344
Brain Research, 140 (1978) 344-348 © Elsevier/North-Holland Biomedical Press
Extracellular spike triggered averaging for plotting synaptic projections
A. TAYLOR, J. A. STEPHENS, G. SOMJEN*, K. APPENTENG and M. J. O'DONOVAN Sherrington School of Physiology, St. Thomas's Hospital, London, SE1 7EH (Great Britain)
(Accepted August 31st, 1977)
Averaging intracellular noise with a computer triggered by the firing of a physiologically identified afferent unit ('spike triggered averaging', STA) has become established as a highly specific and sensitive method for detecting synaptic connectionsS-TA 0. However, the difficulty of securing stable in continuity recording from an identified afferent simultaneously with stable intracellular recording from the appropriate motoneurone has been a limitation to the application of the method. During the course of the above experiments, extracellular averages, taken as controls, commonly revealed small negative fields close to motoneurones receiving excitatory input. These fields, due to single afferents were detectable in the spinal cord over distances comparable with the extent of the motor nuclei, but were remarkably irregular and uncertain as controls for the weakest intracellularly recorded averages 1°. The irregularity of the extracellular fields seems likely to have been due to the non-homogeneity of the neural tissue at the scale of the glass micropipette recording tip. This communication is to show that when recorded with relatively large glass-coated tungsten electrodes, the unit fields are well-defined and apparently useful for determining the extent and strength of a given afferent fibre's projection to a particular nucleus. The system under study was the monosynaptic projection of spindles of the jaw-elevator muscles to the motor nucleus of the fifth nerve in the cat 1. Animals were anaesthetized with pentobarbitone and stimulating wires implanted around the masseter nerve and part of the temporalis nerve. The head was secured in a stereotaxic frame and the mandible to an electromagnetic displacement servo. One glass-coated tungsten electrode (25/~m tip exposed) was inserted through an anterior craniotomy to reach the mesencephalic nucleus of the fifth nerve so as to isolate single units belonging to jaw elevator muscle spindles 2. A second tungsten electrode with 80-100 #m tip exposed, was inserted at a 30 ° angle through the cerebellum to reach the motor nucleus and recordings made via an electrometer amplifier to give a bindwidth of 1.6 Hz to 3.2 kHz and sensitivity of 100 #V/cm. The electrode position was initially adjusted to the point of maximum negative antidromic field potential. Field recordings were then delayed by 3 msec and digitally averaged * Permanent address. Department of Physiology, Duke University Medical Center, Durham, N.C., U.S.A.
345 Depth rn
(~m)_.,p. j,,
i~.j
]6oV !
3500
f
:"
4000
b
e
.,.\f'-
I
4500
:' :
1 d I
I
6000
I
I
Fig. 1. a: extracellular field in motor nucleus V due to single jaw-elevator spindle afferent. Average of 1024 sweeps, b: control for above, triggering from unrelated cell in midbrain. Conditions as in a but for 2 × expanded time scale, c: unit EPSP recorded intracellularly in masseter motoneurone. 1024 sweeps triggered by masseter spindle, d: extracellular control of c. e-i: extracellular averages (1024 sweeps) at successivedepths through the motor nucleus V. Depths indicated in #m relative to arbitrary zero. Triggering from masseter spindle. All records: negative downwards, 2 msec time bar commencing at trigger spike recorded in mesencephalic nucleus.
(Medelec DAV6) with 34 kHz sampling rate and generally 1024 sweeps. The sweep provided 1024 ordinates for 30 msec and the display used 8 point linear interpolation between ordinates. Fig. 1 shows the typical appearance of a record obtained from averaging within the motor nucleus, while triggering from a jaw elevator spindle. When achieving such high effective sensitivity as this by summation of many sweeps it is essential to check for artifacts. Fig. lb shows a control from the same point, triggering instead from a spontaneously active unit in the midbrain unrelated to jaw movement. For comparison, an intracellular record obtained with a glass micropipette, while triggering from a spindle afferent, is shown in Fig. lc. The latencies of the responses are identical and their general from rather similar. The negative polarity of the extracellular response is appropriate for an electrode position close to the sink of excitatory current. Note in this example that the extracellular control (Fig. ld), obtained on withdrawing the pipette from the cell, shows little sign of any extracellular field. This was commonly the case, and it has been noted before 10 that such extracellular fields show irregular fluctuation with small displacements of the pipette tip. By contrast, when recording with the relatively large tungsten electrode, tracking through the nucleus yielded smoothly increasing and decreasing fields (Fig. le-i). The larger lead-off area apparently bridges the short range inhomogeneities of the tissue.
346 Depth(pm) 4
0
4500
a
~
b
C
~
'ii ]5 v
J
l
lmV
500O
6000
, , 2 msec
, , 4 msec
~
, msec
Fig. 2. Each column of records represents averages obtained at successive depths indicated in/~m relative to an arbitrary zero through the motor nucleus V. a: triggering from jaw elevator spindle, 1024 sweeps, b: responses to minute, quick stretches of jaw-elevator muscles, 8 sweeps, c: responses to stimulation of temporalis nerve, 8 sweeps. All records: negative downwards. A conspicuous feature of these records is the frequent appearance of a brief triphasic wave immediately preceding the onset of the unit field, which has been interpreted as due to presynaptic propagation 4. The very short, and sometimes negative, latency of the response relative to the spike in the mesencephalic nucleus is explained as follows. The incoming spindle afferent gives off a collateral to the motor nucleus as it passes close by on the lateral side, and then has to travel between 3 and I 1 mm further to reach its first order cell body. Consequently, excitatory terminals in the motor nucleus can be invaded before the trigger spike is detected. The amplitude of the postsynaptic part of the unit extracellular fields varied with electrode position within the nucleus. In those cases in which systematic exploration was made (n = 43) the maximum values ranged from 1.3 to 13 #V (mean 4.69, S.D. 2.65 #V). The rise times for this group ranged from 0.2 to 2.5 msec (mean 0.72, S.D. 0.46 msec). Their falling phase was approximately exponential with time constants from 0.69 to 5.0 msec (mean 2.27, S.D. 1.24 msec). Fig. 2 shows how extracellular averaging can localize the projection of a single afferent to a part of the motor nucleus (see ref. 3 for a review of field analysis). On tracking through the nucleus a relatively large negative field was obtained by STA at depth 4500 /~m (below an arbitrary zero). Going 500 # m deeper the field had reversed, and thereafter died away. These records indicate that the sink of excitatory current produced by this single afferent must lie between depths of 4000 and 5000, probably centred at about 4500/~m. Interpretation is aided by reference to Fig. 2b showing the total effect of a semi-synchronous input from many spindles produced by
347 minute, quick stretches of the jaw-elevator muscles. In this case the negative field is more widespread, extending from 4000 to 5000 #m, then reversing polarity to become strongly positive at depth 6000/zm. The projection of the single afferent is clearly less extensive than that of the spindle population as a whole. The negative antidromically elicited field potentials may be taken to indicate the region of maximum density of motoneurone cell bodies (in Fig. 2c in the region 5000-5500 #m). The negative 'quick stretch' evoked fields may be taken to indicate the spatial concentration of synaptic input (in Fig. 2b in the region 4000-5000 btm). Histologically it is seen that the motoneurone cell bodies extend over about 1000 # m in the electrode track and that the motor axons extend ventrolaterally from the motor nucleus, while the dendrites are directed generally dorsally. Taken together this is suggestive evidence that the spindle input to this nucleus may be weighted towards the dendrites rather than the cell bodies, which is borne out by the relatively slowly rising and falling shape of intracellular EPSPs found by STA within this nucleus. The unit fields recorded by STA were always within the area of and more restricted than those due to 'quick stretch'. In some cases the STA field was so sharply localized within the other (Fig. 2a) that it appears that the method may be demonstrating a rather restricted projection of single afferents to a part of the motoneurone pool. Note that the maximum quick stretch positive field occurs at a depth (6000 #m) where the antidromic cell response has largely disappeared, leaving only an early positive wave. This is appropriate to a source for the synaptic current at the origins of the motor axons which leave the nucleus ventrolaterally. The extracellular averaging technique is very convenient for rapidly mapping the extent of projection of a single afferent within a nucleus and for examining many afferents for their synaptic effect. It also circumvents two disadvantages of the intracellular technique. First, the bias towards sampling of large cells will be reduced and secondly, dendritic input, which appears greatly attenuated when recorded intracellularly in the soma, should be just as effective as somatic input in producing field potentials recorded extracellularly. The support of the St. Thomas's Hospital Research Endowment Fund is gratefully acknowledged. 1 Appenteng, K., O'Donovan, M. J., Stephens, J. A. and Taylor, A., Synaptic projection of single spindle afferents demonstrated by extra-cellular spike triggered averaging, J. Physiol. (Lond.), Proceedings (1977). 2 Cody, F. W. J., Lee, R. W. H. and Taylor, A., A functional analysis of the components of the mesencephalic nucleus of the fifth nerve in the cat, J. Physiol. (Lond.), 226 (1972) 249-261. 3 Hubbard, J. I., Llin,'ls, F. and Quastel, D. M. J., Electrophysiological Analysis of Synaptic Transmission, Arnold, London, 1969, pp. 265-293. 4 Jankowska, E. and Roberts, W. J., Synaptic actions of single interneurones mediating reciprocal Ia inhibition of motoneurones, J. Physiol. (Lond.), 222 (1972) 623-642. 5 Kirkwood, P. A. and Sears, T. A., Interaction between the monosynaptic EPSP and the central respiratory drive potential of expiratory motoneurones in the cat, J. PhysioL (Lond.), 232 (1973) 38-40P. 6 Kirkwood, P. A. and Sears, T. A., Monosynaptic excitation of motoneurones from secondary endings of muscle spindles, Nature (Lond.), 252 (1974) 243-244.
348 7 Mendell, L. M. and Henneman, E., Terminals of single la fibres : location, density and distribution within a pool of 309 homonymous motoneurones, J. Neurophysiol., 34 (1971) 171-187. 8 Merrill, E. G. and Ainsworth, A., Glass-coated platinum-plated tungsten microelectrodes, Med. biol. Engng., 10 (1972) 662-671. 9 Taylor, A., Stephens, J. A. and Appenteng, K., The synaptic projection of single spindle afferents revealed by extra-cellular spike triggered averaging, Proc. X X V I I Int. Congr. Physiol. Sci. Paris (1977). 10 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, 140 (1978) 344-348 © Elsevier/North-Holland Biomedical Press
Extracellular spike triggered averaging for plotting synaptic projections
A. TAYLOR, J. A. STEPHENS, G. SOMJEN*, K. APPENTENG and M. J. O'DONOVAN Sherrington School of Physiology, St. Thomas's Hospital, London, SE1 7EH (Great Britain)
(Accepted August 31st, 1977)
Averaging intracellular noise with a computer triggered by the firing of a physiologically identified afferent unit ('spike triggered averaging', STA) has become established as a highly specific and sensitive method for detecting synaptic connectionsS-TA 0. However, the difficulty of securing stable in continuity recording from an identified afferent simultaneously with stable intracellular recording from the appropriate motoneurone has been a limitation to the application of the method. During the course of the above experiments, extracellular averages, taken as controls, commonly revealed small negative fields close to motoneurones receiving excitatory input. These fields, due to single afferents were detectable in the spinal cord over distances comparable with the extent of the motor nuclei, but were remarkably irregular and uncertain as controls for the weakest intracellularly recorded averages 1°. The irregularity of the extracellular fields seems likely to have been due to the non-homogeneity of the neural tissue at the scale of the glass micropipette recording tip. This communication is to show that when recorded with relatively large glass-coated tungsten electrodes, the unit fields are well-defined and apparently useful for determining the extent and strength of a given afferent fibre's projection to a particular nucleus. The system under study was the monosynaptic projection of spindles of the jaw-elevator muscles to the motor nucleus of the fifth nerve in the cat 1. Animals were anaesthetized with pentobarbitone and stimulating wires implanted around the masseter nerve and part of the temporalis nerve. The head was secured in a stereotaxic frame and the mandible to an electromagnetic displacement servo. One glass-coated tungsten electrode (25/~m tip exposed) was inserted through an anterior craniotomy to reach the mesencephalic nucleus of the fifth nerve so as to isolate single units belonging to jaw elevator muscle spindles 2. A second tungsten electrode with 80-100 #m tip exposed, was inserted at a 30 ° angle through the cerebellum to reach the motor nucleus and recordings made via an electrometer amplifier to give a bindwidth of 1.6 Hz to 3.2 kHz and sensitivity of 100 #V/cm. The electrode position was initially adjusted to the point of maximum negative antidromic field potential. Field recordings were then delayed by 3 msec and digitally averaged * Permanent address. Department of Physiology, Duke University Medical Center, Durham, N.C., U.S.A.
345 Depth rn
(~m)_.,p. j,,
i~.j
]6oV !
3500
f
:"
4000
b
e
.,.\f'-
I
4500
:' :
1 d I
I
6000
I
I
Fig. 1. a: extracellular field in motor nucleus V due to single jaw-elevator spindle afferent. Average of 1024 sweeps, b: control for above, triggering from unrelated cell in midbrain. Conditions as in a but for 2 × expanded time scale, c: unit EPSP recorded intracellularly in masseter motoneurone. 1024 sweeps triggered by masseter spindle, d: extracellular control of c. e-i: extracellular averages (1024 sweeps) at successivedepths through the motor nucleus V. Depths indicated in #m relative to arbitrary zero. Triggering from masseter spindle. All records: negative downwards, 2 msec time bar commencing at trigger spike recorded in mesencephalic nucleus.
(Medelec DAV6) with 34 kHz sampling rate and generally 1024 sweeps. The sweep provided 1024 ordinates for 30 msec and the display used 8 point linear interpolation between ordinates. Fig. 1 shows the typical appearance of a record obtained from averaging within the motor nucleus, while triggering from a jaw elevator spindle. When achieving such high effective sensitivity as this by summation of many sweeps it is essential to check for artifacts. Fig. lb shows a control from the same point, triggering instead from a spontaneously active unit in the midbrain unrelated to jaw movement. For comparison, an intracellular record obtained with a glass micropipette, while triggering from a spindle afferent, is shown in Fig. lc. The latencies of the responses are identical and their general from rather similar. The negative polarity of the extracellular response is appropriate for an electrode position close to the sink of excitatory current. Note in this example that the extracellular control (Fig. ld), obtained on withdrawing the pipette from the cell, shows little sign of any extracellular field. This was commonly the case, and it has been noted before 10 that such extracellular fields show irregular fluctuation with small displacements of the pipette tip. By contrast, when recording with the relatively large tungsten electrode, tracking through the nucleus yielded smoothly increasing and decreasing fields (Fig. le-i). The larger lead-off area apparently bridges the short range inhomogeneities of the tissue.
346 Depth(pm) 4
0
4500
a
~
b
C
~
'ii ]5 v
J
l
lmV
500O
6000
, , 2 msec
, , 4 msec
~
, msec
Fig. 2. Each column of records represents averages obtained at successive depths indicated in/~m relative to an arbitrary zero through the motor nucleus V. a: triggering from jaw elevator spindle, 1024 sweeps, b: responses to minute, quick stretches of jaw-elevator muscles, 8 sweeps, c: responses to stimulation of temporalis nerve, 8 sweeps. All records: negative downwards. A conspicuous feature of these records is the frequent appearance of a brief triphasic wave immediately preceding the onset of the unit field, which has been interpreted as due to presynaptic propagation 4. The very short, and sometimes negative, latency of the response relative to the spike in the mesencephalic nucleus is explained as follows. The incoming spindle afferent gives off a collateral to the motor nucleus as it passes close by on the lateral side, and then has to travel between 3 and I 1 mm further to reach its first order cell body. Consequently, excitatory terminals in the motor nucleus can be invaded before the trigger spike is detected. The amplitude of the postsynaptic part of the unit extracellular fields varied with electrode position within the nucleus. In those cases in which systematic exploration was made (n = 43) the maximum values ranged from 1.3 to 13 #V (mean 4.69, S.D. 2.65 #V). The rise times for this group ranged from 0.2 to 2.5 msec (mean 0.72, S.D. 0.46 msec). Their falling phase was approximately exponential with time constants from 0.69 to 5.0 msec (mean 2.27, S.D. 1.24 msec). Fig. 2 shows how extracellular averaging can localize the projection of a single afferent to a part of the motor nucleus (see ref. 3 for a review of field analysis). On tracking through the nucleus a relatively large negative field was obtained by STA at depth 4500 /~m (below an arbitrary zero). Going 500 # m deeper the field had reversed, and thereafter died away. These records indicate that the sink of excitatory current produced by this single afferent must lie between depths of 4000 and 5000, probably centred at about 4500/~m. Interpretation is aided by reference to Fig. 2b showing the total effect of a semi-synchronous input from many spindles produced by
347 minute, quick stretches of the jaw-elevator muscles. In this case the negative field is more widespread, extending from 4000 to 5000 #m, then reversing polarity to become strongly positive at depth 6000/zm. The projection of the single afferent is clearly less extensive than that of the spindle population as a whole. The negative antidromically elicited field potentials may be taken to indicate the region of maximum density of motoneurone cell bodies (in Fig. 2c in the region 5000-5500 #m). The negative 'quick stretch' evoked fields may be taken to indicate the spatial concentration of synaptic input (in Fig. 2b in the region 4000-5000 btm). Histologically it is seen that the motoneurone cell bodies extend over about 1000 # m in the electrode track and that the motor axons extend ventrolaterally from the motor nucleus, while the dendrites are directed generally dorsally. Taken together this is suggestive evidence that the spindle input to this nucleus may be weighted towards the dendrites rather than the cell bodies, which is borne out by the relatively slowly rising and falling shape of intracellular EPSPs found by STA within this nucleus. The unit fields recorded by STA were always within the area of and more restricted than those due to 'quick stretch'. In some cases the STA field was so sharply localized within the other (Fig. 2a) that it appears that the method may be demonstrating a rather restricted projection of single afferents to a part of the motoneurone pool. Note that the maximum quick stretch positive field occurs at a depth (6000 #m) where the antidromic cell response has largely disappeared, leaving only an early positive wave. This is appropriate to a source for the synaptic current at the origins of the motor axons which leave the nucleus ventrolaterally. The extracellular averaging technique is very convenient for rapidly mapping the extent of projection of a single afferent within a nucleus and for examining many afferents for their synaptic effect. It also circumvents two disadvantages of the intracellular technique. First, the bias towards sampling of large cells will be reduced and secondly, dendritic input, which appears greatly attenuated when recorded intracellularly in the soma, should be just as effective as somatic input in producing field potentials recorded extracellularly. The support of the St. Thomas's Hospital Research Endowment Fund is gratefully acknowledged. 1 Appenteng, K., O'Donovan, M. J., Stephens, J. A. and Taylor, A., Synaptic projection of single spindle afferents demonstrated by extra-cellular spike triggered averaging, J. Physiol. (Lond.), Proceedings (1977). 2 Cody, F. W. J., Lee, R. W. H. and Taylor, A., A functional analysis of the components of the mesencephalic nucleus of the fifth nerve in the cat, J. Physiol. (Lond.), 226 (1972) 249-261. 3 Hubbard, J. I., Llin,'ls, F. and Quastel, D. M. J., Electrophysiological Analysis of Synaptic Transmission, Arnold, London, 1969, pp. 265-293. 4 Jankowska, E. and Roberts, W. J., Synaptic actions of single interneurones mediating reciprocal Ia inhibition of motoneurones, J. Physiol. (Lond.), 222 (1972) 623-642. 5 Kirkwood, P. A. and Sears, T. A., Interaction between the monosynaptic EPSP and the central respiratory drive potential of expiratory motoneurones in the cat, J. PhysioL (Lond.), 232 (1973) 38-40P. 6 Kirkwood, P. A. and Sears, T. A., Monosynaptic excitation of motoneurones from secondary endings of muscle spindles, Nature (Lond.), 252 (1974) 243-244.
348 7 Mendell, L. M. and Henneman, E., Terminals of single la fibres : location, density and distribution within a pool of 309 homonymous motoneurones, J. Neurophysiol., 34 (1971) 171-187. 8 Merrill, E. G. and Ainsworth, A., Glass-coated platinum-plated tungsten microelectrodes, Med. biol. Engng., 10 (1972) 662-671. 9 Taylor, A., Stephens, J. A. and Appenteng, K., The synaptic projection of single spindle afferents revealed by extra-cellular spike triggered averaging, Proc. X X V I I Int. Congr. Physiol. Sci. Paris (1977). 10 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.
