305
Brain Research, 596 (1992) 305-310 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25434
Observations on morpholo and electrophysiological properties of the normal and axotomlzed facial motoneurons in the cat Yoshihiro Nishimura, Toshihiro Asahara, Tetsuro Yamamoto and Tsutomu Tanaka Department of Physiology, School of Medicine, Mie Unirersity, Tsu, Mie-ken (Japan) (Accepted 18 August 1992)
Key. words: Facial motoneuron; Neuron geometry; Membrane property; Discharge frequency regulation; Axotomy; Cat
The correlation between the morphology of facial motoneurons stained intracellularly with horseradish peroxidase and their physiological parameters was examined in cats following facial nerve section and in cats with intact facial nerve. A certain statistical relationship exists between cell size and excitability in normal neurons. After axotomy, facial neurons showed a slow conduction velocity and a low rheobasic current, but had a normal cell size. Physiological changes include repetitive firing in response to intracellular current injection, reflecting an increase in the excitability in the axotomized neurons.
Although a large number of publications describe the morphological and electrophysiological properties of spinal motoneurons in a variety of mammalian and non-mammalian species, complete information on the properties of the facial nucleus (FN) is unobtainable due to technical limitations. The FN partakes in various reflex reactions of facial muscles and is characterized by wider connections with brain stem structures than the other nuclei of cranial nerves ~. Up to now there have been no intracellular studies of electrophysiological properties of the FN neurons combined with
their intracellular staining. In this study, electrophysiological and morphological properties of the FN neurons of cats were investigated by using both intrace!!u!ar staining with horseradish peroxidase (HRP) and intracellular current injection to find out to what degree their membrane properties correspond to those of spinal motoneurons. As it has long been known that axotomy alters the morphology and physiology of neurons 3'4, attempts were also made to examine whether axotomy of the FN neurons can cause changes in their electric membrane properties. Adult cats were anaesthetized with ketamin (20 mg/kg) intraperitoneaily and supplemented with small
doses (10-15 mg/kg) of pentobarbital intravenously to maintain a relatively deep level of anesthesia (no withdrawal to paw pinch). The animals were immobilized with gallaminc triethiodide and artificially respired. For intracellular recording from FN neurons, the cerebellum was aspired and micro-electrodes filled with 3 M KC! or 5-20% HRP (Toyobo, Grade l-C) in 1.5 M KC! and 0.05 M-Tris buffer at pH 7.3 were inserted through the floor of the fourth ventricle, angle 60° above the horizontal. Passive membrane properties of the FN neurons were studied in normal cats and denervated ones 8-46 days after sectioning of the peripheral facial nerve distal to the stylomastoid foramen. The FN neurons were identified by antidromical invasion from the cut facial nerve in both normal and axotomized preparations. Axonal conduction velocity (CV) was calculated from the latency of antidromic spikes and average conduction distance measured in situ on ten cats. Input resistance of the whole neuron (R n) was measured by the spike-height method. The rheobase (Rh), defined as the minimal current sufficient to elicit a single spike in half of the trials, was measured by injection of short (up to 50 ms) depolarizing current pulses of graded intensity. Afterhyperpolar-
Correspondence: T. Tanaka, Department of Physiology, School of Medicine, Mie University, Tsu, Mie-ken, 514 Japan.
306 of the different variables was evaluated using Student's t-test. Thirty-two physiologically identified FN neurons were intracellularly labeled with HRP and reconstructed. The soma diameters ((long axis + short axis)/2) of these FN neurons ranged from 18.5/zm to 37.5 /zm with an average of 29.1 + 4.7/zm (mean + S.D.). The average SSA was 2587 + 833/,~m 2 ranging from 1011 p , m 2 t o 4239 /~m 2 (n = 32). The SSAs of three stained FN neurons in Fig. 1 ( A - C ) w e r e 3925, p,m 2 2239 p , m 2 and 1413 p , m 2 , respectively. In the present study on dendrites of identified FN neurons, we used a few, easily measured parameters: the number and diameter of dendritic stems. The number of dendritic stems per cell varied between 4 and 10, the mean being 5.8 _+ 1.6 (n = 32). The mean stem-dendrite diameters varied over 2.3-6.5 /Lm, the average being 4.2/~m (n = 24). The relation between the number of the stem dendrites per cell and the SSA yielded a high positive correlation (correlation coefficient r =
izations (AHPs) were studied following spikes evoked by short (less than 1 ms) current pulses. The AHP duration was measured from the spike onset to the point where the AHP crossed the baseline. The repetitive firing was induced by injection of 300 ms long, depolarizing current pulses at a frequency of 0.5/s. Following these physiological measurements, HRP was injected by trains of depolarizing current pulses (0.3-0.5 s pulse duration at a rate of 1 Hz for 3-15 min at a current intensity of 20 nA). The animals were perfused 1-6 h after HRP injections with 10% formaldehyde solution in 0.1 M phosphate buffer through the left ventricle. The sagittal frozen sections were cut at 70/~m and reacted with diaminobenzidine and counterstained with 0.1% Cresyl violet solution. To calculate the somatic surface, cell bodies were approximated by a simple geometrical figure (ellipsoid). The soma surface area (SSA) was estimated by ~r times the product of the maximum and minimum diameters. The level of statistical significance between the means
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Soma surface area Fig. 1. Morphological and electrophysiological properties of cat FN neurons. A-C: projection drawings of three normal FN neurons stained with HRP. Only cell bodies of proximal dendrites are shown. Neurons arranged in order: 3925 ~ m 2 (A), 2238 p,m 2 (B), 1300/~m 2 (C). ax, axon. D: graphic illustration of the relation between the soma surface area and the mean diameter of dendrites. E: graph plotting of the soma surface area and the axonal conduction velocity (CV) in a double-logarithmic coordinate scale. Data points corresponding to the FN neurons (A, B and (2) are indicated by the arrows. F: graphic illustration of the relation between the soma surface area and the reobasic current in a double-logarithmic diagram.
307 0.71., P < 0.001, n = 32). The mean stem-dendrite diameter of a cell was also correlated to its SSA (r = 0.5 1, P < 0.02, N = 23) (Fig. 1D). The Cd3i2 is an important parameter for the analysis of the electrical properties of neurons, where d is dendritic stem diameteri6. Among the FN neurons examined, the mean xd3i2 was (51 f 27) lo-” cm3j2 (range (12.6-105.3) low6 cm3i2, n = 18). There was a fairly strong correlation between zd312 and the SSA (r = 0.91, P < 0.001, y1= 18). This agrees with the observations by Ulfhake and Cullheim on HRP-stained cat lumbar motoneuronsi9. In studies of cells injected with HRP it was demonstrated that there is a fairly strong correlation between axonal conduction velocity (CV) and the diameter of the cell body’,“, whereas Burke et al.2 have demonstrated relatively poor correlation between axonal CV and SSA in cat lumbosacral motoneurons. FN neurons had, on average, smaller cell bodies than those of spinal a-motoneurons (58.3 pm, range 47.5-75.5 Frn in ref. 20). This is in accordance with the slow CV of the FN cells”. Quantitative data relating SSA and CV of stained neurons are shown in Fig. 1E. CV of 22 stained FN neurons ranged from 22.5 m/s to 55.4 m/s with an average of 40.2 m/s (+9.2 m/s). Plotted on a double logarithmic coordinate scale, data points from these FN neurons can be fitted by a straight line. The regression line (calculated by the method of least squares) follows the equation log CV = -0.16 f 0.5 log SSA. The correlation coefficient r is + 0.72 (P < 0.001). CV, an easily measurable parameter, is thus well suited to deduce soma size. Rh values were measured on 20 HRP-stained FN neurons. For neurons with Rh ranging from 1.0 to 7.0 nA, the average was 3.2 nA ( f 1.4 nA). Fig. 1F shows that the relation between log Rh and log SSA can be approximated by a straight line which follows the equation log Rh = -3.5 + I.2 log SSA (r is 0.65, P < 0.01). R, values of stained FN neurons ranged from 1.0 to 5.0 ML! (2.8 + 1.1 Ma, n = 9). For these stained cells, R, was not strongly correlated to SSA (r = - 0.61, P < 0.1). Additional measurements on 68 FN neurons not injected with HRP (antidromic amplitude > 70 mV) gave the same limits for the R, range (1.0-6.5 Mn with an average of 2.4 + 1.0 ML!) in agreement with the reports of others13 . The Rh measured in 116 unstained FN neurons ranged from 1.0 to 9.6 nA, the average being 4.1 f 1.9 nA. As in spinal neurons, the R, is inversely related to the current threshold of FN neurons. Quantitative data relating to the R, and the Rh showed a negative correlation (r = - 0.7, P < 0.001, n = 68). A certain statistical relationship seems to exist between cell size and excitability in normal FN neurons: fast conducting cells with low R, values and high Rh values l
l
were large in size while slow conducting cells with high R, values and low Rh values were small in size. In agreement with previous observations’“, the majority of FN neurons showed a slower CV after axotow. Fig. 2A shows histograms of the frequency of occurrence of FN neurons’as a function of CVs in both control (unoperated) and axotomized cats. The CVs of 235 intact FN neurons ranged from 22.5 to 92.0 m/s with an average of 49.5 & 13.9 m/s. The average CV following axotomy decreased to 37.2 + 14.0 m/s (range; 15.5-75.8 m/s, n = 155). This change was statistically significant (P < 0.05). We have been unable to confirm the findings of Cutmann & Holub6i’ that CV becomes progressively reduced in the central stump after section of the peroneal nerves of rabbits. The CV fell within survival periods of 15-25 days and a further reduction was undetected until nearly 50 days after section. Axotomy decreased the cell body size of primary sensory neurons”, it is then possible that the similar reduction in axon size occurs in axotomized FN neurons, thus reducing conduction velocity. Five axotomized FN neurons were intracellularly labeled with HRP and reconstructed. The axotomized FN neurons had an average SSA of 2587 & 833 pm2 (range 13473768 pm’). There was no significant difference in soma size for normal and axotomized FN neurons. Rh values were evaluated in 117 normal and 112 axotomized FN neurons. The distribution of Rh in the normal FN neurons was from 1.0 to 9.6 nA with an average of 4.1 nA, whereas that in the axotomized FN neurons was from 0.4 to 6.8 nA with a mean value of 1.9 nA (Fig. 2B). Thus, the frequency distributions were somewhat different between the two groups of FN neurons and their mean values were statistically different (P < 0.01). One example of the measurement of the Rh for the axotomized motoneurons 19 days after axotomy is exhibited in Fig. 2C. The value was 0.5 nA. R, was very similar in the 69 normal and 39 axotomized FN neurons evaluated. The mean values (2.3 f 1.0 Ma vs 2.5 f 1.2 ML!, control and axotomized neurons, respectively) were nearly identical, and the frequency distribution characteristics were not statistically different (range 1.0-5.3 ML! vs 0.5-5.0 MJ2, control and axotomized neurons, respectively). The reduction of the Rh of the axotomized FN neurons may be due to a decrease of the threshold level of the neurons, because R, and soma size of the FN neurons did not change markedly after axotomy. A major mechanism for repetitive firing control in motoneurons is the long-lasting AHP following each spike’. This AHP is not invariant but is influenced by a variety of conditions. Following axotomy, both the duration and magnitude of the AI-IP are changed, which
308 results in altered firing characteristics ~. The distribution of the AHP duration for two groups of FN neurons is shown in Fig. 2E. There is a chaage in the distribution pattern following axotomy. The range of values is compressed in axotomized motoneurons (20.5-170 ms vs. 11.5-90 ms, respectively). The average value was clearly higher for the intact FN neurons (81.4 + 38.6 ms, n = 23) than for the axotomized FN neurons (51.2 +_ 19.3 ms, n = 21). This difference is to some extent caused by an absence of AHPs of long duration ( > 100 ms) in the axotomized preparations. The descending phase of the AHP also had a different time course in normal and axotomized neurons. The time to maximum hyperpolarization (time to peak) was longer in normal neurons (22.6 + 7.1 ms, n = 23) than in axotomized neurons (10.9 + 6.9 ms, n = 23). This difference is statistically significant ( P < 0 . 0 0 1 ) . Fig. 2D shows one example of AHP for an axotomized FN neuron 18 days after axotomy. Its duration and the time to maximum AHP are 37.5 ms and 3.2 ms, respec-
tively. In a similar manner as shown previously (Kuno et al. ~4) in the spinal motoneurons, axotomy decreased the A H P duration of FN neurons but had little effect on their input resistance, and there was no proportionality between input resistance and A H P duration. As illustrated in Fig. 3A,B, intracellular injection of a prolonged (300 ms) current pulse induced a burst of action potentials in 44 normal FN neurons. The frequency of the action potentials was determined by the strength of the injected currents. In nitre cells studied after axotomy, all neurons fired repetitively during current passage. The frequency-current (f-l) curve is often used to describe the firing behavior of a neuron; the instantaneous frequency ( f , the inverse of the interspike interval for the two adjacent spikes) is plotted against current intensity. The axotomized FN neurons required less current to fire and gain (f/l) was greater than that of control cells, and they discharged at higher frequencies for any given current (Fig. 3D, E), which is in agreement with the findings that in the
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40 Fig. 2. Axotomy-induced changes in eletrophysiological properties of cat FN neurons. A: frequency-distribution histograms for the CVs of the normal (unhatched) and axotomized (hatched) FN neurons. B: frequency-distribution histograms of Rh for the normal (unhatched) and axotomized (hatched) FN neurons. C: responses to Rh from an axotomized FN neuron. The upper beam indicates the current intensity and the upward direction is depolarizing. D: afterhyperpolarization (AHP) in an axotomized FN neuron. E: frequency-distribution histograms of the AHP values for the normal (unhatched) and axotomized (hatched) FN neurons.
309
axotomized FN neurons AHP duration is reduced and Rh is lowered. With current steps approximately 1.4 times above Rh, firing frequency in most FN neurons was linearly related to the intensity of the injected depolarizing current, within certain limits (primary range firing of Granit and colleagues) 6 (Fig. 3C). Further increase in current intensity caused an abrupt increase in the slope of the f - I curve of these FN neurons, which then entered a linear secondary range. The f-I curve for the steady state did not exhibit an upward deviation at higher frequency and the firing frequency was proportional to the current intensity in its slope. As the FN neurons adapted easily to the long-lasting current, they were probably unable to reach the secondary range in
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Fig. 3. Repetitive firing patterns generated in a normal FN neuron (A-C) and an axotomized FN neuron ( D - F ) by the long lasting (300-400 ms in duration) rectangular current pulse with various intensities (A, 16.5 nA; B, 22 nA; D, 5 nA; E, 7.2 nA). C: firing rate vs. value of injected current ( f - I plot) with a long duration of 300 ms for a normal FN neuron. Reciprocalsof the first (tl), second (t 2) and third (t 3) interspike intervals and adapted rate (steady state) are indicated. The firing frequency in the steady state was the averaged value for firing rate 50 ms after the onset of the current injection. F: f - I plot as C for an axotomized FN neuron. Note that the frequency and current scales are given by different linear measures.
310 ization was evident, possibly indicating a reduction in Ca2+-dependent K + conductance 12. Following section of the FN nerve, FN neurons exhibited a marked decrease in the rheobase, reduction in the A H P duration and a decreased axonal CV. There was no change in mean input resistance. These data are consistent with de-differentiation of motoneuron properties following axotomy ~4. De-differentiation can be regarded as a shift to a growth state for the motoneuron 5. I Brodal, A., Neurological Anatomy in Relation to Clinical Medicine, Oxford University Press, New York, 1981. 2 Burke, R.E., Dum, R.P., Fleshman, J.W., Glenn, L.L., L-Toy, A., O'Donovan, M.J. and Pinter, M.J., An HRP study of the relation between cell size and motor unit type in cat ankle extensor motoneurons, J. Comp. Neurol., 209 (1982) 17-28. 3 Cajal, S. and Ramon, Y., Histologie du SystJme Nervezct de l'Homme et des Vertdbrd's, Vol. l, Maloine, Paris, 1909. 4 Eccles, J.C., Libert, B. and Young, R.R., The behavior of chromatolysed motoneurones studied by intracellular recording, J. Physiol., 143 (1958) 11-40. 5 Faber, D.S. and Zottoli, S.J., Axotomy-induced changes in cell structure and membrane excitability are sustained in a vertebrate central neuron, Bra& Res., 223 (1981) 436-443. 6 Granit, R., Mechanisms Regulating the Discharge of Motoneurons, Liverpool University Press, Liverpool, 1972. 7 Grantyn, R. and Schaaf, P., Conduction velocity, input resistance and size of cat ocular motoneurons stained with Procion yellow, Brain Res., 135 (1977) 167-173. 8 Gustafsson, B., Changes in motoneurone electrical properties following axotomy, J. Physiol., 293 (1979) 197-215. 9 Gutmann, E. and Holubfir, J., Atrophy of nerve fibres in the central stump following nerve section and the possibilities of its prevention, Arch. &t. Stud. Neurology, I (1951) 1-11.
!0 Heyer, C. and Llinfis, R., Control of rhythmic firing in normal and axotomized cat spinal motoneurons, J. Neurophysiol., 40 (1977) 480-488. 11 Kitai, S.T., Tanaka, T., Tsukahara, N. and Yu, H., The facial nucleus of the cat: antidromic and synaptic activation and peripheral nerve representation, Exp. Brain Res., 16 (1972) 161-183. 12 Kelly, M.E.M., Gordon, T., Shapiro, J. and Smith, P.A., Axotomy affects calcium-sensitive potassium conductance in sympathetic neurones, Neurosci. Lett., 67 (1986) 163-168. 13 Kernell, D. and Zwaagstra, B., Size and remoteness: two relatively independent parameters of dendrites, as studied for spinal motoneurones of the cat, J. Physiol., 413 (1989) 233-254. 14 Kuno, M., Miyata, Y. and Mffioz-Martinez, E.J., Differential reaction of fast and slow alpha-motoneurones to axotomy, J. Physiol., 240 (1974) 725-739. 15 Lux, H.D., Schubert, P. and Kreutzberg, G.W., Direct matching of morphological and electrophysiological data in cat spinal motoneurones. In P. Andersen and J.K.S. Jansen (Eds.), Excitatory Synaptic Mechanisms, Universitetsforlaget, Oslo, 1970, pp. 189198. 16 Rail, W., Core conductor theory and cable properties of neurons. In E.R. Kandel (Ed.), Handbook of Physiology, Section 1, Voi. 1, Pat: 1 American Physiological Society, Bethesda, MD, 1977, pp. 39- 97. 17 Risliu~. M., Aldskogius, H. and Hildebrand, C., Effects of sciatic nerve cru~.h on the L7 spinal roots and dorsal root ganglia in kittens, Exp. Neurol., 79 (1983) 176-187. 18 Sunderland, S., Nerves and Nerve Injuries, Churchill Livingstone, Edinburgh, 1978. 19 UIfhake, B. and Culiheim, S., A quantitative light microscopic study of the dendrites of cat spinal y-motoneurons after intracellular staining with horseradish peroxidase, J. Comp. Neurol., 202 (1981) 585-596. 20 UIfhake, B. and Kellerth, J.-O., A quantitative light microscopic study of the dendrites of cat spinal a-motoneurons after intracellular staining with horseradish peroxidase, J. Comp. Neurol., 202 (1981) 571-583.
Brain Research, 596 (1992) 305-310 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25434
Observations on morpholo and electrophysiological properties of the normal and axotomlzed facial motoneurons in the cat Yoshihiro Nishimura, Toshihiro Asahara, Tetsuro Yamamoto and Tsutomu Tanaka Department of Physiology, School of Medicine, Mie Unirersity, Tsu, Mie-ken (Japan) (Accepted 18 August 1992)
Key. words: Facial motoneuron; Neuron geometry; Membrane property; Discharge frequency regulation; Axotomy; Cat
The correlation between the morphology of facial motoneurons stained intracellularly with horseradish peroxidase and their physiological parameters was examined in cats following facial nerve section and in cats with intact facial nerve. A certain statistical relationship exists between cell size and excitability in normal neurons. After axotomy, facial neurons showed a slow conduction velocity and a low rheobasic current, but had a normal cell size. Physiological changes include repetitive firing in response to intracellular current injection, reflecting an increase in the excitability in the axotomized neurons.
Although a large number of publications describe the morphological and electrophysiological properties of spinal motoneurons in a variety of mammalian and non-mammalian species, complete information on the properties of the facial nucleus (FN) is unobtainable due to technical limitations. The FN partakes in various reflex reactions of facial muscles and is characterized by wider connections with brain stem structures than the other nuclei of cranial nerves ~. Up to now there have been no intracellular studies of electrophysiological properties of the FN neurons combined with
their intracellular staining. In this study, electrophysiological and morphological properties of the FN neurons of cats were investigated by using both intrace!!u!ar staining with horseradish peroxidase (HRP) and intracellular current injection to find out to what degree their membrane properties correspond to those of spinal motoneurons. As it has long been known that axotomy alters the morphology and physiology of neurons 3'4, attempts were also made to examine whether axotomy of the FN neurons can cause changes in their electric membrane properties. Adult cats were anaesthetized with ketamin (20 mg/kg) intraperitoneaily and supplemented with small
doses (10-15 mg/kg) of pentobarbital intravenously to maintain a relatively deep level of anesthesia (no withdrawal to paw pinch). The animals were immobilized with gallaminc triethiodide and artificially respired. For intracellular recording from FN neurons, the cerebellum was aspired and micro-electrodes filled with 3 M KC! or 5-20% HRP (Toyobo, Grade l-C) in 1.5 M KC! and 0.05 M-Tris buffer at pH 7.3 were inserted through the floor of the fourth ventricle, angle 60° above the horizontal. Passive membrane properties of the FN neurons were studied in normal cats and denervated ones 8-46 days after sectioning of the peripheral facial nerve distal to the stylomastoid foramen. The FN neurons were identified by antidromical invasion from the cut facial nerve in both normal and axotomized preparations. Axonal conduction velocity (CV) was calculated from the latency of antidromic spikes and average conduction distance measured in situ on ten cats. Input resistance of the whole neuron (R n) was measured by the spike-height method. The rheobase (Rh), defined as the minimal current sufficient to elicit a single spike in half of the trials, was measured by injection of short (up to 50 ms) depolarizing current pulses of graded intensity. Afterhyperpolar-
Correspondence: T. Tanaka, Department of Physiology, School of Medicine, Mie University, Tsu, Mie-ken, 514 Japan.
306 of the different variables was evaluated using Student's t-test. Thirty-two physiologically identified FN neurons were intracellularly labeled with HRP and reconstructed. The soma diameters ((long axis + short axis)/2) of these FN neurons ranged from 18.5/zm to 37.5 /zm with an average of 29.1 + 4.7/zm (mean + S.D.). The average SSA was 2587 + 833/,~m 2 ranging from 1011 p , m 2 t o 4239 /~m 2 (n = 32). The SSAs of three stained FN neurons in Fig. 1 ( A - C ) w e r e 3925, p,m 2 2239 p , m 2 and 1413 p , m 2 , respectively. In the present study on dendrites of identified FN neurons, we used a few, easily measured parameters: the number and diameter of dendritic stems. The number of dendritic stems per cell varied between 4 and 10, the mean being 5.8 _+ 1.6 (n = 32). The mean stem-dendrite diameters varied over 2.3-6.5 /Lm, the average being 4.2/~m (n = 24). The relation between the number of the stem dendrites per cell and the SSA yielded a high positive correlation (correlation coefficient r =
izations (AHPs) were studied following spikes evoked by short (less than 1 ms) current pulses. The AHP duration was measured from the spike onset to the point where the AHP crossed the baseline. The repetitive firing was induced by injection of 300 ms long, depolarizing current pulses at a frequency of 0.5/s. Following these physiological measurements, HRP was injected by trains of depolarizing current pulses (0.3-0.5 s pulse duration at a rate of 1 Hz for 3-15 min at a current intensity of 20 nA). The animals were perfused 1-6 h after HRP injections with 10% formaldehyde solution in 0.1 M phosphate buffer through the left ventricle. The sagittal frozen sections were cut at 70/~m and reacted with diaminobenzidine and counterstained with 0.1% Cresyl violet solution. To calculate the somatic surface, cell bodies were approximated by a simple geometrical figure (ellipsoid). The soma surface area (SSA) was estimated by ~r times the product of the maximum and minimum diameters. The level of statistical significance between the means
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Soma surface area Fig. 1. Morphological and electrophysiological properties of cat FN neurons. A-C: projection drawings of three normal FN neurons stained with HRP. Only cell bodies of proximal dendrites are shown. Neurons arranged in order: 3925 ~ m 2 (A), 2238 p,m 2 (B), 1300/~m 2 (C). ax, axon. D: graphic illustration of the relation between the soma surface area and the mean diameter of dendrites. E: graph plotting of the soma surface area and the axonal conduction velocity (CV) in a double-logarithmic coordinate scale. Data points corresponding to the FN neurons (A, B and (2) are indicated by the arrows. F: graphic illustration of the relation between the soma surface area and the reobasic current in a double-logarithmic diagram.
307 0.71., P < 0.001, n = 32). The mean stem-dendrite diameter of a cell was also correlated to its SSA (r = 0.5 1, P < 0.02, N = 23) (Fig. 1D). The Cd3i2 is an important parameter for the analysis of the electrical properties of neurons, where d is dendritic stem diameteri6. Among the FN neurons examined, the mean xd3i2 was (51 f 27) lo-” cm3j2 (range (12.6-105.3) low6 cm3i2, n = 18). There was a fairly strong correlation between zd312 and the SSA (r = 0.91, P < 0.001, y1= 18). This agrees with the observations by Ulfhake and Cullheim on HRP-stained cat lumbar motoneuronsi9. In studies of cells injected with HRP it was demonstrated that there is a fairly strong correlation between axonal conduction velocity (CV) and the diameter of the cell body’,“, whereas Burke et al.2 have demonstrated relatively poor correlation between axonal CV and SSA in cat lumbosacral motoneurons. FN neurons had, on average, smaller cell bodies than those of spinal a-motoneurons (58.3 pm, range 47.5-75.5 Frn in ref. 20). This is in accordance with the slow CV of the FN cells”. Quantitative data relating SSA and CV of stained neurons are shown in Fig. 1E. CV of 22 stained FN neurons ranged from 22.5 m/s to 55.4 m/s with an average of 40.2 m/s (+9.2 m/s). Plotted on a double logarithmic coordinate scale, data points from these FN neurons can be fitted by a straight line. The regression line (calculated by the method of least squares) follows the equation log CV = -0.16 f 0.5 log SSA. The correlation coefficient r is + 0.72 (P < 0.001). CV, an easily measurable parameter, is thus well suited to deduce soma size. Rh values were measured on 20 HRP-stained FN neurons. For neurons with Rh ranging from 1.0 to 7.0 nA, the average was 3.2 nA ( f 1.4 nA). Fig. 1F shows that the relation between log Rh and log SSA can be approximated by a straight line which follows the equation log Rh = -3.5 + I.2 log SSA (r is 0.65, P < 0.01). R, values of stained FN neurons ranged from 1.0 to 5.0 ML! (2.8 + 1.1 Ma, n = 9). For these stained cells, R, was not strongly correlated to SSA (r = - 0.61, P < 0.1). Additional measurements on 68 FN neurons not injected with HRP (antidromic amplitude > 70 mV) gave the same limits for the R, range (1.0-6.5 Mn with an average of 2.4 + 1.0 ML!) in agreement with the reports of others13 . The Rh measured in 116 unstained FN neurons ranged from 1.0 to 9.6 nA, the average being 4.1 f 1.9 nA. As in spinal neurons, the R, is inversely related to the current threshold of FN neurons. Quantitative data relating to the R, and the Rh showed a negative correlation (r = - 0.7, P < 0.001, n = 68). A certain statistical relationship seems to exist between cell size and excitability in normal FN neurons: fast conducting cells with low R, values and high Rh values l
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were large in size while slow conducting cells with high R, values and low Rh values were small in size. In agreement with previous observations’“, the majority of FN neurons showed a slower CV after axotow. Fig. 2A shows histograms of the frequency of occurrence of FN neurons’as a function of CVs in both control (unoperated) and axotomized cats. The CVs of 235 intact FN neurons ranged from 22.5 to 92.0 m/s with an average of 49.5 & 13.9 m/s. The average CV following axotomy decreased to 37.2 + 14.0 m/s (range; 15.5-75.8 m/s, n = 155). This change was statistically significant (P < 0.05). We have been unable to confirm the findings of Cutmann & Holub6i’ that CV becomes progressively reduced in the central stump after section of the peroneal nerves of rabbits. The CV fell within survival periods of 15-25 days and a further reduction was undetected until nearly 50 days after section. Axotomy decreased the cell body size of primary sensory neurons”, it is then possible that the similar reduction in axon size occurs in axotomized FN neurons, thus reducing conduction velocity. Five axotomized FN neurons were intracellularly labeled with HRP and reconstructed. The axotomized FN neurons had an average SSA of 2587 & 833 pm2 (range 13473768 pm’). There was no significant difference in soma size for normal and axotomized FN neurons. Rh values were evaluated in 117 normal and 112 axotomized FN neurons. The distribution of Rh in the normal FN neurons was from 1.0 to 9.6 nA with an average of 4.1 nA, whereas that in the axotomized FN neurons was from 0.4 to 6.8 nA with a mean value of 1.9 nA (Fig. 2B). Thus, the frequency distributions were somewhat different between the two groups of FN neurons and their mean values were statistically different (P < 0.01). One example of the measurement of the Rh for the axotomized motoneurons 19 days after axotomy is exhibited in Fig. 2C. The value was 0.5 nA. R, was very similar in the 69 normal and 39 axotomized FN neurons evaluated. The mean values (2.3 f 1.0 Ma vs 2.5 f 1.2 ML!, control and axotomized neurons, respectively) were nearly identical, and the frequency distribution characteristics were not statistically different (range 1.0-5.3 ML! vs 0.5-5.0 MJ2, control and axotomized neurons, respectively). The reduction of the Rh of the axotomized FN neurons may be due to a decrease of the threshold level of the neurons, because R, and soma size of the FN neurons did not change markedly after axotomy. A major mechanism for repetitive firing control in motoneurons is the long-lasting AHP following each spike’. This AHP is not invariant but is influenced by a variety of conditions. Following axotomy, both the duration and magnitude of the AI-IP are changed, which
308 results in altered firing characteristics ~. The distribution of the AHP duration for two groups of FN neurons is shown in Fig. 2E. There is a chaage in the distribution pattern following axotomy. The range of values is compressed in axotomized motoneurons (20.5-170 ms vs. 11.5-90 ms, respectively). The average value was clearly higher for the intact FN neurons (81.4 + 38.6 ms, n = 23) than for the axotomized FN neurons (51.2 +_ 19.3 ms, n = 21). This difference is to some extent caused by an absence of AHPs of long duration ( > 100 ms) in the axotomized preparations. The descending phase of the AHP also had a different time course in normal and axotomized neurons. The time to maximum hyperpolarization (time to peak) was longer in normal neurons (22.6 + 7.1 ms, n = 23) than in axotomized neurons (10.9 + 6.9 ms, n = 23). This difference is statistically significant ( P < 0 . 0 0 1 ) . Fig. 2D shows one example of AHP for an axotomized FN neuron 18 days after axotomy. Its duration and the time to maximum AHP are 37.5 ms and 3.2 ms, respec-
tively. In a similar manner as shown previously (Kuno et al. ~4) in the spinal motoneurons, axotomy decreased the A H P duration of FN neurons but had little effect on their input resistance, and there was no proportionality between input resistance and A H P duration. As illustrated in Fig. 3A,B, intracellular injection of a prolonged (300 ms) current pulse induced a burst of action potentials in 44 normal FN neurons. The frequency of the action potentials was determined by the strength of the injected currents. In nitre cells studied after axotomy, all neurons fired repetitively during current passage. The frequency-current (f-l) curve is often used to describe the firing behavior of a neuron; the instantaneous frequency ( f , the inverse of the interspike interval for the two adjacent spikes) is plotted against current intensity. The axotomized FN neurons required less current to fire and gain (f/l) was greater than that of control cells, and they discharged at higher frequencies for any given current (Fig. 3D, E), which is in agreement with the findings that in the
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40 Fig. 2. Axotomy-induced changes in eletrophysiological properties of cat FN neurons. A: frequency-distribution histograms for the CVs of the normal (unhatched) and axotomized (hatched) FN neurons. B: frequency-distribution histograms of Rh for the normal (unhatched) and axotomized (hatched) FN neurons. C: responses to Rh from an axotomized FN neuron. The upper beam indicates the current intensity and the upward direction is depolarizing. D: afterhyperpolarization (AHP) in an axotomized FN neuron. E: frequency-distribution histograms of the AHP values for the normal (unhatched) and axotomized (hatched) FN neurons.
309
axotomized FN neurons AHP duration is reduced and Rh is lowered. With current steps approximately 1.4 times above Rh, firing frequency in most FN neurons was linearly related to the intensity of the injected depolarizing current, within certain limits (primary range firing of Granit and colleagues) 6 (Fig. 3C). Further increase in current intensity caused an abrupt increase in the slope of the f - I curve of these FN neurons, which then entered a linear secondary range. The f-I curve for the steady state did not exhibit an upward deviation at higher frequency and the firing frequency was proportional to the current intensity in its slope. As the FN neurons adapted easily to the long-lasting current, they were probably unable to reach the secondary range in
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Fig. 3. Repetitive firing patterns generated in a normal FN neuron (A-C) and an axotomized FN neuron ( D - F ) by the long lasting (300-400 ms in duration) rectangular current pulse with various intensities (A, 16.5 nA; B, 22 nA; D, 5 nA; E, 7.2 nA). C: firing rate vs. value of injected current ( f - I plot) with a long duration of 300 ms for a normal FN neuron. Reciprocalsof the first (tl), second (t 2) and third (t 3) interspike intervals and adapted rate (steady state) are indicated. The firing frequency in the steady state was the averaged value for firing rate 50 ms after the onset of the current injection. F: f - I plot as C for an axotomized FN neuron. Note that the frequency and current scales are given by different linear measures.
310 ization was evident, possibly indicating a reduction in Ca2+-dependent K + conductance 12. Following section of the FN nerve, FN neurons exhibited a marked decrease in the rheobase, reduction in the A H P duration and a decreased axonal CV. There was no change in mean input resistance. These data are consistent with de-differentiation of motoneuron properties following axotomy ~4. De-differentiation can be regarded as a shift to a growth state for the motoneuron 5. I Brodal, A., Neurological Anatomy in Relation to Clinical Medicine, Oxford University Press, New York, 1981. 2 Burke, R.E., Dum, R.P., Fleshman, J.W., Glenn, L.L., L-Toy, A., O'Donovan, M.J. and Pinter, M.J., An HRP study of the relation between cell size and motor unit type in cat ankle extensor motoneurons, J. Comp. Neurol., 209 (1982) 17-28. 3 Cajal, S. and Ramon, Y., Histologie du SystJme Nervezct de l'Homme et des Vertdbrd's, Vol. l, Maloine, Paris, 1909. 4 Eccles, J.C., Libert, B. and Young, R.R., The behavior of chromatolysed motoneurones studied by intracellular recording, J. Physiol., 143 (1958) 11-40. 5 Faber, D.S. and Zottoli, S.J., Axotomy-induced changes in cell structure and membrane excitability are sustained in a vertebrate central neuron, Bra& Res., 223 (1981) 436-443. 6 Granit, R., Mechanisms Regulating the Discharge of Motoneurons, Liverpool University Press, Liverpool, 1972. 7 Grantyn, R. and Schaaf, P., Conduction velocity, input resistance and size of cat ocular motoneurons stained with Procion yellow, Brain Res., 135 (1977) 167-173. 8 Gustafsson, B., Changes in motoneurone electrical properties following axotomy, J. Physiol., 293 (1979) 197-215. 9 Gutmann, E. and Holubfir, J., Atrophy of nerve fibres in the central stump following nerve section and the possibilities of its prevention, Arch. &t. Stud. Neurology, I (1951) 1-11.
!0 Heyer, C. and Llinfis, R., Control of rhythmic firing in normal and axotomized cat spinal motoneurons, J. Neurophysiol., 40 (1977) 480-488. 11 Kitai, S.T., Tanaka, T., Tsukahara, N. and Yu, H., The facial nucleus of the cat: antidromic and synaptic activation and peripheral nerve representation, Exp. Brain Res., 16 (1972) 161-183. 12 Kelly, M.E.M., Gordon, T., Shapiro, J. and Smith, P.A., Axotomy affects calcium-sensitive potassium conductance in sympathetic neurones, Neurosci. Lett., 67 (1986) 163-168. 13 Kernell, D. and Zwaagstra, B., Size and remoteness: two relatively independent parameters of dendrites, as studied for spinal motoneurones of the cat, J. Physiol., 413 (1989) 233-254. 14 Kuno, M., Miyata, Y. and Mffioz-Martinez, E.J., Differential reaction of fast and slow alpha-motoneurones to axotomy, J. Physiol., 240 (1974) 725-739. 15 Lux, H.D., Schubert, P. and Kreutzberg, G.W., Direct matching of morphological and electrophysiological data in cat spinal motoneurones. In P. Andersen and J.K.S. Jansen (Eds.), Excitatory Synaptic Mechanisms, Universitetsforlaget, Oslo, 1970, pp. 189198. 16 Rail, W., Core conductor theory and cable properties of neurons. In E.R. Kandel (Ed.), Handbook of Physiology, Section 1, Voi. 1, Pat: 1 American Physiological Society, Bethesda, MD, 1977, pp. 39- 97. 17 Risliu~. M., Aldskogius, H. and Hildebrand, C., Effects of sciatic nerve cru~.h on the L7 spinal roots and dorsal root ganglia in kittens, Exp. Neurol., 79 (1983) 176-187. 18 Sunderland, S., Nerves and Nerve Injuries, Churchill Livingstone, Edinburgh, 1978. 19 UIfhake, B. and Culiheim, S., A quantitative light microscopic study of the dendrites of cat spinal y-motoneurons after intracellular staining with horseradish peroxidase, J. Comp. Neurol., 202 (1981) 585-596. 20 UIfhake, B. and Kellerth, J.-O., A quantitative light microscopic study of the dendrites of cat spinal a-motoneurons after intracellular staining with horseradish peroxidase, J. Comp. Neurol., 202 (1981) 571-583.
