Effects of advancing age on the central response of rat facial neurons to axotomyLight microscope morphometry.код для вставкиСкачать
THE ANATOMICAL RECORD 228211-219 (1990) Effects of Advancing Age on the Central Response of Rat Facial Neurons to Axotomy: Light Microscope Morphometry DEBORAH W. VAUGHAN Department of Anatomy, Boston University School of Medicine, 80 East Concord Street, Boston, M A 02118 ABSTRACT Following axotomy, the regrowth of peripheral axons takes longer in older individuals than in young ones. The present study compares central responses of facial motor neurons to a crush injury of the facial nerve in 3-monthold and 15-month-old male rats sampled through 28 days post-crush (dpc). Neuronal somata, nuclei, and nucleoli were measured in 30 pm brain stem sections within subdivisions of the facial nucleus that contain the cell bodies responsible for the movement of the vibrissae. The temporal patterns of change in the size of the three structures were interpreted with reference to the re-establishment of functional connections, i.e., the return of voluntary vibrissae activity, which is delayed by 4 days in the older animals relative to the younger ones. There was no agerelated difference in the pattern of soma1 swelling and recovery, nor was there a n age-related difference in the response of nuclei and nucleoli to axotomy through 4 dpc. Both nuclei and nucleoli increased in size in animals of both age groups, but after 4 dpc in the older animals nuclear enlargement was prolonged and the nucleolar increases were less robust compared to the younger animals. The greatest age difference appeared with the re-establishment of functional connections. In the 3-month-old animals, the resumption of whisker activity coincided with vigorous transient increases in the sizes of nuclei and nucleoli; in the 15-month-old animals, there was little nuclear response to functional recovery and a comparatively small increase in nuclear sizes. Given that signals from the periphery initiate central metabolic changes when the target is reached, this study suggests that the deteriorative effects of advancing may not simply reside in the neuron’s decreased resiliency, but may reside in deficiencies in the signals derived from peripheral supporting cells, the target muscle, or in the reception of those signals. The regeneration of peripheral nerves after injury takes longer in animals of advancing age than in young adults (Drahota and Gutmann, 1961; Black and Lasek, 1979; Pestronk et al., 1980; Komiya, 1981). However, in the central nervous system of the middle-aged adult animal, there is little morphological evidence of advancing age, and the neurons perform their normal functions-those of basic cell maintenance and neurotransmission-quite adequately (e.g., Ishihara and Araki, 1988). The metabolic challenge of axotomy may expose a n age-related deterioration of motor neurons. This report is part of a n analysis of the response of the facial motor system to axon injury in young adult and middle-aged rats and is concerned with the central morphological response of the motor neurons. Axotomy initiates a well-characterized morphological, metabolic, and synthetic reorganization in the cell bodies of extrinsic neurons, referred to a s the axon reaction (e.g., Lieberman, 1971; Grafstein, 1975; Alberghina and Giuffrida Stella, 1987). Reversible axotomy, that in which the axon successfully regrows to the target, involves three successive stages (Brattgard et al., 1957; Engh et al., 1971): Initially, there is a brief 1ci 1990 WILEY-LISS, INC to 2-day period of latency as the cell body responds to the injury and loss of effective contact with its target. During the second stage of the axon reaction, multiple fine axons grow from the site of injury and elongate to the target where they re-establish functional connections. This stage is typically marked by prominent morphological changes, which reflect the neurons’ switch from a mode of homeostatic cell maintenance and neurotransmission to a mode of intense biosynthetic activity necessary to replace the axon (Lieberman, 1971; Grafstein, 1983; Austin, 1985; Alberghina and Giuffrida Stella, 1988). The third stage of the axon reaction begins with the re-establishment of functional contact between the motor neuron and target muscle. At this time, the cell bodies’ synthetic activity produces cytoskeletal proteins for axon maturation and recovery to pre-axotomy diameter (Hoffman et al., 1987; Tetzlaff e t al., 1988) and proteins related to neurotransmission (Kreutzberg et al., 1984) and the repair and reforma- Received November 27, 1989; accepted March 8, 1990. 212 D.W. VAUGHAN tion of receptors to re-establish the motor neurons’ afferent synaptic connections (Sumner, 1975; Rotter et al., 1979; Hoover and Hancock, 1985). The time course and the intensity of the neuronal responses during the axon reaction are influenced by animal species (Cammermeyer, 19691, severity of injury (Soreide, 19811, specific neurons under study (Aldskogius et al., 19801, the animals’ developmental age (Jones and LaVelle, 19861, and the distance between the cell body and the injury (Geist, 1933). In this study, the key to understanding the effects of advancing age on the neuron’s ability to respond to axotomy is to describe and compare the neurons’ response at comparable stages in the axon reaction, not a t specific days post-injury. In the rat facial motor system in both the young and the older animals, all three stages of the axon reaction can be identified within 28 days following crush-induced axotomy. The two age groups of animals used in this study are considered to be “young adult” and “middle aged.” The age of the younger animals in this study, 3 months, represents a n age at which early developmental changes have stabilized (Jones and LaVelle, 1986) and the central nervous system has reached adult dimensions (Peters et al., 1983). In this species, which has a mean lifespan of 25 months (Curcio et al., 19841, the older animals, at 15 months of age, are considered to be middle-aged. These older animals show no outward signs of advancing age other than their increased size. But, despite the weight differences, the skeletal sizes are similar in the 3- and 15-month-old animals, and the distance spanned by the regenerating axons to the denervated muscles is the same (40 mm) in both age groups. ture concentrated fixatives consisting of paraformaldehyde and glutaraldehyde in 0.1 M cacodylate buffer a t pH 7.4 (Reese and Karnovsky, 1967). Following perfusion the animal carcass was refrigerated overnight in a plastic bag to permit fixation to continue in situ, and the brain stem was removed on the following day. The block of brain stem containing the caudal two-thirds of the two facial nuclei was prepared for the serial frozen sections by cryoprotection with 20% buffered glycerol, before sectioning in the transverse plane a t 30 pm. A groove cut into the dorsal surface of the brain stem marked the control side (Fig. 1). Serial sections through the caudalmost extent of the nuclei were mounted in order on glass slides and stained with thionin. Functional Recovery The principal target of the motor neurons examined in this study is the nasolabialis muscle of the whisker pad (Hinrichsen and Watson, 1984; Klein and Rhoades, 1985; Semba and Eggar, 1986). Functional recovery for the purpose of this study was determined by the return of voluntary whisker movement. Morphornetry Neurons in the uninjured facial nucleus served as controls within each animal. Using the caudalmost extent of the right and left facial nuclei in the serial series, individual brain stem sections were selected for the samples of control and injured profiles. Specifically, a level of the nucleus about 15 sections (450 pm) from the caudal end of the nucleus was identified separately on both right and left sides and neuronal profiles located in the lateral and intermediate subdivisions of the facial nucleus were selected for tracing. At least MATERIALS AND METHODS 90% of the neurons in these subdivisions innervate the A total of 28 3-month-old and 30 15-month-old muscles responsible for whisker activity (McCall and Sprague-Dawley-derived male rats (Taconic Farms, Aghajanian, 1979; Semba and Eggar, 1986). Two to Germantown, NY) was used in this study. The survival three sequentially rostra1 sections were examined to period of days post-crush (dpc), with the numbers of attain the designated sample number. For each animal, a t least 45 somata from the control young and old animals, respectively, were as follows: 1 dpc (two 3-month and two 15-month), 2 dpc (three 3- and injured sides were traced with a camera lucida month and five 15-month, respectively), 4 dpc (three attachment to a light microscope and a x 40 objective. 3-month and seven 15-month), 10 dpc (four 3-month Only somata with the nucleus fully within the section and three 15-month), 16 dpc (four 3-month and six 15- were selected for tracing. Since the nucleus does not month), 21 dpc (six 3-month and four 15-month), and shift to a n eccentric position during the facial motor 28 dpc (three 3-month and three 15-month). The ani- neuron axon reaction (Soreide, 19811, it could be exmals of most sample groups were prepared a s part of a t pected that the maximum profile of the soma was inleast two different series so as to avoid unintentional cluded in the 30 pm thick section. Nuclei, and their between-sample variation due to factors other than age nucleoli, were traced using a x 100 oil immersion oband survival times. Within each of the series, surgery jective. Since the diameters of these two structures are was performed on animals of both age groups a t the approximately 18 and 5 pm, respectively, it is possible same “day zero” so that a t the selected survival times, to select only those which are fully within the tissue animals of both age groups were perfused and prepared section. The areas of the traced profiles were subsequently quantified using a graphics tablet and a n Apfor microscopy. The facial nerve on the left side was crushed a t its ple IIe computer in conjunction with morphometry and exit from the stylomastoid foramen between the tips of analytical software designed by Dr. Christine Curcio. Dumont No. 5 forceps for two 10 second periods. With Statistical Methods the first crush the vibrissae were observed to protract, The mean areas for each animal’s injured and control after which they remained paralyzed until axons regrew several days later. For the control operation the somata, nuclei, and nucleoli were subjected to a refacial nerve on the right was exposed but not disturbed. peated measures ANOVA using a n SAS (Statistical At the designated survival time, the animal was Analysis Systems) program. The ANOVA was used to anesthetized and perfused through the aorta with a determine whether a statistically significant difference two-stage perfusion of 38°C dilute and room-tempera- existed in the control values of the three structures, EFFECTS OF AGE ON N E U R O N r m w w E TO AXOTOMY within and between the two age groups, and then to determine the statistical significance of the individual structure's response to the lesion and whether there was significant interaction between the lesion, the animal ages, and the days post-crush. Where the effects of the lesion were found to be significant, paired t-tests were used to determine which individual differences were statistically significant. Since there was no statistically significant difference between the areas of the control structures and no evidence for a contralateral effect, the data have been presented as percent difference from control. RESULTS Functional Recovery The rodents' characteristic rhythmic whisking motion is normally accomplished by the voluntary contraction of intrinsic muscles in the whisker pad which draws the vibrissae forward and, with relaxation of the muscle, the elastic return of the vibrissae back to resting position (Dorfl, 1982). When recovery of function began, such voluntary movement first appeared as the ability to protract the vibrissae partially; that is, the vibrissae swept from their relaxed position against the side of the animal's head through about 45"rather than the normally full arc of about 140". During the 3 to 4 day period following the first signs of vibrissae movement, the animals were able to draw the vibrissae progressively further forward until bilaterally symmetrical whisking behavior was attained. Among the animals of both age groups, the period of recovery was quite uniform. In the 3-month-old animals, this return of function occurred during 12 to 14 dpc. The 15-monthold animals uniformly began recovery at 16 dpc, but full recovery of bilaterally symmetrical whisking was attained by animals on either 19 or 20 dpc. These periods over which recovery progressively occurred are indicated by shaded vertical bars on the graphs in Figures 4 through 6. Light Microscopy During the 28 day period following axotomy, there was no striking qualitative difference in the appearance of the soma, nuclei, or nucleoli other than increases in volume. Nuclear eccentricity did not develop; there was no increase in the number of nucleoli; and there was no apparent diminution of cytoplasmic basophilia. The volume increases were most evident a t 16 dpc and were apparent even a t low-power magnification, as in Figure 1. The changes appeared to be uniform throughout the population of motor neurons in the lateral and intermediate subdivisions of the facial nuclei. Figures 2 and 3 illustrate neuronal cell bodies of a 3-month-old animal (Fig. 2) and a 15-month-old animal (Fig. 3) a t 16 dpc. 213 three levels. Table 2 indicates the sample times (dpc) at which the lesion effects were statistically significant. The response of neuronal cell body areas to axotomy is illustrated in Figure 4.In both the 3-month-old and the 15-month-old animals, following axotomy, the injured neuronal cell body increased in size and reached a peak a t 16 dpc. As evident in Figure 4,there was no age-related difference in the magnitude or in the pattern of this response. As seen in Table 1, a statistically significant interaction existed between the lesion effect and the days post-crush, but the age groups did not influence the significance of the response nor was the interaction of the lesion effects and age and days postcrush statistically significant. There were age-related differences in the patterns of response of both the nucleus and the nucleolus. After 4 dpc both the temporal pattern of the response and the magnitude of the alterations in these two structures were different between the two age groups. As seen in Figure 5, in the 3-month-old animals, the injured nuclei rapidly increased in area during the first 4 days, but afterward they became smaller. Coincident with the first signs of functional connections, the injured nuclei once again increased dramatically in size, and then between 6 and 21 dpc returned toward normal. Such a bimodal response was not present in the 15month-old animals: their injured nuclei showed a gradual increase in area through 10 dpc, followed by a gradual decline. Even a t their maximum size, the older nuclei enlarged no more than the initial increase of the younger ones. Unlike the younger animals' nuclear response, there was little change in the nuclear response coincident with the return of function in the older animals. As seen in Table 1, age itself was not a significant factor in the lesion effect (age may cause little more than the delay in response). The interaction between the lesion, the animals' age, and days post-crush was highly significant. Figure 6 compares the patterns of the nucleolar responses. In the 3-month-old animals the injured nucleoli increased in size gradually through 10 dpc, then dramatically increased through 16 dpc, after which they returned toward control dimensions. The pattern of injured nucleolar change in the 15-month-old animals was bimodal, showing one peak in area a t 4 dpc and a second peak a t 21 dpc, followed by a return toward normal. The two peaks in the size of the 15month-old nucleoli were similar in magnitude and corresponded in magnitude to the peak reached by the 3-month-old nucleolar profiles a t 4 dpc. Table 1 shows that for the nucleoli there was a significant interaction between animal age and the lesion effect. As with the other two structures analyzed, there was highly significant interaction between the lesion and days postcrush, and the lesion, days, and age. Morphometry DISCUSSION The ANOVA of the mean areas of the control somata, nuclei, and nucleoli during the post-crush period showed no statistically significant differences either within or between the two age groups. Table 1 presents, for each of the three structures, the P values derived from the ANOVA for the interactions between the lesion effect and 1)the days post-crush survival, 2) the two animal age groups, and 3) the interaction of all This is the first detailed morphological examination of the effects of advancing age on central changes brought about by peripheral axon injury. Older animals require more time to regenerate injured peripheral axons than young adults, but the reason is uncertain. It has been suggested that advancing age causes a delay in the initial period preceding regrowth (Drahota and Gutmann, 1961) or advancing age causes a 214 D.W. VAUGHAN Figs. 1-3 E F F E C T S OF AGE ON NEURON RESPONSE TO AXOTOMY TABLE 1. P values of interactions Interactions Lesion . day post-crush Lesion . age Lesion day . age ‘ Soma Nucleus Nucleolus <.0001 = .3 = .9 <.0001 = .9 <.0001 <.0001 = ,007 <.0001 reduced rate of regeneration (Black and Lasek, 19791, or age is responsible for both initial delay and reduced rate (Komiya, 1981).With the exception of a study of axotomized facial motor neurons in rabbits by Cammermeyer (1963), in which he reports the early changes were generally more conspicuous and the recovery process less intense in very old animals, we know of no other morphological study of the effects of advancing age on the central changes that accompany the axon reaction. No age difference was detected between the 3-monthold and 15-month-old animals in the initial response of the soma, nuclei, and nucleoli to axotomy. Each of these three structures began increasing in size very soon after axotomy so that by 24 hours each was enlarged. Thus there was no evidence that advancing age, at least through middle age, caused a delay in the neurons’ initial response to injury. However, if there were a n increased latency on the order of that which Drahota and Gutmann (1961) detected in regenerating dorsal root ganglion-1 day versus 1.5 days-the sample points of this study may not have been adequate to detect such a difference. The second stage of the axon reaction, t h a t of axon outgrowth, lasts until the axon re-establishes contact with its target and therefore is of different duration in the two groups of animals. For this study, we roughly equated muscle fiber reinnervation with recovery of function, but recognized that some neurons may reestablish contact with muscle fibers before or after “full recovery” of function. Regardless of that, for the present study we considered that a t the time bilaterally symmetrical whisking occurred the muscle was equivalently reinnervated in both the old and the young animals. This second stage of the axon reaction is the period during which the injured neurons become dedicated to regrowing axons. Acceleration of rRNA synthesis and protein synthesis have been correlated with somal, nuclear, and nucleolar enlargement (Whitnall and Grafstein, 1982; Goessens, 1984; Jones and LaVelle, 1986; Hall and Borke, 1988; Wells and Vaidya, 1989). There Fig. 1. Thirty micrometer section of brain stem from 3-month-old animal, 16 dpc. The notch indicates t h e control side of t h e brain stem. Neuronal cell bodies of the facial nuclei a r e evident in t h e ventrolatera1 regions of the brain stem. The boxed areas indicate the lateral and intermediate regions of the nuclei from which t h e neurons were selected for analysis. x 20. Fig. 2. Neuronal cell bodies from a brain stem section of 3-monthold, 16 dpc animal, photographed with Nomarski optics. a: Control; b: lesion x 4 7 5 . Fig. 3. Neuronal cell bodies from brain stem section of 15-month-old, 16 dpc animal, photographed with Nomarski optics. a: Control; b: lesion. x 475. 215 was no significant age difference detected in the response of the neuronal cell bodies during this time. The unimodal increase and decrease in somal volume have been reported by others (Aldskogius e t al., 1980; Hall and Borke, 19881, although Brattgard and coworkers (1957) find that the rabbit hypoglossal neuronal cell volume did not return to its original value in a linear fashion after attaining maximum volume a t day 6. The volumes of the hypoglossal somata decreased slightly after day 6, then increased a second time, though by a smaller amount, a t 48 days, before regaining original values after 90 days. The pattern of nuclear change during this stage in the 15-month-old animals was similar to that of the 3-month-old animals in that the relative percent increase and subsequent condensation were comparable, but the process was delayed in the older animals. Thus, while nuclei appeared to undergo the same changes in both age groups, they required more time to reach their peak in the older animals. Studies have shown some proteins generated a t this time in the axon reaction are not normally found in the perikaryal cytoplasm of adult neurons, but represent a reappearance of proteins formally present during developmental stages (Griffith and LaVelle, 1971; Skene and Willard, 1981). Furthermore, a P-tubulin mRNA that normally disappears when developmental stages are complete has been found to reappear in regenerating adult dorsal root ganglion cells (Hoffman and Cleveland, 1988) and a n embryonic a-tubulin mRNA has been found to be rapidly induced in regenerating adult facial nuclei, then down-regulated following functional reinnervation (Miller et al., 1989). The ability of regenerating neurons to re-express genes which have been repressed since the end of development is a n intriguing concept as i t relates to this study, for the nuclear differences observed in the regenerating older neurons may be related to a difficulty in re-expressing genes that have been repressed for a long time. The nuclear enlargement observed during this stage could reflect the transient transcription of the mRNA, and the older animals simply take longer to access those parts of the genome in which are located these genes. Age differences in the pattern of nucleolar change during this stage of axon regrowth may be explained by the greater length of this period in the older animals. Enlargement followed by condensation may be a normal nucleolar response that follows the production of necessary ribosomes. Langford and coworkers (1980) described a bimodal pattern in the rate of ribosomal RNA synthesis in the crushed rat nodose ganglion cells: a rapid early increase peaking a t 3 days, a fall to control levels, then a second peak during the second week after axotomy. In this study, the second stage of the axon reaction was about 14 days duration in the 15-month-old animals, but only 10 days duration in the 3-month-old animals. In the young adult animal there may not have been enough time to reveal nucleolar condensation because a new set of demands, signalled by the axon reaching its target, stimulated a second nucleolar expansion shortly after the demands for growing the axon had been met. The third phase of the axon reaction begins with the re-establishment of functional contact between the neuron and target muscle, when the neurons’ synthetic 50 -- 40 -- Soma A r e d A Percent D 1f f e re nc e 30 3 month animals -& 15 m o n t h aniicals -- 70 -- lo -- : . 1 I t 1 , I I I I I I I Nucleus Area 60 50 40 Percent 3o Difference PO 10 70 60 50 40 rt i Nucleolus Area 3 / , , Percent Difference 30 20 10 Fins 4-6 , I , I - - - - A -3- m~ o~n t h animals + 15 month a n i m a l s 217 EFFECTS OF AGE O N N E U R O N RESPONSE TO AXOTOMY TABLE 2. Significance of the lesion at specific days post-crush Soma 3 month 15 month Nucleus 3 month 15 month Nucleolus 3 month 15 month 1 2 ns ns ** ns ns ns ns * *** 4: g *:g :c *: :,: ;*:% ~ :*:,: Day 10 4 :*:,: +. :,:i: ns *** 16 21 s: 1: **1: :*:i: 1: * 1: 1:* *1: :* * ns I: 28 :p:c :j: * * ns ns **.# *:** ns ns, not significant; *P < .05; **P < .01; ***P < .001. activities must switch again. Following functional recovery, many of the parameters found to have changed during the axon reaction begin to return to original levels, such as the mass of the nucleus and its nucleic acid content, and the mass of the cell body and its nucleic acid content (Watson, 1970; Aldskogius et al., 1980). Several investigators have suggested that interaction with the denervated muscle signals a variety of central responses in the neuronal cell body (Brattgard et al., 1957; Engh et al., 1971; Cragg, 1979; Kreutzberg e t al., 1984; Smith et al., 1984; Johnson et al., 1985; Hall and Borke, 1988), including induction of a neurofilament gene that is expressed at this time (Hoffman and Cleveland, 1988) and turning off of the a-tubulin mRNA expression referred to above (Miller et al., 1989). Among the 3-month-old animals of this study, dramatic changes in the nucleus and nucleolus accompanied the re-establishment of functional connections: a vigorous increase in the size of both of these structures followed by a n equally vigorous condensation toward normal levels. Such a n acute morphological response has not previously been reported, but there are a t least three explanations for why this striking response is seen in the present study. 1. Experimental design. Many studies of the axon reaction are designed to reduce or prevent the chance of successful axon regeneration, and so a second peak in the response of central structures simply may not occur because it is the contact with the target that signals the second response (e.g., Engh e t al., 1971). LaVelle Flg. 4. Percent difference in the cross-sectional areas of cell bodies for 3-month-old animals (dotted lines, and 15-month-old animals (solid lines). The data are presented as means 2 S.E.M. The stippled vertical bars represent the periods over which the animals recover bilaterally symmetrical whisking behavior. This occurs between 12 to 14 dpc in the 3-month-old and 16 to 19.5 dpc in the 15-month-old animals. Fig. 5 . Percent difference in the areas of nuclei for 3-month-old (dotted lines) and 15-month-old (solid lines) animals. The data are presented as means i S.E.M. The stippled vertical bars represent the periods of functional recovery as described in Figure 4. Fig. 6. Percent difference in the areas of nucleoli for 3-month-old (dotted lines) and 15-month-old (solid lines) animals. The data are presented as means i S.E M The stippled vertical bars represent the periods of functional recovery a s described for Figure 4. and LaVelle (1958) prevented regeneration after axotomy of hamster facial neurons and they report that the peak central response occurred 4 days after they severed the facial nerve. In the present study, if axons had been prevented from reaching the denervated muscle fibers, the vigorous second response of nuclei and nucleoli would probably not occur, and the peak response would perhaps also occur a t 4 days post-injury. 2. Uniform distance to target. The neuron’s acute response to reaching its target is detectable in this study because the population of neurons examined is relatively uniform with regard to the distance the axons must grow to reach the target. At least 90% of the neurons in the lateral and intermediate subdivisions of the facial nucleus project to the muscles of the whisker pad (Klein and Rhoades, 1985; Semba and Eggar, 19861, and they are furthermore all likely to re-innervate their original targets because the crush injury does not interrupt the external (basal) lamina tubes which guide their direction of growth (e.g., Thomas, 1989). All the motor neurons reach their intended target at about the same time. In a more heterogeneous population of neurons, and with longer distances to grow, as in the case of spinal cord motor neurons responding to sciatic nerve injury, sampling a t a given post-operative time would record a blur of responses from different neurons reaching targets over a broader period of time. 3. Selection of post-operative survival times. In the present study, 1 week after recovery of function, the sizes of nuclei and nucleoli are similar in magnitude to what they were just preceding the reinnervation. The increase in size was detected at about the time of functional recovery. As a n example of this importance of sampling time, Aldskogius and coworkers (1980) may have missed a similar second response in their examination of the transected rat hypoglossal nerve: They sampled up through 14 days post-surgery and then again a t day 28. Reinnervation of the tongue occurs, apparently, by day 21 (Hall and Borke, 1988), and so it is not surprising they detected only unimodal changes in the sizes of the nuclei and nucleoli. In this study, differences attributable to advancing age were most substantial a t the beginning of the final stage of the axon reaction. In the older animals, the peak nuclear increase recorded 2 days following recovery of function was considerably less than that of the 3-month-old animals recorded a t a comparable time. 218 D.W. VAUGHAN Dorfl, J. 1982 The musculature of the mystacial vibrissae of the white mouse. J. Anat., 135:147-154. Engh, C.A., B.H. Schofield, S.B. Doty, and R.A. Robinson 1971 Perikaryal synthetic function following reversible and irreversible peripheral axon injuries as shown by radioautography. J. Comp. Neurol., 142,465-480. Geist, F.D. 1933 Chromatolysis of different neurons. Arch. Neurol. Psychiatry, 29t88-103. Goessens, G. 1984 Nucleolar structure. Int. Rev. Cytol., 87.107-58. Grafstein, B. 1975 The nerve cell body response to axotomy. Exp. Neurol., 48t32-51. Grafstein, B. 1983 Chromatolysis reconsidered: A new view of the reaction of the nerve cell body to axon injury. In: Nerve, Organ and Tissue Regeneration: Research Perspectives. F.J. Seil, ed. Academic Press, New York, pp. 37-50. Griffth, A., and A. LaVelle 1971 Developmental protein changes in normal and chromatolytic facial nerve nuclear regions. Exp. Neurol., 33t360-371. Hall, L.L., and R.C. Borke 1988 A morphometric analysis of the somata and organelles of regenerating hypoglossal motoneurons from the rat. J. Neurocytol., 17t835-844. Hirnrichsen, C.F.L., and C.D. Watson 1984 The facial nucleus of the rat: Representation of facial muscles revealed by retrograde transport of horseradish peroxidase. Anat. Rec., 209t407-415. Hoffman, P.N., and D.W. Cleveland 1988 Neurofilament and tubulin expression recapitulates the developmental program during axonal regeneration: Induction of a specific B-tubulin isotype. Proc. Natl. Acad. Sci. USA, 85t4530-4533. Hoffman, P.N., D.W. Cleveland, J.W. Griffin, P.W. Landes, N.J. Cowan, and D.L. Price 1987 Neuroflament gene expression: A major determinant of axonal caliber. Proc. Natl. Acad. Sci. USA, 84t3472-3476. Hoover, D.B., and J.C. Hancock 1985 Effect of facial nerve transection on acetylcholinesterase, choline acetyltransferase and ['HI quinuclidinyl benzilate binding in rat facial nuclei. Neuroscience, I5t481-487. Ishihara, A,, and H. Araki 1988 Effects of age on the number and histochemical properties of muscle fibers and motoneurons in the ACKNOWLEDGMENTS rat extensor digitorum longus muscle. Mech. Ageing Dev., 45: The author thanks Ms. Ketti K. White, Ms. Claire 213-221. Sethares, and Ms. Chris Waring for assistance in the Johnson, I.P., A.H. Pullen, and T.A. Sears 1985 Target dependence of Nissl body ultrastructure in cat thoracic motoneurons. Neurosci. preparation of this work. This work was supported by Lett., 61:201-205. grant AGO6154 from the National Institute of Aging. Jones, K.J., and A. LaVelle 1986 Differential effects of axotomy on immature and mature hamster facial neurons: A time course LITERATURE CITED study of initial nucleolar and nuclear changes. J. Neurocytol., I5t197-206. Alberghina, M., and A.M. Giuffrida Stella 1987 The relationship between perikaryal synthesis and axonal transport during nerve Klein, B.G., and R.W. Rhoades 1985 Representation of whisker follicle intrinsic musculature in the facial motor nucleus of the rat. J. regeneration. In: Metabolism and Development of the Nervous Comp. Neurol., 232:55-69. System. S. Tucek, ed. J. Wiley and Sons, Prague, pp. 219-228. Komiya, Y. 1981 Axonal regeneration in bifurcating axons of rat dorAlberghina, M., and A.M. Giuffrida Stella 1988 Changes of phosphosal root ganglion. Exp. Neurol., 73t824-826. lipid-metabolizing and lysosomal enzymes in hypoglossal nucleus and ventral horn motoneurons during regeneration of craniospi- Kreutzberg, G.W., W. Tetzlaff, and L. Toth 1984 Cytochemical changes of cholinesterase in motor neurons during regeneration. nal nerves. J. Neurochem., 51:15-20. In: Cholinesterases-Fundamental and Applied Aspects. M. Aldskogius, H., K.D. Barron, and R. Regal 1980 Axon reaction in Brzin, T. Kiauta, and R.A. Barnard, eds. Walter de Gruyter, Berdorsal motor vagal and hypoglossal neurons of the adult rat. lin, pp. 273-288. Light microscopy and RNA-cytochemistry.J. Comp. Neurol., 193: Langford, C.J., J.W. Scheffer, P.L. Jeffrey, and L. Austin 1980 The in 165-177. vitro synthesis of RNA within the rat nodose ganglion following Austin, L. 1985 Molecular aspects of nerve regeneration. In: Altervagotomy. J. Neurochem., 34.531-539. ations of Metabolites in the Nervous System. Handbook of NeuLaVelle, A., and F.W. LaVelle 1958 Neuronal swelling and chromarochemistry, 2nd ed. A. Lajthe, ed. Plenum, New York, vol. 9, pp. tolysis a s influenced by the state of cell development. Am. J. 1-29. Anat. 102:219-241. Black, M.M., and R.J. Lasek 1979 Slowing the rate of axonal regenLieberman, A.R. 1971 The axon reaction. A review of the principal eration during growth and maturation. Exp. Neurol., 63:108features of perikaryal response to axon injury. Int. Rev. Neuro119. biol., 14r49-124. Brattgard, S.-O., J.E. Edstrom, and H. Hyden 1957 The chemical McCall, R.B., and G.K. Aghajanian 1979 Serotonergic facilitation of changes in regenerating neurons. J. Neurochem., 1:316-325. facial motoneuron excitation. Brain Res., 169t11-27. Cammermeyer, J. 1963 Differential response of two neuron types to facial nerve transection in young and old rabbits. J. Neuropathol. Miller, F.D., W. Tetzlaff, M.A. Bisby, J.W. Fawcett, and R.J. Miller Exp. Neurol., 22.594-616. 1989 Rapid induction of the major embryonic a-tubulin mRNA, Tal, during nerve regeneration in adult rats. J. Neurosci., 9. Cammermeyer, J. 1969 Species differences in the acute retrograde 1452-1463. neuronal reaction of the facial and hypoglossal nuclei. J. Hirnforsch., I 1t13-29. Pestronk, A., D.B. Drachman, and J.W. Griffin 1980 Effects of aging on nerve sprouting and regeneration. Exp. Neurol., 70t65-82. Cragg, B.G. 1979 What is the signal for chromatolysis? Brain Res., Peters, A., M.L. Feldman, and D.W. Vaughan 1983 The effect of aging 23: 1-21. on the neuronal population within area 17 of adult rat cerebral Curcio, C., N.A. McNelly, and J.W. Hinds 1984 Variation in longevity cortex. Neurobiol. Aging, 4t273-282. of rats. Evidence for a systematic increase in lifespan over time Exp. Aging Res., I0t137-140. Reese, T.S., and M.S. Karnovsky 1967 Fine structural localization of blood-brain barrier to exogenous peroxidase. J. Cell Biol., 34: Drahota, Z., and E. Gutmann 1961 The influence of age on the course 207-2 18. of reinnervation of muscle Gerontologia, 5t88-109. The nucleolar response in the 15-month-old animal during the same time was also considerably less robust than t h a t of the 3-month-old animals. Thus, when the older neurons had metabolically to switch back and synthesize different materials again, they appeared unable to mount as vigorous a response as the young. The implication from these morphological studies that the older neurons respond less robustly than their younger counterparts to the re-establishment of functional connections has been borne out in a series of our own companion studies of axotomy-induced changes in the activities of acetylcholinesterase (an enzyme related to neurotransmitter functions) and cytochrome oxidase (a mitochondria1 enzyme) within the facial nucleus (Vaughan, 1989).Functional reconnection in the young animals is accompanied by decisive return to normal preaxotomy levels of enzyme activities, while in the older animals there is a delayed and much more gradual return to preaxotomy levels. The conclusion from this study is that the altered response of the aging neurons to axon injury may be in part attributable to a deterioration of the neuron. However, i t cannot be discounted that, on the other hand, the neurons at this stage of the animals' lifespan may be perfectly capable of a full and robust response to axon injury, and the age-related differences detected are attributable to deficiencies in the generation or reception of signals derived from supporting cells in the peripheral nerve or in the muscle fibers of the target. EFFECTS OF AGE ON NEURON RESPONSE TO AXOTOMY Rotter, A,, N.J.M. Birdsall, A.S.V. Bergan, P.M. Field, A. Smolen, and G. Raisman 1979 Muscarinic receptors in the central nervous system of the rat. IV. A comparison of the effects of axotomy and deafferentation on the binding of “H-propylbensilylcholinemustard and associated synaptic changes in the hypoglossal and pontine nuclei. Brain Res. Rev., 1.207-224. Semba, K., and M.D. Eggar 1986 The facial “motor” nerve of the rat: control of vibrissal movement and examination of motor and sensory components. J. Comp. Neurol., 247,144-158. Skene, J.H.P., and M. Willard 1981 Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems. J. Cell Biol., 89t96-103. Smith, C.B., A.M. Crane, M. Kadekaro, B.W. Agranoff, and L. Sokoloff 1984 Stimulation of protein synthesis and glucose utilization in the hypoglossal nucleus induced by axotomy. J. Neurosci., 42489-2496. Soreide, A.J. 1981 Variations in the axon reaction after different types of nerve lesion. Acta Anat., lI0t173-188. Sumner, B.E.H. 1975 A quantitative analysis of the response of pre- 219 synaptic boutons to postsynaptic motor neuron axotomy. Exp. Neurol., 46t605-615. Tetzlaff, W., M.A. Bisby, and G.W. Kreutzberg 1988 Changes in cytoskeletal proteins in the rat facial nucleus following axotomy. J. Neurosci., 8t3181-3189. Thomas, P.K. 1989 Invited review: Facial nerve injury: Guidance factors during axonal regeneration. Muscle Nerve, 12t796-802. Vaughan, D.W. 1989 Age effects on AChE and cytochrome oxidase enzyme activities in axotomized rat facial motor neurons. Anat. Rec., 223.118. Watson, W.E. 1970 Some metabolic responses of axotomized neurons to contact between their axons and denervated muscle. J. Physiol,, 210t321-343. Wells, M.R., and U. Vaidya 1989 Morphological alterations in dorsal root ganglion neurons after peripheral axon injury: Association with changes in metabolism. Exp. Neurol., 104.3-38. Whitnall, M.H., and B. Grafstein 1982 Perikaryal routing of newly synthesized proteins in regenerating neurons: Quantitative electron microscopic autoradiography. Brain Res., 239t41-56.