the thalamus is involved in the relay of sensory information relating to pathophysiological bladder conditions. Interestingly, results from this study provide evidence for an alternative role for these two structures in micturition. 3) PAXINOS & WATSON (1998). 4 th Edition. NY: Academic Press 4) FOWLER et al., (2008). Nat Rev Neurosci., 9(6): /RR40 Studies were conducted on female Sprague-Dawley rats ( g; n=7; Charles River UK) in accordance with United Kingdom legislation and subject to local ethical review. Experiments were conducted under terminal general anaesthesia and animals were euthanased at the end of each study by an overdose of Euthatal. Rats were initially anaesthetised with isoflurane (50:50% N 2 O:O 2 ) and following cannulation of the femoral vein, the gaseous anaesthesia was discontinued and urethane was administered (1.2g kg -1, i.v.). Supplementary doses of urethane (0.1g kg -1 ) were given as required. The trachea was cannulated to maintain a patent pathway. The distal ends of the ureters were tied off and cut to prevent the bladder filling with urine during experiments. A double lumen cuffed cannula was inserted into the bladder for infusion of saline (0.1 ml min -1 ) and the measurement of bladder pressure. Dual-site recording of single-unit and local field potential (LFP) activity using 8- channel microelectrode arrays (NBLabs) were made in the anterior cingulate gyrus of the mPFC (2.7 mm anterior and 0.5 mm lateral to bregma; mm ventral to the brain surface) and ventral posteriolateral (VPL), ventral posteriomedial (VPM) and posterior (Po) thalamic nuclei (2.8 mm posterior and mm lateral to bregma; mm ventrally) 3. Single-unit, LFP activity and bladder contractions were monitored using a Multiple Acquisition Processor system (Plexon Inc). Experimental protocol: Animals were allowed to stabilise for one hour post-surgery. The bladder was subsequently infused with saline until threshold was reached and a bladder contraction evoked. Saline was infused continuously for 15 minutes to ‘prime’ the system and ensure stability of the response. After ‘priming the system’ infusion was discontinued and the bladder emptied. Saline infusion was resumed once more and bladder contractions resulting in successful fluid expulsion from the urethral meatus were evoked at regular intervals. Data analysis: Only data from animals with confirmed mPFC and thalamic electrode placement were analysed. Single-units were sorted using both automatic and manual sorting techniques in Offline Sorter ( Analysis of single-unit and LFP data were conducted using NeuroExplorer ( and customised MATLAB scripts. Effects of bladder contractions on neuronal activity recorded in the mPFC and thalamus was derived from comparisons made between pre-voiding with during/post-voiding conditions. Methods Time (s) P Bladder (mmHg) Counts/bin Figure 2:Representative single-unit population peri-event time histograms (PETHs; average for 8 mPFC and 7 thalamic cells in single experiment) showing suppressed activity shortly (~200ms) after saline infusion-evoked bladder contractions. Time 0 = start of micturition; black line in top panel represents mean firing rate. mPFC Thalamus Time (s) P Bladder (mmHg) Counts/bin Results From a total of 52 neurones encountered in the mPFC, 15% demonstrated bladder- related activity with suppression (≥30% reduction in firing rate) occurring shortly after (~ 200 – 400 ms) saline infusion-evoked bladder contractions. In the thalamus, 28% and 7% of the 54 neurones encountered displayed suppressed and excited activity respectively. Further, mPFC single-units (but not thalamus) exhibited a significant (p<0.05) increase in burst firing during/post-void compared to pre-void conditions. In both regions, the LFP amplitude was significantly (p<0.05) increased during/post void when compared to pre-void conditions. Interestingly, only neurones recorded in the anterior cingulate gyrus (ACG) but not prelimbic cortex (PrL) of the mPFC demonstrated bladder-related activity. Acknowledgements Research funded by Pfizer (Sandwich, UK); YM is under the mentorship of RM, VC & RC. - More information on lab website: LFP amplitude (mV) ** Pre-void During/Post-void LFP amplitude (mV) * Figure 4:Effects of voiding on the LFP signal amplitude in mPFC & thalamus (A) mPFC (B) Thalamus Time (s) P Bladder (mmHg) LFP activity (mV) Time (s) P Bladder (mmHg) LFP activity (mV) p<0.01 p< s P Bladder (mmHg) Unit 1 Unit 2 Unit 3 Unit 4 Unit Pre-void Post-void Bursts/second * p<0.05 Figure 3:Effects of voiding on burst firing pattern in the mPFC. (Left) Representative unit spike rasters and bladder contraction plot from one experiment (Right) Pooled data of spike bursting rate (n = 7 rats). Figure 1:(A) Representative electrode placements (B) Schematic representation of microelectrode array placements in mPFC (top right) and thalamus (bottom right); n=7 rats bregma +2.7 mm (B) Thalamus (A) mPFC bregma mm VPL VPM Po ACG