Effect of phenobarbital on Purkinje cell growth patterns in the rat cerebellum

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Effect of Phenobarbital on Purkinje Cell Growth Patterns in the Rat Cerebellum R. S. HANNAH,

S. H. ROTH, AND A. W. SPIRA’

Departments ofAnatomy and ofPharmacology & Therapeutics, University of Calgary, Calgary, Alberta, Canada T2N 4NI Received May 29, 1987; revision received September 24, 1987 The effects of low level phenobarbital administration (18 postcoitus to 2 1 days postnatal) on Purkinje cell growth and remodeling were studied from 3 to 20 weeks postnatal). The Purkinje cell dendritic trees were analyzed both metrically and topologically using the method of vertex analysis. The total segment length, mean terminal path length, and mean vertex path length were reduced in the treated cells. The pattern of segment frequency as related to equivalent orders was abnormal in the treated cells. The Va/Vb vertex ratios and the levels of trichotomy indicated that the treated cells underwent nonrandom remodeling, unlike the control cells which exhibited dichotomous, random terminal branching. These observations confirm that phenobarbital produces distinct long-term morphologic alterations in Purkinje cells. o 1988 Academic

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INTRODUCTION

Phenobarbital, when administered to prenatal and neonatal rats produces long-term morphologic changes in the cerebellum (6). Our purpose was to examine the growth and plasticity of Purkinje cell dendritic fields that follow early administration of phenobarbital. Most previous investigations on the neonatal effects of phenobarbital used the mouse as the experimental animal together with relatively large (to 60 mg/kg) daily doses of phenobarbital. Morphologic alterations in the mouse cerebellum included a reduction in cerebellar size ( 15, 17), a reduction in the Purkinje and granule cell population (15), and a reduction in the number of Abbreviations: pn-postnatal, VD, VT-di, trichotomous branch points; VR-vertex ratio. ’ The authors gratefully acknowledge Mrs. D. Foster for technical assistance. This work was supported by The Alberta Mental Health Council. 354 0014-4886/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Purkinje cell dendritic spines (16). Only a few investigators have studied this problem using the rat. Shain and Watanabe (12), administering doses of 30 to 60 mg/kg phenobarbital, reported a reduction in rat cerebellar weight with the 60 mg/kg dose. Hannah et al. (6) utilized a much smaller dose (10 mg/ kg) of phenobarbital and reported no weight difference but a 20% reduction in the number of Purkinje cells. In this study we examined the growth and subsequent remodeling of the Purkinje cell dendritic trees under the identical treatment protocol used by Hannah et al. (6). The dendritic trees were analyzed using the method of vertex analysis (2), which not only permits the quantifying of networks but also provides information on modes of both growth and plasticity. METHODS Timed-pregnant Long-Evans hooded rats were utilized in this study. On Day 18 postcoitus, based on Day 1 as the first day, all animals were administered phenobarbital (Phenobarbitone, B.D.H.) (10 mg/kg, diluted with normal saline) or normal saline intramuscularly. Maternal administration of either drug or saline continued on a once daily basis until 2 1 days postnatal (pn). The animals were maintained on rat chow and tap water ad libitum. At birth, the litter size was culled to eight pups to minimize any intralitter nutritional effect or variation in the amount of drug ingested, based on unequal litter size. Entire litters were killed at 3,5,7, or 20 weeks pn. A total of 16 control and 16 treated litters were used in this study. Four treated and four control litters were used at each of four sample times. Four pups from each litter were selected for vertex analysis resulting in a final number of 16 pups in each treated and control group for each sampling time. The pups were anesthetized with pentobarbital (Diabutol), the brains were removed, and processed using Sholl’s Golgi-Cox method ( 13). The cerebella were embedded in celloidin and sectioned at 100 pm. Fully impregnated Purkinje cells were selected from the pyramis. A minimum of 10 cells was analyzed from each control and treated group at every sample time with no more than one cell chosen from any individual animal. The cells were drawn using a camera lucida at a magnification of X 1400. The drawings were digitized using a digitizing tablet linked to a computer (VAX 750). The data were analyzed with a vertex analysis program adapted from that designed by Berry and Flinn (2). Vertex analysis represents a distinct improvement on earlier analytical methods by overcoming the inherent problems of missing branches due to incomplete staining or loss of branches due to sectioning and also allows for inclusion of trichotomous branching. This method permits analysis of dendritic trees on the basis of a small number of nodal vertex types. Dichotomous branch points (VD) are divided into

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FIG. 1. Schematic to demonstrate both the method of ordering and designation of nodal vertices. Beginning at the primary dendrite (l), each successive branch point or nodal vertex is increased by a factor of 1. The three types of dichotomous vertices are Va, Vb, and Vc. Va nodal vertices have two terminal segments, Vb nodal vertices have one terminal segment and one nonterminal segment, and Vc nodal vertices have two nonterminal segments. The four types of trichotomous nodal vertices are also shown (Va’, Vb’, Vc’, and Vd’). Vr or the root vertex represents the axon hillock. r-root vertex (axon hillock), E-nodal vertex (branching point), l pendant vertex (dendrite tip), E-O-pendant arc (terminal segment/branch), E-El-line arc (segment/branch), r--O-root arc (primary dendrite).

three distinct types of nodal vertices, Va, Vb, and Vc, and trichotomous branch points (VT) are divided into four distinct branch points, Va’, Vb’, Vc’, and Vd’ (examples are illustrated in Fig. 1). VTs are arbitrarily defined as a separation distance between two VDs of I pm, or less. Berry and Flinn (2) demonstrated that various types of growth could be determined by evaluating the Va/Vb vertex ratio (VR). Growth by random terminal branching results in a VR = 1, segmental growth produces a VR = 0.5, and collateral growth produces a VR = 0. In order to include trichotomous branches into the Va/Vb ratio, all nodal vertices were transformed into equivalent dichotomous nodes. The vertex analysis method uses a centrifugal ordering system to rank the orders of branches (5). By assigning the root vertex as equivalent order 0 the primary dendrite will be equivalent order 1; with each successive branch point the order number is increased by one (Fig. 1). The term “equivalent orders” means that all segments and vertices of the same order are equivalent in that they are all removed from the root vertex by the same number of segments and vertices. In order to prevent confusion in terminology when discussing vertex analysis we have chosen to utilize the mathematical terms proposed by Berry and Flinn (2). The following definitions are offered: nodal vertex is equivalent to dendritic branch point, root vertex is

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TABLE 1 Effect of Phenobarbital on Purkinje Cell Dendritic Growth”

3 weeks 5 weeks 7 weeks 20 weeks

Control Treated Control Treated Control Treated Control Treated

Mean segment length (w)

Mean total segment length (Nrn)

Total mean no. of segments

VRb

Percentage trichotomy

9.9 (0.6) 8.5 (1.5) 10.0 (0.07) 8.7 (1.5) 10.2 (0.04) 8.9 (1.6) 10.3 (0.05) 9.2 (1.8)

6395 (280) 5 159 (240)* 6470(165) 5684(180)* 6890(176) 5510(198)* 7065 (204) 5466 (190)*

650(12) 590 (21) 720 (19) 578 (15); 725 (21) 585 (25)* 735 (20) 595 (18)*

0.86 (0.03) 0.95 (0.07) 0.92 (0.04) 0.97 (0.06) 0.9 1 (0.03) 1.12 (0.09) 0.94 (0.02) 1.18 (0.06)

6.2 6.0 5.1 4.8 4.7 4.1 4.6 3.1

’ Values are means + (SE). ’ VR-Va/Vb vertex ratio. * Significant to P < 0.0 I compared with values of control animals of the same age (Student’s t test).

equivalent to axon hillock, pendant vertex is equivalent to the terminal of a dendrite, and segment is equivalent to a dendritic branch.

end

RESULTS

SegmentLengths. The mean total segment length (Table 1) is the summation of the length of all segments within the dendritic tree. Both control and treated cells demonstrated a small but significant (P c 0.0 1) increase in mean total segment length between 3 and 20 weeks pn. However, at all time periods examined, the total segment length of the treated cells was significantly (P < 0.0 1) less than comparable control values. Mean terminal path length, which is a measure of the mean length from the tips of all terminal dendrites back to the axon hillock, is an indicator of the overall size of the cell. For control cells at 20 weeks pn this measurement was 179 + 7 pm, with treated cells at the same age being significantly (P < 0.00 1) smaller in size at 16 1 f 11 pm. There was no significant difference in the mean terminal path length between 3 and 20 weeks in either control or treated cells, indicating that other changes observed during this time period, such as the addition of new branches, occurred internally within the tree without increasing the overall circumferential size. The mean segment length for individual branches without regard for equivalent orders is shown in Table 1. The high variation in length of the treated segments negated any statistical difference between groups. However, the high variation was in itself important when segment lengths were plotted

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2. Mean segment length is plotted against equivalent orders for control and treated cells at 20 weeks pn. Compare the near-linear placement of segment length above equivalent order 5 observed in control cells to the highly variable treated-cell values. FIG.

against equivalent order. Figure 2 demonstrates the distribution of segment lengths over equivalent orders for control and treated cells at 20 weeks pn. Although segments proximal to the soma were longer, the lengths in control cells stabilized by the fifth equivalent order and higher equivalent orders at about 10 pm. In contrast, the marked linear symmetry of the control cells was not achieved by the treated cells. The mean vertex path length which represents the mean distance from all vertices of the same equivalent order back to the axon hillock yielded, in this case, a better understanding of what was occurring in the treated cells. It must be noted that the mean vertex path length was not the same as the mean segment length because a cumulative total of all proximal equivalent orders was used in the computation of the mean vertex path length. For example, Fig. 3 compares the mean vertex path length of control and treated cells at 20 weeks pn. Both path lengths were approximately linear, indicating a regular increment in distance from the axon hillock for each equivalent order. However, the increments for the treated cells were significantly (P < 0.05) smaller, thus altering the slope of the line. As with the mean terminal path length, this indicated a smaller field of branching. Segment Frequency. In control cells, the changes in segment frequencies plotted by equivalent orders over time demonstrated a gradual shift to a symmetrical distribution (Fig. 4a). During the period of rapid cell growth (prior

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FIG. 3. Mean vertex path length is the mean distance between vertices of the same equivalent order to vertices of the next successively higher equivalent order beginning at the axon hillock. Each successive equivalent order in control cells is approximately 8 pm and in treated cells the increment is approximately 6 pm. Error bars = SE.

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FIG. 4. Mean frequency of segments plotted against equivalent orders for control (a) and treated (b) cells at 3, 5, 7, and 20 weeks pn. In the control cells the increase in frequency of branches in the middle equivalent orders is coupled with a decrease in the frequency of the higher equivalent order branches or the 3- to 20-week period. In contrast, by 3 to 7 weeks pn the treated cells lose branches in the middle equivalent orders and add branches in the higher equivalent orders.

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FIG. 5. Computer reconstruction of a typical treated and control cell at 20 weeks pn. Equivalent orders 18 and beyond are indicated by heavy lines. Compare the large number of segments in the treated cell which exceed equivalent order 18 with those in the control cell.

to 5 weeks), as the dendritic tree expanded both laterally and to the pial surface, the distribution was skewed with the slope representing the growth front of the tree. After the initial rapid growth phase the cell entered a remodeling phase where the higher equivalent order branches, which were primarily situated at the periphery, were pruned from the tree, with new branches filling out the central portion or middle equivalent order area of the tree. Therefore, by 20 weeks pn the frequency distribution became symmetrical. In contrast (Fig. 4b), the treated cells began with a skewed distribution similar to control cells but then took on a marked bimodal distribution. Specifically, the addition of new branches in the middle equivalent orders progressed normally to and including 5 weeks pn; however, by 7 weeks pn there was an observable decline in middle equivalent order branch numbers and by 20 weeks pn the frequency of branches in the middle equivalent orders was significantly (P < 0.05) reduced below control values. The removal of middle equivalent order branches occurred at the same time as an increase in branches in the higher equivalent orders. This observed pattern of remodeling in the treated cells was the exact reverse of that observed in the control cells where high equivalent order branches were removed and middle equivalent order branches were added. Computer-drawn reconstructions (Fig. 5) of typical dendritic trees from control and treated cells at 20 weeks pn demonstrated that the new branches added to the treated cells were primarily terminal branches arranged around the periphery of the dendritic tree, whereas there was a paucity of these high equivalent order branches in the control

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FIG. 6. Mean distribution of Va, Vb and Vc vertices (including transformed Vts) in 20-weeks pn control (a) and treated(b) cells. Note the bimodal arrangement found in the treated cells(b).

cells. Therefore, it was evident that the treated cell did not lose the high equivalent order branches during remodeling but increased their number. Va/Vb Vertex Ratio (VR) and Trichotomy. The VR is an excellent predictor of specific modes of growth within trees. Table 1 compares the changes in VR and frequency of trichotomy associated with age, between control and treated cells. Both groups demonstrated an overall increase in the VR and a reduction in the frequency of trichotomy. However, at both 7 and 20 weeks pn, the VR observed in the treated cells exceeded 1.0, indicating a change from symmetrical to nonsymmetrical growth. Accompanying this shift was a reduction in the frequency of trichotomy below comparable control values. Figure 6a and b compares the frequency of the three types of dichotomous nodal vertices plotted against equivalent orders for control and treated groups at 20 weeks pn. The symmetrical distribution observed in the control cells (Fig. 6a) was in marked contrast to the bimodal distribution observed in the treated cells (Fig. 6b). In the treated cells the frequencies of the VDs were relatively even in number over the lower equivalent orders. Beginning at approximately equivalent order 18, the second peak possessed a greater proportion of Va’s, which resulted in a VR for the second peak of 1.3 1. DISCUSSION Chronic administration of relatively small doses of phenobarbital to late prenatal and neonatal rats produces abnormal remodeling in Purkinje cell dendritic trees. Berry et al. (1) suggested that the process of growth and re-

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modeling of Purkinje cell dendritic trees was controlled by environmental factors. Two major factors must be considered: (i) the effects on the dendritic tree produced by possible alterations to those neurons and their processes associated with the Purkinje cell, and (ii) the possible direct action of phenobarbital on the dendritic tree. Considering first the possibility of deafferentation, it has been well established that removal of a specific afferent input to the growing Purkinje cell results in distinctive morphologic alterations. However, removal or reduction of parallel fibers (4), climbing fibers (4) or catecholaminergic fibers ( 14) results in changes in Purkinje cell morphology which do not match the pattern of changes observed after phenobarbital administration. This is not meant to imply that these structures are not altered, but that phenobarbital does not act by the simple ablation of one type of input. Other quantitative dendritic field analyses, studies which can be easily compared with the parameters utilized in this study, involved treatments such as undernutrition (9) and chlorpromazine administration (7). It is probable that those types of treatments have a wide effect on many neuronal types, but produce architectural changes different than those observed after phenobarbital treatment. Therefore, it appears as if phenobarbital produces yet another unique modification to the Purkinje cell. The vertex analysis method provides the best means to examine critically and compare the changes occurring in the dendritic fields during and after environmental insult. This study primarily examined the period of remodeling of dendritic fields. Analysis began on Day 2 1 pn, which was the last day of treatment. Purkinje cells at this time are undergoing the last stages of growth and expansion, as indicated by the fairly constant mean terminal path length observed in older cells. From an examination of segment frequency, it is evident that phenobarbital reduced the total number of branches produced (Table 1). The distribution of the branches, in terms of the equivalent orders, was also anomalous. During normal Purkinje cell development in this study and as reported for Purkinje cells during remodeling in other studies (2,7, 1 I), the branches on the higher equivalent orders (above 20) are removed and replaced by new middle equivalent order branches (Fig. 4a). In contrast, the phenobarbitaltreated cells exhibited a progressive loss of the middle equivalent order branches with new branches being added in the higher equivalent orders producing a bimodal distribution. Computer-assisted drawings of the treated cells (Fig. 4b) clearly demonstrated that the new growth in the higher equivalent orders was most intense toward the pial surface. The abnormal increase in the frequency of higher equivalent order branches was reflected in the increased VR. Plotting of the three types of VDs by equivalent order, also revealed a bimodal arrangement in the treated cells (Fig. 6a, b). The abnor-

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mally large numbers of Va type vertices in the second peak drove the overall VR above the value of 1.O, indicating nonsymmetrical growth at the periphery of the field. A similar but transitory increase in VR above the value 1.O occurred in much younger control cells during the growth phase when the tree was rapidly expanding during the first 10 days (2,7, 11). Some of these effects may be related to the disposition of the parallel fibers, as these have been shown to play an important role in shaping of the Purkinje cell dendritic tree (1). During the remodeling phase, the Purkinje cell dendrites must locate new parallel fibers; the most “attractive” parallel fibers, in the case of the treated cells, appear to be in proximity to the pial surface. As to the availability of new parallel fibers being presented to individual dendritic trees during this time, Lauder (8) demonstrated that the parallel fibers continue to elongate as late as 90 days pn. When the remodeling occurs in control cells, the VR is constantly maintained, implying that old terminals are removed and new terminals are added randomly. However, the addition of the new branches in the treated cells appears to be nonrandom, seeming to favor terminal segment addition as manifested by the alteration in the VR to levels above 1.O. One possible explanation of the observed condition may be related to the density of the parallel fibers. According to the hypothesis of Berry et al. (3), the segment length will be inversely proportional to the number of contacts, whereas segment number will be directly proportional to the number of contacts. Therefore, if phenobarbital treatment resulted in a reduction of parallel fiber density or rendered them less recognizable, dendritic branches might have to extend further to establish a suitable contact; one would, therefore, expect to observe a general increase in mean segment length and an overall decrease in segment number (due to a reduction in fibers available for contact). The mean segment length of the treated cells, however, is not increased although they are highly variable compared with that observed in the control cells. The findings of an unchanged mean segment length, albeit variable, and reduced mean vertex path length, however, are inconsistent with an explanation based on quantitative change in the parallel fibers. Even though both chlorpromazine and phenobarbital produce sizable Purkinje cell death (6), the factors regulating subsequent growth and remodeling of dendrites are differentially affected by chlorpromazine compared with phenobarbital. One extrinsic factor, which may be related to this difference is the effect of exercise. Pysh and Weiss (10) established that increased exercise produces Purkinje cells with more branches and an increased overall size. Hannah (unpublished findings), utilizing an activity monitor, found that chlorpromazine-treated animals were hyperactive, whereas phenobarbital-treated animals were in fact markedly hypoactive. In terms of the observed reduction in total mean segment number, total segment length, and

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mean terminal path length, it is obvious that the phenobarbital-treated cell is generally smaller. This contrasts with the effect of chlorpromazine which, like phenobarbital, results in a significant reduction in mean Purkinje cell number (6) but an enlargement of dendritic tree size (7). Thus the two drugs are affecting different growth-regulating processes; phenobarbital treatment, perhaps through effects on activity, retards Purkinje cell growth. REFERENCES 1. BERRY, M., P. BRADLEY, AND S. BORGES. 1978. Environmental and genetic determinants of connectivity in the central nervous system-An approach through dendritic field analysis. Pages 133-146 in M. A. CORNER, R. E. BAKER, N. E. VAN DE POLL, D. F. SWAAB, and H. B. M. UYLINGS, Eds., Progress in Bruin Research, Vol. 48. Elsevier, Amsterdam. 2. BERRY, M., AND R. PLINN. 1984. Vertex analysis of Purkinje cell dendritic trees in the cerebellum of the rat. Proc. R. Sot. Lond. B. 221: 32 l-348. 3. BERRY, M., J. SIEVERS,AND H. G. BAUMGARTEN. 1980. Adaption of the cerebellum to deafferentation. Pages 65-92 in P. S. MCCONNELL, G. J. BOER, H. J. ROMUN, N. E. VAN DE POLL, and M. A. CORNER, Eds., Progress in Brain Research, Vol. 53. Elsevier, Amsterdam. 4. BRADLEY, P., AND M. BERRY. 1976. The effectsof reduced climbing and parallel fibre input on Purkinje cell dendritic growth. Bruin Res. 109: 133- I5 1. 5. COLEMAN, P. D., AND A. H. RIESEN. 1968. Environmental effects on cortical dendritic fields. I. Rearing in the dark. J. Anut. 102: 363-374. 6. HANNAH, R. S., S. H. ROTH, AND A. W. SPIRA. 1982. The effects of chlorpromazine and phenobarbital on cerebellar Purkinje cells. Teratology 26: 21-25. 7. HANNAH, R. S., A. SPIRA, AND S. H. ROTH. 1987. Effect of chlorpromazine on Purkinje cell growth patterns. Dev. Neurol. 9: 190-200. 8. LAUDER, J. M. 1978. The effects of early hypo- and hyperthyroidism on the development of the rat cerebellar cortex. IV. The parallel fibres. Brain Res. 42: 25-40. 9. MCCONNELL, P., AND M. BERRY. 1978. Effects of undernutrition on Purkinje cell dendritic growth in the rat. J. Comp. Neural. 177: I59- 172. 10. PUSH, J. J., AND G. M. WEISS. 1979. Exercise during development induces an increase in Purkinje cell dendritic tree size. Science 206: 230-23 1. 11. SADLER, M. N., AND M. BERRY. 1984. Remodelling during development of the Purkinje cell dendritic tree in the mouse. Proc. R. Sot. Lond. B. 221: 349-368. 12. SHAIN, R. J., AND K. WATANABE. 1975. Effect of chronic phenobarbital administration upon brain growth of the infant rat. Exp. Neurol. 47: 509-5 15. 13. SHOLL, D. A. 1953. Dendritic organization in the neurons of the visual and motor cortices of the cat. J. Amt. 87: 387-406. 14. SIEVERS,J., H. P. KLEMM, S. JENNER,H. G. BAUMGARTEN, AND M. BERRY. 1980. Neuronal and extraneuronal effectsof intracistemally administered 6-hydroxydopamine on the developing rat brain. J. Neurochem. 34: 765-77 1. 15. YANAI, J., AND A. BERGMAN. 198 1. Neuronal deficits in mice following neonatal exposure to barbituates. Exp. Neurol. 73: 199-208. 16. YANAI, J., AND C. ISER. 198 1. Stereologic study of Purkinje cells in mice after early exposure to phenobarbital. Exp. Neurol. 74: 707-7 16. 17. YANAI, J., L. ROSSELI-AUSTIN, AND B. TABAKOFF. 1979. Neuronal deficits in mice following prenatal exposure to phenobarbital. Exp. Neurol. 64: 237-244.