Immunoreactive vasopressin and oxytocin: concentration in individual human hypothalamic nuclei.

Individual hypothalamic nuclei were microdissected from brain tissue of ten human subjects who had died suddenly while in apparent good health. Appreciable amounts of vasopressin and oxytocin immunoreactivity were found by specific radioimmunoassay in six hypothalamic nuclei including supraoptic and paraventricular nuclei. Vasopressin and oxytocin are presumed to be synthesized in supraoptic and paraventricular nuclei for axonal transport to the posterior pituitary for storage and release. Vasopressin and oxytocin in other hypothalamic nuclei may be a part of this system of neurosecretion or may serve some other function.

The ACTH-releasing hormone of the hypothalamus requires a co-factor.

Gel filtration of an extract of rat median eminence tissue in 0.1 N HCl on Sephadex G-25 separated two peaks, both with feeble ACTH-releasing activity. Full activity of the extract was regained when both peaks were recombined. This observation suggests that the ACTH-releasing hormone of the hypothalamus requires a co-factor for activity.

Characterization of tissue and releasable molecular forms of somatostatin-28[1-12]-like immunoreactivity in rat median eminence.

The main purpose of the present study was to characterize the tissue and releasable molecular forms of somatostatin-28[1-12]-like immunoreactivity (S-28[1-12]LI) in rat median eminence (ME) fragments and to compare them with somatostatin-14-like immunoreactivity (S-14 LI) forms. Acetic acid extracts of ME were fractionated on Sephadex G-50 columns (in 6 M urea). The column eluate was monitored for S-28[1-12] LI by RIA with antibody R21 which detects S-28[1-12], S-28, and higher molecular weight forms of S-28[1-12] LI, but not S-14. The S-14 LI RIA utilized recognizes S-14, S-28, and prosomatostatin (pro-S). Rat ME contained 221 +/- 25 pmol S-14 LI/mg protein and 407 +/- 51 pmol S-28[1-12] LI/mg protein. By gel filtration S-14 LI was resolved into three peaks corresponding to S-14, S-28, and a higher mol wt form (14,000) corresponding to pro-S. S-28[1-12] LI consisted of at least five forms corresponding to pro-S, S-28, S-28[1-12], a form which represented pro-S without the S-14 sequence, and a form slightly smaller than S-28[1-12]. Pools of 20 ME incubated in 56 mM K+ solution showed 4.6-fold Ca++-dependent release of S-14 LI and 4-fold release of S-28[1-12] LI. Gel chromatographic analysis of the released material showed all three tissue S-14 LI forms and each of the tissue S-28[1-12] LI forms. HPLC analysis and RIAs further confirmed the release of S-14, S-28, S-28[1-12], and the S-28[1-12] LI form smaller than S-28[1-12]. These data suggest the presence of at least six molecular forms of somatostatin in ME. The release of this large number of peptides, presumably from mature secretory granules in ME in response to depolarization, suggests that they are products of the normal posttranslational processing of pro-S.

Immunoreactive growth hormone-releasing hormone in rat placenta.

It has been shown that immunoreactive and biologically active GH-releasing hormone (GHRH)-like material is present in rat placenta. To investigate the role of placental GHRH, we measured it in human and rat placenta of different gestational stages, using specific RIA systems. GHRH and somatostatin contents in median eminence, pituitary GH contents, and plasma GHRH levels were also quantified in rats. Immunoreactive GHRH was detectable in rat placenta [13 days of gestation, 1.4 +/- 0.4 (+/- SD); 16 days, 1.4 +/- 0.2; 20 days, 1.7 +/- 0.4 ng/g] but not in human placenta (less than 0.06 ng/g in both full-term and mid-term placenta). GHRH concentrations in rat placenta did not change significantly during pregnancy, but total contents increased progressively in relation to placental growth. GHRH and somatostatin contents in median eminence of pregnant rats were not different from those of control female rats. In contrast, rat pituitary GH contents in pregnant rats were significantly lower than those of control female rats. Immunoreactive GHRH was not detectable in plasma of either pregnant rats or nonpregnant rats. Molecular sieve chromatography revealed two peaks of immunoreactive GHRH in rat placental extracts: a major peak eluted in the position of synthetic rat GHRH and a minor peak in the higher molecular weight region. In contrast, a single peak in the position of rat GHRH was observed in rat median eminence extracts. Detection of immunoreactive GHRH in rat placenta but not in human may suggest that the mechanism of GHRH gene expression in placenta is species specific. Failure of detection of immunoreactive GHRH in rat maternal circulation suggests that placental GHRH may not affect the maternal hypothalamic pituitary axis. Presence of high molecular weight materials of immunoreactive GHRH in rat placenta but not in median eminence suggests that posttranslational processing of the GHRH precursor molecule may be different in the two organs. Placental GHRH may have a paracrine function or may be secreted into fetal circulation and contribute to fetal growth.

Neurotensin neurons in the rat hypothalamus: an immunocytochemical study.

Neurotensin was localized in the hypothalamic tissues of adult Sprague-Dawley rats by immunoperoxidase techniques. Visualization of perikarya was greatly enhanced by intraventricular administration of colchicine. Many perikarya containing neurotensin-like immunoreactivity were seen in the medial preoptic area, the periventricular hypothalamus, the parvocellular portion of the paraventricular nucleus, the arcuate nucleus, and the lateral hypothalamus in the perifornical area. There were moderate numbers of cell bodies in the ventral portion of the anterior hypothalamus, the dorsomedial nucleus, and the posterior hypothalamus. No positive cells were seen in the suprachiasmatic, ventromedial, or mammillary nuclei. Reactive fibers were generally distributed in the same regions as cell bodies. Additional dense collections were seen in the lateral part of the zona externa of the median eminence, the pituitary stalk, the posterior mammillary nucleus, and the most lateral portions of the hypothalamus at the medial edge of the crura cerbri. There were smaller numbers of fibers found in the pre-mammillary and posterior hypothalamic nuclei and the posterior pituitary gland. These results indicate that the neurotensin system in the hypothalamus is very extensive and complex, as it is in many other brain regions. Neurons and fibers are found in many hypothalamic areas, including projections to the hypophysial portal system in the median eminence, suggesting that neurotensin may affect neuroendocrine mechanisms at several levels, including the anterior pituitary gland.

Releasing factors in the circumventricular organs in the rat brain.

With a recently developed microdissection technique, four circumventricular organs were removed from the rat brain, and their contents of LHRH and TRH were measured. The subfornical organ, the organon vasculosum lamia terminalis, the subcommissural organ, the area postrema all contained significant quantities of both releasing factors. The concentration of LHRH in the organon vasculosum (OVLT) was 14 ng/mg protein, 58% of that found in the median eminence. The concentration of LHRH in the remaining circumventricular organs ranged from 4.2 to 10.2 ng/mg protein. The concentration of TRH in these structures, however, was considerably lower, ranging from 0.7 to 1.8 ng/mg protein. The concentrations of LHRH and TRH in the tissue immediately adjacent to the organon vasculosum were nearly 50 times and 4 times less, respectively, than the concentration of these two releasing factors within the OVLT itself.

Localization of luteinizing hormone-releasing hormone in the preoptic area and hypothalamus of the rat using radioimmunoassay.

To determine the localization of luteinizing hormone-releasing hormone (LHRH), five brains from adult male rats were serially sectioned in a cryostat at - 10 C in either the frontal, horizontal or sagittal planes. Acetic acid-ethanol extracts of each section were assayed for LHRH using radioimmunoassay (RIA) and in some cases using bioassay as well. Approximately 0.2 ng LHRH was concentrated in medial basal preoptic (MB-PO) tissue overlying the rostral portion of the optic chiasm. This LHRH appears to be associated with the organum vasculosum of the lamina terminalis and/or adjacent neural tissue. Uniform, low levels of LHRH were detected in hypothalamic tissue between the preoptic area (POA) and arcuate-median eminence (ARC-ME) region. In the ARC-ME region 2.7 ng of LHRH were concentrated primarily in the median eminence. The lateral distribution of LHRH in the ARC-ME region extended beyond the median eminence into tissue corresponding to the lateral aspect of the ventromedial nucleus. Concomitant bioassay and RIA determinations of LHRH were highly correlated. Of the sections bioassayed, only those sections containing LHRH released FSH. These results confirm the presence of LHRH in the POA and in the rostral hypothalamus of the rat brain. The possible significance of LHRH in the POA for the regulation of LH release is discussed.

Biosynthesis of thyrotropin-releasing hormone in organ cultures of the guinea pig median eminence.

Guinea pig median eminence tissue was put into organ culture for periods up to 13 days. The cultured tissue was examined in 3 ways at various ages: a) morphologically, by transmission electron microscopy and light microscopic autoradiography; b) biochemically, by determination of the ability of the cultures to accumulate [3H]L-proline and to incorporate this isotope into [3H]TRH; and c) by bioassay, in which the content of TRH in tissue and culture medium was determined by the ability of extracts of both to release radioimmunoassayable TSH from rat hemi-pituitaries incubated in vitro. It was found that the cultures exhibited a high degree of preservation of ependymal cell morphology and a sustained ability to accumulate [3H]L-proline over the entire 13-day time course. Neuronal elements showed a progressive degeneration with time in culture, and the ability of the culture to produce [3H]TRH was lost concomitantly with the loss in neuronal integrity.

The decrease in hypothalamic dopamine secretion induced by suckling: comparison of voltammetric and radioisotopic methods of measurement.

Previous in situ voltammetric microelectrode measurements of median eminence dopamine release during mammary nerve stimulation of anesthetized lactating rats revealed a transient (1-3 min) 70% decline of dopamine concentrations. This dopamine was believed to be destined for secretion into the hypophysial portal circulation, but direct experimental support for this supposition was lacking. Thus, in the present study, [3H]dopamine release into brief sequential samples of hypophysial portal blood was compared with dopamine release in the median eminence measured by voltammetry. Lactating female rats were urethane anesthetized, and the median eminence pituitary region was exposed. [3H]Tyrosine was injected into a jugular cannula (100 microCi) followed by continuous infusion (5 microCi/min). In a preliminary experiment, this regimen produced a steady state level of [3H]dopamine in the portal blood within 45 min. In subsequent experiments, portal blood was collected as sequential 3-min samples, and electrochemical sampling from a microelectrode placed in the median eminence occurred at 1-min intervals. Electrochemical current resulting from the oxidation of dopamine in the medial median eminence was unvarying throughout the 75-min experiment in control rats (n = 4) and during the 30-min control period preceding mammary nerve stimulation in the other group (n = 4). These results were parallel by [3H] dopamine levels in portal blood during the same periods of time. All animals showed simultaneous decreases in oxidation current and [3H]dopamine levels within 1-4 min after initiation of mammary nerve stimulation (respectively, 35 +/- 7% and 62.5 +/- 5.9%, mean +/- SEM). Significant increases in oxidation current, taking the form of brief 2- to 6-min pulses began within an average of 18.5 min after initiation of stimulation. Similar increases in [3H]dopamine levels in portal blood were also observed. These and earlier results demonstrate that mammary nerve stimulation (and by extension, suckling) induces a momentary, but profound, decrease in hypothalamic dopamine secretion which precedes or accompanies the rise in PRL secretion evoked by the same stimulus.

Immunohistochemical identification of a novel substance with human growth hormone-like immunoreactivity in rat brain.

Through use of an antiserum directed against hGH, an immunoreactive hGH-like material has been identified in the rat brain by peroxidase immunohistochemistry. Peroxidase-positive material was found in beaded, neuronal fibers in the external zone of the median eminence, lateral septum, and organum vasculosum of the lamina terminalis and was unaffected by prior hypophysectomy. After pretreatment with intraventricular colchicine, numerous immunoreactive neuronal cell bodies were visualized within the parvocellular medial division of the paraventricular nucleus, periventricular nucleus, dorsomedial nucleus, lateral hypothalamus, and preoptic area. Immunohistochemical staining was completely abolished by preincubation of the antiserum with 10(-6) M hGH, the 20,000 mol wt variant of hGH. hGH dimer, core peptide 20-64/135-167, proteolytically derived hGH fragments 1-134 and 147-91, and human placental lactogen. There was no diminution in staining after preincubation with hGH N-terminal fragment 1-43, hGH C-terminal fragment 171-191, rat GH, rat or human PRL, and numerous other neuropeptides and anterior pituitary hormones. Bilateral electrolytic ablation of the paraventricular nucleus area caused a loss of immunostaining in the median eminence. These results indicate the presence of a hitherto undescribed intrinsic neuronal system in rat brain that contains a substance bearing immunological similarity to the midportion of the hGH molecule and to human placental lactogen. It is proposed that this substance is part of a tuberoinfundibular neuronal system deriving from the parvocellular medial division of the paraventricular nucleus-immunoreactive perikarya and may, therefore, be involved in hypophysial regulation. It may also act as a neuromodulator of limbic lobe structures and other hypothalamic regions.

Effect of hypophysectomy on immunocytochemically demonstrated gonadotropin-releasing hormone in the rat brain.

The objective was to determine the effect of hypophysectomy on the store of gonadotropin-releasing hormone (GnRH) in certain parts of the brain as revealed by immunocytochemistry. The antiserum used was prepared against synthetic GnRH conjugated with limpet hemocyanin. No change was observed in the store of GnRH in the organum vasculosum of the lamina terminalis or in the cephalic segment of the median eminence GnRH was depleted severely from the central and caudal (junction with the infundibular stem) segments of the median eminence. GnRH was not found in the axons of magnocellular neurons that regenerate during repair of the median eminence-pituitary stalk after hypophysectomy.

Oxytocin content of microdissected areas of rat hypothalamus.

Oxytocin content has been measured by radioimmunoassay in microdissected hypothalamic nuclei. Equal concentrations of oxytocin were found in the supraoptic and the paraventricular nuclei, indicating that both are major sources of the hormone. The concentration of oxytocin in the median eminence was more than three times that in either the supraoptic or the paraventricular nuclei, and significant amounts of oxytocin were also found in the arcuate nucleus and in tow anterior hypothalamic nuclei.

Distribution of gonadotropin-releasing hormone in the rat brain as observed with immunocytochemistry.

The distribution of gonadotropin-releasing hormone (GnRH) was studied in the brain of adult female rats with three immunocytochemical techniques using antisera to unconjugated synthetic GnRH and to GnRH conjugated with limpet hemocyanin. GnRH was found in nervous tissue surrounding blood vessels of the organum vasculosum of the lamina terminalis. In the median eminence it occurred in nervous tissue associated primarily with the tuberoinfundibular sulci throughout their extent. Cephalic to the pars tuberalis GnRH often spread across the median eminence from sulcus to sulcus. Caudally, with widening of the median eminence, GnRH occurred dorsal to the tuberoinfundibular sulci, and especially in the external lamina medial to the sulci. A broad median zone of the median eminence was rather free of GnRH. GnRH was most concentrated in the region of continuity between the dorsolateral walls of the infundibulum and floor of the third ventricle where the tuberoinfundibular sulci are deep. Caudal to the infundibulum GnRH was disposed in a flat zone through the cephalic portion of the floor of the mammillary recess. In the median eminence GnRH appeared to be located in axons that terminated there. The amount of demonstrable GnRH varied significantly from rat to rat. The distributions of GnRH as revealed by use of antisera to unconjugated and conjugated GnRH were essentially the same. The apparent order of sensitivity of the immunocytochemical methods was: the peroxidase-antiperoxidase (PAP) (Sternberger et al.) procedure greater than the immunoglobulin-enzyme bridge (Mason et al.) procedure smaller than the conjugated antibody (Nakane and Pierce) procedure.

Reset of feedback in the adrenocortical system: an apparent shift in sensitivity of adrenocorticotropin to inhibition by corticosterone between morning and evening.

There is evidence in man and rats that higher circulating levels of glucocorticoids are required to normalize basal unstimulated ACTH levels at the peak of the circadian rhythm than at the trough. To explore this phenomenon, we tested the inhibitory effect of constant levels of corticosterone on plasma ACTH in the morning (AM) and evening (PM) in young male rats implanted with fused pellets of corticosterone-cholesterol at the time of adrenalectomy (ADX+B) and studied 5 days later. There was a marked shift of the plasma corticosterone-ACTH inhibition curve to the right between AM and PM, demonstrating that the efficacy of corticosterone feedback inhibition of ACTH is less in the PM. Comparison of plasma ACTH and corticosterone levels during 24 h in sham-adrenalectomized rats (SHAM-ADX), adrenalectomized rats (ADX), and ADX+B revealed constantly low ACTH in SHAM-ADX, constantly high ACTH in ADX, and biphasic ACTH levels in ADX+B. Corticosterone levels were biphasic in SHAM-ADX and were constant in the other two groups. These results again showed a shift in corticosterone feedback efficacy as a function of the time of day and also suggested that basal ACTH secretion is maintained in the low normal range in intact rats because of the marked diurnal rhythm in corticosterone. The sensitivity of the pituitary ACTH response to exogenous CRF did not change between AM and PM in either intact or ADX+B showing that the shift in feedback sensitivity to corticosterone does not reside in the pituitary. The response of the entire adrenocortical system to histamine stress was shown to be equivalent in both the AM and PM, suggesting that feedback sensitivity of the entire system to corticosterone does not change as a function of the time of day. We conclude from these results that there is an apparent diurnal change in ACTH sensitivity to corticosterone feedback that can be defined operationally as reset. We believe that the site of feedback being tested shifts solely from the pituitary in the AM (at the nadir of the rhythm) to the brain and the pituitary in the PM (at the peak of the rhythm). The lack of the normally high transients of corticosterone that occur in SHAM-ADX rats results in increased brain drive of the pituitary in ADX+B.

Anterior pituitary type II thyroxine 5'-deiodinase activity is not affected by lesions of the hypothalamic paraventricular nucleus which profoundly depress pituitary thyrotropin secretion.

Bilateral destruction of the hypothalamic paraventricular nuclei (PVN) produced a profound depression of plasma TSH and the median eminence TRH concentration in hypothyroid rats. Anterior pituitary type II iodothyronine 5'-deiodinase (5'-D) activity was consistently lower but not significantly different in sham- and PVN-lesioned rats. Treatment with suboptimal replacement doses of 0.15 and 0.75 micrograms T4/100 g BW.day produced a graded depression of plasma TSH in the PVN (P less than 0.02), but not in the sham (P greater than 0.8) groups. Adenohypophyseal 5'-D was depressed in both sham and PVN groups by the highest T4 dose. Plasma T4 was much lower in PVN than in sham rats given comparable doses of T4 (P less than 0.001), but plasma T3 was not significantly different. This suggests that an increase in peripheral T4 metabolism was produced by PVN lesions. Our data indicate that changes in adenohypophyseal 5'-D activity are not responsible for the decrease in plasma TSH in PVN-lesioned rats and that neither the PVN nor endogenous TRH plays a significant role in the regulation of anterior pituitary 5'-D activity.

Distribution of releasing factors, biogenic amines, and related enzymes in the bovine median eminence.

The bovine median eminence was dissected into eight different subdivisions: rostral, anterior internal, anterior external, middle external medial, middle external lateral, middle internal medial, middle internal lateral, and caudal. Thyrotropin-releasing hormone (TRH) was found in the highest concentrations in the middle external medial and lateral subdivisions; luteinizing hormone-releasing hormone (LHRH) was concentrated in the middle external lateral and anterior internal subdivisions. Among the various neurotransmitters and enzymes assayed, only dopamine and choline acetyltransferase were present in highest concentrations in the same subdivisions of the bovine median eminence found to be rich in TRH and LHRH. The distributions of norepinephrine, dopamine-beta-hydroxylase, serotonin, tryptophan hydroxylase, phenylethanolamine-N-methyltransferase, glutamic acid decarboxylase, and histamine appeared to correlate poorly with the major distributions of TRH and LHRH. These findings suggest that at the level of the median eminence, central neuroendocrine regulation of TRH and LHRH release may involve an interaction only with dopamine and acetylcholine.

Serotonergic innervation of the rat pituitary intermediate lobe: decrease after stalk section.

Serotonin (5-HT)-containing nerve fibers and terminals, but not cell bodies, have been demonstrated by light and electron microscopic immunocytochemistry in the intermediate lobe of the rat pituitary. Seven days after pituitary stalk transection, 5-HT immunoreactive fibers disappeared almost entirely from the intermediate lobe. Intermediate lobe 5-HT and 5-hydroxyindoleacetic acid concentrations, measured by high pressure liquid chromatography coupled to electrochemical detection, fell to 56% and 62% of control, respectively. There were no changes in 5-HT concentrations in anterior or posterior lobes of the pituitary or in the median eminence. These findings, and the failure of sympathectomy to cause a drop in pars intermedia 5-HT, indicate that in the intermediate lobe of the pituitary neuronal 5-HT originates in cells situated in the central nervous system.

Characterization of gamma-melanotropin-like immunoreactivity and its secretion in an adrenocorticotropin-producing mouse pituitary tumor cell line.

gamma MSH, a putative hormone in the N-terminal region of the ACTH/beta-endorphin (beta-EP) precursor protein, was studied by RIA with an antiserum against gamma 3MSH in ACTH-producing mouse pituitary tumor cells, AtT-20/D16v. Serial dilution of the culture medium or the cell extract gave parallel lines to the standard curve in the RIA for gamma MSH. Rat median eminence extracts enhanced the release of gamma MSH-like immunoreactivity (gamma MSH-LI) concomitant with ACTH-like immunoreactivity (ACTH-LI) and beta-EP-like immunoreactivity (beta-EP-LI). Dexamethasone suppressed the release of gamma MSH-LI as well as ACTH-LI and beta-EP-LI. Gel exclusion chromatography of the culture medium and the cell extract has revealed that gamma MSH-LI consists of two peaks; one eluted near the elution position of beta-lipotropin and the other near the elution position of beta-EP. There was no peak corresponding to the elution position of synthetic gamma 3MSH. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has demonstrated that gamma MSH-LI migrated at five positions with molecular weights of 31K, 21-23K, 16-17K, 13-14K, and 3.8K, respectively. The 31K gamma MSH coincided with the migration position of 31K ACTH of 31K beta-EP, and 21-23K gamma MSH coincided with the position of 21-23K ACTH on SDS-PAGE. The 16-17K gamma MSH coincided with the mouse 16K fragment (reported by Eipper and Mains) of ACTH-beta-lipotropin precursor protein in the migration in SDS-PAGE and in immunoreactivity to anti-gamma MSH antiserum. [3H]Glucosamine was incorporated into 16K, 13K, and 3.8K gamma MSH. These results suggest that AtT-20/D16v cells produce gamma MSH-LIs with molecular weights of 31K, 21-23K, 16-17K, 13-14K, and 3.8K, and they are secreted concomitantly with ACTH-LI and beta-EP-LI.

Estrogen decreases rat hypothalamic proopiomelanocortin messenger ribonucleic acid levels.

Ovariectomy and estrogen (E) or E plus progesterone treatment has previously been shown to alter both hypothalamic content and portal plasma levels of beta-endorphin. To determine if these changes were accompanied by changes in beta-endorphin synthesis, we used a RNA dot blot method to quantify proopiomelanocortin (POMC) mRNA levels in the arcuate nucleus-median eminence region of rats. Animals were bilaterally ovariectomized, implanted with Silastic capsules containing E or oil, and killed 1 or 3 days after implantation. Total nucleic acid was isolated from dissections of the arcuate-median eminence by proteinase-K/sodium dodecyl sulfate/phenol extraction, and POMC mRNA was quantified by dot blot analysis. Although 1 day of E treatment had no effect on hypothalamic POMC mRNA levels, 3 days of E treatment caused a significant reduction of approximately 40% of POMC mRNA levels relative to oil controls in two replicate experiments. These results suggest that the decreases in hypothalamic POMC peptide levels after E administration reported previously may be due to a decrease in POMC peptide biosynthesis resulting from a decrease in hypothalamic POMC mRNA.

Tyrosine hydroxylase in the stalk-median eminence and posterior pituitary is inactivated only during the plateau phase of the preovulatory prolactin surge.

This study examined changes in the activity of tyrosine hydroxylase (TH) in the stalk-median eminence (SME) and posterior pituitary (PP) during the preovulatory PRL surge. Immature female rats were injected with PMSG on day 28. Blood PRL levels were low on the morning of day 30, rose to a peak from 1400-1600 h, remained at a lower plateau from 1800-2400 h, and declined to basal levels on the morning of day 31. SME, PP, and striatum were removed from PMSG-treated rats at selected times during the periovulatory period and from age-matched control rats. TH activity was determined in tissue homogenates by a coupled hydroxylation-decarboxylation assay. Apparent Km and maximum velocity values with respect to 6-methyl tetrahydropterine were estimated from substrate saturation curves. The kinetic parameters for TH in either the SME or the PP of control rats were similar at 1100 and 1800 h on day 30. However, the apparent Km in both tissues was significantly lower than that in the striatum. The affinity of TH in the SME and PP was unchanged before and during the peak phase of the PRL surge, reduced significantly during the late plateau, and returned to presurge levels in the morning of day 31. TH activity in the striatum was similar at all times examined. To determine the state of activation of the enzyme, tissue homogenates were preincubated with cAMP, ATP, and magnesium. TH activity in the SME during the peak phase was unchanged by cAMP, and that in the PP was modestly increased. The relatively inactive enzyme in both tissues during the plateau phase was markedly activated by a cAMP-dependent mechanism. The low affinity of striatal TH was greatly increased by cAMP at both times. These data suggest that TH in the SME and PP exists in an activated state most of the time and is transiently inactivated during the plateau phase of the PRL surge. In contrast, TH in the striatum is relatively inactive in the basal state and is not affected by hormonal changes induced by PMSG. We conclude that the peak PRL surge occurs in spite of active dopamine (DA) neurons, suggesting that it is generated by a nondopaminergic mechanism. Decreased TH activity in DA neurons in the SME and PP may prolong the PRL surge during the plateau phase, whereas increased DA activity coincides with the termination of the surge.