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Latency-rise time relationship in unitary postsynaptic potentials
404
Brain Research, 151 (1978)404-408 © Elsevier/North-Holland Biomedical Press
Latency-rise time relationship in unitary postsynaptic potentials
JOHN B. MUNSON and GEORGE W. SYPERT Departments of Neuroscience and Surgery, College of Medicine, University of Florida and VA Hospital, Gainesville, Fla. 32610 (U.S.A.)
(Accepted February 23rd, 1978)
Theoretical 7 and experimentaP, s data suggest that the location of a synapse on the somadendritic membrane of a neuron can be approximated from the characteristics of the excitatory postsynaptic potential (EPSP) generated at that synapse and recorded intracellularly in the cell soma. The characteristics of the EPSP traditionally used for such purposes are the EPSP rise time, half-width, slope (dV/dt) and amplitude 6-s. Theory 7 suggests that another measure may also be useful in such an analysis: the latency from the event which generates the EPSP to the time at which it is recorded in the soma. This latency of the EPSP should vary systematically with the electrotonic distance from the recording site due to delays introduced by electrotonic conduction. Data from two previous studies are pertinent to this question. Watt et al. 12 have plotted the amplitude of about 100 Ia-elicited monosynaptically activated EPSPs as a function of latency from the Ia activity recorded at the dorsal root entry zone. Latency and amplitude appear to be unrelated for this monosynaptically activated subgroup of EPSPs. Mendell and Henneman 5 have plotted the latencies from Ia activity recorded in the dorsal roots to the foot of the EPSP as a function of EPSP rise time for 37 Iaelicited unitary EPSPs. The two measures are clearly related but are complicated by the unknown transmission times from the dorsal root trigger point to the motoneuron under study. A more accurate analysis of the hypothetical relationship between synaptic location and EPSP latency would be possible if a latency measurement could be made between the synaptic event itself and the arrival of the EPSP in the soma. We have approached this problem by measuring the delay from the presynaptic spikeS,9,12 to the EPSP, both recorded with intracellular electrodes in cat spinal motoneurons. The presynaptic spike has been described in numerous previous papers (see ref. 3, p. 632). Most recently, Watt et a l J e described it as "a small, brief, positive-negative diphasic spike shortly preceding the EPSP' (p. 1380). This description is consistent with other observationsa, 9 and with our own (Figs. 1 and 2), all involving unitary EPSPs in spinal motoneurons. The hypothesis of the present work is that EPSPs generated at a distance from the recording site will exhibit longer latencies between the presynaptic spike and the EPSP than will those generated close to the recording site (i.e., the motoneuron soma).
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Fig. 1. A: electrical potentials at successivedepths in lumbar spinal cord occurring synchronously with action potentials in single MG Ia afferent. Numbers refer to depth in microns from surface of spinal cord. All recon Is are extracellular except' 1149', which is an intracellular recording from an MG motoneuron. Calibration square wave: 50/~V, 1 msec. Analysistime: 10.24msec. Bin width: 10/~sec. Number of sweeps: 1024. Presynchronizationtime: 0.5 msec.B: section through lumbar spinal cord showing location of triceps surae motoneuron pool (stipple) and track of microelectrode, marked at 1000 and 2000 #m below cord surface (redrawn with permission from Burke et al.1).
An assumption of the present work is that transmission of the presynaptic spike and of the EPSP to the electrode tip involve two different processes: the presynaptic spike is transmitted by the resistive properties of both the cell and the extracellular space and is recorded essentially simultaneously with its occurrence; and transmission of the EPSP (as recorded) is electrotonic via the resistive and capacitative elements of the cell. Thus the latency of the EPSP (as measured) will consist of the time required for synaptic transmission, the time for electrotonic conduction from the synaptic to the recording site and any time which may be required for activation of the afferent terminals. Data were accumulated from experiments on 12 cats anesthetized with sodium pentobarbital. Surgical and experimental procedures were essentially as described by Scott and Mendel110. Unitary EPSPs were elicited by medial gastrocnemius (MG) Ia single afferent fibers using the spike-triggered averaging technique of Mendell and Henneman 5. Activity was recorded with 3 M KCI filled low impedance bevelled micropipettes (3-5 Mf~), amplified 10 x by a DC preamplifier and then 50 × by an AC am-
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Fig. 2. Intracellular (A, B, D, E) and extracellular (C and F) records from two motoneurons showing presynaptic spikes and EPSPs generated by single MG Ia afferent. Scales expanded 4 x in B and E. Recorded as in 1A except 2048 sweeps. Details presented in text.
406 plifier (bandpass 0.1-30 kHz) before being averaged by a signal averager equipped with a cursor for precise determination of time and amplitude at 10/~sec intervals. Details of the presynaptic spike are illustrated in Fig. 1A. Most commonly, it appeared as a positive-negative spike up to 50 #V peak-to-peak amplitude. When generated by an MG Ia afferent, it was readily recorded both intra- and extracellularly at depths of 1-1.5 mm from the cord surface, a region corresponding to the nucleus of triceps surae motoneurons 1 (Fig. 1A and B) and also to the network of grossly coarsened Ia terminal branches which may generate the presynaptic spike as recorded 11. This analysis is based on records from a group of 78 MG motoneurons and 23 lateral gastrocnemius or soleus (LG/S) motoneurons having antidromic action potentials 60 mV or greater as well as a clearly discernible presynaptic spike and EPSP. Two significant potentials were observed in the averaged intracellular records: the presynaptic spike, followed by a classical EPSP. Examples of these potentials are shown in Fig. 2A and D. The EPSP in Fig. 2A was recorded from an MG motoneuron (action potential 62 mV, resting potential 54 mV, conduction velocity 98 m/sec). Note the presynaptic spike preceding the EPSP. The following values were calculated for this EPSP: latency 0.24 msec (calculated from negative peak of the presynaptic spike to the foot of the EPSp4); 10-50 ~o rise time 0.11 msec; 10-90 ~ rise time 0.27 msec; 10-50 slope 760/~V/msec; amplitude 209/zV. The same potential is shown with expanded amplitude and time base (approximately 4 ×) in Fig. 2B to show details of the presynaptic spike and EPSP rising phase. Fig. 2C shows the electrical activity averaged immediately external to the motoneuron. The presynaptic spike is essentially unchanged. Fig. 2D, E and F shows records obtained similarly from an LG/S motoneuron (action potential 62 mV, resting potential 52 mV; conduction velocity 71 m/sec). Values determined for this EPSP are: latency 0.42 msec; 10-50~ rise time 0.41 msec; 10-90 ~ rise time 1.40 msec; 10-50 ~ slope 243/zV/msec; amplitude 249/zV. Again, the presynaptic spike is seen clearly in the extracellular record (Fig. 2F). Iles 2 has shown that a Ia afferent may terminate in several boutons upon a single motoneuron. Consistent with this observation, many of our averaged records appeared to be comprised of multiple superimposed EPSPs with different shape indexes. Frequently, the rising phase of the EPSP demonstrated two slopes: a rapid rise followed by a slower rise. The rapid rise phase should be produced by the most proximal synapse and should have the shortest latency. Thus, in the case of EPSPs generated by multiple terminals of the triggering afferent, the latency as measured should be correlated most strongly with the earliest component of the EPSP. Accordingly, we have obtained the following correlations with EPSP latency: 10-50~ rise time r = 0.72; 10-90~ rise time r = 0.58; 10-50 ~ slope r --~ --0.45; and amplitude r = --0.31. All correlations are significant by t-test (P < 0.005). Since latency correlated best with 10-50 ~ rise time, of all values measured, we have constructed a scatter diagram showing this relationship (Fig. 3). The data are fit by the relationship RT = 0.66L --0.03. Mean values for latency are: homonymous (MG) 0.37 4- 0.15 msec (SD) and heteronymous (LG/S) 0.49 ± 0.21 msec (SD). Mean values for 10-50 ~ rise time are: homonymous 0.22 4- 0.13 msec (SD) and heteronymous 0.28 4- 0.21 msec (SD). The latency differences are significant (P < 0.005); the
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Fig. 3. Scatter diagram of relation between rise time and latency for M G Ia-elicited EPSPs in 78 M G mot•neurons and 23 LG/S mot•neurons. One LG/S point is not plotted: (1.14, 1.02). Dashed vertical line at L = 0.275 demarks minimum latency recorded for heteronymous (LG/S) ESPSP latencies. One-third of h o m o n y m • u s (MG) latencies are below this value.
rise time differences are not (0.10 > P > 0.05). It is particularly noteworthy that almost one-third of the homonymous latencies are shorter than the shortest heteronymous latency. Rise time values show a similar trend. Scott and Mendell TM have shown that homonymous Ia-motoneuron projections, when compared with heteronymous Ia-motoneuron projections, produce EPSPs of greater amplitude and faster rise time, suggestive of a moIe proximal location for homonymous connections. This contention is strongly supported by our findings of significantly shorter latency for homonymous compared with heteronymous connections, and by the trend in our rise time data. These data confirm the theoretical calculations of Rall a depicting a relationship between EPSP shape index and EPSP latency, measured in the cell soma. Sources of error include the non-uniqueness of factors capable of producing an EPSP of given dimensions: synaptic conductance time course, location of synaptic input and dendritic electrotonic length 7, as well as variable times for terminal activation. Experiments manipulating the location of effective synapses on the soma-dendritic membrane (e.g. axotomy)6 which should predictably alter the presynaptic spike-to-EPSP latency are currently in progress. We thank Drs. L. M. Mendell, W. Rall and D. C. Spray who reviewed earlier manuscripts and made many useful comments. The Szent~gothai referencO1 was brought to our attention by Dr. P. Andersen. This study was supported by NEI grant EY-01264 and the Medical Research Service of the Veterans Administration Hospital.
408 1 Burke, R. E., Strick, P. L., Kanda, K., Kim, C. C. and Walmsley, B., Anatomy of medial gastrocnemius and soleus motor nuclei in cat spinal cord, J. Neurophysiol., 40 (1977), 667-680. 2 lles, J. F., Central terminations of muscle afferents on motoneurons in the cat spinal cord, J. Physiol. (Land.), 262 (1976) 91--117. 3 Jankowska, E. and Roberts, W., Synaptic actions of single interneurons mediating reciprocal Ia inhibition of motoneurons, J. Physiol. (Lond.), 222 (1972) 623-642. 4 Katz, B. and Miledi, R., The measurement of synaptic delay and the time course of acetylcholine release at the neuromuscular junction, Proc. roy. Soc. B., 161 (1965) 483-495. 5 Mendell, L. M. and Henneman, E., Terminals of single Ia fibers : location, density and distribution within a pool of 300 homonymous motoneurons, J. Neurophysiol., 34 (1971) 171-187. 6 Mendell, L. M., Munson, J. B. and Scott, J. L. ,Alterations ofsynapses on axotomized motoneurons, J. Physiol. (Lond.), 255 (1976) 67-79. 7 Rall, W,, Distinguishing theoretical synaptic potentials computed for different soma-dendritic distribution of synaptic input, J. Neurophysiol., 30 (1967) 1138-1168. 8 Rall, W., Burke, R. E., Smith, J. G., Nelson, P. G. and Frank, K., Dendritic location of synapses and possible mechanisms for the monosynaptic EPSP in motoneurons, J. Neurophysiol., 30 (1967) 1169-1193. 9 Rapoport, S., Susswein, A., Uchino, Y. and Wilson, V. J., Synaptic actions of individual vestibular neurones on cat neck motoneurons, J. Physiol. (Lond.), 272 (1977) 367-382. 10 Scott, J. G. and Mendell, L. M., Individual EPSPs produced by single triceps surae la afferent fibers in homonymous and heteronymous motoneurons, J. Neurophysiol., 39 (1976) 679-692. 11 Szentfigothai, J., Synaptic architecture of the motoneuron pool, Electroenceph. clin. Neurophysiol., Suppl. 25 (1967) 4-19. 12 Watt, D. G. D., Stauffer, E. K., Taylor, A., Reinking, R. M. and Stuart, D. G., Analysis of muscle receptor connections by spike-triggered averaging. 1. Spindle primary and tendon organ afferents, J. Neurophysiol., 39 (1976) 1375-1392.
Brain Research, 151 (1978)404-408 © Elsevier/North-Holland Biomedical Press
Latency-rise time relationship in unitary postsynaptic potentials
JOHN B. MUNSON and GEORGE W. SYPERT Departments of Neuroscience and Surgery, College of Medicine, University of Florida and VA Hospital, Gainesville, Fla. 32610 (U.S.A.)
(Accepted February 23rd, 1978)
Theoretical 7 and experimentaP, s data suggest that the location of a synapse on the somadendritic membrane of a neuron can be approximated from the characteristics of the excitatory postsynaptic potential (EPSP) generated at that synapse and recorded intracellularly in the cell soma. The characteristics of the EPSP traditionally used for such purposes are the EPSP rise time, half-width, slope (dV/dt) and amplitude 6-s. Theory 7 suggests that another measure may also be useful in such an analysis: the latency from the event which generates the EPSP to the time at which it is recorded in the soma. This latency of the EPSP should vary systematically with the electrotonic distance from the recording site due to delays introduced by electrotonic conduction. Data from two previous studies are pertinent to this question. Watt et al. 12 have plotted the amplitude of about 100 Ia-elicited monosynaptically activated EPSPs as a function of latency from the Ia activity recorded at the dorsal root entry zone. Latency and amplitude appear to be unrelated for this monosynaptically activated subgroup of EPSPs. Mendell and Henneman 5 have plotted the latencies from Ia activity recorded in the dorsal roots to the foot of the EPSP as a function of EPSP rise time for 37 Iaelicited unitary EPSPs. The two measures are clearly related but are complicated by the unknown transmission times from the dorsal root trigger point to the motoneuron under study. A more accurate analysis of the hypothetical relationship between synaptic location and EPSP latency would be possible if a latency measurement could be made between the synaptic event itself and the arrival of the EPSP in the soma. We have approached this problem by measuring the delay from the presynaptic spikeS,9,12 to the EPSP, both recorded with intracellular electrodes in cat spinal motoneurons. The presynaptic spike has been described in numerous previous papers (see ref. 3, p. 632). Most recently, Watt et a l J e described it as "a small, brief, positive-negative diphasic spike shortly preceding the EPSP' (p. 1380). This description is consistent with other observationsa, 9 and with our own (Figs. 1 and 2), all involving unitary EPSPs in spinal motoneurons. The hypothesis of the present work is that EPSPs generated at a distance from the recording site will exhibit longer latencies between the presynaptic spike and the EPSP than will those generated close to the recording site (i.e., the motoneuron soma).
405
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Fig. 1. A: electrical potentials at successivedepths in lumbar spinal cord occurring synchronously with action potentials in single MG Ia afferent. Numbers refer to depth in microns from surface of spinal cord. All recon Is are extracellular except' 1149', which is an intracellular recording from an MG motoneuron. Calibration square wave: 50/~V, 1 msec. Analysistime: 10.24msec. Bin width: 10/~sec. Number of sweeps: 1024. Presynchronizationtime: 0.5 msec.B: section through lumbar spinal cord showing location of triceps surae motoneuron pool (stipple) and track of microelectrode, marked at 1000 and 2000 #m below cord surface (redrawn with permission from Burke et al.1).
An assumption of the present work is that transmission of the presynaptic spike and of the EPSP to the electrode tip involve two different processes: the presynaptic spike is transmitted by the resistive properties of both the cell and the extracellular space and is recorded essentially simultaneously with its occurrence; and transmission of the EPSP (as recorded) is electrotonic via the resistive and capacitative elements of the cell. Thus the latency of the EPSP (as measured) will consist of the time required for synaptic transmission, the time for electrotonic conduction from the synaptic to the recording site and any time which may be required for activation of the afferent terminals. Data were accumulated from experiments on 12 cats anesthetized with sodium pentobarbital. Surgical and experimental procedures were essentially as described by Scott and Mendel110. Unitary EPSPs were elicited by medial gastrocnemius (MG) Ia single afferent fibers using the spike-triggered averaging technique of Mendell and Henneman 5. Activity was recorded with 3 M KCI filled low impedance bevelled micropipettes (3-5 Mf~), amplified 10 x by a DC preamplifier and then 50 × by an AC am-
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Fig. 2. Intracellular (A, B, D, E) and extracellular (C and F) records from two motoneurons showing presynaptic spikes and EPSPs generated by single MG Ia afferent. Scales expanded 4 x in B and E. Recorded as in 1A except 2048 sweeps. Details presented in text.
406 plifier (bandpass 0.1-30 kHz) before being averaged by a signal averager equipped with a cursor for precise determination of time and amplitude at 10/~sec intervals. Details of the presynaptic spike are illustrated in Fig. 1A. Most commonly, it appeared as a positive-negative spike up to 50 #V peak-to-peak amplitude. When generated by an MG Ia afferent, it was readily recorded both intra- and extracellularly at depths of 1-1.5 mm from the cord surface, a region corresponding to the nucleus of triceps surae motoneurons 1 (Fig. 1A and B) and also to the network of grossly coarsened Ia terminal branches which may generate the presynaptic spike as recorded 11. This analysis is based on records from a group of 78 MG motoneurons and 23 lateral gastrocnemius or soleus (LG/S) motoneurons having antidromic action potentials 60 mV or greater as well as a clearly discernible presynaptic spike and EPSP. Two significant potentials were observed in the averaged intracellular records: the presynaptic spike, followed by a classical EPSP. Examples of these potentials are shown in Fig. 2A and D. The EPSP in Fig. 2A was recorded from an MG motoneuron (action potential 62 mV, resting potential 54 mV, conduction velocity 98 m/sec). Note the presynaptic spike preceding the EPSP. The following values were calculated for this EPSP: latency 0.24 msec (calculated from negative peak of the presynaptic spike to the foot of the EPSp4); 10-50 ~o rise time 0.11 msec; 10-90 ~ rise time 0.27 msec; 10-50 slope 760/~V/msec; amplitude 209/zV. The same potential is shown with expanded amplitude and time base (approximately 4 ×) in Fig. 2B to show details of the presynaptic spike and EPSP rising phase. Fig. 2C shows the electrical activity averaged immediately external to the motoneuron. The presynaptic spike is essentially unchanged. Fig. 2D, E and F shows records obtained similarly from an LG/S motoneuron (action potential 62 mV, resting potential 52 mV; conduction velocity 71 m/sec). Values determined for this EPSP are: latency 0.42 msec; 10-50~ rise time 0.41 msec; 10-90 ~ rise time 1.40 msec; 10-50 ~ slope 243/zV/msec; amplitude 249/zV. Again, the presynaptic spike is seen clearly in the extracellular record (Fig. 2F). Iles 2 has shown that a Ia afferent may terminate in several boutons upon a single motoneuron. Consistent with this observation, many of our averaged records appeared to be comprised of multiple superimposed EPSPs with different shape indexes. Frequently, the rising phase of the EPSP demonstrated two slopes: a rapid rise followed by a slower rise. The rapid rise phase should be produced by the most proximal synapse and should have the shortest latency. Thus, in the case of EPSPs generated by multiple terminals of the triggering afferent, the latency as measured should be correlated most strongly with the earliest component of the EPSP. Accordingly, we have obtained the following correlations with EPSP latency: 10-50~ rise time r = 0.72; 10-90~ rise time r = 0.58; 10-50 ~ slope r --~ --0.45; and amplitude r = --0.31. All correlations are significant by t-test (P < 0.005). Since latency correlated best with 10-50 ~ rise time, of all values measured, we have constructed a scatter diagram showing this relationship (Fig. 3). The data are fit by the relationship RT = 0.66L --0.03. Mean values for latency are: homonymous (MG) 0.37 4- 0.15 msec (SD) and heteronymous (LG/S) 0.49 ± 0.21 msec (SD). Mean values for 10-50 ~ rise time are: homonymous 0.22 4- 0.13 msec (SD) and heteronymous 0.28 4- 0.21 msec (SD). The latency differences are significant (P < 0.005); the
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Fig. 3. Scatter diagram of relation between rise time and latency for M G Ia-elicited EPSPs in 78 M G mot•neurons and 23 LG/S mot•neurons. One LG/S point is not plotted: (1.14, 1.02). Dashed vertical line at L = 0.275 demarks minimum latency recorded for heteronymous (LG/S) ESPSP latencies. One-third of h o m o n y m • u s (MG) latencies are below this value.
rise time differences are not (0.10 > P > 0.05). It is particularly noteworthy that almost one-third of the homonymous latencies are shorter than the shortest heteronymous latency. Rise time values show a similar trend. Scott and Mendell TM have shown that homonymous Ia-motoneuron projections, when compared with heteronymous Ia-motoneuron projections, produce EPSPs of greater amplitude and faster rise time, suggestive of a moIe proximal location for homonymous connections. This contention is strongly supported by our findings of significantly shorter latency for homonymous compared with heteronymous connections, and by the trend in our rise time data. These data confirm the theoretical calculations of Rall a depicting a relationship between EPSP shape index and EPSP latency, measured in the cell soma. Sources of error include the non-uniqueness of factors capable of producing an EPSP of given dimensions: synaptic conductance time course, location of synaptic input and dendritic electrotonic length 7, as well as variable times for terminal activation. Experiments manipulating the location of effective synapses on the soma-dendritic membrane (e.g. axotomy)6 which should predictably alter the presynaptic spike-to-EPSP latency are currently in progress. We thank Drs. L. M. Mendell, W. Rall and D. C. Spray who reviewed earlier manuscripts and made many useful comments. The Szent~gothai referencO1 was brought to our attention by Dr. P. Andersen. This study was supported by NEI grant EY-01264 and the Medical Research Service of the Veterans Administration Hospital.
408 1 Burke, R. E., Strick, P. L., Kanda, K., Kim, C. C. and Walmsley, B., Anatomy of medial gastrocnemius and soleus motor nuclei in cat spinal cord, J. Neurophysiol., 40 (1977), 667-680. 2 lles, J. F., Central terminations of muscle afferents on motoneurons in the cat spinal cord, J. Physiol. (Land.), 262 (1976) 91--117. 3 Jankowska, E. and Roberts, W., Synaptic actions of single interneurons mediating reciprocal Ia inhibition of motoneurons, J. Physiol. (Lond.), 222 (1972) 623-642. 4 Katz, B. and Miledi, R., The measurement of synaptic delay and the time course of acetylcholine release at the neuromuscular junction, Proc. roy. Soc. B., 161 (1965) 483-495. 5 Mendell, L. M. and Henneman, E., Terminals of single Ia fibers : location, density and distribution within a pool of 300 homonymous motoneurons, J. Neurophysiol., 34 (1971) 171-187. 6 Mendell, L. M., Munson, J. B. and Scott, J. L. ,Alterations ofsynapses on axotomized motoneurons, J. Physiol. (Lond.), 255 (1976) 67-79. 7 Rall, W,, Distinguishing theoretical synaptic potentials computed for different soma-dendritic distribution of synaptic input, J. Neurophysiol., 30 (1967) 1138-1168. 8 Rall, W., Burke, R. E., Smith, J. G., Nelson, P. G. and Frank, K., Dendritic location of synapses and possible mechanisms for the monosynaptic EPSP in motoneurons, J. Neurophysiol., 30 (1967) 1169-1193. 9 Rapoport, S., Susswein, A., Uchino, Y. and Wilson, V. J., Synaptic actions of individual vestibular neurones on cat neck motoneurons, J. Physiol. (Lond.), 272 (1977) 367-382. 10 Scott, J. G. and Mendell, L. M., Individual EPSPs produced by single triceps surae la afferent fibers in homonymous and heteronymous motoneurons, J. Neurophysiol., 39 (1976) 679-692. 11 Szentfigothai, J., Synaptic architecture of the motoneuron pool, Electroenceph. clin. Neurophysiol., Suppl. 25 (1967) 4-19. 12 Watt, D. G. D., Stauffer, E. K., Taylor, A., Reinking, R. M. and Stuart, D. G., Analysis of muscle receptor connections by spike-triggered averaging. 1. Spindle primary and tendon organ afferents, J. Neurophysiol., 39 (1976) 1375-1392.