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Many efforts have been made to illuminate the nature of past hydroclimates in semi-arid and arid regions, where current and future shifts in water availability have enormous consequences on human subsistence. Deep desert aquifers, where groundwater is stored for prolonged periods, might serve as a direct record of major paleo-recharge events. To date, groundwater-based paleoclimate reconstructions have mainly focused on a relatively narrow timescale (up to ∼40 kyr), limited by the relatively short half-life of the widely used radiocarbon (5.73 kyr). Here we demonstrate the usage of deep regional aquifers in the arid southeastern Mediterranean as a hydroclimate archive for earlier Mid-to-Late Pleistocene epochs. State-of-the-art dating tools, primarily the 81 Kr radioisotope (t 1/2  = 229 kyr), were combined with other atmosphere-derived tracers to illuminate the impact of four distinguishable wetter episodes over the past 400 kyr, with differences in climatic conditions and paleo-recharge locations. Variations in stable water isotope composition suggest moisture transport from more proximal (Mediterranean) and distal (Atlantic) sources to different parts of the region at distinct times. Large variability in the computed noble gas-based recharge temperature (NGT), ranging ~15-30 °C, cannot be explained by climate variations solely, and points to different recharge pathways, including geothermal heating in the deep unsaturated zone and recharge from high-elevation (colder) regions. The obtained groundwater record complements and enhances the interpretation of other terrestrial archives in the arid region, including a contribution of valuable information regarding the moisture source origin as reflected in the deuterium-excess values, which is unattainable from the common practice analysis of calcitic cave deposits. We conclude that similar applications in other deep (hundred-m-order) regional groundwater systems (e.g., the Sahara desert aquifers) can significantly advance our understanding of long-term (up to 1 Myr) paleo-hydroclimate in arid regions, including places where no terrestrial remnants, such as cave, lake, and spring sediments, are available., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The abundance and isotopic composition of noble gases dissolved in water have many applications in the geosciences. In recent years, new analytical techniques have opened the door to the use of high-precision measurements of noble gas isotopes as tracers for groundwater hydrology, oceanography, mantle geochemistry, and paleoclimatology. These analytical advances have brought about new measurements of solubility equilibrium isotope effects (SEIEs) in water (i.e., the relative solubilities of noble gas isotopes) and their sensitivities to the temperature and salinity. Here, we carry out a suite of classical molecular dynamics (MD) simulations and employ the theoretical method of quantum correction to estimate SEIEs for comparison with experimental observations. We find that classical MD simulations can accurately predict SEIEs for the isotopes of Ar, Kr, and Xe to order 0.01‰, on the scale of analytical uncertainty. However, MD simulations consistently overpredict the SEIEs of Ne and He by up to 40% of observed values. We carry out sensitivity tests at different temperatures, salinities, and pressures and employ different sets of interatomic potential parameters and water models. For all noble gas isotopes, the TIP4P water model is found to reproduce observed SEIEs more accurately than the SPC/E and TIP4P/ice models. Classical MD simulations also accurately capture the sign and approximate magnitude of temperature and salinity sensitivities of SEIEs for heavy noble gases. We find that experimental and modeled SEIEs generally follow an inverse-square mass dependence, which implies that the mean-square force experienced by a noble gas atom within a solvation shell is similar for all noble gases. This inverse-square mass proportionality is nearly exact for Ar, Kr, and Xe isotopes, but He and Ne exhibit a slightly weaker mass dependence. We hypothesize that the apparent dichotomy between He-Ne and Ar-Kr-Xe SEIEs may result from atomic size differences, whereby the smaller noble gases are more likely to spontaneously fit within cavities of water without breaking water-water H-bonds, thereby experiencing softer collisions during translation within a solvation shell. We further speculate that the overprediction of simulated He and Ne SEIEs may result from the neglection of higher-order quantum corrections or the overly stiff representation of van der Waals repulsion by the widely used Lennard-Jones 6-12 potential model. We suggest that new measurements of SEIEs of heavy and light noble gases may represent a novel set of constraints with which to refine hydrophobic solvation theories and optimize the set of interatomic potential models used in MD simulations of water and noble gases.

Two subtypes of alpha (α)subunits, α1and α2, belonging to AP-2 complex have been described in the central nervous system (CNS). The specific role of each subtype is still unclear. In this study, we evaluated the expression and interaction with cell membranes of both subtypes in the postnatal developing cerebral cortex and cerebellum in two rat strains that display distinct developmental features. We observed that α2 displays higher variations than α1 during development, and at lesser extent in the rats with delayed rate of development. Additionally, by in vitro binding assays we evaluated the interaction of α subunits with bovine brain membranes. Both subtypes displayed clear differences in their performance, maximum binding of α1 was higher and α2 reached it faster than α1. In addition, both subtypes displayed different binding to membranes when bivalent cations or nucleotides were added. We conclude that both subtypes interact differently with membranes and that they may play different roles in clathrin-mediated endocytosis in the CNS., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Mantle-derived noble gases in volcanic gases are powerful tracers of terrestrial volatile evolution, as they contain mixtures of both primordial (from Earth's accretion) and secondary (e.g., radiogenic) isotope signals that characterize the composition of deep Earth. However, volcanic gases emitted through subaerial hydrothermal systems also contain contributions from shallow reservoirs (groundwater, crust, atmosphere). Deconvolving deep and shallow source signals is critical for robust interpretations of mantle-derived signals. Here, we use a novel dynamic mass spectrometry technique to measure argon, krypton, and xenon isotopes in volcanic gas with ultrahigh precision. Data from Iceland, Germany, United States (Yellowstone, Salton Sea), Costa Rica, and Chile show that subsurface isotope fractionation within hydrothermal systems is a globally pervasive and previously unrecognized process causing substantial nonradiogenic Ar-Kr-Xe isotope variations. Quantitatively accounting for this process is vital for accurately interpreting mantle-derived volatile (e.g., noble gas and nitrogen) signals, with profound implications for our understanding of terrestrial volatile evolution.

Gas exchange between the atmosphere and ocean interior profoundly impacts global climate and biogeochemistry. However, our understanding of the relevant physical processes remains limited by a scarcity of direct observations. Dissolved noble gases in the deep ocean are powerful tracers of physical air-sea interaction due to their chemical and biological inertness, yet their isotope ratios have remained underexplored. Here, we present high-precision noble gas isotope and elemental ratios from the deep North Atlantic (~32°N, 64°W) to evaluate gas exchange parameterizations using an ocean circulation model. The unprecedented precision of these data reveal deep-ocean undersaturation of heavy noble gases and isotopes resulting from cooling-driven air-to-sea gas transport associated with deep convection in the northern high latitudes. Our data also imply an underappreciated and large role for bubble-mediated gas exchange in the global air-sea transfer of sparingly soluble gases, including O 2 , N 2 , and SF 6 . Using noble gases to validate the physical representation of air-sea gas exchange in a model also provides a unique opportunity to distinguish physical from biogeochemical signals. As a case study, we compare dissolved N 2 /Ar measurements in the deep North Atlantic to physics-only model predictions, revealing excess N 2 from benthic denitrification in older deep waters (below 2.9 km). These data indicate that the rate of fixed N removal in the deep Northeastern Atlantic is at least three times higher than the global deep-ocean mean, suggesting tight coupling with organic carbon export and raising potential future implications for the marine N cycle.

Terrestrial amplification (TA) of land warming relative to oceans is apparent in recent climatic observations. TA results from land-sea coupling of moisture and heat and is therefore important for predicting future warming and water availability. However, the theoretical basis for TA has never been tested outside the short instrumental period, and the spatial pattern and amplitude of TA remain uncertain. Here, we investigate TA during the Last Glacial Maximum (LGM; ~20 thousand years) in the low latitudes, where the theory is most applicable. We find remarkable consistency between paleotemperature proxies, theory, and climate model simulations of both LGM and future climates. Paleoclimate data thus provide crucial new support for TA, refining the range of future low-latitude, low-elevation TA to [Formula: see text] (95% confidence interval), i.e., land warming ~40% more than oceans. The observed data model theory agreement helps reconcile LGM marine and terrestrial paleotemperature proxies, with implications for equilibrium climate sensitivity.

The effects during healthy aging of the tetrodotoxin-resistant voltage-gated sodium channel 1.8 (Nav1.8), the acid-sensing ion channel-3 (ASIC3), the purinergic-receptor 2X3 (P2X3) and transient receptor potential of melastatin-8 (TRPM8) on responses to non-noxious stimuli are poorly understood. These effects will influence the transferability to geriatric subjects of findings obtained using young animals. To evaluate the involvement of these functional markers in mechanical and cold sensitivity to non-noxious stimuli and their underlying mechanisms, we used a combination of immunohistochemistry and quantitation of immunostaining in sub-populations of neurons of the dorsal root ganglia (DRG), behavioral tests, pharmacological interventions and Western-blot in healthy male Wistar rats from 3 to 24 months of age. We found significantly decreased sensitivity to mechanical and cold stimuli in geriatric rats. These behavioural alterations occurred simultaneously with differing changes in the expression of Nav1.8, ASIC3, P2X3 and TRPM8 in the DRG at different ages. Using pharmacological blockade in vivo we demonstrated the involvement of ASIC3 and P2X3 in normal mechanosensation and of Nav1.8 and ASIC3 in cold sensitivity. Geriatric rats also exhibited reductions in the number of A-like large neurons and in the proportion of peptidergic to non-peptidergic neurons. The changes in normal sensory physiology in geriatric rats we report here strongly support the inclusion of aged rodents as an important group in the design of pre-clinical studies evaluating pain treatments., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

It is well established that mantle plumes are the main conduits for upwelling geochemically enriched material from Earth's deep interior. The fashion and extent to which lateral flow processes at shallow depths may disperse enriched mantle material far (>1,000 km) from vertical plume conduits, however, remain poorly constrained. Here, we report He and C isotope data from 65 hydrothermal fluids from the southern Central America Margin (CAM) which reveal strikingly high 3 He/ 4 He (up to 8.9R A ) in low-temperature (≤50 °C) geothermal springs of central Panama that are not associated with active volcanism. Following radiogenic correction, these data imply a mantle source 3 He/ 4 He >10.3R A (and potentially up to 26R A , similar to Galápagos hotspot lavas) markedly greater than the upper mantle range (8 ± 1R A ). Lava geochemistry (Pb isotopes, Nb/U, and Ce/Pb) and geophysical constraints show that high 3 He/ 4 He values in central Panama are likely derived from the infiltration of a Galápagos plume-like mantle through a slab window that opened ∼8 Mya. Two potential transport mechanisms can explain the connection between the Galápagos plume and the slab window: 1) sublithospheric transport of Galápagos plume material channeled by lithosphere thinning along the Panama Fracture Zone or 2) active upwelling of Galápagos plume material blown by a "mantle wind" toward the CAM. We present a model of global mantle flow that supports the second mechanism, whereby most of the eastward transport of Galápagos plume material occurs in the shallow asthenosphere. These findings underscore the potential for lateral mantle flow to transport mantle geochemical heterogeneities thousands of kilometers away from plume conduits., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The magnitude of global cooling during the Last Glacial Maximum (LGM, the coldest multimillennial interval of the last glacial period) is an important constraint for evaluating estimates of Earth's climate sensitivity 1,2 . Reliable LGM temperatures come from high-latitude ice cores 3,4 , but substantial disagreement exists between proxy records in the low latitudes 1,5-8 , where quantitative low-elevation records on land are scarce. Filling this data gap, noble gases in ancient groundwater record past land surface temperatures through a direct physical relationship that is rooted in their temperature-dependent solubility in water 9,10 . Dissolved noble gases are suitable tracers of LGM temperature because of their complete insensitivity to biological and chemical processes and the ubiquity of LGM-aged groundwater around the globe 11,12 . However, although several individual noble gas studies have found substantial tropical LGM cooling 13-16 , they have used different methodologies and provide limited spatial coverage. Here we use noble gases in groundwater to show that the low-altitude, low-to-mid-latitude land surface (45 degrees south to 35 degrees north) cooled by 5.8 ± 0.6 degrees Celsius (mean ± 95% confidence interval) during the LGM. Our analysis includes four decades of groundwater noble gas data from six continents, along with new records from the tropics, all of which were interpreted using the same physical framework. Our land-based result broadly supports a recent reconstruction based on marine proxy data assimilation 1 that suggested greater climate sensitivity than previous estimates 5-7 .

Groundwater is an important source of drinking and irrigation water. Dating groundwater informs its vulnerability to contamination and aids in calibrating flow models. Here, we report measurements of multiple age tracers ( 14 C, 3 H, 39 Ar, and 85 Kr) and parameters relevant to dissolved inorganic carbon (DIC) from 17 wells in California's San Joaquin Valley (SJV), an agricultural region that is heavily reliant on groundwater. We find evidence for a major mid-20th century shift in groundwater DIC input from mostly closed- to mostly open-system carbonate dissolution, which we suggest is driven by input of anthropogenic carbonate soil amendments. Crucially, enhanced open-system dissolution, in which DIC equilibrates with soil CO 2 , fundamentally affects the initial 14 C activity of recently recharged groundwater. Conventional 14 C dating of deeper SJV groundwater, assuming an open system, substantially overestimates residence time and thereby underestimates susceptibility to modern contamination. Because carbonate soil amendments are ubiquitous, other groundwater-reliant agricultural regions may be similarly affected., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Neuropathic and inflammatory pain results from cellular and molecular changes in dorsal root ganglion (DRG) neurons. The type-2 receptor for Angiotensin-II (AT2R) has been involved in this type of pain. However, the underlying mechanisms are poorly understood, including the role of the type-1 receptor for Angiotensin-II (AT1R). Here, we used a combination of immunohistochemistry and immunocytochemistry, RT-PCR and in vitro and in vivo pharmacological manipulation to examine how cutaneous inflammation affected the expression of AT1R and AT2R in subpopulations of rat DRG neurons and studied their impact on inflammation-induced neuritogenesis. We demonstrated that AT2R-neurons express C- or A-neuron markers, primarily IB4, trkA, and substance-P. AT1R expression was highest in small neurons and co-localized significantly with AT2R. In vitro, an inflammatory soup caused significant elevation of AT2R mRNA, whereas AT1R mRNA levels remained unchanged. In vivo, we found a unique pattern of change in the expression of AT1R and AT2R after cutaneous inflammation. AT2R increased in small neurons at 1 day and in medium size neurons at 4 days. Interestingly, cutaneous inflammation increased AT1R levels only in large neurons at 4 days. We found that in vitro and in vivo AT1R and AT2R acted co-operatively to regulate DRG neurite outgrowth. In vivo, AT2R inhibition impacted more on non-peptidergic C-neurons neuritogenesis, whereas AT1R blockade affected primarily peptidergic nerve terminals. Thus, cutaneous-induced inflammation regulated AT1R and AT2R expression and function in different DRG neuronal subpopulations at different times. These findings must be considered when targeting AT1R and AT2R to treat chronic inflammatory pain. Cover Image for this issue: doi: 10.1111/jnc.14737., (© 2019 International Society for Neurochemistry.)

Constraining the magnitude of past hydrological change may improve understanding and predictions of future shifts in water availability. Here we demonstrate that water-table depth, a sensitive indicator of hydroclimate, can be quantitatively reconstructed using Kr and Xe isotopes in groundwater. We present the first-ever measurements of these dissolved noble gas isotopes in groundwater at high precision (≤0.005‰ amu -1 ; 1σ), which reveal depth-proportional signals set by gravitational settling in soil air at the time of recharge. Analyses of California groundwater successfully reproduce modern groundwater levels and indicate a 17.9 ± 1.3 m (±1 SE) decline in water-table depth in Southern California during the last deglaciation. This hydroclimatic transition from the wetter glacial period to more arid Holocene accompanies a surface warming of 6.2 ± 0.6 °C (±1 SE). This new hydroclimate proxy builds upon an existing paleo-temperature application of noble gases and may identify regions prone to future hydrological change.

In 2017, fluid and gas samples were collected across the Costa Rican Arc. He and Ne isotopes, C isotopes as well as total organic and inorganic carbon concentrations were measured. The samples (n = 24) from 2017 are accompanied by (n = 17) samples collected in 2008, 2010 and 2012. He-isotopes ranged from arc-like (6.8 R A ) to crustal (0.5 R A ). Measured dissolved inorganic carbon (DIC) δ 13 C VPDB values varied from 3.55 to -21.57‰, with dissolved organic carbon (DOC) following the trends of DIC. Gas phase CO 2 only occurs within ~20 km of the arc; δ 13 C VPDB values varied from -0.84 to -5.23‰. Onsite, pH, conductivity, temperature and dissolved oxygen (DO) were measured; pH ranged from 0.9-10.0, conductivity from 200-91,900 μS/cm, temperatures from 23-89 °C and DO from 2-84%. Data were used to develop a model which suggests that ~91 ± 4.0% of carbon released from the slab/mantle beneath the Costa Rican forearc is sequestered within the crust by calcite deposition with an additional 3.3 ± 1.3% incorporated into autotrophic biomass.

The Neuropeptide EI (NEI, glutamic acid- isoleucine amide) participates in neuroendocrine function. Previously we demonstrated that NEI concentration is regulated by thyroid hormones in discrete hypothalamic areas in rats. We observed that the thyroid status affects the dopaminergic regulation of the pituitary hormones. In this study we explored possible interactions between NEI and tyrosine hydroxylase (TH) containing elements in selected hypothalamic areas of male rats. Neuronal somas, terminals and boutons were assessed by confocal microscopy, in hypo- and hyperthyroid animals. We observed a remodeling of the contacts between the TH and NEI immunoreactive elements in the incerto-hypothalamic area (IHy, also known as rostromedial zona incerta) according to thyroid function. However, in the dorsolateral zone of the peduncular part of the lateral hypothalamus (DL-PLH) the thyroid hormones affect the dendritic trees of the neurons without perturbing the overall NEI/TH contacts. Also, we demonstrated that TRH Receptor 1 (TRH-R1) is colocalized in NEI immunoreactive neurons in the peduncular part of the lateral hypothalamus (PLH) and NEI precursor mRNA expression increased by hypothyroidism indicating that NEI neurons are responsive to the feedback mechanisms of the Hypothalamic Pituitary-Thyroid Axis (HPT). In conclusion, the hypothyroid status seems to increase the interactions between the NEI neurons and the dopaminergic pathways while hyperthyroidism either decreases or displays no effects. Altogether these observations support the participation of the IHy and PLH NEI as a modulating component of the HPT suggesting that altered neuroendocrine, behavioral and cognitive dysfunctions induced by dysthyroidism could be in part mediated by NEI., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In this study we examined cerebellar alterations in a neonatal rat model of hypoxic-ischemic brain injury with or without hypoxic preconditioning (Pc). Between postnatal days 7 and 15, the cerebellum is still undergoing intense cellular proliferation, differentiation and migration, dendritogenesis and synaptogenesis. The expression of glutamate decarboxylase 1 (GAD67) and the differentiation factor NeuroD1 were examined as markers of Purkinje and granule cells, respectively. We applied quantitative immunohistochemistry to sagittal cerebellar slices, and Western blot analysis of whole cerebella obtained from control (C) rats and rats submitted to Pc, hypoxia-ischemia (L) and a combination of both treatments (PcL). We found that either hypoxia-ischemia or Pc perturbed the granule cells in the posterior lobes, affecting their migration and final placement in the internal granular layer. These effects were partially attenuated when the Pc was delivered prior to the hypoxia-ischemia. Interestingly, whole nuclear NeuroD1 levels in Pc animals were comparable to those in the C rats. However, a subset of Purkinje cells that were severely affected by the hypoxic-ischemic insult--showing signs of neuronal distress at the levels of the nucleus, cytoplasm and dendritic arborization--were not protected by Pc. A monoclonal antibody specific for GAD67 revealed a three-band pattern in cytoplasmic extracts from whole P15 cerebella. A ∼110 kDa band, interpreted as a potential homodimer of a truncated form of GAD67, was reduced in Pc and L groups while its levels were close to the control animals in PcL rats. Additionally we demonstrated differential glial responses depending on the treatment, including astrogliosis in hypoxiated cerebella and a selective effect of hypoxia-ischemia on the vimentin-immunolabeled intermediate filaments of the Bergmann glia. Thus, while both glutamatergic and GABAergic cerebellar neurons are compromised by the hypoxic-ischemic insult, the former are protected by a preconditioning hypoxia while the latter are not.

Background/aims: Few studies address the long-term consequences of perinatal hypoxia (H), a frequent birth complication. Previously we described advanced reproductive senescence (premature loss of regular cyclicity) in female rats subjected to perinatal H or H plus unilateral ischemia (HI) associated with changes in the hypothalamic expression of estrogen and opioid receptors. Our aim is to explore whether hypothalamic inflammation and oxidative damage mediate these reproductive alterations., Methods: Female rats were subjected on postnatal day (PND) 7 to H (6.5% O2 for 50 min) or HI (H + right carotid artery ligature) and inflammation/oxidative damage markers, such as iNOS, nNOS, insulin-like growth factor (IGF) system expression, glial reaction and macrophage invasion in the medial basal hypothalamus-preoptic area (GFAP Western blot and immunohistochemistry, ED1 immunohistochemistry), were determined. The effect of antioxidant treatment with vitamin E (VE; 1.5 mg/rat on PND 4, 6 and 8) was also explored., Results: No significant cellular inflammatory reactions were observed although GFAP protein was significantly increased at early times after injury. Forty-eight hours after injury iNOS, nNOS and IGF-I mRNA decreased in the HI group, and nNOS in the H group. IGFBP-3 mRNA increased in HI rats at 48 h and 30 days, while it fell at 7 days postinjury in both groups. VE treatment prevented the effects of HI on oxidation/inflammation markers, but did not prevent the premature onset of reproductive senescence or the altered hormone receptors expression., Conclusion: These results suggest that the oxidative and inflammatory damage caused by perinatal H or HI may not be responsible for the late-onset reproductive abnormalities., (© 2014 S. Karger AG, Basel.)

The angiotensin II receptor subtype 2 (AT2-R) has been proposed to mediate protective vascular actions after brain injury. In this study we investigated the participation of this peptide in the tolerance to cellular damage induced by preconditioning in a rat model of neonatal hypoxia-ischemia (HI). We found that injured animals present a decreased number of microvessels in the ipsilateral (IPLT) side of the brain while in the contralateral (CNLT) side the microvessel number is increased. On the contrary, in the preconditioned animals the microvessels maintained the same number as in control animals. However these vessels show a remarkable increase of the fluorescent signal when they are labeled with antiFlk-1 (VEGFR2), while the Flt-1 (VEGFR1) signal faded in both the injured and the preconditioned animals. The pharmacological blockade of the AT2-R by the drug PD123319 (1.69 mM in the lateral ventricle) diminished the resilience of the microvasculature to HI injury provided by preconditioning and also the Flk-1 increase that occurred in these animals. In conclusion these results suggest an interaction of the AT2-R with VEGFR2 in the neonatal brain microvasculature that produces protective effects which are associated with injury tolerance., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Angiotensin II (Ang II) binds and activates two major receptors subtypes, namely AT(1) and AT(2). In the fetus, AT(2) receptors predominate in all tissues and decline shortly after birth, being restricted to a few organs including brain. Interpretation of the function of Ang II in the cerebellum requires a thorough understanding of the localization of Ang II receptors. The aim of the present paper is to evaluate the localization of Ang II AT(2) receptors in the Purkinje cell (PC) layer during development. By binding autoradiography, a clear complementary pattern of AT(1) and AT(2) binding labeled by [(125)I] Ang II was observed in young rats within the cerebellar cortex. This pattern was present at the stages P8 and P15, but not at P30 and P60, where AT(2) binding appears low and superimposed with AT(1) binding. We demonstrate that AT(2) antibodies recognized postmitotic Purkinje cells, labeling the somata of these cells at all the stages studied, from P8 to P60, suggesting that PCs express these receptors from early stages of development until adulthood. In P8 and P15 animals, we observed a clear correspondence between immunolabeling and the well-defined layer observed by binding autoradiography. Confocal analysis allowed us to discard the co-localization of AT(2) receptors with glial fibrillary acidic protein (GFAP), a glial marker. Double immunolabeling allowed us to demonstrate the co-localization of Ang II AT(2) receptors with zebrin II, a specific PC marker. Since PCs are the sole output signal from the cerebellar cortex and considering the role of cerebellum in movement control, the specific receptor localization suggests a potential role for Ang II AT(2) receptors in the cerebellar function., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Evidence suggests that Angiotensin II plays an important role in the complex process of renal organogenesis. Rat kidney organogenesis starts between E13-14 and lasts up to 2 weeks after birth. The present study demonstrates histologic modifications and changes in receptor localisation in animals born from mothers treated with Angiotensin II, Losartan or PD123319 (1.0 mg/kg/day) during late pregnancy. Angiotensin II-treated animals exhibited very well developed tubules in the renal medulla in coincidence with higher AT(1) binding. Control animals exhibited angiotensin AT(2) binding in the outer stripe of the outer medulla, while in the Angiotensin II-treated animals binding was observed to the inner stripe. In Angiotensin II-treated 1-week-old animals, the nephrogenic zone contained fewer immature structures, and more developed collecting tubules than control animals. Treatment with Losartan resulted in severe renal abnormalities. For newborn and 1-week-old animals, glomeruli exhibited altered shape and enlarged Bowman spaces, in concordance with a loss of [(125)I]Angiotensin II binding in the cortex. Blockade with PD123319 led to an enlarged nephrogenic zone with increased number of immature glomeruli, and less glomeruli in the juxtamedullary area. Autoradiography showed a considerable loss of AT(1) binding in the kidney cortex of PD123319-treated animals at both ages. The present results show for the first time histomorphological and receptor localisation alterations following treatment with low doses of Losartan and PD123319 during pregnancy. These observations confirm previous assumptions that in the developing kidney Angiotensin II exerts stimulatory effects through AT(1) receptors that might be counterbalanced by angiotensin AT(2) receptors.

Perinatal hypoxia is a frequent birth complication, and although its early consequences on brain development have been well studied, few studies address any long-term effects. Postnatal insults producing small disturbances in endocrine function can have marked and long-lasting effects. In the present work we studied the effects of two types of perinatal brain injury: global hypoxia (H, 6.5% O2 for 50 min) and hypoxia plus ischemia (HI, ligature of the right carotid artery) on female rat reproductive performance and expression of mediobasal hypothalamus-preoptic area (MBH-PO) estrogen, progesterone and micro-opioid receptors at different times after injury, measuring the mRNA (by semiquantitative RT-PCR) and protein (by Western blot). H or HI advanced approximately 3 months after the appearance of blunted preovulatory LH surges and cyclic irregularities (prolonged estrus) characteristic of the early stages of reproductive senescence. 48 h after H or HI we observed decreases in ERbeta, microOR and PR (only in the H group) mRNAs and in total ER and microOR proteins, followed by increased PR levels (mRNA and protein) 7 days post-injury and by increased microOR protein and ERbeta mRNA in the H group and ERalpha, ERbeta and microOR mRNAs and ER protein in the HI group 30 days post-injury. Thus, an episode of hypoxia suffered during early postnatal life induces premature reproductive senescence on the female rats, accompanied by early changes in some MBH-PO hormone receptors (microOR, ER and PR), whose expression is intimately involved in the regulation of gonadotropin secretion and female sexual cyclicity.

We assessed immunoreactivity (IR) in the cerebral cortex (CC), hippocampus (Hipp), and striatum (ST) of a growth-associated protein, GAP-43, and of proteins of the synaptic vesicle fusion complex: VAMP-2, Syntaxin-1, and SNAP-25 (SNARE proteins) throughout postnatal development of rats after submitting the animals to acute global postnatal hypoxia (6.5% O(2), 70 min) at postnatal day 4 (PND4). In the CC only the IR of the SNARE protein SNAP-25 increased significantly with age. The hypoxic animals showed the same pattern of IR for SNAP-25, although with lower levels at PND11, and also a significant increase of VAMP-2. SNAP-25 (control): PND11 P < 0.001 vs. PND18, 25, and 40, SNAP-25 (hypoxic): P < 0.001 vs. PND18, 25, and 40; VAMP-2 (hypoxic): P < 0.05 PND11 vs. PND18, and P < 0.01 vs. PND25 and PND40; one-way ANOVA and Bonferroni post-test. In the Hipp, SNAP-25 and syntaxin-1 increased significantly with age, reaching a plateau at PND25 through PND40 in control animals (one-way ANOVA: syntaxin-1: P = 0.043; Bonferroni: NS; SNAP-25: P = 0.013; Bonferroni: P < 0.01 PND11 vs. PND40). Hypoxic rats showed higher levels of significance in the one-way ANOVA than controls (syntaxin-1: P = 0.009; Bonferroni: P < 0.05 PND11 vs. PND25 and P < 0.001 PND11 vs. PND40). In the ST, GAP-43 differed significantly among hypoxic and control animals and the two-way ANOVA revealed significant differences with age (F = 3.23; P = 0.037) and treatment (F = 4.84; P = 0.036). VAMP-2 expression also reached statistical significance when comparing control and treated animals (F = 6.25, P = 0.018) without changes regarding to age. Elevated plus maze test performed at PND40 indicated a lower level of anxiety in the hypoxic animals. At adulthood (12 weeks) learning, memory and locomotor abilities were identical in both groups of animals. With these results, we demonstrate that proteins of the presynaptic structures of the ST are sensitive to acute disruption of homeostatic conditions, such as a temporary decrease of the O(2) concentration. Modifications in the activity of these proteins could contribute to the long term altered responses to stress due to acute hypoxic insult in the neonatal period.

The neocortex and the striatum are the brain regions most known to be particularly vulnerable to acute insults like hypoxia or ischemia. In this work, we assess the possibility of cellular damage to the substantia nigra (SN) after hypoxia-reoxygenation in the new born rat. The aim of the present paper was to evaluate the expression of growth factor IGF-I, and growth factor binding proteins IGFBP-3 and IGFBP-5 genes and induction of NOS family members (nNOS, eNOS and iNOS) and TNF-alpha genes together with glia activation, in the SN at 5 and 48 h after severe hypoxia in the 7 day-old rat, a model for the term human fetus. At early time, while IGFs remain unchanged, we found a transient increase in eNOS and nNOS. Two days after the injury, nNOS expression remained high, iNOS and TNF-alpha increased and also GFAP protein expression was observed together with a profusion of reactive astrocytes distributed throughout the SN. This study on the acute effects of hypoxia on the developing brain provides additional insights into the vulnerability of the SN, a brain region involved in neurodegenerative pathologies.

Hypoxia-ischemia during the perinatal period causes excitotoxic lesions in sensitive brain areas, such as the striatum. The impact of hypoxia-ischemia on nigral neurons is less well known. Hypoxia alone, a less traumatic event without overt histological sequelae, has neuroprotective properties when used as a preconditioning stimulus. In some pathologies, injured neurons of the nigrostriatal system in the adult may be the result of neurodegenerative processes that originated at early stages of life. The effects of hypoxia on the immunoreactivity to tyrosine hydroxylase of the dopaminergic neurons of the substantia nigra pars compacta and the effects of a period of hypoxia previous to an excitotoxic lesion were examined by means of histological and Western blot methods, at immediate and late periods of the episode. By counting the number of tyrosine hydroxylase-stained neurons and c-fos-positive nuclei a short period after injection of quinolinic acid into the striatum, we observed that hypoxia induced a more marked decrease in the number of tyrosine hydroxylase-stained neurons. On the contrary, c-fos-positive profiles decreased in the substantia nigra pars reticulata of the quinolinic acid-injected animals after the preconditioning hypoxia. Hypoxia alone did not affect the number of tyrosine hydroxylase-positive neurons in the pars compacta nor did hypoxia induce c-fos expression in the pars reticulata. More sensitive Western blot analysis of tissue blocks that included the whole substantia nigra demonstrated the same trend as the immunohistochemical results. We conclude that the responses of the substantia nigra neurons to hypoxia are regionalized and potential neuroprotective effects may depend on the vulnerability of each neuronal type.

1. In the present work we describe the short term effects of mild neonatal hypoxia on the synapse as assessed by the immunoreactivity (IR) of two synaptic proteins: rab 3A and synaptobrevin (VAMP). 2. Using the sensitive methodology of immunoblotting, we measured rab 3A and VAMP-IR in homogenates from the cerebral cortex, hippocampus, and corpus striatum of control (breathing room air) and hypoxiated (breathing 95.5% N2-6.5% O2 for 70 min) 4-day-old rats at 1, 2, and 6 h after the end of the hypoxia. Immunostaining with examination by light microscopy was performed using the synaptic protein-specific antibodies on fixed brain sections from animals belonging to the same litter and submitted to hypoxia. 3. A transient increase of VAMP-IR was observed in the hippocampus and corpus striatum, and for rab 3A in the striatum, 1 h after initiating reoxygenation. At the following time points the values returned to control levels. This effect was less clearly observed in the immunostained sections. 4. Mild hypoxia has an effect on sensitive brain regions, eliciting an increase in the IR of at least two proteins involved in the synaptic vesicle cycle. The transient nature of this effect possibly indicates the activation of endogenous neuroprotective mechanisms.

The NMDA receptor subtype is the major excitatory mediator for glutamate neurotoxicity. To assess its participation in the noxious effects of postnatal hypoxia, we have characterized the binding of the ionophoric marker of NMDA receptor, dizocilpine (MK-801). Binding of (+)-3-[125I]MK-801 to NMDA brain receptors under nonequilibrium conditions was quantified by in vitro autoradiography in rats exposed to hypoxia induced by 93% N2/6.5% O2 exposure for 70 min on Postnatal Day 4. Acute and long-lasting effects were investigated at 4 h after injury and on Postnatal Day 40. At the acute stage, a transient decrease in binding was found in several specific brain areas, hypothalamus, amygdaloid nuclei, entorhinal cortex, perirhinal cortex, and hippocampus, and no differences were found in temporal cortex, thalamus, and geniculate nucleus, when compared to sham-treated animals. At this early age, there was no increase of binding when slices from both groups were incubated in the presence of glutamate and glycine (Glu/Gly), positive allosteric modulators of MK-801 binding. In the 40-day-old brains, the binding to the NMDA receptors of hypoxiatreated animals was not different with respect to controls in most of the areas studied, but the Glu/Gly stimulation of binding in hypoxic rats showed a reduced, or absent, response to the allosteric modulators. In contrast, control rats showed a remarkable increase of the specific binding induced by the presence of the modulators in the incubation buffer. Binding of (+)-3-[125I]MK-801 was also performed at a higher concentration to clarify whether the altered response to Glu/Gly may be due to differences in the number of channels; however, the density of NMDA receptors at this concentration was similar in both control and hypoxia-treated rats. We conclude that the effect of exposure of newborn rats to hypoxia can generate acute and long-lasting effects on the NMDA receptor. The deleterious action of this kind of noxa on the CNS could be exerted by interference with normal glutamatergic transmission and hence over normal growth and development.

Obesity, altered pattern of gonadal hormone secretion, advanced vaginal opening, irregular cycling, altered sexual behavior and infertility are the effects of the neonatal administration of monosodium glutamate (MSG) to rodents. These are the consequences of lesions located mainly in the hypothalamic region. It is believed that the receptors to N-methyl-D-aspartic acid (NMDA) actively participate in the onset and development of such lesions, on the other hand, they may be altered by neuronal dysfunction as well, seriously compromising the glutamatergic pathways that are involved in the neuroendocrine regulation. To clarify the scope of the lesion induced by MSG and its probable effects on the NMDA receptors, we measured them with a very sensitive ligand for autoradiography, (+)-3-[125I]MK-801. Coronal cuts at the level of the arcuate-median eminence of brains from 4-, 8- and 40-day-old rats treated neonatally with MSG (4 mg/g) or saline (controls) were examined. In the normal hypothalamus, NMDA receptor labelling was higher in the young animals than in the 40-day-old animals, and this was observed in both control and treated rats. NMDA receptor labelling of rats at puberty was very low, and no apparent differences were observed between groups. In contrast, in areas where an increase in NMDA binding sites normally occurs with development, a significant impairment of the normal augmentation of MK-801 binding was revealed. In the hippocampal layers, stratum radiatum and stratum oriens and in the cerebral cortex of 40-day-old rats treated with MSG a lower amount of binding was observed, of about 50% fewer sites compared to the untreated controls at the level of CA3 and in the outer layer of the parietal cortex. These results suggest that at an early stage of the MSG lesion the NMDA receptors located in the hypothalamus and other brain areas are apparently expressed normally, but at puberty the effects of the lesion are revealed in the hippocampus and cerebral cortex by a decrease in the density of binding. Thus, the abnormal neuroendocrine and behavioral responses displayed by the MSG-treated rats may be contributed partially by the alteration of the NMDA receptors in these areas.

The aim of our study was to determine the second messenger systems for angiotensin II in the rat median eminence. Angiotensin II AT1 receptors are highly expressed in the median eminence and binding is selectively inhibited by the guanine nucleotide GTP gamma S, indicating possible coupling to G-proteins. In male rats, angiotensin II increased phosphatidylinositol hydrolysis about 45% over basal values, with an EC50 of about 2.7 nM. This effect was antagonized by 10 microM losartan, the selective AT1 antagonist, but not by the AT2 competitor PD 123319. Conversely, angiotensin II, 1 microM, did not alter basal or forskolin-stimulated cAMP production, and failed to influence cGMP production. These results support a role for angiotensin II, through stimulation of AT1 receptors and increased phosphatidylinositol hydrolysis, in the median eminence. Angiotensin II increased the phosphatidylinositol hydrolysis not only in male rats but also in ovariectomized rats, with or without estrogen-progesterone replacement. However, angiotensin II (up to 1 microM) failed to increase the phosphatidylinositol hydrolysis in randomly selected intact female rats. Estrogen treatment did not alter the number or affinity of median eminence AT1 receptors in ovariectomized rats. The increase in phosphatidylinositol hydrolysis resulting from stimulation of median eminence AT1 receptors appears to be sexually dimorphic, but hormonal manipulations failed to point to a role for reproductive hormones in this phenomenon.

1. The present review discusses the proposed roles of the amino acids glutamate and GABA in the central regulation of luteinizing hormone-releasing hormone (LHRH) and in luteinizing hormone (LH) secretion. 2. Descriptions of the mechanisms of action of these neurotransmitters have focused on two diencephalic areas, namely, the preoptic-anterior hypothalamic area where the cell bodies of LHRH neurons are located, and the medial basal hypothalamus which contains the nerve endings of the LHRH system. Increasing endogenous GABA concentration by drugs, GABA agonists, or blockade of glutamatergic neurotransmission by selective antagonists in rats and non-human primates prevents ovulation and pulsatile LH release, and blunts the LH surges induced by estrogen or an estrogen-progesterone combination. In contrast, glutamate and different glutamate agonists such as NMDA, AMPA and kainate, can increase LHRH/LH secretion. 3. The simultaneous enhancement of glutamatergic activity and a decrease of GABAergic tone may positively influence the maturation of the pituitary-gonadal system in rats and non-human primates. Administration of glutamate receptor agonists has been shown to significantly advance the onset of puberty. Conversely, glutamate antagonists or increased endogenous GABA levels may delay the onset of puberty. The physiological regulation of LHRH/LH secretion may thus involve a GABA-glutamate interaction and a cooperative action of the various types of ionotropic glutamate receptors. 4. The inhibitory actions of GABA on LH release and ovulation may be exerted at the level of afferent nerve terminals that regulate LHRH secretion. A likely candidate is noradrenaline, as suggested by the synaptic connections between noradrenergic nerve terminals and GABAergic interneurons in the preoptic area. Recent experiments have provided complementary evidence for the physiological balance between inhibitory and excitatory transmission resulting in modulation of the action of noradrenaline to evoke LHRH release.

We used quantitative autoradiography to investigate distribution of angiotensin II (ANG II) receptor subtypes during development of the kidney in the rat. In fetal, newborn, and 3-day-old rats, immature glomeruli in the form of comma and S-shaped bodies, located in the nephrogenic zone of the renal cortex, expressed only the angiotensin AT2 receptor subtype. Conversely, the juxtamedullary glomeruli, in more advanced developmental stages, expressed only the AT1 subtype. Similarly, maturing and fully developed glomeruli, present in 1-, 2-, and 8-wk-old rats, expressed only AT1 receptors. In the kidney medulla, there was a similar change in ANG II receptor subtype expression, with the AT2 subtype expressed earlier and the AT1 subtype later during development. Our results demonstrate a selective expression of ANG II receptor subtypes during kidney development. We have found glomerular and medullary AT1 receptors only at developmental stages when kidney function has matured. Conversely, AT2 receptors are expressed only in immature structures, suggesting that they may have a role during kidney organogenesis.

The relationship between GABA dynamics and LH release was studied on day 2 after subcutaneous estrogen implant in short-term ovariectomized rats. GABA accumulation, used as an index of GABA turnover, was determined in the medial preoptic nucleus (MPN), medial (MS) and lateral (LS) septal nuclei, median eminence-mediobasal hypothalamus (MBH) and locus ceruleus (LC). Measurements of GABA were performed at two different times of day (11.00 and 15.00 h), 3 h after intraperitoneal administration of gamma-vinyl-GABA (GVG), an irreversible inhibitor of GABA transaminase. Either morning or afternoon ovariectomized rats (OVX) showed a significant increase in GABA accumulation after GVG treatment in all the areas studied. Estrogen-treated OVX rats showed in the morning a lower GABA accumulation in the MPN, MBH and LC, and GABA levels remained unchanged in the LS and MS. In the afternoon, the MPN and LS showed a lower rate of GABA accumulation whereas in the MBH and LC the GABA increase was not observed. In contrast the MS showed a rate of GABA accumulation similar as in the OVX rats. Local administration in the MPN of 20 micrograms GVG, or GABA-A receptor stimulation by muscimol (50 ng), prior to the increase in plasma LH levels, prevented the occurrence of the estradiol-induced LH surge. The effect of muscimol was reversed by bicuculline (30 ng), a GABA-A receptor antagonist. Bicuculline in low doses lacked effect by itself. In conclusion, these results strongly suggest that a decreased GABAergic activity in MPN, MBH and LC precedes the estradiol-evoked LH surges in ovariectomized rats. Moreover, that in septal nuclei, a low GABAergic activity takes place well before the occurrence of plasma LH increase.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of ovariectomy and ovarian steroids on 3H-mepyramine binding, a histamine antagonist considered as a suitable ligand for H1-histamine receptors, were studied. Saturation binding assays and Scatchard plot analyses were performed in membranes from hypothalamus of adult female rats. In the hypothalamus of ovariectomized (OVX) rats, a decrease of the Bmax and a lowering of Kd values as compared to diestrous rats, were found. Estradiol administration to OVX rats as a single SC injection (25 micrograms), or by means of SC Silastic capsules (500 micrograms/ml estradiol benzoate; 10 mm/100 g b.wt.), reversed the effects of OVX, increasing both Bmax and Kd. The addition of progesterone (25 mg) blunted the effects of estradiol (50 micrograms). The present results suggest that hypothalamic H1-histamine receptors may be modulated by the ovarian steroids and provide further evidence of a possible role of histamine in female gonadotropin regulation.

The role of tuberoinfundibular dopamine (DA) in the histamine-induced rise of plasma prolactin was studied in male rats. Right lateral ventricle (icv) histamine injection (30 micrograms/rat) caused a significant rise of plasma prolactin at 15 and 30 min; at 60 min values returned to basal levels. Histamine does not modified steady-state DA concentrations. For turnover evaluation, histamine was icv injected immediately or 30 min after the tyrosine hydroxylase inhibitor alpha-methyltyrosine (250 mg/kg i.p.) and DA concentration in the median eminence was measured at 0, 1 and 2 hours after the inhibitor injection. Rate constants of DA decline and DA synthesis rates were found similar in both controls (cerebrospinal fluid) and histamine-injected rats. These results indicate that the stimulatory action of histamine on prolactin release might not be associated to DA. It would be due mainly to its action on prolactin-releasing factors and to a minor extent, to changes in the dopaminergic system.

The possible role of brain histamine in the release of prolactin, ACTH and corticosterone following acute restraint, was pharmacologically evaluated in adult male rats. Fifteen min of restraint caused marked increases in the plasma levels of these hormones. alpha-Fluoromethyl histidine (FH), a histidine decarboxylase inhibitor which depleted hypothalamic histamine, inhibited the enhancement of plasma prolactin levels. In contrast, plasma ACTH levels were not modified. FH treatment decreased plasma corticosterone concentrations in animals submitted to stress or in rest; this suggests a direct action of FH on the adrenal. Intraventricular (IVT) injection of ranitidine (H2 antagonist) blunted the prolactin response to restraint stress whereas its systemic administration had no effect. On the contrary, pyrilamine (H1 antagonist) given systemically decreased slightly, but significantly, the prolactin rise but when injected IVT it was ineffective. Pyrilamine was also unable to affect the ranitidine action. ACTH and corticosterone levels in plasma of restrained rats were not modified by the histamine antagonists. It is concluded that histamine is involved, mainly through central H2 receptors, in the enhancement of plasma prolactin levels produced by an acute stress. The failure of both antihistaminic compounds and a histamine depletor to alter the ACTH stimulation suggest that histamine has no participation in the hypophysio-corticoadrenal response to acute restraint.