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Laboratories of 1 Neurosciences
2 Genetics, National Institute on Aging, Baltimore, Maryland.
3 Microscope Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Address correspondence to Catherine A. Wolkow, NIA/IRP, 5600 Nathan Shock Drive, Baltimore, MD 21224. E-mail: wolkowca{at}grc.nia.nih.gov
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Abstract |
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One approach for investigating the relationship between age and cellular function is to examine the problem in a genetically tractable organism with a relatively short life span, such as the nematode, Caenorhabditis elegans, which has been widely used for genetic studies of longevity. Genetic screens have identified several genes that determine C. elegans life span, including genes comprising the C. elegans daf-2/insulin-like signaling pathway (1,2). Although the search for genes that determine life span has been fruitful in C. elegans, less is known about how cellular function changes with age in this organism. Body movement declines gradually with increasing age in C. elegans (3–8). Studies (7,8) have shown that the rate of age-related decline in body movement is a good predictor of life span. This correlation suggests that locomotory decline and senescence may share common components in this species. Analysis of cell structure in aged animals showed that muscles, but not neurons, deteriorate significantly in older animals (6). Longitudinal studies (6,9) showed that tissue deterioration correlates well with both locomotory behavior and life span.
One area that has not been investigated is the impact of age on behavioral responses that require the integration of sensory and motor function. C. elegans nematodes display characteristic responses to the presence of chemical attractants and repellents in their environment (10–12). It is not known whether sensory ability declines with age in C. elegans. In addition, the impact of age-related locomotory decline on responses to sensory cues has not been investigated, although an early study (13) reported that aging was associated with reduced movement toward bacterial food. To address these questions, we examined performance of several characteristic C. elegans behaviors during aging between young adulthood and the age of 50% mortality. During this period, mild locomotory deficits appeared which progressed in severity and compromised responses to sensory cues. In contrast, sensory function itself was not apparently affected by aging. Responses to sensory cues were protected from age-related decline in long-lived daf-2/insulin pathway mutants. Interestingly, early age-related locomotory deficits occurred without evidence of significant muscle deterioration, although there was evidence of reduced muscle integrity. These studies reveal that aging in C. elegans manifests early as mild locomotory impairments that impede normal behavioral responses. In addition, they provide several novel and useful approaches for monitoring physiological aging in C. elegans.
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METHODS |
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Age-Synchronized Populations
Aged populations of wild-type and daf-2(e1368) animals were obtained from synchronized egg lays, and the larvae were raised to young adulthood at 15°C with ample bacterial food. For ease of manipulation, some experiments were carried out with fem-1(hc17), an adult sterile strain with the same life span characteristics as wild type. For fem-1(hc17), larval development occurred at 25°C, the nonpermissive temperature for adult fertility. For all strains, the day following the fourth and final larval molt was designated day 0 of adulthood, and animals were transferred to 25°C on fresh medium at densities of 30–50 animals/6 cm plate. We did not expose the aging cohorts to 5-fluorodeoxyuracil, which is sometimes used in life-span studies to suppress progeny production. In control experiments, 5-fluorodeoxyuracil had a deleterious effect on motility, also noted by others (3). For wild-type and daf-2(e1368) strains, adult animals were gently transferred to fresh medium every day while fertile to prevent overcrowding by progeny and approximately every 2–3 days thereafter. Animals damaged during transfers, as reflected by lack of movement within a few minutes of transfer, were excluded from further analysis. Animals were scored as dead when they failed to move in response to gentle prodding with a platinum wire (harsh touch).
Behavioral Assays
Mechanosensation and aversive odor.--
To confirm that all animals were capable of locomotion, only animals that could move away after a harsh touch and could thrash in a drop of buffer were used for behavioral assays. Responses to gentle touch and aversive odor were assayed as described (15,16). In brief, response to gentle touch was assayed by gently stroking the body with an eyebrow hair up to three times and scoring the animal's response as movement away from the stimulus. Different neurons mediate responses to harsh and gentle touch, so it is reasonable to assay these responses independently (15). We tested response to aversive odor by exposing forward-moving animals to an octanol-saturated hair. The times required for animals to (a) stop forward movement and (b) start backing away from the octanol were both measured. Spontaneous stopping time in the absence of octanol was 9 ± 5.4 seconds for day 2 adults and 10.5 ± 4.7 seconds for day 8 adults; time to stop in response to octanol was significantly different (p <.01, t test). Animals that did not back away 
60 seconds after stopping were scored as nonreversers.
Chemotaxis and locomotion.-- Chemotaxis and spontaneous locomotion were assayed at 25°C, essentially as described (10,12,17–19). Animals were washed briefly in a drop of M9 buffer, and 20–30 same-aged animals were placed on the center of a prewarmed 9-cm dish containing 10 mL of solid assay agar (1.6% agar, 1 mM CaCl2, 1 mM MgSO4, 4 µM NH4Cl, 25 mM phosphate buffer) with a 1 µL drop of chemical odorant at one edge. After all animals were transferred onto the agar, a second drop of odorant was added to the same spot as the first along with one drop of 10% sodium azide. Assay plates were gently placed into a 25°C incubator for 1 hour. Chemotaxis was quantified as net displacement away from the odorant, in the general direction of the odorant, as opposed to net displacement from the attractant, because diffusion of the odorant during the assay period would lead to uncertainty in the position of the odorant. Similar results were observed with several attractive (isoamyl alcohol [1%], Cl– [1 M NH4Cl], diacetyl [1%], benzaldehyde [1%]) and noxious (octanol [10%]) odorants. In separate experiments, we examined the effect of a control spot of sodium azide at the opposite edge of the plate from the odorant (12,17), but, in experiments with aging populations, we did not observe a significant difference in outcomes from the presence or absence of the control spot. For locomotion analyses performed in the presence of arecoline, 0.25 mM arecoline was added directly to the molten assay medium before pouring. All experiments were performed with at least three independent populations for wild type and at least two populations for daf-2(e1368) to minimize population effects. Therefore, error bars represent the variation observed between independent populations.
Displacement measurements.-- To determine net distance traveled in chemotaxis and locomotion assays, each animal's position after 1 hour was marked on the bottom of the plate. The marked plate was digitally imaged, and each animal's net displacement from the origin (plate center) was measured using Adobe Photoshop (Adobe Inc., San Jose, CA) or Openlab (Improvision Inc., Lexington, MA) software.
Muscle Analyses
Phalloidin staining.--
For phalloidin staining of actin filaments, fem-1(hc17) adults were transferred onto a lysine-spread slide, freeze-cracked, and fixed in 100% methanol (–20°C) for 4 minutes. Animals were stained with 1–4 µL of 0.1 mM AlexaFluor 488 phalloidin (Molecular Probes, Eugene, OR) and viewed with an Endow GFP filter set (Chroma Technology Corp., Rockingham, VT) on a Nikon E800 microscope. Digital images were collected with a Spot/RT CCD camera (Diagnostic Instruments, Sterling Heights, MI). Similar results were obtained with an alternate protocol in which same-aged animals were washed from an agar plate in M9 or phosphate-buffered saline buffer and fixed for 10 minutes in acetone (–20°C) before staining with FITC-conjugated phalloidin (100 nM). Specimens were mounted on an agarose pad with n-propyl gallate to reduce photobleaching. Similar results were obtained with fem-1(hc17) and N2 wild-type animals.
Quantitation of muscle damage.-- Several images were collected of body-wall muscles in each phalloidin-stained animal. These images were examined for signs of sarcomere damage, such as irregular staining or deformed sarcomeres. Images in which muscles had broken into pieces during the staining process were discarded and did not appear to be more prevalent at older ages. If the majority of an animal's muscles showed signs of damage, the animal was scored as damaged. If the majority of muscles from an animal did not show significant damage, the animal was scored as intact. The fraction of animals containing damaged muscles was determined for each of several independent staining experiments. The average occurrence of animals with damaged muscles was then calculated for all the trials at each age of adult life.
Electron microscopy.-- For electron microscopy, fem-1(hc17) adults were cut transversely in fixative (formaldehyde [2%], gluteraldehyde [2%], CaCl2 [3 mM] in 0.1 M cacodylate buffer, pH 7.2) and incubated for 1 hour at room temperature. Specimens were postfixed in 2% OsO4 in 0.1 M cacodylate buffer (pH 7.2) with 3 mM CaCl2 for 1 hour and stained in 2% uranyl acetate for 30 minutes at room temperature. Cut specimens were aligned and embedded in low-melting-temperature agarose (2.5%; U.S. Biological). The agarose blocks were dehydrated in an ethanol series (5 minutes each in 50%, 70%, 90%, and 100% ethanol) followed by propylene oxide before embedding in Epon. Transverse sections (70 nm) were stained with aqueous 2% uranyl acetate followed by 0.03% lead citrate then examined on a BioTwin CM120 transmission electron microscope (Philips, Eindhoven, the Netherlands) or a Zeiss electron microscope EM10A (Carl Zeiss, Inc., Thornwood, NY). In this study, animals were not segregated by behavioral ability prior to muscle examination, although dead or severely incapacitated animals were removed from the population.
Body-length shrinkage assays.-- For body contraction assays, fem-1(hc17) animals were first photographed on NGM agar plates, transferred to medium supplemented with 0, 50, 100, or 500 µM levamisole (Sigma, St. Louis, MO) and photographed again after 20 minutes. The length of each animal before and after levamisole treatment was determined using software tracing and measuring tools (Openlab 3.1.5; Improvision Inc., Lexington, MA). Similar results were obtained with N2 wild-type animals.
Statistical Analyses
Statistical analyses were performed using Student's t test in a Microsoft Excel (Redmond, WA) spreadsheet to compare results between multiple independent populations.
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RESULTS |
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We examined whether adult age altered the performance of characteristic behaviors in response to several stimuli. The ability of young and old animals to sense gentle touch was measured by movement in response to gentle mechanical stimulation (15). In this assay, any animal failing within 5 seconds to produce detectable movement in response to the stimulus was scored as nonresponsive. The majority of adult day 2 and day 8 animals exhibited appropriate responses to the touch stimulus (Table 1). We noted that the magnitude of response, as judged by distance moved in response to the touch, was reduced in older animals (data not shown).
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To further investigate the effects of age on responses to sensory stimuli, we examined chemotaxis behavior between day 2 and day 10 adults (Figure 1C). When placed on an agar plate containing a small drop of attractive odorant, such as diacetyl (2,3-butanedione), all young adult animals moved rapidly to the attractant, consistent with earlier reports (10,12) (Figure 1D). Increased age was associated with a progressive decline in this response. Adult day 8 and 10 animals exhibited significant declines in movement toward diacetyl (Figure 1D). Nearly half of day 8 and 10 animals did not initiate any significant movement toward diacetyl and instead remained within 0.5 cm of the original position after 1 hour (Figure 1D, white bars). We observed that these animals sometimes moved alternately forward and backward but did not make appreciable progress toward the attractant. Other day 8 and 10 animals were able to respond to diacetyl, consistent with our finding that sensory ability remained intact with age, although these animals did not travel as far as young adult animals during the assay period (Figure 1D, grey bars). The presence of a control spot of sodium azide at the opposite edge of the agar surface did not affect the outcome of these experiments, presumably because animals were able to detect and respond to the presence of the attractant or did not move at all.
In adult day 8 animals, chemotaxis was not significantly improved by altering diacetyl concentration. Animals that had moved slightly toward the attractant in 1-hour assays usually continued progress toward the attractant when assay times were increased to 2 or 3 hours. Animals that had not left the origin in the first hour did not do so after longer incubation times (data not shown). Chemotaxis defects were not odorant-specific; similar results were observed with several attractive (1% isoamyl alcohol, Cl– (1 M NH4Cl), 1% diacetyl, 1% benzaldehyde) and noxious (10% octanol) odorants (data not shown). It should be noted that, by confining behavioral analysis to animals capable of normal movement, these results likely indicate a slightly slower rate of behavioral decline than would be observed in the population as a whole.
Early Age-Associated Behavioral Declines Correlate With Movement Defects
The finding that age had a negative impact on chemotaxis behavior despite apparently normal sensory abilities in older animals suggested that chemotaxis defects in day 8 animals were linked to locomotory deficits. To investigate this possibility, we examined movement at days 2, 4, and 8 of adulthood. Body movement in C. elegans consists of three basic movements. Forward sinusoidal movement is interspersed with reversals, which are spontaneous periods of backward movement and omega turns, in which animals change direction (19). Although many adult day 8 animals were capable of normal forward sinusoidal movement, other aspects of movement behavior were altered in these animals. First, adult day 8 animals moved slower; the number of body waves performed in a 20-second interval declined by half between days 2 and 8 of adulthood (Figure 2A). Day 8 animals also exhibited different movement patterns. When monitored over a 3-minute assay period, day 2 animals moved forward almost continuously (Figure 2B). The duration of forward movement was shorter in day 8 animals, which also did not move for a larger fraction of the assay period. In young animals, forward movement was interspersed by a small number of reversals and omega turns (Figure 2C). In contrast, day 8 animals performed a greater number of reversals during the testing period than did younger animals (Figure 2C). The frequency of omega turns was similar between ages.
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Behavioral Responses Are Protected From Age-Associated Declines in Long-Lived daf-2 Mutants
Longevity in C. elegans is regulated by an insulin-like signaling pathway that includes the gene daf-2 encoding a protein most closely related to vertebrate insulin and insulin-like growth factor-1 receptors (1,20). Regulation of life span by insulin-like pathways appears to be evolutionarily conserved (21). Mutations in daf-2 result in a 2- to 3-fold increase in adult life span (see Figure 1A). Previous studies showed that mutations in daf-2 and age-1, which also functions in the daf-2 pathway to regulate life span, can protect animals from movement declines during aging (5,8,22,23). Locomotory declines were also delayed in long-lived recombinant inbred lines of C. elegans (7).
To determine if daf-2 mutations delayed age-related declines in chemotaxis and locomotion, we examined these behaviors in long-lived daf-2(e1368) animals. Young adult daf-2(e1368) animals exhibited normal chemotaxis responses, and this behavior was better maintained through adult day 10 than in wild-type animals (Figure 3A vs Figure 1D). Similar results were obtained with a more severe daf-2 (e1370) allele (data not shown). By day 20, chemotaxis by daf-2(e1368) animals had significantly declined. Locomotory behavior in daf-2(e1368) animals was relatively normal at adult day 2 and declined over their life span more slowly than in wild-type animals (Figure 3B). Together these results show that long-lived daf-2(e1368) animals were subject to age-related behavioral declines similar to those in wild-type animals, but the progression of these declines was slowed.
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We first examined the overall structure of muscle tissue by staining animals with phalloidin to visualize the actin-containing thin filaments of the body-wall muscles. Sarcomeres in most day 2 adults appeared straight and evenly stained, as expected for intact muscle cells in young adult animals (Figure 4A). Phalloidin-stained muscles at adult days 4 and 6 were similar to those at adult day 2, with a slight increase in the fraction of animals with patched or wrinkled sarcomeres (Figure 4, B and C). In contrast, the majority of day 8 adults contained muscles that appeared patched or wrinkled when stained with phalloidin, indicating that muscles were altered or damaged, either in vivo or as a consequence of the staining process (Figure 4D). We determined the fraction of animals at each age with muscle damage in this assay (see Methods). This analysis showed that day 8 adults were significantly more likely to contain damaged muscles, as visualized by phalloidin, than were day 2 adults (p <.001, t test) (Table 2). Day 4 and 6 adults exhibited a trend toward increased damage, but this difference was not statistically significant in most cases.
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Adult Day 8 Muscles Can Contract Normally in Response to Nicotinic Acetylcholine Receptor Agonists
We next investigated whether specific aspects of muscle function were altered in adult day 8 animals that might be correlated with reduced movement at this age. One marker for muscle function is the ability to contract in the presence of cholinergic agonists. Acetylcholine is the major excitatory neurotransmitter in C. elegans (25). Acetylcholine released by motor neurons triggers muscle contraction via nicotinic acetylcholine receptors in the body-wall muscle. Pharmacologic activation of muscle nicotinic receptors by the nicotinic agonist, levamisole, caused muscle contraction, thus shortening the animal's body length (Figure 5A, upper panel) (26,27). We reasoned that the extent of body-length shrinkage may reflect the relative ability of muscles to contract after levamisole treatment, and this might be useful for assaying muscle function at adult days 2, 4, and 8. When examined over a 10-fold range of levamisole concentration, adult animals of all three ages exhibited similar reductions in body length at most concentrations tested (Figure 5A, lower panel).
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Stimulating Acetylcholine Release From Motor Neurons With Muscarinic Agonists Improved Movement at Days 8 and 10 of Adulthood
Because muscles did not appear to undergo significant structural or functional deterioration between days 2 and 8 of adulthood, we hypothesized that locomotory deficits in older animals may result from deficits in neuromuscular coordination, possibly due to decreased muscle integrity. In this case, stimulating motor neuron acetylcholine release might alleviate the locomotory deficits of day 8 adults. Muscarinic receptors are one pathway for modulating motor neuron acetylcholine release in C. elegans. Arecoline is a muscarinic agonist that can increase acetylcholine release by C. elegans motor neurons (Figure 5B, upper panel) (30). Spontaneous locomotion at days 8 and 10 of adulthood was improved in assays performed in the presence of arecoline (Figure 5B, lower panel). In particular, arecoline treatment of day 10 adults was associated with a significant increase in the fraction of animals that initiated movement away from the original position (Figure 5B, grey bars).
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DISCUSSION |
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Several factors could contribute to the loss of movement between days 2 and 8 of adulthood. Locomotory decline may be a consequence of age-related muscle deterioration, or sarcopenia, which is a feature of aging in C. elegans and other organisms (6). Alternatively, age-related declines in muscle function between days 2 and 8 of adulthood may precede significant structural deterioration. We investigated these possibilities by examining several aspects of muscle structure and function between days 2 and 8 of adulthood.
Our examination of muscle tissue between days 2 and 8 revealed signs of general decline, as evidenced by increased sarcomere damage when day 8 muscles were stained with phalloidin. Significant structural deterioration was absent from the majority of day 8 muscles when examined by electron microscopy, although the relatively small size of the electron microscopy study precludes elimination of the possibility of structural deterioration at day 8. Nevertheless, these findings suggest that day 8 muscles suffered from frailty, or a general decline in cellular integrity, but had not progressed to the state of severe deterioration characteristic of later ages (6). It is possible that day 8 muscles were more sensitive than day 2 muscles to damage from the manipulations for phalloidin staining. It should be noted that similar structural alterations were observed using two different protocols, suggesting that damage in phalloidin-stained day 8 muscles was not the result of a specific protocol, but may reflect general weakness of the tissue.
The day 8 animals examined in this study did not exhibit obvious defects in the ability to perform sinusoidal movement. However, closer inspection revealed that day 8 animals moved more slowly than day 2 and 4 adults, consistent with earlier studies (4,7). Slower movement alone may not necessarily impair chemotaxis behavior, as slow-moving unc-52(e444) animals are reported to chemotax normally, although slowly, to attractants (10). In addition to having slower movement rates, day 8 animals did not maintain forward movement for long intervals. In these experiments, day 8 animals did move in reverse normally. Interestingly, in the aversive odor assay, day 8 animals took significantly longer to start backing away from the odor than did day 2 animals. One possible explanation for this incongruity is that day 8 animals may also suffer from defects in coordinating changes of direction.
We further examined whether a significant decline in muscle function could be detected between days 2 and 8 of adulthood by evaluating muscle contraction by the nicotinic agonist levamisole. Body-length shrinkage by levamisole has been a useful assay for probing postsynaptic components at C. elegans neuromuscular junctions (26,27,30). Our control experiments with unc-52(e444) and unc-54(e190) animals indicated that muscle function can also be measured by relative changes of body length in the presence of levamisole. The finding that levamisole treatment caused similar reductions of body length at days 2 and 8 is consistent with the hypothesis that day 8 muscles have not suffered from significantly structural deterioration or functional decline. Rather, locomotory impairments at day 8 were better correlated with a decline in cellular integrity, possibly a sign of muscle frailty, evident from phalloidin staining of muscle sarcomeres.
Locomotory declines associated with C. elegans aging were partially remedied by treatment with the muscarinic agonist arecoline. In C. elegans, arecoline treatment has been shown to stimulate motor neuron acetylcholine release (30). The finding that arecoline treatment improved locomotory behavior during aging indicates that body movement in aged animals is not limited by muscle function. If age-related functional declines in muscle were the sole limiting factor for locomotory ability, then we would have expected arecoline to have no effect on locomotory behavior in aged animals. Rather, arecoline stimulated locomotion in adult day 8 and 10 animals. One potential explanation for this observation is that aged muscles may function less efficiently than young muscles in the presence of physiological levels of neurotransmitter. Therefore, we hypothesize that stimulation of neuromuscular signaling by arecoline attained a threshold necessary for a more youthful pattern of behavior. Alternatively, aging may negatively affect neuromuscular signaling. Further investigations are necessary to distinguish between these scenarios.
In this study, we have described several changes associated with early stages of aging in C. elegans, before animals take on the deteriorated appearance characteristic of older ages. This time period may be analogous to the period between young adulthood and middle age in mammals, coinciding with the cessation of reproduction and the onset of general decline. An understanding of the cellular changes that occur early during aging is important, as these events may be the basis for subsequent deterioration as aging progresses. This study showed that the early period of C. elegans aging was characterized by a decline in behavioral responses correlated with the onset of locomotory deficits. After day 4 of adulthood, animals were unable to overcome these locomotory deficits, despite the presence of motivational cues, such as attractive odorants. Thus, these locomotory and behavioral alterations are the earliest signs of age-related declines that culminate with the complete loss of mobility at older ages.
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Acknowledgments |
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Footnotes |
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Received April 13, 2003
Accepted July 30, 2004
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  D. Tsui and D. van der Kooy Serotonin mediates a learned increase in attraction to high concentrations of benzaldehyde in aged C. elegans Learn. Mem., October 30, 2008; 15(11): 844 - 855. [Abstract] [Full Text] [PDF]
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3 cm/1 hour. Hatched bars represent animals that moved away from the attractant. At least 20 animals were tested per trial; data represent the average of n trials, each with an independent population; adult day 2, 1 trial; day 4, 2 trials; day 6, 7 trials; day 8, 3 trials; day 10, 2 trials. Error bars represent variation between trials with independent populations
