Effects of opiates on neuronal development in the rat cerebral cortex

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Brain Research Builetin, Vol. 30, pp. 523-527, 1993 Printed in the USA. All rights reserved.

CopyrightQ 1993Pergamon Press Ltd.

Effects of Opiates on Neuronal Development in the Rat Cerebral Cortex JORUEL

V. SEATRIZ

AND

RONALD

P. HAMMER

JR.’

~~part~e~t o~A~ato~~ & Reproductive Biology, Univ~~s~t~of Hawaii School of ~edj~~~e, 1960 East- West Road, Honolulu, HI 96822 Received

6 November

199 1; Accepted

17 June

1992

J. V. AND R. P. HAMMER JR. Effects qfopiates on neuronaldevelopmentin the rat cerebralcortex. BRAIN RES BULL 30(5/6) 523-527, 1993.-Quantitative morphological characteristics of cells in the primary somatosensory cortex of 6-dayold rats were examined following continuous maternal administration of morphine (10 mg/kg/day), naltrexone (10 mg/kg/day), or saline vehicle from gestation day 12. Naltrexone reduced neuronal packing density and significantly increased cortical thickness but had no effect on neuronal number, while morphine reduced neuronal packing density and the number of neurons without affecting cortical thickness. These results suggest that blockade of end~enous opioid function during development enhances neuronal maturation in this brain region, while p&natal morphine administration might act to restrict cortical cell prolife~tion and maturation. Thus, the effect of ontogenetic activation of opioid receptors by endogenous opioid com~unds could be similar to but less severe than the effect of exogenous opiate exposure on cortical cell development. SEATRIZ,

Morphine Opioids Neuronal packing density

Naltrexone Endorphins Cerebral cortex

Opiate receptor

Development

Brain growth

METHOD

ENDOGENOUS and exogenous opiates are known to affect various measures of brain development, including cell proliferation (6,17,23), neurite and dendritic outgrowth (4,8,9), and neuronal density in several brain regions (2 1,22). Both neuronal and glial ( 12,13) development is known to be altered by opiates. Endogenous opioids are thought to inhibit neuronal growth (4,2 1,22) and proliferation (24) by stereospecific, opioid receptordependent mechanisms, while exogenous opiates could produce either specific or generalized neurotoxic effects on brain development (10). Selective regional effects might also exist, whereby differential effects occur depending upon the developmental state of opioid systems during critical periods of opiate exposure. The putative effects of opioid receptor blockade (4) and opiate administration (8) on dendritic growth in somatosensoty neocortex suggest that other morphological indices of cellular growth in this region might also be affected by opiates. Therefore, we examined and compared the effect of continuous naltrexone-induced opioid receptor blockade and morphine administration during the entire period of cortical neurons prolifemtion and growth on cortical cell packing density, thickness, and cell number in specific layers of the primary somatosensory cortex at postnatal day 6. The observed differential laminar effects suggest that endogenous opioid peptide regulation of neuronal growth might occur during a critical period, while the efficacy of exogenous opiates might display a longer time course.

Sprague-Dawley female rats derived from our breeding colony were bred on the evening of proestrus, which was determined by estrous smearing during two consecutive 4day estrous cycles. Osmotic minipumps (Alzet 4ML4; Alza Corp., Palo Alto, CA) that administered either 10 rn~k~~y morphine sulfate, 10 mg/ kg/day naltrexone HCl, or saline vehicle throughout the course of the experiment were subcutaneously implanted on gestation day 12. This size minipump is specified by the manufacturer to administer uniform daily doses across a 28-day period, 1 week longer than required in the present study. The dose and effect of drug administration was not assessed in this experiment, however. All animals utilized in the experiment delivered on gestation day 23, which was designated as postnatal day 1 (Dl). Total maternal weight gain from gestation days 12-23 was calculated, and the number and male-female ratio of pups born was recorded. Litters were culled to eight pups at birth. Between one and three animals were taken from each of the three to four litters in each treatment group; a total of six to eight animals of each sex were examined in each group. Thus, experimental subjects received both gestational and lactational drug exposure via maternal treatment. Similar maternal opiate treatment paradigms have been shown to decrease brain weight and DNA content in offspring (19) suggesting that drug delivery is constant and effective over this period.

’ To whom requests for reprints should be addressed.

523

524

SEATRIZ

On D6, male and female pups were anesthetized by hypothermia and perfused intracardially with isotonic saline followed by 10% neutral buffered formahn (NBF) solution. This time point was selected for comparison with the results of earlier morphometric studies (8) and provides both pm- and postnatal exposure to opiates. Brains were immediately removed and placed into 10% NBF postfix for 24 h, followed by graded sucrose solutions. Brains were then frozen and sectioned at 20 pm on a sliding microtome, and sections were mounted on gelatin-coated glass slides and allowed to dry on a 37°C hotplate. Slide-mounts sections were then stained with buffered thionin, dehydrated, and coverslipped. Primary somatosensory (S,) cortex was first identified by the presence of a distinct layer IV, and the packing density of cells in each layer in the portion of Si cortex immediately adjacent to agranular motor cortex bilaterally was determined using calibrated, high-re~lution microscopy. Specifically. cells were counted at 400X magni~cation in a 10,000 pm2 area defined by an intraocular grid in layers II-III, IV, V, and VI at a brain level corresponding to A 4.7 mm (11). Neurons and glia were differentiated by size, staining characteristics, and presence of nucleoli. Neurons were identified as larger cells (25 Fm), with lightly stained nucleoplasm and distinct nucleoli, while glia were smaller, with dark cytoplasm and/or heterochromatin aggregations. The entire cortex and each laminar boundary in this same region were then drawn using camera lucida at 100X magnification. and laminar thickness was then determined in each layer along a line perpendicular to the cortical surface. The number of neurons per layer was then calculated by multiplying the number of neurons per unit area (packing density) times the laminar thickness. Morphometric data from animals in each litter were combined to yield mean values per litter. and the data were logtransformed whenever variance was heterogeneous. The results were analyzed using three-way analysis of variance (ANOVA) to determine overall effects of treatment, gender, and layer except measurements of total cortical thickness, which were analyzed by two-way ANOVA by treatment and gender. Significant interactions were identified and the loci of differences distinguished using the Scheffe posthoc test. Individual Iaminar analyses were not conducted unless a significant (p I 0.05) interaction of treatment X layer was present. One-way ANOVA was used to assess changes in maternal and pup body weight, total number. and male-female ratio of pups born across treatments. All data are presented as mean vatues + SEM. RESULTS

Drug treatment produced no significant differences in maternal weight gain across gestation or in the total number or male-female ratio of pups born across treatment groups, nor was mean body weight of either male or female pups affected by treatment at D6 (Table 1). In contrast, perinatal morphine and naltrexone treatment affected both qualitative and quantitative parameters of cell number and cortical thickness in S,. Naltrexone treatment produced a noticeable increase of cortical thickness (Fig. f B), while morphine treatment reduced neuronal number (Fig. IC) relative to perinatal saline treatment (Fig. 1A). Other histological characteristics (e.g., cell shape) appeared similar between treatment groups. Quantitative analyses across layers revealed significant (p 5 0.005) overall effects of treatment and layer on neuronal packing density, cortical thickness, and neuronal and glial cell number. However, there were no significant effects of gender for any parameter measured.

AND

HAMMER

JR.

Subsequent analyses illustrated striking effects of perinatal drug treatment on neuronal packing density. A significant (p I 0.00 1) main effect of treatment and treatment X layer interaction was observed on neuronal packing density. Posthoc testing revealed that both morphine and naltrexone reduced neuronal packing density in cell-dense cortical layers (Fig. 2); however, this effect was more robust following morphine treatment, which significantly reduced neuronal packing density by more than 40% in layers II-III, IV. and V. A significant (1) I 0.05) overall treatment X gender interaction was observed: however, posthoc testing revealed no significant gender differences of neuronal packing density in any treatment groups. Measurement of additional parameters further differentiated the effects of morphine from those of naltrexone. Significant main effects of treatment on cortical thickness were observed in the whole cortex (p I 0.005) and among individual layers (p I 0.05), and a significant (p I 0.05) treatment X layer interaction was present. Posthoc testing revealed that naltrexone treatment significantly (11 2 0.05) increased total cortical thickness but produced no significant difference in any individual layers, while morphine treatment had little effect (Fig. 3). A significant (p i 0.001) main effect of treatment and a treatment X layer interaction were observed on neuronal number, and a significant (17 _( 0.05j treatment X gender interaction was present. Glial cell number also showed a significant (p I 0.01) main effect oftreatment; however, no significant treatment X layer interaction was present (see Table 2). Posthoc testing revealed that morphine significantly reduced neuronal number in layers II-III, IV, and V, while naltrexone treatment produced no significant differences (Fig. 4). The apparent treatment X gender interaction of neuronal number seemed attributable to a signi~cant (p 2 0.05) sex difference in saline-treated cases that was not present following other treatments. WYJlSSION

Postnatal naltrexone treatment at a dose known to provide complete opioid receptor blockade (20) has been shown to decrease neuronal packing density and increase cortical thickness without affecting glial cell density in the rat S, cortex (22). These findings were confirmed and extended in the present study using continuous treatment with naltrexone administered at a rate of 10 m~kg/day during the perinatal period. Such continuous naltrexone administmtion in adult rats has been observed to produce consistent upregulation of p- and ij-opioid receptors (15) suggesting that this paradigm provides constant blockade of opioid receptors in the CNS. Continuous perinatal naltrexone treatment in the present experiment appeared to alter the development of Si, producing a slight decrease of neuronal packing density in cell-dense layers and increasing total cortical thickness. However, the number of neurons observed in each layer was unaffected by perinatal naltrexone treatment. Thus, continuous perinatal blockade of opioid receptors had little apparent effect on neuronal number assessed at postnatal day 6, even though these animals were exposed to naltrexone during the entire period of cortical neuron proliferation, which begins at gestation day 13 (2,7). Neuronat number reflects a combination of factors including neuronal proliferation and cell death, while cortical thickness and packing density reflect the additional influence of cell migration and growth of neuronal processes. The effect of opioid receptor blockade on the latter but not the former parameters suggests that endogenous opioids might affect neuronal differentiation more than proliferation in

EFFECTS

OF OPIATES

ON CORTICAL

525

DEVELOPMENT

TABLE

1

EFFECT OF PERINATAL MORPHINE AND NALTREXONE TREATMENT ON PARAMETERS OF MATERNAL AND PUP DEVELOPMENT (MEAN f SEM) Saline

Naltrexone

Morphine

Maternal weight gain

87.0 + 6.3

103.3 f 9.6

86.9 + 20.2

Total number

of pups born Male-female ratio of pups born

12.0 + 0.9

13.3 k 0.8

10.2 f

1.5

1.45 f 0.1

1.25 + 0.5

1.38 f

0.4

Weight of male pups at D6 Weight of female pups at D6

11.1 +0.6 10.5 f 0.4

11.2 +- 1.3 10.5 + 0.2

Parameter

this brain region. In fact, postnatal naltrexone treatment is known to induce elaboration of dendritic branching in cortical pyramidal neurons (4) which could account in part for the increase of interneuronal space (reduced packing density) and cortical thickness observed herein. Alternatively, cortical afferent supply might be enhanced by this treatment. That parameters of neuronal differentiation but not neuronal number are affected by perinatal opioid blockade suggests that endogenous opioid regulation of neuronal development in S, might be limited to a postnatal critical period, which could explain the similarity of the effects of pre- and postnatal naltrexone treatment described herein to those of postnatal treatment alone (22). Moreover, the effect of endogenous opioids during this postnatal critical period could be regionally dependent, as cell proliferation is altered by postnatal opioid receptor blockade in the developing cerebellar cortex (23) wherein most neurons originate postnatally (1). Our results do not indicate which par-

14.0 z!z 2.1 13.1 + 2.1

titular endogenous opioid systems might be involved in regulation of neuronal development in Si as the function of all opioid peptides would be affected by naltrexone-induced receptor blockade. Nevertheless, the possibility that endogenous opioid peptides might act as negative neurotrophic factors during development is supported by these data. Morphine-Induced Alterations in the Somatosensory Cortex Interestingly, morphine effects on the developing S, cortex were not simply the converse of those produced by naltrexone. Rather, perinatal morphine treatment reduced neuronal packing density and also reduced the total number of neurons in layers II-V but did not alter cortical thickness. The morphine-induced reduction of total cell number suggests that perinatal treatment could act to decrease cell proliferation or increase cell death in St in addition to affecting neuronal maturation. Thus, continuous

FlG. 1. Photomicrographs of the left primary somatosensory cortex in Nissl-stained sections from 6day-old male rats treated perinatally with (A) saline vehicle, (B) 10 mg/kg/day naltrexone, or (C) 10 mg/kg/day morphine. Cortical layers II-VI are marked with heavy lines at right in each case in the precise region where quantitative measurements were taken. Nahrexone treatment increased cortical thickness, while morphine reduced the total number of neurons in layers II-V compared to saline treatment. The scale at the upper right shows 500 pm. Original magnification 63X. Quantitative measurements of cell density were conducted using higher magnification to ensure adequate cellular resolution (see the text for details). wm. white matter.

526

SEATRIZ AND HAMMER JR.

n q q

x 150.z ;1” B g loo-

TABLE 2

Saline Naltrexone Morphine

?j a

50-

EFFECT OF PERINATAL MORPHINE AND NALTREXONE TREATMENT ON TOTAL GLlAL CELL NUMBER IN S, CORTEX (CELLS/UNIT j2 i- SEM) Cortkal Layer

Saline

II-IIf

0.10 ‘- 0.01

0.11 io.01

0.07 i 0.0

IV V VI

0.10 * 0.01 0.67 + 0.04 2.60 f 0.08

0.11 + 0.01 0.64 I 0.07 2.42 + 0. I5

0.09 r 0.01 0.63 f 0.06 I.58 ‘-c0.16

Naltrexone

Morphine

I

0-t Layer II-III

Layer IV

Layer v

Layer VI

FIG. 2. Neuronal packing density (cells/mm’ X IO-* 4: SEM) in cortical layers of S, following perinatal saline, naltrexone, or morphine treatment. *Significant (p zz 0.001) difference compared to saline treatment.

p&natal morphine treatment might have similar, albeit more severe, effects to those of endogenous opioid peptides, whose

activity at opioid receptors is blocked by naltrexone. Morphine has been shown to decrease glial cell proliferation in primary glial cell cultures (12) and decrease DNA synthesis in rat brain at Dl and D4 (6), although neuronal and glial proliferation were observed to be unaffected by morphine treatment at D6 in eerebellar cortex (241, su~esting the possible presence of differential regional effects. Moreover, morphine treatment during this same period severely restricts branching and length of basilar dendrites in layer II-III pyramidal neurons of S, measured at D6 (8). That morphine treatment reduced neuronal number and dendritic growth in this layer while cortical thickness was unaffected susests a profound neurotoxic effect in this region that might manifest as decreased cellular connectivity and increased extracellular space. Although the possibility of limited postnatal recovery cannot be excluded as only one time point was assessed in the present study, one might expect morphineinduced alterations such as those observed herein to be long lasting, with a poor prognosis for recovery. It should be noted that the neither morphine level nor behavioral response of offspring to drug exposure or ~thdrawal

were assessed in the present study. However, this paradigm of perinataf maternal morphine administration is known to downregulate opiate receptor density in selected brain regions at D6 (3), and previous work suggests that similar gestational and lactational maternal opiate treatment affects parameters of early brain growth in offspring (19). Thus, this maternal drug dose probably provides continuous exposure of the developing brain to produce these effects. Further, pha~acokinetic studies of maternal cocaine treatment suggest that drug delivery to offspring during lactation might even be enhanced (18). Some variability could occur due to differential feeding patterns of oRspring during lactational exposure, but uniform pup weights across treatment groups (Table 1) suggest that feeding behavior and nutritional level was similar. The similar&y across tr~tments ofgestational parameters also suggests that toxic effects of this drug dose were absent. However, these data do not preclude the possibility of unsustained drug exposure during lactation, which could produce withdrawal, thereby exacerbating ontogenetic effects of drug exposure. In any case, the pharmacological effects of this paradigm on offspring should be further characterized to validate its use. The mechanism underlying these morphine effects in St is unknown; however. the p-opiate receptor is presumably implicated. For example, morphine effects on cortical dendritic development are reversed by concurrent naltrexone treatment (8). Perinatal morphine administration downregulates p-opiate receptors in whole brain homogenates (16) but has no effect in any layers of S, (3,141. Neocortical p-receptors develop during late gestation with additional midco~ical receptors added postnatally (5); thus, these receptors are function-

T

Layer II-IlI Layer II-lTI Layer IV

Layer V

Layer VI

Layer IV

Layer V

n

Saline

Layer VI

Whole

FIG. 3. Cortical thickness (am % SEM) by layer following perinatal saline, naltrexone, or morphine treatment. *Significant (P s 0.05) difference compared to saline treatment.

FIG. 4. Number of neurons (cells/unit g* 2 SEM) in cortical layers of Si following perinatal saline, naltrexone, or mo~hine treatment. ‘Significant (p 2 0.05) difference compared to saline treatment: **significant (p YZ0.001) difference compared to saiine treatment.

EFFECTS OF OPIATES ON CORTICAL

521

DEVELOPMENT

ally available for morphine activation during this period. The possibility of earlier effects of morphine on neuronal progenitors or on fetal glial cells (12) thus affecting neuronal migration and/or survival should be examined in future studies.

ACKNOWLEDGEMENTS

The authors thank Arleen R. Ricalde for technical assistance. This work was supported by USPHS Awards DA0408 I and NSOll61 to R.P.H. J.V.S. was supported by USPHS-MARC Predoctoral Fellowship GM07684.

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