Catecholamine synthesis inhibitors acutely modulate [3H]estradiol binding by specific brain areas and pituitary in ovariectomized rats.

Drugs known to alter endogenous levels of catecholamines were administered to adult ovariectomized rats to assess catecholaminergic effects on estradiol (E2) uptake and binding in nuclear and supernatant fractions of pituitary and specific brain regions and on cytoplasmic E2 receptor numbers and affinities. Specific (i.e. diethylstilbestrol-blockable) binding in vivo was measured 1 h after the iv injection of [3H]E2 (1 micrograms/kg). Administration of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine (alpha MPT) 2 h before [3H]E2, to reduce levels of dopamine (DA), norepinephrine (NE), and epinephrine (Ep), decreased total and specific [3H]E2 binding by 36-56% in the nuclear fraction of the anterior pituitary, basal hypothalamus, and anterior hypothalamus. The dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate (DDC), administered 2 h before [3H]E2 to reduce levels of only NE and Ep, increased the total and specific uptake of [3H]E2 by 62-140% in nuclear and supernatant fractions of the anterior pituitary and also increased uptake in several brain areas. In vitro analysis of hypothalamic and pituitary cytoplasms showed that in vivo administration of DDC increased E2 binding. Scatchard analysis showed that DDC increased receptor numbers 18-29%, with no change in the dissociation constant in pituitary cytoplasms. At the same time, plasma PRL levels were reduced by DDC treatment, indicating that DDC had increased DA output. Phenoxybenzamine (a blocking agent at alpha 1 postsynaptic binding sites) and a high dose of clonidine (a pre- and postsynaptic alpha-receptor agonist) did not significantly alter specific uptake in the cell nuclear fraction of any tissue, suggesting that postsynaptic alpha-receptors do not play a major role in modulating [3H]E2 uptake. No drug altered plasma levels of radioactivity. Because alpha-methyl-p-tyrosine and DDC both inhibit synthesis of NE and Ep, it is suggested that their opposite effects on uptake of [3H]E2 are related to their opposite effects on DA output. This interpretation is compatible with our previous observations that DA agonists increase [3H]E2 uptake in brain and pituitary in ovariectomized rats.

Evidence that decreased function of lymphocyte beta adrenoreceptors reflects regulatory and adaptive processes in panic disorder with agoraphobia.

OBJECTIVE: This study was designed to clarify the nature of the reduced function of the peripheral beta adrenoceptor system observed in panic disorder with agoraphobia. The authors hypothesized that this phenomenon reflected a regulatory and adaptive process. METHODS: Lymphocyte beta adrenoreceptor density and affinity, basal lymphocyte cAMP level, and isoproterenol-stimulated cAMP generation were measured in 27 untreated outpatients with panic disorder with agoraphobia and 24 healthy comparison subjects. Lymphocyte beta receptor attributes were again assessed in patients after 4 weeks of double-blind treatment with adinazolam (slow-release form) or placebo. Panic frequency, agoraphobic symptoms, overall anxiety, and improvement with treatment were assessed with standard rating instruments. RESULTS: Multivariate statistics revealed significantly lower beta receptor density and isoproterenol-stimulated cAMP generation in patients than in comparison subjects. beta receptor density tended to normalize after adinazolam but not after placebo. Pretreatment beta receptor density was lower in treatment responders than nonresponders. Patients with mild agoraphobia had lower cAMP responsivity than patients with moderate or severe agoraphobia. CONCLUSIONS: Decreased function of lymphocyte beta receptors in panic disorder with agoraphobia is expressed as both decreased density and decreased cAMP responsivity. This pattern of changes, and the tendency for receptor density to normalize with treatment, is consistent with an active, regulatory process rather than a structural deficit in the beta receptor system. Preliminary clinical findings suggest that these changes may reflect adaptive processes associated with a favorable clinical course in panic disorder with agoraphobia.

Phosphorylation of Ca2+/calmodulin-dependent protein kinase type ii and the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (ampa) receptor in response to a threonine-devoid diet.

The anterior piriform cortex (APC) functions as a chemosensor for indispensable amino acid deficiency and responds to this deficiency with increased activity, as indicated by observations including averaged evoked-potentials and c-fos expression in the APC. Little is known of the intracellular signaling mechanisms that mediate this deficiency-related increase in neuronal excitability, but previous studies have shown effects on intracellular Ca2+ in deficient APC slices in vitro. In the present study we hypothesized that indispensable amino acid deficiency increases intraneuronal Ca2+, resulting in autophosphorylation of calcium/calmodulin-dependent protein kinase type II (CaMKII) in vivo. Results demonstrated that phosphorylation levels of CaMKII (pCaMKII) in APC neurons increased at 20 and 40 min after a single meal of threonine-devoid diet. Phosphorylation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit (GluR1) at the serine 831 (S831) site was modestly increased in the APC in response to a threonine-devoid meal. The GluR1 subunit also showed increased phosphorylation at the 845 (S845) site, suggesting additional signaling mechanisms. Although phosphorylation of CaMKII was sustained, phosphorylation of the GluR1 subunit returned to control levels by 40 min. These effects of amino acid deficiency did not occur throughout the brain as neither CaMKII nor GluR1 showed increased phosphorylation in the neocortex. These findings support the notion that calcium and glutamate signaling in the APC, but not throughout the brain, are triggered during early responses to amino acid deficiency. They also suggest that longer-term changes in APC neurons in response to such a deficiency may be mediated at least in part by CaMKII.

Alpha 2 noradrenoceptors in the anterior piriform cortex decline with acute amino acid deficiency.

The responses of the brain to the amino acid deficiency that occur after eating imbalanced amino acid diets (IMB) have been associated with decreased concentrations of norepinephrine (NE) and cAMP in the anterior piriform cortex (APC), an area essential for the initial feeding responses to amino acid deficiency. In addition, the anorectic responses to IMB were decreased after injections of the alpha 2 agonist, clonidine, and increased after injections of the alpha 2 antagonist, idazoxan, into the APC. Therefore, to study the role of the alpha 2-noradrenergic receptor further in this model, we measured alpha 2-noradrenergic receptor binding in the APC of rats fed two levels of threonine IMB or a low-protein basal control diet. After basal prefeeding for 10 days, rats were given either a mild IMB, a severe IMB, or the basal diet for 2.5 h. The APC, anterior cingulate cortex (AC), ventromedial hypothalamus (VMH), and lateral hypothalamus (LH) were assayed. Binding of [3H]p-aminoclonidine to alpha 2 receptors determined that alpha 2 binding was decreased the most in APC (P < 0.0003). Binding in APC was significantly correlated with food intake in the anorectic response to IMB (P < 0.001). In AC, binding was also significantly decreased, but less dramatically (P = 0.012), and was not correlated with food intake. There were no significant changes in LH or VMH, although alpha 2-noradrenergic binding in VMH tended to decrease with the severe IMB in a pattern similar to APC. Plasma glucose values did not differ after the same feeding protocol. These data support our hypothesis that NE activity in the APC plays a role in initiating the anorectic response to IMB, perhaps via the alpha 2-noradrenergic receptor.

Temporal-spatial pattern of c-Fos expression in the rat brain in response to indispensable amino acid deficiency. II. The learned taste aversion.

Rats rapidly become anorectic when eating an amino acid-imbalanced diet that induces a deficiency of an indispensable amino acid. Recognition of amino acid deficiency is thought to be a function of the anterior piriform cortex. However, the neuronal circuitry underlying the secondary learned aversion to such diets may involve the amygdala. In this study, Fos immunohistochemistry was employed to identify regions of the brain activated during the learned aversion phase of the response to an amino acid-imbalanced diet. c-Fos expression was examined in the brains of rats at intervals from 1 to 6 h after introduction of a diet imbalanced in threonine, a corrected (amino acid-balanced) diet or a basal (low protein) diet. The study has revealed that, within the time frame associated with the learned aversive response, Fos-immunoreactive (Fos-IR) neurons increased selectively in the central nucleus of the amygdala in animals fed a threonine-imbalanced diet. These results suggest a temporal relationship between changes in neuronal activity in the central nucleus of the amygdala and the learned aversion associated with acute amino acid deficiency.

Temporal-spatial pattern of c-fos expression in the rat brain in response to indispensable amino acid deficiency. I. The initial recognition phase.

Rats reduce their food intake after ingestion of a small amount of an amino acid imbalanced (AA-IMB) diet that induces a pronounced amino acid deficiency. Two hours after ingesting a threonine-IMB diet, just when food intake is depressed significantly, the concentration of threonine is decreased in some but not all brain areas. Neural recognition of this decrease in the limiting amino acid is thought to be the first step in the anorectic responses to AA-IMB diets. To identify the regions of the brain that may be activated upon recognition of an AA-IMB diet, we examined the temporal-spatial distribution of Fos immunoreactive neurons at intervals after introduction of either threonine-IMB or control diets. We found that Fos immunoreactivity in the anterior piriform cortex and immediately surrounding areas, along with the infralimbic cortex, was increased selectively early (by 2 h) after introduction of the AA-IMB diet, and remained elevated through 3 h. The anterior piriform cortex is believed to function in neural recognition of amino acid deficiency. Fos immunoreactivity in the AA-IMB group increased over the control diet groups somewhat later in the dorsomedial nucleus of the hypothalamus. We hypothesize that these areas in the rostral forebrain may serve as neural relays in the early phases of the anorectic responses that occur upon recognition of amino acid deficiency.

Neuropeptide Y and somatostatin in the anterior piriform cortex alter intake of amino acid-deficient diets.

Neuropeptides affect food intake via peripheral and brainstem mechanisms, but their roles in mediating feeding via the cerebral cortex have received little attention. The anterior piriform cortex (APC) appears to play a critical role in neuroperception of deficiencies of essential amino acids (AA) and the anorectic response to such deficiencies. The neural circuitry underlying the role of this paleocortex in these events is not understood. We have shown that neurons containing neuropeptide Y (NPY) and somatostatin (SOM) are cytoarchitecturally in positions to relate synaptically to the neurons of the APC which may mediate responses to AA. Thus, we hypothesized that NPY and SOM administered intracortically to the APC would directly affect food intake in a threonine-imbalanced model. We determined that NPY at 1-1.5 nmol decreased intake of the AA-deficient diet for 3 h, with a cumulative effect that extended through 6 h. SOM had a dual effect; at 1 pmol it increased intake of the AA-deficient diet for 3 h; at 2 nmol, SOM decreased intake of the AA-deficient diet for over 9 h, with a cumulative effect that persisted through 12 h. In the first 3 h, intake of animals receiving 1 pmol of SOM differed significantly from those receiving 2 nmol. These results suggest that NPY and SOM affect the cortical circuitry responsible for recognition of deficiencies in nutritionally essential AA, and that the timing of the cortical responses to the peptides may be related to the time course of the anorectic responses.

Peptide fragments released from Phe-caseinomacropeptide in vivo in the rat.

The aim of this study was to investigate the pharmacokinetics of bovine Phe-caseinomacropeptide (Phe-CMP) in the rat after oral administration. This polypeptide was monophosphorylated and mainly nonglycosylated: Phe-CMP-1P. During gastrointestinal digestion and absorption, Phe-CMP-1P was degraded. Intact Phe-CMP-1P and CMP-1P were rapidly released from the stomach. In contrast, partial hydrolysis by pancreatic enzymes was observed. In vitro hydrolysis by brush-border membrane vesicles also indicated that the peptide was degraded. In the blood, "CMP-immunoreactive material" appeared rapidly, reaching a maximum level of 5.5 microg/ml at 60 min.

Effects of threonine injections in the lateral hypothalamus on intake of amino acid imbalanced diets in rats.

Previous work from this laboratory suggests that animals decrease their intake of an amino acid imbalanced diet (IMB), due in part to a drop in the concentration of the dietary limiting amino (DLAA) in the anterior piriform cortex (APC). Administration of the DLAA, but not of a non-limiting amino acid into the APC, blocks the anorectic response to IMB. To our knowledge, the effects of DLAA injections on intake of a diet devoid of the DLAA (DEV), have not been examined in areas outside the APC. We hypothesized that the LH is a potential chemosensory area for DLAA. Our objectives were: (1) to determine whether injections of the DLAA threonine into the lateral hypothalamus (LH) alter intake of a threonine-devoid diet (DEV); and (2) to examine the dose-response effects of threonine injections into the LH on intake of threonine-corrected diet (COR). Administration of threonine into the LH stimulated DEV intake during the first 6 h at the 0.25 and 1-nmol doses by approximately 26 and 24%, respectively. Threonine (0.25, 2.5 nmol) did not alter COR intake at any time during the first 12 h. Our results suggest that: (1) the LH, along with the APC, likely acts as a chemosensory brain area for indispensable amino acids; and (2) both the APC and LH are part of a circuit that is involved in the short term anorectic response to amino acid imbalanced diets.

Inhibition of norepinephrine release in the rat ventromedial hypothalamic nucleus in essential amino acid deficiency.

Effects of dietary amino acid deficiency on interstitial levels of norepinephrine (NE) were assessed in the ventromedial hypothalamic nucleus (VMH). Microdialysates, collected from the VMH, were analyzed using high pressure liquid chromatography with electrochemical detection (HPLC-EC). Ingestion of an amino acid imbalanced diet, which causes a rapid deficiency of the limiting amino acid, induced a significant decrease in the NE concentration from the VMH. The changes in the NE concentration appeared 60 min after diet ingestion and the lowest NE level was observed at 180 min. The present results suggest that ingestion of an amino acid imbalanced diet inhibits NE release in the VMH and support the hypothesis that the VMH plays a role in the integration of signals for the feeding responses to changes in essential amino acid availability.

Liver denervation, 5-HT3 receptor antagonist, and intake of imbalanced amino acid diet.

The serotonin3 receptor antagonist ICS 205-930 (ICS) may act peripherally to attenuate the anorectic response of rats given an imbalanced amino acid (IMB) diet. Rats were divided into four groups: SHAM+saline (sal); SHAM+ICS; total liver denervation (TLD) + sal; and TLD+ICS. Rats were then given a purified basal diet for 16 days. Next, the groups were injected with sal or 9 mg/kg BW of ICS at 0800 h and at 0900 h (lights out) an isoleucine IMB diet was presented. By 12 h postinjection, the food intake (FI) of TLD and SHAM rats receiving ICS was similarly higher (p < 0.02) than sal-injected counterparts whose FI was also similar; BW followed FI. By day 3, the SHAM groups had similar low FI, whereas the FI of the TLD groups was increasing. The above study was repeated with similar results. Liver innervation is not required for ICS attenuation of IMB diet-induced hypophagia. Also, while sal-injected TLD rats show a normal attenuation of consumption of the IMB diet on the first day of exposure, they subsequently consume more of the IMB diet than SHAM rats. The reason for this difference in TLD rats is not clear but may be related to metabolism of the IMB diet or possibly learning.

Decreased lymphocyte beta-adrenoreceptor function correlates with less agoraphobia and better outcome in panic disorder.

Previous studies have demonstrated reduced function of peripheral beta-adrenoreceptors in panic disorder with agoraphobia (PDA). We recently reported that decreased lymphocyte beta-receptor function was associated with milder agoraphobia and better treatment response in PDA. We now report on lymphocyte beta-receptor function in 12 additional patients with PDA. Lower cyclic AMP responses to isoproterenol were significantly correlated with milder agoraphobia and better response to naturalistic treatment. Lower beta-receptor density tended to correlate similarly with agoraphobia and treatment response. These findings further support the hypothesis that decreased peripheral beta-receptor function in PDA reflects an adaptive process associated with greater resistance to agoraphobia and greater capacity for recovery with treatment.

Acetylcholinesterase activity in the brain of rat pups and dams after exposure to lead via the maternal water supply.

Acetylcholinesterase (AChE) activity was measured in 11 brain areas of control and lead-exposed pups and dams. Lead exposure of pups was by way of the milk of dams drinking 0.2% lead acetate solution as drinking water starting at parturition. Of the brain areas studied, the cerebellum showed the greatest increase in weight (11-fold) and AChE activity per brain area (42-fold per cerebellum) from birth to 20 days, and was the only area in the pups in which AChE activity was consistently affected by the treatment. AChE activity in the cerebellum was 13% less at 10 days of age and 8% less at 20 days in lead-exposed than in control rats. This agrees with observations by others that the developing cerebellum is particularly sensitive to the effects of lead, and may be consistent with our previous report of an effect on motor coordination, i.e., slightly delayed appearance of the air righting reflex, in similarly treated pups. At weaning AChE activity in septum and olfactory tubercles was lower in lead-exposed than in control dams by 15% and 29%, respectively, and the increase in activity in the hypothalamus expected in lactating dams was attenuated by the lead exposure. Thus, lead exposure may affect AChE activity in brain areas of dams, as well as in pups, and so the potential for effects of lead exposure on the dam should not be discounted.

Dopaminergic agonists increase [3H]estradiol binding in hypothalamus of female rats, but not of males.

Specific nuclear binding of [3H]estradiol in the hypothalamus was increased by acute dopaminergic treatment in female, but not in male, gonadectomized-adrenalectomized rats. In the female this increase could be blocked by the dopaminergic receptor blocker perphenazine and was noted from 1 to 3 hours after injection of [3H]estradiol. Binding was not different in male and female rats in the absence of dopaminergic treatment. These results suggest that acute dopaminergic stimulation may modulate estradiol binding in neural areas known to be important in endocrine function.

Basolateral and central amygdaloid lesions leave aversion to dietary amino acid imbalance intact.

Expression of c-fos is increased in the central amygdaloid nucleus (CE) of rats ingesting a diet with a severely imbalanced essential amino acid profile (IMB), at a time associated with development of a conditioned taste aversion (CTA). The CE and the basolateral amygdaloid nucleus (BL) both are reported to be involved in the development of CTA. Large amygdaloid lesions involving CE and BL mitigate the normal decrease in intake of IMB; this treatment also impairs CTA to a flavor cue associated with gastrointestinal discomfort. To differentiate their potential roles in aversive responses to IMB, we electrolytically lesioned CE and BL separately. Neither lesion attenuated IMB-induced anorexia, or prevented the avoidance of flavored solutions previously paired with IMB. In contrast, after saccharin-LiCl pairing, CE-lesioned animals showed attenuated CTA to saccharin solution in a two-bottle test. We conclude that neither the CE nor the BL is essential for the reduction of IMB intake, or for CTA associated with IMB. Furthermore, these results suggest that the aversive consequences of IMB intake do not involve gastrointestinal malaise-evoked neurotransmission involving the CE.

Dietary amino acid imbalance and neurochemical changes in three hypothalamic areas.

The impact of feeding imbalanced amino acid diets on monoamine, metabolite and amino acid concentrations was measured in the ventromedial hypothalamus (VMH), lateral hypothalamus (LH) and paraventricular nucleus (PVN). After rats were fed either an isoleucine imbalanced diet, a threonine imbalanced diet, or the appropriate basal or corrected control diets, regional differences were found in neurochemical concentrations. Contrary to our expectations, the limiting amino acid was unchanged in the imbalanced groups, tending to be decreased only in the isoleucine imbalanced-diet group in the PVN. This is the first report that the limiting amino acid was not reduced uniformly in the brain after imbalanced amino acid feeding. In the VMH, norepinephrine (NE) was increased by 22% and 63% in the threonine and isoleucine imbalanced-diet groups, respectively. Since the concentration of NE was affected even before the decrease in feeding, both in the VMH, and, as previously reported, in the prepyriform cortex, the NE system may be involved in very early responses to imbalanced amino acid diets.

Norepinephrine and amino acids in prepyriform cortex of rats fed imbalanced amino acid diets.

Monoamines and amino acids were measured in anterior prepyriform cortex (PPC) and anterior cingulate cortex (CC) of male Sprague-Dawley rats after they were offered basal, imbalanced (IMB) or corrected amino acid diets, limited in threonine (THR) or isoleucine (ILE). In the THR study, brains were taken after 2.5 hr of feeding, when intake of THR-IMB was just depressed. In the ILE study the brains were taken after 3.5 hr on ILE-IMB, a less severely imbalanced ration, before the onset of food intake depression. The PPC has been shown to be involved in the acute response of animals to imbalanced amino acid diets. In the PPC from the IMB diet groups, NE was reduced by 30%, but the other monoamines were unchanged. In CC, an area involved in the adaptive, but not the acute feeding response to imbalanced diets, the monoamines were unchanged in the IMB diet groups. In both studies, in both tissues, the limiting amino acids were decreased in the IMB groups, although the decrease of ILE in the CC failed to reach significance. The remaining indispensable amino acids, added to create the imbalance, were slightly reduced in the THR-IMB group, but not in the ILE-IMB group in both tissues. Thus, the amino acid patterns were altered in the PPC and CC, as they are in whole brains from animals fed imbalanced amino acid diets. These results also suggest that the concentration of NE in the PPC may be associated with the initial food intake response of animals to imbalanced amino acid diets.

Amino acid imbalance, a nutritional model: serotonin3 mediation of aversive responses.

The feeding responses to essential dietary amino acid (AA) disproportion have been useful in nutritional studies. These responses involve first: recognition of the imbalance and second: rejection of the diet, likely via development of a learned aversion. In the rat, we have studied the role of the limiting AA and protein synthesis in the recognition phase by replacement of the limiting AA into a brain area essential for the initial depressed feeding response. We have also reported a reciprocal relationship between serotonin (5HT) activity and intake of imbalanced diets. High doses of the 5HT3 receptor antagonist, ICS 205-930 (ICS; 9 mg/kg IP), restored food intake to 85% or more of control intake. In the present experiments, similar treatment with ICS blocked the classical conditioned taste aversion to a saccharin solution paired with lithium chloride. These results suggest that the increased intake of AA imbalanced diets after ICS may be due to 5HT-mediated blockade of a learned aversion.

Timing and dose of amino acids injected into prepyriform cortex influence food intake.

The effects of time before feeding and dose of dietary-limiting amino acids (DLAA) injected into the prepyriform cortex (PPC) on intake of amino acid-imbalanced diets were evaluated. Intake of imbalanced diet was increased from approximately 50% to approximately 75% of baseline when an optimal amount of DLAA (1 nmol L-isoleucine or 2 nmol L-threonine) was injected immediately prior to feeding. Injections made several hours prior to feeding were more effective, increasing intake of imbalanced diets to approximately 85% of baseline. Delivering two half-optimal doses of DLAA, several hours apart, increased intake of imbalanced diet only to the same level as a single injection of the optimal dose immediately prior to feeding. The increase in intake of a threonine-imbalanced diet after injecting 2 nmol threonine 6 h prior to feeding was abolished if an additional 2 nmol threonine was injected immediately prior to feeding. It appears that it is the sum of the changes in tissue DLAA concentrations in the PPC that are recognized and influence food intake when amino acid imbalanced diets are fed.

Learned preference and aversion for complete and isoleucine-devoid diets in rats.

Choice tests using flavored solutions were conducted to demonstrate a learned preference or aversion in rats fed replete (complete) or isoleucine (ILE)-devoid diets. In the first learning trial, rats demonstrated a preference for the flavored solutions paired with the replete diet at 6 and 24 h (p < 0.001), and an aversion for the solutions paired with the ILE-devoid diet at 6 h (p < 0.05). In the second trial, using a different concentration of tastant, rats ingested significantly more of the flavored solution paired with the replete diet at 24 h (11.4 +/- 3.0 g vs. 5.4 +/- 1.0 g, respectively; p < 0.05), and significantly more of a novel flavored solution than of the solution paired with the ILE-devoid diet at 24 h (12.4 +/- 2.4 g vs. 1.7 +/- 0.6 g, respectively; p < 0.001). These results suggest that learning contributes to the rat's ability to self-select diets that promote growth, and that a learning paradigm pairing flavored solutions with complete and indispensible amino acid-devoid diets may be used to demonstrate learned preferences and aversions for such diets in rats.