Opiate receptor development in midbrain and forebrain of posthatch chicks

668

Developmental Brain Research, 3 (1982) 668-673

Elsevier Biomedical Press

Opiate receptor development in midbrain and forebrain of poathatch chicks

M. T. BARDO, R. K. BHATNAGAR, G. F. GEBHART and R. A. HUGHES Department oJ'Pharmacology, University of Iowa, lowa City, 1.4 52242 and ( R.A.H.) Department of Psychology, Iowa State University, Ames, IA 50011 (U.S.A.)

(Accepted November 30th, 1981) Key words: opiate receptor - - naloxone - - brain development - - chicken

Specific binding of [3H]naloxone to midbrain and forebrain was examined from chicks which were either 1, 7, 14, 21 or 28 posthatch days of age. While there was a significant age-related increase in the total number of [aH]naloxone binding sites in both brain regions, the increase was greater in magnitude in forebrain than in midbrain. In both brain regions, there was a concomitant age-related decrease in the density of receptor sites without any alteration in receptor affinity for naloxone. The decrease in receptor density was more rapid in onset and was greater in magnitude in midbrain than in forebrain. Young chickens are used often as subjects to assess the effects of opiates and opioid peptides on various behaviors, including responses to painful stimuli2, 20, tonic immobility s,15Aa,23 and social behaviors16,17. The opiate receptors which mediate the behavioral responses to opiates and opioid peptides in chickens were first demonstrated in birds sacrificed 2 days after hatching is. More recently, it has been shown that opiate receptors are evident as early as 10 days of gestation in chicks 7 and that a substantial increase in the number of opiate receptors occurs during embryonic development n. However, little is known about the development of opiate receptors during the early posthatch period when behavioral responses to opiates and opioid peptides are often assessed. In the present report, we examined the development of opiate receptors in both midbrain and forebrain regions of chicks during the first 4 posthatch weeks of life. White Leghorn cockerels were obtained one day after hatching from a local poultry supplier (Welp, Bancroft, IA) and were housed together in a thermostatically controlled commercial brooder with food (Wayne pullet starter) and water continously available. In a preliminary experiment, we sought to demonstrate that specific binding of [3H]naloxone to chick brain was saturable. Seven-day-old chicks were decapitated and the whole brain minus cerebellum was frozen on dry-ice and stored at - - 8 0 °C for 4-5 months. Following storage, brains were thawed and homogenized with a Polytron (15 s, setting 7) in 50 vols. of ice-cold 0.05 M Tris buffer (pH 7.4) containing 100 m M NaC1. Tissue homogenates were then diluted with Tris buffer to a final concentration (w :v) of either 1:100, 1:200 or 1:400. Portions (0.95 ml) of each tissue dilution were incubated at 0 °C for 180 min with varying concentrations (0.1-5.0 nM) 0165-3806/82/0000-0000/$02.75 © Elsevier Biomedical Press

669 of [aH]naloxone (New England Nuclear, spec. act. 50.0 Ci/mmol) in the presence or absence of 100 nM levallorphan tartrate (Hoffman-La Roche). The final incubation volume was 1 ml. Following incubation, samples were filtered under vacuum pressure on Whatman GF/B glass fiber circles and washed with two 5-ml vols. of ice-cold Tris buffer. Filters with washed tissue fragments were placed in glass vials with 10 ml Aquasol-2 (New England Nuclear) and counted by liquid scintillation spectrometry with a counting efficiency of 35 ~o. Specific binding was calculated as the radioactivity obtained in the absence of levallorphan (total binding) minus the radioactivity obtained in the presence of levallorphan (nonspecific binding). All samples were assayed in duplicate. The results of the saturation experiment are summarized in Fig. 1. Specific binding of [3H]naloxone in 7-day-old whole chick brain was saturable at a reasonably low nanomolar concentration of radioligand and was proportional to the concentration of tissue in the incubation mixture. These results indicate that the brain tissue contained only a limited number of receptor sites. In contrast to specific binding, nonspecific binding of [3H]naloxone was linear within the ranges of radioligand and tissue concentrations used. In subsequent experiments, chicks were decapitated at either 1, 7, 14, 21 or 28 posthatch days of age and the brains were rapidly removed and stored at --80 °C for 4-5 months. Each brain was then thawed and dissected on a cold plate (Thermoelectric, TCP-2) into midbrain and forebrain regions using the optic nerves as the ventral demarcation and the deep fissure between the lateral tecta (optic lobes) and anterior extent of the cortical hemispheres as the dorsal demarcation. Midbrain samples included hypothalamus, thalamus and tecta, whereas forebrain samples included cortical hemispheres, striatum, hippocampus and amygdala. Each tissue sample was homogenized in 200

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670 TABLE I Developmental changes in wet weight and protein in midbrain and forebrain regions of posthatch chicks

Each mean based on 4 chicks. Posthatch age (days)

1 7 14 21 28

Wet weight (meang 4- S.E.M.)

Protein concentration (mean mg/g wet weight 4- S.E.M.)

Midbrain

Forebrain

Midbrain

Forebrain

0.25 & 0.01 0.28 4- 0.01 0.33 4- 0.03 0.37 4- 0.04 0.46 4- 0.01

0.44 i 0.01 0.57 ± 0.01 0.71 ± 0.02 0.88 4- 0.02 1.01 4- 0.01

72.7 ± 1.7 80.7 4- 1.3 83.5 ± 3.7 96.1 4- 14.6 92.3 4- 6.1

77.2 i 2.5 85.3 ± 0.7 86.8 ± 0.8 87.1 4- 5.6 89.3 4- 2.0

vols. of ice-cold Tris buffer and assayed for specific binding of 1 nM [aH]naloxone as described above. For Scatchard analyses, the concentration of [3H]naloxone was varied between 0.1 and 2.0 nM. Tissue protein concentrations were determined by the method of Lowry et al. x4. The data were analyzed by split-plot analyses of variance and, in instances where significant (P < 0.05) interactions occurred, subsequent tests of simple main effects were performed 1°. For Scatchard analyses, Ks and Bmax values were estimated by least-squares linear regression. During the late embryonic period, previous reports have demonstrated that chick forebrain hemispheres undergo a greater developmental increase in wet weight than midbrain optic lobes, although both brain regions undergo a parallel increase in protein concentration21,2L During the early posthatch period, we found a similar pattern of development (Table I). From 1 to 28 posthatch days of age, forebrain wet weight increased 130 ~ , whereas midbrain wet weight increased only 84 ~. During this same period, there was a parallel increase in the concentration of protein in both midbrain and forebrain regions. There were no significant differences between midbrain and forebrain regions in the concentration of protein evident at each age. From 1 to 28 days, there was a significant increase in the total number of binding sites in midbrain and forebrain areas (top of Fig. 2). In contrast to the increase in total number of binding sites per brain area, there was a significant decrease in the density of specific binding sites per milligram of protein during this same developmental period (bottom of Fig. 2). The age-related decrease in receptor density was different in midbrain and forebrain regions. From 1 to 14 posthatch days, the midbrain exhibited a 2 5 ~ reduction in the concentration of ligand binding, whereas the forebrain exhibited no significant change in the concentration of ligand binding. In contrast, from 14 to 28 posthatch days, both midbrain and forebrain regions exhibited a parallel decrease (12 ~ ) in the concentration of ligand binding. In total, from 1 to 28 posthatch days, specific binding of [3H]naloxone was decreased 34 ~ in midbrain and 14 ~ in forebrain. Thus, during the posthatch period, the age-related decrease in opiate receptor density in midbrain was more rapid in onset and greater in magnitude than in forebrain.

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AGE (days) Fig. 2. Specific binding of [ZH]naloxone to midbrain and forebrain regions of chicks from 1 to 28 posthatch days of age. Each mean and S.E.M. based on 4-6 chicks.

In order to determine that the age-related decrease in specific binding of [ZH]naloxone reflected a decrease in the number of opiate receptors rather than a change in receptor affinity for naloxone, Scatchard analyses were performed on midbrain and forebrain tissue samples pooled from chicks which were either I or 28 posthatch days of age. In both midbrain and forebrain regions, there was a parallel shift in the linear Scatchard plots obtained at 1 and 28 days of age (Fig. 3). The Bmax values, which reflect the density of receptor sites, were decreased by approximately 25 Y/oin 28-day-old midbrain and forebrain regions relative to 1-day-old midbrain and forebrain regions. In contrast, the Ka values, which reflect the receptor affinity for the radioligand, were essentially unchanged in 28-day-old midbrain and forebrain regions relative to 1-day-old midbrain and forebrain regions. These data indicate that the agerelated change in specific binding of [ZH]naloxone in chick brain reflects a change in the number of binding sites rather than an alteration in receptor affinity for naloxone. At least 2 interpretations of the age-related decrease in opiate receptor density in posthatch chicks are possible. First, while the total number of opiate receptors increases with age, the density of opiate receptors may decrease because the growth of opioid neuronal elements is exceeded by the growth of nonopioid neuronal elements. Second, it is possible that some of the neuronal elements upon which opiate receptors are located may degenerate with age. It is known that sprouting neuronal elements in developing brain may degenerate if they fail to make functional connections 13. In chicks, the rate of neuronal sprouting declines soon after hatching, as the availability and uptake of free amino acids which are essential for dendritic arborization rapidly

672

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Fig. 3. Scatchard plots from midbrain and forebrain regions of chicks at either 1 or 28 posthatch days of age. Tissue was pooled from 4 chicks for each Scatchard plot. The Ka values are expressed in nM and the Bmaxvalues are expressed in pmol/g tissue. declines during this periodll.lL Thus, the degeneration of neuronal elements which do not receive afferent input after hatching may result in an overall decrease in the density of synaptic junctions which bear opiate receptors. Regardless of the interpretation of the decrease in opiate receptor density, the present results indicate that the number of postsynaptic sites for opiates in chicks are relatively mature at birth when compared to guinea pigs 4 and rats. In rats, there is an approximately 8-fold increase in the total number of opiate receptors in whole brain from 1 to 28 days of age and a concomitant 2-fold increase in their densityl,L The postnatal increase in opiate receptors in whole rat brain largely reflects an increase in the density of receptor sites in rostral brain regions which are less mature at birth than caudal brain regions 8. However, in caudal brain regions and in spinal cord, the rat is born with virtually its full complement of opiate receptors, and these regions may actually exhibit an age-related decrease in receptor density after birth 9 in a manner similar to that observed in the present report with chick midbrain and forebrain. The relatively mature nature of opiate receptors in chick midbrain at birth perhaps suggests that midbrain opioid systems subserve early postnatal food-seeking behaviors. We gratefully acknowledge the assistance of Bhavna Chatterjee, Elaine Herink, and Razia Khan in performing the receptor binding and protein assays. Supported by USPHS Grants NS 12121, DA 02879, and M H 15172.

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