K .E. Hall et al. Brain Research 888 2001 128 –137
129
factor in models of neuronal injury and cell death cally prepared from young, middle-aged and aged rats
[5,25,32]. using techniques described previously [18]. Following
Both in-vivo and in-vitro experiments demonstrate that dissection and enzymatic dissociation, thoracic and lumbar
dorsal root ganglion DRG neurons require neurotrophic DRG neurons were cultured in the following serum-free
support from nerve growth factor NGF for survival defined medium modified from that used by Higgins et al.
[22,36], neurite outgrowth [23] and neuronal phenotypic [21] to culture neonatal sensory neurons: 500 ml 50
expression [24,36]. Neuronal cell death is triggered by DMEM 50 Ham’s F12 GIBCO, Grand Island, NY
withdrawal of NGF from cultured DRG neurons, impair- supplemented with 5 mg ml bovine insulin, 50 mg ml
ment of NGF production, treatment with antisense oligo- human transferrin, 100 mM putrescine dihydrochloride, 30
mers directed against NGF receptors [1], or antibodies nM sodium selenite, 50 IU ml penicillin 50 mg ml strep-
directed against NGF [17]. The majority of studies ex- tomycin, 500 mg ml bovine serum albumin Fraction V-
amining the actions of NGF have utilized tissues from fatty acid free, 16 mM NaHCO and 28 mM
D
-glucose.
3 21
neonatal or young adult animals cultured in the presence of The final osmolality was 320 mosm kg
. Dissociated growth medium supplemented either with serum, or with
neurons from each animal were divided into two equal fibroblast growth factor [7]. Serum contains growth fac-
volumes and re-suspended in either serum-free medium tors, some of which have not yet been defined. The
with 100 ng ml 7S mouse NGF NGF-treated: Calbioch- addition of serum to growth medium complicates interpre-
em, San Diego, or serum-free medium without NGF tation of studies designed to examine the specific actions
controls. In some experiments, control medium contained of NGF. To avoid the confounding effects of culture
antibodies directed against 7S NGF Anti-NGF; Calbioch- medium supplemented with serum, our studies were per-
em. Cell density of the two aliquots was assessed by formed in a defined serum-free medium.
counting the number of DRG neurons per high power field The present study was designed to assess the response of
in 10 ml of cell suspension, and was within 62. The primary sensory neurons from aged animals to treatment
treated neurons were plated onto 2 duplicate calf collagen with NGF on three neuronal functions known to be
Calbiochem-coated sterile glass coverslips and cultured affected by this neurotrophin, namely, calcium influx,
in 35 mm sterile culture dishes with 95 air15 CO at
2
neurite outgrowth, and neuronal survival. In addition to 378C for 96 h. During culture, media was replaced every
effects on calcium metabolism, NGF also increases expres- 24 h by fresh media6NGF. To control the effects of
sion and activation of sodium currents in DRGs [44,14], an plating density on neuronal survival, neuron suspensions
effect that is protective in models of axotomy [10]. We from youthful and middle-aged animals were diluted to
chose to focus on the effect of NGF on calcium currents, achieve plating densities analogous to those obtained from
rather than on other ion channels, because of prior reports aged animals.
documenting specific age-associated decreases in calcium influx [30]. We addressed the questions: 1 does exposure
to NGF in a serum-free growth medium restore age- 2.3. Measurement of NGF concentration in culture
associated decrease in calcium currents and neurite out- media
growth to youthful levels, and 2 does treatment with NGF improve survival of aged neurons?
One ml aliquots of the medium from each culture dish were obtained at 24 h, treated with 100 ml of the protease
inhibitor Trasylol Bayer, West Haven, CN, flash-frozen
2. Materials and methods in liquid nitrogen, and stored at 2708C for subsequent
NGF assay. Samples were shipped at 2208C on dry ice by 2.1. Animal model
overnight shipping to the laboratory of Dr. Jeremy Tuttle at the University of Virginia for subsequent radioimmunoas-
Prior approval for these experiments was obtained from say of NGF using methods described previously [38].
the University of Michigan Committee on Use and Care of Animals Authorization [5353A, according to NIH
guidelines. Male Fisher 3443Brown Norway F1 Hybrid 2.4. Measurement of neuronal survival and neurite
rats were provided by the Pepper Center Aged Rodent outgrowth
Core Facility at the University of Michigan. Experiments were performed on tissue from animals in 3 age groups:
Isolated, dissociated DRG neurons were prepared as young 4–6 months, middle-aged 12–16 months and
described above, and cultured in serum-free medium with aged .22 months.
and without supplemental NGF for up to 96 h. Each experiment comprised DRG neurons from at least two age
2.2. Cell dissociation groups cultured under identical conditions in duplicate or
triplicate. Freshly-dissociated neurons from each animal Isolated, acutely dissociated DRG neurons were asepti-
were plated onto two or three coverslips, incubated with
130 K
medium for 1 h, then rinsed with fresh medium to remove 2.6. Stimulation parameters
non-adherent cells. There was no significant difference between age groups in non-adherence rates. The resultant
Neurons were voltage-clamped at a holding potential plating densities ranged between |200–1000 adherent
V 5290 mV. Immediately after patch rupture, 10 de-
h
neurons coverslip. To determine neuronal survival, adher- polarizing calibration voltage steps 15 mV; 12 ms dura-
ent DRG soma were counted on the day of preparation 0 tion were applied at 1 s intervals, averaged and the whole
h, and at 24, 48, 72 and 96 h after plating. The total cell capacitance calculated as described previously [18].
number of remaining neurons on all coverslips was de- High-threshold calcium currents I were elicited at 30 s
Ca
termined, and expressed as a percentage of the number at 0 intervals by depolarization from V 5290 mV to 110 mV
h
h. At least three separate animals were tested in each age for 100 ms. During each depolarization command, leak
group. To assess neurite outgrowth, the number of neurons currents were generated by hyperpolarizing commands of
demonstrating neurite outgrowth of lengththe diameter of equal value to those used to depolarize the cell, and were
the DRG soma was expressed as a percentage of the total not significantly different between age groups |35–50
number of surviving neurons at that time. Neurons with pA. After subtraction of leak current, I
was divided by
Ca
neuronal projections that came into close proximity ,1 capacitance to yield calcium current density I
; pA
DCa 21
mm to other DRG soma were identified and expressed as a pF
. As membrane capacitance is a function of cell percentage of total surviving neurons. Average6S.E.M. of
surface, this maneuver normalized current amplitude from numbers of surviving neurons, percentage of neurons with
cells of varying size [18]. Peak calcium current density neurite outgrowth, and percentage with neurites in close
was plotted and calculated for neurons from young, proximity to other DRG soma were determined for each
middle-aged and aged animals using the pCLAMP soft- age group, with and without NGF supplementation.
ware program CLAMPEX Axon Instruments. 2.7. Determination of calcium current subtypes
2.5. Whole-cell voltage-clamp recordings Calcium currents were recorded using the stimulation
Whole cell patch-clamp recordings of high-threshold parameters described above in DRG neurons from young,
calcium currents I were performed at room temperature
middle-aged and aged animals. Following measurement of
Ca
on 20–40 mm diameter DRG neurons using techniques the maximum calcium current density after runup of the
described previously [18]. Recording electrode resistance current, neurons were exposed to pharmacologic antago-
was 1–2 MV, and seal resistance greater than 1 GV. nists directed against N-type v-conotoxin GVIA; 10 mm
Experiments were performed in a non-perfused culture and L-type nifedipine; 10 mm. Drugs were applied using
dish containing the following external bath solution: mM compressed air via a glass tube in close proximity to the
5 CaCl , 67 choline Cl, 100 tetraethylammonium chloride, cell soma, as described previously [19]. Current amplitude
2
5.6 glucose, 5.3 KCl, 10 HEPES and 0.8 MgCl pH
in the presence of antagonist was determined, and ex-
2 21
7.3–7.4, 320–330 mosm kg . Recording electrodes were
pressed as a percentage of the pre-treatment amplitude. filled with mM 140 cesium Cl, 10 Hepes, 10 EGTA, 5
The percent inhibition thus obtained was defined as the MgATP and 0.1 LiGTP Sigma. The pH was adjusted to
percent current attributable to that channel subtype. After 7.2–7.3 with 1 M CsOH after addition of ATP and GTP,
subtraction of both N-, and L-type current percentage, the
21
and the final osmolality 280–290 mosm kg was
residual was presumed to reflect the contribution of other adjusted to 10–15 below that of the external recording
subtypes P Q R previously described in DRG neurons. solution using distilled water. High-threshold calcium
currents were elicited by depolarizing voltage steps gener- 2.8. Measurement of neuronal apoptosis
ated using the program CLAMPEX pCLAMP, Axon Instruments, Foster City, CA. Currents were recorded
Coverslips containing neurons cultured as described using an Axopatch 220A patch-clamp amplifier Axon
were washed in phosphate buffered saline PBS for 5 Instruments with an input resistance of 1–3 MV, filtered
min32, fixed with 4 neutral buffered formalin on ice for with a Bessel filter at 5 kHz 23 decibels, sampled at 20
20 min, re-washed with PBS and fixed with 100 metha- kHz and stored on hard disk as binary data files. The total
nol for 10 min. After a final wash in PBS, coverslips were duration of recording was between 10 and 20 min from the
stored at 2708C until stained. All chemicals were obtained time of patch rupture. In a subset of neurons, the per-
from Sigma St. Louis, MI. Apoptosis was assessed using centage of the high threshold calcium current attributable
a commercially-available kit ApopTag E Plus In Situ
to specific calcium channel subtypes was assessed using Apoptosis detection kit, Oncor, Gaithersburg, MD em-
pharmacologic antagonists v-conotoxin GVIA N-type, ploying the TUNEL method [39]. This method uses
nifedipine L-Type, and electrophysiologic criteria T- terminal deoxynucleotidyl transferase to extend the 39-OH
type applied by an air-pressure puffer system described terminals of apoptotic DNA fragments by adding digox-
previously [18]. igenin-bound dUTP nucleotides. Neurons were exposed to
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
