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DEVELOPMENT AND TISSUE PLASTICITY
A. Széll,1,21Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261; 3Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214; and 2Departments of Pediatrics and Physiology, Health Science Center, University of Debrecen, Debrecen, Hungary H4012
Submitted 16 October 2002 ; accepted in final form 10 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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0.5
Hz was detected at 3-5 wk of age. Atropine but not tetrodotoxin decreased the
amplitude of spontaneous contractions, whereas carbachol, a muscarinic
agonist, unmasked or stimulated spontaneous activity. These data suggest that
slow rhythmic activity observed previously in neonatal whole bladders is
generated by pacemaker cells in the bladder base or dome. The emergence of
faster activity in bladders from older animals may reflect the development of
multiple pacemaker sites, which would reduce coordination within the bladder
wall and improve storage function in the mature bladder. spontaneous contractions; fast Fourier transformation; nonlinear analysis
Excitatory and inhibitory neural mechanisms also change during development (1, 22, 23, 25). For example, neurally evoked bladder contractions were mediated entirely by cholinergic mechanisms in the bladder strips from 1-wk-old rats but became primarily purinergic in strips obtained from 2-wk-old animals (13, 14). Inhibitory neural mechanisms driven by tonic out-flow from the spinal cord have been detected in neonatal rat spinal cord-bladder preparations in vitro (22, 23, 25). Inhibitory responses were also elicited by electrical field stimulation in bethanechol-contracted in vitro fetal bovine bladders and bladder strips. These inhibitory responses were not detectable in strips from postnatal and adult animals (10).
The neonatal rat bladder also exhibits prominent changes in activity in response to alterations in temperature (24). In bladders from 1- to 2-wk-old animals the amplitude of spontaneous contractions is maximal at body temperature and decreases as the temperature is reduced. On the other hand, in bladders from neonatal animals older than 3 wk of age and from adult animals, spontaneous contractions are of low amplitude at body temperature and increase in amplitude at lower temperatures. This dramatic change in temperature sensitivity occurs during the developmental period when central micturition pathways are maturing (1, 5, 11, 12, 14). Thus under physiological conditions the neonatal bladder is capable of generating large-amplitude intrinsic contractions, which presumably reflect pacemaker activity and efficient mechanisms for conducting this activity throughout the bladder (22, 24). This activity may be necessary to promote voiding when the neural control of the bladder is immature. Conversely, the bladder of mature animals exhibits minimal intrinsic activity, which improves the urine storage capabilities but makes voiding entirely dependent on neural mechanisms.
The present study was undertaken to examine the changes in spontaneous activity in the dome and base of the neonatal rat bladder during postnatal development and to determine the influence of cholinergic mechanisms on this activity.
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METHODS |
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Contractile measurement. The initial tension was set to 5 mN for 1- to 2-wk-old and to 10 mN for 3- to 5-wk-old bladders. The isometric contractions of the strips were measured with strain-gauge transducers (Grass AstroMed, West Warwick, RI) and recorded with a computerized data-acquisition program (Windaq, DATAQ Instruments, Akron, OH), using a 20-Hz sampling rate.
Experimental protocol. When the spontaneous activity was stable, carbachol was applied (30-40 min) and then washed out. When the regular spontaneous activity returned (30-50 min), either TTX or atropine was applied. In three experiments we applied TTX and then atropine also. However, the atropine and TTX were not applied in every experiment where the spontaneous activity was detected. It means the numbers of animals are different in Figs. 1 and 2 compared with Fig. 5.
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Drugs. Atropine sulfate (RBI), carbamylcholine chloride (carbachol, Sigma), and TTX (Sigma) were injected into the Krebs solution in the organ bath.
Data analysis. Spontaneous activity of bladder strips was analyzed by the following methods: 1) fast Fourier transform (FFT) algorithm (2), 2) nonlinear cross-prediction test (NLCP; 19, 20), 3) renormalized Shannon entropy (4), and 4) approximate entropy (ApEn; Ref. 16). For data analysis the following computer programs were applied: WindaqEx, Microsoft Excel, and Microcal Origin.
For frequency analysis the hanning version of FFT was used by collecting 16,384 data points for each analysis and then plotting the magnitude level against the frequency of the spontaneous activity.
For evaluation of the regularity of spontaneous contractions three nonlinear mathematical models were used: the NLCP, the renormalized Shannon entropy test, and the ApEn. The computational method for NLCP was written in Java programming language. The Shannon entropy and ApEn programs were written on C-analytic software. Generally, 4,500 points were used for the nonlinear tests. The change in system complexity was determined by a correlation dimension algorithm (7) or a fractal dimension algorithm (8). A new mathematical model, the NLCP, was used to determine either linear or nonlinear system activity, or low-dimensional chaos in our preparations using the method of Stam and Pritchard (20). This model characterizes the time series on the basis of predictability, amplitude asymmetry (ama; A), or time asymmetry (tir; B), where A and B measure the nonlinearity in the system. Because in the case of A and B the time series used for constructing the model and the time series that has to be predicted are different, the two series can be considered as a method to estimate NLCP. The NLCP (19) determines whether 1) the time series are asymmetric around their mean values, e.g., the predictability of A will be less than that of the original time series, and 2) the time series are time irreversible, e.g., the predictability of B will be less than that of the original time series. Stam et al. (19) obtained simplified quantitative estimates by averaging the correlation coefficient of 20 prediction steps (pred). In this context, ama signifies the difference between the average correlation coefficient for the original time series and A, and tir signifies the difference between the average correlation coefficient for the original time series and B.
In this paper we analyze our data both with a newer method, NLCP, and a more traditional nonlinear method, the Shannon entropy measurement. The latter method has been successfully applied in cardiology (18), in neurology (4), as well is in molecular biology (21). The renormalized version of the Shannon entropy was used because it is amplitude independent (4). Our data were also analyzed by a new statistical entropy method, the ApEn (16).
For statistical analysis, all the data are expressed as means ± SE. The two-tailed Student's t-test was used to compare unpaired data between dome and base in the same age. For multiple comparisons of the age-dependent changes within either dome or base, ANOVA was used. P < 0.05 was taken to indicate statistical significance. In Figs. 2, 5, and 6, the asterisk (*) shows the significant differences when Student's t-test was used and the pound sign (#) shows when ANOVA was used for the comparison.
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RESULTS |
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In bladder strips from 3- and 4-wk-old animals (n = 14), small-amplitude (<0.5 mN), high-frequency spontaneous contractions (fast component) (0.43 ± 0.07 and 0.41 ± 0.05 Hz, base and dome, respectively, P > 0.05) were superposed on high-amplitude (2-7 mN), low-frequency contractions (0.14 ± 0.03 and 0.1 ± 0.01 Hz, base and dome, respectively, P > 0.05) (Fig. 1C and Fig. 2). The fast component was present in 70% of the base strips but in only 20% of the dome strips. Since there was no difference in the data from 3- and 4-wk-bladders, these two groups were pooled together, and in this way we obtained four age groups: 1 wk old (3-7 day old), 2 wk old, 3-4 wk old, and 5 wk old. In bladder strips from 5-wk-old rats (n = 13), the amplitude of the slow activity was reduced to 1-2 mN, and the fast component was present more consistently in the dome (70%). In the FFT spectrum the magnitude of the slow peak was reduced and the fast peak was more prominent (Fig. 1D).
Effect of TTX and atropine on the frequency and amplitude composition of spontaneous bladder contraction. TTX (1 µM) did not affect the spontaneous activity of bladder strips or the FFT curves from base or dome strips at any age (Fig. 3, n = 6 in the 2- to 3-wk-old group, n = 9 in the 4- to 5-wk-old group). Atropine (1 µM) reduced the amplitude of spontaneous contractions by 65 ± 8% in dome strips and by 39 ± 15% in base strips from 2- to 3-wk-old rats (Fig. 4, n = 9). In 4- to 5-wk-old bladders atropine was less effective, reducing the amplitude of the contractions in the dome and base by 8 ± 3 and 23 ± 6%, respectively (n = 11). Atropine reduced the basal tone of the muscle strips. This effect of atropine was more prominent in 2- to 3-wk-old (63 ± 10%, 58 ± 9%, dome and base, respectively) than in 4- to 5-wk-old bladders (22 ± 6%, 44 ± 5%, dome and base, respectively). The magnitude of the slow peak in the FFT spectrum was significantly reduced in the 2- to 3-wk-old rats (Fig. 4, A and B) but not in 4- to 5-wk-old rats. Effect of carbachol on the amplitude and frequency of spontaneous bladder contractions. Carbachol (1 µM), a cholinergic agonist, induced large tonic contractions of bladder strips from 1- to 5-wk-old animals and also changed the amplitude and frequency of the spontaneous contractions. In strips from 1-wk-old rats carbachol (1 µM) induced vigorous spontaneous activity in 60% of the strips (Fig. 5A). The peak frequency of slow activity was significantly (P < 0.05) increased in the base (from 0.21 ± 0.03 to 0.41 ± 0.08 Hz) and in the dome (from 0.08 ± 0.01 to 0.23 ± 0.03 Hz), and a fast (1.17 ± 0.18 Hz) component was unmasked in the base of 1- and 2-wk-old rats but not the dome after carbachol (Fig. 5, B and C). After carbachol the frequency of the slow component was significantly (P < 0.05) higher in the base than in the dome (Fig. 5, B and C) as noted before carbachol administration (to facilitate comparisons the data from Fig. 2 are also shown in Fig. 5, B and C). In 2-wk-old rats, carbachol increased the frequency of the slow component in strips from the base but not from the dome (Fig. 5, B and C). Also, carbachol induced a fast component in the base but not the dome of 1- and 2-wk-old rats. In bladder strips from 3- to 4-wk-old rats, carbachol also significantly stimulated the slow component in the base but not in the dome, whereas in strips from 5-wk-old rats the drug did not significantly change the slow component in the base or the dome (0.19 ± 0.04 and 0.16 ± 0.04 Hz, respectively; P > 0.05). However, in strips from 5-wk-old rats after carbachol treatment, the frequency of the fast component was significantly higher in the base than in the dome (0.75 ± 0.08 and 0.58 ± 0.07 Hz, respectively, P < 0.05) (Fig. 5, B and C). This difference was not noted in untreated strips (Figs. 2 and 5).
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Nonlinear analysis of spontaneous bladder contraction. The pattern of spontaneous bladder activity was analyzed by the NLCP (Fig. 6A) in tissues from animals of different ages. In the 1-wk-old age group, tissue from a total of 13 animals was studied. Although spontaneous activity was only detected in bladder strips from three animals (5-7 days old), it was clear that the activity was more regular than in older animals. Analysis of records from individual strips showed that both major parameters, i.e., ama and tir, were near zero and that both dome and bladder strips showed regular contractions [i.e., prediction number (pred) was near 1]. In 2-wk-old bladders the "pred" of dome strips was decreased (0.75 ± 0.04) compared with the base strips (0.93 ± 0.03), indicating that the regularity of spontaneous activity in dome strips was reduced. The reason for this irregular muscle activity was revealed in the ama analysis, which showed higher values in dome strips (0.27 ± 0.07) than in base strips (0.15 ± 0.03). This indicates a greater variability in the amplitude of individual contractions in the dome versus the base. The analysis of individual records showed that 70% of dome-strips had irregular time series (tir, Fig. 6A). In the 3-wk-old animals the ama was further reduced both in the dome and the base (0.13 ± 0.03 and 0.1 ± 0.04, respectively), and this reduction was statistically significant for the dome strips (P < 0.05). The tir values were increased, but this elevation was not significant. A further tendency of ama reduction and tir elevation was observed in 4- and 5-wk-old bladder strips where the tir elevation was more prominent in tissues from the dome. In strips from 5-wk-old rats, the regularity (pred) was also reduced in dome strips; however, the reason for this irregular activity was the increased variation in intervals between contractions as measured by the tir, which was significantly increased (P < 0.05) from 0.07 ± 0.02 in 2-wk-old bladders to 0.15 ± 0.024 in 5-wk-old bladders. The analysis of individual preparations showed that 80% of the dome strips have an irregular time series, but this was only detected in 25% of base strips. These differences between dome and base were eliminated after treatment with carbachol (1 µM); e.g., there was no difference in pred, and the tir was not elevated.
The data were also evaluated by a more traditional method, the renormalized
Shannon entropy, which is amplitude independent. The frequency-dependent
complexity of contractile activity of bladder strips was more regular in the
1-wk-old bladders (Fig.
6B). The renormalized Shannon entropy was smaller than 2
in both the dome and the base. As noted by Bondarenko
(4), high Shannon entropy
indicates a more irregular activity. In the muscle strips from older animals,
the Shannon entropy significantly increased
(Fig. 6B), indicating
that the low-complexity, synchronous muscle activity disappeared and that the
activity in 3- to 4-wk-old and 5-wk-old bladder strips was becoming more
complex and more asynchronous. At these ages the renormalized Shannon entropy
value was 3 (Fig.
6B). Generally, there was no significant difference
between the dome and base muscle strips at any age. This developmental entropy
change was masked in presence of 1 µM carbachol
(Fig. 6B). Similar
tends in the results were detected using by the new statistical entropy
method, ApEn (data not shown).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Previous studies revealed that the whole neonatal rat bladder preparation
in vitro during the first 2-3 postnatal weeks exhibited rhythmic spontaneous
contractions that were larger in amplitude and slower in frequency than those
in adult rat whole bladder preparations
(24), suggesting that the
immature bladder exhibits synchronized activity that originates at a pacemaker
site. The activity then spreads in a coordinated manner throughout the bladder
muscle. Our present findings on bladder smooth muscle strips provide evidence
that the highest frequency pacemaker is located in the region of the bladder
base. FFT analysis of spontaneous contractions of bladder strips from 1- to
2-wk-old animals indicated a peak of activity occurring at slow frequencies
ranging from 0.08 to 0.21 Hz. The slow rhythmic activity was detected in both
the bladder dome and base but occurred at a higher frequency in the base,
indicating that even though cells in both regions are capable of generating
coordinated contractions, the cells in the bladder base by virtue of their
higher pacing frequency may generate the pattern of rhythmic activity in the
whole bladder, assuming that the data obtained in strips are applicable to the
intact organ. Although whole bladders and strips are studied under different
experimental conditions, it should be noted that the frequency of spontaneous
activity in these two preparations at 1-2 wk of age is not markedly different.
This is suggested by the fact that intercontraction intervals in strips
calculated from the FFT peaks in the present study ranged from 5 to 12 s and
the peak intercontraction interval in whole bladders
(24) was 20 s. Another
study (15) of neonatal bladder
strips (1-3 days old) reported an even better match (e.g., an intercontraction
interval of 22 s) with the whole bladder measurements. The difference in
results between the two bladder strip studies could be related to differences
in the methods used, including orientation and size of the strips as well as
the basal tension. Nevertheless, there seems to be a reasonably good
correlation between strip and whole bladder studies in regard to the
spontaneous contractile activity during the first two postnatal weeks.
The similarity between bladder strip and whole bladder data was also apparent in the changes, which occurred later in development. In the whole bladder the frequency of spontaneous contractions increased at 3-5 wk of age, amplitude decreased, and bladder activity became more chaotic. FFT analysis revealed a similar change in bladder strips from the dome and base. At 3 wk of age a second peak representing faster activity (0.4 Hz) appeared in the FFT. This peak became more prominent in the base strips at 5 wk of age and also was detectable in the FFT of dome strips. It is noteworthy that the intercontraction interval for this fast activity (2.5 s) is very similar to the interval (1.9 s) between small spontaneous movements visible on the surface of the mature bladder (24).
The emergence of faster activity in whole bladders and in bladder strips from older animals may reflect the development of multiple and independent pacemaker sites. This conclusion is consistent with the results of the nonlinear analysis of spontaneous contractile activity. In a small number of strips from the 1-wk age group, both dome and base strips revealed amplitude and time asymmetry measurements near zero and a prediction number near 1, indicating regular contractions. Similar measurements in strips from older animals revealed irregular time and amplitude characteristics, which were more prominent in the dome than in the base. When data were evaluated by a more traditional method, the renormalized Shannon entropy, which is independent of amplitude measurements, the results indicated that activity in muscle strips from 1- to 2-wk-old animals was synchronous and less complex, whereas the activity in strips from older animals was asynchronous and more complex. These observations suggest that contractile activity is more chaotic in older bladders, particularly in the region of the dome, and that intercellular communication mechanisms, possibly via gap junctions, may be downregulated during development. Preliminary studies using optical imaging methods and calcium-sensitive and voltage-sensitive dyes in whole bladder preparations from the neonatal rat have detected electrical activity moving in a coordinated manner from localized regions over the entire bladder (9). On the other hand, only chaotic activity originating at multiple sites was detected in adult bladders.
To determine if the spontaneous activity of the neonatal bladder is modulated by neural mechanisms, the effect of the sodium channel blocker TTX and the muscarinic antagonist atropine was evaluated. TTX did not alter the amplitude or the pattern of spontaneous contractile activity, indicating that neural firing was not important in regulating the contractions of bladder strips. Different results were obtained in the in vitro neonatal spinal cord-bladder preparation in which TTX increased the spontaneous bladder contractions (22). These as well as other experiments (25) indicate that the neonatal rat bladder receives a tonic inhibitory neural input from the spinal cord, which suppresses the intrinsic pacemaker activity of the smooth muscle.
When postjunctional muscarinic receptors were blocked with atropine, a significant reduction in the amplitude but not the frequency of the spontaneous contractions of strips from 2-wk-old rats was observed. Maggi et al. (14) also reported a small inhibitory effect of atropine on spontaneous activity in bladder strips from newborn (1-3 days old) but not adult rats. These results suggest that the smooth muscle contractions are modulated by the spontaneous release of acetylcholine from cholinergic nerve endings. Because atropine did not alter the frequency of contractile activity, it seems reasonable to conclude that neurally released acetylcholine does not alter the properties of the pacemaker cells but rather promotes the spread of activity from these cells to other parts of the bladder.
Activation of postjunctional muscarinic receptors with carbachol induced a large-amplitude muscle contraction and changed the pattern of spontaneous activity. Carbachol increased the frequency of the slow activity in the base and dome and also unmasked fast activity in the base but not the dome in 1-wk-old rats. This indicates that pacemaker activity can be enhanced either indirectly by raising the tension in the bladder wall or directly by stimulation of muscarinic receptors in the pacemaker cells. The induction of fast activity by carbachol could also be due to an indirect effect to increase wall tension and in turn induce the emergence of multiple pacemaker sites throughout the bladder. However, it is interesting that even in the presence of carbachol, the base exhibited a higher frequency of activity than the dome.
In summary, the neonatal rat bladder exhibits large-amplitude coordinated contractions that occur in the absence of neural input but which are modulated by spontaneous release of acetylcholine, presumably from cholinergic nerve terminals. FFT analysis revealed that cells in the bladder base have a significantly higher rate of contractile activity than cells in the bladder dome, raising the possibility that this higher level of activity in the base contributes to continence mechanisms by maintaining bladder neck closure or that in spontaneous contractions of the whole bladder might be controlled by pacemaker activity arising in the base and then spreading to the remainder of the bladder. Coordinated, large-amplitude, low-frequency contractile activity declines in strips from older animals and is replaced by low-amplitude, high-frequency, more irregular activity that appears to reflect the emergence of multiple pacemaker sites. This change in intrinsic activity coincides in time with the development of the central neuronal mechanisms that mediate voluntary voiding in adult animals (5, 6, 25). The change in the intrinsic properties of the bladder probably reflects the appearance of the mature storage function of the organ, which appears to depend at least in part on the disruption of the intercellular smooth muscle communication and emergence of asynchronous, chaotic muscle activity.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-amino-3-hydroxy-4-isoxazolepropionate (LY215490) receptor antagonists
on the voiding reflex induced by perineal stimulation in the neonatal rat.
Neuroscience 90:
1415-1420, 1999.[ISI][Medline]This article has been cited by other articles:
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Y. Ikeda, C. Fry, F. Hayashi, D. Stolz, D. Griffiths, and A. Kanai Role of gap junctions in spontaneous activity of the rat bladder Am J Physiol Renal Physiol, October 1, 2007; 293(4): F1018 - F1025. [Abstract] [Full Text] [PDF]
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