300
Brain Research, 570 (1992) 300-306 O 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17388
Passive electrical properties of motoneurons in aged cats following axotomy Jack Yamuy, John K. Engelhardt, Francisco R. Morales and Michael H. Chase Department of Physiology, Department of Anatomy and Cell Biology and the Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.) (Accepted 17 September 1991)
Key words: Aged; Cat; Motoneuron; Axotomy; Electrophysiology
The objective of this study was to determine whether the aging process influences the changes in the electrophysiological properties of motoneurons that occur as a consequence of axotomy. Accordingly, using intracellular recording and stimulating techniques, the basic electrical properties of control (unaxotomized) and axotomized spinal cord motoneurons of aged cats were determined. Compared with control motoneurons, axotomized motoneurons exhibited increases in input resistance (Ri=), membrane time constant (rb) and the equalizing time constant (re). While the electrotonic length (L) remained unchanged, axotomy induced a decrease in the total cell capacitance (Cceu). The post-axotomy reduction of C¢~n indicates that the motoneuron surface area was reduced and the increased membrane time constant indicates that there was an increase in membrane resistivity (Rm). The post-axotomy conservation of L accompanied by an increase in R m suggests that aged axotomized motoneurons undergo geometrical changes. Furthermore, calculations based on cable theory suggest that the diameter of the equivalent cylinder (d) decreased following axotomy, whereas the equivalent cylinder length (1) remained unaffected. It is concluded that axotomy produces significant alterations in the soma-dendritic portion of aged spinal motoneurons, as indicated by the changes found in their passive electrophysiological properties, and that the pattern of the response that occurs in axotomized motoneurons of adult cats is also present in axotomized motoneurons of aged animals. INTRODUCTION
rons. It was c o n c l u d e d that t h e r e was an a g e - d e p e n d e n t increase in m e m b r a n e resistance and an a g e - d e p e n d e n t
It has long b e e n k n o w n that a x o t o m y alters the m o r -
d e c r e a s e in cell surface a r e a of old cat m o t o n e u r o n s .
p h o l o g y and physiology of n e u r o n s 5'6. Spinal c o r d mo-
B e c a u s e a x o t o m y is k n o w n to p r o d u c e changes in these
t o n e u r o n s , particularly of the cat, h a v e b e e n extensively
basic electrical p r o p e r t i e s of m o t o n e u r o n s , we wished to
investigated after chronic axonal section (for r e v i e w see
e x a m i n e the e x t e n t to which the aging process impacts on
T i t m u s and Faber26). T h e y r e s p o n d to a x o t o m y with a
the r e a c t i o n of aged m o t o n e u r o n s to a x o t o m y . Prelimi-
series of changes classically k n o w n as the ' a x o n reac-
nary d a t a h a v e b e e n p u b l i s h e d as an abstract 8.
tion '~9. A m o n g the c h a n g e s o b s e r v e d are increases in input resistance (Rin) and m e m b r a n e t i m e c o n s t a n t (rb) 9'11. A x o t o m i z e d m o t o n e u r o n s also s h o w a d e c r e a s e in the calculated v a l u e of total cell c a p a c i t a n c e (Cce,) which has b e e n i n t e r p r e t e d as a d e c r e m e n t in the total surface a r e a of the cell 13'22. T h e s e changes in passive
MATERIALS AND METHODS
Animals Five cats, ranging in age from 13 to 16.5 years, were utilized in this study. All animals were in good health at the time of the experiment as determined by the UCLA Division of Laboratory Animal Medicine.
electrical p r o p e r t i e s h a v e led different authors to prop o s e that a x o t o m i z e d m o t o n e u r o n s u n d e r g o an increase in m e m b r a n e resistance and a d e c r e a s e in cell size which are a c c o m p a n i e d by changes in cell g e o m e t r y 1~-13'22. R e c e n t l y , the basic electrical p r o p e r t i e s of m o t o n e u rons f r o m aged cats w e r e d e s c r i b e d 7'2°. C o m p a r e d with a control p o p u l a t i o n of adult cat m o t o n e u r o n s , aged cat m o t o n e u r o n s e x h i b i t e d increases in Rin and rb, while Ccell was r e d u c e d . N o significant differences w e r e f o u n d bet w e e n resting potentials of adult and old cat m o t o n e u -
Surgical procedures Axotomy was performed while the animals were anesthetized with halothane. A small incision was made in the popliteal fossa of the left hind-limb and the nerve to the gastrocnemius medialis muscle (GM) and the tibialis (Tib) nerve were dissected, ligated at their distal end, and cut. In one cat the gastrocnemius lateralis and soleus (GL-S) nerves were also cut distally. In addition, the following procedures were performed in order to avoid subsequent reinnervation. The silk from the ligature, along with the distal 3-4 mm of the nerve, was threaded through a 5 mm polyethylene tube. Each nerve was fixed inside its tube by sealing the end of the tube and the thread with a hot glass rod. Afterwards, using the same
Correspondence: J. Yamuy, Brain Research Institute, UCLA Center for the Health Sciences, 43-367 CHS, Los Angeles, CA 90024-1751, U.S.A. Fax: (1) (213) 206-3499.
301 thread, the tube was anchored to the overlying fat in the popliteal fossa approximately 2 cm from the nerve's target muscle. The surgical wound was closed with a skin suture and antibiotic ointment was applied. Prophylactic antibiotics were administered for 7 days after surgery. After a period of 43 days (4 cats) or 54 days (1 cat), the animals were anesthetized with pentobarbital (30 mg/kg) and prepared for intraceUular recording from lumbar motoneurons. The trachea was cannulated and the right carotid artery and external jugular vein were catheterized. The arterial blood pressure and end-tidal pCO 2 were monitored continuously and maintained within physiological limits. The skin along the posterior surface of the left hindlimb was incised and the formerly axotomized nerves (GM, Tib, GL-S) were individualized and dissected centrally. The following nerves were then dissected and excised distally: hamstring (HAM), GL-S (in the animals in which it was not axotomized) and common peroneal (CP). These nerves, including the trunk of the sciatic, were then placed on stimulating electrodes. A laminectomy was performed from vertebra L4-LT; the dura was then cut and retracted and the left dorsal roots Ls-S 1 were cut distally. Spinal and hindlimb pools were constructed with skin flaps and filled with warm mineral oil. An infrared lamp and heating pad were used to keep the animal and mineral oil pools between 38°C and 390C.
Recording and analysis Intracellular recordings were obtained using broken-tip glass micropipette electrodes filled with 3 M KC1 (1-1.5 #m, approximate tip diameter). The resistance of the electrodes ranged from 4 to 12 megohms. An Axoclamp-2A amplifier (Axon Instruments, Inc.) was used in the bridge configuration to inject current through the recording electrode. The intracellular data were recorded at low gain (x 10) and high gain (x 100) using a Vetter Model D instrumentation tape recorder for subsequent analysis. A DC offset circuit was employed to eliminate the membrane potential bias at the input of the high gain channel. Membrane potential values were determined by utilizing as a zero reference the DC potential that was recorded immediately after withdrawing the microelectrode from the cell. Motoneurons were identified by antidromic stimulation of their axons in peripheral nerves. The population of control motoneurons was distributed among the nerves as follows: CP (8), GL-S (6), HAM (18), SC (2). For the axotomized motoneurons the distribution was: GM (23), GL-S (9), SC (3) and Tib (7). Input resistance was determined by injecting small current pulses through the microelectrode (50 ms duration, _+ 1.4 nA) and recording the change in voltage. The data were digitized off-line and 30-50 samples for each current value were averaged on a Macintosh II computer using specially designed software. The cell's Rin was measured by the 'direct' method 1'2°'29. Values for Rin were obtained for each current injected and the Ri, for each motoneuron was defined as the average of the samples taken from the linear portion of the I - V curve near resting potential. Fig. 1 illustrates the responses obtained from representative control (A) and axotomized (B) aged motoneurons following the application of a depolarizing current pulse of 1 nA. The upper traces are single sweep responses, whereas the lower traces are the respective averaged responses from which Rin was measured. In the present study, we analyzed two time constants, namely: r b (the membrane time constant) and 3¢ (the first equalizing time constant24). They were obtained as described by Engelhardt et al. 7 based on methods repQrted by Ito and Oshima 16 and Zengel et al. 29. The voltage changes evoked by current pulses (50 ms, + 1-4 nA) were digitized and analyzed off-line. By means of specially designed software, the samples were averaged and the different time constants were obtained from the decay of the membrane potential that followed the cessation of the current pulse. Fig. 2A and B illustrate the process of analysis of time constants from the averaged voltage responses to 1 nA current pulses of representative
control (HAM) and axotomized (G-M) motoneurons, respectively. Time constants were estimated by selecting two extreme points of the linear portion of the respective semilogarithmic plots followed by a linear regression using all of the data between the selected values. As depicted in Figs. 2A2 and 2B2, 3 exponential terms, za (the slow process described by Ito and OshimaX6), r b and re, were extracted by .successively peeling the exponential term with the longest time constant from semilogarithmic plots of voltage or dV/dt vs time. The electrotonic length of the equivalent cylinder model of the motoneuron was estimated using the following formula: L = :t/[(rbh:c)-l] lr2 23
Total cell capacitance was estimated using the following formula:
C=n = rbLl[ R j a n h ( L )] This formula was first proposed by Gustafsson and Pinter 12. It uses parameters which are measured experimentally (Rin , l"b and to) and the parameter L which is calculated using zb and re. The diameter of the equivalent cylinder was calculated using formulas in Ral124. Rall's formula for the input resistance of a cylinder of length L was solved for diameter (d) using a value for intracellular resistivity (Ri) of 70 ~'~ cm 2'1. d = [(4 RmRi)(coth L)2/(:rRin)2] lr3 This value of d was used in the following formula to calculate the space constant (3.). 3` = [ ( R , , , d ) l ( 4 R i ) ]
1'2
This value for 3` was employed, together with the estimated value of L, to calculate the length (/) of the equivalent cylinder. l = L3` A.
Control
(~. Axotornized
Raw data /
\
:
~.,.~.-~..,.~.~
Averaged data
S\ ~
]
2 mv
50 ms
Fig. 1. Voltage responses from control (A) and axotomized (B) aged motoneurons to injected current steps. Digitized single sweeps (upper traces) were averaged (lower traces) and from these averaged responses the input resistance of motoneurons was measured. The parameters of the stimulating pulses were + 1 nA and 50 ms for both cells. The control motoneuron which belonged to the hamstring pool had an input resistance of 1.4 MQ. The axotomized cell was a medial gastrocnemius motoneuron; its input resistance was_ 2.9 MfL Data were digitized at 50 #s/bin. Lower traces are averages of 50 single sweeps.
302 The level of statistical significance between the means of the different variables was evaluated using the Student's t test and/or the Mann-Whitney U test. The criterion chosen to discard the null hypothesis was P < 0.05. The P values reported below are from the Mann-Whitney U test, unless otherwise specified.
A.
Control
B. A x o t o m i z e d
1. Input resistance
_ _ 1 1 mv 5 0 ms 2. T i m e constant analysis
0 4]
50
100
~b= 6.1ms
0 4]
50
100
lrb : 10.3 ms
c
4 1.....
25
50
rc= 0.6 ms
41
-1 / .....'i..... 0
0
25
50
"rc= 1.14 ms
-I j ................
2.5
.5
0 Time (ms)
2.5
Data were obtained from 34 unaxotomized motoneurons and 42 axotomized motoneurons. All motoneurons selected for this study exhibited action potential amplitudes which were greater than 60 mV. Of the 34 control cells, 91% were classified as being type F motoneurons on the basis of their A H P duration 7'29. Therefore, in this study, we will be comparing the present results with comparable data from adult and aged type F motoneurons. There was a 4.4 mV difference between the mean resting potentials of control and axotomized motoneurons (-67.3 + 1.4 mV vs -62.9 + 1.5 mV, respectively). This difference was statistically significant according to the Student's t test (P < 0.05), although it was not statistically significant according to the Mann-Whitney U test. The means of + S.E.M.s of the electrophysiological data which are the focus of this study are presented in Table I in the order in which they are reported below. Since these data are pooled, individual values of the measured electrophysiological parameters and the calculated electrotonic length from a representative sample of motoneurons are presented in Table II. Input resistance
o
0
RESULTS
5
Fig. 2. Input resistance study of control and axotomized aged motoneurons (A1 and B1). The control cell belonged to the hamstring pool; its input resistance was 1.8 MQ. The axotomized motoneuron belonged to the medial gastrocnemius pool and its input resistance was 2.4 Mfl. Note that the bridges were slightly unbalanced for the control and axotomized cells. The parameters of the current pulse were + 1 nA and 50 ms for both cells. Data were digitized at 50 /xs/bin. The traces are averages of 50 single sweeps. Time constant analysis for the same control (left) and axotomized (right) motoneurons is illustrated in A2 and B2. The upper traces are semilogarithmic plots of the In (-voltage) vs time of the undershoot that followed the cessation of the current pulse. Note that the polarity of these plots is reversed. From these plots the slow process exponential was obtained. When this slow process exponential function was peeled from the raw data, a second exponential term was obtained which is depicted in the middle traces. The time constant of this exponential term corresponds to the membrane time constant (rb). Finally, the peeling of the latter exponential resulted in a third exponential function (bottom traces) from which the equalizing time constant (re) was obtained. The vertical dashed lines in each plot indicate the extreme points of the linear portion selected for the peeling process of the exponential terms. As shown for the representative motoneurons of Fig. 2B, % and r c increased following axotomy. For further details see Materials and Methods.
The m e a n Rin increased 127% following axotomy (1.1 + 0.1 M ~ vs 2.5 + 0.2 Mr2, control and axotomized motoneurons, respectively). Examples of the axotomyinduced change in Rin are illustrated in the recordings of Figs. 1 and 2A. The frequency distribution of Rin from control and axotomized motoneurons are presented in Fig. 3. Following axotomy, the entire distribution was shifted to the right by approximately 1.5 ME. Quantitatively similar increases have been reported to occur after axotomy in motoneurons of presumed adult cats 9'11, Membrane time constant
Axotomized aged motoneurons exhibited longer mem-
TABLE I
Summary of motoneuron properties Control 1.1+ 0.1 (34) Input resistance (Mr2)* Membrane time constant (ms)* 6.5+ 0.3 (34) Equalizing time constant (ms)* 0.7+ 0.05 (30) 1.1+ 0.03 (30) Eletrotonic length (2) 9.5+ 0.8 (30) Total cell capacitance (nF)** Equivalent cylinder diameter 54.2+ 0.3 (30) Cum)* Equivalent cylinder length 2.6+ 0.01 (30) (mm)
Axotomized 2.5+ 9.6+ 1.1+ 1.1+ 6.0+
0.2 0.4 0.08 0.04 0.4
35.0+ 0.1
(42) (40) (40) (40) (40) (40)
2.7+ 0.01 (40)
Values are means + S.E.M.'s. Numbers in parentheses indicate sample size. *P < 0.0001; **P < 0.0005.
303 TABLE II Data for a representative sample o f motoneurons Values of input resistance, membrane time constant, equalizing time constant and the calculated value of the electrotonic length for 10 representative control and axotomized aged motoneurons. The values of the electrotonic length were calculated according to the equation: L = :r/[(%/rc)-l] lt2. Control
Axotomized
Cell (CA)
R i. (M~)
% (ms)
rc (ms)
L (2)
Cell (CA)
R i. (Mf~)
rb (ms)
rc (ms)
L (2)
1 2 3 4 5 6 7 8 9 10
0.8 1.8 1.1 1.1 0.8 1.5 0.8 0.7 0.5 0.9
5.0 5.8 8.8 6.1 4.2 6.1 8.1 5.3 3.5 5.6
0.9 0.5 0.6 0.7 0.6 0.4 0.9 0.6 0.4 0.6
1.5 1.0 0.9 1.1 1.3 0.9 1.1 1.1 1.1 1.1
1 2 3 4 5 6 7 8 9 10
2.0 3.0 3.6 1.6 2.1 2.5 1.7 3.7 2.0 3.5
7.9 8.4 10.5 7.1 8.4 10.9 8.6 12.7 6.6 10.8
1.1 1.2 1.0 0.7 0.9 1.2 1.2 1.2 0.7 0.8
1.3 1.3 1.0 1.0 1.1 1.1 1.3 1.0 1.0 0.9
brane time constants than control aged motoneurons (Fig, 2B, middle traces). Distribution histograms of the values obtained for % are presented in Fig. 4. There was a 48% increase in the mean membrane time constant following axotomy (6.5 + 0.3 ms, vs 9.6 + 0.4 ms,
A.
control and axotomized cells, respectively). If we assume that the specific membrane capacitance is constant, the observed increase in % can be interpreted as indicating a similar percentage increase in the specific membrane resistance of axotomized cells.
Control
A
Control
12
16. 12.
8 8' 4 4. o
0
0
2
4
c 0
8
12
1'6
2'0
16
20
E B.
Axotomized
Axotomized
16
e~
Z
8 8 4
4. 0
~ 0
2
4
O
~ 6
g
Input resistance (M.Q)
Fig. 3. Frequency distribution of input resistance in control (A) and axotomized (B) motoneurons. The arrows indicate the mean values of each population (1.1 MQ and 2.5 MQ for the control and axotomized cells, respectively). Further statistical details in the text and in Table I.
0
,
.
,
4
8
12
Membrane time constant (ms)
Fig. 4. Frequency distribution of the membrane time constant in control (A) and axotomized (B) motoneurons. The arrows point to the mean values of each histogram (6.5 ms and 9.6 ms for control and axotomized cells, respectively). Additional statistical details in the text and in Table I.
304
Equalizing time constant
Total cell capacitance
The mean r~ increased 57% following axotomy (0.7 + 0.05 ms vs 1.1 + 0.8 ms, control and axotomized cells, respectively). Fig. 2B, bottom traces, shows an example of this axotomy-induced change in r~. This time constant is related to the redistribution of the injected charge along the soma-dendritic tree of the cell and its alterations after axotomy may indicate changes in the geometry and/or specific resistances of the cell membrane or cytoplasm24.
The mean total cell capacitance decreased by 37% after axotomy (9.5 + 0.8 nF vs 6.0 + 0.4 nF, control and axotomized cells, respectively). Distribution histograms of Cce. estimates are presented in Fig. 5. The mode of the distribution shifts to smaller values of total cell capacitance following axotomy. The decrease in Cecil is similar to that reported by others in adult cats 13. The 37% reduction in total cell capacitance that is observed following axotomy can be used to estimate the change in cell surface area if one assumes that the membrane capacitance is constant 12. The loss of cell surface area would be expected to increase the cell input resistance, but the magnitude of this contribution would depend on which portions of the soma-dendritic tree were affected241
Electrotonic length In spite of the major changes in other cell electrical properties, the electrotonic length o f the motoneuron remained remarkably stable forlowing axotomy (1.1 _+ 0.03 2 vs 1.1 + I}.04 2, control and axotomized motoneurons, respectively). This stability in L, which is a function of rt,/r¢, reflects the fact that this ratio decreased by only 8% following axotomy. The increase in r b described above, accompanied by the constant electrotonic length, implies that axotomized motoneurons in aged cats undergo geometrical (shape) changes in their somadendritic tree that are similar to those previously reported for axotomized motoneurons in adult cats 13.
Equivalent cyfinder diameter and length The equivalent cylinder diameter (see Materials and Methods) decreased by 35% following axotomy (54.2 + 0.3/~m vs 35.0 + 0.1 #m, control and axotomized motoneurons, respectively). However, axotomy did not produce a significant change in l. These results are presented in the scattergrams of Fig. 6. Note that the data points
A.
Control
5 A.
Control
4
16
O0 0 0 O~
3
12,
2
8
1
0
00
0 0 0
0
O00
0
o
0 o
o
o
-7 0
5
10
15
20
25
30
2'0
4'0
6'0
80
160
6'0
~o
~8o
E
==
O
E
B.
B.
Axotomized
Axotomized
5.
b
.13
16.
4.
•
E Z
12,
3-
~lm
8. 1
4,
•
0 0
o
s
~o
~5
2b
~
3'0
Ccell (nF)
Fig. 5. Frequency distribution of the calculated total cell capacitance in control (A) and axotomized (B) motoneurons. The arrows point to the mean values of each histogram (9.5 nF and 6.0 nF for control and axotomized cells, respectively). Additional statistical details in the text and in Table I.
o
2'0
4'0
Diameter (#m)
Fig. 6. Scattergrams illustrating the relationship between the diameter and the length of the equivalent cylinder of control (A) and axotomized (B) motoneurons. Note that while the length appears unaffected after axotomy, the diameters from axotomized motoneurons shift to the left and cluster in a narrower range compared to that exhibited by control motoneurons.
305 from axotomized motoneurons are clustered within a narrower range of diameters than that exhibited by the data from control cells.
Rm11'13'22. In order to test this hypothesis, we calculated the electrotonic length and the total cell capacitance of motoneurons.
DISCUSSION
Electrotonic length Following axotomy, L was unchanged in aged cat motoneurons. This conservation of L was associated with a large increase in r b. These results indicate that there were changes in the soma-dendritic geometry of aged motoneurons following axotomy x3'22.
The results presented here demonstrate that aged cat motoneurons exhibited significant alterations in their passive electrical properties following axotomy. We will discuss the changes in each parameter and compare them to the alterations induced by axotomy in adult cat motoneu-
rons9,11,13,17,22. Input resistance In the present study the mean Rin of the control aged motoneurons was 1.1 Mf~, which is similar to the value reported by Engelhardt et al. 7 for Type F motoneurons in the aged cat. Following axotomy, the mean Rin increased to 2.5 MQ (127%). This increase can be caused by changes in certain electrical properties of the motoneuron 3. To a first approximation, we can model the motoneuron's passive electrical properties with an equivalent cylinder model in which the input resistance is given by the following equation: Rin -------(Rm/An)[L[tanh(L)] (ref. 2) where Rm is the specific membrane resistance, L is the electrotonic length of the equivalent cylinder, and A n is the total membrane area. The variables in the above equation that can affect the input resistance of the cell are Rm, A n and the electrotonic length. In an effort to determine the relative contribution of these variables to the observed increase in input resistance, we measured the membrane time constant and calculated the electrotonic length and total cell capacitance of the motoneurons.
Membrane time constant The membrane time constant is directly proportional to the product of the R m and the membrane capacitance (Cm). The membrane capacitance is assumed to be a constant under the conditions of the present experiment, therefore any changes observed in rb are taken as reflecting proportional changes in R m. The 48% increase in rb found in the old cat axotomized motoneurons is therefore believed to reflect a proportional increase in the R m of aged motoneurons following axotomy. Although large, the magnitude of the increase in R m cannot account by itself for the 127% increase in input resistance. It is therefore likely that aged motoneurons react to axotomy with changes in cell surface area and/or geometry in addition to the observed increase in
Total cell capacitance and cell geometry Total cell capacitance was calculated because this electrical property should vary in direct proportion with the total surface area of the motoneuron assuming that the membrane capacitance is constant 12. Estimated values of motoneuron surface area obtained from C ~ calculation 7'12 have been shown to agree well with surface area data reported from anatomical studies. The 37% decrease in Co:n indicates that axotomized motoneurons in aged cats have lost 37% of their soma-dendritic membrane. Furthermore, calculations based on cable theory allow one to determine the length and diameter of the cylinder model which is equivalent to the soma-dendritic tree of the aged motoneurons (see Materials and Methods). These calculations indicate that almost all of the changes in surface area are attributable to a 35% decrease in diameter of the equivalent cylinder (see Fig. 6). These results are consistent with axotomy-induced retraction of dendritic branches which is similar to that which occurs in brainstem motoneurons 25. In addition, the present data suggest that the population of axotomized aged motoneurons is rather homogeneous in size because the diameter distribution is compressed following axotomy. The relation between the geometric and total surface area changes in the soma-dendritic tree (which explain part of the reduction in Rin ) and those in the axon (which explain the reduction in axonal conduction velocity observed by Engelhardt et al. 8 in the same population of motoneurons) is not known. However, neurofilament content and interneurofilament spacing have been related to axonal diameter in myelinated fibers ~4'~8, which is an important determinant of axonal conduction velocity. Neurofilament synthesis and the axonal content of neurofilaments have also been shown to be reduced following axotomy ~°'15'28. On the other hand, the geometry and size of dendrites have been linked to their content of microtubules, membrane related organelles and microtubule-associated proteins (MAPs), but not with their neurofilament content 4'2~'27. It is therefore possible that axotomy-induced changes in the synthesis and/or content of these cytoskeletal components are responsible for the altered geometry of the cell's soma-dendritic tree.
306 A s d e t e r m i n e d by e l e c t r o p h y s i o l o g i c a l m e a s u r e m e n t s ,
physiological p r o p e r t i e s as a result of the aging process.
m o t o n e u r o n s in a g e d cats r e s p o n d to a x o t o m y to an e x t e n t that is similar to that which takes place in m o t o n e u r o n s in adult cats in spite of the fact that m a j o r changes h a v e already o c c u r r e d in t h e s e s a m e electro-
REFERENCES 1 Barret, J.N. and Crill, W.E., Specific membrane properties of cat motoneurons, J. Physiol., 239 (1974) 301-325. 2 Burke, R.E., Motor units: anatomy, physiology and functional organization. In Handbook of Physiology. The Nervous System. Motor Control, Vol. 2, Sect. 1, Am. Physiol. Soc., Bethesda, Maryland, 1981, pp. 345-422. 3 Burke, R.E. and Rudomin, P., Spinal neurons and synapses. In Handbook of Physiology. The Nervous System. Cellular Biology of Neurons. Vol. 1, Part 2, Am. Physiol. Soc., Bethesda, Maryland, 1977, pp. 877-944. 4 C~iceres, A., Busciglio, J., Ferreira, A. and Steward, O., An immunocytochemical and biochemical study of the microtubuleassociated protein MAP-2 during post-lesion dendritic remodelling in the central nervous system of adult rats, Mol. Brain Res., 3 (1988) 233-246. 5 Cajal, S. and Ramon, Y., Histologie du systdme nerveux de l'homme et des vertdbrds, Vol. 1, Maloine, Paris, 1909. 6 Eccles, J.C., Libet, B. and Young, R.R., The behavior of chromatolysed motoneurons studied by intracellular recording, J. Physiol., 143 (1958) 11-40. 7 Engelhardt, J.K., Morales, ER., Yamuy, J. and Chase, M.H., Cable properties of spinal cord motoneurons in adult and aged cats, J. Neurophysiol., 61 (1989) 194-201. 8 Engelhardt, J.K., Yamuy, J., Morales, ER. and Chase, M.H., Changes in electrophysioiogical properties of aged cat lumbar motoneurons following axotomy, Soc. Neurosci. Abstr., 16 (1990) 843. 9 Foehring, R.C., Sypert, G.W. and Munson, J.B., Properties of self-reinnervated motor units of medial gastrocnemius of cat. II. Axotomized motoneurons and time course of recovery, J. Neurophysiol., 55 (1986) 947-965. 10 Goldstein, M.E., Weiss, S.R., Lazzarini, R.A., Schneidman, ES., Lees, J.E and Schlaepfer, W.W., mRNA levels of all three neurofilament proteins decline following nerve transection, Mol. Brain Res., 3 (1988) 287-292. 11 Gustafsson, B., Changes in motoneurone electrical properties following axotomy, J. Physiol., 293 (1979) 197-215. 12 Gustafsson, B. and Pinter, M.J., Relations among passive electrical properties of lumbar a-motoneurons of the cat, J. Physiol., 356 (1984) 401-431. 13 Gustafsson, B. and Pinter, M.J., Effects of axotomy on the distribution of passive electrical properties of cat motoneurons, J. Physiol., 356 (1984) 433-442. 14 Hoffman, EN., Cleveland, D.W., Griffin, J.W., Landes, P.W., Cowan, N.J. and Price, D.L., Neurofilament gene expression: a major determinant of axonal caliber, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 3472-3476. 15 Hoffrnan, P.N., Thompson, G.W., Griffin, J.W. and Price, D.L.,
Acknowledgements. This research was supported by USPHS Grant AGO4307.
Changes in neurofilament transport coincide temporally with alterations in the caliber of axons in regenerating motor fibers, Z Cell Biol., 101 (1985) 1332-1340. 16 Ito, M. and Oshima, T., Electrical behavior of the motoneurone membrane during intracellularly applied current steps, J. Physiol., 180 (1965) 607-635. 17 Kuno, M., Miyata, Y. and Nufioz-Martinez, E.J., Differential reaction of fast and slow a-motoneurons to axotomy, J. Physiol., 240 (1974) 725-739. 18 Lasek, R.J., Oblinger, M.M. and Drake, P.F., Molecular biology of neuronal geometry: expression of neurofilament genes influences axonal diameter, Cold Spring Harb. Syrup. Quant. Biol., 48 (1983) 731-744. 19 Lieberman, A.R., The axon reaction: a review of the principal features of perikaryal responses to axon injury, Int. Rev. Neurobiol., 14 (1971) 49-124. 20 Morales, ER., Boxer, P.A., Fung, S.J. and Chase, M.H., Basic electrophysiological properties of spinal cord motoneurons during old age in the cat, J. Neurophysiol., 58 (1987) 180-194. 21 Parhad, I.M., Clark, A.W. and Griffin, J.W., The effects of changing of neurofilament content on caliber of small axons. The fl,fl'-imminodipropionitrile (IDPN) model, J. Neurosci., 7 (1987) 2256-2263. 22 Pinter, M.J. and Vanden Noven, S., Effects of preventing reinnervation on axotomized spinal motoneurons in the cat. I. Motoneurons electrical properties, J. Neurophysiol., 62 (1989) 311324. 23 Rail, W., Time constants and electrotonic length of membrane cylinders and neurons, Biophys. J., 9 (1969) 1483-1508. 24 Rail, W., Core conductor theory and cable properties of neurons. In Handbook of Physiology. The Nervous System. Cellular Biology of Neurons. Vol. 1, Part 1, Am. Physiol. Soc. Bethesda, Maryland, 1977, p. 39-97. 25 Sumner, B.E.H. and Watson, W.E., Retraction and expansion of dendritic trees of motoneurones of adult rats induced in vivo, Nature, 233 (1971) 273-275. 26 Titmus, M.J. and Faber, D.S., Axotomy-induced alterations in the electrophysiological characteristics of neurons, Prog. Neurobiol., 35 (1990) 1-51. 27 Tucker, R.P., The roles of microtubule-associated proteins in brain morphogenesis: a review, Brain Res. Rev., 15 (1990) 101120. 28 Wong, J.K. and Oblinger, M.M., Changes in neurofilament gene expression occur after axotomy of dorsal root ganglion neurons: an in situ hybridization study, Metabol. Brain Dis., 2 (1987) 291-303. 29 Zengel, J.E., Reid, S.A., Sypert, G.W. and Munson, J.B., Membrane electrical properties and prediction of motor-unit type of medial gastrocnemius motoneurons in the cat, J. Neurophysiol., 53 (1985) 1323-1344.
Brain Research, 570 (1992) 300-306 O 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17388
Passive electrical properties of motoneurons in aged cats following axotomy Jack Yamuy, John K. Engelhardt, Francisco R. Morales and Michael H. Chase Department of Physiology, Department of Anatomy and Cell Biology and the Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.) (Accepted 17 September 1991)
Key words: Aged; Cat; Motoneuron; Axotomy; Electrophysiology
The objective of this study was to determine whether the aging process influences the changes in the electrophysiological properties of motoneurons that occur as a consequence of axotomy. Accordingly, using intracellular recording and stimulating techniques, the basic electrical properties of control (unaxotomized) and axotomized spinal cord motoneurons of aged cats were determined. Compared with control motoneurons, axotomized motoneurons exhibited increases in input resistance (Ri=), membrane time constant (rb) and the equalizing time constant (re). While the electrotonic length (L) remained unchanged, axotomy induced a decrease in the total cell capacitance (Cceu). The post-axotomy reduction of C¢~n indicates that the motoneuron surface area was reduced and the increased membrane time constant indicates that there was an increase in membrane resistivity (Rm). The post-axotomy conservation of L accompanied by an increase in R m suggests that aged axotomized motoneurons undergo geometrical changes. Furthermore, calculations based on cable theory suggest that the diameter of the equivalent cylinder (d) decreased following axotomy, whereas the equivalent cylinder length (1) remained unaffected. It is concluded that axotomy produces significant alterations in the soma-dendritic portion of aged spinal motoneurons, as indicated by the changes found in their passive electrophysiological properties, and that the pattern of the response that occurs in axotomized motoneurons of adult cats is also present in axotomized motoneurons of aged animals. INTRODUCTION
rons. It was c o n c l u d e d that t h e r e was an a g e - d e p e n d e n t increase in m e m b r a n e resistance and an a g e - d e p e n d e n t
It has long b e e n k n o w n that a x o t o m y alters the m o r -
d e c r e a s e in cell surface a r e a of old cat m o t o n e u r o n s .
p h o l o g y and physiology of n e u r o n s 5'6. Spinal c o r d mo-
B e c a u s e a x o t o m y is k n o w n to p r o d u c e changes in these
t o n e u r o n s , particularly of the cat, h a v e b e e n extensively
basic electrical p r o p e r t i e s of m o t o n e u r o n s , we wished to
investigated after chronic axonal section (for r e v i e w see
e x a m i n e the e x t e n t to which the aging process impacts on
T i t m u s and Faber26). T h e y r e s p o n d to a x o t o m y with a
the r e a c t i o n of aged m o t o n e u r o n s to a x o t o m y . Prelimi-
series of changes classically k n o w n as the ' a x o n reac-
nary d a t a h a v e b e e n p u b l i s h e d as an abstract 8.
tion '~9. A m o n g the c h a n g e s o b s e r v e d are increases in input resistance (Rin) and m e m b r a n e t i m e c o n s t a n t (rb) 9'11. A x o t o m i z e d m o t o n e u r o n s also s h o w a d e c r e a s e in the calculated v a l u e of total cell c a p a c i t a n c e (Cce,) which has b e e n i n t e r p r e t e d as a d e c r e m e n t in the total surface a r e a of the cell 13'22. T h e s e changes in passive
MATERIALS AND METHODS
Animals Five cats, ranging in age from 13 to 16.5 years, were utilized in this study. All animals were in good health at the time of the experiment as determined by the UCLA Division of Laboratory Animal Medicine.
electrical p r o p e r t i e s h a v e led different authors to prop o s e that a x o t o m i z e d m o t o n e u r o n s u n d e r g o an increase in m e m b r a n e resistance and a d e c r e a s e in cell size which are a c c o m p a n i e d by changes in cell g e o m e t r y 1~-13'22. R e c e n t l y , the basic electrical p r o p e r t i e s of m o t o n e u rons f r o m aged cats w e r e d e s c r i b e d 7'2°. C o m p a r e d with a control p o p u l a t i o n of adult cat m o t o n e u r o n s , aged cat m o t o n e u r o n s e x h i b i t e d increases in Rin and rb, while Ccell was r e d u c e d . N o significant differences w e r e f o u n d bet w e e n resting potentials of adult and old cat m o t o n e u -
Surgical procedures Axotomy was performed while the animals were anesthetized with halothane. A small incision was made in the popliteal fossa of the left hind-limb and the nerve to the gastrocnemius medialis muscle (GM) and the tibialis (Tib) nerve were dissected, ligated at their distal end, and cut. In one cat the gastrocnemius lateralis and soleus (GL-S) nerves were also cut distally. In addition, the following procedures were performed in order to avoid subsequent reinnervation. The silk from the ligature, along with the distal 3-4 mm of the nerve, was threaded through a 5 mm polyethylene tube. Each nerve was fixed inside its tube by sealing the end of the tube and the thread with a hot glass rod. Afterwards, using the same
Correspondence: J. Yamuy, Brain Research Institute, UCLA Center for the Health Sciences, 43-367 CHS, Los Angeles, CA 90024-1751, U.S.A. Fax: (1) (213) 206-3499.
301 thread, the tube was anchored to the overlying fat in the popliteal fossa approximately 2 cm from the nerve's target muscle. The surgical wound was closed with a skin suture and antibiotic ointment was applied. Prophylactic antibiotics were administered for 7 days after surgery. After a period of 43 days (4 cats) or 54 days (1 cat), the animals were anesthetized with pentobarbital (30 mg/kg) and prepared for intraceUular recording from lumbar motoneurons. The trachea was cannulated and the right carotid artery and external jugular vein were catheterized. The arterial blood pressure and end-tidal pCO 2 were monitored continuously and maintained within physiological limits. The skin along the posterior surface of the left hindlimb was incised and the formerly axotomized nerves (GM, Tib, GL-S) were individualized and dissected centrally. The following nerves were then dissected and excised distally: hamstring (HAM), GL-S (in the animals in which it was not axotomized) and common peroneal (CP). These nerves, including the trunk of the sciatic, were then placed on stimulating electrodes. A laminectomy was performed from vertebra L4-LT; the dura was then cut and retracted and the left dorsal roots Ls-S 1 were cut distally. Spinal and hindlimb pools were constructed with skin flaps and filled with warm mineral oil. An infrared lamp and heating pad were used to keep the animal and mineral oil pools between 38°C and 390C.
Recording and analysis Intracellular recordings were obtained using broken-tip glass micropipette electrodes filled with 3 M KC1 (1-1.5 #m, approximate tip diameter). The resistance of the electrodes ranged from 4 to 12 megohms. An Axoclamp-2A amplifier (Axon Instruments, Inc.) was used in the bridge configuration to inject current through the recording electrode. The intracellular data were recorded at low gain (x 10) and high gain (x 100) using a Vetter Model D instrumentation tape recorder for subsequent analysis. A DC offset circuit was employed to eliminate the membrane potential bias at the input of the high gain channel. Membrane potential values were determined by utilizing as a zero reference the DC potential that was recorded immediately after withdrawing the microelectrode from the cell. Motoneurons were identified by antidromic stimulation of their axons in peripheral nerves. The population of control motoneurons was distributed among the nerves as follows: CP (8), GL-S (6), HAM (18), SC (2). For the axotomized motoneurons the distribution was: GM (23), GL-S (9), SC (3) and Tib (7). Input resistance was determined by injecting small current pulses through the microelectrode (50 ms duration, _+ 1.4 nA) and recording the change in voltage. The data were digitized off-line and 30-50 samples for each current value were averaged on a Macintosh II computer using specially designed software. The cell's Rin was measured by the 'direct' method 1'2°'29. Values for Rin were obtained for each current injected and the Ri, for each motoneuron was defined as the average of the samples taken from the linear portion of the I - V curve near resting potential. Fig. 1 illustrates the responses obtained from representative control (A) and axotomized (B) aged motoneurons following the application of a depolarizing current pulse of 1 nA. The upper traces are single sweep responses, whereas the lower traces are the respective averaged responses from which Rin was measured. In the present study, we analyzed two time constants, namely: r b (the membrane time constant) and 3¢ (the first equalizing time constant24). They were obtained as described by Engelhardt et al. 7 based on methods repQrted by Ito and Oshima 16 and Zengel et al. 29. The voltage changes evoked by current pulses (50 ms, + 1-4 nA) were digitized and analyzed off-line. By means of specially designed software, the samples were averaged and the different time constants were obtained from the decay of the membrane potential that followed the cessation of the current pulse. Fig. 2A and B illustrate the process of analysis of time constants from the averaged voltage responses to 1 nA current pulses of representative
control (HAM) and axotomized (G-M) motoneurons, respectively. Time constants were estimated by selecting two extreme points of the linear portion of the respective semilogarithmic plots followed by a linear regression using all of the data between the selected values. As depicted in Figs. 2A2 and 2B2, 3 exponential terms, za (the slow process described by Ito and OshimaX6), r b and re, were extracted by .successively peeling the exponential term with the longest time constant from semilogarithmic plots of voltage or dV/dt vs time. The electrotonic length of the equivalent cylinder model of the motoneuron was estimated using the following formula: L = :t/[(rbh:c)-l] lr2 23
Total cell capacitance was estimated using the following formula:
C=n = rbLl[ R j a n h ( L )] This formula was first proposed by Gustafsson and Pinter 12. It uses parameters which are measured experimentally (Rin , l"b and to) and the parameter L which is calculated using zb and re. The diameter of the equivalent cylinder was calculated using formulas in Ral124. Rall's formula for the input resistance of a cylinder of length L was solved for diameter (d) using a value for intracellular resistivity (Ri) of 70 ~'~ cm 2'1. d = [(4 RmRi)(coth L)2/(:rRin)2] lr3 This value of d was used in the following formula to calculate the space constant (3.). 3` = [ ( R , , , d ) l ( 4 R i ) ]
1'2
This value for 3` was employed, together with the estimated value of L, to calculate the length (/) of the equivalent cylinder. l = L3` A.
Control
(~. Axotornized
Raw data /
\
:
~.,.~.-~..,.~.~
Averaged data
S\ ~
]
2 mv
50 ms
Fig. 1. Voltage responses from control (A) and axotomized (B) aged motoneurons to injected current steps. Digitized single sweeps (upper traces) were averaged (lower traces) and from these averaged responses the input resistance of motoneurons was measured. The parameters of the stimulating pulses were + 1 nA and 50 ms for both cells. The control motoneuron which belonged to the hamstring pool had an input resistance of 1.4 MQ. The axotomized cell was a medial gastrocnemius motoneuron; its input resistance was_ 2.9 MfL Data were digitized at 50 #s/bin. Lower traces are averages of 50 single sweeps.
302 The level of statistical significance between the means of the different variables was evaluated using the Student's t test and/or the Mann-Whitney U test. The criterion chosen to discard the null hypothesis was P < 0.05. The P values reported below are from the Mann-Whitney U test, unless otherwise specified.
A.
Control
B. A x o t o m i z e d
1. Input resistance
_ _ 1 1 mv 5 0 ms 2. T i m e constant analysis
0 4]
50
100
~b= 6.1ms
0 4]
50
100
lrb : 10.3 ms
c
4 1.....
25
50
rc= 0.6 ms
41
-1 / .....'i..... 0
0
25
50
"rc= 1.14 ms
-I j ................
2.5
.5
0 Time (ms)
2.5
Data were obtained from 34 unaxotomized motoneurons and 42 axotomized motoneurons. All motoneurons selected for this study exhibited action potential amplitudes which were greater than 60 mV. Of the 34 control cells, 91% were classified as being type F motoneurons on the basis of their A H P duration 7'29. Therefore, in this study, we will be comparing the present results with comparable data from adult and aged type F motoneurons. There was a 4.4 mV difference between the mean resting potentials of control and axotomized motoneurons (-67.3 + 1.4 mV vs -62.9 + 1.5 mV, respectively). This difference was statistically significant according to the Student's t test (P < 0.05), although it was not statistically significant according to the Mann-Whitney U test. The means of + S.E.M.s of the electrophysiological data which are the focus of this study are presented in Table I in the order in which they are reported below. Since these data are pooled, individual values of the measured electrophysiological parameters and the calculated electrotonic length from a representative sample of motoneurons are presented in Table II. Input resistance
o
0
RESULTS
5
Fig. 2. Input resistance study of control and axotomized aged motoneurons (A1 and B1). The control cell belonged to the hamstring pool; its input resistance was 1.8 MQ. The axotomized motoneuron belonged to the medial gastrocnemius pool and its input resistance was 2.4 Mfl. Note that the bridges were slightly unbalanced for the control and axotomized cells. The parameters of the current pulse were + 1 nA and 50 ms for both cells. Data were digitized at 50 /xs/bin. The traces are averages of 50 single sweeps. Time constant analysis for the same control (left) and axotomized (right) motoneurons is illustrated in A2 and B2. The upper traces are semilogarithmic plots of the In (-voltage) vs time of the undershoot that followed the cessation of the current pulse. Note that the polarity of these plots is reversed. From these plots the slow process exponential was obtained. When this slow process exponential function was peeled from the raw data, a second exponential term was obtained which is depicted in the middle traces. The time constant of this exponential term corresponds to the membrane time constant (rb). Finally, the peeling of the latter exponential resulted in a third exponential function (bottom traces) from which the equalizing time constant (re) was obtained. The vertical dashed lines in each plot indicate the extreme points of the linear portion selected for the peeling process of the exponential terms. As shown for the representative motoneurons of Fig. 2B, % and r c increased following axotomy. For further details see Materials and Methods.
The m e a n Rin increased 127% following axotomy (1.1 + 0.1 M ~ vs 2.5 + 0.2 Mr2, control and axotomized motoneurons, respectively). Examples of the axotomyinduced change in Rin are illustrated in the recordings of Figs. 1 and 2A. The frequency distribution of Rin from control and axotomized motoneurons are presented in Fig. 3. Following axotomy, the entire distribution was shifted to the right by approximately 1.5 ME. Quantitatively similar increases have been reported to occur after axotomy in motoneurons of presumed adult cats 9'11, Membrane time constant
Axotomized aged motoneurons exhibited longer mem-
TABLE I
Summary of motoneuron properties Control 1.1+ 0.1 (34) Input resistance (Mr2)* Membrane time constant (ms)* 6.5+ 0.3 (34) Equalizing time constant (ms)* 0.7+ 0.05 (30) 1.1+ 0.03 (30) Eletrotonic length (2) 9.5+ 0.8 (30) Total cell capacitance (nF)** Equivalent cylinder diameter 54.2+ 0.3 (30) Cum)* Equivalent cylinder length 2.6+ 0.01 (30) (mm)
Axotomized 2.5+ 9.6+ 1.1+ 1.1+ 6.0+
0.2 0.4 0.08 0.04 0.4
35.0+ 0.1
(42) (40) (40) (40) (40) (40)
2.7+ 0.01 (40)
Values are means + S.E.M.'s. Numbers in parentheses indicate sample size. *P < 0.0001; **P < 0.0005.
303 TABLE II Data for a representative sample o f motoneurons Values of input resistance, membrane time constant, equalizing time constant and the calculated value of the electrotonic length for 10 representative control and axotomized aged motoneurons. The values of the electrotonic length were calculated according to the equation: L = :r/[(%/rc)-l] lt2. Control
Axotomized
Cell (CA)
R i. (M~)
% (ms)
rc (ms)
L (2)
Cell (CA)
R i. (Mf~)
rb (ms)
rc (ms)
L (2)
1 2 3 4 5 6 7 8 9 10
0.8 1.8 1.1 1.1 0.8 1.5 0.8 0.7 0.5 0.9
5.0 5.8 8.8 6.1 4.2 6.1 8.1 5.3 3.5 5.6
0.9 0.5 0.6 0.7 0.6 0.4 0.9 0.6 0.4 0.6
1.5 1.0 0.9 1.1 1.3 0.9 1.1 1.1 1.1 1.1
1 2 3 4 5 6 7 8 9 10
2.0 3.0 3.6 1.6 2.1 2.5 1.7 3.7 2.0 3.5
7.9 8.4 10.5 7.1 8.4 10.9 8.6 12.7 6.6 10.8
1.1 1.2 1.0 0.7 0.9 1.2 1.2 1.2 0.7 0.8
1.3 1.3 1.0 1.0 1.1 1.1 1.3 1.0 1.0 0.9
brane time constants than control aged motoneurons (Fig, 2B, middle traces). Distribution histograms of the values obtained for % are presented in Fig. 4. There was a 48% increase in the mean membrane time constant following axotomy (6.5 + 0.3 ms, vs 9.6 + 0.4 ms,
A.
control and axotomized cells, respectively). If we assume that the specific membrane capacitance is constant, the observed increase in % can be interpreted as indicating a similar percentage increase in the specific membrane resistance of axotomized cells.
Control
A
Control
12
16. 12.
8 8' 4 4. o
0
0
2
4
c 0
8
12
1'6
2'0
16
20
E B.
Axotomized
Axotomized
16
e~
Z
8 8 4
4. 0
~ 0
2
4
O
~ 6
g
Input resistance (M.Q)
Fig. 3. Frequency distribution of input resistance in control (A) and axotomized (B) motoneurons. The arrows indicate the mean values of each population (1.1 MQ and 2.5 MQ for the control and axotomized cells, respectively). Further statistical details in the text and in Table I.
0
,
.
,
4
8
12
Membrane time constant (ms)
Fig. 4. Frequency distribution of the membrane time constant in control (A) and axotomized (B) motoneurons. The arrows point to the mean values of each histogram (6.5 ms and 9.6 ms for control and axotomized cells, respectively). Additional statistical details in the text and in Table I.
304
Equalizing time constant
Total cell capacitance
The mean r~ increased 57% following axotomy (0.7 + 0.05 ms vs 1.1 + 0.8 ms, control and axotomized cells, respectively). Fig. 2B, bottom traces, shows an example of this axotomy-induced change in r~. This time constant is related to the redistribution of the injected charge along the soma-dendritic tree of the cell and its alterations after axotomy may indicate changes in the geometry and/or specific resistances of the cell membrane or cytoplasm24.
The mean total cell capacitance decreased by 37% after axotomy (9.5 + 0.8 nF vs 6.0 + 0.4 nF, control and axotomized cells, respectively). Distribution histograms of Cce. estimates are presented in Fig. 5. The mode of the distribution shifts to smaller values of total cell capacitance following axotomy. The decrease in Cecil is similar to that reported by others in adult cats 13. The 37% reduction in total cell capacitance that is observed following axotomy can be used to estimate the change in cell surface area if one assumes that the membrane capacitance is constant 12. The loss of cell surface area would be expected to increase the cell input resistance, but the magnitude of this contribution would depend on which portions of the soma-dendritic tree were affected241
Electrotonic length In spite of the major changes in other cell electrical properties, the electrotonic length o f the motoneuron remained remarkably stable forlowing axotomy (1.1 _+ 0.03 2 vs 1.1 + I}.04 2, control and axotomized motoneurons, respectively). This stability in L, which is a function of rt,/r¢, reflects the fact that this ratio decreased by only 8% following axotomy. The increase in r b described above, accompanied by the constant electrotonic length, implies that axotomized motoneurons in aged cats undergo geometrical (shape) changes in their somadendritic tree that are similar to those previously reported for axotomized motoneurons in adult cats 13.
Equivalent cyfinder diameter and length The equivalent cylinder diameter (see Materials and Methods) decreased by 35% following axotomy (54.2 + 0.3/~m vs 35.0 + 0.1 #m, control and axotomized motoneurons, respectively). However, axotomy did not produce a significant change in l. These results are presented in the scattergrams of Fig. 6. Note that the data points
A.
Control
5 A.
Control
4
16
O0 0 0 O~
3
12,
2
8
1
0
00
0 0 0
0
O00
0
o
0 o
o
o
-7 0
5
10
15
20
25
30
2'0
4'0
6'0
80
160
6'0
~o
~8o
E
==
O
E
B.
B.
Axotomized
Axotomized
5.
b
.13
16.
4.
•
E Z
12,
3-
~lm
8. 1
4,
•
0 0
o
s
~o
~5
2b
~
3'0
Ccell (nF)
Fig. 5. Frequency distribution of the calculated total cell capacitance in control (A) and axotomized (B) motoneurons. The arrows point to the mean values of each histogram (9.5 nF and 6.0 nF for control and axotomized cells, respectively). Additional statistical details in the text and in Table I.
o
2'0
4'0
Diameter (#m)
Fig. 6. Scattergrams illustrating the relationship between the diameter and the length of the equivalent cylinder of control (A) and axotomized (B) motoneurons. Note that while the length appears unaffected after axotomy, the diameters from axotomized motoneurons shift to the left and cluster in a narrower range compared to that exhibited by control motoneurons.
305 from axotomized motoneurons are clustered within a narrower range of diameters than that exhibited by the data from control cells.
Rm11'13'22. In order to test this hypothesis, we calculated the electrotonic length and the total cell capacitance of motoneurons.
DISCUSSION
Electrotonic length Following axotomy, L was unchanged in aged cat motoneurons. This conservation of L was associated with a large increase in r b. These results indicate that there were changes in the soma-dendritic geometry of aged motoneurons following axotomy x3'22.
The results presented here demonstrate that aged cat motoneurons exhibited significant alterations in their passive electrical properties following axotomy. We will discuss the changes in each parameter and compare them to the alterations induced by axotomy in adult cat motoneu-
rons9,11,13,17,22. Input resistance In the present study the mean Rin of the control aged motoneurons was 1.1 Mf~, which is similar to the value reported by Engelhardt et al. 7 for Type F motoneurons in the aged cat. Following axotomy, the mean Rin increased to 2.5 MQ (127%). This increase can be caused by changes in certain electrical properties of the motoneuron 3. To a first approximation, we can model the motoneuron's passive electrical properties with an equivalent cylinder model in which the input resistance is given by the following equation: Rin -------(Rm/An)[L[tanh(L)] (ref. 2) where Rm is the specific membrane resistance, L is the electrotonic length of the equivalent cylinder, and A n is the total membrane area. The variables in the above equation that can affect the input resistance of the cell are Rm, A n and the electrotonic length. In an effort to determine the relative contribution of these variables to the observed increase in input resistance, we measured the membrane time constant and calculated the electrotonic length and total cell capacitance of the motoneurons.
Membrane time constant The membrane time constant is directly proportional to the product of the R m and the membrane capacitance (Cm). The membrane capacitance is assumed to be a constant under the conditions of the present experiment, therefore any changes observed in rb are taken as reflecting proportional changes in R m. The 48% increase in rb found in the old cat axotomized motoneurons is therefore believed to reflect a proportional increase in the R m of aged motoneurons following axotomy. Although large, the magnitude of the increase in R m cannot account by itself for the 127% increase in input resistance. It is therefore likely that aged motoneurons react to axotomy with changes in cell surface area and/or geometry in addition to the observed increase in
Total cell capacitance and cell geometry Total cell capacitance was calculated because this electrical property should vary in direct proportion with the total surface area of the motoneuron assuming that the membrane capacitance is constant 12. Estimated values of motoneuron surface area obtained from C ~ calculation 7'12 have been shown to agree well with surface area data reported from anatomical studies. The 37% decrease in Co:n indicates that axotomized motoneurons in aged cats have lost 37% of their soma-dendritic membrane. Furthermore, calculations based on cable theory allow one to determine the length and diameter of the cylinder model which is equivalent to the soma-dendritic tree of the aged motoneurons (see Materials and Methods). These calculations indicate that almost all of the changes in surface area are attributable to a 35% decrease in diameter of the equivalent cylinder (see Fig. 6). These results are consistent with axotomy-induced retraction of dendritic branches which is similar to that which occurs in brainstem motoneurons 25. In addition, the present data suggest that the population of axotomized aged motoneurons is rather homogeneous in size because the diameter distribution is compressed following axotomy. The relation between the geometric and total surface area changes in the soma-dendritic tree (which explain part of the reduction in Rin ) and those in the axon (which explain the reduction in axonal conduction velocity observed by Engelhardt et al. 8 in the same population of motoneurons) is not known. However, neurofilament content and interneurofilament spacing have been related to axonal diameter in myelinated fibers ~4'~8, which is an important determinant of axonal conduction velocity. Neurofilament synthesis and the axonal content of neurofilaments have also been shown to be reduced following axotomy ~°'15'28. On the other hand, the geometry and size of dendrites have been linked to their content of microtubules, membrane related organelles and microtubule-associated proteins (MAPs), but not with their neurofilament content 4'2~'27. It is therefore possible that axotomy-induced changes in the synthesis and/or content of these cytoskeletal components are responsible for the altered geometry of the cell's soma-dendritic tree.
306 A s d e t e r m i n e d by e l e c t r o p h y s i o l o g i c a l m e a s u r e m e n t s ,
physiological p r o p e r t i e s as a result of the aging process.
m o t o n e u r o n s in a g e d cats r e s p o n d to a x o t o m y to an e x t e n t that is similar to that which takes place in m o t o n e u r o n s in adult cats in spite of the fact that m a j o r changes h a v e already o c c u r r e d in t h e s e s a m e electro-
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