Introduction

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The current literature regarding the effects of the neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] on behavior is considerable. Recent advances in both molecular biology of multiple serotonin receptor sites and the development of selective drugs for these receptors are refining our basic understanding of the functional correlates of serotonergic receptors. To date, at least seven different families of 5-HT receptors are recognized (5-HT_1-7), and many of these receptors possess two or more subtypes (Hoyer et al., 1994). For example, the 5-HT₁ receptor family consists of 5-HT_1A, 5-HT_1B, 5-HT_1D, 5-HT_1E, and 5-HT_1F sites, whereas the 5-HT₂ group consists of 5-HT_2A, 5-HT_2B, and 5-HT_2C receptor sites.

The administration of either 5-HT precursors (L-tryptophan or 5-hydroxytryptophan) or 5-HT releasers (e.g., parachloroamphetamine or d-fenfluramine), which dramatically increase the synaptic concentration of 5-HT, produce a series of behaviors in rodents that are known collectively as the 5-HT behavioral syndrome (SS) (for reviews, see Glennon and Lucki, 1989; Heal et al., 1992). The clearest definition of the 5-HT behavioral syndrome was proposed by Jacobs (1976), who defined the syndrome as the simultaneous display of four to six symptoms: hindleg abduction (HLA), forepaw treading (FPT), lateral head-weaving (LHW), resting tremor (T), rigidity, and straub tail (ST). If rats showed four of the six signs, they were rated as showing the syndrome in an all-or-none fashion. In an attempt to quantify drug-induced alterations on the symptoms of SS, more recently, many investigators have provided continual rating of the intensity of each symptom because individual symptoms may respond separately to pharmacological manipulations (Dickinson et al., 1983; Tricklebank et al., 1985; Eison and Wright, 1992). The SS underwent a renaissance during 1980s with the findings that administration of the selective 5-HT_1A receptor agonist 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT) can produce the various components of this response in rats (for reviews, see Glennon and Lucki, 1989; Heal et al., 1992). Moreover, silent and highly selective 5-HT_1A receptor antagonists such as (S)-UH 301 or WAY 100635 potently antagonize the various symptoms of the syndrome produced by 8-OH-DPAT (Björk et al., 1991; Forster et al., 1995). Stimulation of 5-HT_1A receptors also induces hypothermia in rodents, and the hypothermic effect of 8-OH-DPAT can be potently blocked by the cited 5-HT_1A antagonists (Björk et al., 1991; Forster et al., 1995).

Activation of 5-HT_2A receptors either by direct (selective or nonselective) or indirect (5-HT precursors or releasers) 5-HT agonists produce the head-twitch response (HTR) in mice and its behavioral homolog, the wet-dog shakes (WDS), in rats (for reviews, see Heal et al., 1992; Glennon and Lucki, 1989). Furthermore, administration of the selective 5-HT_2A/C agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) not only produces the latter corresponding behaviors in mice (Darmani et al., 1990) and rats (Schreiber et al., 1995) but also induces the ear-scratch response (ESR) in mice (Darmani, 1992) and back muscle contractions (BMC) in rats (Pranzatelli, 1990). Although it is well accepted that the HTR and WDS are mediated via the activation of 5-HT_2A sites, as yet the 5-HT₂ receptor subtype responsible for the production of ESR and BMC is not fully defined. However, limited antagonist studies suggest that activation of 5-HT_2C sites may be responsible for the production of the latter behaviors in mice (Darmani, 1992) and rats (Pranzatelli, 1990), respectively.

A closer inspection of the current literature reveals that most investigators only consider drug effects on the most prominent components of the SS (FPT, HLA, and LHW). Preliminary studies in this laboratory have indicated that other symptoms of SS [flat body posture (FBP), ST, and T] exhibit robust intensities during early age but become less intense in adulthood. Because the long-term sequential ontogenic development of different components of SS in the rat has not yet been reported, the initial goal of this study was to determine the ability of two doses of 8-OH-DPAT (1.25 and 5 mg/kg) to induce the different symptoms of the SS on postnatal days (PD) 7, 14, 18, 22, 28, 35, 60, 90, and 120. Because activation of 5-HT_1A receptors also leads to production of hypothermia in rodents, the second goal of this investigation was to study the comparative ontogenic development of 8-OH-DPAT-induced hypothermia on the cited postnatal ages. Currently, there also is a lack of long-term developmental behavioral studies on the production of WDS and BMC in rats. Thus, the final goal of the present study was to determine the ability of the 5-HT_2A/C agonist, DOI, to induce the latter behaviors on the cited postnatal ages in the rat.

	Methods

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Animals and Drugs. Mature male and female Sprague-Dawley rats were purchased from Hilltop Laboratory Animals Inc. (Scotsdale, PA). Breeder group (30 pairs) were kept as one male and one female per cage, and the offspring were culled to eight on the day of birth. The breeding pairs were kept together to continuously provide new pups. The pups were weaned at the age of 21 days. The weaned male and female offspring were kept in separate cages in groups of three or four. The male offspring were used during their adulthood in another study, whereas the female offspring were used throughout the present study. Only one female pup from a particular litter was used per dose for a given age group for a 5-HT_1A or 5-HT₂ receptor-mediated effect. At maximum, only two female pups from a particular litter were used before weaning, and each animal was used only once during the investigation. The animals were kept on a 12-h light/dark cycle at a room temperature of 22°C with free access to food and water. All animals received care according to the "Guide for the Care and Use of Laboratory Animals" (DHHS Publication, Revised, 1985). The facilities are certified by the American Association of Accreditation of Laboratory Care. These studies were approved by the Institutional Animal Care and Use Committee of KCOM. (±)-DOI HCl and (±)-8-OH-DPAT HBR were purchased from Research Biochemicals Inc. (Natick, MA). Drugs were dissolved in distilled water and given i.p. at a volume of 10 ml/kg.

Measurement of SS, WDS, BMC, and Core Body Temperature. The 5-HT_1A receptor-mediated components of SS as well as the 5-HT₂ receptor-induced events are defined in Table 1. The frequency of two components of SS (LHW and RO) induced by 8-OH-DPAT was determined by an experienced observer via use of multiple tally counters. These behaviors were scored continuously at 5-min durations for 20 min immediately after the i.p. injection of 8-OH-DPAT. The intensities of other symptoms of the induced SS (FPT, FBP, HLA, T, and ST) were also individually scored during 5-min intervals continuously for 20 min with the following intensity scale based on the method of Arnt and Hyttel (1989): absent (score 0), periodic (score 1), semicontinuous (score 2), or continuous (score 3). The scores for each SS symptom was collected over the 20-min observation period, and the total cumulative mean score (±S.E.M.) for each age group was subsequently calculated. The core body temperature (CBT) was measured with a Labcroft digital thermometer both just before 8-OH-DPAT injection and immediately after the termination of SS observation. The difference between the two temperature readings (°C) was considered as a temperature change. For the CBT determination, a rat rectal probe was inserted 3 cm into the colon of rats older than 22 days. In the younger rat pups, a mouse rectal probe was inserted 2 cm into the colon. The probes were lubricated with mineral oil before each temperature determination.

Experimental Protocols. On the test day, the animals were transferred to the experimental room and were allowed to acclimate for at least 1 h before experimentation. The fume hood was turned on to produce a constant white noise during the experimental procedures. From our preliminary dose-response study in 35-day-old rat pups, it was decided that a relatively small (1.25 mg/kg) and a larger dose of 8-OH-DPAT (5 mg/kg) could be used for the production of different symptoms of SS in the present ontogenic study. To habituate the rat pups to the test environment, each animal was transferred randomly 20 min before treatment to a 45 × 25 × 20 cm plastic holding cage lined with a thin layer of wood chips. Thus, different groups of female rat pups at varying postnatal ages (7, 14, 18, 22, 28, 35, 60, 90, and 120 days old, n = 6 or 7 per dose for each age group) received either vehicle (i.p.) or the cited doses of 8-OH-DPAT (i.p.), and after the injection, each rat was transferred to an observation cage of similar dimensions. Depending on the SS symptom, either the frequency or the intensity of each symptom of 8-OH-DPAT-induced SS was scored for the next 20 min immediately after injection, as was described earlier. The 8-OH-DPAT-induced hypothermia was also measured in these animals as described above.

Statistical Analysis. The data were analyzed by a two-way analysis of variance (ANOVA) (with age and drug dose as factors) followed by Bonferroni's (with control) multiple-comparison test as post hoc analysis. The statistical significance was set at p < .05 for all analysis.

	Results

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Ontogenic Development of SS. The vehicle-injected control groups throughout the postnatal test days produced either no or weak intensities of different symptoms of SS. The various components of 8-OH-DPAT-induced syndrome underwent three different patterns of development as age progressed:

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Effect of aging on the ability of two doses of 8-OH-DPAT [1.25 () and 5 () mg/kg i.p.] in producing the classic symptoms of SS relative to age-matched vehicle-injected controls () in female rats. The data depict either the frequency (LHW) or the intensity (FPT, HLA, FBP, T, and ST) of different components of SS. Values represent mean ± S.E.M. (n = 6 or 7 per group). *Significantly different from age-matched vehicle-treated controls. +Significant difference between the cited doses of 8-OH-DPAT. P < .05, two-way ANOVA followed by Bonferroni's multiple comparisons test.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Ontogenic onset and disappearance of ROs in female rat pups produced by different doses of 8-OH-DPAT [0 (), 1.25 (), and 5 () mg/kg i.p.) as function of age. Values represent mean ± S.E.M. (n = 6 or 7 per group). *Significantly different from age-matched vehicle-injected controls. +Significant difference between the two doses of 8-OH-DPAT. P < .05, two-way ANOVA followed by Bonferroni's multiple comparisons test.

Ontogenic Development of 8-OH-DPAT-Induced Hypothermia. Seven-day-old, vehicle-injected, control rat pups exhibited an hypothermic response (2.18 ± 0.57°C) after the administration of saline (Fig. 3). However, from day 14, no significant temperature change was observed after saline injection in control animals. On the 7th day of life, the 1.25-mg/kg dose of 8-OH-DPAT also caused a hypothermic effect, but its intensity was not significantly different from that of the saline-injected age-matched control group. However, the 5-mg/kg dose significantly reduced rat pup CBT (F_2,135 = 377, p < .000001). Both doses of 8-OH-DPAT caused similar degrees of hypothermia in the developing rat pups throughout the cited test days (F_8,135 = 4.70, p < .00004). There also was an interaction between age and dose (F_16,135 = 3.75, p < .00001). The intensity of hypothermic effects of the two doses of 8-OH-DPAT were not altered to a great extent during development.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Ontogeny of CBT () and the ability of two doses of 8-OH-DPAT [1.25 () and 5 () mg/kg i.p.] to induce hypothermia as function of age. Values represent mean ± S.E.M. (n = 6 or 7 per group). *Significantly different from age-matched vehicle-injected controls. P < .05, two-way ANOVA followed by Bonferroni's multiple comparisons test.

Ontogenic Development of DOI-Induced WDS. Figure 4 represents the developmental appearance of WDS in rats. A two-way ANOVA of WDS for age and dose exhibited highly significant differences between ages (F_9,232 = 12.61, p < .000001) and the doses (F_2,232 = 95.6, p < .000001). The administration of 0.5- and 4-mg/kg doses of DOI in the 7- and 10-day-old pups failed to produce WDS. The onset of the appearance of DOI-induced WDS occurs between PD 14 and 18. Bonferroni's test showed, relative to vehicle control groups, the 4-mg/kg dose of DOI produced significantly greater WDS frequencies from day 18 throughout the postnatal testing days, except day 90. On the other hand, the smaller dose of DOI (0.5 mg/kg) exhibited significant increases in WDS frequency on PD 14 and 28 only. The maximal response to 5 mg/kg DOI remained relatively constant up to PD 60 and was significantly reduced on PD 90, which was not different from vehicle control.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effects of aging (PD 7-120) on the ability of varying doses of DOI [0 (), 0.5 (), and 4 () mg/kg i.p.] to induce WDS in female rats. Results are presented as mean ± S.E.M. (n = 8-13 per group). *Significantly different from age-matched vehicle-injected controls. +Significant difference between the two doses of DOI. P < .05, two-way ANOVA followed by Bonferroni's multiple comparisons test.

Ontogenic Development of DOI-Induced BMC. Figure 5 shows the ontogenic appearance and maturation of DOI-induced BMC in rats. A two-way ANOVA of BMC for age and dose exhibited highly significant differences between ages (F_9,233 = 28.9, p < .000001) and among doses (F_2,233 = 96.1, p < .000001). Relative to vehicle-exposed age-matched control groups, a significant enhancement in BMC frequency for either doses of DOI was not seen until PD 28. On this day, similar frequencies of BMC were produced by the cited doses of DOI. The BMC frequency attained maximum levels by age 35 and remained relatively constant thereafter. Both doses of DOI produced similar BMC frequencies throughout the ontogenic test days, except for day 90. 

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of aging (PD 7-120) on the ability of varying doses of DOI [0 (), 0.5 (), and 4 () mg/kg i.p.] to induce BMC in female rats. Data are given as mean ± S.E.M. (n = 8-13 per group). *Significantly different from age-matched vehicle-injected controls. +Significant difference between the two doses of DOI. P < .05, two-way ANOVA followed by Bonferroni's multiple comparisons test.

	Discussion

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The first finding of the present study is that the onset of functional development of different components of 8-OH-DPAT-induced SS can vary by as much as 2 weeks. Indeed, the onset of appearance of most symptoms of SS occurred within the first week of life, whereas the commencement of LHW and FPT required a developmental lag phase of 14 to 18 days. The frequency of LHW symptom rapidly developed at PD 14 to 18 and attained an initial maximal intensity during 18 to 28 days of age, which then further increased as pups aged. Maximal FPT intensity was observed on PD 18 and then persisted throughout adulthood. Ontogeny of 5-hydroxytryptophan (5-HTP)-induced FPT appears to be opposite to the current study because its maximal score on PD 3 rapidly diminished during early development (Mokler et al., 1992). Unlike the latter report, other studies also show robust intensities of FPT in adult rats in response to 8-OH-DPAT (Arnt and Hyttel, 1989) or 5-HT precursors (Dickinson and Curzon, 1986). Other components of 8-OH-DPAT-induced syndrome (FBP, HLA, RO, ST, and T) were already present on the earliest day of testing (i.e., PD 7). Moreover, significant degrees of HLA, RO, ST, and T appear to develop as early as PD 3 in response to 5-HTP administration (Mokler et al., 1992). The FBP in response to 8-OH-DPAT administration has been reported to be apparent on the day of birth (Pranzatelli, 1992). The present findings further show that the lag time required for the development of maximal intensity of different symptoms of SS can vary by many weeks. For example, the maximal LHW frequency occurred on PD 90 and maximal FPT occurred on PD 18. Moreover, ST required 14 days of development to exhibit its maximal intensity, whereas FBP, HLA, T, and RO symptoms exhibited maximal intensities on PD 7.

The second finding of this study was that the rate of maturational development of LHW and FPT symptoms rapidly rose during early age (PD 7-18), but later their intensities either increased more slowly (LHW) or did not change (FPT). The other symptoms of SS became steadily less intense in nature as age progressed. Indeed, as pups became older, some symptoms assumed a lower mean steady intensity (e.g., FBP) or, depending on the 8-OH-DPAT dose used, other SS symptoms either completely disappeared or exhibited a fraction of their maximal scores (e.g., ST and T). The ontogenic appearance and disappearance of RO seem to be unique because the behavior did not occur in response to 8-OH-DPAT after the 7th day of life. A similar phenomenon occurs in response to 5-HTP administration (Mokler et al., 1992). The developmental progression of HLA is also extraordinary in that its maximal intensity on day 7 progressively decreased to minimal values by age 35 days and then subsequently reappeared to 30 to 80% of its 7-day maximal value as rats aged further. Unlike the 8-OH-DPAT-induced age- and dose-dependent variations in maturation and disappearance of different components of SS, the hypothermic effects of both doses of 8-OH-DPAT were relatively stable throughout development. Thus, the present results suggest that the ability of 5-HT_1A receptors to induce hypothermia does not change as animals age. This conclusion is further supported by the finding that the hypothermic effect of 8-OH-DPAT was similar in 3-4-month-old rats relative to aged rats (18-19 months) (Robson et al., 1993). Vehicle-injected rat pups were unable to sufficiently thermoregulate when they were removed from dams on the 7th day of life, but they acquired this function by PD 14. Indeed, the thermoregulatory system of rats has been reported not to be fully developed until 2 weeks of life (Adolph, 1957; Prosser, 1973).

The DOI-induced WDS in rats and its behavioral homolog, the HTR in mice, are mediated via the activation of 5-HT_2A receptors (Darmani et al., 1990; Schreiber et al., 1995). The onset of DOI-induced WDS in rats occurred at PD 14 to 18. The ability of 5-HTP to produce the latter behavior in rats has also been reported to significantly increase after PD 14 (Mokler et al., 1992). In addition, in the mouse, the onset of appearance of HTR also occurs at PD 14 to 18 (Darmani et al., 1996; Eble and Goodrich, 1987). In the present study, the maximal frequency of DOI-induced WDS in rats occurred at PD 18 and persisted up to age 60 days, after which it declined by about 40% of its maximal value. A similar ontogenic phenomenon occurs in the maturation of HTR in mice (Eble and Goodrich, 1987; Darmani et al., 1996). On the other hand, the onset of a significant enhancement in DOI-induced BMC occurred on PD 28. Thus, the developmental onset of DOI-induced WDS and BMC in rats varies by as much as 2 weeks. A similar differential profile occurs for the maturational onsets of DOI-induced HTR and ESR in mice (Darmani et al., 1996). The rates of maturation and attainment of maximal frequencies of DOI-induced WDS and BMC are also different. Indeed, at the time WDS attained its maximal intensity (i.e., at PD 14-18), the onset of production of BMC was not yet developed, and by the time BMC gained its maximal intensity (at PD 90-120), the HTR frequency subsided to its lowest frequency due to aging. Moreover, there was no significant difference in the ability of the two doses of DOI to produce BMC throughout the study, whereas for WDS, significant differences between the doses were observed on PDs 18, 35, and 60.

The present and the discussed behavioral findings suggest that 5-HT_1A receptors become fully functional in the first 2 weeks of life, whereas the onset and maturation of 5-HT_2A/C receptor function commence after the end of second week of life in rodents. Published biochemical studies seem to support this view. Undoubtedly, the 5-HT_1A receptor is present in several different loci of the rat pup brain after birth (Daval et al., 1987; Zifa et al., 1988). However, 5-HT_1A receptor density in different brain areas undergoes differential maturation. In the cortex, hippocampus, and dente gyrus, the density of 5-HT_1A sites steadily increases by 3- to 8-fold at PD 14 to 16 and reaches adult levels by 21 to 23 days of age. This developmental pattern fits well with the ontogenic increase in the intensities of LHW and FPT observed in the present study. An opposite pattern of development of 5-HT_1A receptor density occurred in the cerebellum because ³H-8-OH-DPAT binding was high as soon as PD 5 but then declined progressively to undetectable levels in both 2-week-old and adult rats (Daval et al., 1987). In some brain areas (thalamus and olfactory cortex), 5-HT_1A receptor density appeared to be already as high on the first PD as in adult animals. Furthermore, a bell-shaped developmental pattern was seen in the lateral lemniscus. The latter three examples of differential changes in 5-HT_1A receptor density during development correlate well with the ontogenic behavioral variations observed with the other components of SS (FBP, HLA, RO, ST, and T). In addition, it should be realized that the functional competence of different muscle groups responsible for the production of these behaviors may also develop in a differential manner. Overall, the discussed data suggest that although the anatomical origin of SS is the hind brain and spinal cord (for reviews, see Glennon and Lucki, 1989; Heal et al., 1992), postsynaptic 5-HT_1A receptors in different brain loci may control the production of various components of SS.

The onset of immunocytochemical expression of 5-HT_2A receptors in several areas of rat brain occurs relatively late during development at PD 5 (Morilak and Ciaranello, 1993; Morilak et al., 1994). This onset is followed by a period of hyperexpression lasting 2 weeks. Receptor binding studies show that 5-HT_2A receptor is present in rat pup brain before birth, and its density significantly increases at PD 5 and reaches its maximal concentration at ages 12 to 17 days (Roth et al., 1991). This pattern of 5-HT_2A receptor overexpression after the second week of life correlates well with the onset and maturation of WDS in the present study. In a comparative receptor binding study using specific areas of rat brain, it has been shown that 5-HT_2C receptors can be detected as early as PD 1 (Pranzatelli, 1992). However, like 5-HT_2A sites, the 5-HT_2C receptor density underwent differential ontogenic development. For example, in the cortex, diencephalon, and spinal cord, significant increases in the density of 5-HT_2C sites were evident at PD 14 to 21 and attained their maximal densities during ages 21 to 28 days. On the other hand, the 5-HT_2C receptor number did not significantly change during development in the hippocampus and brain stem. The pattern of development in those brain loci that exhibit developmental increases in 5-HT_2C receptor density correlates more closely with the late developmental onset and maturation of DOI-induced BC in the present investigation.

In summary, the present study investigated the relative ontogenic developments of 5-HT_1A receptor-mediated responses (i.e., 8-OH-DPAT-induced SS and hypothermia) as well as 5-HT_2A/C receptor-mediated events (i.e., DOI-induced WDS and BC). The results show (1) most components of SS appeared within 14 day of development but matured differentially; (2) unlike developmental changes in 8-OH-DPAT-induced SS, at the doses used, the hypothermic effect of 8-OH-DPAT remained relatively constant during aging; (3) the time of onset and developmental maturation of WDS was intermediate in nature (i.e., between the second and third week of life); and (4) the onset and progress to full maturation of BMC required 1-2 months of development.