K .E. Hall et al. Brain Research 888 2001 128 –137
131
an anti-digoxigenin antibody–fluorescein solution, and variance ANOVA for multiple comparisons, and defined
yellow–green fluorescence measured at 494 nm. Prop- as a P value ,0.05 [31].
idium iodide counterstaining red was used as a counters- tain to identify the total number of surviving neurons.
Percent apoptosis was calculated by dividing the number
3. Results
of apoptotic neurons by the total number of surviving neurons.
3.1. Neuronal survival was diminished in DRG neurons from aged rats compared to youthful and middle-age
2.9. Statistical analysis animals
Statistical analysis was performed using GraphPad DRG neurons from youthful, middle-aged and aged
Prism and Instat GraphPad Software, San Diego, CA. animals were cultured in serum-free medium with and
Significance was determined using 1 or 2 way analysis of without NGF for up to 96 h. Fig. 1 comprises representa-
Fig. 1. Photomicrographs of DRG neurons from aged 26 month rats cultured in serum-free medium without NGF 2NGF and with supplemental NGF at a concentration of 100 ng ml 1NGF. Compared to non-NGF-treated neurons, neurons cultured in the presence of NGF had more neurites longer than
1 somal diameter, and many more neurites that were in close proximity to other DRG soma. All micrographs were taken at the same magnification 30 mm scale bar below D and H.
132 K
tive photomicrographs of DRG neurons from aged animals cultured for 24–96 h with, and without NGF supple-
mentation. When cultured in serum-free medium without NGF supplementation Fig. 2: Y-NGF; O-NGF, the
percentage of surviving neurons decreased with time in culture. The initial 25 decrease in survival during the
first 24 h appeared to have a substantial component of necrosis, as demonstrated by inability to exclude trypan
blue, and was not significantly different between age groups. During the subsequent 72 h, neurons unable to
exclude trypan blue were rare. Compared to youthful DRG neurons cultured in serum-free media without NGF Y-
NGF, DRGs from aged animals exhibited significantly diminished survival P,0.01 by 48 h. Survival of neurons
from middle-aged animals was almost identical to that of youthful DRGs not shown. Apoptosis was assessed by
measurement of DNA fragmentation using TUNEL stain-
Fig. 3. Apoptosis measured by the TUNEL method at 48 h culture in
ing at 48 h, and expressed as a percentage of total neurons
non-NGF-containing medium 2NGF was significantly increased in
Fig. 3. This method, which identifies neurons in the later
DRG neurons from aged animals, compared to youthful controls. Addi-
stages of apoptosis, demonstrated a significant 4-fold
tion of 100 ng ml NGF to the culture medium 1NGF was associated with significantly decreased apoptosis at 48 h in all age groups when
increase in the percentage of apoptotic neurons in cultures
compared to age-matched, non-NGF-treated neurons. With NGF treat-
from aged animals. In previous studies of apoptosis in
ment, apoptosis rates were still elevated P,0.05 in the aged group
diabetic neuropathy, we have documented that rates of
when compared with NGF-treated youthful controls. Results are
apoptosis in cultured neurons of 4 at a single time point
mean6S.E.M. of three separate experimental animals age group, with a
obtained using TUNEL staining correspond to much higher
minimum of 500 total DRG neurons counted animal. P,0.01, P, 0.005.
cumulative rates approaching 20, when compared with other methods such as flow cytometry [39].
percentage of neurons with identifiable neurites of length the diameter of the corresponding cell soma, and per-
3.2. DRG neurons from aged animals demonstrated centage of neurons with neurites growing in close proximi-
decreased neurite outgrowth compared to youthful and ty to other DRG soma. Cultures of aged neurons had
middle-age animals significantly fewer neurites longer than one somal diam-
eter, and fewer neurites that extended into close proximity Neurite outgrowth was assessed by determining the
with other DRG soma Table 1. Neurite growth in cultures of middle-aged neurons was similar to that of
youthful preparations.
3.3. Aging was associated with a decrease in neuronal calcium currents
Whole cell voltage-clamp recordings of high-threshold calcium currents were performed on isolated DRG neurons
from youthful, middle-aged and aged animals cultured for 24–96 h as described in the methods. Neurons cultured for
.48 h in NGF-containing medium formed extensive
Fig. 2. Percent survival of DRG neurons cultured in serum-free medium
neuronal processes, causing considerable space clamp
with and without NGF. Freshly-dissociated DRG neurons from young
artifact with depolarization. This resulted in poor quality
n54 and aged n55 animals were plated and counted at time50, and
whole-cell recordings in NGF-treated DRG neurons cul-
at 24 h intervals up to 96 h, as described in the methods. The initial
tured longer than 48 h. Similar difficulties were ex-
decrease in neuron numbers during the first 24 h appeared to be due to necrosis, and was unchanged with NGF treatment. From 48 to 96 h,
perienced in attempting to record whole cell currents in
significantly P,0.01 fewer DRG neurons from aged animals O-NGF
non-NGF-treated cultures after 72 h. For this reason, the
were present in dishes, compared to cultures from youthful Y-NGF
results presented in Table 2 and Fig. 4 compare the effect
animals. NGF treatment improved neuronal survival significantly in the
of NGF treatment on whole cell calcium current recordings
aged group O1NGF. Survival of NGF-treated and non-NGF-treated
in DRG neurons cultured for 24–48 h. Calcium currents
neurons from middle-aged animals was similar to that observed for youthful neurons, and for clarity is omitted from this figure.
were elicited as described in the Methods, and calcium
K .E. Hall et al. Brain Research 888 2001 128 –137
133 Table 1
a
Effect of aging on neurite growth in short-term DRG culture Hours in
with neurite lengthsdiameter of soma with neurites close to other DRG soma
culture 24 h
48 h 72 h
96 h 24 h
48 h 72 h
96 h Young 4
864 1768
4369 6468
362 1263
2869 3267
Middle-aged 4 963
2164 29611
53611 461
865 23615
29610 Old 5
664 1665
2269 3767
663 966
1366
a
DRG neurons from young, middle-aged and aged rats were cultured in serum-free medium for 96 h. At 24, 48, 72 and 96 h after plating the number of neurons demonstrating neurite outgrowth longer than 1 somal diameter, or growing in close proximity to another DRG soma were expressed as a
percentage of the total number of neurons counted see methods. Compared to young animals, neurons from aged animals plated at equivalent initial densities had significantly decreased numbers of cells with neurites longer than 1 somal diameter, and decreased neurites that came into close proximity to
other DRG soma. Values are expressed as the mean6S.E.M. of n experiments. P,0.05. P,0.01.
Table 2
following patch rupture ‘runup’ to a maximum amplitude
Calcium current density I recorded in DRG neurons from young,
DCa
[18,19]. There was no significant difference in the rate of
a
middle-aged and aged rats from 24 to 72 h in culture
current ‘runup’ between age groups. The average maxi-
Animal age 24 h
48 h 72 h
mum high-threshold I recorded in neurons from the
DCa
Young 4 97610
108613 110611
three age groups for cultures up to 72 h are shown in Table
Middle-Aged 4 108618
113614 120619
2 and Fig. 4. Compared to currents recorded in neurons
Old 5 61617
64620 65626
from youthful animals, maximum I was significantly
DCa
a
I current density calculated as described in methods. Number of
decreased in DRG neurons from aged animals. Under these
DCa
animals indicated in parentheses. Each value is the mean6S.E.M. of
recording conditions, following the point of maximum
recordings from a minimum of six DRG neurons from each of n
I , calcium current density decreased at a steady rate
DCa
animals. P,0.05.
‘rundown’ during the remainder of the recording [18]. Current rundown also occurred at a similar rate in the three
age groups. Using methods described previously [18,19], current density I
; pA pF determined by dividing the we did not observe a shift in the peak current to higher
DCa
current by the whole cell capacitance to minimize the voltages in the current–voltage IV relationship, or an
effect of varying cell size on current amplitude [18]. I increase in steady-state inactivation that would account for
DCa
increased 20–30 in amplitude during the initial 2–5 min the decrease in I
with aging. Using pharmacologic
DCa
antagonists directed against N-type v-conotoxin GVIA; 10 mm and L-type nifedipine; 10 mm calcium channels,
the percentage of current attributable to N-, L- and residual non-N, -L-subtypes of calcium channels was determined in
neurons incubated for 48 h Fig. 5. Compared to youthful neurons, there was a significant increase P,0.05 in the
percentage of L-type calcium currents in aged neurons.
Fig. 4. Effect of age on peak DRG neuron calcium current density I
Dca
recorded at 48 h in culture. Representative current tracings are shown above the corresponding average peak calcium current density I
: Fig. 5. Calcium currents from aged DRG neurons exhibited a larger
DCa
pA pF. For each animal, recordings were performed in a minimum of 4 percentage of L-type current. The percentage of current attributable to N-,
DRG neurons. Recordings obtained in 4 young, 4 middle-aged and 5 aged L-, and residual P Q R-type channels was determined using antagonists
rats were combined and the average current density1S.E.M. calculated as as described in the methods. A significantly P,0.05 larger component
described in the methods. Compared to recordings in young and middle of current recorded in aged DRG neurons was inhibited by nifedipine,
aged animals, significantly P,0.05 smaller currents were obtained in indicating that L-type currents comprised a larger proportion of the total
recordings from aged DRG neurons. current in aged neurons.
134 K
Table 3
a
Effect of NGF treatment 100 ng ml on neurite outgrowth of cultured DRG neurons from young, middle-aged and old rats Hours in
with neurite lengthsdiameter of soma with neurites close to other DRG soma
culture 24 h
48 h 72 h
96 h 24 h
48 h 72 h
96 h Young 4
1962 5269
87613 9862
565 34611
61612 84613
Middle-aged 4 1463
44614 7866
9366 262
1669 48617
70623 Old 5
1563 3967
82619 9068
866 2967
6269 79618
a
DRG neurons from young, middle-aged, and aged animals were plated at equivalent densities and cultured in serum-free medium with 100 ng ml NGF for up to 96 h. At 24, 48, 72 and 96 h after plating, the number of neurons demonstrating neurite outgrowth from the soma of lengthdiameter of the
corresponding soma, and percent with neurites in close proximity to another soma, were expressed as a percentage of the total number of neurons counted. There was no significant difference between the three age groups in the numbers of neurons that grew neurites or demonstrated interconnections in the
presence of NGF. Values are expressed as the mean6S.E.M. of n experiments.
3.4. NGF treatment significantly increased survival of trations were negligible, namely 0.760.3 n53, 0.860.4
cultured aged DRG neurons n53, and 0.560.4 n53 respectively.
Percentage survival Fig. 2: Y1NGF; O1NGF and 3.5. Treatment with NGF increased high-threshold
neurite outgrowth Table 3 of cultured aged sensory calcium current amplitude
neurons was significantly increased by NGF supple- mentation, approaching that of youthful cultures. There
Compared to untreated cultures, DRG neurons treated was a concomitant decrease in apoptosis measured at 48 h
with NGF 100 ng ml for 48 h demonstrated a significant in NGF-treated cultures of aged neurons Fig. 3. There
elevation in maximum I in all age groups Fig. 6. This
DCa
was a modest but non-significant increase in survival of effect was particularly striking in neurons from aged
youthful DRGs with NGF supplementation. NGF con- animals exposed to NGF. There was no significant differ-
centration pg ml in the media of cultures supplemented ence in the proportion of N-, L-, and residual P Q R-
with NGF was measured by radioimmunoassay at 24 h type currents in NGF-treated cultures from the three age
incubation, and found to be 478675 n53, 326644 groups. As untreated aged DRG neurons appeared to have
n53, and 565682 n53 for neurons from young, a greater component of L-type current Fig. 5, this
middle-aged and aged rats respectively. This difference suggested that NGF treatment might cause a preferential
was not significant. In untreated dishes, NGF concen- increase in other calcium current subtypes in aged neurons.
The increase in current amplitude observed with NGF treatment was accompanied by a significant increase in
peak amplitude of the current–voltage IV relationship. There was no significant shift in the voltage range of
activation or steady-state inactivation of the calcium channels activated by the stimulation parameters used,
suggesting that the change in current amplitude might be due to recruitment of additional channels, rather than a
shift in the individual channel kinetics.
4. Discussion
