COX-1-derived PGE2 and PGE2 type 1 receptors are vital for angiotensin II-induced formation of reactive oxygen species and Ca(2+) influx in the subfornical organ.

Regulation of blood pressure by angiotensin II (ANG II) is a process that involves the reactive oxygen species (ROS) and calcium. We have shown that ANG-II type 1 receptor (AT1R) and prostaglandin E2 (PGE2) type 1 receptors (EP1R) are required in the subfornical organ (SFO) for ROS-mediated hypertension induced by slow-pressor ANG-II infusion. However, the signaling pathway associated with this process remains unclear. We sought to determine mechanisms underlying the ANG II-induced ROS and calcium influx in mouse SFO cells. Ultrastructural studies showed that cyclooxygenase 1 (COX-1) codistributes with AT1R in the SFO, indicating spatial proximity. Functional studies using SFO cells revealed that ANG II potentiated PGE2 release, an effect dependent on AT1R, phospholipase A2 (PLA2) and COX-1. Furthermore, both ANG II and PGE2 increased ROS formation. While the increase in ROS initiated by ANG II, but not PGE2, required the activation of the AT1R/PLA2/COX-1 pathway, both ANG II and PGE2 were dependent on EP1R and Nox2 as downstream effectors. Finally, ANG II potentiated voltage-gated L-type Ca(2+) currents in SFO neurons via the same signaling pathway required for PGE2 production. Blockade of EP1R and Nox2-derived ROS inhibited ANG II and PGE2-mediated Ca(2+) currents. We propose a mechanism whereby ANG II increases COX-1-derived PGE2 through the AT1R/PLA2 pathway, which promotes ROS production by EP1R/Nox2 signaling in the SFO. ANG II-induced ROS are coupled with Ca(2+) influx in SFO neurons, which may influence SFO-mediated sympathoexcitation. Our findings provide the first evidence of a spatial and functional framework that underlies ANG-II signaling in the SFO and reveal novel targets for antihypertensive therapies.

New insights into the role of hilar ectopic granule cells in the dentate gyrus based on quantitative anatomic analysis and three-dimensional reconstruction.

The dentate gyrus is one of two main areas of the mammalian brain where neurons are born throughout adulthood, a phenomenon called postnatal neurogenesis. Most of the neurons that are generated are granule cells (GCs), the major principal cell type in the dentate gyrus. Some adult-born granule cells develop in ectopic locations, such as the dentate hilus. The generation of hilar ectopic granule cells (HEGCs) is greatly increased in several animal models of epilepsy and has also been demonstrated in surgical specimens from patients with intractable temporal lobe epilepsy (TLE). Herein we review the results of our quantitative neuroanatomic analysis of HEGCs that were filled with Neurobiotin following electrophysiologic characterization in hippocampal slices. The data suggest that two types of HEGCs exist, based on a proximal or distal location of the cell body relative to the granule cell layer, and based on the location of most of the dendrites, in the molecular layer or hilus. Three-dimensional reconstruction revealed that the dendrites of distal HEGCs can extend along the transverse and longitudinal axis of the hippocampus. Analysis of axons demonstrated that HEGCs have projections that contribute to the normal mossy fiber innervation of CA3 as well as the abnormal sprouted fibers in the inner molecular layer of epileptic rodents (mossy fiber sprouting). These data support the idea that HEGCs could function as a "hub" cell in the dentate gyrus and play a critical role in network excitability.

The neurokinin-3 (NK3) and the neurokinin-1 (NK1) receptors are differentially targeted to mesocortical and mesolimbic projection neurons and to neuronal nuclei in the rat ventral tegmental area.

Tonic activation of neurokinin-3 (NK(3)) receptors in dopamine neurons of the ventral tegmental area (VTA) has been implicated in the pathophysiology of schizophrenia. This psychiatric disorder is associated with a dysfunctional activity in VTA projection neurons that can affect cognitive function at the level of the medial prefrontal cortex (mPFC) as well as motor and motivational states controlled in part by mesolimbic output to the nucleus accumbens (Acb). To determine the relevant sites for NK(3) receptor activation within this neuronal network, we used confocal and electron microscopy to examine NK(3) receptors (Cy5; immunogold) and retrograde labeling of fluorogold (FG, FITC; immunoperoxidase) in the VTA of rats receiving either Acb or mPFC injections of FG. Comparison was made with neurokinin-1 (NK(1)) receptors, which are also present, but less abundant then NK(3) receptors, in dopaminergic and GABAergic VTA neurons. There were no observable differences between NK(3) and NK(1) receptors in their primary locations in the cytoplasm and on the plasma membrane of VTA somata and dendrites with or without FG. Dendrites labeled with FG retrogradely transported from mPFC, however, contained more NK(3) or less NK(1) immunogold particles (plasmalemmal + cytoplasmic) then those retrogradely labeled following FG injection in the Acb. Moreover, only the NK(3) receptors were detected in neuronal nuclei in the VTA and in the nuclei of human HEK-293T NK(3)-transfected cells. The enrichment of NK(3) receptors in mesocortical projection neurons and nuclear distribution of these receptors may provide insight for understanding the selective antipsychotic effectiveness of NK(3) antagonists.

Mossy cell axon synaptic contacts on ectopic granule cells that are born following pilocarpine-induced seizures.

Granule cell neurogenesis increases following seizures, and some newly born granule cells develop at abnormal locations within the hilus. These ectopic granule cells (EGCs) demonstrate regular bursts of action potentials that are synchronized with CA3 pyramidal cell burst discharges and the bursts of hilar neurons, including mossy cells. Such findings suggest that mossy cells may participate in circuits that activate EGCs. Electron microscopic immunolabeling was therefore used to determine if mossy cell axon terminals form synapses with hilar EGC dendrites, using animals that underwent pilocarpine-induced status epilepticus. Pilocarpine was administered to adult male rats, and those which developed status epilepticus were perfused 5-7 months later, after the period of EGC genesis. Hippocampal sections were processed for dual electron microscopic immunolabeling (using calcitonin gene-related peptide (CGRP) as a marker for mossy cells and calbindin (CaBP) as a marker for EGCs). Light microscopic analysis revealed large CGRP-immunoreactive cells in the hilus, with the appearance and distribution of mossy cells. Electron microscopic analysis revealed numerous CaBP-immunoreactive dendrites in the hilus, some of which were innervated by CGRP-immunoreactive terminals. The results suggest that mossy cells participate in the excitatory circuits which activate EGCs, providing further insight into the network rearrangements that accompany seizure-induced neurogenesis in this animal model of epilepsy.

Sensory-Derived Glutamate Regulates Presynaptic Inhibitory Terminals in Mouse Spinal Cord.

Circuit function in the CNS relies on the balanced interplay of excitatory and inhibitory synaptic signaling. How neuronal activity influences synaptic differentiation to maintain such balance remains unclear. In the mouse spinal cord, a population of GABAergic interneurons, GABApre, forms synapses with the terminals of proprioceptive sensory neurons and controls information transfer at sensory-motor connections through presynaptic inhibition. We show that reducing sensory glutamate release results in decreased expression of GABA-synthesizing enzymes GAD65 and GAD67 in GABApre terminals and decreased presynaptic inhibition. Glutamate directs GAD67 expression via the metabotropic glutamate receptor mGluR1ß on GABApre terminals and regulates GAD65 expression via autocrine influence on sensory terminal BDNF. We demonstrate that dual retrograde signals from sensory terminals operate hierarchically to direct the molecular differentiation of GABApre terminals and the efficacy of presynaptic inhibition. These retrograde signals comprise a feedback mechanism by which excitatory sensory activity drives GABAergic inhibition to maintain circuit homeostasis.

Hippocampal mossy fiber leu-enkephalin immunoreactivity in female rats is significantly altered following both acute and chronic stress.

Research indicates that responses to stress are sexually dimorphic, particularly in regard to learning and memory processes: while males display impaired cognitive performance and hippocampal CA3 pyramidal cell dendritic remodeling following chronic stress, females exhibit enhanced performance and no remodeling. Leu-enkephalin, an endogenous opioid peptide found in the hippocampal mossy fiber pathway, plays a critical role in mediating synaptic plasticity at the mossy fiber-CA3 pyramidal cell synapse. Estrogen is known to influence the expression of leu-enkephalin in the mossy fibers of females, with leu-enkephalin levels being highest at proestrus and estrus, when estrogen levels are elevated. Since stress is also known to alter the expression of leu-enkephalin in various brain regions, this study was designed to determine whether acute or chronic stress had an effect on mossy fiber leu-enkephalin levels in females or males, through the application of correlated quantitative light and electron microscopic immunocytochemistry. Both acute and chronic stress eliminated the estrogen-dependence of leu-enkephalin levels across the estrous cycle in females, but had no effect on male levels. However, following acute stress leu-enkephalin levels in females were consistently lowered to values comparable to the lowest control values, while following chronic stress they were consistently elevated to values comparable to the highest control values. Ultrastructural changes in leu-enkephalin labeled dense core vesicles paralleled light microscopic observations, with acute stress inducing a decrease in leu-enkephalin labeled dense core vesicles, and chronic stress inducing an increase in leu-enkephalin labeled dense-core vesicles in females. These findings suggest that alterations in leu-enkephalin levels following stress could play an important role in the sex-specific responses that females display in learning processes, including those important in addiction.

ER stress in the brain subfornical organ mediates angiotensin-dependent hypertension.

Although endoplasmic reticulum (ER) stress is a pathologic mechanism in a variety of chronic diseases, it is unclear what role it plays in chronic hypertension (HTN). Dysregulation of brain mechanisms controlling arterial pressure is strongly implicated in HTN, particularly in models involving angiotensin II (Ang II). We tested the hypothesis that ER stress in the brain is causally linked to Ang II-dependent HTN. Chronic systemic infusion of low-dose Ang II in C57BL/6 mice induced slowly developing HTN, which was abolished by co-infusion of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) into the lateral cerebroventricle. Investigations of the brain regions involved revealed robust increases in ER stress biomarkers and profound ER morphological abnormalities in the circumventricular subfornical organ (SFO), a region outside the blood-brain barrier and replete with Ang II receptors. Ang II-induced HTN could be prevented in this model by selective genetic supplementation of the ER chaperone 78-kDa glucose-regulated protein (GRP78) in the SFO. These data demonstrate that Ang II-dependent HTN is mediated by ER stress in the brain, particularly the SFO. To our knowledge, this is the first report that ER stress, notably brain ER stress, plays a key role in chronic HTN. Taken together, these findings may have broad implications for the pathophysiology of this disease.

Morphometry of hilar ectopic granule cells in the rat.

Granule cell (GC) neurogenesis in the dentate gyrus (DG) does not always proceed normally. After severe seizures (e.g., status epilepticus [SE]) and some other conditions, newborn GCs appear in the hilus. Hilar ectopic GCs (EGCs) can potentially provide insight into the effects of abnormal location and seizures on GC development. Additionally, hilar EGCs that develop after SE may contribute to epileptogenesis and cognitive impairments that follow SE. Thus, it is critical to understand how EGCs differ from normal GCs. Relatively little morphometric information is available on EGCs, especially those restricted to the hilus. This study quantitatively analyzed the structural morphology of hilar EGCs from adult male rats several months after pilocarpine-induced SE, when they are considered to have chronic epilepsy. Hilar EGCs were physiologically identified in slices, intracellularly labeled, processed for light microscopic reconstruction, and compared to GC layer GCs, from both the same post-SE tissue and the NeuroMorpho database (normal GCs). Consistently, hilar EGC and GC layer GCs had similar dendritic lengths and field sizes, and identifiable apical dendrites. However, hilar EGC dendrites were topologically more complex, with more branch points and tortuous dendritic paths. Three-dimensional analysis revealed that, remarkably, hilar EGC dendrites often extended along the longitudinal DG axis, suggesting increased capacity for septotemporal integration. Axonal reconstruction demonstrated that hilar EGCs contributed to mossy fiber sprouting. This combination of preserved and aberrant morphological features, potentially supporting convergent afferent input to EGCs and broad, divergent efferent output, could help explain why the hilar EGC population could impair DG function.

Degenerating processes identified by electron microscopic immunocytochemical methods.

The application of electron microscopic immunolabeling techniques to the identification and analysis of degenerating processes in neural tissue has greatly enhanced the ability of researchers to examine apoptosis and other degenerative disease mechanisms. This is particularly true for the early stages of such mechanisms. Traditionally, degenerating processes could only be identified at the ultrastructural level after significant cellular atrophy had occurred, when subcellular detail was obscured and synaptic relationships altered. Using immunocytochemical labeling procedures, degenerating neural and glial processes are first identified through the use of antibodies directed against a variety of degenerative markers, such as proapoptotic effectors (i.e., cytoplasmic cytochrome c), pathological components (i.e., beta amyloid deposits), or inflammatory agents (i.e., Iba1). Both the subcellular distribution of the marker within the process and the relationship of the labeled process to surrounding elements can then be carefully characterized. The information obtained can be further refined through the use of dual immunolabeling, which can provide additional data on the phenotype of the degenerating process and inputs to the process.

The influences of reproductive status and acute stress on the levels of phosphorylated mu opioid receptor immunoreactivity in rat hippocampus.

Opioids play a critical role in hippocampally dependent behavior and plasticity. In the hippocampal formation, mu opioid receptors (MOR) are prominent in parvalbumin (PARV) containing interneurons. Previously we found that gonadal hormones modulate the trafficking of MORs in PARV interneurons. Although sex differences in response to stress are well documented, the point at which opioids, sex and stress interact to influence hippocampal function remains elusive. Thus, we used quantitative immunocytochemistry in combination with light and electron microscopy for the phosphorylated MOR at the SER375 carboxy-terminal residue (pMOR) in male and female rats to assess these interactions. In both sexes, pMOR-immunoreactivity (ir) was prominent in axons and terminals and in a few neuronal somata and dendrites, some of which contained PARV in the mossy fiber pathway region of the dentate gyrus (DG) hilus and CA3 stratum lucidum. In unstressed rats, the levels of pMOR-ir in the DG or CA3 were not affected by sex or estrous cycle stage. However, immediately following 30 minutes of acute immobilization stress (AIS), males had higher levels of pMOR-ir whereas females at proestrus and estrus (high estrogen stages) had lower levels of pMOR-ir within the DG. In contrast, the number and types of neuronal profiles with pMOR-ir were not altered by AIS in either males or proestrus females. These data demonstrate that although gonadal steroids do not affect pMOR levels at resting conditions, they are differentially activated both pre- and post-synaptic MORs following stress. These interactions may contribute to the reported sex differences in hippocampally dependent behaviors in stressed animals.

Sex differences in angiotensin signaling in bulbospinal neurons in the rat rostral ventrolateral medulla.

Sex differences may play a significant role in determining the risk of hypertension. Bulbospinal neurons in the rostral ventrolateral medulla (RVLM) are involved in the tonic regulation of arterial pressure and participate in the central mechanisms of hypertension. Angiotensin II (ANG II) acting on angiotensin type 1 (AT(1)) receptors in RVLM neurons is implicated in the development of hypertension by activating NADPH oxidase and producing reactive oxygen species (ROS). Therefore, we analyzed RVLM bulbospinal neurons to determine whether there are sex differences in: 1) immunolabeling for AT(1) receptors and the key NADPH oxidase subunit p47 using dual-label immunoelectron microscopy, and 2) the effects of ANG II on ROS production and Ca(2+) currents using, respectively, hydroethidine fluoromicrography and patch-clamping. In tyrosine hydroxylase-positive RVLM neurons, female rats displayed significantly more AT(1) receptor immunoreactivity and less p47 immunoreactivity than male rats (P < 0.05). Although ANG II (100 nM) induced comparable ROS production in dissociated RVLM bulbospinal neurons of female and male rats (P > 0.05), an effect mediated by AT(1) receptors and NADPH oxidase, it triggered significantly larger dihydropyridine-sensitive long-lasting (L-type) Ca(2+) currents in female RVLM neurons (P < 0.05). These observations suggest that an increase in AT(1) receptors in female RVLM neurons is counterbalanced by a reduction in p47 levels, such that ANG II-induced ROS production does not differ between females and males. Since the Ca(2+) current activator Bay K 8644 induced larger Ca(2+) currents in females than in male RVLM neurons, increased ANG II-induced L-type Ca(2+) currents in females may result from sex differences in calcium channel densities or dynamics.

Cellular and subcellular localization of androgen receptor immunoreactivity relative to C1 adrenergic neurons in the rostral ventrolateral medulla of male and female rats.

In male and female rats, high androgen levels can increase blood pressure. The C1 area of the rostral ventrolateral medulla (RVLM), which is crucial for blood pressure regulation, contains estrogen receptors (ERs) in pre- and postsynaptic neuronal compartments and is modulated by estrogens (Wang et al. [2006] Brain Res 1094:163-178). In this study, the cellular and subcellular localization of androgen receptors (ARs) in the C1 area was examined in sections from male, proestrus (high estrogen) and diestrus (low estrogen) female rat brains that were immunocytochemically labeled for AR and tyrosine hydroxylase (TH). By light and electron microscopy, AR-labeled nuclei were scattered among TH-labeled somata in the RVLM; significantly more AR-labeled nuclei were seen males compared to females. Electron microscopy revealed that extranuclear AR-immunoreactivity (ir) was in similar profile types in male and female rats. AR-ir was almost exclusively in myelinated and unmyelinated axons and in glia. Rarely, AR-ir was in axon terminals that contacted TH-containing dendrites. AR-labeled axon terminals had large diameters and contained numerous dense-core vesicles, resembling peptide-containing hypothalamic or solitary tract inputs. No nuclear or extranuclear AR-ir was found in TH-labeled perikarya and dendrites although a few non-TH- labeled dendrites contained AR-ir. Qualitatively, more axonal profiles appeared to be present in males compared to females. These studies suggest that, unlike ERs, ARs in male and female rats are almost exclusively positioned on afferents and glia, suggesting that androgens modulate RVLM C1 neurons, and thus blood pressure, through presynaptic and glial signaling.

Stereological methods reveal the robust size and stability of ectopic hilar granule cells after pilocarpine-induced status epilepticus in the adult rat.

Following status epilepticus in the rat, dentate granule cell neurogenesis increases greatly, and many of the new neurons appear to develop ectopically, in the hilar region of the hippocampal formation. It has been suggested that the ectopic hilar granule cells could contribute to the spontaneous seizures that ultimately develop after status epilepticus. However, the population has never been quantified, so it is unclear whether it is substantial enough to have a strong influence on epileptogenesis. To quantify this population, the total number of ectopic hilar granule cells was estimated using unbiased stereology at different times after pilocarpine-induced status epilepticus. The number of hilar neurons immunoreactive for Prox-1, a granule-cell-specific marker, was estimated using the optical fractionator method. The results indicate that the size of the hilar ectopic granule cell population after status epilepticus is substantial, and stable over time. Interestingly, the size of the population appears to be correlated with the frequency of behavioral seizures, because animals with more ectopic granule cells in the hilus have more frequent behavioral seizures. The hilar ectopic granule cell population does not appear to vary systematically across the septotemporal axis, although it is associated with an increase in volume of the hilus. The results provide new insight into the potential role of ectopic hilar granule cells in the pilocarpine model of temporal lobe epilepsy.

Mossy fibers are the primary source of afferent input to ectopic granule cells that are born after pilocarpine-induced seizures.

Granule cell (GC) neurogenesis increases following seizures, and some newborn GCs develop in abnormal locations within the hilus. These ectopic GCs (EGCs) display robust spontaneous and evoked excitatory activity. However, the pattern of afferent input they receive has not been fully defined. This study used electron microscopic immunolabeling to quantitatively evaluate mossy fiber (MF) input to EGCs since MFs densely innervate the hilus normally and undergo sprouting in many animal models of epilepsy. EGC dendrites were examined in tissue from epileptic rats that had initially been treated with pilocarpine to induce status epilepticus and subsequently had spontaneous seizures. MF terminals were labeled with a zinc transporter-3 antibody, and calbindin immunoreactivity was used to label hilar EGCs and GC layer GCs. The pattern of input provided by sprouted MF terminals to EGC dendrites was then compared to the pattern of MF input to GC dendrites in the inner molecular layer (IML), where most sprouted fibers are thought to project. Analysis of EGC dendrites demonstrated that MF terminals represented their predominant source of afferent input: they comprised 63% of all terminals and, on average, occupied 40% and 29% of the dendritic surface in the dorsal and ventral dentate gyrus, respectively, forming frequent synapses. These measures of connectivity were significantly greater than comparable values for MF innervation of GC dendrites located in the IML of the same tissue sections. Thus, EGCs develop a pattern of synaptic connections that could help explain their previously identified predisposition to discharge in epileptiform bursts and suggest that they play an important role in the generation of seizure activity in the dentate gyrus.