Seasonal changes of SP and NKA in frontal cortex, striatum and testes in the rat. Role of maternal pineal gland

Peptides 25 (2004) 997–1004

Seasonal changes of SP and NKA in frontal cortex, striatum and testes in the rat. Role of maternal pineal gland N. Vázquez Moreno a , L. Debeljuk b , E. D´ıaz Rodr´ıguez a , C. Fernández Alvarez a , B. D´ıaz López a,∗ a

Dpto. Biolog´ıa Funcional, Area Fisiolog´ıa, Facultad de Medicina, Universidad de Oviedo, C/Julian Claver´ıa, No. 6, 33006 Oviedo, Spain Department of Health Care Professions, College of Applied Sciences and Arts, Southern Illinois University, Carbondale, IL 62901, USA

b

Received 11 June 2003; accepted 19 March 2004

Abstract The concentrations of neurokinin A (NKA) and substance P (SP), members of tachykinins family, have been studied in all seasons of the year in frontal cortex, striatum and testes of male offspring 21-, 31-, or 60 days old of mother Wistar rats: control, pinealectomized (PIN-X) and pinealectomized + melatonin during pregnancy (PIN-X + MEL) kept under 12 h:12 h L:D. Control-offspring: in spite of having been kept under constant environmental conditions throughout the year, had marked differences in tachykinin concentrations. The highest tachykinin concentrations in the frontal cortex were found in summer and fall and the lowest in winter and spring. Maternal PIN-X resulted in alterations of this developmental pattern, mainly in PIN-X- and PIN-X + MEL-offspring in which the highest tachykinin concentrations at 21 and 31 days of age were only observed during summer. The alterations were observed up to 60 days of age for both tachykinins, when at this age control-offspring showed similar NKA concentrations. Seasonal variations were still observed in PIN-X- and PIN-X + MEL-offspring. In striatum and testes no mayor modifications throughout the four seasons of the year were found, with very few exceptions. PIN-X did not alter tachykinin concentrations, neither treatment with melatonin did it. In conclusion, our data clearly indicate for the first time that NKA and SP do indeed have seasonal rhythms in frontal cortex and that the maternal pineal gland plays a role in their entrainment already during fetal life. © 2004 Elsevier Inc. All rights reserved. Keywords: NKA; SP; Seasonal variations; Maternal pineal gland; Frontal cortex; Striatum; Testes

1. Introduction Neurokinin A (NKA) is a decapeptide with structural characteristics, biological activities, and localizations similar to those of substance P (SP). NKA and SP, as members of the tachykinin group, represent one of the largest families of peptides present in all animal species, from lower invertebrate to large mammals. Tachykinins are widely distributed in the central and peripheral nervous system. In the central nervous system (CNS) tachykinins occur in large amounts, particularly in areas involving the central control of several peripheral autonomic functions including vital functions (e.g., drinking behaviour), affective and emotional life (e.g., motility, anxiety, aggression and pain), along with higher cerebral functions such as learning and memory. In neuronal cells, they act as neurotransmitters/neuromodulators, ∗

Corresponding author. Tel.: +34-985102713; fax: +34-985103534. E-mail address: [email protected] (B. D´ıaz L´opez).

0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.03.022

while in nonneuronal cells they act as autocrine, paracrine or endocrine regulators [31]. Less is known regarding the distribution of NKA is the rat brain. It appears to be similar to that of SP with some differences [3,4,11,34]. SP and NKA, however, have been reported in the rat testes [1,7]. Three doses of tachykinins stimulated the release of lactic acid and transferrin by Sertoli cells into the culture medium in incubations lasting for 24 h. Lactate and transferrin are an index of the secretory activity of Sertoli cells involved in the progression of spermatogenesis [18]. The mammalian pineal gland, acting through the circadian secretion rhythm of melatonin, reaches higher activity during the night, and in turn mediates the photoperiodic entrainment of endogenous circadian and seasonal rhythms [13]. Endogenous cyclic changes, in absent of photoperiod or temperature cues, have also been described along with the continued presence of annual rhythms in body mass, locomotor activity, and hibernation bouts of ground squirrels [23]. In rats without photoperiodic or temperature changes,

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other authors have noted circannual variations in serum concentrations of several hormones [35], hypothalamic neuropeptides [2], ventromedial hypothalamic nucleus [25] and the size of the cell nuclei of the ventrolateral part of the suprachiasmatic nucleus (SCN) [26]. The laboratory rat, although a nonphotoperiodic rodent, acts in much the same manner as a seasonal breeder, with the photoperiodic information being transduced from the pineal gland of the mother to the fetus [17,18]. However, no modification in litter size and weight of male pups was observed between sham-operated and PIN-X mother rats [18]. Others have found an influence of maternal pineal gland upon prepubertal rat ovarian oocyte development, including the somatic postnatal development of male rat offspring and postnatal ontogeny of the hormones involved in the neuroendocrine-reproductive axis in both female and male developing rats [9,10,12]. We suspect similar findings, i.e., postnatal seasonal variations may be dependent upon an intact maternal melatonin rhythm. The rat pineal gland generates the melatonin circadian rhythm on days 9 and 11 after birth in serum and pineal gland, respectively [33]. The possibility that the source of melatonin for the neonate may come from the mother has been demonstrated in rats [19], Rhesus monkeys [28] and sheep [36]. While the biological time clock of rat pups seem to be determined by their mothers [8], no influence of maternal pineal gland on postnatal seasonal variations has been reported. Neither, seasonal variations in tachykinin concentrations in the CNS in laboratory animals kept in a controlled environment. In the present study, we investigate whether or not tachykinins are found in laboratory rats showing seasonal rhythms in areas of the CNS (specifically the frontal cortex and striatum), or in peripheral tissue. To prove an involvement of the maternal pineal gland in the mechanism entraining such seasonal rhythms, maternal pinealectomy and melatonin treatment were performed.

2. Material and methods 2.1. Laboratory animals Female Wistar rats from our colony of the Faculty of Medicine, University of Oviedo, Spain, were housed under controlled environmental conditions such as 12 h:12 h

photoperiod (lights on at 08.00 a.m.), at a room temperature of approximately 23 ◦ C in a humidity-controlled environment, and they were fed standard rat chow and water ad libitum. Mother rats (Table 1) were divided into the following groups: control, pinealectomized (PIN-X) and pinealectomized + melatonin treatment (PIN-X + MEL). Pinealectomy was carried out two weeks before mating. The study was performed during the four seasons of the year. The experiment was carried out from fall 1999 until winter 2001. Mother rats were mating pairs of one male with two females, possible pregnancy was monitored by the presence of vaginal spermatozoa. During pregnancy mother rats were kept individually in polypropylene cages, at the beginning of each season, on day 20 of December, March and June and 22 of September. Since pregnancy in the rat lasts 21 days, with this timetable offspring up to 60 days of age could be studied in the corresponding 3 months for each season, offspring were studied in the last 2 months of each season. At delivery, litter sex and number of pups were recorded and, in order to obtain uniformity in the development of the pups, on the day of birth each litter was adjusted to 12 pups per dam by cross-fostering some pups from larger litters within treatment groups. Pups remained with the mother until weaning on day 21 (birth = day 0). To study male offspring we followed the classification [22] used for postnatal maturation: (a) juvenile or prepubertal period, from 21 to 35 days of age; animals were studied on the 21st and 31st day; (b) pubertal period, from day 35 to 55–60 days of age, animals being examined on day 60. At the mentioned ages the animals were brought between 09:30 and 10:30 h from the adjacent animal room in the polypropylene cage to a laboratory and were decapitated with a guillotine. After head removal, the sink, guillotine, and counter top were rinsed with copious amounts of water to remove the odor of blood before the next animal was brought in. 2.2. Tissue extraction and processing After decapitation, the skull was quickly opened, the brain was removed and placed on dry ice, then transferred to an ultra-low freezer (−80 ◦ C) until peptide determinations were performed. Brain areas were cut in a single fragment of approximately 20–60 mg of the same part of the frontal cortex, of 4.6–42.9 mg for the striatum and fragments of 52.6–181.9 mg of testes were also removed. Tissues from each animal were immersed in 0.5 ml of ice-cold 2N acetic

Table 1 Body weight (g) and number of mother rats (raised on a 12 h:12 h L:D cycle) control, pinealectomized (PIN-X) or pinealectomized plus melatonin (PIN-X + MEL) (100 ␮g/100 g body weight) during pregnancy, in the four seasons of the year

Control (g) PIN-X (g) PIN-X + MEL (g)

Spring

Summer

Fall

Winter

Total

274–420 (n = 8) 260–340 (n = 7) 244–367 (n = 9)

260–454 (n = 8) 265–376 (n = 6) 274–373 (n = 9)

212–365 (n = 9) 226–416 (n = 9) 252–362 (n = 10)

250–407 (n = 12) 233–370 (n = 7) 240–378 (n = 12)

212–454 (n = 37) 226–416 (n = 29) 240–378 (n = 39)

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acid contained in Eppendorf tubes. The tissues were homogeneized by sonication, the suspensions centrifuged for 10 min at 11,000 rpm and the supernatants aspirated. The tissues were then heated in a boiling water bath for 7 min to inactivate proteolytic enzymes, and the supernatants aspirated, again centrifuged for 30 min at 17,000 rpm and the supernatant lyophilized, kept at −80 ◦ C until assayed. 2.2.1. Melatonin treatment Considering previous findings [19] in which 20 ␮Ci of 3 H-acetyl-melatonin was administered to pregnant rats, and that each fetus contained slightly more than 0.1% (20 nCi), of the injection dose, 100 ␮g MEL/100 g body weight were used as a standard dose in the present study. Melatonin (M-5250, Sigma Chemical Co., St. Louis, MO) was dissolved in a small volume of absolute ethanol (0.04 ml) and diluted in 0.9% NaCl to a dose of 100 ␮g/100 g body weight. Melatonin treatment was given by s.c. injections at the end of the light phase to mother rats, and daily throughout pregnancy. Control and PIN-X mother rats received ethanol/saline alone. 2.3. Pinealectomy Pinealectomy was carried out according to the procedure previously described [24]. Briefly, rats were anaesthetized with chloral hydrate (1 cm3 /100 g body weight); a longitudinal cut was made 2 cm in front to 1 cm behind the inion, and the periostium was removed from skull between both temporal lines. Using a cylindrical trephan powered by a dentist’s drill, a circular bone disc was cut centered on the lambda point and removed, after which an incision of the duramater was made, with the aid of an ophthalmic scalpel along the lower edge of both lateral sinuses. The right transverse sinus was then cut after halting blood circulation with a vacuum pump. The procedure allowed us to remove the pineal gland and immediately to replace the duramater and the bone disc. With this surgical procedure the venous return was not subjected to any disturbance because blood was able to flow through the intact left sinus to the jugular vein. The survival rate of the animals was very high and only conditioned by the skills of the investigator, during the surgical procedure. After surgery, the animals were allowed to recover for 15 days. All mothers used in the study were examined after weaning to verify the complete pinealectomy. Offspring of the mothers with failed pinealectomies were removed, not taken into consideration for the experiment, and new mothers were prepared for the next year at the same season. 2.4. Peptide quantification Following the methodology previously used in the determinations of the values for NKA and SP in the hypothalamus and anterior pituitary [3–5], we determined the concentrations of these tachykinins in extracts of frontal

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cortex and striatum. In the testes only SP was determined. NKA and SP were determined by double antibody radioimmunoassays previously described [3]. The characterization of the antisera as well as the specificity of the assay and its validation was investigated using HPLC. The methodology used was previously described in reports from our laboratory [27,34]. Briefly the characterization of NKA in the anterior pituitary (AP) extracts was carried out using HPLC (Beckman, Ultrasphere ODS 5 ␮m; 4.6 cm × 25 cm). A main peak coincident with peak of synthetic NKA was observed when AP extracts were applied to the column. Using the same methodology, rat hypothalamic extracts, synthetic NKA, and synthetic neuropeptide K, a peptide that contains the whole sequence of NKA and therefore strongly cross-reacts with the anti-NKA serum used, were subjected to HPLC [34]. Synthetic SP was submitted to the same procedure, a single peak corresponding to SP was detected in the hypothalamic extracts [5]. SP was determined using an anti-SP serum obtained in rabbits. The specificity of the assays was confirmed by the presence of a single immunoreactive peak (corresponding to SP) in extracts of hypothalami and anterior pituitaries, purified by HPLC [5]. NKA was determined in frontal cortex and striatum extracts by a radioimmunoassay method using an antiserum that we produced by immunizing rabbits with NKA coupled to bovine thyrogobulin. It must be pointed out that neuropeptide K (NPK) and neuropeptide gamma (NP␥) contain the whole sequence of NKA and therefore cross-react with this antiserum. This antiserum should be therefore considered to be able to bind NKA plus NKA contained in NPK and NP␥. This was confirmed by performing NKA assays in extracts of hypothalami and anterior pituitaries, purified by HPLC [4,34]. Before being assayed, each cortex extract was redissolved in 0.4 ml of 0.1N acetic acid. Fifty microlitres of this extracts was aliquoted into test tubes in duplicates, the volume was completed up to 0.5 ml with 0.5% BSA in phosphate-saline buffer. In the case of testicular extracts, before performing the assays, the lyophilized extracts were suspended in 500 ␮l of assay buffer (0.5% BSA, Sigma Chemical Co.; bacitracin 20 ␮M, PBS). The choice of bovine serum albumin may be critical, since some albumins apparently contain proteolytic activity, which may render the assay unsuccessful. In this assays bovine serum albumin isolated by heat shock (Sigma Chemical Co.) was used with good results. The solution was centrifuged to eliminate insoluble particles, and 500 ␮g aliquots were dispensed into each assay tube. Synthetic SP and NKA (Cambridge Research Biochemicals, Wilmington, DE) were used as standards preparations. Standard curves with synthetic NKA and SP were set up with doses ranging from 2.5 to 1250 pg/tube. The antisera were diluted in 1% normal rabbit serum in EDTA-PBS and dispensed in 200 ␮l/tube. Bolton-Hunter labelled 125 I-SP or 125 I-NKA (Amersham Corp., Arlington Heights., IL) were used as tracers. The labelled peptides were added to each tube in a volume of 100 ␮l containing 10,000 cpm. The

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incubation was carried out for 4 days at 4 ◦ C and the separation between bound and free NKA or SP was achieved with the addition of a second antibody. The results were expressed as pg of synthetic NKA or SP (Cambridge Research Biomedicals, Wilmington, DE)/mg tissue. All samples were run in the same assay for each neuropeptide to avoid interassay variations. 2.5. Statistical analysis Data of each age group were adjusted to a normal distribution test before being used in the statistical analysis. A 99 percentage of accuracy to normal distribution was required. Statistical analysis was performed using the SPSS version 10.0 (SPSS Inc., Chicago, IL). Sample size was indicated as follows: the lower and higher number of cases for each age in the three groups in every season. When two ages had the same number of cases they were pooled. Results were expressed as mean ± S.E.M. Comparisons among data of tachykinin concentrations at each time point studied were determined by ANOVA; individual comparisons were then made by Tukey test. Kruskal–Wallis test was used whenever it was not possible to apply ANOVA test. Probability values lower than P < 0.05 were considered significant. Comparisons among the four seasons for each age and group were noted by # P < 0.01 and ## P < 0.05.

3. Results 3.1. NKA 3.1.1. Frontal cortex (Fig. 1) Comparing NKA concentrations in the four seasons, significant variations were observed. In control-offspring at 21 days of age the highest values were observed during summer and fall with significant differences (P < 0.01) as compared to the low values observed during winter and spring. At 31 days of age the highest NKA concentrations were observed in summer and fall with significant differences (P < 0.01, P < 0.05) when compared to spring or winter. Concentrations during winter were significantly higher (P < 0.01) in comparison to spring. At 60 days of age control-offspring showed no significant differences among seasons in NKA concentrations. In PIN-X-offspring at 21 days of age the highest NKA concentrations appeared during summer and fall, showing significantly higher (P < 0.01, P < 0.05) values comparing summer to winter and spring, and comparing fall (P < 0.05) to winter. At 31 days of age the highest values were found in summer, with significant differences (P < 0.01) as compared to fall, winter and spring. At 60 days of age, differences among seasons were evident, with the highest values observed during spring and summer and showing statistically significant differences (P < 0.01) when compared to fall and winter.

Fig. 1. Frontal cortex NKA concentrations throughout the four seasons of the year of male offspring of mother rats: control-, PIN-X, or PIN-X+MEL during pregnancy (100 µg/100 g body weight). Limit of sample sizes, spring: 21 and 31 days (n = 13–15), 60 days (n = 9–14); summer: 21 days (n = 12–15), 31 days (n = 14–15), 60 days (n = 7–12); fall: 21 days (n = 13–15), 31 days (n = 15), 60 days (n = 11–15); winter: 21 days (n = 13–15), 31 days (n = 14–15), 60 days (n = 6–12). Values are presented as mean ± S.E.M. Comparisons: control-offspring: 21 days old, # P < 0.01 vs. spring and winter; 31 days old, # P < 0.01 vs. spring, ## P < 0.05 vs. winter. PIN-X-offspring: 21 days old, # P < 0.01 vs. winter; ## P < 0.05 summer vs. spring; fall vs. winter; 31 days old, # P < 0.01 vs. spring, fall and winter; 60 days old, # P < 0.01 vs. fall and winter. PIN-X + MEL-offspring: 21 days old, # P < 0.01 summer vs. spring, fall and winter; spring and fall vs. winter; 31 days old, # P < 0.01 vs. spring, fall and winter, ## P < 0.05 vs. fall; 60 days old, # P < 0.01 spring vs. winter; summer vs. fall and winter.

In PIN-X + MEL-offspring at 21 days of age the highest values were found in summer, with significantly higher differences (P < 0.01) when compared to fall, winter and spring. Values in spring and fall were significantly higher (P < 0.01) than in winter. At 31 days of age the highest values were observed during summer, with significant differences (P < 0.01) when compared to the other three seasons studied. In spring NKA values were significantly higher (P < 0.05) than in fall. At 60 days of age differences among

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seasons were still evident, with the highest values observed in spring and summer and with statistically significant differences (P < 0.01) in spring versus winter and in summer versus fall and winter.

with significant differences (P < 0.05) when compared to spring.

3.1.2. Striatum (Fig. 2) Only minor seasonal changes were observed in striatal NKA concentrations. In control-offspring at 31 days of age the highest values were observed during winter with significantly higher differences (P < 0.01) when compared to spring and summer and (P < 0.05) fall. In PIN-X-offspring at 60 days of age the highest values were observed in spring with significant differences (P < 0.01) when compared to summer. In PIN-X + MEL-offspring at 31 days of age the highest values were observed in summer,

3.2.1. Frontal cortex (Fig. 3) Comparisons among seasons indicate that for controloffspring at 21 days of age, the highest values were observed during summer and fall, with significantly higher differences (P < 0.01) when compared to winter and spring and at 31 days of age significantly higher (P < 0.01) values during summer, fall and winter when compared to those during spring. Also, rats at 60 days of age during summer had

Fig. 2. Striatal NKA concentrations throughout the four seasons of the year in the same groups as those shown in Fig. 1. Limit of sample sizes, spring: 21 days (n = 12–15), 31 days (n = 13–14), 60 days (n = 7–13); summer: 21 days (n = 12–15), 31 days (n = 14–15), 60 days (n = 7–11); fall: 21 days (n = 13–14), 31 days (n = 14–15), 60 days (n = 10–15); winter: 21 days (n = 12–15), 31 days (n = 12–14), 60 days (n = 7–11). Values are presented as mean ± S.E.M. Comparisons: control-offspring: 31 days old, # P < 0.01 vs. spring and summer, ## P < 0.05 vs. fall. PIN-X-offspring: 60 days old, # P < 0.01 vs. summer. PIN-X + MEL-offspring: 31 days old, ## P < 0.05 vs. spring.

3.2. SP

Fig. 3. Frontal cortex SP concentrations throughout the four seasons of the year in the same groups as those in Fig. 1. Limit of sample sizes, spring: 21 days (n = 12–15), 31 days (n = 13–15), 60 days (n = 9–14); summer: 21 days (n = 12–15), 31 days (n = 14–15), 60 days (n = 7–12); fall: 21 days (n = 13–15), 31 days (n = 14–15), 60 days (n = 10–14); winter: 21 and 31 days (n = 13–15), 60 days (n = 8–13). Values are presented as mean ± S.E.M. Comparisons: control-offspring: 21 days old, # P < 0.01 vs. spring and winter; 31 days old, # P < 0.01 vs. spring; 60 days old, ## P < 0.05 vs. spring. PIN-X-offspring: 21 days old, # P < 0.01 vs. spring and winter; 31 days old, # P < 0.01 vs. spring, fall and winter; 60 days old, # P < 0.01 vs. fall and winter. PIN-X + MEL-offspring: 21 days old, # P < 0.01 vs. spring, fall and winter; 31 days old, # P < 0.01 vs. spring, fall and winter; 60 days old, # P < 0.01 vs. fall and winter.

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significantly higher (P < 0.05) values than in spring. Maternal PIN-X resulted in alterations of the seasonal rhythms with the highest values observed only during summer at 21 days of age, showing significantly differences in comparison to spring and winter (P < 0.01). At 31 days of age significantly higher (P < 0.01) SP concentrations were found during summer in comparison to spring, fall and winter. At 60 days of age SP concentrations observed during spring and summer were significantly higher (P < 0.01) than those observed during fall and winter. The influence of maternal PIN-X+MEL produced similar alterations in the offspring as PIN-X alone. Differences between both treatments were only observed at 21 days of age showing PIN-X+MEL-offspring significantly higher (P < 0.01) values during summer when compared not only with spring and winter but also

with fall. At 60 days of age in summer, SP concentrations were significantly higher (P < 0.01) than in fall and winter.

Fig. 4. Striatal SP concentrations throughout the four seasons of the year in the same groups as those shown in Fig. 1. Limit of sample sizes, spring: 21 days (n = 12–14), 31 days (n = 13–14), 60 days (n = 7–13); summer: 21 days (n = 10–13), 31 days (n = 13–15), 60 days (n = 7–11); fall: 21 days (n = 12–14), 31 days (n = 14–15), 60 days (n = 10–15); winter: 21 days (n = 11–15), 31 days (n = 13–14), 60 days (n = 7–13). Values are presented as mean ± S.E.M. Comparisons: control-offspring: 21 days old, # P < 0.01 vs. winter; 60 days old, # P < 0.01 vs. winter, ## P < 0.05 vs. winter. PIN-X-offspring: 60 days old, # P < 0.01 vs. summer and winter. PIN-X + MEL: 21 days old, ## P < 0.05 vs. fall.

Fig. 5. Testicular SP concentrations throughout the four seasons of the year in the same groups as those shown in Fig. 1. Limit of sample sizes, spring: 21 days (n = 12–14), 31 days (n = 13–15), 60 days (n = 8–14); summer: 21 days (n = 10–15), 31 days (n = 13–15), 60 days (n = 6–11); fall: 21 and 31 days (n = 13–15), 60 days (n = 8–14); winter: 21 days (n = 12–15), 31 days (n = 12–14), 60 days (n = 7–13). Values are presented as mean ± S.E.M. Comparisons: control-offspring: 31 days old, # P < 0.01 vs. spring. PIN-X-offspring: 31 days old, # P < 0.01 vs. fall, ## P < 0.05 vs. winter. PIN-X+MEL: 21 days old, # P < 0.01, ## P < 0.05 vs. spring.

3.2.2. Striatum (Fig. 4) Comparisons among the four seasons showed few differences. In control-offspring at 21 and 60 days of age, significantly higher (P < 0.01, P < 0.05) SP concentrations during spring and summer were found, as compared with those during winter. In PIN-X-offspring during spring at 60 days of age, SP concentrations were significantly higher (P < 0.01) than in summer and winter. In PIN-X + MEL-offspring during spring at 21 days of age, SP concentrations were significantly higher (P < 0.05) than in fall.

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3.2.3. Testes (Fig. 5) Comparing testicular concentrations in the four seasons only control-offspring at 31 days of age had significantly higher (P < 0.01) values during fall and winter than in spring. In PIN-X-offspring at 31 days of age during summer significantly higher (P < 0.01, P < 0.05) values than during fall and winter were found. PIN-X + MEL-offspring at 21 days of age during summer and winter had significantly higher values (P < 0.01, P < 0.05) than in spring. 4. Discussion The results presented here confirm the presence of tachykinins in the frontal cortex, striatum, and, at a much lower magnitude, in the testes of prepubertal and young adult rats. Very remarkable differences, throughout the four seasons of the year, were found in the rats. This occurred in spite of the fact that the rats were kept under strictly controlled environmental conditions throughout the year. Highest tachykinin concentrations (in the frontal cortex) were found in summer and fall, while the lowest were noted in winter and spring. At this time, the mechanisms of the process are unclear, i.e., causes of the observed fluctuations in the frontal cortex, considering the periodicity of photoperiod and constant laboratory conditions, have not been determined. It is nevertheless evident that a biological clock must exist in these animals and that this biological clock is independent of the unchanged environmental conditions provided in a vivarium setting. It is well known that the SCN of the hypothalamus constitutes an important centre in the CNS regulating biological rhythms [14,37], however, our results indicate that some regulatory input from the frontal cortex may also participate in these mechanisms. These seasonal differences in the tachykinin concentrations in the frontal cortex may well be a direct expression of the SCN activity. Others have reported seasonal variations of neuropeptides such CRF, TRH, neurotensin and neuromedin in the rat hypothalamus [2], as well as vasopressin-immunoreactivity in human SCN [14] and VIP-LI in SCN of Jerboa (Jaculus orientalis) [20]. No data have been reported on seasonal changes for NKA or SP, nor for concentrations of NKA in frontal cortex. In spite of the indications noted in our results, few reports describe nocturnal increase of hypothalamic SP content in rats [21], in SCN of Djungarian hamsters [29] or anterior pituitary NKA in Siberian hamster [6] suggesting that these tachykinins may be involved in the regulation of circadian or seasonal rhythms. It is also interesting to note that our study found no major modifications of tachykinin concentrations in the striatum, throughout the four seasons of the year. Furthermore, It is evident that whatever mechanism was instrumental in inducing seasonal rhythms in frontal cortex tachykinin levels, that mechanism was not operative in the striatum. Previous findings in adult rat and macaque monkey (Macaca mulatta),

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showed that SP receptors mediate a complex combination of post- and presynaptic functions in the mammalian striatum and may modulate the release of striatal neurotransmitters [16]. Data on the normal developmental pattern from infantile up to the end of pubertal period (i.e., 61 days of age) have shown low tachykinin concentrations. Only during the prepubertal period are concentrations found higher than in the infantile or pubertal period [11] (although in this case, the season of the year was not taken into consideration). Our results indicate that NKA and SP show significant fluctuations in the cerebral cortex. However, this is not the case in the striatum, in spite of the fact that higher tachykinin concentrations were found. Considering that environmental information first reaches the frontal part of the animal, the frontal cortex may play a more important role on the seasonal rhythms than the striatum itself. It is well understood that tachykinins are present in the human, hamster and rat testes. Their localizations are found mainly in the nerve fibres and in the Leydig cells of the testicular interstitium [1,7,30] and may act as modulators of the secretory activity of Leydig and Sertoli cells [7,27]. In the present investigation, no major seasonal modification could be observed. PIN-X and treatment with melatonin only caused minor and inconsistent modifications in testicular SP levels. No previous data exist on the influence of maternal PIN-X on the development of testicular neuropeptides. Evidence exists, however, that the mother’s pineal gland may play a part in the control of hormone [17,18] and postnatal testicular development [10]. Overall, our results are in agreement with previous data observed some years ago regarding the question of the role played by the maternal pineal gland on fetal biological clock setting. In the rat, the biological clock for circadian N-acetyl-transferase activity is set in independently of the light schedule [8]. Under such circumstances the clock time of the pups is determined by their mothers so as to synchronize to their own rhythms. Results obtained in Montane voles revealed that postweaning body growth and reproductive tract weights are modulated by the photoperiod to which the vole mothers were exposed while pregnant. This would appear to indicate that the effect is a result of factors acting in utero rather than on maternal effects during lactation [15]. Nevertheless, the mechanisms by which the maternal pineal gland plays a role in the entrainment of seasonal changes in the offspring still remains unclear. In the present study, the offspring of the three groups studied were intact, not being submitted to PIN-X in any case. Consequently, they experienced a normal circadian rhythmicity. Since it is known that the photic regulation entrainment of the circadian rhythm of pineal melatonin production becomes functional at 10–15 days of postnatal life [32], the observed disturbances of seasonal changes (up to adult age), suggest an influence of maternal pineal gland acting in utero. In conclusion, our data clearly indicate for the first time that NKA and SP do indeed show seasonal rhythms in the

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frontal cortex, but not in striatum and in the testes. When these seasonal rhythms are present, the maternal pineal gland likely plays a role in their entrainment.

[17]

[18]

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