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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 dKaren 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; ICa 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: 1
1-734-761-from the proposed homeostatic calcium ‘set-point’ over
7489.
E-mail address: [email protected] (K.E. Hall). prolonged periods has been implicated as a contributing
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, 100mM putrescine dihydrochloride, 30 mers directed against NGF receptors [1], or antibodies nM sodium selenite, 50 IU / ml penicillin / 50mg / 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 mM3 D-glucose.
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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 at2
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 100ml 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 at2208C 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
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 )h 5290 mV. Immediately after patch rupture, 10
de-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 sCa
termined, and expressed as a percentage of the number at 0 intervals by depolarization from Vh5290 mV to 110 mV 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 length$the 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, ICa was divided by neuronal projections that came into close proximity (,1 capacitance to yield calcium current density (IDCa; pA
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 (ICa) were performed at room temperature middle-aged and aged animals. Following measurement of 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; 10mm) Experiments were performed in a non-perfused culture and L-type (nifedipine; 10mm). 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,2 cell soma, as described previously [19]. Current amplitude 5.6 glucose, 5.3 KCl, 10 HEPES and 0.8 MgCl2 (pH in the presence of antagonist was determined, and
ex-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
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
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
Table 1
a
Effect of aging on neurite growth in short-term DRG culture
Hours in % with neurite lengths$diameter 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* 0 663 966* 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 (IDCa) recorded in DRG neurons from young, [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
IDCa (current density) calculated as described in methods. Number of decreased in DRG neurons from aged animals. Under these 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)
IDCa, calcium current density decreased at a steady rate
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 (IDCa; pA / pF) determined by dividing the we did not observe a shift in the peak current to higher 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]. IDCa increase in steady-state inactivation that would account for increased 20–30% in amplitude during the initial 2–5 min the decrease in IDCa with aging. Using pharmacologic antagonists directed against N-type (v-conotoxin GVIA; 10 mm) and L-type (nifedipine; 10mm) 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 (IDca) recorded at 48 h in culture. Representative current tracings are shown
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 lengths$diameter 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 length$diameter 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 IDCa in all age groups (Fig. 6). This 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
Compared to DRG neurons from youthful or middle-aged animals, neurons from middle-aged rats cultured in serum-free medium demonstrated significantly decreased calcium currents, decreased neurite outgrowth, fewer neuronal projections in close proximity to other neuronal soma, and
Fig. 6. NGF treatment significantly increased calcium current density in
diminished survival. Treatment of DRG cultures from aged
aged DRG neurons. Neurons cultured in growth medium supplemented
with 100 ng / ml NGF demonstrated increased IDCa in all age groups, animals with NGF-containing serum-free medium
im-compared to age-matched, non-NGF-treated controls. The increase in IDCa proved survival, with neurite formation approximating that
over youthful neurons treated with NGF was significant in recordings observed in youthful cultures. Calcium currents were from the middle-aged and aged group.2NGF: number of recordings as in
increased by NGF treatment, particularly in aged neurons.
Fig. 4.1NGF: Results expressed as mean1S.E.M. of recordings obtained
This may have been due to a relative increase in
non-L-from a minimum of 4 DRG neurons non-L-from 10 young, 8 middle-aged, and 6
seen in non-NGF-treated aged cultures was not observed in NGF from cultured sympathetic neurons [27]. Neuro-NGF-treated aged DRGs. These results suggest that aged protection was correlated with enhancement of intracellular neurons are capable of exhibiting trophic responses to calcium release, as use of calcium chelators in the latter NGF treatment, as assessed by the parameters of neuronal study abolished the protective effect of K1-dependent function measured in these studies. To minimize the depolarization. On the other hand, substantial increases in contribution of undefined growth factors to our studies of calcium influx observed with exposure to serum from NGF response, we used a serum-free medium for this individuals with Type 2 diabetes mellitus with neuropathy study. Bovine and equine serum are routinely added to promoted calcium-dependent apoptosis in neurons from culture media to promote growth, and serum contains a young rats [39]. This suggests that the neuronal responses variety of trophic factors, some of which are as yet to changes in calcium signaling may change with age. The
undefined. mechanism by which NGF enhanced high-threshold
cal-Our results suggest that aging appears to diminish the cium currents could be due to increased expression of ability of neurons to survive adverse conditions, such as calcium channels, as has been described in pheochromocy-culture in serum-free medium without neurotrophic stimu- toma cell lines [43]. Other possible mechanisms for lation. Early neuronal loss by necrosis was observed in increased survival of DRG neurons with NGF treatment cultures from all age groups. After 48 h, decreased include increased activity of other ion channels, particu-neuronal survival was correlated with an increase in the larly sodium [10,14,44]. This may be the preferred protec-rate of induction of apoptosis, suggesting that diminished tive pathway in neonatal superior cervical ganglion neu-survival under these circumstances could be a result of rons subjected to NGF withdrawal [41], particularly in the programmed cell death. This effect was more pronounced setting of sodium channel activation by veratridine [28]. in DRG neurons from aged animals, suggesting that aged The effect of aging on neurotrophic pathways is of neurons were more susceptible to induction of apoptosis considerable interest, as exposure to exogenous neuro-caused by lack of neurotrophin stimulation. trophins may improve neuronal function [34]. Various Altered calcium homeostasis has been implicated as a models of aging are associated with decreased synthesis contributing factor in several models of neuronal injury and transport of NGF in the brain under basal conditions [32]. Neurons from aged animals cultured in serum-free [8,26,29,33], or following injury [37]. Diminished availa-medium demonstrated a significant decrease in maximum bility of NGF in aging appears to have pathogenic signifi-calcium currents compared to youthful neurons. A sig- cance, as exogenous NGF infused into the CNS decreases nificantly greater proportion of this smaller current was the severity of memory impairment and synaptic loss in rat attributable to L-type currents in aged neurons. We did not models of aging [4,13]. Acetyl-L-carnitine-induced
eleva-observe a shift in the current–voltage relationship or tion of NGF increased choline acetyltransferase activity in voltage-dependent steady-state inactivation to explain the aged rats [40]. Similar improvement in neuronal function diminished currents in aged DRGs. This also appears to be has been observed with NGF treatment of peripheral the case in the diminution of calcium currents that occurs nerves [12]. NGF responses could be impaired at the during early development [7]. Other mechanisms, such as cellular level by decreased expression of high-affinity decreased expression of calcium channels, or decreased TrkA receptors implicated in neuronal development and open duration or open frequency of individual channels survival [3]. Decreased expression of neurotrophin re-could account for the decrease in calcium currents. The ceptors with aging has also been described in the medial latter has been implicated in voltage-dependent inhibition septum [8], and in the cortex of aged microencephalic of calcium channels by agonists such as opiates or animals [6]. However, despite this, our study suggests that norepinephrine [2]. Decreased calcium currents may con- aged neurons retain the ability to respond to NGF in-vitro, tribute to aging-associated changes in cytosolic calcium indicating that the functional and trophic changes seen in buffering [11], as there is a correlation between the degree aged neurons are not irreversible. Finally, age-dependent of calcium influx and subsequent cytosolic calcium release changes in the target tissues of sensory nerves could also in DRG neurons [42]. Our studies indicate that treatment play a role in age-associated neuronal dysfunction [9]. of DRG neurons with NGF at concentrations that promoted Studies investigating the ability of NGF to stimulate re-survival and neurite growth was associated with elevations innervation of ocular grafts by iridial nerves in-vitro have in peak high-threshold calcium current amplitude in all age shown that the ability of aged nerves to re-innervate grafts groups, and particularly in neurons from aged animals. As was dependent on the age of the graft [16].
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