Inhibition of GH in maternal separation may be mediated through altered serotonergic activity at 5-HT2A and 5-HT2C Receptors

Pergamon

Psychoneuroendocrinology, Vol. 21, No. 2, pp. 219-235, 1996 Copyright © 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 031)6-4530/96 $15,{X} + .0~1

0306-4530(95)00043-7

INHIBITION OF GH IN MATERNAL SEPARATION MAY BE MEDIATED THROUGH ALTERED SEROTONERGIC ACTIVITY AT 5-HT2A AND 5-HT2c RECEPTORS Lawrence M. Katz, Lauren Nathan, Cynthia M. Kuhn and Saul M. Schanberg Box 3813, Duke University Medical Center, Durham, NC 27710, USA

SUMMARY The hyposecretion of growth hormone (GH) in maternal separation (MS) of rat pups is remarkably similar to the specific suppression of GH secretion to evocative tests in infants diagnosed with Reactive Attachment Disorder of Infancy (RADI). Growth hormone-releasing factor (GRF) and somatostatin (SS) provide opposing regulation of GH secretion, and both are modified by noradrenergic and serotonergic stimuli in neonatal and adult rats. In this study, GRF administration reversed MS-induced suppression of GH secretion in 10-day-old pups, but this action of GRF was prevented by pretreatment with cyproheptadine (Cypro), a serotonergic antagonist. The normalization of GH secretion after return to the dam was not altered by pretreatment with SS. Indirect 5-HT agonists, fluoxetine (FLX) and 5-HTP, both stimulated GH secretion in 10-day-old pups. All mixed serotonin- and 5-HTiA-receptor agonists suppressed GH secretion in 10-day-old pups. Antagonists Cypro and ketanserin (Ket) suppressed FLX-induced GH secretion. In contrast, only Cypro suppressed 5-HTP-induced GH secretion. Maternal separation inhibited GH secretion stimulated by 5HTP, but not by FLX. The serotonergic pathway acting on 5-HT2A receptors may be obligatory for GRF-mediated stimulation and is sensitive to inhibition by Cypro. In addition, a Ket-sensitive serotonergic parallel pathway acting on 5-HT2c receptors may also stimulate GH secretion by acting on GRF or SS. However, only the obligate 5-HT2A pathway appears to be suppressed in MS. These data and observations by others indicate that specific suppression of GH secretion in MS may derive from a reduction in GRF release through noradrenergic neurons, possibly impinging upon serotonergic terminals in the hypothalamus. This study may also provide insight into mechanisms by which GH secretion is suppressed in humans with RADI. Copyright © 1996 Elsevier Science Ltd Keywords--Maternal separation; Serotonin; 5-HT2A receptor; Rats: Fluoxetine; 5-HTP.

INTRODUCTION The Syndrome of Maternal Deprivation (Anderson et al., 1994) is defined as growth retardation, weight below the third percentile for height, social withdrawal, a rigid, awkward interpersonal relatedness and inhibited verbal communication. In the current psychiatric literature, the equivalent syndrome is termed Reactive Attachment Disorder of Infancy (RADI) (Shaffer & Campbell, 1994). Early on, researchers such as Harlow and Zimmermann (1959) and Hinde and Spencer-Booth (1971) described many associated phenomena in maternally deprived rhesus monkeys (Macaca mulatta). Patton and Gardner (1963) published one of the earliest descriptions of RADI in six children with retardation of Address correspondence and reprint requests to: Saul M. Schanberg, Box 3813 D U M C , Durham, NC 27710, U S A (Tel: 919-684-5187; Fax: 919-681-8609). 219

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bone growth, short stature, and who were living under emotional deprivation. Powell et al. (1967a, b) first reported endocrine abnormalities in relationship to RADI, and similar endocrine findings were reported by others (Frasier & Rallison, 1972; Rayner & Rudd, 1973). Originally diagnosed with idiopathic hypopituitarism, these children reportedly suffered from short stature, impaired insulin- or arginine-stimulated growth hormone (GH) and metyrapone-stimulated adrenocorticotrophic hormone (ACTH) secretion (Powell et al., 1967a, b), and sometimes elevated cortisol levels (Rayner & Rudd, 1973). These children had adequate nutrition and unexpectedly recovered without any medications during hospitalization (Powell et al., 1967a, b), or after placement into a caring environment (Frasier & Rallison, 1972; Rayner & Rudd, 1973). In sum, these reports indicate that psychosocial factors predominate in the production of RADI. Short-term maternal separation (MS) in pre-weanling rats has been shown to be a model for RADI (Butler & Schanberg, 1977; Butler et al., 1978; Kuhn et al., 1979, 1983, 1991b; Pauk et al., 1986; Scafidi et al. 1990; Schanberg & Field, 1987; Schanberg & Kuhn, 1980; Schanberg et al., 1984). Schanberg et al. (1984) have demonstrated that maternal care is a critical regulator of pup physiology and behavior during the first 14 days postnatally. Maternal separation has been characterized in this laboratory as a combination of declines in (1) activity of the enzyme ornithine decarboxylase activity (ODC), a sensitive index of tissue growth, and its products putrescine, spermidine, spermine and proteins; (2) synthesis of DNA; (3) GH secretion; and (4) tissue sensitivity to GH (Bartolome et al., 1994; Kuhn et al., 1983, 1990; Slotkin & Bartolome, 1986). The specific suppression of GH is one of the hallmarks of this condition (Kuhn et al., 1979). Growth hormone secretion is restored by return to the dam, or by a specific pattern of active tactile stimulation (Evoniuk et al., 1979; Pauk et al., 1986). Although touch maintains secretion in neonatal rats, the neural mechanism through which this occurs has not been defined. Growth hormone secretion is controlled in adult rats by the opposing actions of growth hormone-releasing factor (GRF) secreted from neurons which originate in the arcuate nucleus (AN) of the hypothalamus to stimulate GH release and by somatostatin (SS) deriving from neurons arising in the periventricular area (PVN) of the hypothalamus (MOiler et al., 1977; Tannenbaum, 1991). By neonatal day 3, both GRF and SS contribute to regulation of GH secretion but SS predominates (Baird et al., 1984; Cella et al., 1990; Jansson et al., 1987; Oliver et al., 1982; Rieutort, 1981). Several neurotransmitter systems which may modify GRF and SS secretion have been identified from ontogenetic studies of GH secretion. Noradrenergic, opioid and serotonergic stimuli have all been shown to stimulate GH in neonatal rats (Cocchi et al., 1977; Kacs6h et al., 1993; Kuhn & Schanberg, 1981; Lamberts & MacLeod, 1979). However, the functionality of the noradrenergic system which acts through GRF can be demonstrated in MS but not control rats (Celia et al., 1985, 1988, 1990; Cocchi et al., 1977; Kacs6h et al., 1993; Kuhn & Schanberg, 1981). In addition, our preliminary studies of opioid involvement in MS have not supported a role for either the stimulatory/~ or inhibitory K receptors in the GH response (unpublished data). In contrast to studies which question the role of noradrenergic and opioid neurons in the GH response to MS, a role for serotonergic neurons in control and MS rat pups is supported by studies reporting 5-HT stimulation of GH secretion in neonatal rats (Cocchi et al., 1977; Kacs6h et al., 1993). Stimulation of GH secretion in adult rats may involve both stimulation of GRF and inhibition of SS secretion by 5-HT (Aghajanian, 1972; Arimura & Fishback, 1981). Multiple serotonin receptors have been implicated in these effects. However, only stimulation has

GH Inhibition in MS Through 5-HT2Aand 5-HT2c Receptors

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been reported in pups (Kacs6h et al., 1993), suggesting that serotonergic neurons primarily contribute to stimulatory and not inhibitory control of GH secretion in early development. The present investigation focuses on the neuroendocrine regulation of GH secretion by GRF and SS in control and MS rat pups. As the role of serotonin receptor subtypes in control of GH secretion has not been explored, we used GRF, SS, indirectly acting agents fluoxetine (FLX) and 5-HTP (the serotonin precursor), as well as relatively selective 5-HTl and 5-HT2 agonists and antagonists. These drugs were tested under three conditions, including the ability of (1) GRF and SS to influence GH secretion during MS and during return to the dam; (2) serotonin agonists to stimulate GH secretion in normal and MS pups; and (3) serotonin antagonists to block the rise in GH during return to the dam.

METHODS

Mixed-sex litters of 10-day-old rat pups with lactating dams were obtained from Charles River Laboratories (Raleigh, NC, USA). Litters were brought to the laboratory the evening before each experiment to lessen stress induced by environmental changes (Kuhn & Schanberg, 1981) and were culled to 10 pups per group with random redistribution among the dams. Litters were kept in an ambient temperature of 22°C. All experiments were completed between 0700 h and 1300 h. Nonseparated pups remained with the dams except during injections; separated pups in groups of 10 were placed in plastic containers with wood shavings into an Air Shields 'Isolette' humidified, ventilated incubator at an ambient 2 7 C . After injections, pups were transferred to the dam's cage in groups of twos or threes, or kept in the container for transfer to the incubator. Except as otherwise noted, handling was minimized to reduce stimulatory effects on pup GH secretion (Eck & Kuhn, 1992; Smythe et al., 1975). Equivalent volumes of drug or vehicle were injected in each experiment. Fluoxetine (FLX), a serotonin-specific re-uptake inhibitor, was obtained from Eli Lilly and Co., Inc. (Indianapolis, IN, USA). The MK-212, a 5-HTIB,ZC agonist (Hoyer, 1988), was obtained from Merck, Inc. (Rahway, N J, USA). The 5-HTIA agonist, 8-OH-DPAT, quipazine (Quip), a nonspecific 5-HT1B,ZA,2C,3 agonist and possibly a 5-HTm antagonist, mCPP, a 5-HTm.2c agonist, DOI, a 5-HT2A,2C agonist, ketanserin (Ket), a serotonergic antagonist with greater affinity for 5-HT2c compared to 5-HT2A receptors, and S(-)-pindolol (Pin), a selective 5-HTIA, m antagonist, were all obtained from RBI (Natick, MA, USA). The L-5-HTP, cyproheptadine (Cypro), a nonspecific 5-HT antagonist, and SS were purchased Table I. 5-HTP and FLX (fluoxetine) dose-responses for GH (growth hormone) secretion in 10-day-old pups 30 rain after 5-HTP IP, FLX SC, or equal volumes of vehicle (IP or SCI are presented

Dose (mg/kg)

5-HTP

0 5 10 20 3(/

12 _+ l 29 + 10 38 +_4 -38 _+8

GH (ng/ml) n

FLX

n

69 23 24 -5

ll _+ 1 18 + 2 25 _+4 22 _+2 --

17 5 21 5 --

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L.M. Katz et al.

Table II. Effects o f 5 - H T agonists on G H (growth hormone) secretion in 10-day-old pups are expressed as means _+ SEM. * indicates p < .05 or better versus control level after treatment with various doses o f four different agonists Agonist

Dose (mg/kg)

GH (ng/ml) _+ SEM

n

m-CPP

0 0.25 0.5 1.0 5.0 0 0.03 0.3 1.0 4.5 10.0 0 0.03 0.1 0.3 1.0 0 0.25 0.5 1.0 10.0

13 _+ 4 11 _-23 6.7 _+ 0.4 6.9 _+ 0.8 6.0 _+ 0.4* 13 _+ 2 11 _+ 2 13 _+ 3 9 _+ 3 5.9 + 0.4* 2.3 _+ 0.1' 13 _+ 1 8.3 _+ 0.9 5.5 _+ 0.6* l l _+ 1 6 + 1' 13 _+ 2 13 _+ 2 9 _+ 1 7.4 _+ 0.9 10 _+ 2

16 5 5 11 4 26 4 4 11 5 5 10 4 5 4 4 15 5 5 10 5

MK-212

8-OH-DPAT

Quipazine

from Sigma Chemical Co., Inc. (St Louis, MO, USA). The G R F was purchased from American Peptides Co. (Sunnyvale, CA, USA).

Hormone Assays Trunk blood was collected after rapid decapitation into polystyrene vials, chilled on ice, then centrifuged for 2-2.5 min. Serum was individually pipetted into new polystyrene tubes, frozen on dry ice for less than 30 min, then stored at - 8 0 ° C until the time o f assays. Growth hormone was quantified by radioimmunoassay (RIA) using standard RP2 G H and antisera

Table III. G H (growth hormone) responses to 5 - H T antagonists in 10-day-old pups are presented 1 h after injections of vehicle, cyproheptadine (Cypro) 10 mg/kg or ketanserin (Ket) 1 mg/kg SC. *indicates p < .02 or better versus control, and I" indicates p < .02 versus Ket Drug Vehicle Cypro*t Ket*

GH (ng/ml) -+ SEM

n

12.8 _+ 0.9 3.9 + 0.5 8.6 _+ 1.3

33 19 16

GH Inhibition in MS Through 5-HT2A and 5-HT2c Receptors

223

GH Response to Antagonists and 5-HTP 80 70 60 50 40 '-r(.9 3O

20 10

Treatment

Fig. 1. Mean GH (growth hormone) and SEM in 10-day-old pups are plotted after 5-HT agonistantagonist combinations. Pups were injected with either vehicle, cyproheptadine (Cypro) 10 mg/kg (n = 5), or ketanserin (Ket) 1 mg/kg SC (n = 9), and then injected 1 h later with vehicle (n = 61) or 5-HTP 30 mg/kg IP (n = 13). Decapitation followed 30 min later.

supplied by the NIAMDD Rat Pituitary Hormone Distribution program. The 125I-GH was synthesized in our laboratories. Sensitivity of this RIA is 0.16 ng/ml with inter- and intraassay variability each being 9%. The GH levels were statistically analyzed across groups with one-, two- or three-way ANOVAs, depending on the number of independent variables, followed by individual ANOVAs. Post-hoc comparisons were completed with the Student's-Newman-Keuls t tests for parametric data. For nonparametric data, two- or three-way Scheirer-Ray-Hare extension ANOVAs were followed by Kruskal-Wallis comparisons and post-hoc Student's-Newman-Keuls t tests or Dunn's tests. Kruskal-Wallis test or Student's t test was applied for single pairwise comparisons depending on the normalcy and variance testing of data. Replicated sets at each time point were analyzed together, with outliers (defined as beyond 2 SD from the mean) removed before combination of the data for parametric distributions. Statistical significance was designated at p < .05.

RESULTS Pups with Dams 5-HTP and FLX. Table I shows that GH secretion is at maximal stimulation and nearly equivalent at 10 and 30 mg/kg. In the following series of experiments either 15 or 30 mg/kg

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GH Response to Antagonists and FLX 35

30

25

2o

7-

l

1()

5

o

J Treatment

Fig. 2. Mean GH (growth hormone) and SEM in 10-day-old pups are presented after FLX (fluoxetine) antagonist combinations. Vehicle, cyproheptadine (Cypro) 10 mg/kg (n = 11), or ketanserin (Ket) 1 mg/kg SC (n = 9) was given 1 h before vehicle (n = 61) or FLX (n = 14) and pups were decapitated 30 min later.

5-HTP IP was used. Growth hormone was also measured 30 min after various FLX doses. The GH response was nearly equivalent with any of the doses.

Agonists. Administration of 5-HT agonists m-CPP, MK-212, 8-OH-DPAT and Quip were associated with either no effect or declines in GH at higher doses, as shown in Table II. When the 5-HT2A,2C agonist DOI was administered, no significant differences were found in GH for vehicle injections vs. 0.3 (low) and 3.65 (high) mg/kg doses. The GH secretion after the high dose tended to be lower compared to vehicle or the low dose (data not shown). Antagonists. Table III illustrates significant inhibitory effects of Cypro 10 mg/kg and Ket 1 mg/kg on basal GH secretion (t = 8.64, dr= 78, p < .001 for Cypro; t = 2.66, dr= 92, p < .02 for Ket). Nonparametric analysis of the data in Fig. 1 showed a main effect for treatment with 5-HTP [H(1,84)= 49.7, p < .001] and for treatment with an antagonist [H(2,84) = 17.8, p < .001]. After post-hoc tests, it was determined that 30 mg/kg 5-HTP significantly stimulated GH secretion (vs. vehicle p < .05) and that the effect of 5-HTP was inhibited by Cypro 10 mg/kg pretreatment 1 h earlier (vs. no antagonistp < .05) but not by 1 mg/kg Ket pretreatment. The inhibitory effect of Cypro was significantly different from the effect of Ket (p < .05). As shown in Fig. 2, FLX treatment significantly stimulated GH secretion [H(1,91) = 24.6, p < .001] and the pretreatment with an antagonist reversed this stimulation by FLX [/-/(2,91)= 26.3, p < .001]. Post-hoc comparisons revealed that the

GH Inhibition in MS Through 5-HT2A and 5-HT2c Receptors

225

GH R e s p o n s e to SS in Normal and R e t u r n e d Pups 30

25

T

r-

20 3: (.9

15

10

! Treatment

Fig. 3. Mean GH (growth hormone) and SEM are presented for 10-day-old pups after MS (maternal separation). Pups were separated or remained with dams for 4 h, then injected with either vehicle (n = 14 for MS and n = 5 for normal pups) or somatostatin (SS) 1 mg/kg SC before remaining with dams (n = 5), continued separation (n = 10) or before returning to dams (n = 14) for an additional 1 h prior to decapitation. secretion of GH by FLX was significantly inhibited by 1 h pretreatment with Cypro or Ket (p < .05 in both cases). In this case also, the inhibitory effect of Cypro was significantly greater than of Ket (p < .05). The 5-HT1A,1B antagonist Pin had no significant effect on GH secretion compared to vehicle in separated and nonseparated 10-day-old pups (data not shown).

Somatostatin and growth hormone-releasing factor. The SS did not suppress basal GH levels in control pups (Fig. 3) nor did it prevent normalization of GH levels in returned pups having undergone MS. The main effects of MS and returning the pups to the dams in this three-way nonparametric ANOVA were significant [H(1,44)= 6.32, p < .03 for MS; H(1,44) = 17.1, p < .001 for returning]. The follow-up Kruskal-Wallis tests confirmed significance only for the effect of returning pups (H = 10.5, dr= 1,p = .001; p < .05 for the post-hoc). The GRF did not stimulate GH secretion in normal pups, but MS tended to lower GH (Fig. 4). Two-way nonparametric ANOVA analysis revealed a strong tendency for GRF to stimulate GH secretion independent of MS status [H(1,52) = 3.76, p = .058], apparently due only to the induction of GRF in MS pups. Since indicating a trend towards significance, this result was followed by a Kruskal-Wallis test which was applied to the GRF normal/MS data without the vehicle data included; GRF administration 15 min before sacrifice did reverse the inhibition of GH secretion by MS (H = 5.15, dr= 1, p = .02 overall; p < .05 by the post-hoc test).

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GH Response to GRF in Normal and MS Pups 6O

50

40

"130

20

10

i Treatment

Fig. 4. The GH (growth hormone) is expressed as mean and SEM for 10-day-old pups after MS (maternal separation) and GRF (growth hormone-releasing factor). Pups were separated or remained with dams for 2 h, then injected with vehicle (n = 14 in both normal and MS pups) or GRF 1/2g/kg SC 15 min prior to decapitation (n = 14 for normal and n = 15 for MS pups).

Pups FollowingMaternal Separation Agonists. Maternal separation (Fig.

5) was associated with a significant decrease in vehicle and 5-HTP stimulated GH secretion (t = 2.40, dr= 21, p < .03; t = 3.25, df= 21, p < .005, respectively). In contrast, MS did not inhibit FLX-stimulated GH secretion.

Antagonists. Comparison with a three-way nonparametric ANOVA of Cypro vs. vehicle in normal, MS and returned pups revealed main effects both for Cypro [H(1,67) = 38.0, p < .001] and for return of pups to dams [H(1,67) = 11.2, p < .001]. Follow-up Kruskal-Wallis testing and post-hoc analysis showed that Cypro robustly inhibited GH secretion in both normal and MS pups (H = 26.0, df= 1, p < .0001; p < .05 for the post-hoc) (Fig. 6). Returning the pups also stimulated GH secretion without respect to drug treatment (H = 9.02, dr= 1, p < .003; p < .05 for the post-hoc). When Ket was substituted for Cypro (Fig. 7), a similar three-way nonparametric ANOVA indicated that the main effects of MS and of returning pups to dams were significant [MS: H(1,130) = 19.1, p < .001; returning: H(1,130) = 42.3, p < .001]. The follow-up Kruskal-Wallis tests did not indicate a significant effect of MS, but returning pups to dams significantly stimulated GH secretion independent of drug status (H = 18.2, dr= 1, p < .0001; p < .05 by the post-hoc test). However, Ket caused no additional GH suppression in MS pups and did not prevent the increase in GH in returned pups.

GH Inhibition in MS Through 5-HT2A and 5-HT2c Receptors

227

MS inhibition of 5-HTP or FLX-Stimulated GH vehicle

100

5-HTP 15 mg/kg FLX 10 rng/kg

80

60

"r

o

40

20

Nonseparated

MS

Fig. 5. The GH (growth hormone) response to 5-HTP or fluoxetine (FLX) in non-separated and maternally separated (MS) 10-day-old pups is presented as means and SEM. Pups were treated with either vehicle SC, 5-HTP 15 mg/kg IP or FLX 10 mg/kg SC for 30 min preceding decapitation (n = 19-20 in each case). In the MS group, pups were separated in the incubator from dams for 4 h before injections.

Nonparametric two-way ANOVA analysis indicated that the interactive effect of Cypro and GRF treatments was nonsignificant but that main effects for each drug vs. vehicle were significant [Cypro: H(1,36) = 29.7, p < .001; GRF: H(1,36) = 13.3, p < .001]. As shown in Fig. 8, in MS pups GRF markedly stimulated GH secretion and Cypro pretreatment dramatically blocked this effect (p < .05 for each post-hoc comparison). Cypro also inhibited GH secretion in MS pups not given GRF (p < .05 vs. vehicle).

DISCUSSION The similarities between neuroendocrine consequences of RADI in humans and MS in rat pups are striking. The GH response of humans to parental deprivation recapitulates the specific GH response in pups (Kuhn et al., 1979, 1990; Powell et al., 1967a, b; Schanberg & Field, 1987). In addition, both RADI children (Frasier & Rallison, 1972; Rayner & Rudd, 1973) and separated rat pups (Kuhn et al., 1979) are refractory to exogenous GH. Finally, improving the environment for deprived children (Frasier & Rallison, 1972; Rayner & Rudd, 1973) or providing active tactile stimulation for separated pups (Butler et al., 1978; Pauk et al., 1986) and deprived preterm infants (Field et al., 1986) reverses many effects of both syndromes. The present study indicates that in the rat pup, active tactile stimulation or maternal care may stimulate GH secretion through serotonergic influences on GRF and SS secretion (Fig. 9).

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L . M . Katz et al.

GH Response to Cypro in MS and Returned Pups 12

I

v '1"

(.9

0

0 Treatment

Fig. 6. Mean GH (growth hormone) responses and SEM after MS (maternal separation), return and Cypro (cyproheptadine). Pups were either separated or remained with dams for 3 h prior to injection of vehicle (n = 12 for MS and n = 18 for normal) or Cypro 10 mg/kg SC (n = 14 in both cases) and then either remained separated or returned to dams for another 1 h before decapitation. Both serotonin antagonist and agonist experiments demonstrated a stimulatory role for serotonin in control of GH secretion in normal and MS rat pups. As shown previously (Smythe et al., 1975; Smythe & Lazarus, 1973), the serotonergic agonist 5-HTP and the serotonin uptake inhibitor FLX increased GH secretion in pups. Furthermore, both the nonselective antagonist Cypro and the selective 5-HT2c antagonist Ket lowered basal GH secretion. The suppression of GH by these antagonists is consistent with reports that 5-HT2A and 5-HT2c receptors on GRF neurons are stimulatory to GH secretion (Di Sciullo et al., 1990; Meller & Bohmaker, 1994; MOiler & Nistic6, 1989; Pompeiano et al., 1994; Pritchette et al., 1988; Willoughby et al., 1987). Studies in both adults and pups (Arnold & Fernstrom, 1981; Kacs6h et al., 1993; Murakami et al., 1986) suggest that the primary serotonergic control of GH secretion is mediated by actions on GRF neurons. This serotonergic neuron may be an obligate mediator of the actions of norepinephrine (NE) at ~2-adrenergic receptors in animals 10 days or older (Arnold & Fernstrom, 1981; Kacs6h et al., 1993). The present results are mainly consistent with this postulated mechanism (Fig. 9). The variable actions of DOI were unexpected, but may reflect in part known cardiovascular responses to noradrenergic stimulation (Dedeoglu & Fisher, 1991; Helke et al., 1991; Rittenhouse et al., 1991). The added suppression produced by Cypro in MS pups suggests that there might be an additional stimulatory serotonergic mechanism contributing to control of GH secretion in neonatal rats. Similarly, the report of Kacs6h et al. (1993) in rat pups in vivo provides

GH Inhibition in MS Through 5-HT2A and 5-HTec Receptors

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GH Response to KET in MS and Returned Pups

4° 1 35

30 4

i i

i i

25 20

•

"1"

C9 15

10

! S Treatment

Fig. 7. Mean GH (growth hormone) responses and SEM for 10-day-old pups after MS (maternal separation) and Ket (ketanserin). Pups were treated as in Fig. 7, except for an initial separation of only 2 h, and were injected with ketanserin (Ket) 1 mg/kg (n = 33 for normal control pups; n = 26 for normal Ket pups; n = 18 for MS control pups; n = 20 for MS Ket pups and for MS returned pups; and n = 17 for MS returned Ket pups). evidence of dual serotonergic mechanisms. The study of adult hypothalamus in vitro by Richardson et al. (1981) specifically shows effects of 5-HT on both GRF and SS secretion. It is probable, therefore, that a serotonergic mechanism acting through 5-HT2A receptors (insensitive to Ket), as well as an additional mechanism, are primarily active in neonatal rats. However, suppression of pituitary release of GH by Cypro represents an alternative interpretation. The ability of GRF to stimulate GH secretion was impaired by Cypro, which has been reported to prevent prolactin release from lactotrophs (Lamberts et al., 1985). However, a single study in somatotrophs in adult rats showed no effects of Cypro (Lamberts 8,: MacLeod, 1979). Therefore, inhibition at 5-HT2c receptors and not at the pituitary may be the more likely mechanism. The greater maximal GH response caused by 5-HTP compared to FLX and the inability of Ket to inhibit the actions of 5-HTP provides additional evidence in support of two serotonergic mechanisms, and suggests that 5-HTP stimulates both. The reported ability of 5-HTP to release catecholamines (adult rat) represents one such possibility. It is likely that 5-HTP stimulates GH through two additive mechanisms: stimulation of ct2-adrenergic receptors by endogenously released NE, and stimulation of the serotonin receptors described above that may be downstream from this adrenergic neuron (Fig. 9). Previous studies in neonatal rats support this conclusion, as the ~2-antagonist phentolamine is reported to prevent the actions of 5-HTP on GH secretion in neonatal rats (Stuart et al., 1976). In addition, the study by Chihara et al. (1979) suggests that NE may alter SS secretion. The

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L . M . Katz et al.

GH Response to Cypro and GRF in MS Pups 60

50

40

"r"

30

T 20

10

J Treatment

Fig. 8. Mean GH (growth hormone) and SEM in 10-day-old MS (maternal separation) pups treated with Cypro (cyproheptadine). All pups were deprived for 2 h, injected with vehicle or cyproheptadine (Cypro) 10 mg/kg SC, and then injected with vehicle or growth hormone-releasing factor (GRF) 1 #g/kg SC after 1 h (n = 10 for each combination). Pups were decapitated 15 min later. inability of Ket to prevent 5-HTP induced GH secretion suggests that 5-HTP might activate two serotonin receptor populations: 5-HT2A and 5-HT2c. In the presence of Ket, it is possible that stimulation of the alternative pathway maintains secretion. While the results with 5-HTP could be interpreted to reflect additive effects at two serotonin receptor populations, it is not clear why FLX did not act similarly at both; FLX should raise synaptic serotonin levels at all innervated receptor populations (Fuller & Wong, 1977; Schmidt et al., 1988). There are several explanations for this discrepancy. The 5-HTP could stimulate serotonin receptor populations that are not yet innervated. Although innervation of the hypothalamus occurs fairly early in gestation (Aghajanian, 1972; Artigas, 1993; Lesch et al., 1993; Martin & Sanders-Bush, 1982; Mosko & Jacobs, 1977), serotonin receptor populations may exist on postnatal day 10 that are not well innervated. Therefore, 5-HT (from 5-HTP) would stimulate secretion, while an indirectly acting agent dependent upon neuronal release would not. Alternatively, marked differences in impulse flow of specific populations of serotonergic neurons may occur in the hypothalamus, modifying the efficacy of FLX but not of high dose 5-HTP. Finally, 5-HTP and FLX are known to affect several non-serotonergic systems. These include noradrenergic, adrenergic, dopaminergic, neuropeptide and opioid systems (Aguilar et al., 1994; Carr et al., 1991; Celia et al., 1988; Hagan & Moss, 1993; Holtzman & Steinfels, 1994; Hynes et al., 1985). Clearly, considerably more experimentation will be needed to resolve this discrepancy.

GH Inhibition in MS Through 5-HT2A and 5-HT2c Receptors

~

231

13-endorphin

NE 5 -HT

5 - HT. a []

I

5 - H T . c []

CRF

+ +

I

[

ss

J.

]

Pituitary Growth hormone

Fig. 9. Hypothetical model of serotonergic and noradrenerglc regulation of GRF (growth hormonereleasing factor) and SS (somatostatin). Bold lines and arrows designate the serotonergic systems in neonatal rats altered in this study.

The present results do not support a strong role for 5-HTtA or 5-HTIB receptors in stimulatory control of GH secretion in neonatal rats. Only suppression, not stimulation, of GH secretion was observed after administration of the 5-HT1A agonist 8-OH-DPAT (Hjorth & Sharp, 1991; Hoyer, 1988; Middlemiss & Fozard, 1983) and mixed agonists MK-212, mCPP and quipazine (Alhaider et al., 1993; Hoyer, 1988; Milburn & Peroutka, 1989; Schechter & Simansky, 1988; Sills et al., 1984). Furthermore, the 5-HT1A antagonist Pin had no effect on GH secretion in either control or MS pups. However, these agents probably have multiple sites of action, including actions on presynaptic sites which regulate cell firing in the raphe, as well as postsynaptic receptor sites in the hypothalamus. Therefore, it is possible that multiple opposing actions might have obscured potentially significant effects. The findings with GRF and SS as well as serotonergic agents suggest that GH secretion falls during MS due to withdrawal of stimulation through a serotonergic intermediate (see Fig. 9). Several findings support this hypothesis. The GRF only stimulated GH secretion during MS (p = .058), while SS failed to block the rise when pups were returned to dams. The stimulation of GH release by exogenous GRF only in deprived pups may be explained by MS-induced withdrawal of a maximal rate of GRF secretion and compensatory decrease in SS release. The ability of Cypro but not Ket to block this rise further indicates that 5-HT2A and not 5-HT2c receptors might provide the final common pathway that is influenced by MS. Regarding this strong tendency (p = .058) for change in GH secretion in MS pups 15 min after treatment with GRF compared to controls, it has been shown that the handling effect itself stimulates GH secretion within 15 min in the 10-day-old rat pup (Kuhn et al., 1991a; Eck & Kuhn, 1992). Therefore, any GRF-elicited increase was likely obscured by handling itself. It is possible that the suppression of GH by MS is so great that changes are maintained despite stimulatory handling. This handling effect is reflected in the elevation in GH in both control and MS pups in this experiment relative to GH secretion in other experiments.

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The mechanism by which MS suppressed 5-HTP induced GH secretion remains indeterminate. It is possible that 5-HTP elicits less synaptic catecholamine release during MS or that the ct2-receptor system is down-regulated. However, numerous studies showing that :t2-receptor stimulation can be detected only during MS indicate that this receptor and its signal transduction pathway is functional during MS (Cella et al., 1985, 1988, 1990; Cocchi et al., 1977; Kacs6h et al., 1993; Kuhn & Schanberg, 1981). Alternatively, the specific serotonin receptor population stimulated uniquely by 5-HTP but not by FLX may be functionally impaired during MS. Again, if this population is obligatory, as hypothesized for :t2-adrenergic receptor stimulation of GH, this seems implausible. Regardless of the mechanism, this suppressed response is clearly comparable to the specific suppression of GH secretion to evocative stimuli that occurs during RADI in human populations (Powell et al., 1967a, b). Future studies of this problem should provide insight into possible mechanisms by which GH secretion is impaired during RADI in humans. In summary, the data presented in this study and observations of others illustrate that GH hyposecretion during MS may derive from a reduction in GRF release through noradrenergic neurons which impinge upon serotonin terminals in the hypothalamus. The profile of antagonist specificity suggests that a 5-HT2A receptor may be critically involved in this process. A parallel serotonergic pathway involving 5-HT2c receptors, GRF or perhaps SS, may also contribute to control of GH secretion in neonatal rats.

Acknowledgements:The work presented in this paper was partly funded by NeurobehavioralSciences Research Training Program grant number NIH MH 15177. The program is administered by Everett Ellinwood, MD, at Duke University Medical Center. The work was also funded by grant number NIH MH 13688. Saul Schanberg, MD, PhD, is the principle investigator for this psychopharmacologyand molecular biology laboratory. We express appreciation to Judy G. Johnston, BS, for assistance throughout the study and in the preparationof this manuscript.

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