The development of cholecystokinin in the interpeduncular nucleus of rats

Developmental Brain Research, 65 (1992) 275-279 ~) I992 Elsevier Science PublishersB.V. All rights reserved. 0165-3806/92/$05.00

275

BRESD 60436

The development of cholecystokinin in the interpeduncular nucleus of rats :

M y r a P. J o y c e 1 a n d G o r d o n

A . B a r r 1'2

t Biopsychology Doctoral Program, City University of New York, Department of Psychology, Hunter College of the City University of New York, New York, NY 10021 (U.S.A.) and ZDivision of Developmental Psychobiology, Department of Psychiatry, New York State Psychiatric Institute, Columbia University, College of Physiciar.s and Surgeons, New York, NY 10027 (U.S.A.)

(Accepted l October 1991) Key words: Interpeduncular nucleus; Cholecystokinin;[mmunoreactivity

This study describes the development and late disappearance of cholecystokinin-likeimmunoreactivity(CCK-LI) in the interpeduncular nucleus (IPN) of rats. Between one and 7 days of age, CCK-positivefiber labelingwas sparse and restricted to the lateral subnucleiof the caudal IPN. By 28 days of age the densityof CCK-positivefibersincreasedand labelingwas found in the lateral, rostral and apical subnuclei, and medial to the dorsolateral subnuclei. At 35 days of age CCK-LI fiber labeling was absent in the lateral subnuclei and medial to the dorsolateral subnuclei. No additional changes in fiber labeling were observed after 35 days of age. These results suggest that significantanatomical or biochemicalreorganizationmay occur in projections to the IPN between 28 and 35 days of age. During early postnatal development, cholecystokinin (CCK) mRNA and CCK-immunoreactivity (CCK-IR) are initially detected at different ages in different sites in the central nervous system. Fluctuations of concentrations of the peptide and its mRNA vary regionally during development and, at some sites, peak between 28 and 40 days of age2-4'9'ts. The interpeduncular nucleus (IPN), an anatomically complex structure, is located in the ventral midbrain. It is subdivided into 4 paired and 3 unpaired subnuclei7. lmmunohistochemical studies have shown that the IPN contains a variety of classic neurotransmitters and neuropeptides. These neurochemicals have distinct distributions within specific subnuclei of the IPN. With the exception of substance pl,20, ChAT and serotonin 1, the development of the concentration and distribution of the neurochemicals in the IPN has not been examined. Although the complex anatomy of the IPN is apparent at birth, synapse formation and the maturation of synapse morphology continue postnatally. The process is largely completed by 28 days of age n, although some synaptic modification continues through 90 days of age 13. Thus significant anatomical growth and restructuring in the IPN occurs during the time that the peptide CCK undergoes concentration changes in the CNS. The present study was undertaken to determine if the development of CCK in the IPN conforms to the general development of CCK in the CNS where peak concentrations of CCK are attained between 28 and 40 days

of age and subsequently decline, and if the changes in density and distribution of CCK in the IPN coincide with the previously described anatomical changes that occur in the IPN. Subjects were 45 male and female Long-Evans hooded rats. Pups were defined as zero days of age on the day of birth, There was no sex difference noted in CCK-IR labeling. Pups were sacrificed at 1, 7, 14, 21, 28, 35, 60 and 100 days of age for CCK immunocytochemistry by an overdose of sodium pentobarbital i.p. and subsequently perfused transcardially with 0.9% saline, pH 7.2, followed by 4% buffered paraformaldehyde, pH 7,2. The brains were removed and placed in fixative for 48 h and then in sucrose for 48-72 h. Frozen 30-/~m floating sections were stained using the Vectastain Elite ABC kit (Vector Labs.). The primary antibody, rabbit-anti-CCK (Cambridge Research Biochemicals, Inc.) was diluted to 1:20,000. The protocol of the Vectastain Kit was followed with the exception that the tissue was incubated in the primary antibody overnight at room temperature on a shaker. Control sections were subjected to the same treatment except that in some cases the antibody was preadsorbed to the sulfated octapeptide diluted 1:20,000 prior to incubation with the tissue or the primary antibody was omitted from the procedure. Cholecystokinin-positive cell bodies were not observed in the IPN at any age. Since colchicine treatment was not used in this study, it cannot be stated with certainty that CCK synthesizing neurons are not present in the

Correspondence: M.E Joyce, Department of Psychology,Hunter College, 695 Park Ave., New York, NY 10021, U.S.A.

276 IPN, Staining was not seen in the tissue in which the primary antibody was preadsorbed to the peptide nor in the tissue in which the primary antibody was omitted from the procedure. Fig. 1 shows photomicrographs of Cresyl violet stained sections of the IPN a~ the rostral, middle and caudal levels. Fibers showing CCK-IR were apparent at l and 7 days of age (data not shown). Extremely sparse CCK-IR punctate fibers were observed in the dorsal portion of the rostral subnucleus. The CCK-positive fiber labeling extended along the lateral aspects of the rostral subnucleus toward the ventral surface of the IPN. In the caudal IPN, at the level above the pons, very sparse fiber labeling was observed in the lateral aspects and medial portion of the apical subnucleus. In the lateral subnuclei and medial to the dorsolateral subnuclei, punctate CCK-IR fibers were slightly more dense than in the rostral subnucleus. Fig. 2 shows rostral, medial and caudal levels of CCKlabeled sections of the IPN of 14-, 28- and 35-day-old rats. At 14 days of age, both punctate and a small number of varicose CCK-IR fibers were observed throughout the rostral subnucleus. The dorsal portion of the rostral subnucleus showed moderately dense labeling. The CCK-positive fiber labeling that extended in the ventral direction along the lateral aspects of the IPN was slightly more dense than that in the dorsal part of the rostral subnucleus. At a more caudal level, sparse CCK-IR was present throughout the apical subnucleus. In the middle of the lateral subnuclei and in the sites medial to the dorsolateral subnuclei, CCK-positive fibers show a substantial increase in density relative to the 7-day.old rat pup, At both 21 and 28 days of age, the distribution of CCK-IR fiber labeling was quite similar to that observed in the 14-day-old animal but the density of CCK-positire fibers increased substantially and the proportion of varicose fibers relative to the number of punctate fibers increased. Fiber density showed the greatest increase in the lateral subnuclei and medial to the dorsolateral sub. nuclei. In addition, the size of the area showing fiber labeling at these sites was more extensive relative to that seen in the 14-day-old pup. By 28 days of age, the CCK content in the IPN, as indicated by the density and distribution of fiber labeling, reached its maximum. The most dramatic change observed in CCK-IR fiber labeling in the IPN occurred between 28 and 35 days of age. CCK-IR fibers that had densely labeled the lateral subnuclei and the sites medial to the dorsal lateral subnuclei in the 28-day-old rat were completely absent at 35 days. The density of CCK-positive fiber labeling increased slightly in the dorsal part of the rostral subnucleus between 28 and 35 days of age. Ventrally, CCK-IR

fibers were much more sparse relative to the dorsal aspect of the IPN. In general, at this rostral level of the IPN, the

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Fig. i . Cresy] violet stained sections of the IPN in an adult rat at rostral (a), medial (b), and caudal (c) levels• The subnuclei are labeled as described in ref. 13: apical (A); central (C); dorsolateral (DL); intermediate (I); lateral (L); and rostral (R). Bar = 200 #m.

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Fig. 2. CCK-IRin rostral, medial, and caudal levels of 14-day-old(a-c), 28-day-old(d-f) and 35-day.old(g-i) rats. Note that in the caudal section of the IPN, the dense fiber labelingin the lateral subnucleusthat was present in the 28-day-oldis completelyabsent in the 35-day-old rat. Bar .. 200 ~m.

distribution and density of CCK-IR fibers were similar to the 28-day.old animals. The punctate and varicose fibers labeled for CCK were distributed evenly through. out the apical subnucleus. Neither the density nor the distribution of CCK-positive fibers in the IPN changed after 35 days of age. The presence and distribution of CCK-IR in the IPN were first described by Hamill et al. a. The distribution of CCK-IR described in that study differs from that observed in the present experiment in that Hamill showed dense CCK fiber labeling in the lateral subnuclei and medial to the dorsal lateral subnuclei. In our study, only pups 28 days of age and younger showed the distribution described by Hamill. In that earlier study, the ages of the subjects were not reported. There were procedural differences such as the use of different strains of rats and the use of immunofluorescence immunocytochemistry rather than the avidin-biotin horseradish peroxidase method that might account for these differences.

In the whole brain, a rapid rise in CCK-LI is observed after 10 days of age, peaks between 28 and 40 clays and remains stable through 60 days of age. Between 60 and 180 days of age, CCK concentrations decrease by 40% 2. Through 28 days of age, the development of CCK-LI in the IPN conforms to the pattern of development of CCK-L!I in whole brain assays. In the IPN, CCK-LI increases in area and density after 7 days and peaks by 28 days of age. By 35 days, though, the sites that showed the densest labeling at 28 days, the lateral subnuclei and the areas ventral and medial to the dorsal lateral subnuclei, contain virtually no observable CCK-positive fibers• This is a rather unusual occurrence in general since most anatomical reorganization, including synaptogenesis, competition for synaptic sites, and cell proliferation and cell death occurs before 28 days of ages'~''15, with some exceptions 17. This loss of CCK-IR may be indicative of a reduction in the number of CCK synthesizing cells or an example or late transient phenotypic expression of a peptide in the brain. While synaptic morphology in the

278 28-day-old rat is similar to the adult animal, left-right pairing of axons at crest synapses arising from projections of the medial habenula are still being modified t3. One mechanism that could account for the loss 6f CCKpositive labeling in the 35-day-old animal is replacement of the CCK system with another, late maturing system. The identity of that system is not known. Several studies have shown regional differences in fluctuations of C C K m R N A concentrations through development. Most significant is the finding that CCK m R N A content decreases precipitously in several areas of the central nervous system around 28 days of age, particularly in the frontal cortex and the striatum TM. In spite of the decrease in m R N A content, the CCK-IR appeared unchanged. The evidence does indicate, though, that developmental changes occur in the transcription of the gene encoding CCK in several brain regions and therefore disappearance of the peptide at specific projection sites might not be unexpected. It is possible that CCK synthesis stops between 28 and 35 days at the source of the input to the lateral subnuclei of the IPN. During this period another neurochemical may make its initial appearance, or alternatively, CCK may be co-localized with another neurochemical and CCK synthesis ceases between these ages. Double labeling experiments have shown that C C K - I R is co-lo-

calized with other neuropeptides and neurotransmitters within neurons in several areas in the central nervous system. The neurochemicals found to be co-localized with CCK include vasoactive intestinal peptide 14, enkephalin 6, tyrosine hydroxylase ~6. substance p19, and galanin 1°. All of these substances are found in the IPN, although their distributions do not overlap with CCK. This does not preclude the possibility that C C K is colocalized with an as yet undetermined neurochemical in the IPN through 28 days of age, and between 28 and 35 days, CCK synthesis ceases at the origin of the projection. In summary, C C K - L I fiber labeling increases in density and area of distribution in the IPN between the ages of 1 and 28 days of age. The lateral subnuclei and the sites medial to the dorsolateral subnuclei contain the highest density of CCK-positive fiber labeling at 28 days of age. By 35 days, these sites contain virtually no CCK-LI fibers. This disappearance of C C K - L I between 28 and 35 days of age suggests that either a major anatomical or biochemical reorganization occurs during that period.

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We thank Dr. Marion Murray for her critical reading of an early version of the manuscript. This work was supported in part by funds from the Biopsychology Doctoral Program and Hunter College.

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