Brain Research, 586 (1992) 67-71 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
67
BRES 17844
Somatostatin receptors increase in the olfactory bulb of developing pups after perinatal exposure to cocaine M.N. R o d r l g u e z - S ~ n c h e z a n d E. Arilla Molecular Neuroendocrinology Unit, Department of Biochemistry and Molecular Biology, Medical School, Unn'ersity of AIcald, AIcald de Henares, Madrid (Spain) (Accepted, 29 January 1992)
Key words: Cocaine; Somatostatin receptor; Olfactory bulb; Rat
The effects of chronic prenatal and/or postnatal exposure to cocaine on somatostatin concentration and receptors were studied in the olfactory bulbs of rat pups at birth and at 15 days old. Wistar rats were injected subcutaneously with single daily doses of 40 mg cocaine hydrochloride/kg from days 7 to 19 of gestation, from day 7 of gestation to day 15 postpartum or from parturation to day 15 postpartum. Fetal exposure to cocaine decreased SS concentrations in the olfactory bulb of the newborn pups while prenatal-plus-postnatal exposure increased this parameter. Administration of cocaine only during lactation did not induce any change. Exposure during gestation or during nursing induced an increase in the total number of somatostatin receptors and a decrease in the affinity constant in the olfactory bulb of newborn and 15-day-old pups. These results suggest that the development of somatostatin receptors in the olfactory bulb can be altered by prenatal and/or nursing period exposure to cocaine.
INTRODUCTION
The dramatic increase in cocaine use among young adults of child-bearing age has resulted in a rise in its use during pregnancy z'7'u. In laboratory investigations cocaine has been shown to penetrate the pli~,centa and accumulate in fetal tissues at concentrations greater than those observed in maternal plasma 42'u. In addition, cocaine can be passed to offspring through breast feeding 52. Early studies of gestational cocaine exposure in laboratory animals focused predominantly on possible teratogenic effects ~3'26, whereas more recent studies have assessed neurobehavioral function in exposed offspring 43'4s. Spear et al.45 have recently reported that prenatally cocaine-exposed offspring showed marked impairment in learning an odor/milk association. It should be kept in mind that the sense of smell is important for developing animals, and especially for neonatal rats, which rely on olfactory cues to locate their mother's nipple 3. Evidence is accumulating to the
effect that the mammalian olfactory bulb (OB) contains various neuroactive substances including peptides 1"~'27,41. Somatostatin (SS), originally detected in the hypothalamus 4, is associated with several different classes of neurons and neuronal processes in the OB that are known to interact in the integration of olfactory information 2~'27'4L4~.In addition, SS receptors have been described in OB ~1'2°'34, Previous studies from our laboratory have indicated that acute cocaine administration influences the SS binding in the OB of mature rats 35. These previous reports raise the possibility that cocaine might be able to influence the development of somatostatinergic receptors or neurons in the immature OB. As an initial approach to examining this possibility, we studied the effects of administering cocaine to pregnant and/or nursing rats on SS binding in the OB's of their developing offspring at 0 and 15 days old. The study also includes the determination of the concentration of somatostatin-like immunoreactivity (SLI) in this brain area.
Correspondence: E. Arilla, Molecular Neuroendocrinology Unit, Department of Biochemistry and Molecular Biology, Medical School, University of AIcahl, 28871 AIcahl de Henares, Madrid, Spain.
68 15-day-old rats, the olfactory bulbs were excised from ten offspring from each experimental group. In both cases the olfactory bulba were analyzed individually.
MATERIALS AND METHODS
Materials Synthetic Tyrlt-SS and SS-I4 were purchased from Universal Biologicals Ltd. (Cambridge, UK). cocaine hydrochloride was from Alcaliber S.A. (Madrid, Spain) and bovine serum albumin (BSA) was from Sigma (St. Louis, M e , USA), carrier-free Na1251 (IMS 30; 100 mCi/ml) was from the Radiochemical Centre (Amersham, UK). Tyrtt-SS was radioiodinated by the chloramine-T method 21, The tracer was purified on a Sephadex G-25 coarse column (I × 100) which had been equilibrated with 0.l M acetic acid containing bovine ,serum albumin. The rabbit antibody used in the radioimmunoassay technique was purchased from the Radiochemical Centre (Amersham. UK). This antiserum was raised in rabbits against SS-14 conjugated to bovine serum albumin and is specific for SS but. since SS-14 constitutes the C-terminal portions of both SS-25 and SS-28, the a n t i , rum does not distinguish between these three forms. Animals Nulliparous female Wistar rats from our own colony weighing 250-280 g at the initiation of mating were used. All animals received a stock diet and tap water ad libitum. Room temperature was kept at 22°C and a day-night cycle of 12 h. Animals were mated overnight and were afterwards randomly assigned to control and experimental groups. The morning on which a sperm plug was found was designated gestation day 0. The animals were divided into four groups. The first group of rats were injected subcutaneously with a single daily doses of 40 mg of cocaine hydrochloride/kg dissolved in saline from day 7 through 19 of gestation. The major periods for organogenesis and the development of the central nervous system (CNS) in the rat occur during gestational days 7-19 'pj, The cocaine dose was selected on the basis of the literature a't'~''s, Recent studies have found substantial amounts of cocaine in fetal circul:ttion and in the brain after s.c. administralion of 40 mg/k8 of cocaine, suggesting that this is an adequate administration route 4~, Some pups from females treated with cocaine were cross.fostered with litters from untreated (control) mothers within one day of delivery. In a second group the mothers received the sl,me treatment as above except that it was extended beyond parturition through to the fifteenth postnatal tiny. In a third group maternal cocaine administration was started ju~st after birth and continued during fifteen days I~)st partum, The site of the injection on the dorsal surface was varied daily to avoid injecting the cocaine solution into necrotic sites, Control animals received saline instead of cocaine. Since females are more sensitive to cocaine than males t~'tM, the present study was restricted to females. The female offspring of the treated and control rats were sacrificed by decapitation at 0 or 15 days old, their brains were rapidly removed at 4°C, and the OBs were immediately dissected on ice Iq. The olfactory bulbs were excised from six or seven newborn rat pups from control and cocaine-treated rats, respectively, in the case of the
Tissue extraction and somatostatin radioimmunoassay SS was extracted from each OB pool following the method of Patel and Reichlin "~°, and the yield of the overall extraction procedure was about 85%. Protein concentration was determined by the method of Lowry et al. 23 using bovine serum albumin (BSA) as standard. SL! levels were measured by a modified specific radioimmunoassay method ~°, with a sensitivity limit of l0 pg/ml, Incubation tubes prepared in triplicate contained 100 pl samples of unknown or standard solutions of 0-500 pg cyclic SS tetradecapeptide diluted in phosphate buffer (0.05 M, pH 7.2, containing 0,3% BSA, 0.01 M EDTA), 200 ~l appropriately diluted anti-SS serum, and 100 /,tl freshly prepared 1251-TyrlLSS diluted in buffer to give a final volume of 0.8 ml. All reagents and assay tubes were kept chilled on ice until the 48 h incubation at 4°C. separation of bound and free hormone was accomplished by the addition of 1 ml dextran-coated charcoal (dextran 0 2% w/v; charcoal 2% w/v). Dilution curves for OB were parallel to the standard curve. The intra- and inter-assay variation coefficients were 6.5% and 8.7% respectively.
Binding assay on cell preparations Cells from each pool of the OBs of newborn or 15-day-old rats pups were dissociated by mechanical means as described previously 3~. Briefly, brains were rapidly removed and placed on ice, After the blood vessels and pin mater had been carefully removed, the OB were minced into small chunks (I mm "~) and transferred to a 50 ml tube for suspension in 0.9% ice-cold saline solution. The tissue was mashed with the help of a I ml pipette and mixed using a Vortex for I rain at maximum speed. The tissue suspension was first filtered through a Nitex nylon filter of 355 tim pore size, then through another with a pore size of 80 ~tm. The resulting cell suspension was centrifuged at 1,0(~)x R for 10 rain and suspended in phosphatebuffered saline. Cell viability was always greater than 9O% as measured by exchision of Trypan blue dye, Cell protein wits estimated by the method of l~wry et al, ~'~, Expurlmental conditions for SS binding were essentially as previously described q, Briefly, dissociated cells from rat OB (I mg pro. t~in/ml) were incubated in 0.5 ml of 50 mM Tris-llCI buffer (pit 7.5) containing 5 mM MgCI,, 30 mM NaCI, I% BSA, 0.1% bacitracin and II~) pM (t'~Sl-Tyr*t-SS) in the absence or presence of 0,01-10 nM unlabeled SS, After 60 rain incubation at 25°C, cellbound peptide was isolated by centrifugation ~t 13,000× r for 1,5 rain, and radioactivity determined in a Kontron gamma counter. Non-specific binding was obtained from the amount of radioactivity bound in the presence of 10-* M SS and represented about 20% of the binding observed in the absence of unlabeled peptide, This non-specific component was subtracted from the total bound radioactivity in order to obtain the corresl~nding specific binding,
TABLE I
Effect of eta'ainu administration to prt'gnant and~or m~rsing rats on the somalosmtin.like #mmmoreactirity (SLI) #~ olfactory bulb in de~,eloping offspnn~ Results are me,ms± S,E,M. They correspond to the mean values of all treatment periods for each animal, in each of the experiments determinations were made in duplicate,
Gro~q;s
Saline Cocaine-exposure during pregnancy Cocaine-exposure during nursing Cocaine-exposure during pregnancy and nursing
SLi c(mcentratkm (rig /mR protein)
0 Days
n
15 Days
n
3.19 ~ 0.17 2.31 + 0.25 * * -
6 7
4.9O :!:0.37 5.53 + 0.47 5,07 :[:0,66
10 10 10
6,76 + 0.47 *
10
_
Statistical cow,qarison versus controls: * P < 0.05; * * P < 0.005; n = number of animals.
69 El,aluation of radiolabeled peptide degradation To determine the extent of tracer degradation during incubation, we measured the ability of preincubated peptide to bind to fresh cells as previously described 3'~. Briefly, 12SI-TyrlLSS (100 pM) was incubated with dissociated cells from rat OB (1 mg protein/ml) for 60 rain at 25°C. After this preincubation aliquots of the medium were added to fresh cells and incubated for an additional 60 rain at 25°C. The fraction of the added radiolabeled peptide which was specifically bound during the second incubation was measured and expressed as a percentage of the binding that had been obtained in control experiments performed in the absence of cells during the preincubation period.
"~ 100 o
so !
Analysis data Results were given in all cases as the mean +S.E.M. Student's t-test for unpaired variables was employed to assess differences between control and experimental groups, as indicated in the text and figures. The number of receptors and affinity constants in the Scatchard plots "~a were calculated by linear regression analysis,
0
J
ao 11
10 9 (SSi,-IogM
8
Fig. 1. Competitive inhibition of specific 12Sl-TyrlLsomatostatin (12S|-Tyrl!-SS, 100 pM) binding by unlabeled somatostatin to dissociated ceils of the olfactory bulb 15-day-old offspring of control (o), rats treated with cocaine during pregnancy and sacrificed at 15 days of age (o) or during 15 days postpartum (El) or during pregnancy plus 15 first days postpartum ( ,~ ). Dissociated cells (1 mg protein/ml) were incubated for 60 rain at 25°C in the presence of 100 pM 12SI-Tyrll - SS and increasing concentrations of native peptide. Each point corresponds to the mean values of all treatment periods for each animal. The determinations were made in duplicate. For the sake of clarity, standard error is not represented but it was always below 10% of the mean values.
RESULTS
Cocaine administration to pregnant rats resulted in decreased SLI concentration in the OB of the offspring on the day of birth. While prenatal plus postnatal exposure increased this parameter, maternal cocaine administration only during lactation (i.e. begun after birth) produced no changes in SLI level in the OB of the offspring as compared with the control groups on day 15 (Table l). Dissociated cells from the OB from both control and cocaine-exposed rats of 0 and 15 days old bound ~2S-Tyr~-SS in a time-dependent process; an apparent equilibrium was observed between 50 and 120 rain at 25°C (data not shown). All subsequent binding experiments were therefore conducted at 25°C for 60 rain. Peptide degradation was determined in all the preparations to rule out the possibility of different SS de-
grading activities which might have affected the interpretation of the results. Dissociated cells from the OB showed a similar peptide degradation capacity and the values varied by no more than 10% in all the experimental groups, Increasing concentrations of unlaueled SS competitively inhibited the specific binding of ~SI-Tyr~-SS to dissociated cells in the preparations from the offspring of both control and cocaine.exposed rats (Fig. 1). The administration of cocaine during either pregnancy or
TABLE !i Effect of cocaine adtninistration to pregnant attd / or nursing rats on ecluilibrium parumeters for somatostatm Omdmx to dis',vociated cells from olfactory bulb in det,eloping o])'spring K o is the dissociation constant, nM; Bm,~x is the binding capacity, from fmol of SS bound per mg protein. Results are means+ S.E.M. They correspond to the mean values of all treatment periods for each animal.
Groups
Olfactory bulb 0 Days
n ,
Saline Ko Bmux
Cocaine-exposure during pregnancy Kd Bmax Cocaine-exposure during nursing Kd
6 0.32 + 0.03 89 4-7
n 10
0.47 4- 0.08 127 :i-20 7
0.86+0,07 * * * * 150 -*'5 * * * *
10 0.90+ 0.12 * 250 -k14 *** 10 0,71+ 0.16 * 269 +57 **
Bmllx
Cocaine-exposure during pregnancy and nursing Kd Bmag
15 Days
,,
i0 M M
0.34 + 0.06 150 + 26
Statistical comparison versus controls: * P < 0.05; * * P < 0.025; * * * P < 0.0025; * * * * P < 0.0001; n = number of animals.
70 nursing modified the binding of ~2"~l-Tyrl~-SS in dissociated cells from the OB in the 0- and 15-day-old offspring (Fig. 1). Data were analyzed by the Scatchard method 3a and these studies revealed that there was an increase in the number of specific SS receptors and a decrease in the affinity constant in the OB in the 0- to 15-day-old offspring of the rats exposed to cocaine during either pregnancy or nursing, but not in those continuously exposed to the drug from conception until 15 days (Table !1). DISCUSSION The administration of cocaine during either gestation or lactation increased the number of SS receptors and decreased the affinity constant in dissociated cells from the OB in the 0- and 15-day-o!d offspring. Continued maternal administration of cocaine until 15 days post partum did not affect the SS binding in the brain area studied. The change in SL! content between 0- and 15-dayold offspring of the control rats shown here is similar to that seen in previous studies 2a'2~. Since SS activity is known to be prcsynaptically regulated by dopamine 4H, a decrease in SL! content could be a secondary reaction to the cocaine-induced inhibition of dopamine re.uptake 4~. The increase reported in the number of SS receptors in offspring of control rats parallels the development of specific somatostatinergic pathways :M. it should be mentioned that Scatchard analysis demonstrated the existence of only one type of SS receptor. This feature agrees with some studies z°,lz4r', but differs from
others ~'~'~t. It is conceivable that the use of small SS analogs "~'~ or their I,beli;~, with different isotopes 51 might explain these differences. The fact that exposure to cocaine during gestation and the first 15 days post partum (throughout lacta. tion) did not modify SS receptors in the offspring may be related to tolerance which occurs after repeated cocaine administration 37. The mechanism responsible for the increase in the SS receptor number observed in the present study has not yet been identified. Several studies have shown anatomical and functional intetconnections between the dopaminergie and somatostatinergic systems t''~'tt''4°'4x. We have recently reported that blocking dopamine receptors with haloperidoi decreased the number of SS receptors in the rat brain 3~. After haloperidol is withdrawn, the number of dopamine receptors ~i~es~', and the number of SS receptors returns to cor~trol values, although the actual number of
receptors remains slightly higher 32. Recently, it has been shown that perinatal cocaine exposure leads to an increase in the number of dopamine receptors in the rat brain t4. These previous findings together with the present results may suggest that the effects of prenatal or postnatal cocaine exposure on SS receptors may be mediated in part by the effects of the drug on dopaminergic neuronal activity, although they do not preclude the possibility that other factors may be involved as well. These results suggest that the development of SS receptors in the rat OB can be altered by prenatal a n d / o r nursing-period exposure to cocaine. Since SS and its receptor are present in the fetal OB 2°'2s and this neurotransmitter is associat~! with several different classes of OB neurons and neuronal processes that are known to interact in the integration of olfactory information 22'27'4t'49, it is tempting to speculate that an early alteration of the somatostatinergic system could contribute at least in part to the impaired sensorial information processed by the OB that has been reported in prenatal cocaine-exposed offspring 4"~. Acknowledgements. This work was supported by grants from the
Comisi0n lnterministerial de Ciencia y Tecnologia(PB87-0753) and the Fondo de investigaeionesSanitarias de la Seguridad Social of Spain (88/0903). The authors thank Carol F. Warren, from the Alcalti de llenares UniversityInstitute of Education Sciencesfor her editorial help.
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32 Perez-Oso, E., L6pez-Ruiz, M.P. and Arilla, E., Effect of haloperidol withdrawal on somatostatin level and binding in rat brain, Biosci. Rep, 10 (1990) 15-22. 33 Reubi, J.C., Evidence for two somatostatin-14 receptor types in rat brain cortex, Neurosci. Lett., 49 (1984) 259-263. 34 Reubi, J.C. and Maurer, R., Autoradiographic mapping of somatostatin receptors in the rat central nervous system and pituitary, Neuroscience, 15 (1985) 1183-1193. 35 Rodr[guez-Sfinchez, M.N. and Arilla, E., Effects of acute and chronic cocaine administration on somatost~tin level and binding in the rat brain, Neuropeptides, 16 (1990) 1-7. 36 Roeder, L.M., Tildon, J.T. and Williams, I.B., Transport of 2-deoxy-D-glucose by dissociated brain cells, Brain Res., 345 (1985) 298-305. 37 Roy, S.N., Bhattacharyya, A.K., Pradhan, S. and Pradhan, S.N., Behavioural and neurochemical effects of repeated administration of cocaine in rats, Neuropharmacology, 17 (1978) 559-564. 38 Scatchard, G., The attractions of proteins for small molecules and ions, Ann. NYAcad. Sci., 51 (1949) 660-671. 39 Schonbrunn, A., RorstaJ, Q*.P., Westendorf, J.M. and Martin, J.B., Somatostatin analogs: correlation between receptor binding affinity and biological potency in GH pituitary cells, Endocrinology, 113 (1983) 1559-1567. 40 Salin, P., Mercugliano, M. and Chesselet, M.F., Differential effects of chronic treatment with haloperidol and clozapine on the level of preprosomatostatin mRNA in the striatum, nucleus accumbens, and frontal cortex of the rat, Cell. Mol. NeurobioL, 10 (1990) 127-144. 41 Seroogy, K., H6kfelt, T., Buchan, A., Brown, J.C., Terenius, L., Norman, A.W. and Goldstein, M., Somatostatin-like immunoreactivity in rat main olfactory bulb: extent of coexistence with neuropeptide Y-, tyrosine hydroxylase- and vitamin D-dependent calcium binding protein-like immunoreactivities, Brain Res., 496 (1989) 389-396. 42 Shah, N.S., May, D.A. and Yates, J.D., Disposition of levo[3H]cocaine in pregnant and non-pregnant mice, Toxicol. Appl. PharnmcoL, 53 (1980) 279-284. 43 Smith, R.F., Mattran, K.M., Kurkjian, M.F. and Kurtz, S.L., Alterations in offspring behavior induced by chronic prenatal cocaine dosing, Nel4rotoxicoL Teratol., I ! (1989) 35-38. 44 Spear, L.P., Frambes, N.A. and Kirstein, C.L., Fetal :rod maternal brain and plasma levels of cocaine and benzoylecgonine following chronic subcutaneous administration of cocaine during gestation in rats, P.~ychopharmacology, 97 (1989) 427-431, 45 Spear, L.P., Kirstein, C.L., Bell, J., Yoottanasumpun, V., Greenbaum, R., O'Shea, J., Hoffmann, H. and Spear, N.E., Effects of prenatal cocaine exposure on behavior during the early postnatal period, Net~rotoxicol. TeratoL, I I (1989) 57-63. 46 Srikant, C.B. and Patel, Y.C., Somatostatin receptors: identification and characterization in rat brain membranes, Proc. Natl. Ac.~d. Sci. USA, 78 (1981) 3930-3934. 47 Taylor, D. and Ho, B.T., Comparison of ;nhibition of monoamine uptake by cocaine, methylphenidate and amphetamine, Res. Commun. Chem. Pathol. PharmacoL, 21 (1978)67-75. 48 Thai, L.J., Laing, K., Horowitz, S.G. and Makman, M.lt., Dopamine stimulates rat cortical somatostatin release, Brain Res., 372 (1986) 205-209. 49 Tuchmann-Duplessis, H., Drug Effects on the Fetus, ADIS Press, Sydney, 1975. 50 Vincent, S.R., Mclntosh, C.H.S., Buchan, A.M.J. and Brown, J.C., Central somatostatin systems revealed with monoclonal antibodies, J. Comp. NeuroL, 238 (1985) 169-186. 51 Weightman, D.R., Whitford, C.A., Snell, C.R., Hirst, B.H., Brundish, D.E. and Kendall-Taylor, P.A., Rat brain membranes possess two high-affinity binding sites for ['~H]somatostatin, Ne,rosci. Lett., 55 (1985) 161-166. 52 Wiggins, R.C., Rolsten, C., Ruiz, B. and Davis, C.M., Pharmacokinetics of cocaine: basic studies of route dosage, pregnancy, and lactation, Neurotoxicology, 10 (1989) 367-382.
67
BRES 17844
Somatostatin receptors increase in the olfactory bulb of developing pups after perinatal exposure to cocaine M.N. R o d r l g u e z - S ~ n c h e z a n d E. Arilla Molecular Neuroendocrinology Unit, Department of Biochemistry and Molecular Biology, Medical School, Unn'ersity of AIcald, AIcald de Henares, Madrid (Spain) (Accepted, 29 January 1992)
Key words: Cocaine; Somatostatin receptor; Olfactory bulb; Rat
The effects of chronic prenatal and/or postnatal exposure to cocaine on somatostatin concentration and receptors were studied in the olfactory bulbs of rat pups at birth and at 15 days old. Wistar rats were injected subcutaneously with single daily doses of 40 mg cocaine hydrochloride/kg from days 7 to 19 of gestation, from day 7 of gestation to day 15 postpartum or from parturation to day 15 postpartum. Fetal exposure to cocaine decreased SS concentrations in the olfactory bulb of the newborn pups while prenatal-plus-postnatal exposure increased this parameter. Administration of cocaine only during lactation did not induce any change. Exposure during gestation or during nursing induced an increase in the total number of somatostatin receptors and a decrease in the affinity constant in the olfactory bulb of newborn and 15-day-old pups. These results suggest that the development of somatostatin receptors in the olfactory bulb can be altered by prenatal and/or nursing period exposure to cocaine.
INTRODUCTION
The dramatic increase in cocaine use among young adults of child-bearing age has resulted in a rise in its use during pregnancy z'7'u. In laboratory investigations cocaine has been shown to penetrate the pli~,centa and accumulate in fetal tissues at concentrations greater than those observed in maternal plasma 42'u. In addition, cocaine can be passed to offspring through breast feeding 52. Early studies of gestational cocaine exposure in laboratory animals focused predominantly on possible teratogenic effects ~3'26, whereas more recent studies have assessed neurobehavioral function in exposed offspring 43'4s. Spear et al.45 have recently reported that prenatally cocaine-exposed offspring showed marked impairment in learning an odor/milk association. It should be kept in mind that the sense of smell is important for developing animals, and especially for neonatal rats, which rely on olfactory cues to locate their mother's nipple 3. Evidence is accumulating to the
effect that the mammalian olfactory bulb (OB) contains various neuroactive substances including peptides 1"~'27,41. Somatostatin (SS), originally detected in the hypothalamus 4, is associated with several different classes of neurons and neuronal processes in the OB that are known to interact in the integration of olfactory information 2~'27'4L4~.In addition, SS receptors have been described in OB ~1'2°'34, Previous studies from our laboratory have indicated that acute cocaine administration influences the SS binding in the OB of mature rats 35. These previous reports raise the possibility that cocaine might be able to influence the development of somatostatinergic receptors or neurons in the immature OB. As an initial approach to examining this possibility, we studied the effects of administering cocaine to pregnant and/or nursing rats on SS binding in the OB's of their developing offspring at 0 and 15 days old. The study also includes the determination of the concentration of somatostatin-like immunoreactivity (SLI) in this brain area.
Correspondence: E. Arilla, Molecular Neuroendocrinology Unit, Department of Biochemistry and Molecular Biology, Medical School, University of AIcahl, 28871 AIcahl de Henares, Madrid, Spain.
68 15-day-old rats, the olfactory bulbs were excised from ten offspring from each experimental group. In both cases the olfactory bulba were analyzed individually.
MATERIALS AND METHODS
Materials Synthetic Tyrlt-SS and SS-I4 were purchased from Universal Biologicals Ltd. (Cambridge, UK). cocaine hydrochloride was from Alcaliber S.A. (Madrid, Spain) and bovine serum albumin (BSA) was from Sigma (St. Louis, M e , USA), carrier-free Na1251 (IMS 30; 100 mCi/ml) was from the Radiochemical Centre (Amersham, UK). Tyrtt-SS was radioiodinated by the chloramine-T method 21, The tracer was purified on a Sephadex G-25 coarse column (I × 100) which had been equilibrated with 0.l M acetic acid containing bovine ,serum albumin. The rabbit antibody used in the radioimmunoassay technique was purchased from the Radiochemical Centre (Amersham. UK). This antiserum was raised in rabbits against SS-14 conjugated to bovine serum albumin and is specific for SS but. since SS-14 constitutes the C-terminal portions of both SS-25 and SS-28, the a n t i , rum does not distinguish between these three forms. Animals Nulliparous female Wistar rats from our own colony weighing 250-280 g at the initiation of mating were used. All animals received a stock diet and tap water ad libitum. Room temperature was kept at 22°C and a day-night cycle of 12 h. Animals were mated overnight and were afterwards randomly assigned to control and experimental groups. The morning on which a sperm plug was found was designated gestation day 0. The animals were divided into four groups. The first group of rats were injected subcutaneously with a single daily doses of 40 mg of cocaine hydrochloride/kg dissolved in saline from day 7 through 19 of gestation. The major periods for organogenesis and the development of the central nervous system (CNS) in the rat occur during gestational days 7-19 'pj, The cocaine dose was selected on the basis of the literature a't'~''s, Recent studies have found substantial amounts of cocaine in fetal circul:ttion and in the brain after s.c. administralion of 40 mg/k8 of cocaine, suggesting that this is an adequate administration route 4~, Some pups from females treated with cocaine were cross.fostered with litters from untreated (control) mothers within one day of delivery. In a second group the mothers received the sl,me treatment as above except that it was extended beyond parturition through to the fifteenth postnatal tiny. In a third group maternal cocaine administration was started ju~st after birth and continued during fifteen days I~)st partum, The site of the injection on the dorsal surface was varied daily to avoid injecting the cocaine solution into necrotic sites, Control animals received saline instead of cocaine. Since females are more sensitive to cocaine than males t~'tM, the present study was restricted to females. The female offspring of the treated and control rats were sacrificed by decapitation at 0 or 15 days old, their brains were rapidly removed at 4°C, and the OBs were immediately dissected on ice Iq. The olfactory bulbs were excised from six or seven newborn rat pups from control and cocaine-treated rats, respectively, in the case of the
Tissue extraction and somatostatin radioimmunoassay SS was extracted from each OB pool following the method of Patel and Reichlin "~°, and the yield of the overall extraction procedure was about 85%. Protein concentration was determined by the method of Lowry et al. 23 using bovine serum albumin (BSA) as standard. SL! levels were measured by a modified specific radioimmunoassay method ~°, with a sensitivity limit of l0 pg/ml, Incubation tubes prepared in triplicate contained 100 pl samples of unknown or standard solutions of 0-500 pg cyclic SS tetradecapeptide diluted in phosphate buffer (0.05 M, pH 7.2, containing 0,3% BSA, 0.01 M EDTA), 200 ~l appropriately diluted anti-SS serum, and 100 /,tl freshly prepared 1251-TyrlLSS diluted in buffer to give a final volume of 0.8 ml. All reagents and assay tubes were kept chilled on ice until the 48 h incubation at 4°C. separation of bound and free hormone was accomplished by the addition of 1 ml dextran-coated charcoal (dextran 0 2% w/v; charcoal 2% w/v). Dilution curves for OB were parallel to the standard curve. The intra- and inter-assay variation coefficients were 6.5% and 8.7% respectively.
Binding assay on cell preparations Cells from each pool of the OBs of newborn or 15-day-old rats pups were dissociated by mechanical means as described previously 3~. Briefly, brains were rapidly removed and placed on ice, After the blood vessels and pin mater had been carefully removed, the OB were minced into small chunks (I mm "~) and transferred to a 50 ml tube for suspension in 0.9% ice-cold saline solution. The tissue was mashed with the help of a I ml pipette and mixed using a Vortex for I rain at maximum speed. The tissue suspension was first filtered through a Nitex nylon filter of 355 tim pore size, then through another with a pore size of 80 ~tm. The resulting cell suspension was centrifuged at 1,0(~)x R for 10 rain and suspended in phosphatebuffered saline. Cell viability was always greater than 9O% as measured by exchision of Trypan blue dye, Cell protein wits estimated by the method of l~wry et al, ~'~, Expurlmental conditions for SS binding were essentially as previously described q, Briefly, dissociated cells from rat OB (I mg pro. t~in/ml) were incubated in 0.5 ml of 50 mM Tris-llCI buffer (pit 7.5) containing 5 mM MgCI,, 30 mM NaCI, I% BSA, 0.1% bacitracin and II~) pM (t'~Sl-Tyr*t-SS) in the absence or presence of 0,01-10 nM unlabeled SS, After 60 rain incubation at 25°C, cellbound peptide was isolated by centrifugation ~t 13,000× r for 1,5 rain, and radioactivity determined in a Kontron gamma counter. Non-specific binding was obtained from the amount of radioactivity bound in the presence of 10-* M SS and represented about 20% of the binding observed in the absence of unlabeled peptide, This non-specific component was subtracted from the total bound radioactivity in order to obtain the corresl~nding specific binding,
TABLE I
Effect of eta'ainu administration to prt'gnant and~or m~rsing rats on the somalosmtin.like #mmmoreactirity (SLI) #~ olfactory bulb in de~,eloping offspnn~ Results are me,ms± S,E,M. They correspond to the mean values of all treatment periods for each animal, in each of the experiments determinations were made in duplicate,
Gro~q;s
Saline Cocaine-exposure during pregnancy Cocaine-exposure during nursing Cocaine-exposure during pregnancy and nursing
SLi c(mcentratkm (rig /mR protein)
0 Days
n
15 Days
n
3.19 ~ 0.17 2.31 + 0.25 * * -
6 7
4.9O :!:0.37 5.53 + 0.47 5,07 :[:0,66
10 10 10
6,76 + 0.47 *
10
_
Statistical cow,qarison versus controls: * P < 0.05; * * P < 0.005; n = number of animals.
69 El,aluation of radiolabeled peptide degradation To determine the extent of tracer degradation during incubation, we measured the ability of preincubated peptide to bind to fresh cells as previously described 3'~. Briefly, 12SI-TyrlLSS (100 pM) was incubated with dissociated cells from rat OB (1 mg protein/ml) for 60 rain at 25°C. After this preincubation aliquots of the medium were added to fresh cells and incubated for an additional 60 rain at 25°C. The fraction of the added radiolabeled peptide which was specifically bound during the second incubation was measured and expressed as a percentage of the binding that had been obtained in control experiments performed in the absence of cells during the preincubation period.
"~ 100 o
so !
Analysis data Results were given in all cases as the mean +S.E.M. Student's t-test for unpaired variables was employed to assess differences between control and experimental groups, as indicated in the text and figures. The number of receptors and affinity constants in the Scatchard plots "~a were calculated by linear regression analysis,
0
J
ao 11
10 9 (SSi,-IogM
8
Fig. 1. Competitive inhibition of specific 12Sl-TyrlLsomatostatin (12S|-Tyrl!-SS, 100 pM) binding by unlabeled somatostatin to dissociated ceils of the olfactory bulb 15-day-old offspring of control (o), rats treated with cocaine during pregnancy and sacrificed at 15 days of age (o) or during 15 days postpartum (El) or during pregnancy plus 15 first days postpartum ( ,~ ). Dissociated cells (1 mg protein/ml) were incubated for 60 rain at 25°C in the presence of 100 pM 12SI-Tyrll - SS and increasing concentrations of native peptide. Each point corresponds to the mean values of all treatment periods for each animal. The determinations were made in duplicate. For the sake of clarity, standard error is not represented but it was always below 10% of the mean values.
RESULTS
Cocaine administration to pregnant rats resulted in decreased SLI concentration in the OB of the offspring on the day of birth. While prenatal plus postnatal exposure increased this parameter, maternal cocaine administration only during lactation (i.e. begun after birth) produced no changes in SLI level in the OB of the offspring as compared with the control groups on day 15 (Table l). Dissociated cells from the OB from both control and cocaine-exposed rats of 0 and 15 days old bound ~2S-Tyr~-SS in a time-dependent process; an apparent equilibrium was observed between 50 and 120 rain at 25°C (data not shown). All subsequent binding experiments were therefore conducted at 25°C for 60 rain. Peptide degradation was determined in all the preparations to rule out the possibility of different SS de-
grading activities which might have affected the interpretation of the results. Dissociated cells from the OB showed a similar peptide degradation capacity and the values varied by no more than 10% in all the experimental groups, Increasing concentrations of unlaueled SS competitively inhibited the specific binding of ~SI-Tyr~-SS to dissociated cells in the preparations from the offspring of both control and cocaine.exposed rats (Fig. 1). The administration of cocaine during either pregnancy or
TABLE !i Effect of cocaine adtninistration to pregnant attd / or nursing rats on ecluilibrium parumeters for somatostatm Omdmx to dis',vociated cells from olfactory bulb in det,eloping o])'spring K o is the dissociation constant, nM; Bm,~x is the binding capacity, from fmol of SS bound per mg protein. Results are means+ S.E.M. They correspond to the mean values of all treatment periods for each animal.
Groups
Olfactory bulb 0 Days
n ,
Saline Ko Bmux
Cocaine-exposure during pregnancy Kd Bmax Cocaine-exposure during nursing Kd
6 0.32 + 0.03 89 4-7
n 10
0.47 4- 0.08 127 :i-20 7
0.86+0,07 * * * * 150 -*'5 * * * *
10 0.90+ 0.12 * 250 -k14 *** 10 0,71+ 0.16 * 269 +57 **
Bmllx
Cocaine-exposure during pregnancy and nursing Kd Bmag
15 Days
,,
i0 M M
0.34 + 0.06 150 + 26
Statistical comparison versus controls: * P < 0.05; * * P < 0.025; * * * P < 0.0025; * * * * P < 0.0001; n = number of animals.
70 nursing modified the binding of ~2"~l-Tyrl~-SS in dissociated cells from the OB in the 0- and 15-day-old offspring (Fig. 1). Data were analyzed by the Scatchard method 3a and these studies revealed that there was an increase in the number of specific SS receptors and a decrease in the affinity constant in the OB in the 0- to 15-day-old offspring of the rats exposed to cocaine during either pregnancy or nursing, but not in those continuously exposed to the drug from conception until 15 days (Table !1). DISCUSSION The administration of cocaine during either gestation or lactation increased the number of SS receptors and decreased the affinity constant in dissociated cells from the OB in the 0- and 15-day-o!d offspring. Continued maternal administration of cocaine until 15 days post partum did not affect the SS binding in the brain area studied. The change in SL! content between 0- and 15-dayold offspring of the control rats shown here is similar to that seen in previous studies 2a'2~. Since SS activity is known to be prcsynaptically regulated by dopamine 4H, a decrease in SL! content could be a secondary reaction to the cocaine-induced inhibition of dopamine re.uptake 4~. The increase reported in the number of SS receptors in offspring of control rats parallels the development of specific somatostatinergic pathways :M. it should be mentioned that Scatchard analysis demonstrated the existence of only one type of SS receptor. This feature agrees with some studies z°,lz4r', but differs from
others ~'~'~t. It is conceivable that the use of small SS analogs "~'~ or their I,beli;~, with different isotopes 51 might explain these differences. The fact that exposure to cocaine during gestation and the first 15 days post partum (throughout lacta. tion) did not modify SS receptors in the offspring may be related to tolerance which occurs after repeated cocaine administration 37. The mechanism responsible for the increase in the SS receptor number observed in the present study has not yet been identified. Several studies have shown anatomical and functional intetconnections between the dopaminergie and somatostatinergic systems t''~'tt''4°'4x. We have recently reported that blocking dopamine receptors with haloperidoi decreased the number of SS receptors in the rat brain 3~. After haloperidol is withdrawn, the number of dopamine receptors ~i~es~', and the number of SS receptors returns to cor~trol values, although the actual number of
receptors remains slightly higher 32. Recently, it has been shown that perinatal cocaine exposure leads to an increase in the number of dopamine receptors in the rat brain t4. These previous findings together with the present results may suggest that the effects of prenatal or postnatal cocaine exposure on SS receptors may be mediated in part by the effects of the drug on dopaminergic neuronal activity, although they do not preclude the possibility that other factors may be involved as well. These results suggest that the development of SS receptors in the rat OB can be altered by prenatal a n d / o r nursing-period exposure to cocaine. Since SS and its receptor are present in the fetal OB 2°'2s and this neurotransmitter is associat~! with several different classes of OB neurons and neuronal processes that are known to interact in the integration of olfactory information 22'27'4t'49, it is tempting to speculate that an early alteration of the somatostatinergic system could contribute at least in part to the impaired sensorial information processed by the OB that has been reported in prenatal cocaine-exposed offspring 4"~. Acknowledgements. This work was supported by grants from the
Comisi0n lnterministerial de Ciencia y Tecnologia(PB87-0753) and the Fondo de investigaeionesSanitarias de la Seguridad Social of Spain (88/0903). The authors thank Carol F. Warren, from the Alcalti de llenares UniversityInstitute of Education Sciencesfor her editorial help.
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