Plasticity of catecholaminergic neurons in aged rat brain: Reinnervation and functional recovery after axotomy

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Plasticity of Catecholaminergic Neurons in Aged Rat rairx Reinnervation and Functions Recovery After ~otomy’ CAROL J. PHELPS2 AND JOHN R. SLADEK, JR.3 department

ofAnatomy, ~~iversi~ ofRochester School of medicine and Dentistry, Rochester, NY 14442 Received 3 December 1984

PHEZLPS,C. J. AND 1. R. SLADEK, JR. Plusticity of coteddantiwrgic neurons in aged rat bruin: Reinnervation ami functiod recmwy after axotomy. BKAIN RES BULL 13(6) 1727-736, 1984.-Rcgencrzttive growth at the lesion site, rcinmuvntion of 8 target uuckua and fimctioual lessons of recovery were studied in aged (20 and 30 months old) rats Mjcctcd to loq@crm trauscction of catecholamiaergic (CA) fibers which contact and inftuencc neurous of the supmoptic nucleus(SON).SmallbUstersIknife cuts were piaced sMuMaxically just causal and medial to the SON. CA histofluoresctnct, induced by f~~hy~~u~&by~ (FAGLU) or ~~~-fo~&hyde (ALFA) methods, ~88 examined in bypotbabkmus at 2,14,21 and 60 days postsurgiudly. Water consumption, and urine volume and osmo&dity, were monitomd presu&My. and through survival times. Subtotal CA dencrvatioa in the SON, and @pical axonal tmnsmitter “pile-up” at the lesion site, were evident two days a&er surgery. Among these degenerative profilea, which persi&xJ for up to three weeks, fine-sized new fibcn were apparent at the lesion, bc@nning betweca 2 and 14 days, and persisting throqhout tbc period studied. At 21 days, and progressivelythsrssftcr,SON acuronswere rimmedwith fluoresctat varicosites. Water ~ns~~on initkdly was depressed, but returned to pnsuxgical mean k&s by tie days. Urine volume ramed to normal by 32 days. Urine os~l~ty showed a recovery by ~~~1~ three weeks. These functional parameters r&ounded to levels higher than presurgical means smog 20 month old, but not 30 month otd, rats beyond 6 weeks survival, coucurrent with a morphological hypcrinncrvation. The results reaffhm mo@&ogical mgcneration, and support reinnervation and functional rcsovery, which extend considcmbly into the aging process. Catechokimiuc Histofluorescence

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Regeneration

Reinnervation

Lesion

in catechokminergic systems is uniquely demonstrable at the light microscopic level, using higbly sensitive histofluoresceuce techniques such as glyoxyk acid treatment [48] or aqueous fo~~hyd~~y& fixation [ 151.These methods were used, respectively, in previous reports from this laboratory of CA w across mechanical knife cuts in the dopamhtergic turn region C4S)and in the retrochiasmatic medial forebrain bundle 1331. To what extent CA neurons retain a caplecity for re-

THE constnrctive plasticity exhibited by axonal growth during development is classically regarded as absent from the adult mamma&n central nervous system [3S]. Growth following axotomy in the adult has been shown to be limited to localized arborizatioas from severed fibers [4,8] or to sprouting of spared fibers within a lesioned tract 122,343.In particular, the existence of a coilagenous or glial scar at a lesion site has been shown to block axon regrowth [18, 28, SO].Conversely, several receut reports have demonstrated extensive axousl growth across me&anicaI lesions which occur in the course of tissue impkntation [I, 5,29,39,43,44]. Foerster

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process requims fmthcr elucidation. Jew ami e [ 17] found that restoration of CA varkosites in the hail after intraanbraveatricular neurotoxk (6-bydroxydopamine) was compromised in senescent rats, but that such reinnervation did occur. We have reported a considerable degree of repatteming amoug hypo&&mk-a@m CA fibers [33,40] in aged brain, and that regrowth at a ksion site may occur in 20- and even 30 months oki (m.o.) rats [33]. An actor question is whether appn@a& ninnervation and r&ted &st&onal restoration may occur at?er severance of

[14] has shown thst reconstructin of central tracts by severed and tibcqmtly regcmrat& axons may occur even

in the presence of a chronic physical barrier, by means of extensive mwth around the periphery of such a device. Monoamine* systems may be especially robust in theii capacity for plasticity in the aduit CNS [5J. Regrowth by means of co?Iateml sprouting or axonal regeneration, or both, have been demonstrated following chemical [9,17,493 or electro&tic [ 19,231lesions. ft is possible that new growth

“Tbcstudywas supportedby PHS Graut AG a0847 (JKS). ‘Form&y Car& Tutpen. ‘Requestsfor reprints shoukt be addnssuI to Johu R. Slrrdek, Jr., Ph.D., Department of Auatomy, University of Kochester S&toot of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY M42.

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central CA fiber tracts. Functional restoration after axotomy has been reported for neuroendocrine regulation [31,51]; functional and morphological restoration also has been reported to be compromised at hippocampal targets in the aged brain [38]. The present study extends our previous findings [33], where regenerative morphology at a lesion site was observed within 14 days postsurgically in the brains of aged rats. Thus, surgery consisted of bilateral knife cuts designed to interrupt CA axons which are afferent to the supraoptic nuclei (SON) [25, 26, 371. Brains of 20 and 30 m.o. rats were examined for histofluorescence at the lesion sites and at SON after survival intervals of up to 60 days after surgery. Adequate evidence indicates that catecholaminergic afferents to SON influence the secretion of vasopressin, although whether that influence is facilitatory or inhibiting [l, 2, 11, 20, 27, 461 remains controversial. Therefore, experimental aged rats in the present study were monitored for functional indices (daily water intake, and urine volume and osmolality) of vasopressin secretion before lesion placement and during the period of recovery.

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Male Fischer 344 rats were obtained from Charles River Labs, through the National Institute on Aging, at 20 or 30 months of age (months old-m.o.). The animals were allowed at least one week of acclimation to light (12: 12) and temperature (73rTF&controlled quarters, with food and water ad lib, prior to pre-surgical monitoring for water balance function. Neurosurgical procedures were performed after a minimum of four days of such monitoring. Rats were housed individually, in “metabolic” cages, throughout the entire study. The experiments were performed in two separate studies. The initial study included five 20 m.o. and five 30 m.o. rats; the second study used eight 20 m.o. rats.

FIG. 1. Drinking and urine excretion in 20 month old rats after lesion of the medial forebrain bundle. Water intake volume and urine volume were measured prior to, and for 2 to 21 days after lesion placement. Mean (cS.E.M.) 24 hour volumes for intact and shamoperated rats (n=8 at day 1) are shown by the stippled area; individual daily values are represented by lines/symbols as they depart from the mean of remaining rats.

Water Balance Monitoring

Histojluorescencr

Water and urine volumes were measured, to the nearest ml, for the duration of the experiment. Volumes were corrected to 24 hour values, and are reported for days of collection (Figs. 1 and 3). Urine osmolality was determined for each collection day using a vapor pressure osmometer (Wescar Model 5100B); a double measurement was performed on each sample. Osmolality was recorded as mOsm/kg water.

At selected intervals after lesion placement (2. 14, 21 and 60 days), brains of the experimental animals were prepared by induction of native CA fluorescence by one of two methods: formaIdehyde/glutaraldehyde perfusion (FAGLU) using the technique designed by Fumess and colleagues [IS], or aluminum/formaldehyde perfusion (ALFA), as originally described by Loren and associates [21]. For FAGLU preparations, sections were cut in the coronal or sagittal planes, at 30 pm; every 6th section was stained with Luxol fast blue/cresyl violet (Nissl) for brightfield microscopic veritication of lesion placement and for orientation; sections at comparable intervals were examined for histofluorescence. Sections prepared by the ALFA method were freeze-dried [12], paraffin-embedded, and cut at 8 pm in the coronal or sagittal plane; every 10th section was Nissl-stained. and comparable intervals were examined for histofluorescence. Perfusions were all performed on rats deeply anesthetized with pentobarbital (Nembutal, 3m mg/lOO g b.w.).

Neurosurgery Bilateral transection of CA fibers in the medial forebrain bundle (mfb) which contact neurons in the SON was performed. This was accomplished using 135” rotation of a handhewn knife placed stereotaxically caudal and medial to the SON. With each rat’s head held in a position such that the base of the brain was horizontal (nose bar at -3.3 mm), the knife was lowered at 2.0 mm caudal to bregma, 1.2 mm lateral to the superior sagittal sinus, and 10.2 mm ventral to dorsal dura matter; the coordinates were established according to the three-dimensional atlas of Paxinos and Watson [30], taking into consideration the adjustments necessary for cranial enlargement in aged rats. Sham-operated rats were 20 m.o.; sham surgery consisted of anesthesia, securing the animal to the stereotaxic apparatus (tympanic puncture), skin incision and drilling through the calvarium as for knife placement. All neurosurgery was performed under pen-

tobarbitah’chloralhydrate (Equithesin, 3.0 ml/kg b.w.) anesthesia, supplemented with ether as necessary. The method has been described previously [33].

Experimental

Design

Two separate investigations were performed: the first study extended previous findings [33), using an identical histofluorescence technique (FAGLU); the second study was a re-examination of previous observations, using ALFAinduced fluorescence. For investigation of long-term regen-

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FIG. 2. Urine osmolality in 20 month old rats after medial forebrain bundle lesion. Symbols and representation of presurgical/shamoperated mean are as described for Fii. 1. Osmolality of urine aliquots was measured for samples collected every four days of the period monitored. Taken together, the data of Figs. 1 and 2 indicate return within 2 weeks, to levels of water consumption, and to normal volume and concentration of urine.

eration and reinnervation, five 20 m.o. and five 30 m.o. rats (five each) were simultaneously subjected to neurosurgery. Sham-operated animals of each age (2 at 20 m.o., 2 at 30 m.0.) also were performed; these animals were sacrificed at 48 hrs and 21 days after surgery. Among animals designated for long-term survival, 3 of the 30 m.o. rats died prior to planned (60 days) survival time-l prior to, and 2 subsequent to, surgery. Kidney failure (osmolalities were very low in these 30 m.o. animals, and fibrosing nephritis with proteinuria was found), and “typical” aging phenomena, such as testicular inter&al cell tumors, lymphocytic granulation in lung bronchioles, and blood vascular congestion, were diagnosed by veterinary pathology. The reported results (Figs. 3 and 4) include only monitoring of two 30 m.o. rats which survived to 60 days postsurgically. In the second study, 20 m.o. animals were monitored, beginning at an identical date; surgery was performed sequentially according to projected survival time (2, 14, 21 days), and all animals were perfused by the ALFA method, on the same date.

FIG. 3. Drinking and mine excretion in aged rats monitored for 60 days after mfb lesion placement. Points represent mean for 24 hour volumes of groups of 20 month old (n=5) and 30 month old (n=2) rats. Vertical bars at each point show S.E.M. for each group. Presurgical volumes were measured for four days; arrow on the horizontal axis shows day of surgery.

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FIG. 4. Urine osmolality in aged rats monitored for 60 days after mtb lesion placement. Points and vertical bars as described for Pi. 3. Horizontal broken lines represent extension of presurgical means. Osmolality was measured in urine ahquots collected at one to four days for the period covered. The collective data (Figs. 3 and 4) indicate rapid (within 2 weeks) return to presurgical water consumption and urine volume and concentration, and, chronically, a state of negative water balance.

RESULTS

Water Balance The data from pre- and postsutgical monitoring are shown in Figs. l-4; values for 20 m.o. rats monitored up to 21 days postsurgically are given in Figs. 1 and 2. Immediately after lesion placement, water and urine volumes dropped markedly (Fig. 1) to approximately 50%, of mean volumes before surgery, or of the remaining intact control rats. Water intake by lesioned animals returned to normal range by 9 to 10 days after surgery; urine volumes regained presurgical levels by 5

to 8 days. Urine osmolality (Fig. 2) showed a more transient change, rising above group means immediately after surgery, but returning to the less concentrated range measured in intact controls, by 4-5 days. Water and urine values for sham-operated controls were included in the means shown for all rats prior to surgery. Water and urine vahtes for animals sacritked two days after lesion placement are not shown because the changes noted for 2 postsurgical days probably would reflect the effect of anesthesia and surgery.

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IN AGED BRAIN

In Fig. 3, mean volumes of 20 m.o. (5) and 30 m.o. (2 surviving) rats are shown as separate groups. As in the experiment which examined shorter survival intervals, 24 hour water and urine values dropped markedly after surgery. Water intake returned to presurgical levels by 6-7 days. Among 20 m.o. rats, water intake continued to rise for the survival period; a rise above presurgical levels also was noted among 30 m.o. rats, but not until 6 weeks after surgery. Urine volumes, however, for both age groups remained similar, returning to presurgical levels by approximately four weeks postsurgically, and continuing to increase steadily thereafter. Urine osmolalities (Fig. 4) showed a transient fall, then rise, and, after considerabk variability, dropped steadily for the survival period. Dilute urine was particularly notable at six weeks or longer after surgery, when water and urine volumes were elevated. It should be noted that osmolality data for 20 m.o. and 30 m.o. rats was similar, regardless of possible renal pathological deterioration. A calculation of “water conservation*’ was performed with data from 20 m.o. rats, wherein water intake minus urine volume was divided by total water intake: water volume-urine volume x lOO = Y0 ,,conse.vation.. water volume Water conservation maintained a stable level of 67-70% presurgically, increased dramatically to 9% within 4 days, maintained and, in fact, increased conservation to 95-97% at 3 weeks after surgery, and dropped precipitously thereafter, reaching SO-S% water conservation by 60 postsurgical days. Hisrojluorescence Postsurgical patterns of CA fluorescence among rats sacrificed 2 to 2 1 days after surgery were similar to those examined previously, using the FAGLU technique [33]. At the lesion site, typical axonal degenerative profiles of transmitter “pile-up” were evident at two days. Such swollen axons were present rostral and medial as well as caudal and lateml, to the knife cut, which was represented by a tract of yellow (non-CA) fluorescence, typical of inflammation. The degenerative profiles persisted at the 14 and 21 days intervals. In addition, fine-sized fluorescent fibers were present in the region of the lesion at these survival times. Numbers of such small axons increased progressively through the period studied. In the SON, fluorescence density decreased markedly two days after surgery, compared with fluorescence patterns in sham-operated rats. Fluorescence density was increased qualitatively (Fig. S), by 21 days after lesioning in comparison to shorter survival times. SON fluorescence five weeks after surgery was similar to patterns observed in intact and sham-operated rats: varicose fibers entered the nucleus from medial and caudal aspects, showing linearity in both coronal and parasagittal planes. Fluorescent varicosites

appeared in apposition to magnocellular neurons in all areas of the nucleus, including lateral regions which most consistently lost fluorescence after lesion placement. Histofluorescence density in the SON (Fig. 6) was 4-5+, equal to or greater than that seen in intact aged animals. Magnocellular neurons consistently were rimmed with fluorescent varicosities which at 60 days reflected a hyperinnervation. Fine-sized fibers were traceable from the region dorsal to that lateral optic chiasm into the medial SON. The fluorescence pattern in the wet nucleus was qualitatively denser in the nuclei of 20 m.o. than in 30 m.o. experimental animals. This pattern of sprouting at the lesion site, and return of CA fluorescence in the SON, was extended among animals sacrificed 60 days a&r surgery. An abundance of fme-calibre fibers surrounded and traversed the wound site (Fig. 7A). The fibers followed rostral and medial courses over the optic chiasm (Fig. 7B) toward the SON. The numbers of such profiles often exceeded patterns observed in intact and sham-operated animals. DISCUSSION The current observations support the hypothesis that plasticity in neuronal connectivity persists in aged, as well as young adult, mammalian brain. Although aging is generally associated with degenerative phenomena such as neuronal loss [47], decline in certain synaptic terminal fields (7,401 and decreased transmitter synthesis or transport [13, 16, 24.36, 411, evidence for continued dendritic growth [6] and increased target innervation [33,40] also exists. In the present report, the histofluorescence evidence for new growth at a wound site, and restoration of patterns of terminal varicosities at a target nucleus following long-term survival were significant. The lesion site usually was well demarcated, even 60 days after surgery. In each animal, a rich plexus of new fiber growth was apparent at the wound site. The arborization qualitatively was more extensive in 20 m.0. than in 30 m.0. animals. A consistent feature of this new growth was the fmding of fine-sized fibers traversing the knife cut, at which yellow fluorescence indicative of inflammation and moderate gliosis demarcated the wound site. Such traversing fibers were evident by 14 days after lesion placement and their frequency increased with survival time (Fig. 7A). A large number of such fine-sized fibers were observed consistently in the area between the knife cut and the target nucleus (i.e., SON), curving over the rostro-lateral region of the optic chiasm (Fig. 7B). Likewise, the reestablishment of CA fiber arborization in the target nucleus was progressive according to postsurgical interval (see Figs. 5 and 6). The SON in aged rat brain has been shown to lose CA histofluorescence density in lateral and ventral areas which are rich in vasopressin-containing magnocellular neurons [40]. These areas of SON were con-

FACING PAGE FIG. 5. Catecholamine fluorescence is depicted in the SON of sham operated (A), two day post-lesion (B) and 21 day post-lesion (C) rats at 20 months of age. The pattern of varicosities seen in A is typical of that seen in aged brain, i.e., the density of fibers (4) is reduced in comparison to young animals (not pictured). The lipofuscin autofluorescence of magnocelMar neurons is granular and yellow which allows easy distinction from the blue-green fluorescence of the CA varicosities. B. Two days after interruption of the ascending CA fibers, a sharp reduction in fiber density is seen in the SON; a few fibers are seen (+), but are less abundant than in the aged, sham lesioned animal. C. At 21 days following transection the fiber density in the SON is higher than that seen in sham operated animals and is elevated above that reported for aged, non-lesioned rats. Varicosities are so abundant (+) that they tend to obscure the lipofuscin fluorescence in tar8et neurons. OC=optic chiasm. A,B,C x300.

PHELPS

FIG. 6. The pattern of catecholamine fiber hyperinnervation is seen in the frontal plane at middle (A) and caudal (B) levels of the SON at 60 days post lesion. Delicate strands of CA fibers (+) appear in the neuropil surrounding magnocellular neurons. Some target neurons are heavily rimmed with varicosities 0). The fine size of the fibers is suggestive of regenerative growth or sprouting. OC-optic chiasm. A.Bx3.50.

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FIG. 7. A. Delicate, fine-sized fibers (-+) are seen in parasa&ttal sections in the region of the knife cut at 60 days following surgery. The hqe, yellow structures are characteristic of the lesion site. Some swollen catecholamine tibers are present (@ and serve to refiect the regenerative process also. B. Linear pro&s of catechohunhte fibers (+) are seen in a position between the lesion site and the target nucleus. The directionality of these regenerative axons suggests some attempt by the lesioned fibers to reinnervate the SON, although collateral sprouting from remaining fibers in the target region also may contribute to the robust hyperinnervation depicted in Figs. 5-6. A,B,x350.

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sistently further deprived of CA varicosities after the “deafferentation” cut; ventromedial and basal laminal dendritic zones of the SON most often retained some CA innervation after lesion placement (Fig. 5B). Afferents to these areas appear to reach magnocellular neurons via a “transchiasmatic” route; extension of the knife ventrally into the optic chiasm was deliberately avoided. It should be noted that complete deafferentation of the SON by parenchymal injection of ~hydroxydopa~ne consistently has led to animal death within five days [lo]; such a procedure obviates study of morphological or functional recovery. Thus, collateral sprouting probably played a significant part in reinnervation in the present study where partial denervations were performed. Magnocellular neurons in all the regions of SON appeared heavily rimmed with fluorescent varicosities, beginning at 14 days postsurgically (Fig. SC) and continuing progressively to 60 days (Fig. 6). The fluorescence density assessed at 60 days postsurgically was qualitatively denser than the patterns of fluorescence observed in 20 m.o. sham-operated or intact rats. Such a suggested hyperinnervation has been observed in the SON of young adult rats after chemical lesions of the medial forebrain bundle 19,321. The ~ont~bution of peripheral CA innervation to such a pattern has not yet been assessed; sympathetic afferents from superior cervical ganglia remained intact in the present study. Madison and Davis [23] observed that CA reinnervation of hippocampal targets after electrolytic medial septal lesions reflected contribution from both central and peripheral NE systems, and that the cont~bution from central NE neurons stabilized when normal NE concentrations were attained. Ingrowth of peripheral sympathetic inputs to the area continued, reaching (biochemical) concentrations of NE that were well beyond levels found in intact, unlesioned rats. The abundance of CA varicosities found in SON at 60 days postsurgically, compared with SON histofluorescence patterns at shorter postsurgical intervals, may well be the result of a similar phenomenon. The significant contribution of ascending CA fibers is, however, well-established by morphological observations in the vicinity of, and distal to, lesion sites. Functional indices of vasopressin (VP) secretion, rather than actual circulating VP levels, were deliberately employed. Invasive procedures, such as periodic blood sampling in aged animals, were avoided because of possible further effects on vasopressin secretion 2421. The data collected by chronic monitoring reflect mutual influence among parameters: increased water intake was followed by increased daily urine volume; urine osmolality simultaneously was decreased; however, the changes observed within in

AND SL.AL)t;.K

water balance in lesioned animals extended beyond the transient effects of surgical stress. Daily water intake returned quickly to preoperative or nonoperative means among 20 m.o. animals; water intake remained depressed in 30 m.o. rats. Urine volume, however, showed no significant differences between 20 and 30 m.o. animals; it returned to the range exhibited by unoperated rats slowly, but consistently, by 4-5 weeks after lesioning. Beyond 4-5 weeks survival, water consumption and urine vofume increased in all rats; urine osmolality, which reached a peak concentration at this time interval, subsequently began to fall precipitously, reaching relatively low concentration ( 1200 to 1400 mOsm/kg) by 60 days postsurgically. Although water balance in the peripheral renal system of aged rats may be compromised due to common renal pathology. the data indicate negative water balance during chronic postsurgical survivai time. It should be noted that data for urine volume {Fig. 3) and osmolaltiy (Fig. 4) are parallel and nearly identical for 20 and 30 m.o. rats. Such a loss in homeostatic water balance suggests decreasing influence of vasopressin (VP), and consequently, decreased VP secretion by a major brain VP source which normally is influenced by NE. The change in functional parameters corresponded chronologically to increased histofluorescence density in SON. Taken together, the morphological and functional data support the thesis of an inhibitory modulation of VP by NE in SON. It should be noted that the morphological effect of the lesion on the paraventricular nuclei probably was minimal; histofhiorescence assessment indicated a qualitatively decreased density in PVN, but the effect of the knife cut on CA afferents to PVN was slight. The continued influence of VP-secreting PVN neurons thus must be considered in water balance assessment. Moreover, it should be considered that the present data constitute a unique assessment of NE influence upon VP neurons, namely, their influence following regenerative phenomena in aged rats. In conclusion, the capacity for regenerative growth and/or collateral sprouting of CA axons is retained to a significant and remarkable extent in the hypothalamus of aged rats. The morphological phenomena reported here also influence functional parameters indicative of a role for NE on VP secretion.

ACKNOWLEDGEMENTS The authors would like to acknowledge the technical contributions of Ms. Barbara Blanchard, in histofluorescence preparation, and Ms. Janice White, for collecting data on water balance. Ms. Patricia Weilert prepared the final manuscript.

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