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A study of facilitation in vesical parasympathetic ganglia of the cat using intracellular recording techniques
388
Brain Research, 169 (1979) 388-392 ((~)Elsevier/North-HollandBiomedicalPress
A study of facilitation in vesicai parasympathetic ganglia of the cat using intraceltutar recording techniques
A. M. BOOTH and W. C. DeGROAT Department of Pharmacology, Medical School, University of Pittsburgh, Pittsburgh, Pa. 1.5261 (U. S.A.)
(Accepted February 15th, 1979)
Previous studies of transmission in vesical parasympathetic ganglia of the cat revealed that continuous stimulation of preganglionic axons in the pelvic nerves at frequencies between 1-20 Hz elicited responses in vesical postganglionic nerves that gradually increased in amplitude 7,a. Depending upon the frequency of stimulation, the amplitude of the discharge increased 2-20 times over control levels obtained at a frequency of 0.25 Hz. Thus it was proposed that the safety factor for transmission in vesical ganglia is low and that activation of the entire presynaptic input to the ganglion with single shocks only elicits firing in a small percentage of cells. The ganglia seem to act as filters in the efferent pathway to the bladder blocking excitatory input when preganglionic firing is low, but facilitating the neural input to the bladder during mieturition when preganglionic activity is high4,z. In the present study we have employed an in vitro preparation and intracellular recording to examine the mechanisms underlying facilitation in vesical ganglia. A preliminary report of these observations has been presented3. Cats were anesthetized with pentobarbitol (30 mg/kg) or a mixture of diallylbarbituric acid (70 mg/kg), urethane (280 mg/kg), and monoethylurea (280 mg/kg). Ganglia from the surface of the urinary bladder and the pelvic plexus were removed along with preganglionic and postganglionic nerves. The tissue was pinned out i n s bath having a silicone rubber base and perfused at 3-5 ml/min with a Krebs-Ringer solution saturated with 95 ~ oxygen and 5 ~ carbon dioxide. Suction electrodes were attached to pre- and postganglionic fibers for stimulation and recording. Glass pipettes filled with 3 M potassium chloride or 1.4 M potassium citrate, having impedences of between 30 and 70 Mf~ were connected to a preamplifier bridge circuit allowing simultaneous recording and stimulation of the impaled cells. Temperature was maintained between 23-26 °C during some experiments and between 35-37°C for others. Data were obtained from 130 cells in 25 ganglia. Recording from postganglionic fibers established that the in vitro preparation displayed the same prominent facilitation to repetitive stimulation as previously reported for the in vivo preparation s. This is illustrated in Fig. I.
389
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,,jlllllllllllll11 1111II 1 II1 1111111 Fig. 1. Compound action potentials recorded from postganglionic nerves. A: stimulation train of 1 Hz to preganglionic fiber. B: preganglionic stimulation at 5 Hz. C: averages of 10 compound action potentials from another preparation with preganglionic stimulation of 0.25 Hz (upper) and 2.5 Hz (lower). Marker dots indicate stimulus artifact followed by through fiber response and ganglion response. Calibration marks represent 40 t~V and 1 sec (A and B) and 40#V and 10 msec (C).
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Fig. 2. EPSPs recorded from vesical ganglion cells while stimulating the pelvic nerve. A : plot of the increase in EPSP amplitude with respect to time at stimulation frequencies from 0.5 to 10 Hz. Each point is an average of 7 cells except at I 0 Hz where only 4 cells were used. Standard error of the mean ~:~ 0.36. Insert 1 shows single EPSP superimposed upon a hyperpolarizing pulse (50 msec) of sutficient amplitude to prevent cell firing. Insert 2 superimposes the responses to a train of 6 stimuli delivered at a frequency of 5 Hz. B: recordings of EPSP amplitude in response to stimulus trains of I Hz (upper) and 5 Hz (lower). Up arrow indicates offset of 0.25 Hz stimulus train and onset of test train. Down arrow denotes offset of test train and resumption of 0.25 Hz stimulation. Calibration for the insert is 10 mV and 20 msec and for B is 10 mV and 1 sec.
390 Ganglion cells which were studied at a bath temperature of 23-26 '~C had mean resting membrane potentials of 46:k l mV and action potentials of 7l. 1:_~2.5, whereas cells examined at 35-37 °C exhibited membrane and action potentials of 50.8 ;t: 1.2 and 76.8zk2 mV respectively. Membrane resistance, calculated only at 35-37 °C, was 25.5~1.8 M~q and the time constant was 5.9~0.3 msec. Preganglionic nerve stimulation at frequencies greater than 0.25 Hz and at intensities that were below threshold to evoke postsynaptic spikes elicited EPSPs that increased in amplitude with successive stimuli. In the majority of cells any stimulus intensity that resulted in a detectable EPSP at 0.25 Hz would result in firing when the frequency of stimulation was raised to 2.5 Hz and maintained for 10-15 sec. Thus it was necessary to block cell firing to study facilitation at high frequencies of stimulation. This was accomplished by hyperpolarizing the cells with either pulsed or continuous intracellular current injection. We established that hyperpolarization that was sufficient to block spike generation did not alter EPSP amplitude. Fig. 2 illustrates the relationship between stimulus frequency and EPSP amplitude. At frequencies ranging from 0.5-10 Hz EPSP amplitude reached maximum at a particular frequency after 15-20 stimuli and remained at this maximum for the duration of the stimulus train. EPSP amplitude returned to control levels within 60 sec when stimulation frequencies were lowered to 0.25 Hz (Fig. 3). At higher frequencies ( > 10 Hz) there was a decline in EPSP amplitude after 60-70 stimuli. Since most ganglion ceils could be excited by more than one preganglionic nerve entering the ganglia and the EPSPs produced by these separate inputs could be summated with appropriate stimulation, it was possible to examine the influence of homosynaptic and heterosynaptic conditioning volleys on synaptic transmission. Homosynaptic conditioning with 1-5 stimuli (5-10 Hz) produced a facilitation of transmission lasting for several hundred msec, whereas heterosynaptic conditioning did not produce detectable facilitation.
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Fig. 3. Decay of EPSP amplitude following offset of preganglionic stimulation at 7.2 Hz. Standard error of the mean <~ 0.23. Average of 11 cells.
39l In summary, facilitation of transmission in bladder ganglia with repetitive stimulation appears to be due entirely to enhanced presynaptic release of transmitter. Within the limits of our test conditions (0.5-10 Hz) the EPSP amplitude increased over the first 15-20 stimuli and remained at or near the maximum amplitude at that frequency for the duration of the stimulation. The time course of facilitation observed when monitoring action potentials on a postganglionic nerve whether in vitro or in situ s appeared to be identical to the time-course of facilitation obtained with intracellular recording of EPSPs. Facilitation was elicited by homosynaptic conditioning but not by heterosynaptic conditioning volleys, was not associated with the occurrence of slow EPSPs and was not blocked by atropine s. These observations indicate that facilitation is mediated by a presynaptic rather than a postsynaptic mechanism. Presynaptic mechanisms have also been implicated in facilitation seen at other peripheral synapses such as the neuromuscular junction10,11,1z the guinea pig superior cervical ganglion 9 and the sympathetic chain ganglia s. Facilitation in other abdominal ganglia, such as the inferior mesenteric ganglia s and the colonic parasympathetic ganglia of the cat 6 and the pelvic ganglia of the guinea pig I is less prominent than facilitation in bladder ganglia. This variation in the magnitude of facilitation may reflect differences in the function of these pathways. For example in the inferior mesenteric ganglion which transmits tonic activity in vasomotor and inhibitory pathways transmission occurs with a high safety factor, and facilitation is minimal. These ganglia seem to serve primarily as relay centers transmitting subtle changes in preganglionic activity to the effector organs. On the other hand, bladder ganglia modulate the neural input to the effector organ, blocking input when preganglionic activity is low and amplifying transmission when preganglionic activity is high. This 'high pass filter' promotes urinary continence while the bladder is filling, yet contributes to large, well sustained bladder contractions during micturition when preganglionic activity is intense. This investigation was supported in part by Grant NB 07923 N I N D S to W. C. de G. and 5430 for research training in the biological sciences N I M H to A. M. B.
1 Blackman, J. G., Crowcroft, P. J., Devine, C. E., Holman, M. E. and Yonemura, K., Transmission from preganglionic fibers in the bypogastric nerve to peripheral ganglia of male guineapigs, J. Physiol. (Lond.), 201 (1969) 723-743. 2 Blackman, J. G. and Purves, R. D., Intracellular recordings from ganglia of the thoracic sympathetic chain of the guinea-pig, J. Physiol. (Lond.), 203 (1969) 173-198. 3 Booth, A. M. and DeGroat, W. C., A study of recruitment in vesical parasympathetic ganglia of the cat using intracellular recording techniques, Fed. Proc., 37 (1978) 526. 4 DeGroat, W. C. Nervous control of the urinary bladder of the cat, Brain Research, 87 (1975) 201 211. 5 DeGroat, W. C., Booth, A. M. and Krier, J. Interaction between sacral parasympathetic and lumbar sympathetic inputs to pelvic ganglia. In C. M. Brooks, K. Koizumi and A. Sato (Eds.), Integrative Functions of the Autonomic Nervous System, University of Tokyo Press, in press. 6 DeGroat, W. C. and Krier, J., An electrophysiologicalstudy of the sacral parasympathetic pathway to the colon of the cat, J. Physiol. (Lond.), 260 (1976) 425-445. 7 DeGroat, W. C. and Ryall, R. W., Reflexes to sacral parasympathetic neurones concerned with micturition in the cat, J. Physiol. (Lond.), 200 (1969) 87-108.
392 8 DeGroat, W. C. and Saum, W. R., Synaptic transmission in parasympathetic ganglia in the urinary bladder of the cat, J. PhysioL (Lond.), 256 (1976) 137-158. 9 McLachlan, E. M., An analysis of the release of acetytcholine from preganglionic nerve terminals, J. Physiol. (Lond.), 245 (1975) 447-466. 10 Magleby, K. L., The effect of repetitive stimulation on facilitation of transmitter release at the frog neuromuscular junction, J. Physiol. (Lond.), 234 (1973) 327-352. 11 Magleby, K. L., The effect of tetanic and post-tetanic potentiation on facilitation of transmitter release at the frog neuromuscular junction, J. Physiol. (Lond.), 234 (1973) 353-371~ 12 Wernig, A., Changes in statistical parameters during facilitation at the crayfish neuromuscular junction, J. Physiol. (Lond.), 226 (1972) 751-759.
Brain Research, 169 (1979) 388-392 ((~)Elsevier/North-HollandBiomedicalPress
A study of facilitation in vesicai parasympathetic ganglia of the cat using intraceltutar recording techniques
A. M. BOOTH and W. C. DeGROAT Department of Pharmacology, Medical School, University of Pittsburgh, Pittsburgh, Pa. 1.5261 (U. S.A.)
(Accepted February 15th, 1979)
Previous studies of transmission in vesical parasympathetic ganglia of the cat revealed that continuous stimulation of preganglionic axons in the pelvic nerves at frequencies between 1-20 Hz elicited responses in vesical postganglionic nerves that gradually increased in amplitude 7,a. Depending upon the frequency of stimulation, the amplitude of the discharge increased 2-20 times over control levels obtained at a frequency of 0.25 Hz. Thus it was proposed that the safety factor for transmission in vesical ganglia is low and that activation of the entire presynaptic input to the ganglion with single shocks only elicits firing in a small percentage of cells. The ganglia seem to act as filters in the efferent pathway to the bladder blocking excitatory input when preganglionic firing is low, but facilitating the neural input to the bladder during mieturition when preganglionic activity is high4,z. In the present study we have employed an in vitro preparation and intracellular recording to examine the mechanisms underlying facilitation in vesical ganglia. A preliminary report of these observations has been presented3. Cats were anesthetized with pentobarbitol (30 mg/kg) or a mixture of diallylbarbituric acid (70 mg/kg), urethane (280 mg/kg), and monoethylurea (280 mg/kg). Ganglia from the surface of the urinary bladder and the pelvic plexus were removed along with preganglionic and postganglionic nerves. The tissue was pinned out i n s bath having a silicone rubber base and perfused at 3-5 ml/min with a Krebs-Ringer solution saturated with 95 ~ oxygen and 5 ~ carbon dioxide. Suction electrodes were attached to pre- and postganglionic fibers for stimulation and recording. Glass pipettes filled with 3 M potassium chloride or 1.4 M potassium citrate, having impedences of between 30 and 70 Mf~ were connected to a preamplifier bridge circuit allowing simultaneous recording and stimulation of the impaled cells. Temperature was maintained between 23-26 °C during some experiments and between 35-37°C for others. Data were obtained from 130 cells in 25 ganglia. Recording from postganglionic fibers established that the in vitro preparation displayed the same prominent facilitation to repetitive stimulation as previously reported for the in vivo preparation s. This is illustrated in Fig. I.
389
C
A
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~i iii,_ L L L LI
B
j
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,,jlllllllllllll11 1111II 1 II1 1111111 Fig. 1. Compound action potentials recorded from postganglionic nerves. A: stimulation train of 1 Hz to preganglionic fiber. B: preganglionic stimulation at 5 Hz. C: averages of 10 compound action potentials from another preparation with preganglionic stimulation of 0.25 Hz (upper) and 2.5 Hz (lower). Marker dots indicate stimulus artifact followed by through fiber response and ganglion response. Calibration marks represent 40 t~V and 1 sec (A and B) and 40#V and 10 msec (C).
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5
10
20
50
100
TIME {5EC)
B =~,
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I 1 , I j j 1 I I j j I j , =
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Fig. 2. EPSPs recorded from vesical ganglion cells while stimulating the pelvic nerve. A : plot of the increase in EPSP amplitude with respect to time at stimulation frequencies from 0.5 to 10 Hz. Each point is an average of 7 cells except at I 0 Hz where only 4 cells were used. Standard error of the mean ~:~ 0.36. Insert 1 shows single EPSP superimposed upon a hyperpolarizing pulse (50 msec) of sutficient amplitude to prevent cell firing. Insert 2 superimposes the responses to a train of 6 stimuli delivered at a frequency of 5 Hz. B: recordings of EPSP amplitude in response to stimulus trains of I Hz (upper) and 5 Hz (lower). Up arrow indicates offset of 0.25 Hz stimulus train and onset of test train. Down arrow denotes offset of test train and resumption of 0.25 Hz stimulation. Calibration for the insert is 10 mV and 20 msec and for B is 10 mV and 1 sec.
390 Ganglion cells which were studied at a bath temperature of 23-26 '~C had mean resting membrane potentials of 46:k l mV and action potentials of 7l. 1:_~2.5, whereas cells examined at 35-37 °C exhibited membrane and action potentials of 50.8 ;t: 1.2 and 76.8zk2 mV respectively. Membrane resistance, calculated only at 35-37 °C, was 25.5~1.8 M~q and the time constant was 5.9~0.3 msec. Preganglionic nerve stimulation at frequencies greater than 0.25 Hz and at intensities that were below threshold to evoke postsynaptic spikes elicited EPSPs that increased in amplitude with successive stimuli. In the majority of cells any stimulus intensity that resulted in a detectable EPSP at 0.25 Hz would result in firing when the frequency of stimulation was raised to 2.5 Hz and maintained for 10-15 sec. Thus it was necessary to block cell firing to study facilitation at high frequencies of stimulation. This was accomplished by hyperpolarizing the cells with either pulsed or continuous intracellular current injection. We established that hyperpolarization that was sufficient to block spike generation did not alter EPSP amplitude. Fig. 2 illustrates the relationship between stimulus frequency and EPSP amplitude. At frequencies ranging from 0.5-10 Hz EPSP amplitude reached maximum at a particular frequency after 15-20 stimuli and remained at this maximum for the duration of the stimulus train. EPSP amplitude returned to control levels within 60 sec when stimulation frequencies were lowered to 0.25 Hz (Fig. 3). At higher frequencies ( > 10 Hz) there was a decline in EPSP amplitude after 60-70 stimuli. Since most ganglion ceils could be excited by more than one preganglionic nerve entering the ganglia and the EPSPs produced by these separate inputs could be summated with appropriate stimulation, it was possible to examine the influence of homosynaptic and heterosynaptic conditioning volleys on synaptic transmission. Homosynaptic conditioning with 1-5 stimuli (5-10 Hz) produced a facilitation of transmission lasting for several hundred msec, whereas heterosynaptic conditioning did not produce detectable facilitation.
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Fig. 3. Decay of EPSP amplitude following offset of preganglionic stimulation at 7.2 Hz. Standard error of the mean <~ 0.23. Average of 11 cells.
39l In summary, facilitation of transmission in bladder ganglia with repetitive stimulation appears to be due entirely to enhanced presynaptic release of transmitter. Within the limits of our test conditions (0.5-10 Hz) the EPSP amplitude increased over the first 15-20 stimuli and remained at or near the maximum amplitude at that frequency for the duration of the stimulation. The time course of facilitation observed when monitoring action potentials on a postganglionic nerve whether in vitro or in situ s appeared to be identical to the time-course of facilitation obtained with intracellular recording of EPSPs. Facilitation was elicited by homosynaptic conditioning but not by heterosynaptic conditioning volleys, was not associated with the occurrence of slow EPSPs and was not blocked by atropine s. These observations indicate that facilitation is mediated by a presynaptic rather than a postsynaptic mechanism. Presynaptic mechanisms have also been implicated in facilitation seen at other peripheral synapses such as the neuromuscular junction10,11,1z the guinea pig superior cervical ganglion 9 and the sympathetic chain ganglia s. Facilitation in other abdominal ganglia, such as the inferior mesenteric ganglia s and the colonic parasympathetic ganglia of the cat 6 and the pelvic ganglia of the guinea pig I is less prominent than facilitation in bladder ganglia. This variation in the magnitude of facilitation may reflect differences in the function of these pathways. For example in the inferior mesenteric ganglion which transmits tonic activity in vasomotor and inhibitory pathways transmission occurs with a high safety factor, and facilitation is minimal. These ganglia seem to serve primarily as relay centers transmitting subtle changes in preganglionic activity to the effector organs. On the other hand, bladder ganglia modulate the neural input to the effector organ, blocking input when preganglionic activity is low and amplifying transmission when preganglionic activity is high. This 'high pass filter' promotes urinary continence while the bladder is filling, yet contributes to large, well sustained bladder contractions during micturition when preganglionic activity is intense. This investigation was supported in part by Grant NB 07923 N I N D S to W. C. de G. and 5430 for research training in the biological sciences N I M H to A. M. B.
1 Blackman, J. G., Crowcroft, P. J., Devine, C. E., Holman, M. E. and Yonemura, K., Transmission from preganglionic fibers in the bypogastric nerve to peripheral ganglia of male guineapigs, J. Physiol. (Lond.), 201 (1969) 723-743. 2 Blackman, J. G. and Purves, R. D., Intracellular recordings from ganglia of the thoracic sympathetic chain of the guinea-pig, J. Physiol. (Lond.), 203 (1969) 173-198. 3 Booth, A. M. and DeGroat, W. C., A study of recruitment in vesical parasympathetic ganglia of the cat using intracellular recording techniques, Fed. Proc., 37 (1978) 526. 4 DeGroat, W. C. Nervous control of the urinary bladder of the cat, Brain Research, 87 (1975) 201 211. 5 DeGroat, W. C., Booth, A. M. and Krier, J. Interaction between sacral parasympathetic and lumbar sympathetic inputs to pelvic ganglia. In C. M. Brooks, K. Koizumi and A. Sato (Eds.), Integrative Functions of the Autonomic Nervous System, University of Tokyo Press, in press. 6 DeGroat, W. C. and Krier, J., An electrophysiologicalstudy of the sacral parasympathetic pathway to the colon of the cat, J. Physiol. (Lond.), 260 (1976) 425-445. 7 DeGroat, W. C. and Ryall, R. W., Reflexes to sacral parasympathetic neurones concerned with micturition in the cat, J. Physiol. (Lond.), 200 (1969) 87-108.
392 8 DeGroat, W. C. and Saum, W. R., Synaptic transmission in parasympathetic ganglia in the urinary bladder of the cat, J. PhysioL (Lond.), 256 (1976) 137-158. 9 McLachlan, E. M., An analysis of the release of acetytcholine from preganglionic nerve terminals, J. Physiol. (Lond.), 245 (1975) 447-466. 10 Magleby, K. L., The effect of repetitive stimulation on facilitation of transmitter release at the frog neuromuscular junction, J. Physiol. (Lond.), 234 (1973) 327-352. 11 Magleby, K. L., The effect of tetanic and post-tetanic potentiation on facilitation of transmitter release at the frog neuromuscular junction, J. Physiol. (Lond.), 234 (1973) 353-371~ 12 Wernig, A., Changes in statistical parameters during facilitation at the crayfish neuromuscular junction, J. Physiol. (Lond.), 226 (1972) 751-759.