Brain Research 888 2001 128–137 www.elsevier.com locate bres
Research report
Treatment of aged rat sensory neurons in short-term, serum-free culture with nerve growth factor reverses the effect of aging on
neurite outgrowth, calcium currents, and neuronal survival
a , b
c d
Karen E. Hall , Huaibao C. Sheng , Shanthi Srinivasan , John M. Spitsbergen ,
e f
a
Jeremy B. Tuttle , William D. Steers , John W. Wiley
a
Department of Internal Medicine , Ann Arbor VA Medical Center, University of Michigan, GRECC 11G, D-318, Ann Arbor, MI 48105-2399, USA
b
Department of Pathology , University of Louisville, Louisville, KY, USA
c
Washington University in St . Louis, St. Louis, MO, USA
d
Department of Biological Sciences , Western Michigan University, Kalamazoo, MI, USA
e
Department of Neurobiology , University of Virginia School of Medicine, Charlottesville, VA, USA
f
Department of Urology , University of Virginia School of Medicine, Charlottesville, VA, USA
Accepted 26 September 2000
Abstract
Impaired NGF production and release has been documented in aged animals, suggesting that decreased NGF receptor stimulation may be one factor contributing to neuronal dysfunction with aging. Other studies have suggested that aging may be associated with impaired
intracellular responses to NGF. Because aging-associated neuronal dysfunction contributes to morbidity and mortality in the geriatric population, it is important to determine whether the effects of aging on sensory neuron function and survival are reversible. In the present
study, we observed significantly decreased neurite outgrowth and neuronal survival in short-term cultures 0–96 h of dorsal root ganglion DRG neurons from aged .22 months Fisher 3443Brown Norway F1 hybrid rats, compared to young 4–6 month and middle-aged
14 month animals. From 24 to 96 h in culture, diminished survival of aged neurons appeared to be due to an increased rate of apoptotic cell death. DRG neurons from aged animals also exhibited significantly decreased whole cell, high-threshold voltage-dependent calcium
currents, with a larger proportion of L-type current, compared to youthful and middle-aged animals. Treatment of aged DRG neurons with NGF restored neurite outgrowth, neuronal survival and calcium current amplitude and subtype distribution to those observed in youthful
DRG neurons.
2001 Elsevier Science B.V. All rights reserved.
Theme : Development and regeneration
Topic : Aging process
Keywords : Aging; Nerve growth factor; Calcium signaling; Calcium currents; Neuronal development; Apoptosis
1. Introduction been documented in specific areas, such as the hippocam-
pus [20]. Decreased neuronal calcium influx appears to Age-associated diminution in calcium influx and in-
underlie the decrease in neurotransmitter release described tracellular calcium regulation has been documented in
previously [35]. In addition to impairment of calcium entry many models of aging [11,30], although increased calcium
at the plasma membrane, aging is also associated with influx due to increased expression of calcium channels has
alterations in the ability of neurons to release and re- sequester calcium in various intracellular pools [11].
Impaired re-sequestration of calcium released by depolar-
Abbreviations: DRG, dorsal root ganglion; I , calcium current; I ,
Ca DCa
ization can result in prolonged elevation of cytosolic
calcium current density
calcium. This is of potential concern, as modest deviations
Corresponding author. Tel.: 11-734-761-5564; fax: 11-734-761-
from the proposed homeostatic calcium ‘set-point’ over
7489. E-mail address
: kehallumich.edu K.E. Hall.
prolonged periods has been implicated as a contributing
0006-8993 01 – see front matter
2001 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 3 0 3 8 - 9
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
