Quantitative histochemical and microchemical changes in the adult mouse central nervous system after section of the infraorbital and optic nerves

Brain Research, 406 (1987) 157-170

157

Elsevier BRE 12426

Quantitative histochemical and microchemical changes in the adult mouse central nervous system after section of the infraorbital and optic nerves V.S. Yip 1, W.-P. Zhang 2., T.A. Woolsey 2 and O.H. Lowry 1 1Department of Pharmacology, Washington UniversitySchool of Medicine, Saint Louis, MO 63110 (U.S.A.) and 2.]. L. O'Leary Division of Experimental Neurology and Neurosurgery and McDonnell Centerfor Studies of Higher Brain Function, Washington UniversitySchool of Medicine, Saint Louis, MO 63110 (U.S.A.) (Accepted 5 August 1986)

Key words: Whisker; Oxidative enzyme; Denervation; Trigeminal system; Visual system

The sensory projections from the whiskers of mice and other rodents synapse somatotopicaily in 3 subnuclei in the brainstem trigeminal complex, in the ventrobasal complex of the thalamus and in the somatosensory cortex. Deafferentation of the whiskers in adult animals results in qualitative and quantitative changes in activities of the metabolic enzymes in the somatosensory cortex (e.g.J. Neurosci., 1 (1981) 929-935). We determined the time course and extent of changes in the subcortical trigeminal centers of adult mice after deafferentation. The right infraorbital nerve was sectioned in mice under surgical anesthesia; the animals survived for periods up to 26 weeks. The optic nerve was also cut to evaluate the effects of central tract section. Some brains were prepared histochemically for the mitochondrial enzymes cytochrome oxidase (CO) and succinic dehydrogenase (SDH), and some were prepared for microchemical analysis of the enzymes citrate synthase (CS), malate dehydrogenase (MDH) and phosphorylase. All deafferented and intact nuclei were examined in each animal quantitatively. The oxidative enzymes (CO, SDH, CS and MDH) that were analyzed by histochemical and microchemical approaches showed a decrease in activities as early as 3 weeks postdeafferentation, a trend that continued up to 12 weeks in all the subcortical trigeminal stations and lateral geniculate nucleus (LGN) when compared with the intact side. By 25 weeks postlesion, the levels were comparable to the intact side except that in the LGN, the levels remained depressed. The phosphorylase levels increased at around 3 weeks postoperation and remained elevated 25 weeks postlesion. Each case provided results on the effects of deafferentation at a given time point throughout the trigeminal pathway. Direct quantitative correlation of histochemical and microchemical approaches for glycolytic enzymes is consistent with a coordinate regulation of these molecules. The changes in enzyme levels in all nuclei occur simultaneously and to a similar degree. This strongly suggests that neuronal activity plays an important role in regulating metabolic machinery throughout this pathway in adults.

INTRODUCTION Metabolic processes underlie the functional characteristics of the nervous system and the two a p p e a r to be tightly coupled. R e g i o n a l correlations between various metabolic indicators and nervous system activity have been d e m o n s t r a t e d in m a n y species and neural systems in developing and m a t u r e brains. A l tering nervous system function over a long time peri-

od m a y induce changes in the levels of metabolic enzymes which could in turn be used as a measure of integrated activity over time. Histochemical staining and direct biochemical analysis are two a p p r o a c h e s for studying long-term changes in metabolic enzymes. Histochemical methods (e.g. refs. 7, 16) have been e m p l o y e d widely as metabolic markers. F o r example, succinic dehydrogenase and cytochrome oxidase recently have

* Present address: Shanghai Institute of Physiology, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai, China, People's Republic of China. Correspondence: V.S. Yip, Department of Pharmacology, Box 8103, Washington University School of Medicine, 660 South Euclid Avenue, Saint Louis, MO 63110, U.S.A. 0006-8993/87/$03.50 (~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)

158 been used to study the trigeminal 6,27 and visual pathways 1°~'2~'25. For a variety of technical reasons, it is relatively difficult to use histochemistry for a quantitative estimate of actual enzyme activities and for determination of the activities of a number of related enzymes from the same histological specimen. Not all enzymes can be demonstrated histochemically, Microchemical methods can provide quantitative measurement of a variety of enzymes, substrates and other materials from small samples. One goal of this study was to compare the two approaches quantitatively. The trigeminal system of the rodent has a number of features which make it particularly favorable to study long-term metabolic consequences of altered functional activity. The sensory pathways from the whiskers in the mouse are highly ordered. Vibrissal representations can be demonstrated histologically in the caudal and interpolar parts of the spinal nucleus of V (nVc and nVi), principal sensory nucleus of V (nVpv), ventrobasal complex of the dorsal thalamus (VB) and layer IV of the primary somatosensory cortex (e.g. refs. 1, 20, 28). In the mature animal, section of the infraorbital nerve has little or no effect on the morphological integrity in the involved structures of the trigeminal pathways. Previous studies have shown that sensory deprivation and deafferentation of the whiskers significantly alters the metabolic status of the somatosensory cortex 3,27. As this cortex is 3 synapses removed from the sensory periphery, a second goal was to demonstrate metabolic changes in the ascending pathway. The present study assessed the biochemical effects of infraorbital nerve section on the cortical and subcortical relay stations of the trigeminal pathway. For comparative purposes, the optic nerve was sectioned also and the effects of cutting this central tract on the dorsal lateral geniculate nucleus (LGN) examined. The effects of deafferentation were demonstrated thus: (1) the activities of the oxidative enzymes cytochrome oxidase (CO) and succinic dehydrogenase (SDH) were assessed by histochemical staining, and (2) the activities of the energy-related enzymes, malate dehydrogenase (MDH), citrate synthase (CS) and glycogen phosphorylase, were measured quantitatively by microchemistry. The results from the histological and direct biochemical analyses were highly correlated in time

course and extent. The findings indicate remarkable metabolic plasticity of the central nervous system of adult animals. MATERIALS AND METHODS

Animals One hundred adult Swiss Webster mice of both sexes weighing 20-30 g were used. Ten were normal controls; the balance had the right infraorbital and optic nerves divided surgically. The operations were successful in 55 of these animals. The operated animals were divided into 3 groups, roughly equal in number, for SDH, for CO and for microchemical analyses. Initially, one animal was examined by each approach at 1, 2, 3, 4, 5, 6, 8, 10 and 12 weeks and 6 months (22-26 weeks) after the lesions to ascertain the time courses of the changes. From this survey, postlesion time points of 1, 3, 6, 8 and 12 weeks and 6 months were selected for further analysis. For the quantitative studies: (1) all CNS loci were recovered from the same specimen; and (2) the plane of section was perpendicular to the midline in order to obtain symmetrical sections. We obtained good specimens for each histochemical method and for microchemical analyses at each time point.

Surgical procedures Surgery was performed under anesthesia with antisepsis using a dissecting microscope. The right eye was removed after ligating the optic nerve and the eyelids sutured. The infraorbital branch of the trigeminai nerve on the right side was isolated at the infraorbital foramen, tied twice before being cut between proximal and distal ties and the incision was closed. The animals survived for 1-26 weeks; they were examined daily for signs of infection at suture lines. Animals prepared for histochemistry were anesthetized prior to systemic perfusion; those studied with microchemical methods were decapitated. After the brain was removed, all heads were fixed in 10% formaldehyde by immersion. Postmortem examination of the orbit and face was made of every case. At longer survival times, the proximal ligature on the infraorbital nerve was found embedded in a neuroma and the distal branches of the nerve were barely visible as connective tissue ghosts. For the cases described in this paper, the division of the infraor-

159 bital and optic nerves was complete and no evidence of regeneration was found with the dissecting microscope.

to those on the intact side were obtained from normal animals and from animals at 1, 3, 6, 12 and 24 weeks postdeafferentation.

Histochemistry

Microchemistry

Details of the succinic dehydrogenase (SDH) and cytochrome oxidase (CO) histochemical methods have been described 6'11. For SDH, animals were anesthetized and perfused transcardially with 0.9% saline followed by 10% glycerin in 0.5% formaldehyde. After removal, the brains were immediately frozen by immersion in heptane, cooled in dry ice. Fifty-/~m transverse serial sections were cut in a cryostat at - 2 0 °C. Sections were collected on slides, dried on a hot plate at 37 °C, and stained with 0.55 mM nitroblue tetrazolium-0.05 M sodium succinate in 0.05 M phosphate buffer at pH 7.2. For CO, animals were perfused with 0.1 M sodium phosphate buffer (pH 7.4), followed by a mixture of 1.5% glutaraldehyde, 2.4% paraformaldehyde, and 4% sucrose in 0.1 M phosphate buffer at pH 7.4. The brains were sunk in 30% sucrose in phosphate buffer, sectioned on a freezing microtome at 50 p m and the sections incubated in a cytochrome-c-diaminobenzidine-sucrose solution 24 for 20-40 min. The sections were then mounted on slides, air dried, defatted and cover-slipped.

The animals were decapitated and the brains removed and quickly frozen in liquid nitrogen. The tissue blocks were sectioned in a cryostat at -20 to -25 °C at a thickness of 20 pm; the sections were dried under vacuum at -35 °C and stored at - 7 0 °C. Special effort was made to dissect samples which were free of major fiber bundles. We assayed samples which covered an average area of 80 × 80/~m and weighed only 20-40 ng. The coefficient of variation for samples from the same nucleus averaged 10%, i.e. the standard error for averages of 5 samples was about 5% of the mean. The initial steps of the enzyme analyses were carried out under oil in Teflon 'oil well' racks 13. The analytical methods are based directly or indirectly on pyridine nucleotide enzyme reactions 2. The method for CS (EC 4.1.3.7.) is modified from that of Dietrich et al. 3, chiefly by doubling the substrate concentrations, increasing the ZnC12 concentration to 0.4 mM, and increasing the citrate lyase concentration to 50pg/ml. The MDH (EC 1.1.1.37) method is essentially that of Hintz et al. 9, except that KCI was omitted from the preincubation reagent. The phosphorylase (EC 2.4.1.1.) method was adapted from one used for analysis of muscle fibers 12. Appropriate internal standards were included in each assay. Assays for the different enzymes were performed on the same or adjacent serial sections. Control values for these 3 enzymes were obtained from unopcrated adult mice (n = 3). For the experimental animals, 4-5 samples from both the deafferented and the intact sides were paired for each animal.

Evaluation of the histochemistry All histochemical preparations were evaluated qualitatively on a section-by-section basis. Sections were scored as to whether the stain in the deafferented structures was less dense, the same as or denser than that on the intact side. Other structures, i.e. striatum, connected to the trigeminal and visual pathways, often showed systematic histochemical changes in response to the deafferentation. The optical density (OD) of the 6 brain areas was measured with a densitometer attached to a Leitz Ortholux II microscope. A rectangular window was positioned over the brain region and its size was adjusted so that the area of interest was covered (see Fig. 1). A 490-nm monochromatic filter (blue) was employed to increase contrast of the CO-stained sections. Transmittances of 10 sections from each side of the brain were averaged and optical density was calculated. The ratios of OD's of the deafferented side

Numerical analyses Photometric data are plotted as simple normalized averages (%) obtained from 10 sections from each location in each specimen. Measurements of enzyme activities were grouped according to brain locus, days postdeafferentation and enzyme studied. For each brain 3-5 samples were used to compute a mean and S.E.M. Enzyme levels on the intact side of lesioned animals (n = 7) were compared to those from control animals (n = 3)

160 by Student's t-test (see Table I). Linear regression analyses were used to evaluate the correlation between the experimentally induced changes shown in Figs. 4 and 6. Data were paired according to the postoperative survival time and then values from one data set plotted against those from the second data set and fitted with a regression line. Results obtained with a particular method at different brain loci and results obtained with different methods at a particular brain locus were compared (Figs. 7 and 8). In all, 91 such pair-wise comparisons were made and selected examples are illustrated in Fig. 7. The results of these calculations for each method in different brain loci are tabulated in Fig. 8. The correlations between the different microchemical assays and the two histochemical assays at different times across all anatomical loci were determined by calculating a rank-correlation coefficient. The significance was determined from Student's tdistribution 5. RESULTS

Qualitative histochemistry Sections stained histochemically for CO and S D H at different levels of adult mouse brains were examined in animals 1-26 weeks after right infraorbital nerve section and optic nerve section. The deafferented and intact sides of the relevant parts of the brain were compared on the same section. Fig. 1 shows the staining pattern of the two histochemical methods at 6 weeks postoperation, and depicts the relevant brain structures. All brain loci examined are easily recognized with these stains. There is decrease in staining density in all structures directly related to the lesions for both histochemical methods. Figs. 2

and 3 illustrate the time courses of these changes in staining density in nVpv and VB, respectively. CO staining in nVpv changes as a function of time after the infraorbital nerve lesion. The CO staining intensity for nVpv is paler on the experimental side at 1, 3 and 6 weeks after lesioning (Fig. 2). Dorsal nVpd receives input from the mandibular division of the trigeminal nerve and is not affected by the infraorbital nerve section. This feature is an internal control along the trigeminal pathway (cf. Fig. 2, week 3). As a rule, the relative intensity of the stains fluctuates from section to section. Therefore, from a qualitative standpoint, it is critical to evaluate all sections transversing the structure. Extreme examples of this are shown for the 25-week-old animals in Figs. 2 and 3 where the intact side shown is lighter than the deafferented side. The sections were not reversed in mounting. Regardless of brain area, normal animals show obvious differences in staining density between the two sides in about 20% of the sections as confirmed by O D measurements. These differences are most clearly related to changes in brain architecture rather than to artifacts of the histochemical methods. For nVpv interspersed time points, i.e. 2, 4, 5, 8 and 10 weeks, show appropriate changes. Throughout the trigeminal system the same decrease in staining intensity over the first weeks with a return to normal density half a year after deafferentation was observed. For the L G N the result is different; even 25 weeks after the optic nerve lesion, the staining on the experimental side was less than on the intact side. A series of SDH-stained sections of the ventrobasal complex of the thalamus is shown in Fig. 3. The lighter staining density of the deafferented side was detected at 3 weeks postoperation and was more pronounced in animals after 6 weeks. Like the CO-

Fig. 1. Photomicrographs of histochemicaUy stained transverse sections at different levels of adult mouse brains 6 weeks after division of the right infraorbital and optic nerves. A brain stained for SDH is depicted on the left (A-F); one stained for CO is depicted on the right (A'-F'). Both specimens are reproduced at the same magnification. The scale in A' applies to panels A and A'; that in F' to the remaining panels. The CO sections are smaller due to fixation shrinkage. All panels show histochemical changes (decreased staining intensity) in structures related to the deafferentation relative to the intact structure on the opposite side. The boxes on the unretouched photomicrographs indicate approximate size, location and name (broad arrows) of the areas measured photometrically. In all panels the right side of the section is the right side of the animal. Dorsal is up. A and A': somatosensory cortex. The boxed-in area on the left is within the deafferented barrel cortex. B and B': the dorsal lateral geniculate nuclei which receive a largely crossed direct input from the eye. C and C': the ventrobasal complex, the medial or arcuate portion of which receives input from the contralateral trigeminal nuclear complex in the brainstem. D and D': the principal nucleus of the trigeminal nerve. This nucleus as the other portions of the brainstem trigeminal complex receives direct input from the three divisions of the ipsilateral trigeminal nerve. E and E': the interpolar part of the brainstem trigeminai nuclear complex. F and F': the caudal part of the brainstem trigeminal nuclear complex.

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162 stained materials, all S D H - s t a i n e d trigeminal centers showed a similar time course of changes after deaf-

ferentation with recovery occurring 12-26 weeks postlesion. Like the CO preparations, the S D H materials from normal animals show r i g h t - l e f t differences for ca. 20% of the sections regardless of brain region, in the S D H material there was no recovery of the LGN.

Quantitative histochemistry ( O D measurements) Sections were m e a s u r e d photometrically. The relative O D ' s from the deafferented sides are shown in Fig. 4. S D H density was depressed at 3 weeks postlesion on the deafferented side in all 6 regions. This trend continued for up to 12 weeks but the difference between the sides had d i s a p p e a r e d by 25 weeks except in the case of L G N . The brainstem nuclei, on the other hand, showed a decrease in O D for S D H by 3 weeks, but the staining intensity a p p e a r e d to return to normal by 12 weeks. C O showed similar trends. All the affected nuclei had an initial decrease in O D by 3 weeks, and were similar to those on the intact side by 25 weeks postsection. In nVpv, nVi and VB the staining densities returned to normal as early as 12 weeks postdeafferentation. As with S D H , the densities of C O in the L G N were depressed initially at 3 weeks and r e m a i n e d so even 25 weeks postoperation. The same time course of these quantitative changes is found by section scoring (see Materials and Methods).

Quantitative microchemistry Unfixed, unstained, freeze dried sections used for these analysis are depicted in Fig. 5. The relevant structures are readily identifiable under p r o p e r illumination. The enzyme activities (Table I, Fig. 6) of the deafferented side are expressed in Fig. 6 as a percentage of those of the intact side of the same animal. D a t a for the somatosensory cortex are taken from Dietrich et al. 3. The effects of deafferentation on CS and M D H were very similar in both extent and time course (Fig. 6, Table I). On the deafferented side,

Fig. 2. Photomicrographs of CO-stained transverse sections through the pons of a normal adult mouse and mice at differing survivals after section of the right infraorbital nerve. All panels shown at the same magnification. The division of the nVp into nVpd and nVpv divisions, as indicated in the panel for right side of the 3-week animal, is based on architectural and connectional information ~4. Dorsal is up and medial is along the line dividing left and right panels.

163 there was a decrease in activities of both enzymes from 3 to 12 weeks which averaged 20% for CS and

15% for M D H . The decrease in CS was statistically significant in all nuclei, and that of M D H significant in all nuclei but L G N (Table I). For M D H and CS the maximal decrease in activities was 1 5 - 2 5 % and 2 0 - 3 0 % respectively in all loci except for the somatosensory cortex where the decreases reached 40%. The values had returned to normal 24 weeks postoperation. Phosphorylase activities, on the other hand, demonstrated an opposite trend (Fig. 6, Table I). All deafferented loci studied had increased phosphorylase activity 3 - 6 weeks postdeafferentation which remained elevated even 24 weeks after deafferentation except in the case of LGN. The average phosphorylase increase for 3 - 1 2 weeks combined was 21% and ranged from 13% for L G N to 25% for nVi (Table I). The greatest increases were in nVc at 12 weeks and in VB at 24 weeks (Fig. 6.) Average CS levels on the deafferented side of the operated animals were almost the same as in normal controls, except that in nVc the activity exceeded that of normals by 11% (P ~< 0.05; Table I). M D H was consistently higher than normal in the intact nuclei by 11-36% (average 20%). The average difference for all 5 areas was highly significant (P ~< 0.01). Therefore it is likely that M D H activity increases are a genuine consequence of the deafferentation of the posite nuclei.

Correlation between brain loci The microchemical results for oxidative enzymes M D H and CS are similar to each other both in time course and extent and are similar to O D changes in the histochemical stains for the oxidative enzymes S D H and CO. As with the enzymes assessed histochemically, the changes observed with the microchemical methods occur in all parts of the trigeminal system at about the same time. The correlations for CS

Fig. 3. Photomicrographs of SDH-stained transverse sections through thalami to show VB (boxes) of a normal adult mouse and mice at differing survival times after section of the right infraorbital nerve which is related to the left thalamus. All panels shown at the same magnification. The white band in most of the sections coursing from dorsolateral to ventromedial separates the arcuate and external divisions of the nucleus which are related to the trigeminal and spinal inputs to this nucleus respectively. The ventrolateral spinal region and the dorsomedial trigeminal regions of the nucleus serve as internal controls for the changes produced by infraorbital nerve lesions. Orientation as in Fig. 2.

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Fig. 4. Graphs showing the results of photometer readings at the different anatomical locations from one animal at each time point (SDH above and CO below). The values of the deafferented side are expressed as a percentage of the values obtained from the intact side, and are plotted as a function of time after deafferentation. Results from the 3 brainstem trigeminal nuclei are on the left and those from the thalamus and cerebral cortex are on the right.

between nVpv and the other nuclei are shown in Fig. 7. All the correlations with other subcortical trigeminal stations are significant (P ~< 0.05). The results of all 50 possible correlations (5 methods, 5 areas) are summarized in Fig. 8. Eighteen (36%) of these were significant at the P ~< 0.05 level and 34 (68%) had correlation at a P <~ 0.20 level. If the L G N is not included, 53% of correlations are significant at P ~< 0.05. When correlation in time course and extent are compared for each e n z y m e , the changes with CS correlate at the P ~< 0.05 level for all parts of the subcortical trigeminal system; CO is next best (67%) followed by M D H (50%), phosphorylase (33%) and

S D H (17%) at the P ~< 0.05 level. Correlations are best in the brainstem trigeminal complex where all nuclei receive inputs from the same source. Taken together, the correlations suggest that for the trigeminal system most of the deafferentation-related enz y m e changes, regardless of the e n z y m e , occur at about the same time and to the same extent.

Correlation between methods To evaluate the correspondence of the different methods, the r a n k - o r d e r correlation was used across all observations taken with each method, histochemical and/or microchemical, in all 10 possible combinations. The best correlations were observed between

165 pairs of m e t h o d s for oxidative enzymes, i.e. CS, M D H , C O and S D H as follows: CS vs M D H , P ~< 0.005; CO vs S D H , P ~< 0.005; CS vs S D H , P ~<

0.025; and CS vs C O , P ~< 0.025. M D H did not correlate significantly with either of the histochemical methods. Phosphorylase c o r r e l a t e d negatively with all of the oxidative enzymes but was not statistically significant. DISCUSSION The levels of the metabolic enzymes were determined in the subcortical trigeminal stations and the L G N . In normal animals CS and M D H levels were almost identical in all the regions examined, but this was not true of phosphorylase, which was twice as high in the 3 brainstem nuclei as in VB and L G N (Table I). A s the brainstem trigeminal nuclei are the first synaptic stations in the trigeminal pathways they may be m o r e likely to mobilize stored glycogen when there are rapid and p r o l o n g e d changes in primary afferent stimulation, than m o r e central structures are. Previous studies have shown that vibrissal deafferentation or whisker hair removal result in significant biochemical changes of the barrel field in the somatosensory cortex 3,4'27. The present p a p e r examined the effects of infraorbital nerve lesion in adult animals on enzyme levels in the subcortical nuclei related to the whiskers. A l t h o u g h there are no m a j o r morphological alterations in the whisker pathway as a result of infraorbital nerve lesions in m a t u r e animals, the related nuclei are metabolically plastic as demonstrated by C O and S D H staining and quantitative measurements of CS, M D H and phosphorylase activities. The changes in the subcortical nuclei are similar to findings in the barrel cortex 3,27.

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Fig. 5. Twenty-pm, cryostat-cut, freeze-dried, unstained sections through the mouse brain to show the locations of structures sampled for microchemical assays. All panels reproduced at the same magnification; scale shown in E. A: horizontal section through the forebrain at the level of the dorsal thalamus. Anterior is up. In this section VB is seen on the left and LGN on the right as the section plane was not perfectly horizontal. B: transverse section through the pons at the level of the facial nerve. The facial nerve, trigeminal tract and the cerebellar peduncles outline the nVp. Dorsal is up. C: the section in B after dissection of small (20-40 ng) samples for chemical analysis. The arrows show the sites from which the tissue was taken. D: transverse section through the open medulla at the level of the nVi. The location of this nucleus is determined in part from the typical cross-sectional profile of the medulla at this level. Dorsal is up. E: transverse section through the closed medulla below the obex. The pd serves as a useful internal landmark to aid in the identification of the caudal part of the nVc. Dorsal is up.

166 TABLE 1 Comparisons of absolute values of enzyme activities in brain structures in normal animals and from the intact and deafferented sides of experimental animals

The data are from 3 normal (not operated) animals and 7 operated animals, two each at 6, 8 and 12 weeks postoperation and one at 3 weeks. Four or 5 samples were assayed from each nucleus of each animal. Activities were measured at 20 °C. The average differences are shown for all nuclei combined between normal and intact and between intact and deafferented. Values are in mol/kg dry wt./h, mean _+S.E.M. Enzyme

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17.9 + 2.43 19.2 + 3.14 16.5 + 1.26 17.9 + 2.02 17.2 + 0.86 +3.56 + 0.68*

Glycogen phosphorylase

nVc nVi nVp VB LGN Average difference

0.57 0.07 0.16 0.84 0.90

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10.6 + 0.30 10.7 + 0.34 10.3 + 0.49* 10.3 + 0.63 10.2 + 0.62 +0.46 + 0.32 -2.00 + 0.05** 21.9 + 0.90 21.6 + 0.68 22.5 + 1.30" 20.9 + 0.99 19.6 + 0.65* -3.28 + 0.68**

8.5 + 0.34** 8.6 + 0.43** 8.5 + 0.58** 8.3 _+0.43** 8.2 + 0.43** 18.0 + 0.52** 17.6 + 0.80** 18.2 + 1.27"* 17.3 + 0.67** 19.0 + 0.67

0.741 + 0.030 0.752 + 0.030 0.941 + 0.678 + 0.012 0.682 + 0.030 0.859 + 0.657 + 0.020 0.724 + 0.034 0.851 + 0.270 +- 0.020 0.288 + 0.021 0.331 + 0.367 + 0.023 0.445 + 0.037 0.521 + +0.036 + 0.015 +0.121 + 0.029'*

0.047** 0.050** 0.028** 0.021 0.018"*

* Significantat P ~<0.05 for intact vs normal. ** Significantat p ~<0.05 for deafferented vs intact.

The initial decrease in oxidative enzyme activities

response to the need for an increase in oxidative ca-

after infraorbital nerve lesion, when compared with

pacity, with a trade-off in glycolytic capacity. The

the intact side, may be an adaptive response to decreased n e u r o n a l activity as suggested by Wong-Riley 23-25. After long-term survival, the enzyme actiw-

fact that changes in the opposite direction occur in deafferented brain nuclei fits exactly with this interpretation. The changes are such as to leave the brain

ties return to levels comparable to those on the intact side. In preliminary electron microscopic studies (Ma, unpublished observations in our laboratory)

less able to maintain a steady high level of metabolism, yet still able to respond to short-term demands by converting glycogen to lactate. Deafferentation could disrupt transport of 'trophic' factors and n e u r o n a l uptake which may be important in maintaining ' n o r m a l ' metabolic functions. The trigeminal brainstem nuclei are the first central synaptic target for the trigeminal ganglion neurons. The ventrobasal complex receives afferents from the opposite brainstem. The data presented do not indicate a sequential decrease or increase in the enzyme activities. Rather, the changes observed are simultaneous in all nuclei. This could indicate that the long term metabolic functions are activity-related21-26. However, simultaneity does not rule out a role for the effects of putative trophic factors because they

there is no evidence of sprouting in the brainstem trigeminal complex. The restoration of ' n o r m a l ' activities suggests that the metabolic machinery has reequilibrated by maintaining a balance between synthesis and degradation of a group of enzymes resulting in steady-state levels close to normal. The 4 oxidative enzymes we examined in the brain all respond similarly to deafferentation. In skeletal muscle, for example, continuous low-frequency stimulation of a fast-twitch muscle results in a coordinated increase in enzymes of oxidative metabolism, accompanied by a decrease in enzymes of glycogenolysis, including phosphorylase TM. This is an adaptive

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Fig. 6. Graphs showing the results of microchemical analyses at different anatomical locations in deafferented nuclei. T h e results after different survival periods are plotted as the ratio of the levels found on the side related to the deafferentation to the levels found for the contralateral intact structure of the same brains. T h e data are for two mice at 6, 8 and 12 weeks and one m o u s e at 1, 3 and 24 weeks postoperation. Values from the 3 brainstem trigeminal nuclei are shown to the left and those from the thalamus and cortex are shown to the right. (Values for the cortex are taken from Dietrich et al.3.)

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could act in c o n j u n c t i o n with n e u r o n a l activity. T h e vibrissae are a m a j o r sensory i n p u t for the m o u s e . W i t h d e a f f e r e n t a t i o n o n o n e side of t h e face, the m o u s e m a y c o m p e n s a t e for the deficit by using the intact side m o r e , as s u g g e s t e d by D i e t r i c h et al. 4 to explain the e n z y m e c h a n g e s in t h e c o r t e x on t h e intact side after u n i l a t e r a l w h i s k e r clipping. T h e present results s h o w o n l y a c o n v i n c i n g i n c r e a s e in M D H in subcortical stations and a significant i n c r e a s e in CS in n V p v . P r e l i m i n a r y m i c r o c h e m i c a l d a t a for t h e barrel c o r t e x h a v e n o t s h o w n an i n c r e a s e in o x i d a t i v e enz y m e levels o n the intact side after infraorbital n e r v e section.

Fig. 8. Correlations similar to those shown in Fig. 7 for all methods (left) between all anatomical loci (top and left). Key at the bottom of the figure indicates the P levels determined by the Student's t-test, calculated from the correlation coefficients with 4 or 5 degrees of freedom. Each correlation is plotted twice so that the results obtained with each method in each locus can be read across and down the figure. Of all 50 correlations, 36% are significant at P ~< 0.05. The correlations are highest between structures related to the trigeminal system and for the CS, CO and MDH methods. Changes in the LGN correlate poorly with those observed in the trigeminal system. In general, these results suggest that the time course and extent of change after infraorbital nerve section observed with a particular method are very similar at all levels of the subcortical trigeminal pathway.

169 The LGN is the first synaptic relay for retinal ganglion cells, as is the brainstem for the trigeminal ganglion cells. The LGN and brainstem both show initial decreases in MDH, CS, SDH and CO activities after deafferentation and increase in phosphorylase activities at the earlier time points. Half a year postoperation the initial changes in the LGN persist. Division of the optic nerve severs a central nervous system tract, whereas the division of the infraorbital nerve cuts a peripheral nerve. After peripheral lesions of the trigeminal nerve there is modest central terminal degeneration 8 and no evidence for transneuronal cell degeneration in the brainstem (Ma, personal communication); optic nerve section results in prompt degeneration of retinogeniculate terminals 15 and a profound transneuronal atrophy of the relevant neurons in the LGN 19. Whether the differences we observe in the long-term metabolic responses after deafferentation of the LGN and the brainstem are related to these factors, to the different patterns of inputs to the LGN and brainstem from the periphery or elsewhere, or to how the rodent uses these two different sensory channels is unclear. Our findings, in the LGN are consistent with those of Onoda et a1.17who reported that 6 months post-olfactory bulbectomy in rabbits, which interrupts a central tract, the CO stain density of the olfactory cortex remained depressed. We have described the metabolic consequences of primary afferent denervation throughout the trigeminal system and have some evidence on the time-dependent characteristics of the LGN after deafferentation. Measurements taken on these pathways using a combination of microchemical and of histochemical methods for 4 different but metabolically related oxidative enzymes are largely similar in the direction and extent of the changes produced. The present study is the first, to our knowledge, that has documented these metabolic changes in the brain using a combination of quantitative approaches. The corre-

LIST OF ABBREVIATIONS CB cc cp DG dLGN fx hc

cerebellum corpus callosum cerebral peduncle dentate gyrus dorsallateral geniculate nucleus fornix hippocampalcommissure

spondence is useful information for a fuller understanding of the metabolic consequences of altered functional activity throughout a central nervous system pathway. The mechanism for these changes and particularly their coordination is a matter for speculation. Is it the rate of enzyme synthesis or of breakdown or both that is altered? Is there a single message generated having opposite effects on the oxidative and glycolytic enzyme groups? We point out these obvious possibilities here only because the relevant technology for proving one mechanism or the other is now available. As it appears from our data that long-term changes in the 'house keeping' proteins of neurons in different parts of the nervous system are susceptible to change, and that these changes can occur in adult animals, it would seem to be interesting to investigate the mechanisms of these changes in both the normal economy of the central nervous system and in relation to altered central nervous system function in disease.

ACKNOWLEDGEMENTS This work was supported by grants from the United States Public Health Service (NS07129, NS08862, EY02294 and P01 NS17763), the American Cancer Society (BC4-29) the Sarah and O. William Lowry Fund, the McDonnell Center for Studies of Higher Brain Function and the McKnight Foundation. W.-P.Z. was partially supported by the Division of Biology and Biomedical Sciences of Washington University as a visiting scholar. We wish to thank Mike Noble and Spencer Countess for preparing some of the animals, Qiao Yan for early analysis of some of the histological materials, Dr. R. Collins for the microscope photometer, Richard Robb for computing, Joe Hayes for photography and Margo Gross for secretarial assistance.

hpt ic IOC ml NA nTB nnVIII nVc nVi

habenulopedunculartract internal capsule inferior olivarycomplex medial lemniscus nucleusambiguus nucleusof the trapezoid body cochlearnuclei spinaltrigeminalcomplex interpolar part of spinal trigeminaicomplex

170 nVIl nVmot nVp nVpd nVpv nX nXII

facial nucleus trigeminal motor nucleus principal nucleus of the trigeminal nerve dorsal nVp ventral nVp dorsal motor nucleus of the vagus nerve hypoglossal nucleus

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pd SEPT Sml ST VB vii vLGN

pyramidal decussation septal nuclei first somatosensory neocortex corpus striatum ventrobasal complex seventh nerve ventrallateralgeniculate nucleus

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