Evidence for intraventricular secretion of angiotensinogen and angiotensin by the subfornical organ using transgenic mice.

Direct intracerebroventricular injection of angiotensin II (ANG II) causes increases in blood pressure and salt and water intake, presumably mimicking an effect mediated by an endogenous mechanism. The subfornical organ (SFO) is a potential source of cerebrospinal fluid (CSF), ANG I, and ANG II, and thus we hypothesized that the SFO has a secretory function. Endogenous levels of angiotensinogen (AGT) and renin are very low in the brain. We therefore examined the immunohistochemical localization of angiotensin peptides and AGT in the SFO, and AGT in the CSF in two transgenic models that overexpress either human AGT (A+ mice), or both human AGT (hAGT) and human renin (SRA mice) in the brain. Measurements were made at baseline and following volumetric depletion of CSF. Ultrastructural analysis with immunoelectron microscopy revealed that superficially located ANG I/ANG II and AGT immunoreactive cells in the SFO were vacuolated and opened directly into the ventricle. Withdrawal of CSF produced an increase in AGT in the CSF that was accompanied by a large decline in AGT immunoreactivity within SFO cells. Our data provide support for the hypothesis that the SFO is a secretory organ that releases AGT and possibly ANG I/ANG II into the ventricle at least under conditions when genes that control the renin-angiotensin system are overexpressed in mice.

Activation of the renin-angiotensin system, specifically in the subfornical organ is sufficient to induce fluid intake.

Increased activity of the renin-angiotensin system within the brain elevates fluid intake, blood pressure, and resting metabolic rate. Renin and angiotensinogen are coexpressed within the same cells of the subfornical organ, and the production and action of ANG II through the ANG II type 1 receptor in the subfornical organ (SFO) are necessary for fluid intake due to increased activity of the brain renin-angiotensin system. We generated an inducible model of ANG II production by breeding transgenic mice expressing human renin in neurons controlled by the synapsin promoter with transgenic mice containing a Cre-recombinase-inducible human angiotensinogen construct. Adenoviral delivery of Cre-recombinase causes SFO-selective induction of human angiotensinogen expression. Selective production of ANG II in the SFO results in increased water intake but did not change blood pressure or resting metabolic rate. The increase in water intake was ANG II type 1 receptor-dependent. When given a choice between water and 0.15 M NaCl, these mice increased total fluid and sodium, but not water, because of an increased preference for NaCl. When provided a choice between water and 0.3 M NaCl, the mice exhibited increased fluid, water, and sodium intake, but no change in preference for NaCl. The increase in fluid intake was blocked by an inhibitor of PKC, but not ERK, and was correlated with increased phosphorylated cyclic AMP response element binding protein in the subfornical organ. Thus, increased production and action of ANG II specifically in the subfornical organ are sufficient on their own to mediate an increase in drinking through PKC.

Hypertension in mice with transgenic activation of the brain renin-angiotensin system is vasopressin dependent.

An indispensable role for the brain renin-angiotensin system (RAS) has been documented in most experimental animal models of hypertension. To identify the specific efferent pathway activated by the brain RAS that mediates hypertension, we examined the hypothesis that elevated arginine vasopressin (AVP) release is necessary for hypertension in a double-transgenic model of brain-specific RAS hyperactivity (the "sRA" mouse model). sRA mice experience elevated brain RAS activity due to human angiotensinogen expression plus neuron-specific human renin expression. Total daily loss of the 4-kDa AVP prosegment (copeptin) into urine was grossly elevated (≥8-fold). Immunohistochemical staining for AVP was increased in the supraoptic nucleus of sRA mice (~2-fold), but no quantitative difference in the paraventricular nucleus was observed. Chronic subcutaneous infusion of a nonselective AVP receptor antagonist conivaptan (YM-087, Vaprisol, 22 ng/h) or the V(2)-selective antagonist tolvaptan (OPC-41061, 22 ng/h) resulted in normalization of the baseline (~15 mmHg) hypertension in sRA mice. Abdominal aortas and second-order mesenteric arteries displayed AVP-specific desensitization, with minor or no changes in responses to phenylephrine and endothelin-1. Mesenteric arteries exhibited substantial reductions in V(1A) receptor mRNA, but no significant changes in V(2) receptor expression in kidney were observed. Chronic tolvaptan infusion also normalized the (5 mmol/l) hyponatremia of sRA mice. Together, these data support a major role for vasopressin in the hypertension of mice with brain-specific hyperactivity of the RAS and suggest a primary role of V(2) receptors.

Ablation of the leptin receptor in the hypothalamic arcuate nucleus abrogates leptin-induced sympathetic activation.

RATIONALE: The hypothalamic arcuate nucleus (ARC) is considered a major site for leptin signaling that regulates several physiological processes. OBJECTIVE: To test the hypothesis that leptin receptor in the ARC is required to mediate leptin-induced sympathetic activation. METHODS AND RESULTS: First, we used the ROSA Cre-reporter mice to establish the feasibility of driving Cre expression in the ARC in a controlled manner with bilateral microinjection of adenovirus-expressing Cre-recombinase (Ad-Cre). Ad-Cre microinjection into the ARC of ObR(flox/flox) mice robustly reduced ObR expression and leptin-induced Stat3 activation in the ARC but not in the adjacent nuclei, confirming the efficacy and selectivity of the ARC deletion of ObR. Critically, deletion of ObR in the ARC attenuated brown adipose tissue and renal sympathetic nerve responses to leptin. We also examined whether ObR in the ARC is required for the preserved leptin-induced increase in renal sympathetic activity in dietary obesity. We found that deletion of ARC ObR abrogated leptin-induced increases in renal sympathetic discharge and resolved arterial pressure elevation in diet-induced obese ObR(flox/flox) mice. CONCLUSIONS: These data demonstrate a critical role for ObR in the ARC in mediating the sympathetic nerve responses to leptin and in the adverse sympathoexcitatory effects of leptin in obesity.

Dimensions of subcortical infarcts associated with first- to third-order branches of the basal ganglia arteries.

BACKGROUND: It has been described that lacunar infarct is characterized by its smallish size (15-20 mm) in the axial plane. However, the size of the basal ganglia artery responsible for this type of infarct is uncertain. Detection of small arterial occlusion is not possible with current angiography, hindering correlation of arterial occlusion with subcortical infarct size. Recently, investigators have published microangiographic templates of arteries supplying the basal ganglia. These templates display first-order (proximal) to third-order (distal) branching of these arteries and can help with estimating the likely site of arterial disease in subcortical infarcts. We correlated the dimensions of subcortical infarcts with the order of arterial branching described in a microangiographic template. Such data may provide further clues about the type of arteries associated with subcortical infarcts and assist in refining the concept of lacunar infarction. METHOD: Patients with subcortical infarcts on MR imaging (MRI) admitted to our institution between 2009 and 2011 were included in the study. Infarcts were manually segmented and registered to a standard brain template. These segmented infarcts were scaled and overlapped with published microangiographic templates, and used by 6 raters who independently estimated the branching order of arterial disease that might result in these infarcts. We used regression analysis to relate these ratings to infarct dimensions. RESULTS: Among 777 patients, there were 33 (58% male) patients with subcortical infarcts. The mean age was 63.1 ± 15.1 years. Infarct dimensions for the groups were as follows: group 1 (first-order branch): height 37.6 ± 7.4 mm, horizontal width 21.2 ± 11.6 mm, anterior-posterior length 36.8 ± 20.1 mm; group 2 (second-order branch): height 25.2 ± 7.9 mm, horizontal width 16.6 ± 22.8 mm, anterior-posterior length 16.1 ± 8.0 mm; group 3 (third-order branch): height 11.6 ± 5.7 mm, axial width 5.3 ± 3.1 mm, anterior-posterior length 5.5 ± 3.8 mm. Increasing vessel branching order (from large to small vessels) was linearly and negatively associated with infarct height (ß = -16.7 mm per change in branching order disease, 95% CI -20.3, -13.1 mm, p < 0.01) and anterior-posterior length (ß = -16.8 mm per change in branching order disease, 95% CI -23.2, -10.5 mm, p < 0.01). DISCUSSION: Based on MRI infarct dimensions and a microangiographic template, it may be possible to estimate the branching order of the artery involved in subcortical infarcts. Further, our small data set suggests that reliance on an axial dimension of 15-20 mm may not be the best approach to classifying lacunar infarct. This finding needs to be confirmed in a larger data set.

Neuron- or glial-specific ablation of secreted renin does not affect renal renin, baseline arterial pressure, or metabolism.

The renin-angiotensin system (RAS), known for its roles in cardiovascular, metabolic, and developmental regulation, is present in both the circulation and in many individual tissues throughout the body. Substantial evidence supports the existence of a brain RAS, though quantification and localization of brain renin have been hampered by its low expression levels. We and others have previously determined that there are two isoforms of renin expressed in the brain. The classical isoform encoding secreted renin (sREN) and a novel isoform encoding intracellular renin (icREN), the product of an alternative promoter and first exon (exon 1b). The differential role that these two isoforms play in cardiovascular and metabolic regulation remains unclear. Here we examined the physiological consequences of neuron- and glia-specific knockouts of sREN by crossing mice in which the sREN promoter and isoform-specific first exon (exon-1a) is flanked by LoxP sequences (sREN(flox) mice) with mice expressing Cre-recombinase controlled by either the neuron-specific Nestin promoter or the glia-specific GFAP promoter. Resulting offspring exhibited selective knockout of sREN in either neurons or glia, while preserving expression of icREN. Consistent with a hypothesized role of icREN in the brain RAS, neuron- and glia-specific knockout of sREN had no effect on blood pressure or heart rate; food, water, or sodium intake; renal function; or metabolic rate. These data demonstrate that sREN is dispensable within the brain for normal physiological regulation of cardiovascular, hydromineral, and metabolic regulation, and thereby indirectly support the importance of icREN in brain RAS function.

Local production of angiotensin II in the subfornical organ causes elevated drinking.

The mechanism controlling cell-specific Ang II production in the brain remains unclear despite evidence supporting neuron-specific renin and glial- and neuronal-specific angiotensinogen (AGT) expression. We generated double-transgenic mice expressing human renin (hREN) from a neuron-specific promoter and human AGT (hAGT) from its own promoter (SRA mice) to emulate this expression. SRA mice exhibited an increase in water and salt intake and urinary volume, which were significantly reduced after chronic intracerebroventricular delivery of losartan. Ang II-like immunoreactivity was markedly increased in the subfornical organ (SFO). To further evaluate the physiological importance of de novo Ang II production specifically in the SFO, we utilized a transgenic mouse model expressing a floxed version of hAGT (hAGT(flox)), so that deletions could be induced with Cre recombinase. We targeted SFO-specific ablation of hAGT(flox) by microinjection of an adenovirus encoding Cre recombinase (AdCre). SRA(flox) mice exhibited a marked increase in drinking at baseline and a significant decrease in water intake after administration of AdCre/adenovirus encoding enhanced GFP (AdCre/AdEGFP), but not after administration of AdEGFP alone. This decrease only occurred when Cre recombinase correctly targeted the SFO and correlated with a loss of hAGT and angiotensin peptide immunostaining in the SFO. These data provide strong genetic evidence implicating de novo synthesis of Ang II in the SFO as an integral player in fluid homeostasis.

A knockin mouse model of the Bardet-Biedl syndrome 1 M390R mutation has cilia defects, ventriculomegaly, retinopathy, and obesity.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder that results in retinal degeneration, obesity, cognitive impairment, polydactyly, renal abnormalities, and hypogenitalism. Of the 12 known BBS genes, BBS1 is the most commonly mutated, and a single missense mutation (M390R) accounts for approximately 80% of BBS1 cases. To gain insight into the function of BBS1, we generated a Bbs1(M390R/M390R) knockin mouse model. Mice homozygous for the M390R mutation recapitulated aspects of the human phenotype, including retinal degeneration, male infertility, and obesity. The obese mutant mice were hyperphagic and hyperleptinemic and exhibited reduced locomotor activity but no elevation in mean arterial blood pressure. Morphological evaluation of Bbs1 mutant brain neuroanatomy revealed ventriculomegaly of the lateral and third ventricles, thinning of the cerebral cortex, and reduced volume of the corpus striatum and hippocampus. Similar abnormalities were also observed in the brains of Bbs2(-/-), Bbs4(-/-), and Bbs6(-/-) mice, establishing these neuroanatomical defects as a previously undescribed BBS mouse model phenotype. Ultrastructural examination of the ependymal cell cilia that line the enlarged third ventricle of the Bbs1 mutant brains showed that, whereas the 9 + 2 arrangement of axonemal microtubules was intact, elongated cilia and cilia with abnormally swollen distal ends were present. Together with data from transmission electron microscopy analysis of photoreceptor cell connecting cilia, the Bbs1 M390R mutation does not affect axonemal structure, but it may play a role in the regulation of cilia assembly and/or function.

Co-localization of caldesmon and calponin with cortical afferents, metabotropic glutamate and neurotrophic receptors in the lateral and central nuclei of the amygdala.

Caldesmon (Cd) and calponin (Cp) are two actin/calmodulin-binding proteins involved in 'actin-linked' regulation of smooth muscle and non-muscle Mg(2+) actin-activated myosin II ATPase activity. However, in the brain, Cd and Cp are associated with the regulation of the neuronal cytoskeleton. In this study we investigated the subcellular distribution of Cd and Cp in the amygdala and their possible relationship to metabotropic glutamate (mGluR1 alpha and 5) and TrkB receptors which interact with inputs from the cortex and are involved in associative learning. Cd and Cp immunoreactivity (IR) was mainly found in dendritic spines, along dendritic microtubules, and in neuronal perikarya but never in axon terminals. Punctate labeling representing spine labeling was restricted to small patches in the lateral nucleus of amygdala, intercalated cell masses (ICM), and the lateral subdivision of central nucleus. This restricted distribution may reflect local afferent activation. In addition, Cd, Cp, mGluR1 alpha and cortical afferents are co-distributed in the ICM distributed in the lateral nucleus and lateral capsular division of the central nucleus, and the lateral division of the central nucleus itself. Consistent with our previous studies, TrkB IR in the central nucleus was associated with Cd and Cp-immunoreactive spines whereas mGluR1 alpha IR and mGluR5 IR were almost exclusively associated with the PSDs of asymmetric synapses, in most cases apposed by cortical terminals. mGluR1 alpha and TrkB immunoreactivities were invariably associated with each other. Overall, these findings suggest that caldesmon and calponin in the amygdala are closely associated with afferents and receptors that have been strongly implicated in associative learning.

The vascular supply of the functional compartments of the human striatum.

The basal ganglia (BG) contain several functional compartments and multiple, parallel segregated circuits processing different cortical information through cortical-BG-thalamus-cortical loops. Three zones of corticostriatal input are present: sensorimotor, associative and limbic, which correspond to poor, intermediate and strong calbindin (CB) labelling, respectively. Other functional compartments, such as striosomes, extrastriosomal matrix and matrisomes, also convey segregated projections. Microvascular territories in the human BG are spatially consistent with little overlap and few anastomoses. A high percentage of lacunar infarcts occur in the BG, yet the relationship between lacunae and functional compartments is unknown. We determined the relationship between microvascular territories and functional compartments within the human striatum. Microvascular territories were labelled by co-injection of diffusible dye, radio-opaque substance and gelatin into parental vessels and by sectioning each BG co-planar with the Talairach system. Sections underwent immunocytochemistry or histochemistry and the overlap of microvascular and functional territories was examined. CB staining of the arterial-injected striatum matched the functional compartments reported previously and overlay of microvascular territories revealed a correspondence between (i) the lateral lenticulostriate arteries (LSA) and sensorimotor zone; (ii) the medial LSA and associative zone; and (iii) the recurrent artery of Heubner (RAH) and limbic zone. A greater number of large vessels and capillaries were found in the matrix compared to striosomes, and a likely correspondence exists between high-density arteriole envelopes and matrisomes. The higher number of non-anastomotic vessels and capillary beds within the matrix predisposes these regions to both large lesions and small lacunar infarcts, creating specific symptoms based on striatal circuitry.

Obesity-induced hepatic steatosis is mediated by endoplasmic reticulum stress in the subfornical organ of the brain.

Nonalcoholic fatty liver disease (NAFLD), characterized by an excess accumulation of hepatic triglycerides, is a growing health epidemic. While ER stress in the liver has been implicated in the development of NAFLD, the role of brain ER stress - which is emerging as a key contributor to a number of chronic diseases including obesity - in NAFLD remains unclear. These studies reveal that chemical induction of ER stress in the brain caused hepatomegaly and hepatic steatosis in mice. Conversely, pharmacological reductions in brain ER stress in diet-induced obese mice rescued NAFLD independent of body weight, food intake, and adiposity. Evaluation of brain regions involved revealed robust activation of ER stress biomarkers and ER ultrastructural abnormalities in the circumventricular subfornical organ (SFO), a nucleus situated outside of the blood-brain-barrier, in response to high-fat diet. Targeted reductions in SFO-ER stress in obese mice via SFO-specific supplementation of the ER chaperone 78-kDa glucose-regulated protein ameliorated hepatomegaly and hepatic steatosis without altering body weight, food intake, adiposity, or obesity-induced hypertension. Overall, these findings indicate a novel role for brain ER stress, notably within the SFO, in the pathogenesis of NAFLD.

Neurotrophic factors in the central nucleus of amygdala may be organized to provide substrates for associative learning.

The central nucleus of amygdala was examined to identify the ultrastructural distribution of neurotrophins responsible for the complex of neuronal signaling processes which regulate synaptic transmission and neuronal plasticity, and possibly underlie memory formation. We investigated at the electron microscopic level the cellular organization of brain-derived neurotrophic factor (BDNF) and its receptor, tyrosine kinase receptor B (TrkB), in the extended amygdala (CE). We also investigated the interaction between cortical inputs to CE and BDNF and TrkB. Our results indicate the presence of pro-BDNF and BDNF in terminals in the CE which show a strong association with immunoreactive postsynaptic densities. TrkB receptor immunoreactivity was localized to postsynaptic densities of asymmetric synapses on dendrites and dendritic spines. Cortical terminals formed asymmetric synapses with dendritic shafts and spines, but were not BDNF immunoreactive. TrkB receptors were observed opposed to cortical terminals. These data also suggest that one potential substrate for associative learning may be the interaction of different cortical inputs with neurotrophin-containing terminals ending on dendritic spines and other neuronal structures of CE.

The brain renin-angiotensin system in transgenic mice carrying a highly regulated human renin transgene.

We previously reported the generation of 2 novel transgenic mouse models containing the human renin (hREN) gene encoded on P1 artificial chromosomes (PAC) containing large amounts of 5'-flanking DNA. These mice exhibit a very narrow tissue-specific expression profile and exhibit tightly regulated expression in kidney in response to physiological cues. In brain, transcription of hREN occurs from an alternative upstream promoter, causing translation to initiate within exon-II and potentially generating an intracellular form of active renin. Double transgenic mice containing a PAC transgene and the human angiotensinogen (hAGT) gene (P+/A+) are moderately hypertensive. We tested whether increased RAS activity in the brain contributes to the mechanism of hypertension in P+/A+ double transgenic mice. Expression of hREN mRNA in brain was confirmed in 4 independent PAC transgenic lines and utilization of the alternative transcription start site in brain was confirmed in each line. Human REN immunostaining was observed in the dorsal cochlear nucleus, hypothalamus, and cortex. P+/A+ mice exhibited a greater fall in mean arterial pressure after intracerebroventricular injection of losartan than controls. P+/A+ mice exhibited a greater drop in arterial pressure after intravenous injection of a vasopressin V(1) receptor antagonist, and an equivalent drop in arterial pressure after intravenous injection of a ganglion blocker compared with controls. These results support the hypothesis that renin is endogenously expressed in the brain and suggest that increased brain RAS activity may contribute to the maintenance of moderate hypertension in P+/A+ transgenic mice at least in part by a vasopressin-dependent mechanism.

Selective Deletion of the Brain-Specific Isoform of Renin Causes Neurogenic Hypertension.

The renin-angiotensin system (RAS) in the brain is a critical determinant of blood pressure, but the mechanisms regulating RAS activity in the brain remain unclear. Expression of brain renin (renin-b) occurs from an alternative promoter-first exon. The predicted translation product is a nonsecreted enzymatically active renin whose function is unknown. We generated a unique mouse model by selectively ablating the brain-specific isoform of renin (renin-b) while preserving the expression and function of the classical isoform expressed in the kidney (renin-a). Preservation of renal renin was confirmed by measurements of renin gene expression and immunohistochemistry. Surprisingly, renin-b-deficient mice exhibited hypertension, increased sympathetic nerve activity to the kidney and heart, and impaired baroreflex sensitivity. Whereas these mice displayed decreased circulating RAS activity, there was a paradoxical increase in brain RAS activity. Physiologically, renin-b-deficient mice exhibited an exaggerated depressor response to intracerebroventricular administration of losartan, captopril, or aliskiren. At the molecular level, renin-b-deficient mice exhibited increased expression of angiotensin-II type 1 receptor in the paraventricular nucleus, which correlated with an increased renal sympathetic nerve response to leptin, which was dependent on angiotensin-II type 1 receptor activity. Interestingly, despite an ablation of renin-b expression, expression of renin-a was significantly increased in rostral ventrolateral medulla. These data support a new paradigm for the genetic control of RAS activity in the brain by a coordinated regulation of the renin isoforms, with expression of renin-b tonically inhibiting expression of renin-a under baseline conditions. Impairment of this control mechanism causes neurogenic hypertension.

Nervous System Expression of PPARγ and Mutant PPARγ Has Profound Effects on Metabolic Regulation and Brain Development.

Peroxisome proliferator activated receptor (PPARγ) is a nuclear receptor transcription factor that regulates adipogenesis and energy homeostasis. Recent studies suggest PPARγ may mediate some of its metabolic effects through actions in the brain. We used a Cre-recombinase-dependent (using NestinCre) conditionally activatable transgene expressing either wild-type (WT) or dominant-negative (P467L) PPARγ to examine mechanisms by which PPARγ in the nervous system controls energy balance. Inducible expression of PPARγ was evident throughout the brain. Expression of 2 PPARγ target genes, aP2 and CD36, was induced by WT but not P467L PPARγ in the brain. Surprisingly, NesCre/PPARγ-WT mice exhibited severe microcephaly and brain malformation, suggesting that PPARγ can modulate brain development. On the contrary, NesCre/PPARγ-P467L mice exhibited blunted weight gain to high-fat diet, which correlated with a decrease in lean mass and tissue masses, accompanied by elevated plasma GH, and depressed plasma IGF-1, indicative of GH resistance. There was no expression of the transgene in the pancreas but both fasting plasma glucose, and fed and fasted plasma insulin levels were markedly decreased. NesCre/PPARγ-P467L mice fed either control diet or high-fat diet displayed impaired glucose tolerance yet exhibited increased sensitivity to exogenous insulin and increased insulin receptor signaling in white adipose tissue, liver, and skeletal muscle. These observations support the concept that alterations in PPARγ-driven mechanisms in the nervous system play a role in the regulation of growth and glucose metabolic homeostasis.

FGF21 Mediates Endocrine Control of Simple Sugar Intake and Sweet Taste Preference by the Liver.

The liver is an important integrator of nutrient metabolism, yet no liver-derived factors regulating nutrient preference or carbohydrate appetite have been identified. Here we show that the liver regulates carbohydrate intake through production of the hepatokine fibroblast growth factor 21 (FGF21), which markedly suppresses consumption of simple sugars, but not complex carbohydrates, proteins, or lipids. Genetic loss of FGF21 in mice increases sucrose consumption, whereas acute administration or overexpression of FGF21 suppresses the intake of both sugar and non-caloric sweeteners. FGF21 does not affect chorda tympani nerve responses to sweet tastants, instead reducing sweet-seeking behavior and meal size via neurons in the hypothalamus. This liver-to-brain hormonal axis likely represents a negative feedback loop as hepatic FGF21 production is elevated by sucrose ingestion. We conclude that the liver functions to regulate macronutrient-specific intake by producing an endocrine satiety signal that acts centrally to suppress the intake of "sweets."

Acid-sensing ion channel 1 is localized in brain regions with high synaptic density and contributes to fear conditioning.

The acid-sensing ion channel, ASIC1, contributes to synaptic plasticity in the hippocampus and to hippocampus-dependent spatial memory. To explore the role of ASIC1 in brain, we examined the distribution of ASIC1 protein. Surprisingly, although ASIC1 was present in the hippocampal circuit, it was much more abundant in several areas outside the hippocampus. ASIC1 was enriched in areas with strong excitatory synaptic input such as the glomerulus of the olfactory bulb, whisker barrel cortex, cingulate cortex, striatum, nucleus accumbens, amygdala, and cerebellar cortex. Because ASIC1 levels were particularly high in the amygdala, we focused further on this area. We found that extracellular acidosis elicited a greater current density in amygdala neurons than hippocampal neurons and that disrupting the ASIC1 gene eliminated H+-evoked currents in the amygdala. We also tested the effect of ASIC1 on amygdala-dependent behavior; ASIC1-null mice displayed deficits in cue and context fear conditioning, yet baseline fear on the elevated plus maze was intact. These studies suggest that ASIC1 is distributed to regions supporting high levels of synaptic plasticity and contributes to the neural mechanisms of fear conditioning.

The Vulnerable Ventral Tegmental Area in Parkinson's Disease.

INTRODUCTION: The involvement of dopaminergic neurons in the ventral tegmental area (VTA) in Parkinson's disease (PD) has not been universally recognized by neuroscientists and neurologists. Here, we conduct a review of previous research documenting dopaminergic neuronal loss in both the substantia nigra pars compacta (SNpc) and VTA and add three new post-mortem PD cases to the literature. METHODS: PD and control brains were sectioned, stained for tyrosine hydroxylase, and cells in the SNpc and VTA were counted. RESULTS: Based on the review, we report two main results: 1) the VTA does degenerate in PD, and 2) the VTA degenerates less than the SNpc. CONCLUSION: Inconsistent clinical information about these cases limits our ability to interpret how the VTA contributes to PD symptoms. However, our data in combination with prior PD neuropathological cases in the literature unequivocally establish that the VTA is involved in PD, and could be relevant for future investigation of non-motor symptoms in PD.

Brain endoplasmic reticulum stress mechanistically distinguishes the saline-intake and hypertensive response to deoxycorticosterone acetate-salt.

Endoplasmic reticulum stress has become an important mechanism in hypertension. We examined the role of endoplasmic reticulum stress in mediating the increased saline-intake and hypertensive effects in response to deoxycorticosterone acetate (DOCA)-salt. Intracerebroventricular delivery of the endoplasmic reticulum stress-reducing chemical chaperone tauroursodeoxycholic acid did not affect the magnitude of hypertension, but markedly decreased saline-intake in response to DOCA-salt. Increased saline-intake returned after tauroursodeoxycholic acid was terminated. Decreased saline-intake was also observed after intracerebroventricular infusion of 4-phenylbutyrate, another chemical chaperone. Immunoreactivity to CCAAT homologous binding protein, a marker of irremediable endoplasmic reticulum stress, was increased in the subfornical organ and supraoptic nucleus of DOCA-salt mice, but the signal was absent in control and CCAAT homologous binding protein-deficient mice. Electron microscopy revealed abnormalities in endoplasmic reticulum structure (decrease in membrane length, swollen membranes, and decreased ribosome numbers) in the subfornical organ consistent with endoplasmic reticulum stress. Subfornical organ-targeted adenoviral delivery of GRP78, a resident endoplasmic reticulum chaperone, decreased DOCA-salt-induced saline-intake. The increase in saline-intake in response to DOCA-salt was blunted in CCAAT homologous binding protein-deficient mice, but these mice exhibited a normal hypertensive response. We conclude that (1) brain endoplasmic reticulum stress mediates the saline-intake, but not blood pressure response to DOCA-salt, (2) DOCA-salt causes endoplasmic reticulum stress in the subfornical organ, which when attenuated by GRP78 blunts saline-intake, and (3) CCAAT homologous binding protein may play a functional role in DOCA-salt-induced saline-intake. The results suggest a mechanistic distinction between the importance of endoplasmic reticulum stress in mediating effects of DOCA-salt on saline-intake and blood pressure.

Neuron-specific expression of human angiotensinogen in brain causes increased salt appetite.

The brain renin-angiotensin system (RAS) has an important role in the regulation of cardiovascular function. In the brain, angiotensinogen (AGT) is expressed mainly in astrocytes (glia) and in some neurons in regions controlling cardiovascular activities. Because of the inability to dissect the functional role of astrocyte- vs. neuron-derived AGT in vivo by pharmacological approaches, the exact role of neuron-derived AGT in the regulation of blood pressure (BP) and fluid and electrolyte balance remains unclear. Therefore, we generated a transgenic mouse model overexpressing human AGT under the control of a neuron-specific (synapsin I) promoter (SYN-hAGT). These mice exhibited high-level expression of human AGT mRNA in the brain, with lower expression in the kidney and heart. Human AGT was not detected in plasma, but in the brain it was expressed exclusively in neurons. Intracerebroventricular (30 ng) but not intravenous (500 ng) injection of purified human renin (hREN) caused a pressor response, which was prevented by intracerebroventricular preinjection of the angiotensin II type 1 receptor antagonist losartan, indicating an AT(1) receptor-dependent functional role of neuron-derived AGT in the regulation of BP in response to exogenous REN. Double transgenic mice expressing both the hREN gene and SYN-hAGT transgene exhibited normal BP and water intake but had an increased preference for salt. These data suggest that neuronal AGT may play an important role in regulating salt intake and salt appetite.