Articles in PresS. J Neurophysiol (September 9, 2009). doi:10.1152/jn.00700.2009
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Brain Switch for Reflex Micturition Control
Detected by fMRI in Rats
Changfeng Tai,1 Jicheng Wang, 1 Tao Jin, 2 Ping Wang, 2
Seong-Gi Kim, 2 James R. Roppolo, 3 and William C. de Groat 3
1. Department of Urology, University of Pittsburgh, Pittsburgh, PA, USA
2. Department of Radiology, University of Pittsburgh, Pittsburgh, PA, USA
3. Department of Pharmacology and Chemical Biology, Pittsburgh, PA, USA
Running Head: Brain switch for micturition control
Correspondence to:
Changfeng Tai, Ph.D.
Department of Urology
University of Pittsburgh
700 Kaufmann Building
Pittsburgh, PA 15213, USA
Phone: 412-692-4142
Fax: 412-692-4380
Email: [email protected]
Copyright © 2009 by the American Physiological Society.
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ABSTRACT
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The functions of the lower urinary tract are controlled by complex pathways in
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the brain that act like switching circuits to voluntarily or reflexly shift the activity of
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various pelvic organs (bladder, urethra, urethral sphincter and pelvic floor muscles) from
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urine storage to micturition. In this study, functional MRI (fMRI) was employed to
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visualize the brain switching circuits controlling reflex micturition in anesthetized rats.
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The fMRI images confirmed the hypothesis based on previous neuroanatomical and
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neurophysiological studies that the brainstem switch for reflex micturition control
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involves both the periaqueductal gray (PAG) and the pontine micturition center (PMC).
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During storage, the PAG was activated by afferent input from the urinary bladder while
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the PMC was inactive. When bladder volume increased to the micturition threshold, the
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switch from storage to micturition was associated with PMC activation and enhanced
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PAG activity. A complex brain network that may regulate the brainstem micturition
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switch and control storage and voiding was also identified. Storage was accompanied by
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activation of the motor cortex, somatosensory cortex, cingulate cortex, retrosplenial
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cortex, thalamus, putamen, insula, and septal nucleus. On the other hand micturition was
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associated with: (1) increased activity of the motor cortex, thalamus, putamen, (2) a shift
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in the locus of activity in the cingulate and insula and (3) the emergence of activity in the
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hypothalamus, substantia nigra, globus pallidus, hippocampus and inferior colliculus.
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Understanding brain control of reflex micturition is important for elucidating the
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mechanisms underlying neurogenic bladder dysfunctions including frequency, urgency,
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and incontinence.
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Keywords: brain; bladder; micturition; fMRI, rat
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INTRODUCTION
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Storage and periodic elimination of urine can be controlled involuntarily in
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infants or voluntarily in adults by neural pathways in the brain and spinal cord
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(Barrington 1921; de Groat and Ryall 1969; de Groat 1975; de Groat et al. 1993; Kuru
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1965). During storage when urine slowly accumulates in the bladder, bladder afferent
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activity in the pelvic nerve gradually increases and is transmitted via the sacral spinal
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cord to the brain to provide information about the extent of bladder filling. When the
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bladder is controlled involuntarily and volume is below the threshold level for triggering
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micturition, the brain switch for reflex micturition is turned off and the bladder is
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quiescent promoting the storage of urine. However, when bladder volume reaches the
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micturition threshold, activation of a brain switching circuit sends an excitatory signal
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through the spinal cord to the bladder to induce a sustained bladder contraction and an
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inhibitory signal to induce a reciprocal relaxation of the external urethral sphincter (EUS)
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leading to the release of urine.
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Electrophysiological studies in animals indicate that the neural switching circuit
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controlling reflex micturition is located in the rostral brain stem (de Groat and Ryall
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1969; de Groat 1975; Kuru 1965; Noto et al. 1991). After midcollicular decerebration
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reflex micturition is maintained; however destruction of a region in the dorsolateral pons
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(termed the pontine micturition center, PMC) or transection of the neuraxis at any level
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below the PMC blocks reflex micturition (Barrington 1921; Ruch and Tang 1956).
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Recordings of neural firing in the PMC in cats revealed all-or-none patterns of activity
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that correlate with the storage and voiding phases of bladder activity (de Groat et al.
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1998; Sakakibara et al. 2002; Sasaki 2002, 2005; Sugaya et al. 2003; Tanaka et al. 2003;
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Willette et al. 1988). In addition microinjections of inhibitory agents into the PMC in
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decerebrate cats increase the micturition volume threshold or completely block
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micturition (Mallory et al. 1991).
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Neuroanatomical studies in cats (Blok and Holstege 1994, 1996, 1998) indicate
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that afferent input from the bladder is received in the periaqueductal grey (PAG) and then
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transmitted to the PMC which in turn sends motor signals back to the spinal cord to
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induce micturition. This spinobulbospinal switching circuit is modulated by inputs from
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the forebrain which control reflex micturition (de Groat et al. 1993; Kuru 1965; Ruch and
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Tang 1956; Yokoyama et al. 2002) and mediate voluntary voiding. Sensory input from
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the bladder to the forebrain very likely passes through relays in the PAG (Holstege 2005)
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as well as in the thalamus (Craig 1996, 2002; Mayer et al. 2006).
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Functional brain imaging technologies including fMRI and positron emission
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tomography (PET) have been employed in humans to identify brain regions activated
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during bladder filling and voluntary control of micturition (Athwal et al. 2001; Blok et al.
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1997a, 1997b, 1998, 2006; Dasgupta et al. 2005; Di Gangi Herms et al. 2006; Griffiths et
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al. 2005, 2007; Kitta et al. 2006; Kuhtz-Buschbeck et al. 2005, 2007; Matsuura et al.
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2002; Mehnert et al. 2008; Nour et al. 2000; Sakakibara et al. 1999; Seseke et al. 2006,
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2008; Zhang et al. 2005). Under these conditions the neural mechanisms controlling
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micturition are not only influenced by the peripheral sensory input from the bladder, but
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also by conscious brain processes including attention, expectation, decision, emotion, etc
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(Mayer et al. 2006). Since these processes can vary at different times in the same
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individual or between individuals this complicates the interpretation of brain imaging
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data (Mayer et al. 2006). In order to separate the basic micturition neural circuitry that
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mediates reflex control from the conscious control of bladder function, it was recently
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suggested that human brain fMRI studies might be performed under anesthesia (Naliboff
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and Mayer 2006). Information about the reflex control of voiding is important clinically
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because many dysfunctions of the lower urinary tract (e.g., urinary incontinence) are
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mediated by involuntary neural mechanisms.
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In this study, brain fMRI imaging was performed on anesthetized rats in order to
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identify neuronal circuitry involved in reflex micturition. In the rat the PAG and PMC
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appear to have roles in the reflex control of micturition similar to those identified in the
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cat. Electrophysiological studies showed that stimulation of bladder afferent nerves
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evoked action potentials in PAG at shorter latencies than the action potentials in the PMC
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suggesting that bladder afferent input from the spinal cord is processed first in the PAG
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and then relayed to the PMC (Noto et al. 1991). However ascending projections from the
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lumbosacral spinal cord have been detected with axonal tracing techniques in the PMC as
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well as in the PAG (Ding et al. 1997); raising the possibility that bladder afferent input
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may also go directly to the PMC. Electrical stimulation in the PAG as well as the PMC
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evoked firing in parasympathetic efferent pathways to the bladder and bladder
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contractions demonstrating a connection of neurons in both of these areas with the
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efferent outflow to the urinary bladder (de Groat 1975; Kruse et al. 1990; Noto et al.
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1989, 1991; Taniguchi et al. 2002). This was confirmed by retrograde transneuronal virus
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tracing in the rat (Grill et al. 1999; Marson 1997; Nadelhaft et al. 1992; Sugaya et al.
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1997; Vizzard et al. 1995) which revealed that both the PMC and PAG were labeled after
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injection of virus into the bladder or the urethra. Assuming that the micturition switch is
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located in the PMC in the rat then it is reasonable to hypothesize that, fMRI imaging
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should detect a signal in the PAG area but not in the PMC in response to bladder
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distension during urine storage. However during micturition both regions should exhibit
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an increased signal.
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How neurons in other brain centers participate in the switch from storage to
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micturition is largely unknown. A recent neuroanatomical study in the cat (Kuipers et al.
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2006) showed that with the exception of the hypothalamus and PAG, no other brain
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structures have direct monosynaptic access to the PMC. Thus control of the PMC
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switching circuit by the brain must be primarily indirect via relays through the PAG and
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hypothalamus. The goal of this study was to use fMRI to determine how activity in brain
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regions, which are potentially involved in micturition control, changes during filling of
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the bladder and after the initiation of reflex micturition in anesthetized animals.
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METHODS
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Animal Preparation
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A total of 9 male Sprague-Dawley rats (300-400g) were used in this study. All
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protocols involving the use of animals were approved by the Animal Care and Use
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Committee at the University of Pittsburgh. The animals were initially anesthetized with
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5% isoflurane. Following intubation for mechanical ventilation (RSP-1002; Kent
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Scientific, CT, USA) isoflurane was reduced to 2-3% during surgical preparation. The
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femoral artery and vein were catheterized for blood pressure monitoring, blood gas
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sampling, and fluid/drug administration. Via an abdominal incision a tube (PE 50) was
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inserted into the bladder through the bladder dome and secured by a ligature. Through a
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T-connector the tube was connected to a syringe pump and a pressure transducer to infuse
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the bladder with saline and record the bladder pressure. A ligature was tied around the
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base of the penis to prevent urine release into the MRI scanner. The animal was then
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placed in a custom-built plastic cradle with the head secured by two ear bars and a bite
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bar in order to reduce motion. A neuromuscular blocking agent (pancuronium bromide,
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0.6-0.8 mg/kg/hr, i.v.) was administered during the experiment to further reduce head
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motion. After completing the surgery, urethane (1.2 g/kg initial dose followed by a
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supplemental dose of 0.4 g/kg every 4-5 hours) was injected subcutaneously to replace
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isoflurane anesthesia. The arterial blood pressure and breathing pattern were continuously
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monitored with a multi-channel recording unit (AcKnowledge; Biopak, CA, USA). End-
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tidal CO2 was monitored (Stat profile pHOx; Nova Biomedical, MA, USA) and
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maintained at 3.5-4% by varying ventilation volume and frequency. The animal’s body
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temperature was maintained at 37.5°C ± 0.5°C with a feedback-controlled warm-water
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pad using a rectal thermal probe. The animal was given saline with 5% dextrose (1.5-2
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ml/kg/hr) intravenously.
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MRI Scanning Protocol
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All MRI experiments were performed on a 9.4 T/31 cm magnet (Magnex, UK),
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interfaced to a Unity INOVA console (Varian, Palo Alto, CA). The actively shielded 12-
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cm diameter gradient insert (Magnex, UK) operates at a maximum gradient strength of 40
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gauss/cm with a rise time of 130 µs. A rectangular surface coil (2.5 cm x 2 cm) was
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positioned on top of the animal’s head for both excitation and reception. fMRI images
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were acquired using the spin-echo echo planar imaging (EPI) technique with repetition
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time/echo time (TR/TE) = 600/32 ms, field of view (FOV) = 2.8 cm x 2.1 cm, and matrix
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size = 64x48. Scanning the entire brain required 5.4 seconds with a total of 9 coronal
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slices (2 mm thickness of each slice). Anatomical images were acquired using
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TURBOFLASH sequence with TR/TE = 20/5.5 ms and matrix size = 192x144.
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For each animal 10-12 fMRI experimental trials of bladder filling (i.e.,
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cystometrogram) were performed for blood oxygen level dependent (BOLD) imaging.
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During each trial the rat brain was scanned continuously (see Fig.1). The bladder was
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initially empty during the continuous fMRI scanning in order to collect the control data
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(control 1 in Fig.1) that included at least 20 fMRI images (each image included 9 coronal
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slices). Then, the bladder was slowly infused with saline (0.1-0.3 ml/min) until a large
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bladder contraction was induced (see Fig.1). If the bladder contraction was not
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maintained for at least 2 min, additional bladder infusion was immediately started to
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maintain the micturition reflex so that at least 20 fMRI images could be acquired. At the
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end of the continuous fMRI scanning, the bladder was emptied by withdrawing the saline
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via the bladder catheter. Another 20 fMRI images were acquired after bladder emptying
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in order to collect control data (control 2 in Fig.1). After a 10 minute resting period for
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the reflex pathways to recover, the same fMRI experiment trial was repeated. A total of
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105 experimental trials were performed on the 9 animals.
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After acquiring BOLD images, MION (monocrystalline iron oxide nanoparticles,
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10 mg/kg) was administered intravenously to the animal. Then the fMRI scanning
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protocol as shown in Fig.1 was repeated for 10 experimental trials on each animal (total
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70 trials on 7 animals) with a faster infusion rate of 1 ml/min. The CBV-weighted
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(cerebral blood volume-weighted) MION imaging has been shown to increase the
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functional sensitivity compared to the BOLD technique (Jones 2002; Mandeville et al.
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1998; Zhao et al. 2006). The purpose of MION fMRI experiment was to further confirm
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the brain activation detected by BOLD fMRI during a micturition reflex.
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fMRI Data Processing and Analysis
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The MRI image series acquired during each experimental trial in an individual
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animal was extracted to form a new image series (see Fig.1). 20 images were extracted
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from each of the following periods: during the initial empty bladder period (control 1 in
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Fig.1), during saline infusion before a bladder contraction (storage in Fig.1), during
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micturition contraction (contraction in Fig.1), and during the period after bladder
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emptying (control 2 in Fig.1). The series of images extracted during the same time period
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in repeated trials were averaged to increase the signal-to-noise ratio. Then, the averaged
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image series was re-organized according to different box cars (see Fig.1) for detecting
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brain activation during either the storage phase or contraction phase. The newly formed
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MRI image series from each individual animal was used to determine brain activation by
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SPM5 software (available at http://www.fil.ion.ucl.ac.uk/spm).
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The MRI images were preprocessed in SPM5 software by: (1) realignment for
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subtle motion correction, (2) co-registration of functional and anatomical images, (3)
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spatial normalization to a standard rat brain template image, (4) high-pass-filtered (cut off
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period of 128 s) to remove low-frequency drift, and (5) spatial smoothing. After the
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preprocessing of the MRI data, statistical analysis was performed in 2 steps. First,
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individual statistical analysis was performed on each animal using a box-car function
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(either for storage or for contraction) and a general linear model to calculate the statistical
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parametric map (SPM). A contrast threshold of P < 0.01 for each voxel and an extend
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threshold of ≥ 5 contiguous supra-threshold voxels per cluster were used. In the second
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step, group statistical analysis (i.e., one-sample t-test) was performed on the fMRI images
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from the 9 animals. Different P values (0.002-0.0001) with a cluster size of minimal 2
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contiguous voxels were used to threshold the fMRI images and display the significantly
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activated brain regions. Due to the weak MRI signal in the brainstem area, a region of
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interest (ROI) analysis was performed in this region in order to detect the activation of
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PAG and PMC neurons. One-sample t-test was performed in the brainstem area using P <
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0.03 with a cluster size of minimal 2 contiguous voxels.
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The final fMRI images were superimposed on the anatomical MRI template
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images and on the standard rat brain template drawings (Paxinos and Watson 2005) that
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correspond to the Bregma coordinates in the anterior-posterior direction as 2.28, 0.24, -
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1.80, -3.84, -5.88, -7.80, and -9.84 mm (see Fig.2). The standard template drawings were
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normalized to the corresponding anatomical MRI template images by aligning the brain
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midline and ventricles and by maximally fitting the brain outline curvatures. Bregma
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coordinates (Paxinos and Watson 2005) were used to indicate the center of the activated
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brain region. The size of the activated brain area was indicated by the maximal lengths in
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3 directions, i.e., LR – left(-)/right(+), DV – dorsal/ventral, and AP – anterior/posterior.
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The activation intensity of each brain region was indicated by the peak t value.
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RESULTS
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Brainstem Switch for Micturition Reflex
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Due to the small size of the neuronal groups and the weak fMRI signal, ROI
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analysis was performed on the brainstem area to detect changes in activity during the
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switch from storage to micturition. As shown in Fig.2, the brainstem switch for the
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micturition reflex involved both PAG and PMC (P < 0.03) confirming previous results of
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neurophysiolgical and neuroanatomical studies (de Groat 1975; Blok and Holstege 1996,
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1998; Noto et al. 1991; Sasaki 2005). During storage the PAG was activated, but the
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PMC was inactive (Fig. 2 left column). During the micturition reflex, the PMC was
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activated and PAG activation was enhanced further (Fig.2 right column). In addition, the
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ROI analysis also identified activation of the parvicellular reticular nucleus (PCRt) and
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inferior colliculus during the micturition reflex (Table 1 and Fig.2).
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Brain Network Switch for Micturition Reflex
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During bladder filling (ie., the storage phase) multiple sites in the brain (Fig.3)
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were activated including: motor cortex, primary and secondary somatosensory cortices,
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cingulate cortex, restrosplenial cortex, thalamus, putamen, insula, and septal nucleus
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(Table 2 and Fig.3 left column, P < 0.002). Activation of motor cortex, as well as primary
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and secondary somatosensory cortices only occurred on the right side of the brain (Fig.3
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A-C during storage).
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When the bladder volume was increased to the micturition threshold (0.65±0.23
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ml) and induced a large amplitude bladder contraction (48±10 cmH2O), many brain
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regions that were activated during storage exhibited enhanced activation. The most
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strongly activated brain regions during micturition are shown in Fig.3 right column (P <
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0.0001). A comparison of the left and right columns in Fig.3 reveals that activation of the
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motor cortex, thalamus, and putamen was enhanced. In addition the hippocampus which
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was not activated during storage was activated during micturition (also see Table 2 and
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Table 3). Other brain regions that were activated during storage (Fig.3 left column) but
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showed more modest activation during micturition (Fig.3 right column) were identified
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by ROI analysis (Fig.4, P < 0.002). These regions include the primary somatosensory
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cortex, cingulate cortex, retrosplenial cortex, and septal nucleus. Activation in the
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posterior area of the right secondary somatosensory cortex was decreased, but activation
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in the right anterior area was increased (Fig.4 B-C).
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In some brain regions which exhibited activity during storage and micturition the
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area of activation shifted during micturition (summarized in Table 4). For example
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activation of cingulate cortex extended from a posterior area to more anterior area when
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switching from storage to micturition, while activation in the retrosplenial cortex
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expanded from an anterior area into a more posterior area (Table 4 and Fig.4). The
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activation of insula shifted from a posterior area to an anterior area (Table 4 and Fig.3).
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Although the predominant activation of both primary and secondary somatosensory
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cortices remained on the right side during the micturition reflex (Table 4 and Fig.4), the
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motor cortex was activated bilaterally (Table 4 and Fig.3).
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Brain Network Activation during Micturition Reflex – MION images
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The activated brain regions during a micturition reflex were further confirmed by
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MION fMRI images (Fig.5). Similar to BOLD images, brain activations were observed in
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motor cortex, primary somatosensory cortex, cingulate cortex, retrosplenial cortex,
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thalamus, putamen, insula, and hippocampus (Table 4 and Table 5). In addition, MION
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fMRI also detected activation of hypothalamus, substantia nigra, and globus pallidus
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during a micturition contraction (Table 5).
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Average MRI Signal Intensity
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During a micturition contraction the average MRI signal from the activated brain
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regions changed about 2% (Fig.6). The BOLD signal change which was positive was
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opposite of the MION signal change (negative) due to the underlying physics. Compared
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to the average BOLD signal during a micturition contraction, the BOLD signal during
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storage was weaker and had a larger variation (compare black and red lines in Fig.6),
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indicating that the brain activation during storage was less intense than the activation
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during micturition (Fig.3-4).
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DISCUSSION
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This study provides support for the hypothesis based on previous neuroanatomical
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and neurophysiological studies (Barrington 1921; Blok and Holstege 1996, 1998; de
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Groat 1975; Noto et al. 1991) that in the anesthetized state a micturition switching circuit
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involving both the PAG and PMC exists in rostral brainstem (Fig.2). During the storage
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phase when the bladder was filling, the PMC was inactive but the PAG was activated
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indicating that this region receives afferent input from the bladder prior to micturition.
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However, when bladder volume reached the micturition threshold, the PMC was
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activated and PAG activation was increased further (Fig.2 right column) consistent with
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the idea that the PMC is the site for initiation of the micturition reflex (Barrington 1921;
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de Groat 1975; de Groat et al. 1993; Kuru 1965). Our studies also revealed that various
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sites in the forebrain were activated in parallel with activation of the PAG before
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micturition indicating that a complex brain network processes afferent signals from the
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bladder, some of which may be relayed first through the PAG (Blok and Holstege 1994;
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Blok et al. 1995; Holstege 2005; Noto et al. 1991). Activation of other forebrain areas
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occurred in parallel with the activation of the PAG/PMC during micturition. These areas
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in the forebrain may subserve multiple functions related to the reflex control of the lower
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urinary tract including: (1) processing sensory input from the bladder, (2) modulating
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efferent storage mechanisms, (3) regulating the initiation of micturition and (4) regulating
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the coordination between bladder and urethra activity during micturition. The data are
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consistent with the conclusions from human fMRI and PET brain imaging studies
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(Athwal et al. 2001; Blok et al. 1997a, 1997b, 1998, 2006; Dasgupta et al. 2005; Di
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Gangi Herms et al. 2006; Griffiths et al. 2005, 2007; Kitta et al. 2006; Kuhtz-Buschbeck
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et al. 2005, 2007; Matsuura et al. 2002; Mehnert et al. 2008; Nour et al. 2000; Sakakibara
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et al. 1999; Seseke et al. 2006, 2008; Zhang et al. 2005) that various forebrain regions
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participate in the control of urine storage and voiding.
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However in contrast to previous human fMRI brain imaging experiments, the
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present study imaged brain activation during a continuous slow infusion of the bladder in
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an attempt to mimic the physiological accumulation of urine. In addition differences in
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brain activity were evaluated in three conditions: (1) empty bladder, (2) partially filled
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bladder and (3) reflexly active bladder to determine in the same animal which brain
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regions participated in storage and voiding functions. In some human fMRI studies
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(Griffiths et al. 2005, 2007; Kuhtz-Buschbeck et al. 2005; Mehnert et al. 2008) the
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bladder was filled to a volume causing a strong desire to void and micturition was
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voluntarily inhibited because the subjects were not allowed to urinate in the MRI scanner.
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In other experiments bladder afferents were activated by rapidly and repeatedly infusing
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and withdrawing a small amount of saline from the bladder to obtain multiple
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measurements during urine storage that could in turn be averaged to detect small changes
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in brain activity (Griffiths et al. 2005, 2007; Mehnert et al. 2008). However these
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techniques did not mimic physiological distention of the bladder and only measured brain
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activation during the storage phase.
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Early PET studies (Blok et al. 1997b, 1998; Nour et al. 2000) investigated brain
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activation during both storage and micturition in the same subject, but very few brain
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regions were activated during the storage phase and PAG activation was not significant.
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Later PET studies only imaged the brain either during the bladder storage phase (Athwal
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et al. 2001; Blok et al. 2006; Dasgupta et al. 2005; Matsuura et al. 2002) or during
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overactive bladder contractions (Kitta et al. 2006). Most fMRI studies investigated brain
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activation during voluntary pelvic floor muscle control (Di Gangi Herms et al. 2006;
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Kuhtz-Buschbeck et al. 2005, 2007; Seseke et al. 2006, 2008; Zhang et al. 2005). A few
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fMRI studies investigated bladder distention, but the bladder was only infused to the
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volume causing strong desire to void and micturition was not allowed (Griffiths et al.
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2005, 2007; Kuhtz-Buschbeck et al. 2005; Mehnert et al. 2008). An fMRI brain imaging
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study to reveal how brain activation switches from storage to micturiton in human is
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currently not available.
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Human studies have been performed under awake conditions; whereas our studies
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in rats were conducted under urethane anesthesia to examine reflex mechanisms. Under
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urethane anesthesia, rats exhibit normal urine storage at low intravesical pressures and
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coordinated activity of the bladder and urethral sphincter during voiding (Maggi et al.
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1986; Yoshiyama et al. 1994). It has been proposed that urethane anesthetized rats might
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be a useful model for neurogenic detrusor overactivity (NDO) because C-fiber bladder
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afferents which have been implicated in NDO in humans, act in combination with A-fiber
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afferents to modulate reflex voiding in these animals. On the other hand, only A-fiber
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bladder afferents initiate voluntary voiding in awake rats (Chuang et al. 2001). Reflex
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control of micturition is clinically relevant because neurogenic bladder dysfunctions
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including bladder overactivity, urgency, and incontinence caused by brain disorders (de
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Groat et al. 1993; Fowler 1999) are often generated by reflex mechanisms that are
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resistant to voluntary control. Studies in awake humans are complicated by the
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contribution and interaction of a variety of brain processes including attention,
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expectation, decision, and emotion (Mayer et al. 2006) that very likely influence voiding
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function. These processes may also influence voiding in awake rats but should be
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minimized under anesthesia.
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In our study we used BOLD and MION techniques to identify activated brain
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areas during micturition, whereas the BOLD technique is routinely used in human
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studies. Although these two methods identified similar brain centers the exact locations
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of activation were not always same. This is probably due to difference in sensitivity and
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the underlying physics of the BOLD and MION techniques. MION which has a long
383
half-life in the blood, can be detected by fMRI due to increased local cerebral blood
384
volume that causes an increase in the local concentration of iron oxide (i.e. MION) (Jones
385
2002; Mandeville et al. 1998; Zhao et al. 2006). In contrast, a BOLD signal results from a
386
decrease in the concentration of deoxyhaemoglobin as venous blood becomes more
387
oxygenated in brain areas of increased blood flow and volume. MION can enhance fMRI
388
sensitivity approximately five times compared with BOLD. However the fMRI images
389
obtained with either technique only show a change in hemodynamics that reflects a net
16
390
change of total neuronal activity. The change in activity could be due to alterations in
391
afferent information arising in the bladder or due to changes in motor commands
392
resulting from the initiation of a micturition reflex. Thus fMRI imaging does not provide
393
insights into the functions of activated regions in regard to regulation of bladder activity.
394
Possible functions of the brain regions identified in our study can be inferred from
395
previous electrophysiological and neuroanatomical experiments of other investigators.
396
For example it is well known that bladder afferent signals can be processed in the PAG as
397
well as the thalamus and then pass to more distal sites in the anterior cingulate cortex and
398
the insula before reaching the orbitofrontal cortex (Craig 1996, 2002; Mayer et al. 2006).
399
These areas were activated during bladder filling in our experiments. The primary and
400
secondary somatosensory cortical areas were also activated during bladder filling.
401
Activation of these areas was unexpected because they are not assumed to play a role in
402
visceral sensation.
403
The afferent signals generated in the bladder during filling occur in low threshold
404
(non-nociceptive) mechanoreceptive afferents; but during micturition they could also
405
arise in high threshold (nociceptive) as well as low threshold afferents because the
406
bladder outlet was occluded in our experiments thereby generating high pressures during
407
the reflex bladder contractions. The high bladder pressure (40-60 cmH2O) might have
408
activated nociceptive bladder afferents during the micturition reflex, although the bladder
409
was maintained at a volume just slightly above the micturition threshold volume (see
410
Fig.1). Measurements of immediate early gene (IEG) expression (c-fos) in the brain after
411
noxious visceral stimulation (Clement et al. 1996) or bladder irritation with
412
cyclophosphamide which activates nociceptive bladder afferents (Bon et al. 1996)
17
413
identified IEG expression in several brain regions that were also identified in the present
414
experiments during micturition. Thus these regions including the thalamus,
415
periaqueductal grey, inferior colliculus and hypothalamus (identified using MION
416
method only) pathways could have been activated by nociceptive bladder afferent nerves.
417
Many brain regions identified in this study have been identified in transneuronal
418
pseudorabies virus (PRV) tracing studies in which PRV was injected into the rat urinary
419
bladder or urethra and then transported in a retrograde manner along peripheral and
420
central efferent pathways to the spinal cord and then to the brain (Grill et al. 1999;
421
Marson 1997; Nadelhaft et al. 1992; Sugaya et al. 1997; Vizzard et al. 1995). It is
422
believed that the PRV initially labels neurons in the motor limb of the micturition reflex
423
and then later labels neurons in sensory and modulatory circuits that send information to
424
the motor pathways. Areas identified by both PRV labeling and using fMRI in the present
425
studies include the PMC, PAG, hypothalamus, substantia nigra, cerebral cortex. Thus
426
these areas may be part of the efferent limb of the micturition reflex pathway
427
Sites in the motor cortex, basal ganglia (putamen, globus pallidus) and substantia
428
nigra which may play a role in bladder-striated sphincter coordination were also activated
429
during the micturition reflex. Motor cortex, somatosensory cortex, cingulate cortex,
430
insula, putamen, thalamus, and hypothalamus (Fig.3-5 and Table 2-5) are the brain
431
regions most frequently reported to be involved in human voluntary micturition control
432
(Fowler and Griffiths 2009; Griffiths and Tadic 2008; Kavia et al. 2005). It is worth
433
noting that the activated region of cingulate cortex extended from a posterior region to a
434
more anterior region when storage switched to the micturition (Table 4 and Fig.3-4). A
435
similar posterior-anterior shift in the activation of the cingulate cortex was also observed
18
436
in previous human studies during micturition (Athwal et al. 2001; Blok et al. 1997b,
437
1998; Matsuura et al. 2002). It was proposed that in humans the posterior region of
438
cingulate cortex might be involved in processing afferent signals during storage and in
439
turn contribute to the generation of bladder filling sensations, while the anterior region
440
might be involved in the perception of changes in bladder volume rather than the process
441
of micturition (Blok et al. 1997b, 1998; Kavia et al. 2005). In this study the micturition
442
contraction occurred under isovolumetric conditions, therefore it is reasonable to
443
conclude that the activation of anterior cingulate cortex was not related to the change of
444
bladder volume, but rather directly related to the micturition reflex. This speculation is
445
also in agreement with the proposal suggesting an “evaluation” function for the posterior
446
cingulate cortex but an “executive” function for the anterior cingulate cortex (Vogt et al.
447
1992).
448
A similar posterior-anterior activation was also observed in right insula (Table 2-4
449
and Fig.3), indicating that different regions of the insula might play different roles in
450
processing sensory input during storage or micturition. Activation of right insula was
451
identified by human brain imaging studies during both storage and micturition (Blok et
452
al. 1998; Griffiths et al. 2005, 2007; Kitta et al. 2006; Matsuura et al. 2002; Nour et al.
453
2000), but the posterior-anterior activation was not reported.
454
Activation of the retrosplenial cortex which was observed in our studies during
455
storage and micturition has not been reported in human imaging experiments nor
456
identified as an area of interest for bladder function in physiological or anatomical
457
experiments in animals. In humans the retrosplenial cortex which is considered to be part
458
of the posterior cingulate cortex sends projections to the hippocampus and is thought to
19
459
be involved in emotional processes and memory (Maddock 1999; Wyss and Van Groen
460
1992). During micturition the area of activation in the retrosplenial region shifted from
461
anterior to posterior (Table 4 and Fig.4) which coincided with the activation of the
462
hippocampus (using both BOLD and MION imaging) an area that was inactive during
463
filling (Table 3-5). Hippocampal activation during micturition was identified in human
464
imaging studies (Blok et al. 2006; Griffiths and Tadic 2008; Zhang et al. 2005; Matsuura
465
et al. 2002). The hippocampus was also labeled in a tracing study after injection of the rat
466
bladder with pseudorabies virus (Grill et al. 1999). The relatively small size of
467
hippocampal activation in this study (Fig.3 and Fig.5) might reflect the effect of
468
anesthesia. It is probable that only the reflex component of the emotional brain system
469
was activated under anesthesia.
470
Although rats do not have a prefrontal cortex and their frontal cortex is not as
471
fully developed as in primates and humans (Preuss 1995), behaviour observations suggest
472
that rats do exhibit voluntary control of micturition that can be influenced by their
473
environment. For example, normal Sprague-Dawley rats urinate ubiquitously throughout
474
their cage but rats exposed to chronic social stress urinate less frequently and always at
475
the corner of the cage (Wood et al. 2009) indicating that they do exhibit to some extent
476
“social continence”. Social continence mechanisms have been linked with activation of
477
the frontal cortex which has frequently been reported in human brain imaging studies of
478
micturtion control (Fowler and Griffiths 2009; Griffiths and Tadic 2008; Kavia et al.
479
2005). Activation of frontal cortex was not detected in our study of reflex micturition;
480
however this might reflect the fact that voluntary control of micturition was lost due to
481
anesthesia. On the other hand primary and secondary somatosensory cortices were
20
482
activated during both storage and micturition (Table 4 and Fig.4). Only a few human
483
brain imaging studies (Athwal et al. 2001; Di Gangi Herms et al. 2006; Nour et al. 2000)
484
detected the activation of somatosensory cortex during micturition control. These studies
485
attributed the activation to either the presence of urethral catheter or the contraction of
486
pelvic floor muscle. In this study, a ligature was applied to the base of the penis to
487
prevent fluid release from the bladder during micturition contraction. The increased
488
urethral pressure during micturition contraction might contribute to the enhanced
489
activation of the somatosensory cortex in this study. Neuronal responses to visceral
490
stimulation have also been recorded in the somatosensory cortex in experimental animals
491
(Bruggemann et al. 1994, 1997). Another possibility is that the concomitant contraction
492
of the external urethral sphincter (EUS) during both bladder filling and contraction in the
493
rat might cause the activation. The EUS of rats contracts intermittently during micturition
494
to facilitate voiding. This is different from the inhibition of EUS activity in humans and
495
cats and may explain the enhanced activation of motor cortex during micturition in the rat
496
(Table 4 and Fig.3).
497
The septal nucleus which was activated during storage was less activated during
498
micturition. Electrical stimulation in this area in animals (Hess 1947) evokes bladder
499
contractions, whereas injury to this area in humans due to aneurysms (Andrews and
500
Nathan 1964; Nathan 1976) produces urgency, frequency and incontinence. Thus neurons
501
in the septal area may play a role in the control of motor pathways to the bladder.
502
MION imaging identified more brain regions than BOLD imaging including
503
substantia nigra and globus pallidus (Table 5). Parkinson’s disease associated with
504
selective degeneration of dopaminergic neurons in substantia nigra causes urinary
21
505
disorders (Araki et al 2000). Lesions in rat substantia nigra cause bladder overactivity
506
(Yoshimura et al. 2003). In the cat electrical stimulation of substantia nigra (Lewin et al.
507
1967; Yoshimura et al. 1992) or the globus pallidus (Lewin et al. 1965, 1967) inhibited
508
bladder activity; whereas activation of globus pallidus has been observed in human brain
509
imaging during micturition (Nour et al. 2000).
510
Predominant activation on the right side of the brain was noticeable (Fig. 3 and
511
Table 4). Previous human brain imaging studies (Blok et al. 1997b, 1998; Mehnert et al.
512
2008) also reported right side predominance. Although only male rats were used in this
513
study, PET imaging studies have shown that the right side predominance exists in both
514
men and women (Blok et al. 1997b, 1998). Gender difference of voluntary micturition
515
control was not observed by human brain fMRI (Kuhtz-Buschbeck et al. 2007; Seseke et
516
al. 2008)
517
fMRI has been used previously in several animal studies (Angenstein et al. 2009;
518
Colonnese et al. 2008; Westlund et al. 2009). However, our study is the first to apply
519
fMRI to imaging brain control of micturition in an animal model. Although fMRI is non-
520
invasive and easily applicable to investigate the human brain, the use of fMRI in an
521
animal model of reflex micturition provides a wide range of opportunities for invasive
522
physiological and pharmacological studies. The model could be used to investigate
523
questions such as how brain switching for reflex micturition is influenced by damaging a
524
specific brain region, and how it is changed by centrally acting drugs. Animal brain fMRI
525
is a very useful tool to monitor neuronal activation in a large brain area compared to
526
traditional single unit electrical recordings.
22
527
This study confirmed the hypothesis based on previous neuroanatomical and
528
neurophysiological studies that the brainstem switch for reflex micturition control
529
involves both PAG and PMC. It also revealed a complex brain network that probably
530
modulates the activation of the brainstem switch to initiate micturition. Understanding
531
brain control of reflex micturition is very important for elucidating the underlying
532
mechanisms of neurogenic bladder dysfunctions including bladder overactivity,
533
frequency, urgency, and incontinence caused by brain disorders.
534
535
536
537
GRANTS
This work was supported by the National Institutes of Health grants DK-068566,
DK-077783, NS-045078, and EB-003375.
538
539
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759
FIGURE LEGENDS
760
Fig.1: fMRI experimental protocol for an individual animal. MRI images acquired during
761
each continuous scanning was extracted and averaged according to the different box cars
762
for detecting brain activation during bladder storage or contraction.
763
Fig.2: BOLD images showing brainstem activation associated with switching from the
764
bladder storage phase to the bladder contraction phase. The locations of coronal brain
765
sections (A-G) are indicated in the saggital brain image at the bottom, which correspond
766
to the Bregma coordinates in the anterior-posterior direction as 2.28, 0.24, -1.80, -3.84, -
767
5.88, -7.80, and –9.84 mm. Region of interest (ROI) analysis was performed on the
768
brainstem at coronal sections F and G in order to detect the activation. The PAG and
769
PMC are indicated by the blue arrows. The color scale bars indicate the t value.
770
Fig.3: BOLD images showing brain activation associated with switching from the bladder
771
storage phase to the bladder contraction phase. The locations of coronal brain sections
33
772
(A-G) are indicated in the saggital brain image at the bottom, which have the same
773
Bregma coordinates as in Fig.2. The color scale bars indicate the t value.
774
Fig.4: Region of interest analysis (ROI) of brain regions that did not show enhanced
775
activation in Fig.3. The 5 brain regions analyzed are marked by arrows. The locations of
776
coronal brain sections (A-G) are same as in Fig.2. Referencing the color scale bars in
777
Fig.3 for the t value.
778
Fig.5: MION images showing brain activation during the bladder contraction phase. The
779
locations of coronal brain sections (A-E) are same as in Fig.2. The color scale bars
780
indicate the t value.
781
Fig.6: MRI signal change during the bladder storage phase or during the bladder
782
contraction phase.
783
Table 1: Activated brainstem regions during storage or bladder contraction detected by
784
BOLD images and ROI analysis (P < 0.03).
785
Table 2: Activated brain regions during storage detected by BOLD images (P < 0.002).
786
Table 3: Activated brain regions during bladder contraction detected by BOLD images (P
787
< 0.0001).
788
Table 4: Changes of the brain network activations when switching from storage to
789
bladder contraction.
790
Table 5: Activated brain regions during bladder contraction detected by MION images (P
791
< 0.005).
792
Stop bladder
infusion
25 cmH2O
Empty
bladder
Start bladder
infusion
Bladder
Pressure
1 min
Continuous MRI Scanning
10-12 trials
MRI
Images
control 1
storage
contraction
control 1
storage
contraction
control 2
Averaged
MRI
Images
control 2
contraction
storage
Box
Cars
control 1
control 2
detecting brain activation during storage
control 1
control 2
detecting brain activation during contraction
Fig.1: fMRI experimental protocol for an individual animal. MRI images acquired during
each continuous scanning was extracted and averaged according to the different box cars
for detecting brain activation during bladder storage or contraction.
Storage
Brainstem Switch
Contraction
F
PAG
PMC
G
2.24
8.06
2.19
11.33
A B C D E F G
Fig.2: BOLD images showing brainstem activation associated with switching from the bladder storage phase to the bladder contraction
phase. The locations of coronal brain sections (A-G) are indicated in the saggital brain image at the bottom, which correspond to the Bregma
coordinates in the anterior-posterior direction as 2.28, 0.24, -1.80, -3.84, -5.88, -7.80, and –9.84 mm. Region of interest (ROI) analysis was
performed on the brainstem at coronal sections F and G in order to detect the activation. The PAG and PMC are indicated by the blue
arrows. The color scale bars indicate the t value.
Storage
Brain Switch
Contraction
A
B
C
D
E
4.23
6.45
8.06
11.33
A B C D E F G
Fig.3: BOLD images showing brain activation associated with switching from the bladder storage
phase to the bladder contraction phase. The locations of coronal brain sections (A-G) are indicated
in the saggital brain image at the bottom, which have the same Bregma coordinates as in Fig.2. The
color scale bars indicate the t value.
Brain Switch
Storage
A
B
Contraction
Cingulate
cortex
Primary
somatosensory
cortex
Septal
nucleus
C
D
Secondary
somatosensory
cortex
Retrosplenial
cortex
E
A B C D E F G
Fig.4: Region of interest analysis (ROI) of brain regions that did not show
enhanced activation in Fig.3. The 5 brain regions analyzed are marked by
arrows. The locations of coronal brain sections (A-G) are same as in Fig.2.
Referencing the color scale bars in Fig.3 for the t value.
A
B
C
D
E
3.71
16.39
Fig.5: MION images showing brain activation during the bladder contraction phase.
The locations of coronal brain sections (A-E) are same as in Fig.2. The color scale
bars indicate the t value.
BOLD signal during storage
BOLD signal during contraction
MION signal during contraction
MRI Signal Change (%)
3
2
1
control 1
control 2
storage
or
contraction
0
-1
-2
-3
0
100
200
300
Time (sec)
Fig.6: MRI signal change during the bladder storage phase
or during the bladder contraction phase.
Table 1: Activated brainstem regions during storage or bladder contraction
detected by BOLD images and ROI analysis (P < 0.03).
Activated
Bregma coordinates (mm) Activation size (mm)
Peak
Brain Region
LR
DV
AP
LR DV
AP t value
During storage
PAG
-0.5
5.9
-7.80 1.0
0.8
2
3.50
PCRt
2.6
8.5
-9.84 0.4
0.7
2
3.10
During micturition contraction
PAG
0.8
6.5
-7.80 1.8
2.0
2
5.20
PMC
1.3
7.8
-9.84 1.3
0.8
2
2.76
-0.9
7.4
-9.84 0.5
0.3
2
2.54
Inferior
-2.0
5.9
-7.80 3.0
0.8
2
2.57
Colliculus
Table 2: Activated brain regions during storage detected by BOLD images (P <
0.002).
Activated
Bregma coordinates (mm) Activation size (mm)
Peak
Brain Region
LR
DV
AP
LR DV
AP t value
Motor cortex
1.6
2.2
2.28 0.5
0.3
2
4.65
1.7
2.3
0.24 0.4
0.4
2
4.71
Primary
3.4
2.2
0.24 1.4
0.6
2
5.35
Somatosensory
Cortex
Secondary
6.7
5.5
0.24 0.9
0.7
2
5.01
Somatosensory 5.8
6.2
-1.80 1.6
2.0
2
8.06
Cortex
Cingulate
0.4
2.3
0.24 1.5
0.6
2
4.60
Cortex
Retrosplenial
-0.2
2.2
-1.80 1.4
1.4
2
6.11
Cortex
-0.2
2.1
-3.84 0.5
0.5
2
4.85
Thalamus
1.8
5.0
-1.80 0.9
0.5
2
5.21
-0.7
5.6
-1.80 0.5
0.4
2
4.93
Putamen
2.0
5.2
0.24 0.7
0.7
2
5.62
-1.8
5.2
0.24 0.8
0.3
2
5.20
Insula
5.8
6.5
-1.80 1.6
0.9
2
7.98
Septal nucleus -0.6
6.0
0.24 0.8
1.0
2
6.41
1.3
5.2
0.24 0.5
0.6
2
5.62
-1.4
5.4
0.24 0.4
0.3
2
5.33
Table 3: Activated brain regions during bladder contraction detected by BOLD
images (P < 0.0001).
Activated
Bregma coordinates (mm) Activation size (mm)
Peak
Brain Region
LR
DV
AP
LR DV
AP t value
Motor cortex
1.8
2.0
2.28 0.9
1.3
2
11.33
1.9
1.7
0.24 0.9
1.0
2
7.65
-1.6
1.7
2.28 0.9
0.7
2
9.18
-1.8
2.1
0.24 1.3
1.3
2
7.89
Secondary
6.0
5.5
0.24 0.4
1.1
2
7.25
Somatosensory
Cortex
Cingulate
0.9
2.4
2.28 0.9
0.7
2
7.52
Cortex
-0.9
2.1
2.28 0.5
0.6
2
6.95
-0.3
3.2
0.24 0.9
0.7
2
8.67
Retrosplenial
1.3
2.7
-5.88 1.0
0.7
2
8.21
Cortex
Thalamus
2.3
4.9
-1.80 1.8
1.0
2
9.82
-3.2
5.5
-1.80 1.4
0.7
2
9.25
1.5
6.0
-3.84 0.8
1.1
2
7.87
-3.1
5.3
-3.84 1.4
1.1
2
7.31
Putamen
2.8
4.8
0.24 1.8
1.1
2
10.35
-2.0
4.4
0.24 0.9
0.6
2
7.31
4.6
5.5
-1.80 1.4
0.7
2
9.51
Insula
5.6
6.0
2.28 0.9
0.6
2
8.17
Hippocampus -0.8
4.0
-1.80 1.0
0.6
2
7.85
2.2
3.4
-5.88 0.9
0.8
2
9.74
Table 4: Changes of the brain network activations when switching from
storage to bladder contraction.
Brain Regions
Storage
Bladder Contraction
Motor cortex
x (right)
n (left + right)
Primary Somatosensory
x (right)
n (right)
Cortex
Secondary Somatosensory x (right anterior)
n (right anterior)
Cortex
x (right posterior)
p (right posterior)
Cingulate Cortex
x (posterior)
n (anterior + posterior)
Retrosplenial Cortex
x (anterior)
n (anterior + posterior)
Thalamus
x
n
Putamen
x
n
Insula
x (right posterior)
x (right anterior)
Septal nucleus
x
n
Hippocampus
x
activated (x); not activated (-); increased (n); decreased (p)
Table 5: Activated brain regions during bladder contraction detected by MION
images (P < 0.005).
Activated
Bregma coordinates (mm) Activation size (mm)
Peak
Brain Region
LR
DV
AP
¨LR
¨DV
¨AP t value
Motor cortex
1.4
2.6
0.24 1.3
1.6
2
6.87
Primary
-6.6
4.0
2.28 1.0
0.4
2
5.56
Somatosensory 5.3
4.5
0.24 1.4
1.0
2
5.93
Cortex
-5.8
4.3
0.24 0.9
0.4
2
5.43
-6.0
4.3
-1.80 0.3
0.3
2
4.95
Cingulate
-1.1
3.9
2.28 1.7
1.0
2
16.39
Cortex
-0.4
3.6
0.24 0.4
0.4
2
6.15
1.0
2.8
0.24 0.6
0.4
2
4.62
Retrosplenial
1.0
0.9
-5.88 1.5
0.4
2
4.16
Cortex
-0.9
0.9
-5.88 1.3
0.4
2
4.53
0.1
2.4
-1.80 1.3
1.1
2
4.18
Thalamus
2.6
5.7
-1.80 1.4
0.9
2
6.12
-2.0
5.0
-1.80 2.0
1.3
2
6.33
1.0
7.1
-1.80 0.5
0.6
2
5.14
2.4
6.0
-3.84 2.5
2.7
2
9.33
-2.9
5.4
-3.84 1.2
1.1
2
6.99
-1.5
7.0
-3.84 0.9
0.7
2
5.56
Hypothalamus 0.3
7.1
-3.84 1.7
1.3
2
5.49
1.6
8.3
-1.80 0.7
0.3
2
5.14
Putamen
5.0
4.9
-1.80 0.8
0.7
2
5.23
5.2
5.4
-3.84 0.8
0.3
2
4.40
Hippocampus
0.4
3.4
-3.84 2.0
1.0
2
6.77
2.9
2.0
-3.84 0.9
0.7
2
5.10
Insula
5.4
6.0
0.24 0.7
0.4
2
4.30
Substantia
-3.0
7.7
-5.88 1.5
0.6
2
6.91
Nigra
Globus
3.7
6.9
-1.80 1.8
0.7
2
5.51
Pallidus
-3.8
6.0
-1.80 0.5
0.4
2
4.12