Direct determination of dopamine D4 receptors in normal and schizophrenic postmortem brain tissue: a [3H]NGD-94-1 study.

Using an indirect subtraction binding technique and human postmortem tissue, several laboratories reported finding increases in dopamine D4 receptors in caudate nuclei of schizophrenic patients, although others have not replicated these findings. NGD-94-1 is a selective D4 antagonist with low affinity for the D2 and D3 receptors. [3H]NGD-94-1 has been used in this study to directly determine the density of D4 receptors in normals (n = 13) and schizophrenic subjects (n = 7) off antipsychotic drugs for at least 3 months prior to death, or on antipsychotic (n = 7) drugs at the time of death. Human postmortem coronal brain sections were incubated with [3H]NGD-94-1 and autoradiograms developed; and binding in pertinent regions was quantified. In normals, the highest density of [3H]NGD-94-1 binding was in the hippocampus (68 fmol mg(-1), temporal (33), insular (30), and entorhinal cortices (24.9). Significant increases in [3H]NGD-94-1 density in schizophrenics (n = 14) vs normals (n = 13) were observed in the entorhinal cortex (46%) at both low and high magnifications. The increases observed in the schizophrenics were found in both schizophrenics off antipsychotic drugs for at least 3 months prior to death and those on antipsychotic drugs at the time of death. Thus, the changes may be disease-related and not a consequence of pharmacological treatment. No significant differences were found between the two schizophrenic groups in any brain area studied.

In vitro autoradiography of GTPgamma[35S] binding at activated NPY receptor subtypes in adult rat brain.

Guanylyl 5'-[gamma[35S]thio]-triphosphate (GTPgamma[35S]) binding to NPY receptor-activated G-proteins was measured in adult rat brain sections in order to determine the neuroanatomical distribution of NPY receptor subtypes. Using the pharmacological specificity of the NPY receptor subtypes, differential stimulation of GTPgamma[] binding by subtype-specific agonists was used to demonstrate the differential distribution of these subtypes in rat brain. Treatment of rat brain slices with selective agonists for the NPY receptor subtypes in the presence of 2000 microM GDP was used to discriminate populations of NPY receptor subtypes. Activation of a NPY Y1 receptor subtype by human [Leu31Pro34]NPY stimulated GTPgamma[35S] binding in the rank order: frontal cortex>dentate gyrus>inferior colliculus>/=thalamus>hypothalamus. In contrast, NPY Y2/Y5 peptide agonist, human PYY(3-36), stimulated GTPgamma[35S] binding in the rank order: hypothalamus>substantia nigra>hippocampus>frontal cortex>/=inferior colliculus. Stimulation of NPY Y5 receptor subtypes by a NPY Y5 selective agonist, rat/human D-Trp, was shown to stimulate GTPgamma[35S] binding in the hypothalamus and discrete nuclei of the thalamus. Little GTPgamma[35S] binding in the dentate gyrus, frontal cortex, or inferior colliculus was measured following stimulation with D-Trp. Stimulation of GTPgamma[35S] binding by [Leu31Pro34]NPY, but not by the other NPY receptor agonists, was blocked by the selective NPY Y1 receptor antagonist, BIBP 3226. In conclusion, functional coupling at NPY receptor subtypes can be shown in rat brain and populations of NPY receptor subtypes can be anatomically discriminated by NPY agonist stimulation of GTPgamma[35S] binding in rat brain.

NGB 2904 and NGB 2849: two highly selective dopamine D3 receptor antagonists.

N-(4-[4-¿2, 3-dichlorophenyl¿-1-piperazinyl]butyl)-3-fluorenylcarboxamide and N-(4-[4-¿2, 3-dichlorophenyl¿-1-piperazinyl]butyl)-2-biphenylenylcarboxamide were prepared in several steps from 2,3-dichloroaniline. These compounds were identified as highly selective dopamine D3 receptor antagonists.

Autoradiographic localization of CRF1 and CRF2 binding sites in adult rat brain.

The regional distribution of corticotropin-releasing factor1 (CRF1) and CRF2 binding sites was assessed autoradiographically in adult rat brain. The differential pharmacological profiles of the CRF1 and CRF2 receptor subtypes were used for the discrimination of the CRF1 and CRF2 receptor subtypes in rat brain. Pharmacological characterization at the human CRF1 receptor subtype, expressed in baculovirus-infected Sf9 cells, showed high affinity binding (Ki < or = 10.0 nM) for the peptide agonists sauvagine, urotensin I, rat/human CRF, and ovine CRF. Pharmacological characterization at the rat CRF2 receptor subtype expressed in CHO cells showed a rank order affinity for the peptide agonists such that sauvagine, urotensin I and rat/human CRF showed high affinity binding whereas ovine CRF had a Ki value of 300 nM. Based on this differential binding affinity for ovine CRF, [125I]sauvagine binding in the presence of increasing concentrations of ovine CRF was used to discriminate CRF1 from CRF2 receptor subtypes in rat brain. The CRF1 receptor subtype was found to be localized to various regions of the cerebellum, as well as to several cortical areas. The CRF2 receptor subtype was shown to be localized to the lateral septal nucleus, entorhinal cortex, and to amygdaloid and hypothalamic regions. The present autoradiographic findings provide evidence that each subtype has a distinct regional distribution, thus strengthening the suggestion that CRF1 and CRF2 receptors serve different roles in mediating CRF function. Such data suggest that the development of CRF receptor subtype selective antagonists should help to delineate the role of CRF1 and CRF2 receptor subtypes in central nervous system function.

II. Localization and characterization of dopamine D4 binding sites in rat and human brain by use of the novel, D4 receptor-selective ligand [3H]NGD 94-1.

The dopamine D4 selective ligand, [H]NGD 94-1, was used in these studies to characterize binding sites in rat and human brain tissue by membrane binding and autoradiography techniques. Autoradiographic analysis of rat brain showed that specific [3H]NGD 94-1 binding was greatest in entorhinal cortex, lateral septal nucleus, hippocampus and the medial preoptic area of the hypothalamus. This nonstriatal distribution of [3H]NGD 94-1 binding was distinct from the autoradiographic distribution of dopamine D2 and D3 receptor subtypes. In homogenate preparations from rat brain regions, [3H]NGD 94-1 binding sites were low in density (<30.0 fmol/mg protein). The low density of D4 binding sites was corroborated by autoradiographic comparisons in which binding density for D4 receptors as measured by [3H]NGD 94-1 was only 1/7 of D2 and 1/5 of D3 receptor densities, despite corrections for differing radioligand binding characteristics. Pharmacological evaluation showed high affinity at rat [3H]NGD 94-1 binding sites for compounds with known D4 receptor affinity and little displacement by compounds with affinity for dopamine D1/D2/D3 receptor subtypes. Specific, high-affinity [3H]NGD 94-1 binding was also present in several human brain regions, including hippocampus, hypothalamus, dorsal medial thalamus, entorhinal cortex, prefrontal cortex and lateral septal nucleus. High-affinity [3H]NGD 94-1 binding was not present in any human striatal region examined. The pharmacological profile of [3H]NGD 94-1 binding sites in human brain was consistent with that previously demonstrated for cloned human D4 receptors expressed in mammalian cells. These findings suggest that specific, high-affinity [3H]NGD 94-1 binding exists in rat and human brain and that these sites reflect populations of dopamine D4 receptors with a distribution unique among dopamine receptor subtypes.

I. NGD 94-1: identification of a novel, high-affinity antagonist at the human dopamine D4 receptor.

NGD 94-1 was evaluated for selectivity and in vitro functional activity at the recombinant human D4.2 receptor stably expressed in Chinese hamster ovary cells. NGD 94-1 showed high affinity for the cloned human D4.2 receptor (Ki = 3.6 +/- 0.6 nM) and had greater than 600-fold selectivity for the D4.2 receptor subtype compared with a wide variety of monoamine or other neurotransmitter receptor or modulatory sites except for 5-HT1A and 5-HT3 receptors, in which NGD 94-1 was approximately 50- and 200-fold selective, respectively, for the D4.2 receptor. In measures of in vitro functional activity, NGD 94-1 showed an antagonist profile at the cloned human D4.2 receptor subtype. NGD 94-1 completely reversed the decrease in forskolin-stimulated cAMP levels produced by the dopamine receptor full agonist quinpirole. Furthermore, NGD 94-1 produced a complete reversal of GTPgamma35S binding induced by quinpirole, but was unable on its own to affect GTPgamma35S binding. These data suggest that NGD 94-1 functions as an antagonist rather than a full or partial agonist at the human D4.2 receptor. In addition, NGD 94-1 binding affinity at the D4.2 receptor subtype was unaffected by G-protein activation by GTP, consistent with the binding affinity seen for other antagonists at the D4 receptor. The binding of tritiated NGD 94-1 was saturable and of high affinity at cloned human D4.2 receptors. Furthermore, the binding of [3H]NGD 94-1 to cloned human D4.2 receptors expressed in Chinese hamster ovary cells displayed a pharmacological profile similar to that observed with the nonselective dopamine receptor ligand [3H]YM 09151-2. Saturation and pharmacological analyses of [3H]NGD 94-1 binding at cloned human D4.2, D4.4 and D4.7 receptor variants showed no difference between the three variants. NGD 94-1 is a novel, high-affinity, D4 receptor-selective antagonist. The clinical use of this subtype-specific compound should permit direct evaluation of the role of D4 receptors in psychiatric disorders.

Allosteric uncoupling after chronic benzodiazepine exposure of recombinant gamma-aminobutyric acid(A) receptors expressed in Sf9 cells: ligand efficacy and subtype selectivity.

By using the baculovirus expression system, we report decreases in allosteric coupling at individual gamma-aminobutyric acid (GABA)(A) receptor subtypes (alpha-1, beta-2 and gamma-2, alpha-2, beta-3 and gamma-2 and alpha-5, beta-3 and gamma-2) after chronic benzodiazepine exposure that replicate coupling changes measured in rat cortical membranes after in vivo benzodiazepine exposure. The appearance of uncoupling was time-dependent and the magnitude of uncoupling at expressed GABA(A) receptor subtypes after chronic exposure was dependent upon the efficacy of the ligand in a subtype-specific manner. In addition, the expression of uncoupling was not accompanied by changes in benzodiazepine receptor number or affinity at any expressed GABA(A) subtype examined. The specificity of the coupling change was further shown by the ability of a brief exposure to the benzodiazepine receptor antagonist, Ro15-1788, to reverse the uncoupling induced by chronic benzodiazepine exposure. These findings suggest that alterations at the GABA(A) receptor complex after chronic benzodiazepine exposure are mediated directly by agonist effects at the GABA(A) receptor complex and are not the product of the changes in the surrounding neuronal environment. Furthermore, the present study shows that drug efficacy, and not simply affinity, plays a critical role in determining the degree of uncoupling, and perhaps, in the development of tolerance and dependence.

Effect of subunit composition on GABAA receptor complex characteristics in a baculovirus expression system.

A baculovirus expression system was used to produce functional human recombinant GABAA receptors in Sf-9 insect cells in order to study the biochemistry, pharmacology and functional characteristics of this receptor complex. We have identified and characterized various factors which influence the level of receptor expression in multiple virus infections. We have shown that the level of expression of the GABAA receptor complex varies with the levels of expression of the individual subunits. We have also shown that the assembly process has a defined timecourse, and it is dependent upon the ratio of the number of infectious virus particles (MOI ratio) of each subunit in multi-virus infections. In multiple infections, the capacity for expression of the infected cell is shared proportionally by entering virus particles and, there is a direct correlation between the amounts of subunit mRNA and levels of subunit protein expression, and the amount of ligand binding to expressed protein. Finally, reinfection of previously infected cells does not result in subsequent protein expression. Knowledge of these various factors allows us to construct recombinant GABAA receptor complexes with reproducibility and flexibility with regard to subunit composition. By co-expression of alpha, beta, and gamma subunits, both the recognition site for GABA and the allosteric (benzodiazepine) modulatory site are formed and appear to reproduce the pharmacology of endogenously expressed receptors as measured in mammalian CNS. Only a single receptor is produced irrespective of the expression levels of the subunits, showing that GABAA receptor assembly is highly regulated.

Cocaine regulation of brain preprothyrotropin-releasing hormone mRNA.

Clinical and preclinical evidence supports a possible role for thyrotropin-releasing hormone (TRH) in cocaine action. However, the interaction between cocaine and TRH has not been directly examined. In the following report we describe a solution hybridization RNase protection assay that can sensitively detect mRNA for the TRH precursor, prepro-TRH (ppTRH). Using this assay, we examined ppTRH mRNA levels in rat brain regions implicated in cocaine reinforcement, including the nucleus accumbens, hypothalamus, amygdala, hippocampus, and thalamus. Acute cocaine treatment (15 mg/kg) resulted in significant decreases in ppTRH mRNA levels in the amygdala and hippocampus, but not in the hypothalamus, nucleus accumbens, or thalamus, 45 min postinjection. Chronic cocaine treatment (15 mg/kg twice daily for 14 days) resulted in marked regulation in all regions but the thalamus. Regulation was strongly dependent on the length of cocaine withdrawal and persisted up to 72 h postinjection in the amygdala. These studies support the hypothesis that TRH or other ppTRH-derived peptides are involved in cocaine action, especially in the extrahypothalamic regions of the amygdala and hippocampus.

Developmental profile of polyadenylated and non-polyadenylated GABAA receptor subunit mRNAs.

The ratio of mRNA not selected for polyadenylation (non-poly(A)+ selected) to mRNA selected for polyadenylation (poly(A)+) for the beta 1, alpha 1 and gamma 2 subunits of the GABAA receptor complex was examined in rats as a function of age. RNA was extracted from whole brain of rats that were either 0, 1, 3, 5 or over 60 days of postnatal age. Poly(A)+ mRNA was purified by oligo(dT)-cellulose chromatography. Non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor beta 1, alpha 1 and gamma 2 subunits were examined by Northern blot analysis using cDNA probes specific for these subunits. Levels of GABAA receptor beta 1 subunit mRNA were also examined by solution hybridization analysis with a beta 1 riboprobe. Analysis of Northern blots revealed that levels of poly(A)+ beta 1 subunit mRNA were highest at 0 days of age, but decreased and reached adult levels by 5 days of postnatal age. However, levels of the beta 1 subunit message extracted from non-poly(A)+ selected mRNA were not significantly different at any of the ages examined, suggesting the existence of a population of beta 1 subunit mRNA that is not polyadenylated. The age-related discrepancy between beta 1 subunit levels measured in non-poly(A)+ selected mRNA and poly(A)+ mRNA was also observed using solution hybridization analysis. In contrast, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the alpha 1 subunit of the GABAA complex increased from 0 days of age to adulthood. Similarly, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor gamma 2 subunit increased with age.(ABSTRACT TRUNCATED AT 250 WORDS)

GABAA receptor subunit mRNA levels are differentially influenced by chronic FG 7142 and diazepam exposure.

Levels of mRNA for the alpha 1, gamma 2 and beta 1 subunits of the GABAA receptor complex were examined in rats maintained on a chronic, continuous schedule of exposure to the benzodiazepine inverse agonist FG 7142. The effect of chronic exposure to the benzodiazepine agonist diazepam was also examined on levels of gamma 2 subunit mRNA. FG 7142 (2 mg/ml of 100% dimethyl sulfoxide (DMSO) or vehicle (100% DMSO) was administered continuously for 8 days in the right ventricle via an osmotic minipump. At the end of the eighth day of exposure, the brain was removed and cerebral cortex, cerebellum and hippocampus were dissected and mRNA prepared from each region. Levels of GABAA alpha 1 and gamma 2 subunit mRNA were examined by Northern blot analysis with cDNA probes specific for these subunits. A significant increase in alpha 1 mRNA was measured in both cortex and hippocampus, but not in cerebellum, of rats chronically exposed to FG 7142 relative to vehicle-treated rats. A significant increase in gamma 2 subunit mRNA in cortex was also evident in drug-treated rats; however, no change in gamma 2 subunit mRNA was observed in either the hippocampus or cerebellum. Examination of GABAA beta 1 subunit mRNA by solution hybridization using a beta 1 riboprobe revealed no effect of chronic FG 7142 treatment on this subunit in either cortex, hippocampus or cerebellum. In rats chronically exposed to diazepam (21 days via silastic implants), levels of gamma 2 subunit mRNA were significantly decreased in cortex, but not changed in either hippocampus or cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadal status and pubertal age influence the responsiveness of the benzodiazepine/GABA receptor complex to environmental challenge in male rats.

Pubertal age and gonadal status in male rats influenced the functional response of the benzodiazepine (BZD)/GABA receptor chloride channel complex to an environmental challenge, i.e., encountering a stranger in familiar versus an unfamiliar environment. Chloride (Cl-) enhancement of [3H]flunitrazepam [( 3H]Flu) binding was facilitated by exposure to an unfamiliar environment relative to that measured in response to a familiar environment in gonadally intact adult rats but not in prepubertal rats (28 days). Enhancement of [3H]Flu by Cl- at 28 days was not differentially affected by the two environments and did not differ from that measured in non-handled control rats at this age. While a differential effect of the two environments on Cl- enhancement of [3H]Flu binding was also present in adult rats castrated as juveniles (at 19 days), the relationship of environmental challenge to Cl- facilitation was the reverse of that measured in intact adult rats. In addition. GABA-mediated 36Cl- uptake was facilitated in intact adult rats tested in either the familiar or unfamiliar environment relative to non-handled intact adult rats. This change in GABA-gated chloride channel function in response to environmental challenge, however, was not present in adult rats castrated as juveniles nor in 28-day-old rats. Therefore, both pubertal age and gonadal status influenced the responsiveness of the BZD/GABA receptor complex to environmental challenge. These findings suggest that pubertal development and adult hormonal status are important determinants of the functional responsiveness of the BZD/GABA receptor complex to environmental challenge.

Gestational exposure to diazepam increases sensitivity to convulsants that act at the GABA/benzodiazepine receptor complex.

Experiments examining seizure sensitivity were conducted on adult male offspring exposed to diazepam at 1.0 or 2.5 mg/kg per day in utero over gestational days 14-20. Threshold dosages to facial clonus, myoclonic jerk, clonic seizure, and extensor tonus were determined via i.v. infusion of bicuculline, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), picrotoxin, pentylenetetrazol, caffeine and strychnine. Relative to uninjected and vehicle-exposed adult male offspring, prenatal diazepam administration reduced the threshold for bicuculline- and DMCM-induced facial clonus and myoclonic jerk by 40-50%. The threshold dosages to facial clonus, myoclonic jerk and clonic seizure from picrotoxin infusion were similarly reduced in animals exposed to diazepam in utero. In contrast, seizure thresholds to pentylenetetrazol, caffeine and strychnine were not affected by early developmental exposure to diazepam. In parallel biochemical studies, an increased sensitivity to the antagonistic effects of bicuculline methiodide on gamma-aminobutyrate (GABA)-stimulated chloride influx was observed in cortical synaptoneurosomes from adult male progeny of diazepam-treated dams. The results are interpreted to reflect a long-lasting alteration in the function of the GABA/benzodiazepine receptor complex by prenatal diazepam exposure that is manifest at the behavioral and neurochemical level in a pharmacologic specific manner.

Experience influences environmental modulation of function at the benzodiazepine (BZD)/GABA receptor chloride channel complex.

Function of the benzodiazepine (BZD)/GABA receptor chloride channel complex was selectively altered by specific aspects of an environmental challenge, i.e. encountering a stranger in a familiar versus an unfamiliar environment. Chloride (Cl-) enhancement of [3H]flunitrazepam [( 3H]flu) binding was facilitated in rats tested in an unfamiliar environment relative to that in rats tested in a familiar environment. Basal [3H]flu binding (binding in the absence of NaCl) also was greater in rats tested in the unfamiliar environment than in rats tested in the familiar environment, and Scatchard analysis of [3H]flu binding indicated that increased [3H]flu binding in the unfamiliar environment reflected an increase in both binding affinity and maximal binding capacity. In addition, both the sensitivity of [3H]flu binding to Cl- and the affinity of BZD recognition sites were decreased in handled control rats relative to non-handled control rats as well as to environmentally-challenged (prehandled) rats, suggesting that the experience of daily handling as well as familiarization with the environment modulates function at the BZD/GABA receptor complex. GABA-mediated 36Cl- uptake was facilitated by testing in either the familiar or unfamiliar environment relative to that measured in non-handled control rats. Thus, changes in GABA-gated chloride channel function may reflect a more fundamental response of this complex to challenging situations. These findings suggest that components of the BZD/GABA receptor complex are differentially influenced by specific aspects of an environmental challenge. Furthermore, function at the BZD recognition site/chloride channel component of this receptor complex is influenced by both repeated and single exposure to specific environments.

Sexually dimorphic influence of prenatal exposure to diazepam on behavioral responses to environmental challenge and on gamma-aminobutyric acid (GABA)-stimulated chloride uptake in the brain.

Early developmental exposure to diazepam (DZ) via administration of the drug to the pregnant rat (1.0 or 2.5 mg/kg) over gestational days 14 to 20 altered both behavior of adult progeny on two tests of anxiety and function of the benzodiazepine/gamma-aminobutyric acid (GABA) receptor complex (a neural substrate of anxiety-related behavior) in a sexually dimorphic manner. Adult male rats (60-90 days) exposed in utero to DZ spent significantly more time on the open arm of the elevated plus-maze than male rats exposed to vehicle, whereas plus-maze performance in female rats was unaffected by the early drug exposure. Similarly, early exposure to DZ markedly altered environment-specific social interaction in male rats, leading to increased social interaction in the unfamiliar environment and decreased social interaction in the familiar environment. Social interaction in adult female rats is not normally environment-specific; however, female rats exposed in utero to DZ at 2.5 mg/kg demonstrated a significant effect of the novel environment on social interaction, thus responding like unmanipulated male rats. The sensitivity of GABA-mediated 36chloride uptake to GABA was enhanced in synaptoneurosomes from male rats exposed in utero to DZ at 2.5 mg/kg and early exposure to either dose of DZ prevented the facilitative effect of DZ added in vitro on GABA-mediated chloride uptake. Function of the receptor complex was not altered in female rats by early DZ exposure. Thus, perinatal insults at the molecular level may underlie gender-related behavioral disorders in the young adult.

Gonadal hormones during puberty organize environment-related social interaction in the male rat.

This study examined the role of gonadal androgens during puberty on the development of environment-related social interaction (SI) in male rats. SI in an unfamiliar environment versus SI in a familiar environment was evaluated in young adult rats as a function of sex and gonadal status. Intact male rats at 60 days of age exhibited a differential response to the two environments, whereas SI in intact female rats at 60 days was equivalent in the two environments. Furthermore, male rats castrated as juveniles and tested for SI at 60 days displayed a pattern of environment-related SI similar to SI in intact adult female rats. This effect of juvenile castration on SI in male rats was prevented by chronic exposure to testosterone propionate (TP) over Days 30 through 60. SI in male rats castrated in adulthood, on the other hand, was not altered either 2 or 4 weeks postcastration. The results from this study indicate that pubertal secretions of gonadal androgen(s) are necessary for the development of environment-related SI in male rats. In contrast, secretions of gonadal androgens in adulthood do not appear to be critical for the continued expression of environment-related SI, as suggested by the observation that environment-related SI in male rats remains unchanged by castration in adulthood.

Developmental influence of gonadal function on the anxiolytic effect of diazepam on environment-related social interaction in the male rat.

This study examined the anxiolytic action of diazepam (DZ) on environment-induced changes in social interaction (SI) in rats as a function of pubertal age. SI in pairs of male rats was measured in a familiar and an unfamiliar environment. Control (gonadally-intact) male rats received daily injections of vehicle or DZ (0.5 or 1.0 mg/kg) for 5 consecutive days and then were tested for SI at 28, 35, or 60 days of age. A differential impact of the two environments on SI was evident in control rats at 35 and 60 days, but not at 28 days. However, DZ modified SI only at 60 days. Based on these findings, the influence of gonadal androgens on the development of DZ modulation of SI was then evaluated. The anxiolytic action of DZ on SI was present in precocious rats tested at 35 days and in rats castrated in adulthood. These results suggest that gonadal androgens are necessary for the development of modifiability by DZ of SI, but not for the anxiolytic effect of DZ in the adult rat. Furthermore, while castration of rats as juveniles (day 19) altered the effect of DZ on SI in adult rats, testosterone replacement during puberty (days 30 to 60) reinstated the ability of DZ to modify SI. The results of these studies demonstrate that gonadal function during puberty is necessary for the development of specific neural systems underlying SI in the adult. These neural systems, perhaps unlike those underlying environment-specific SI at younger ages, are influenced by DZ. Finally, gonadal androgens appear to exert an organizational influence but not an activational effect on DZ modulation of SI.

Pubertal-related changes influence the development of environment-related social interaction in the male rat.

The influence of pubertal age on the differential effect of a familiar versus an unfamiliar environment on social interaction (SI) in pairs of male rats was evaluated. The decrease in SI induced by the unfamiliar environment in adult rats is considered an anxiety-related response. Intact male rats and male rats castrated at 19 days were tested for SI at 28, 35, and 60 days of age. The results revealed that in the intact rats, decreased SI in an unfamiliar environment was evident at 35 and 60 days but not at 28 days. The behavioral composition of the environment-induced response at 35 days was different from that measured at 60 days. Prepubertal castration prevented the decrease in SI in the unfamiliar environment at both 35 and 60 days. This study demonstrated the emergence with the onset of puberty of a specific behavioral response to an anxiogenic condition. Furthermore, the development of this environment-related behavioral response was influenced by gonadal secretion(s), suggesting the importance of gonadal function to the emergence of this response.

Changes in macronutrient selection as a function of dietary tryptophan.

It has been hypothesized that the serotonergic system is involved in the regulation of carbohydrate and/or protein intake. Tests of this hypothesis using added dietary tryptophan and diets varying in the ratio of carbohydrate/protein resulted in depressed intakes of high carbohydrate/low protein diets, elevated intakes of low carbohydrate/high protein diets, and a reduction of total caloric intake. The present studies gave rats increased options for adjusting to added tryptophan by providing them with separate sources of protein, carbohydrate, and fat. The results showed the expected decrease in carbohydrate intake, but also increases in fat intake and, to a lesser extent, protein intake. Total caloric intake was conserved. Hypothalamic concentrations of serotonin and 5-hydroxyindole acetic acid indicated increased activity of the serotonergic system. These results lend support to serotonin's involvement in nutrient selection, in that carbohydrate consumption decreased in response to tryptophan loading, but indicate that other nutrients may also be affected. Given the option of altering fat intake, the animals maintained a constant caloric intake despite the reduction of carbohydrate consumption.