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Firing patterns of micturition-related neurons in the pontine storage centre in cats
Autonomic Neuroscience: Basic and Clinical 99 (2002) 24 – 30 www.elsevier.com/locate/autneu
Firing patterns of micturition-related neurons in the pontine storage centre in cats Ryuji Sakakibara a,*, Ken Nakazawa b, Keisuke Shiba c, Yoshio Nakajima b, Tomoyuki Uchiyama a, Mitsuharu Yoshiyama a, Tomonori Yamanishi d, Takamichi Hattori a a Department of Neurology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana Chuo-ku, Chiba 260-8670, Japan Department of Integrative Neurophysiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana Chuo-ku, Chiba 260-8670, Japan c Department of Otolaryngology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana Chuo-ku, Chiba 260-8670, Japan d Department of Urology, Dokkyo Medical College, Tochigi, Japan
b
Received 26 December 2001; received in revised form 20 March 2002; accepted 25 March 2002
Abstract The pontine storage centre (PSC) and the pontine micturition centre (PMC) are known to be critical for urinary filling and emptying, respectively. In the present study, firing patterns of 45 neurons in the PSC area where electrical stimulation induced inhibition of the micturition reflex were analyzed in 20 male decerebrated and paralyzed cats. The electrically determined PSC area was widespread in the dorsolateral pontine reticular formation (P0 – P4), ventrolateral to the PMC. Four major types of neurons were detected according to urinary storage/micturition cycles: tonic storage neurons (38%), phasic storage neurons (40%), tonic micturition neurons (9%) and phasic micturition neurons (13%). These four types of neurons were intermingled in the PSC. However, the tonic and phasic micturition neurons tended to be located within a limited area (P2 – P3). These neurons were further classified into augmenting, constant and decrementing firing patterns. Some increased their firing prior to the storage/micturition phase initiation. Such preceding pattern was more frequently found in the tonic neurons than in the phasic neurons. In conclusion, the PSC neurons with diverse heterogeneous discharge patterns suggest that these neurons may organize a complex neuronal circuitry, which is critical in the neural control of the urinary continence. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Pontine storage centre (PSC); Pontine micturition centre (PMC); Urinary continence; Single-unit recording
1. Introduction Previous anatomical and physiological studies have revealed that the micturition reflex is dependent on neural circuitry in the pons (de Groat et al., 1993; Blok and Holstege, 1999). Two major structures involved in the micturition reflex exist in the area. One is known as the pontine micturition centre (PMC) (de Groat et al., 1993), Barrington’s nucleus (Barrington, 1921, 1925; Valentino et al., 1999) or M region (Holstege et al., 1986; Blok and Holstege, 1999), which is located in or adjacent to the locus coeruleus in cats, dogs, possibly in humans (Betts et al., 1992; de Groat et al., 1993; Nishizawa and Sugaya, 1994; Sakakibara et al., 1996; Blok et al., 1997; Valentino et al., 1999; Blok and Holstege, 2000) and in the laterodorsal tegmental nucleus in rats (Satoh et al., 1978a,b; to et al., *
Corresponding author. Tel.: +81-43-226-2129; fax: +81-43-226-2160. E-mail address: [email protected] (R. Sakakibara).
1989). Lesions in the PMC result in severe urinary dysfunction such as urinary retention in experimental animals (de Groat et al., 1993; Blok and Holstege, 1999) as well as in humans (Betts et al., 1992; Sakakibara et al., 1996). Electrical or chemical stimulation of the PMC initiates micturition reflex (Noto et al., 1989; Mallory et al., 1989, 1991; Griffiths et al., 1990) by activating a descending pathway to the sacral parasympathetic preganglionic nucleus, which innervates the urinary bladder muscles (Holstege and Kuypers, 1982; Valentino et al., 1999; Blok and Holstege, 2000). The other area is known as the pontine storage centre (PSC) (Nishizawa et al., 1987; de Groat et al., 1993), or L region (Holstege et al., 1986; Blok and Holstege, 1999), which is located ventrolateral to the PMC. Bilateral lesions in the PSC give rise to an inability to store urine; bladder capacity is reduced and urine is expelled prematurely by excessive detrusor activity accompanied by urethral relaxation (Blok and Holstege, 1996). The PSC activation terminates or inhibits the micturition reflex together with an
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 ( 0 2 ) 0 0 0 5 5 - 3
R. Sakakibara et al. / Autonomic Neuroscience: Basic and Clinical 99 (2002) 24–30
excitation of the pelvic floor musculature (Griffiths et al., 1990; Blok and Holstege, 1999) including the urethral sphincter innervated by the sacral Onuf’s nucleus (Holstege and Tan, 1987; Blok et al., 1998). Neurophysiological studies have demonstrated that the PMC neurons discharge with several firing patterns, including neurons that fire predominantly during micturition and predominantly during urinary storage (Bradley and Conway, 1966; Okada and Yamane, 1974; de Groat et al., 1998). In contrast, neuronal activities in the PSC have yet to be investigated. Thus, this study aimed to examine discharge patterns of the PSC neurons that may be crucial for the urinary continence.
2. Materials and methods 2.1. Experimental preparation Experiments were done on 20 adult male cats (3.2 –4.0 kg) that were decerebrated at the precollicular level. Surgi-
25
Table 1 Classification of the PSC neurons according to their firing patterns n
*
Augmenting * Constant
Tonic storage 17 6 (13%) Phasic storage 18 3 (7%) Tonic micturition 4 0 (0%) Phasic micturition 6 1 (2%)
4 7 (16%) 1 10 (23%) – 2 (4%) – 3 (7%)
* Decrementing * 5 – 2 2
4 5 2 2
(9%) (11%) (4%) (4%)
2 1 1 –
Asterisk indicates neurons with preceding firing pattern.
cal preparation was performed under anesthesia with halothane vaporized in nitrous oxide and oxygen. The trachea was intubated and catheters were placed in the femoral artery to monitor blood pressure and in the femoral veins for drug administration. A double-lumen urinary catheter was inserted into the bladder transurethrally to measure bladder pressure and to regulate bladder volume. The animals were positioned in a stereotaxic frame. After decerebration and completion of all the surgical procedures, anesthesia was discontinued, which was done at least 1 h
Fig. 1. (A) Typical responses of bladder pressure to electrical stimulation of the pontine micturition centre (PMC) (a) and the pontine storage centre (PSC) (b). Duration of repetitive stimulation (0.2-ms pulses at 100 Hz, 100-AA pulse amplitude) is indicated by thick lines. (Aa) PMC stimulation immediately initiated the micturition reflex. (Ab) PSC stimulation immediately terminated the micturition reflex. (B) The location of the PMC and the PSC areas according to Horsley – Clarke coordinates, determined by the response patterns to electrical stimulation. LC, locus coeruleus; DTN, dorsal tegmental nucleus; CTF, central tegmental field; PTF, paraleminiscal tegmental field; GTF, gigantocellular tegmental field; LTF, lateral tegmental field.
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Fig. 3. Location of the PSC neurons. (A) Tonic and phasic storage neurons. (B) Tonic and phasic micturition neurons. Filled, tonic; open, phasic; triangle, augmenting; circle, constant; square, decrementing.
prior to data collection. The animals were paralyzed by intravenous infusion of pancuronium bromide (initial infusion of 0.2 mg/kg, additional infusion of 0.02 mg/kg, when necessary), and artificial ventilation was applied. End-tidal C02 was maintained at 3 –5%. Rectal temperature was kept at 37 – 39 jC using a heating lamp. At the end of the experiment, the animals were given an overdose of pentobarbital sodium. 2.2. Recording and stimulation Rhythmic isovolumetric bladder contraction was generated by infusion of a certain amount of saline (20 –50 ml) into the bladder, which resulted in continuous cycles consisting of bladder contractions against a closed outlet and relaxations. A micturition phase was defined as a period between onset and offset of the increase of the
bladder pressure. A storage phase was defined as a period between micturition phases. Electrical stimulation was delivered in the rostral pons to determine the locations of the PMC and PSC. Monopolar tungsten microelectrodes (FHC #25-05-3; tip diameter 25 Am, tip impedance 9 – 12 MV) were inserted stereotaxically into the rostral pons using the Horsley – Clarke coordinates (Berman, 1968), and moved in 0.5-mm steps while applying stimulus trains (stimulus parameters: 0.2-ms duration, 50 – 100 AA, 100 Hz). The sites where stimulation initiated and terminated the micturition reflex were identified as the PMC and PSC, respectively. Extracellular recordings were then obtained from neurons in the PSC with the tungsten microelectrodes used for stimulation. At the end of each recording, stimulation was applied to the exact recording site with the same electrode to verify if the stimulation inhibited the micturition reflex. At the end of the experiment, electrolytic
Fig. 2. Examples of various firing patterns of the PSC neurons. Each of the top panels indicates instantaneous frequencies of the discharges shown in the middle panels. The bottom panels indicate bladder pressure. (A) Tonic storage neuron (augmenting). (B) Tonic storage neuron (constant). (C) Tonic storage neuron (decrementing). (D) Phasic storage neuron (decrementing). (E) Tonic micturition neuron (constant). (F) Phasic micturition neuron (decrementing). Horizontal thick lines indicate 10 s.
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lesion (30 AA, 1 min) were made to mark the location of the recording sites. The recording data from neurons together with bladder pressure were stored using a Cambridge Electronic Design (CED) 1401-plus data interface and Spike 2 software.
3. Results The location of the PMC and PSC was identified by the responses of bladder contractions to electrical stimulation. The typical responses of bladder pressure to PMC and PSC stimulation are seen in Fig. 1Aa and Ab, respectively. The PMC was located within a relatively limited area in the vicinity of the locus coeruleus (Fig. 1B). In contrast, the PSC was distributed in a broader area, mainly ventrolateral to the PMC, extended rostrocaudally (P0– P4) overlapping to the dorsolateral pontine reticular formation including the central, paraleminiscal, gigantocellular and lateral tegmental fields (Fig. 1B). A total of 45 neurons were recorded in the PSC that were related to urinary storage/micturition cycles. Every recording site was confirmed to be within the PSC area by stimulation after recording. These neurons were classified into four major types according to their firing patterns (Table 1): (1) tonic storage neurons that were continuously active throughout storage/micturition cycles with storage phase predominance (17 neurons; 38%) (Fig. 2A, B and C), (2) phasic storage neurons that were only active during the storage phases (18 neurons; 40%) (Fig. 2D), (3) tonic micturition neurons that were continuously active throughout storage/micturition cycles with micturition phase predominance (four neurons; 9%) (Fig. 2E) and (4) phasic micturition neurons that were only active during the micturition phases (six neurons; 13%) (Fig. 2F). These four types of neurons were intermingled in the PSC area (Fig. 3). The majority of the tonic and phasic micturition neurons (9/10) were concentrated within the midcaudal area (P2 –P3), and also the majority of the tonic storage neurons (16/17) were located in the caudal part (P2 –P4). In contrast, phasic storage neurons were extensively existed in the PSC. These neuronal types were further subclassified into augmenting (Fig. 2A and D), constant (Fig. 2B and E) and decrementing (Fig. 2C and F) neurons according to their temporal discharge rate change during the storage and micturition phases for the storage and micturition neurons, respectively. As seen in Table 1, all of these neuronal subtypes were found in each of the four major types, except absence of augmenting neurons in the tonic micturition type, presumably due to sampling insufficiency. With respect to the anatomical location, the augmenting neurons were concentrated in the caudal part (P2 – P4), whereas the constant and decrementing neurons were widely distributed in the PSC. Some of the recorded neurons either increased (for tonic type) or launched (for phasic type) their firing prior to the storage/micturition phase initiation (Fig. 2B and E) (Table 1, asterisk). This preceding firing pattern was
more frequently found in the tonic storage neurons (11/17; 65%) and tonic micturition neurons (3/4; 75%) than in the phasic storage neurons (2/18; 11%) and phasic micturition neurons (2/6; 33%). Average interval between the onset of preceding neuronal firing and the phase initiation was 6.5 s (range 2– 10 s) in the tonic storage neurons, 6.0 s (range 2– 8 s) in the phasic storage neurons, 8.3 s (range 7 – 10 s) in the tonic micturition neurons and 4.0 s (range 3 – 5 s) in the phasic micturition neurons. The neurons with the preceding firing pattern were intermingled with those without such pattern.
4. Discussion The location of the PSC has been investigated in both anatomical and physiological manners. Morphological studies have revealed that the sacral Onuf’s nucleus, which innervates the urethral sphincter, receives a direct projection from a discrete area located ventrolateral to the PMC, named the L region (Holstege and Kuypers, 1982; Holstege et al. 1986; Holstege and Tan, 1987). On the other hand, the detrusor relaxation and the external sphincter contraction are elicited by electrical stimulation of an extensive area of the dorsolateral reticular formation, which Griffiths et al. (1990) called a ribbon-like band, running mainly from the L region to the PMC. The present study demonstrated that the PSC area, defined by electrical stimulation, was widespread in the dorsolateral reticular formation at the level of P0 – P4, which is consistent with the observation of Griffiths et al. (1990). This electrically determined PSC area is considered to contain neurons involved in urinary continence as well as their pathways because electrical stimulation activates soma as well as axons. The neurons involved in urinary continence, therefore, might be located in a more restricted region of the PSC area observed in this study. Indeed, inhibition of the micturition reflex is elicited by chemical stimulation of a relatively limited area, the PoO region, which is ventromedial to the PMC (Sugaya et al., 1987). Nevertheless, if we assume that the neuronal discharges recorded in this study were mainly obtained from the soma, our result may indicate that micturition-related neurons, which could contribute to the maintenance of urinary continence, are extensively existed in the PSC. This study demonstrated that the PSC neurons exhibited a variety of firing patterns according to the storage/micturition cycles. Of these neurons, the majority was the tonic and phasic storage neurons (78%) while the remaining was the tonic and phasic micturition neurons (22%). The proportion of the storage type neurons observed in this study was somewhat higher than that obtained from the PMC area, reported by de Groat et al. (1998), showing that the proportion of the micturition and storage neurons were 21% and 51%, respectively. Predominance of the storage neurons, which may actively be involved in the neural control of the urinary system during the storage phase, might be appropriate for contributing to the maintenance of
R. Sakakibara et al. / Autonomic Neuroscience: Basic and Clinical 99 (2002) 24–30
the urinary storage as well as the suppression of the micturition reflex. On the other hand, the micturition neurons, which seemed to be concentrated in the midcaudal part of the PSC (P2 – P3) in this study, might form a local circuit that inactivates the urinary continence system in the PSC during the micturition phase. In addition to the storage/micturition classification, the neurons showed diverse discharge patterns: the tonic and phasic as well as the augmenting, constant, and decrementing. The neurons with decrementing firing frequency had its peak at the start of firing, which could be called as switch-on neurons. The neurons with augmenting firing frequency had its peak at the end of firing, which could be called as switch-off neurons. If these neurons are storage-related neurons, it is likely that these neurons play an essential role in the initiation (switch-on) and termination (switch-off) of the urinary storage, respectively. Similarly, if these neurons are micturition-related neurons, it is likely that these neurons play a role in the initiation and termination of the micturition reflex, respectively, most probably via an inhibitory neural circuit. The neurons with constant firing frequency may be involved in maintaining urinary storage. Whereas the augmenting neurons were concentrated in the caudal part (P2 –P4), the constant and decrementing neurons were widely distributed in the pons. To elucidate the exact functional role of the neurons, however, further studies in their electrophysiological properties (e.g. excitatory or inhibitory) as well as their functional and morphological connections will be necessary. Some of the neurons recorded in this study exhibited preceding discharge pattern, i.e., either increase or start of firing 2 –10 s prior to the storage/micturition phase initiation. This preceding time interval is considered much larger than the delay time from neuronal phase initiation at pontine level to contraction or relaxation response of the bladder muscle, which can be determined based on neuronal and muscular properties, e.g., it has been reported that central delay for activation of the sacral preganglionic neurons following pontine stimulation is 60 – 75 ms in cats (Mallory et al., 1989). Such neurons with preceding firing pattern, therefore, could be involved in neural control of the storage/micturition phase initiation. In this study, this preceding pattern was more frequently observed in the tonic storage and tonic micturition neurons than in the phasic storage and phasic micturition neurons, indicating a possibility of difference in functional role between the tonic neurons and the phasic neurons, though this dissociation may simply be produced by the difference in the thresholds of membrane excitability between the tonic and phasic neurons.
5. Conclusions The existence of a variety of heterogeneous discharge patterns observed in the PSC neurons suggest that these neurons may organize a complex neuronal circuitry, which is critical in the neural control of the urinary continence and the micturition/storage phase initiation.
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Firing patterns of micturition-related neurons in the pontine storage centre in cats Ryuji Sakakibara a,*, Ken Nakazawa b, Keisuke Shiba c, Yoshio Nakajima b, Tomoyuki Uchiyama a, Mitsuharu Yoshiyama a, Tomonori Yamanishi d, Takamichi Hattori a a Department of Neurology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana Chuo-ku, Chiba 260-8670, Japan Department of Integrative Neurophysiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana Chuo-ku, Chiba 260-8670, Japan c Department of Otolaryngology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana Chuo-ku, Chiba 260-8670, Japan d Department of Urology, Dokkyo Medical College, Tochigi, Japan
b
Received 26 December 2001; received in revised form 20 March 2002; accepted 25 March 2002
Abstract The pontine storage centre (PSC) and the pontine micturition centre (PMC) are known to be critical for urinary filling and emptying, respectively. In the present study, firing patterns of 45 neurons in the PSC area where electrical stimulation induced inhibition of the micturition reflex were analyzed in 20 male decerebrated and paralyzed cats. The electrically determined PSC area was widespread in the dorsolateral pontine reticular formation (P0 – P4), ventrolateral to the PMC. Four major types of neurons were detected according to urinary storage/micturition cycles: tonic storage neurons (38%), phasic storage neurons (40%), tonic micturition neurons (9%) and phasic micturition neurons (13%). These four types of neurons were intermingled in the PSC. However, the tonic and phasic micturition neurons tended to be located within a limited area (P2 – P3). These neurons were further classified into augmenting, constant and decrementing firing patterns. Some increased their firing prior to the storage/micturition phase initiation. Such preceding pattern was more frequently found in the tonic neurons than in the phasic neurons. In conclusion, the PSC neurons with diverse heterogeneous discharge patterns suggest that these neurons may organize a complex neuronal circuitry, which is critical in the neural control of the urinary continence. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Pontine storage centre (PSC); Pontine micturition centre (PMC); Urinary continence; Single-unit recording
1. Introduction Previous anatomical and physiological studies have revealed that the micturition reflex is dependent on neural circuitry in the pons (de Groat et al., 1993; Blok and Holstege, 1999). Two major structures involved in the micturition reflex exist in the area. One is known as the pontine micturition centre (PMC) (de Groat et al., 1993), Barrington’s nucleus (Barrington, 1921, 1925; Valentino et al., 1999) or M region (Holstege et al., 1986; Blok and Holstege, 1999), which is located in or adjacent to the locus coeruleus in cats, dogs, possibly in humans (Betts et al., 1992; de Groat et al., 1993; Nishizawa and Sugaya, 1994; Sakakibara et al., 1996; Blok et al., 1997; Valentino et al., 1999; Blok and Holstege, 2000) and in the laterodorsal tegmental nucleus in rats (Satoh et al., 1978a,b; to et al., *
Corresponding author. Tel.: +81-43-226-2129; fax: +81-43-226-2160. E-mail address: [email protected] (R. Sakakibara).
1989). Lesions in the PMC result in severe urinary dysfunction such as urinary retention in experimental animals (de Groat et al., 1993; Blok and Holstege, 1999) as well as in humans (Betts et al., 1992; Sakakibara et al., 1996). Electrical or chemical stimulation of the PMC initiates micturition reflex (Noto et al., 1989; Mallory et al., 1989, 1991; Griffiths et al., 1990) by activating a descending pathway to the sacral parasympathetic preganglionic nucleus, which innervates the urinary bladder muscles (Holstege and Kuypers, 1982; Valentino et al., 1999; Blok and Holstege, 2000). The other area is known as the pontine storage centre (PSC) (Nishizawa et al., 1987; de Groat et al., 1993), or L region (Holstege et al., 1986; Blok and Holstege, 1999), which is located ventrolateral to the PMC. Bilateral lesions in the PSC give rise to an inability to store urine; bladder capacity is reduced and urine is expelled prematurely by excessive detrusor activity accompanied by urethral relaxation (Blok and Holstege, 1996). The PSC activation terminates or inhibits the micturition reflex together with an
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 ( 0 2 ) 0 0 0 5 5 - 3
R. Sakakibara et al. / Autonomic Neuroscience: Basic and Clinical 99 (2002) 24–30
excitation of the pelvic floor musculature (Griffiths et al., 1990; Blok and Holstege, 1999) including the urethral sphincter innervated by the sacral Onuf’s nucleus (Holstege and Tan, 1987; Blok et al., 1998). Neurophysiological studies have demonstrated that the PMC neurons discharge with several firing patterns, including neurons that fire predominantly during micturition and predominantly during urinary storage (Bradley and Conway, 1966; Okada and Yamane, 1974; de Groat et al., 1998). In contrast, neuronal activities in the PSC have yet to be investigated. Thus, this study aimed to examine discharge patterns of the PSC neurons that may be crucial for the urinary continence.
2. Materials and methods 2.1. Experimental preparation Experiments were done on 20 adult male cats (3.2 –4.0 kg) that were decerebrated at the precollicular level. Surgi-
25
Table 1 Classification of the PSC neurons according to their firing patterns n
*
Augmenting * Constant
Tonic storage 17 6 (13%) Phasic storage 18 3 (7%) Tonic micturition 4 0 (0%) Phasic micturition 6 1 (2%)
4 7 (16%) 1 10 (23%) – 2 (4%) – 3 (7%)
* Decrementing * 5 – 2 2
4 5 2 2
(9%) (11%) (4%) (4%)
2 1 1 –
Asterisk indicates neurons with preceding firing pattern.
cal preparation was performed under anesthesia with halothane vaporized in nitrous oxide and oxygen. The trachea was intubated and catheters were placed in the femoral artery to monitor blood pressure and in the femoral veins for drug administration. A double-lumen urinary catheter was inserted into the bladder transurethrally to measure bladder pressure and to regulate bladder volume. The animals were positioned in a stereotaxic frame. After decerebration and completion of all the surgical procedures, anesthesia was discontinued, which was done at least 1 h
Fig. 1. (A) Typical responses of bladder pressure to electrical stimulation of the pontine micturition centre (PMC) (a) and the pontine storage centre (PSC) (b). Duration of repetitive stimulation (0.2-ms pulses at 100 Hz, 100-AA pulse amplitude) is indicated by thick lines. (Aa) PMC stimulation immediately initiated the micturition reflex. (Ab) PSC stimulation immediately terminated the micturition reflex. (B) The location of the PMC and the PSC areas according to Horsley – Clarke coordinates, determined by the response patterns to electrical stimulation. LC, locus coeruleus; DTN, dorsal tegmental nucleus; CTF, central tegmental field; PTF, paraleminiscal tegmental field; GTF, gigantocellular tegmental field; LTF, lateral tegmental field.
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Fig. 3. Location of the PSC neurons. (A) Tonic and phasic storage neurons. (B) Tonic and phasic micturition neurons. Filled, tonic; open, phasic; triangle, augmenting; circle, constant; square, decrementing.
prior to data collection. The animals were paralyzed by intravenous infusion of pancuronium bromide (initial infusion of 0.2 mg/kg, additional infusion of 0.02 mg/kg, when necessary), and artificial ventilation was applied. End-tidal C02 was maintained at 3 –5%. Rectal temperature was kept at 37 – 39 jC using a heating lamp. At the end of the experiment, the animals were given an overdose of pentobarbital sodium. 2.2. Recording and stimulation Rhythmic isovolumetric bladder contraction was generated by infusion of a certain amount of saline (20 –50 ml) into the bladder, which resulted in continuous cycles consisting of bladder contractions against a closed outlet and relaxations. A micturition phase was defined as a period between onset and offset of the increase of the
bladder pressure. A storage phase was defined as a period between micturition phases. Electrical stimulation was delivered in the rostral pons to determine the locations of the PMC and PSC. Monopolar tungsten microelectrodes (FHC #25-05-3; tip diameter 25 Am, tip impedance 9 – 12 MV) were inserted stereotaxically into the rostral pons using the Horsley – Clarke coordinates (Berman, 1968), and moved in 0.5-mm steps while applying stimulus trains (stimulus parameters: 0.2-ms duration, 50 – 100 AA, 100 Hz). The sites where stimulation initiated and terminated the micturition reflex were identified as the PMC and PSC, respectively. Extracellular recordings were then obtained from neurons in the PSC with the tungsten microelectrodes used for stimulation. At the end of each recording, stimulation was applied to the exact recording site with the same electrode to verify if the stimulation inhibited the micturition reflex. At the end of the experiment, electrolytic
Fig. 2. Examples of various firing patterns of the PSC neurons. Each of the top panels indicates instantaneous frequencies of the discharges shown in the middle panels. The bottom panels indicate bladder pressure. (A) Tonic storage neuron (augmenting). (B) Tonic storage neuron (constant). (C) Tonic storage neuron (decrementing). (D) Phasic storage neuron (decrementing). (E) Tonic micturition neuron (constant). (F) Phasic micturition neuron (decrementing). Horizontal thick lines indicate 10 s.
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lesion (30 AA, 1 min) were made to mark the location of the recording sites. The recording data from neurons together with bladder pressure were stored using a Cambridge Electronic Design (CED) 1401-plus data interface and Spike 2 software.
3. Results The location of the PMC and PSC was identified by the responses of bladder contractions to electrical stimulation. The typical responses of bladder pressure to PMC and PSC stimulation are seen in Fig. 1Aa and Ab, respectively. The PMC was located within a relatively limited area in the vicinity of the locus coeruleus (Fig. 1B). In contrast, the PSC was distributed in a broader area, mainly ventrolateral to the PMC, extended rostrocaudally (P0– P4) overlapping to the dorsolateral pontine reticular formation including the central, paraleminiscal, gigantocellular and lateral tegmental fields (Fig. 1B). A total of 45 neurons were recorded in the PSC that were related to urinary storage/micturition cycles. Every recording site was confirmed to be within the PSC area by stimulation after recording. These neurons were classified into four major types according to their firing patterns (Table 1): (1) tonic storage neurons that were continuously active throughout storage/micturition cycles with storage phase predominance (17 neurons; 38%) (Fig. 2A, B and C), (2) phasic storage neurons that were only active during the storage phases (18 neurons; 40%) (Fig. 2D), (3) tonic micturition neurons that were continuously active throughout storage/micturition cycles with micturition phase predominance (four neurons; 9%) (Fig. 2E) and (4) phasic micturition neurons that were only active during the micturition phases (six neurons; 13%) (Fig. 2F). These four types of neurons were intermingled in the PSC area (Fig. 3). The majority of the tonic and phasic micturition neurons (9/10) were concentrated within the midcaudal area (P2 –P3), and also the majority of the tonic storage neurons (16/17) were located in the caudal part (P2 –P4). In contrast, phasic storage neurons were extensively existed in the PSC. These neuronal types were further subclassified into augmenting (Fig. 2A and D), constant (Fig. 2B and E) and decrementing (Fig. 2C and F) neurons according to their temporal discharge rate change during the storage and micturition phases for the storage and micturition neurons, respectively. As seen in Table 1, all of these neuronal subtypes were found in each of the four major types, except absence of augmenting neurons in the tonic micturition type, presumably due to sampling insufficiency. With respect to the anatomical location, the augmenting neurons were concentrated in the caudal part (P2 – P4), whereas the constant and decrementing neurons were widely distributed in the PSC. Some of the recorded neurons either increased (for tonic type) or launched (for phasic type) their firing prior to the storage/micturition phase initiation (Fig. 2B and E) (Table 1, asterisk). This preceding firing pattern was
more frequently found in the tonic storage neurons (11/17; 65%) and tonic micturition neurons (3/4; 75%) than in the phasic storage neurons (2/18; 11%) and phasic micturition neurons (2/6; 33%). Average interval between the onset of preceding neuronal firing and the phase initiation was 6.5 s (range 2– 10 s) in the tonic storage neurons, 6.0 s (range 2– 8 s) in the phasic storage neurons, 8.3 s (range 7 – 10 s) in the tonic micturition neurons and 4.0 s (range 3 – 5 s) in the phasic micturition neurons. The neurons with the preceding firing pattern were intermingled with those without such pattern.
4. Discussion The location of the PSC has been investigated in both anatomical and physiological manners. Morphological studies have revealed that the sacral Onuf’s nucleus, which innervates the urethral sphincter, receives a direct projection from a discrete area located ventrolateral to the PMC, named the L region (Holstege and Kuypers, 1982; Holstege et al. 1986; Holstege and Tan, 1987). On the other hand, the detrusor relaxation and the external sphincter contraction are elicited by electrical stimulation of an extensive area of the dorsolateral reticular formation, which Griffiths et al. (1990) called a ribbon-like band, running mainly from the L region to the PMC. The present study demonstrated that the PSC area, defined by electrical stimulation, was widespread in the dorsolateral reticular formation at the level of P0 – P4, which is consistent with the observation of Griffiths et al. (1990). This electrically determined PSC area is considered to contain neurons involved in urinary continence as well as their pathways because electrical stimulation activates soma as well as axons. The neurons involved in urinary continence, therefore, might be located in a more restricted region of the PSC area observed in this study. Indeed, inhibition of the micturition reflex is elicited by chemical stimulation of a relatively limited area, the PoO region, which is ventromedial to the PMC (Sugaya et al., 1987). Nevertheless, if we assume that the neuronal discharges recorded in this study were mainly obtained from the soma, our result may indicate that micturition-related neurons, which could contribute to the maintenance of urinary continence, are extensively existed in the PSC. This study demonstrated that the PSC neurons exhibited a variety of firing patterns according to the storage/micturition cycles. Of these neurons, the majority was the tonic and phasic storage neurons (78%) while the remaining was the tonic and phasic micturition neurons (22%). The proportion of the storage type neurons observed in this study was somewhat higher than that obtained from the PMC area, reported by de Groat et al. (1998), showing that the proportion of the micturition and storage neurons were 21% and 51%, respectively. Predominance of the storage neurons, which may actively be involved in the neural control of the urinary system during the storage phase, might be appropriate for contributing to the maintenance of
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the urinary storage as well as the suppression of the micturition reflex. On the other hand, the micturition neurons, which seemed to be concentrated in the midcaudal part of the PSC (P2 – P3) in this study, might form a local circuit that inactivates the urinary continence system in the PSC during the micturition phase. In addition to the storage/micturition classification, the neurons showed diverse discharge patterns: the tonic and phasic as well as the augmenting, constant, and decrementing. The neurons with decrementing firing frequency had its peak at the start of firing, which could be called as switch-on neurons. The neurons with augmenting firing frequency had its peak at the end of firing, which could be called as switch-off neurons. If these neurons are storage-related neurons, it is likely that these neurons play an essential role in the initiation (switch-on) and termination (switch-off) of the urinary storage, respectively. Similarly, if these neurons are micturition-related neurons, it is likely that these neurons play a role in the initiation and termination of the micturition reflex, respectively, most probably via an inhibitory neural circuit. The neurons with constant firing frequency may be involved in maintaining urinary storage. Whereas the augmenting neurons were concentrated in the caudal part (P2 –P4), the constant and decrementing neurons were widely distributed in the pons. To elucidate the exact functional role of the neurons, however, further studies in their electrophysiological properties (e.g. excitatory or inhibitory) as well as their functional and morphological connections will be necessary. Some of the neurons recorded in this study exhibited preceding discharge pattern, i.e., either increase or start of firing 2 –10 s prior to the storage/micturition phase initiation. This preceding time interval is considered much larger than the delay time from neuronal phase initiation at pontine level to contraction or relaxation response of the bladder muscle, which can be determined based on neuronal and muscular properties, e.g., it has been reported that central delay for activation of the sacral preganglionic neurons following pontine stimulation is 60 – 75 ms in cats (Mallory et al., 1989). Such neurons with preceding firing pattern, therefore, could be involved in neural control of the storage/micturition phase initiation. In this study, this preceding pattern was more frequently observed in the tonic storage and tonic micturition neurons than in the phasic storage and phasic micturition neurons, indicating a possibility of difference in functional role between the tonic neurons and the phasic neurons, though this dissociation may simply be produced by the difference in the thresholds of membrane excitability between the tonic and phasic neurons.
5. Conclusions The existence of a variety of heterogeneous discharge patterns observed in the PSC neurons suggest that these neurons may organize a complex neuronal circuitry, which is critical in the neural control of the urinary continence and the micturition/storage phase initiation.
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