G protein-dependent presynaptic inhibition mediated by AMPA receptors at the calyx of Held

The alpha-amino-3-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) is an ionotropic receptor mediating excitatory synaptic transmission, but it can also interact with intracellular messengers. Here we report that, at the calyx of Held in the rat auditory brainstem, activation of AMPARs induced inward currents in the nerve terminal and inhibited presynaptic Ca2+ currents (I(pCa)), thereby attenuating glutamatergic synaptic transmission. The AMPAR-mediated I(pCa) inhibition was disinhibited by a strong depolarizing pulse and occluded by the nonhydrolyzable GTP analog GTPgammaS loaded into the terminal. We conclude that functional AMPARs are expressed at the calyx of Held nerve terminal and that their activation inhibits voltage-gated Ca2+ channels by an interaction with heterotrimeric GTP-binding proteins (G proteins). Thus, at a central glutamatergic synapse, presynaptic AMPARs have a metabotropic nature and regulate transmitter release by means of G proteins.     

Spontaneous transmitter release is critical for the induction of long-term and intermediate-term facilitation in Aplysia

Long-term plasticity can differ from short-term in recruiting the growth of new synaptic connections, a process that requires the participation of both the presynaptic and postsynaptic components of the synapse. How does information about synaptic plasticity spread from its site of origin to recruit the other component? The answer to this question is not known in most systems. We have investigated the possible role of spontaneous transmitter release as such a transsynaptic signal. Until recently, relatively little has been known about the functions of spontaneous release. In this paper, we report that spontaneous release is critical for the induction of a learning-related form of synaptic plasticity, long-term facilitation in Aplysia. In addition, we have found that this signaling is engaged quite early, during an intermediate-term stage that is the first stage to involve postsynaptic as well as presynaptic molecular mechanisms. In a companion paper, we show that spontaneous release from the presynaptic neuron acts as an orthograde signal to recruit the postsynaptic mechanisms of intermediate-term facilitation and initiates a cascade that can culminate in synaptic growth with additional stimulation during long-term facilitation. Spontaneous release could make a similar contribution to learning-related synaptic plasticity in mammals.     

Postsynaptic GluA1 enables acute retrograde enhancement of presynaptic function to coordinate adaptation to synaptic inactivity

Prolonged blockade of AMPA-type glutamate receptors in hippocampal neuron cultures leads to homeostatic enhancements of pre- and postsynaptic function that appear correlated at individual synapses, suggesting some form of transsynaptic coordination. The respective modifications are important for overall synaptic strength but their interrelationship, dynamics, and molecular underpinnings are unclear. Here we demonstrate that adaptation begins postsynaptically but is ultimately communicated to presynaptic terminals and expressed as an accelerated turnover of synaptic vesicles. Critical postsynaptic modifications occur over hours, but enable retrograde communication within minutes once AMPA receptor (AMPAR) blockade is removed, causing elevation of both spontaneous and evoked vesicle fusion. The retrograde signaling does not require spiking activity and can be interrupted by NBQX, philanthotoxin, postsynaptic BAPTA, or external sequestration of BDNF, consistent with the acute release of retrograde messenger, triggered by postsynaptic Ca(2+) elevation via Ca(2+)-permeable AMPARs.     

Responses of serum corticosterone and corticosteroid-binding globulin to acute and prolonged stress in the rat

Responses of serum corticosterone (B) and corticosteroid-binding globulin (CBG) to ether anesthesia (a “classic” acute stress) and to a number of stressors influencing metabolic homeostasis—fasting, physical exercise, cold exposure, and water deprivation—were studied in male and female rats. Metabolic stressors included placing in an ice bath, physical exercise (swimming), fasting for 2 d, swimming after fasting for 2 d, cold-room (4°C) exposure for 2 d, fasting in combination with cold-room exposure for 1 d, and water deprivation for 2 d. The study demonstrated clear differences between males and females in basal B levels and B responses to some stressors. Only ether anesthesia and fasting resulted in similar B levels in males and females whereas in control and all other groups serum B levels were higher in females. Serum CBG was considerably higher in females. In females, ether, swimming, swimming after fasting, fasting, and fasting during cold exposure resulted in a decrease in circulating CBG. Ice bathing and cold exposure did not influence CBG, and water deprivation elevated serum CBG. In males, animals subjected to fasting and fasting during cold exposure had CBG levels lower than control animals. Other groups did not differ from the control. Higher CBG levels in females counterbalanced higher total B in setting circulating free B: significant sex differences in free B were observed only after swimming or fasting during cold exposure. Stress-responsive changes in CBG levels seem to contribute little to changes in free B; the main contributing factor is the rise in total B. However, CBG may play a special role, independent of the functions of corticosteroids. It is proposed that the need for substantial mobilization of spare fuel (as it takes place during physical exercise or fasting) is critical in involving CBG in the stress response.     

Distinct forms of Gq-receptor-dependent plasticity of excitatory transmission in the BNST are differentially affected by stress

Long-term depression (LTD) is an important synaptic mechanism for limiting excitatory influence over circuits subserving cognitive and emotional behavior. A major means of LTD induction is through the recruitment of signaling via G_q-linked receptors activated by norepinephrine (NE), acetylcholine, and glutamate. Receptors from these transmitter families have been proposed to converge on a common postsynaptic LTD maintenance mechanism, such that hetero- and homosynaptic induction produce similar alterations in glutamate synapse efficacy. We report that in the dorsolateral and ventrolateral bed nucleus of the stria terminalis (BNST), recruitment of G_q-linked receptors by glutamate or NE initiates mechanistically distinct forms of postsynaptically maintained LTD and these LTDs are differentially regulated by stress exposure. In particular, we show that although both mGluR5- and α₁-adrenergic receptor (AR)-dependent LTDs involve postsynaptic endocytosis, the α₁-AR-initiated LTD exclusively involves modulation of signaling through calcium-permeable AMPA receptors. Further, α₁-AR- but not mGluR5- dependent LTD is disrupted by restraint stress. α₁-AR LTD is also impaired in mice chronically exposed to ethanol. These data thus suggest that in the BNST, NE- and glutamate-activated G_q-linked signaling pathways differentially tune glutamate synapse efficacy in response to stress.     

Metabotropic glutamate and GABAB receptors contribute to the modulation of glucose-stimulated insulin secretion in pancreatic beta cells

Aims/hypothesis: The neurotransmitters glutamate and γ-aminobutyric acid (GABA) could participate in the regulation of the endocrine functions of islets of Langerhans. We investigated the role of the metabotropic glutamate (mGluRs) and GABA_B (GABA_BRs) receptors in this process. Methods: We studied the expression of mGluRs and GABA _BRs in rat and human islets of Langerhans and in pancreatic α-cell and beta-cell lines using RT-PCR and immunoblot analysis. Effects of mGluR and GABA _B R agonists on insulin secretion were determined by radioimmunoassays and enzyme-linked immunoadsorbent assays (ELISAs). Results: We detected mGluR3 and mGluR5 (but not mGluR1, 6 and 7) mRNAs in all of the samples examined. Trace amount of mGluR2 was found in MIN6 beta cells; mGluR4 was identified in rat islets; and mGluR8 expression was detected in rat islets, RINm5F and MIN6 cells. GABA _BR1 a/b and 2 mRNAs were identified in islets of Langerhans and MIN6 cells. The expression of mGluR3, mGluR5, GABA_BR1 a/b and GABA_BR2 proteins was confirmed using specific antibodies. Group I (mGluR1/5) and group II (mGluR2/3) specific mGluR agonists increased the release of insulin in the presence of 3 to 10 mmol/l or 3 to 25 mmol/l glucose, respectively, whereas a group III (mGluR4/6–8) specific agonist inhibited insulin release at high (10–25 mmol/l) glucose concentrations. Baclofen, a GABA_BR agonist, also inhibited the release of insulin but only in the presence of 25 mmol/l glucose. Conclusion/interpretation: These data suggest that mGluRs and GABA_BRs play a role in the regulation of the endocrine pancreas with mechanisms probably involving direct activation or inhibition of voltage dependent Ca²⁺-channels, cAMP generation and G-protein-mediated modulation of K_ATP channels. [Diabetologia (2002) 45: 242–252] Received: 18 September 2001 and in revised form: 5 November 2001     

Effects of short and long duration hypothyroidism and hyperthyroidism on the plasma adrenocorticotropin and corticosterone responses to ovine corticotropin-releasing hormone in rats.

We report here a study of the plasma ACTH and corticosterone responses to synthetic ovine CRH (oCRH) in hypothyroid and hyperthyroid rats studied 7, 15, and 60 days after either thyroidectomy or the administration of pharmacological doses of T4. The purpose of this study was to further clarify the time-dependent effects of alterations in thyroid status on the functional integrity of the hypothalamic-pituitary-adrenal axis and to aid in the interpretation of the oCRH stimulation test in hypo- and hyperthyroid states. Our data demonstrate that hypothyroid rats have a significant reduction in the cerebrospinal fluid (CSF) levels of corticosterone and a significant decrease in adrenal weight in association with significant increases in the plasma ACTH response to oCRH. On the other hand, the corticosterone response to the ACTH released during the oCRH stimulation test was significantly reduced in hypothyroidism. With increasing duration of thyroidectomy-induced hypothyroidism, there was a progressive fall in CSF corticosterone levels, a progressive increase in the plasma ACTH response to oCRH, and a gradual normalization of the corticosterone responses to the ACTH released during oCRH stimulation. Our findings in hyperthyroid rats were generally the converse of those seen in hypothyroidism. Hence, there was a significant increase in the CSF levels of corticosterone and a significant increase in adrenal weight in association with an initial slight decrease in the ACTH response to oCRH. On the other hand, the corticosterone response to the ACTH released during oCRH stimulation was significantly increased. There was a gradual increase in the magnitude of the rise in CSF corticosterone levels with time, as well as a gradual normalization of adrenocortical responses during oCRH stimulation. The ACTH plasma clearance rates were similar in hypo-, hyper-, and euthyroid rats. Our data do not permit definitive identification of the precise locus in the hypothalamic-pituitary-adrenal axis that is principally affected by experimentally induced alterations in thyroid status. However, these data are most compatible with a subtle hypothyroid-induced centrally mediated adrenal insufficiency and a subtle hyperthyroid-induced centrally mediated hypercortisolism. These data also suggest that alterations in hypothalamic-pituitary-adrenal function in states of disturbed thyroid function become somewhat more pronounced as the duration of thyroid dysfunction increases. The fact that pituitary-adrenal responses to oCRH are consistently altered in states of thyroid dysfunction may be relevant to the clinical interpretation of oCRH stimulation tests.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of temperature on urinary corticosterone metabolite responses to short-term capture and handling stress in the cane toad (Rhinella marina)

Extreme temperature can cause metabolic, immune and behavioural changes in amphibians. Short-term stress hormonal response via increased secretion of corticosterone enables amphibians to make necessary physiological and behavioural adjustments for coping with stressors. The effect of temperature on short-term corticosterone responses has not been studied in amphibians. In this study, this relationship was evaluated in adult male cane toads (Rhinella marina). We acclimated male toads (n=24 toads per group) at low, medium and high temperature (15, 25 or 35°C) under controlled laboratory conditions for a 14day period. After thermal acclimation, short-term corticosterone responses were evaluated in the toads subjected to a standard capture and handling stress protocol over a 24h period. Corticosterone metabolites in toad urine were measured via enzyme-immunoassay. During acclimation, mean baseline urinary corticosterone level increased after transfer of the toads from wild into captivity and returned to baseline on day 14 of acclimation for each of the three temperatures. At the end of the 14days of thermal acclimation period, baseline corticosterone level were highest for toad group at 35°C and lowest at 15°C. All toads generated urinary corticosterone responses to the standard capture and handling stressor for each temperature. Both individual and mean short-term corticosterone responses of the toads were highest at 35°C and lowest at 15°C. Furthermore, Q(10) values (the factor by which the reaction rate increases when the temperature is raised by 10°) were calculated for mean corrected integrated corticosterone responses as follows; (15-35°C) Q(10)=1.51, (15-25°C) Q(10)=1.60; (25-35°C) Q(10)=1.43. Both total and corrected integrated corticosterone responses were highest for toads at 35°C followed by 25°C and lowest for the 15°C toad group. Overall, the results have demonstrated the thermodynamic response of corticosterone secretion to short-term capture and handling stress in an amphibian species.     

The effects of the menopause on calcitriol and parathyroid hormone: responses to a low dietary calcium stress test

Calcium, calcitriol and PTH levels were studied in 11 premenopausal women, aged 41 +/- 3 yr (mean +/- SD), and 11 postmenopausal women, aged 42 +/- 3 yr, at baseline and in 9 women from each group during a 4-day low calcium diet. Serum dialyzable calcium and urinary hydroxyproline excretion were higher in the postmenopausal women at baseline and throughout the low calcium diet. Baseline calcitriol levels were significantly lower in the postmenopausal women, but these were associated with lower vitamin D-binding protein levels; the calculated free calcitriol index was not different between the two groups. After the low calcium diet calcitriol levels rose in both groups to similar levels. PTH levels were not different in the two groups at baseline and rose to the same level on the low calcium diet. These data indicate that estrogen deficiency at the menopause is not associated with deficient calcitriol or PTH reserve, although basal calcitriol levels may be reduced secondarily due to lower vitamin D-binding protein levels or relative hypercalcaemia.     

Effects of prenatal exposure to alcohol on the release of adenocorticotropic hormone, corticosterone, and proinflammatory cytokines

Prenatal alcohol exposure has been shown to produce hyperresponsiveness of the hypothalamic-pituitary-adrenal (HPA) axis to immune challenges. Because cytokines, which are released in response to immune challenges, are known to activate the HPA axis, this study determined whether altered release of cytokines contribute to the HPA hyperresponsiveness to immune challenges observed after prenatal alcohol exposure. Pregnant dams were exposed to alcohol vapors (6-7 hr daily) between days 7 and 18 of gestation. At postnatal days 45 and 60, control (C) and prenatal alcohol-exposed (E) offspring were subjected to three different types of immune challenges: injections of interleukin-1beta or endotoxin (lipopolysaccharide), or turpentine-induced tissue injury. We observed the expected higher plasma adrenocorticotropic hormone and corticosterone levels in E compared with C rats, and this HPA hyperresponsiveness was greater in E females compared with E males. Plasma tumor necrosis factor-alpha or interleukin-6 responses were comparable in the C and E groups. Females exhibited significantly higher corticosterone, tumor necrosis factor-alpha, and interleukin-6 responses than males. These results indicate that (1) prenatal alcohol exposure produces HPA hyperresponsiveness to immune challenges; (2) prenatal alcohol treatment does not influence the release of cytokines to immune challenges; and (3) there are gender differences in the secretory pattern of corticosterone and cytokines to immune challenges. Therefore, these data do not support the hypothesis that cytokines play a role in the hyperresponsiveness of the HPA axis to immune challenges observed after prenatal alcohol exposure.     

Morphologic responses of endothelium to shear stress: reorganization of the adherens junction

Shear stresses induce marked morphologic responses from endothelium which include alterations to cell shape and orientation and changes to cytoskeletal organization. These morphologic changes necessitate remodeling of cell-cell adhesion complexes that are important to control of endothelial cell physiology. Reorganization of endothelial adherens junctions has been characterized, and there are some data that pertain to the signaling pathways that regulate this reorganization. Shear-induced activation of Src, mitogen-activated protein (MAP) kinase (ERK1/2 and p38), and PI 3'-kinase pathways are important candidate pathways, and there is evidence for a role for the Rho GTPases. Very little is known concerning shear-dependence of other junctional complexes, but available data indicates a high degree of shear sensitivity. Given the continuous changes in hemodynamics which occur physiologically in vivo, sensitivity of endothelial cell-cell adhesion complexes to shear will likely prove important to vascular pathophysiology.     

Differential responsiveness of MCF-7 human breast cancer cell line stocks to the pineal hormone, melatonin

The estrogen receptor (ER)-positive MCF-7 human breast cancer cell line has been used extensively for the study of estrogen-responsive human breast cancer. However, various levels of estrogen responsiveness have been described in different stocks of MCF-7 cells. Because we have previously shown that the pineal hormone, melatonin, inhibits proliferation of MCF-7 cells and can modulate ER expression and transactivation, we investigated if various stocks of MCF-7 cells exhibit a differential responsiveness to the anti-proliferative effects of melatonin and the possible mechanisms involved. The MCF-7 stocks (M, O, H) were examined for: (1) mitogenic response to estradiol; (2) steady-state ER mRNA levels; (3) expression of the mt1 melatonin membrane receptor; (4) growth inhibition by melatonin; and (5) melatonin's modulation of expression of the ER and the estrogen-regulated genes, PgR, TGFbeta and pS2. For all of these parameters, there was a stock-specific response which showed: MCF-7M > MCF-7O > MCF-7H. These results demonstrate that there are significant differences in the responsiveness of various stocks of MCF-7 breast cancer cells to the growth-inhibitory effects of melatonin which can be correlated with both the level of ER mRNA expression and the degree of estrogen-responsiveness. These findings suggest that not only may these differences have some impact on the cells' estrogen-response pathway, but also that the primary growth-inhibitory effects of melatonin are transduced through the membrane-associated G-protein coupled mt1 melatonin receptor.