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We established a novel brain slice assay to test the ability of acetylcholinesterase (AChE) reactivators to prevent ACh-induced M1 muscarinic acetylcholine receptor (mAChR) dependent hyperexcitability observed after exposure to the organophosphate (OP)-based AChE inhibitor and sarin surrogate 4-nitrophenyl isopropyl methylphosphonate (NIMP). Whole-cell patch clamp recordings were used to evaluate the response of pyramidal neurons in the rat basolateral amygdala (BLA) to brief (1 min) bath application of ACh (100 μM), either in control conditions, or after exposure to NIMP ± an AChE reactivator. Bath application of ACh produced atropine- and pirenzepine-sensitive inward currents in voltage clamped BLA pyramidal neurons, and increased the frequency of spontaneous EPSCs, suggesting robust activation of M1 mAChRs. Responses to ACh were increased ~3-5 fold in slices that had been preincubated in NIMP, and these effects were reversed in a concentration dependent manner by exposure to a commercially available AChE reactivator. The current work outlines a simple assay that can be used to evaluate the efficacy of both known and novel AChE reactivators in an area of the limbic system that likely contributes to seizures after acute exposure to OP-based AChE inhibitors.

We examined the contribution of N-methyl-D-aspartate receptor (NMDAR) subunits in the redox-mediated decline in NMDAR function during aging. GluN2A and GluN2B selective antagonists decreased peak NMDAR currents to a similar extent in young and aged animals, indicating that a shift in diheteromeric GluN2 subunits does not underlie the age-related decrease in the NMDAR synaptic function. Application of dithiothreitol (DTT) in aged animals, increased peak NMDAR synaptic currents, prolonged the decay time, and increased the sensitivity of the synaptic response to the GluN2B antagonist, ifenprodil, indicating that DTT increased the contribution of GluN2B subunits to the synaptic response. The DTT-mediated increase in NMDAR function was inhibited by partial blockade of NMDARs, and this inhibition was rescued by increasing Ca2+ concentration in the recording medium. The results indicate that DTT-mediated potentiation requires Ca2+ influx through NMDAR activity. Finally, redox regulation of NMDAR function depends on the activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII). The results indicate that activity-dependent NMDAR synaptic plasticity is suppressed by redox-mediated inhibition of CaMKII activation during aging. The redox regulation of NMDARs represents a suppression of a metaplasticity mechanism, which can disrupt synaptic plasticity and cognition associated with neurological or psychiatric diseases, and aging.

Social recognition, the ability to recognize individuals that were previously encountered, requires complex integration of sensory inputs with previous experience. Here, we use a variety of approaches to discern how oxytocin-sensitive neurons in the PFC exert descending control over a circuit mediating social recognition in mice. Using male mice with Cre-recombinase directed to the oxytocin receptor gene (Oxtr), we revealed that oxytocin receptors (OXTRs) are expressed on glutamatergic neurons in the PFC, optogenetic stimulation of which elicited activation of neurons residing in several mesolimbic brain structures. Optogenetic stimulation of axons in the BLA arising from OXTR-expressing neurons in the PFC eliminated the ability to distinguish novel from familiar conspecifics, but remarkably, distinguishing between novel and familiar objects was unaffected. These results suggest that an oxytocin-sensitive PFC to BLA circuit is required for social recognition. The implication is that impaired social memory may manifest from dysregulation of this circuit.SIGNIFICANCE STATEMENTUsing mice, we demonstrate that optogenetic activation of the neurons in the PFC that express the oxytocin receptor gene (Oxtr) impairs the ability to distinguish between novel and familiar conspecifics, but the ability to distinguish between novel and familiar objects remains intact. Subjects with autism spectrum disorders (ASDs) have difficulty identifying a person based on remembering facial features; however, ASDs and typical subjects perform similarly when remembering objects. In subjects with ASD, viewing the same face increases neural activity in the PFC, which may be analogous to the optogenetic excitation of oxytocin receptor (OXTR) expressing neurons in the PFC that impairs social recognition in mice. The implication is that overactivation of OXTR-expressing neurons in the PFC may contribute to ASD symptomology.

We recently found indicators of hypothalamic inflammation and neurodegeneration linked to the loss of neuroprotective factors including insulin-like growth factor (IGF-1) and IGF binding protein-2 (IGFBP-3) in mice made diabetic using streptozotocin (STZ). In the current work, a genetic model of type-1 diabetes (Ins2(Akita) mouse) was used to evaluate changes in neuronal activity and concomitant changes in the proinflammatory mediator high-mobility group box-1 (HMBG1). We found basal hypothalamic neuronal activity as indicated by manganese-enhanced magnetic resonance imaging (MEMRI) was significantly decreased in 8 months old, but not 2 months old Ins2(Akita) diabetic mice compared to controls. In tissue from the same animals we evaluated the expression of HMBG1 using immunohistochemistry and confocal microscopy. We found decreased HMBG1 nuclear localization in the paraventricular nucleus of the hypothalamus (PVN) in 8 months old, but not 2 months old diabetic animals indicating nuclear release of the protein consistent with an inflammatory state. Adjacent thalamic regions showed little change in HMBG1 nuclear localization and neuronal activity as a result of diabetes. This work extends our previous findings demonstrating changes consistent with hypothalamic neuroinflammation in STZ treated animals, and shows active inflammatory processes are correlated with changes in basal hypothalamic neuronal activity in Ins2(Akita) mice.

Purpose: Since loss of retinal SST and circulating SST occurs in DR, we asked if loss of hypothalamic SST production would impact the innervation and function of the BM and influence DR in T2D rat model. Methods: Brain sections of BBZ/Wor rats with 4 months of diabetes and age-matched nondiabetic lean BBZ rats were immuno-stained for SST, Iba-1 and Neu-N. To examine the neuronal connection between the BM and pre-sympathetic brainstem and hypothalamic areas, both control and diabetic rats were injected in a blinded manner with GFP-transgenic pseudorabies virus (PRV-152) into their femurs. Four days later rats were euthanized and brain sections prepared for colocalization of GFP with SST. DR was assessed by enumeration of acellular capillaries in trypsin-digested retinas. Results: Injection of PRV into femur showed the presence of active virus in neurons of the paraventricular nucleus of the hypothalamus (PVN), rostral ventrolateral medulla and nucleus tractus solitarius of the brain of control rats. Colocalization studies showed that periventricular hypothalamic SST neurons were also GFP+ and directly innervated the BM. Quantitation showed that diabetic rats had significantly fewer GFP+ neurons in the PVN (n=6, *P Conclusions: These studies support the notion that periventricular hypothalamic SST neurons directly innervate BM and that preservation of this small, but important population is relevant to prevention of diabetes induced-BM dysfunction and development of DR. Disclosure A. Longhini: None. J. Thinschmidt: None. M. Boulton: None. M.A. King: None. M.B. Grant: None.

Neuroinflammation and neurodegeneration have been observed in the brain in type 1 diabetes (T1D). However, little is known about the mediators of these effects. In T1D mice with 12- and 35-wk duration of diabetes we examined two mechanisms of neurodegeneration, loss of the neuroprotective factors insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) and changes in indoleamine 2,3-dioxygenase (IDO) expression in the brain, and compared the response to age-matched controls. Furthermore, levels of matrix metalloproteinase-2 (MMP-2), nucleoside triphosphate diphosphohydrolase-1 (CD39), and ionized calcium-binding adaptor molecule 1 (Iba-1) were utilized to assess inflammatory changes in astrocytes, microglia, and blood vessels. In the diabetic hypothalamus (HYPO), we observed 20% reduction in neuronal soma diameter ( P < 0.05) and reduced neuronal expression of IGFBP-3 (−32%, P < 0.05) and IGF-I (−15%, P < 0.05) compared with controls at 35 wk. In diabetic HYPO, MMP-2 expression was increased in astrocytes (46%, P < 0.01), and IDO+cell density rose by (62%, P < 0.05). CD39 expression dropped by 30% ( P < 0.05) in microglia and blood vessels. With 10 wk of systemic treatment using minocycline, an anti-inflammatory agent that crosses the blood-brain barrier, MMP-2, IDO, and CD39 levels normalized ( P < 0.05). Our results suggest that increased IDO and early loss of CD39+protective cells lead to activation of inflammation in sympathetic centers of the CNS. As a downstream effect, the loss of the neuronal survival factors IGFBP-3 and IGF-I and the neurotoxic products of the kynurenine pathway contribute to the loss of neuronal density observed in the HYPO in T1D.

The widespread nature of diabetes affects all organ systems of an individual including the bone marrow. Long-term damage to the cellular and extracellular components of the bone marrow leads to a rapid decline in the bone marrow-hematopoietic stem/progenitor cells (HS/PCs) compartment. This review will highlight the importance of bone marrow microenvironment in maintaining bone marrow HS/PC populations and the contribution of these key populations in microvascular repair during the natural history of diabetes. The autonomic nervous system can initiate and propagate bone marrow dysfunction in diabetes. Systemic pharmacological strategies designed to protect the bone marrow-HS/PC population from diabetes induced-oxidative stress and advanced glycation end product accumulation represent a new approach to target diabetic retinopathy progression. Protecting HS/PCs ensures their participation in vascular repair and reduces the risk of vasogdegeneration occurring in the retina.

Currently, the only effective therapy for acute ischemic stroke is the thrombolytic agent recombinant tissue plasminogen activator. α1-Antitrypsin, an endogenous inhibitor of serine proteinases and a primary acute phase protein with potent anti-inflammatory, anti-apoptotic, antimicrobial and cytoprotective activities, could be beneficial in stroke.. The goal of this study was to test whether α1-antitrypsin could improve ischemic stroke outcome in an established rat model. Middle cerebral artery occlusion was induced in male rats via intracranial microinjection of endothelin-1. Five to ten minutes following stroke induction rats received either intracranial or intravenous delivery of human α1-antitrypsin. Cylinder and vibrissae tests were used to evaluate sensorimotor function before and 72 hours after middle cerebral artery occlusion. Infarct volumes were examined via either 2,3,5-triphenyltetrazolium chloride assay or magnetic resonance imaging 72 hours after middle cerebral artery occlusion. Despite equivalent initial strokes, at 72 hours the infarct volumes of the human α1-antitrypsin treatment groups (local and systemic injection) were statistically significantly reduced by 83% and 63% (p

Electroacupuncture (EA) performed in rats and humans using limb acupuncture sites, LI-4 and LI-11, and GV-14 and GV-20 (humans) and Bai-hui (rats) increased functional connectivity between the anterior hypothalamus and the amygdala and mobilized mesenchymal stem cells (MSCs) into the systemic circulation. In human subjects, the source of the MSC was found to be primarily adipose tissue, whereas in rodents the tissue sources were considered more heterogeneous. Pharmacological disinhibition of rat hypothalamus enhanced sympathetic nervous system (SNS) activation and similarly resulted in a release of MSC into the circulation. EA-mediated SNS activation was further supported by browning of white adipose tissue in rats. EA treatment of rats undergoing partial rupture of the Achilles tendon resulted in reduced mechanical hyperalgesia, increased serum interleukin-10 levels and tendon remodeling, effects blocked in propranolol-treated rodents. To distinguish the afferent role of the peripheral nervous system, phosphoinositide-interacting regulator of transient receptor potential channels (Pirt)-GCaMP3 (genetically encoded calcium sensor) mice were treated with EA acupuncture points, ST-36 and LIV-3, and GV-14 and Bai-hui and resulted in a rapid activation of primary sensory neurons. EA activated sensory ganglia and SNS centers to mediate the release of MSC that can enhance tissue repair, increase anti-inflammatory cytokine production and provide pronounced analgesic relief.

By using pseudorabies virus expressing green fluorescence protein, we found that efferent bone marrow-neural connections trace to sympathetic centers of the central nervous system in normal mice. However, this was markedly reduced in type 1 diabetes, suggesting a significant loss of bone marrow innervation. This loss of innervation was associated with a change in hematopoiesis toward generation of more monocytes and an altered diurnal release of monocytes in rodents and patients with type 1 diabetes. In the hypothalamus and granular insular cortex of mice with type 1 diabetes, bone marrow-derived microglia/macrophages were activated and found at a greater density than in controls. Infiltration of CD45(+)/CCR2(+)/GR-1(+)/Iba-1(+) bone marrow-derived monocytes into the hypothalamus could be mitigated by treatment with minocycline, an anti-inflammatory agent capable of crossing the blood-brain barrier. Our studies suggest that targeting central inflammation may facilitate management of microvascular complications.

In this study, we assessed whether Per2 clock gene–mutant mice exhibit a vascular phenotype similar to diabetes. Per2 (B6.129-Per2tm1Drw/J) or wild-type control mice 4 and 12 months of age were used. To evaluate diabetes-like phenotype in Per2 mutant mice, retina was quantified for mRNA expression, and degree of diabetic retinopathy was evaluated. Bone marrow neuropathy was studied by staining femurs for tyrosine hydroxylase (TH) and neurofilament 200 (NF-200). The rate of proliferation and quantification of bone marrow progenitor cells (BMPCs) was performed, and a threefold decrease in proliferation and 50% reduction in nitric oxide levels were observed in Per2 mutant mice. TH-positive nerve processes and NF-200 staining were reduced in Per2 mutant mice. Both retinal protein and mRNA expression of endothelial nitric oxide synthase were decreased by twofold. Other endothelial function genes (VEGFR2, VEGFR1) were downregulated (1.5–2-fold) in Per2 mutant retinas, whereas there was an upregulation of profibrotic pathway mediated by transforming growth factor-β1. Our studies suggest that Per2 mutant mice recapitulate key aspects of diabetes without the metabolic abnormalities, including retinal vascular damage, neuronal loss in the bone marrow, and diminished BMPC function.

We sought to determine the impact of long-standing type 1 diabetes on haematopoietic stem/progenitor cell (HSC) number and function and to examine the impact of modulating glycoprotein (GP)130 receptor in these cells.Wild-type, gp130(-/-) and GFP chimeric mice were treated with streptozotocin to induce type 1 diabetes. Bone marrow (BM)-derived cells were used for colony-formation assay, quantification of side population (SP) cells, examination of gene expression, nitric oxide measurement and migration studies. Endothelial progenitor cells (EPCs), a population of vascular precursors derived from HSCs, were compared in diabetic and control mice. Cytokines were measured in BM supernatant fractions by ELISA and protein array. Flow cytometry was performed on enzymatically dissociated retina from gfp(+) chimeric mice and used to assess BM cell recruitment to the retina, kidney and blood.BM cells from the 12-month-diabetic mice showed reduced colony-forming ability, depletion of SP-HSCs with a proportional increase in SP-HSCs residing in hypoxic regions of BM, decreased EPC numbers, and reduced eNos (also known as Nos3) but increased iNos (also known as Nos2) and oxidative stress-related genes. BM supernatant fraction showed increased cytokines, GP130 ligands and monocyte/macrophage stimulating factor. Retina, kidney and peripheral blood showed increased numbers of CD11b(+)/CD45(hi)/ CCR2(+)/Ly6C(hi) inflammatory monocytes. Diabetic gp130(-/-) mice were protected from development of diabetes-induced changes in their HSCs.The BM microenvironment of type 1 diabetic mice can lead to changes in haematopoiesis, with generation of more monocytes and fewer EPCs contributing to development of microvascular complications. Inhibition of GP130 activation may serve as a therapeutic strategy to improve the key aspects of this dysfunction.

Oxidative stress in the brain is implicated in increased sympathetic drive, inflammatory status, and vascular dysfunctions, associated with development and establishment of hypertension. However, little is known about the mechanism of this impaired brain-vascular communication. Here, we tested the hypothesis that increased oxidative stress in the brain cardioregulatory areas, such as the paraventricular nucleus of the hypothalamus, is driven by mitochondrial reactive oxygen species and leads to increased inflammatory cells (ICs) and decreased/dysfunctional endothelial progenitor cells (EPCs), thereby compromising vasculature repair and accelerating hypertension. Chronic angiotensin II infusion resulted in elevated blood pressure and sympathetic vasomotor drive, decreased spontaneous baroreflex gain, and increased microglia activation in the paraventricular nucleus. This was associated with 46% decrease in bone marrow (BM)-derived EPCs and 250% increase in BM ICs, resulting in 5-fold decrease of EPC/IC ratio in the BM. Treatment with mitochondrial-targeted antioxidant, a scavenger of mitochondrial O 2 −· , intracerebroventricularly but not subcutaneously attenuated angiotensin II–induced hypertension, decreased activation of microglia in the paraventricular nucleus, and normalized EPCs/ICs. This functional communication between the brain and BM was confirmed by retrograde neuronal labeling from the BM with green fluorescent protein–tagged pseudorabies virus. Administration of green fluorescent protein–tagged pseudorabies virus into the BM resulted in predominant labeling of paraventricular nucleus neurons within 3 days, with some fluorescence in the nucleus tractus solitarius, the rostral ventrolateral medulla, and subfornical organ. Taken together, these data demonstrate that inhibition of mitochondrial reactive oxygen species attenuates angiotensin II–induced hypertension and corrects the imbalance in EPCs/ICs in the BM. They suggest that an imbalance in vascular reparative and ICs may perpetuate vascular pathophysiology in this model of hypertension.

In this study, the role of CX3CR1 in the progression of diabetic retinopathy (DR) was investigated. The retinas of wild type (WT), CX3CR1 null (CX3CR1gfp/gfp, KO) and heterozygous (CX3CR1+/gfp, Het) mice were compared in the presence and absence of streptozotocin (STZ) induced diabetes. CX3CR1 deficiency in STZ-KO increased vascular pathology at 4 months of diabetes, as a significant increase in acellular capillaries was observed only in the STZ-KO group. CX3CR1 deficiency and diabetes had similar effects on retinal neurodegeneration measured by an increase in DNA fragmentation. Retinal vascular pathology in STZ-KO mice was associated with increased numbers of monocyte-derived macrophages in the retina. Furthermore, compared to STZ-WT, STZ-KO mice exhibited increased numbers of inflammatory monocytes in the bone marrow and impaired homing of monocytes to the spleen. Induction of retinal IL-10 expression by diabetes was significantly less in KO mice, and when bone marrow-derived macrophages from KO mice were maintained in high glucose they expressed significantly less IL-10 and more TNF-α in response to LPS stimulation. These findings support that CX3CR1 deficiency accelerates the development of vascular pathology in DR through increased recruitment of proinflammatory myeloid cells that demonstrate reduced expression of anti-inflammatory IL-10.

A family of 20 tris-azaaromatic quaternary ammonium (AQA) compounds were tested for their inhibition of alpha7 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. The potency of inhibitory activity was related to the hydrophobic character of the tris head groups. Two tris-AQA compounds were studied in detail: the highly effective inhibitor 1,3,5-tri-[5-(1-quinolinum)-pent-1-yn-1-yl]-benzene tribromide (tPyQB) and the less potent antagonist 1,3,5,-tri-{5-[1-(2-picolinium)]-pent-1-yn-1-yl}benzene tribromide (tPy2PiB). In addition, we evaluated 1,2,4,5-tetra-{5-[1-(3-benzyl)pyridinium]pent-1-yl}benzene tetrabromide (tkP3BzPB), a tetrakis-AQA with very hydrophobic headgroups. We compared the activity of the AQA compounds to the frequently used alpha7-antagonist methyllycaconitine (MLA). Both tPyQB and tkP3BzPB were selective antagonists of alpha7. However, although inhibition by tPyQB was reversible within 5 min, the recovery time constant for tkP3BzPB inhibition was 26.6 +/- 0.8 min, so that the equilibrium inhibition in the prolonged presence of nanomolar concentrations of tkP3BzPB was nearly 100%. The potency, selectivity, and slow reversibility of tkP3BzPB were comparable with or greater than that of MLA. The inhibitory actions of tPyQB, tPy2PiB, and tkP3BzPB were evaluated on the acetylcholine (ACh)-evoked responses of native nAChRs in rat brain slices. The alpha7-mediated responses of hippocampal interneurons were effectively reduced by 1 microM tPyQB and tkP3BzPB but not tPy2PiB. In rat medial septum, tkP3BzPB produced a greater inhibition of ACh-evoked responses of cells with fast inward currents (type I) than of cells with predominantly slow kinetics (type II), suggesting that tkP3BzPB can block alpha7 yet preserve the responsiveness of non-alpha7 receptors. These agents might be helpful in elucidating complex receptor responses in brain regions with mixed populations of nAChRs.

Brain alpha7 nicotinic receptors have become therapeutic targets for Alzheimer's disease (AD) based on their memory-enhancing and neuroprotective actions. This study investigated the feasibility of increasing neuronal alpha7 receptor functions using a gene delivery approach based on neuron-selective recombinant adeno-associated virus (rAAV)-derived vectors. In order to determine whether alpha7 receptor-mediated cytotoxicity was dependent on receptor density, rat alpha7 nicotinic receptors were expressed at high concentrations in GH4C1 cells as measured with nicotine-displaceable [3H]methyllycaconitine (MLA) binding. The potency of GTS-21 (an alpha7 receptor agonist) to induce cell loss was similar in these cells to that seen in pheochromocytoma (PC12) cells expressing nine-times-lower receptor levels, suggesting that cytotoxicity was more dependent on agonist concentration than receptor density. Hippocampal transduction with rat alpha7 nicotinic receptors increased [3H]MLA binding in this region in wild type and alpha7 receptor-knockout (KO) mice without apparent cytotoxicity. No difference was observed in Kd values for MLA binding between endogenous and transgenic receptors. Single cell recordings demonstrated that dentate granule cells that normally have no alpha7 receptor response did so following alpha7 receptor gene delivery in wild type mice. Recovery of alpha7 function was also observed in stratum oriens and stratum radiatum neurons of KO mice following gene delivery. Wild type mice exhibited improved acquisition performance in the Morris water task 1 month after bilateral hippocampal transductions with the rat alpha7 receptor gene compared with green fluorescent protein-transduced controls. However, both groups reached similar training levels and there was no difference in subsequent probe performance. Finally, this gene delivery approach was used to test whether alpha7 receptors affect tau-phosphorylation. Chronic (i.e. 2 month but not 2 week) expression of high levels of alpha7 receptors in hippocampus increased AT8 staining characteristic of hyperphosphorylated tau in that region, indicating that endogenous agonist-mediated receptor activation may be able to modulate this process.

In this study, the role of CX3CR1 in the progression of diabetic retinopathy (DR) was investigated. The retinas of wild-type (WT), CX3CR1 null (CX3CR1gfp/gfp, KO), and heterozygous (CX3CR1+/gfp, Het) mice were compared in the presence and absence of streptozotocin (STZ)-induced diabetes. CX3CR1 deficiency in STZ-KO increased vascular pathology at 4 months of diabetes, as a significant increase in acellular capillaries was observed only in the STZ-KO group. CX3CR1 deficiency and diabetes had similar effects on retinal neurodegeneration measured by an increase in DNA fragmentation. Retinal vascular pathology in STZ-KO mice was associated with increased numbers of monocyte-derived macrophages in the retina. Furthermore, compared to STZ-WT, STZ-KO mice exhibited increased numbers of inflammatory monocytes in the bone marrow and impaired homing of monocytes to the spleen. The induction of retinal IL-10 expression by diabetes was significantly less in KO mice, and when bone marrow-derived macrophages from KO mice were maintained in high glucose, they expressed significantly less IL-10 and more TNF-α in response to LPS stimulation. These findings support that CX3CR1 deficiency accelerates the development of vascular pathology in DR through increased recruitment of proinflammatory myeloid cells that demonstrate reduced expression of anti-inflammatory IL-10. • CX3CR1 deletion in STZ-diabetic mice accelerated the onset of diabetic retinopathy (DR). • The early onset of DR was associated with increased retinal cell apoptosis. • The early onset of DR was associated with increased recruitment of bone marrow-derived macrophages to the retina. • Bone marrow-derived macrophages from CX3CR1 KO diabetic mice expressed more TNF-α and less IL-10. • The role of IL-10 in protection from progression of DR is highlighted.

This study tested the hypothesis that peripheral immune challenges will produce predictable activation patterns in the rat brain consistent with sympathetic excitation. As part of examining this hypothesis, this study asked whether central activation is dependent on capsaicin-sensitive C-fibres. We induced skin contact sensitivity immune responses with 2,4-dinitrochlorobenzene (DNCB), in the presence or absence of the acute C-fibre toxin capsaicin (8-methyl-N-vanillyl-6-nonenamide) to trigger immune responses with and without diminished activity of C-fibres. Innovative blood-oxygen-level-dependent functional magnetic resonance imaging data revealed that the skin contact sensitivity immune responses induced with DNCB were associated with localized increases in brain neuronal activity in treated rats. This response was diminished by pre-treatment with capsaicin 1 week before scans. In the same animals, we found expression of the immediate early gene c-Fos in sub-regions of the amygdala and hypothalamic sympathetic brain nuclei. Significant increases in c-Fos expression were found in the supraoptic nucleus, central amygdala and medial habenula following immune challenges. Our results support the idea that selective brain regions, some of which are associated with sympathetic function, process or modulate immune function through pathways that are partially dependent on C-fibres. Together with previous studies demonstrating the motor control pathways from brain to immune targets, these findings indicate a central neuroimmune system to monitor host status and coordinate appropriate host responses.

The hippocampus receives substantial input from the medial septum/diagonal band of broca (MS/DB) via the fibria-fornix (FF). Projections from the MS/DB innervate hippocampal interneurons that express alpha7 nicotinic receptors and regulate excitation in principal cell populations. In the present report we used stereotaxic surgery, whole-cell patch clamping, and immunohistochemical techniques to evaluate the effects of FF and MS/DB lesions on alpha7 nicotinic receptors in stratum radiatum interneurons. Focal somatic application of ACh (1 mM) evoked methyllycaconitine (MLA)-sensitive currents that were markedly reduced following aspirative lesions of the FF. Reductions in current amplitudes were prevented or restored to levels not significantly different from controls following in vivo treatment with the alpha7-selective agonist GTS-21, and GTS-21 treatment did not change current amplitudes measured in tissue from unlesioned animals. MS/DB injections of the selective cholinergic neurotoxin 192 IgG-saporin did not affect alpha7 receptor currents, although MS/DB ChAT and hippocampal AChE immunolabeling were significantly reduced. In contrast, kainic acid lesions of the MS/DB, potentially more selective for GABAergic projection neurons, produced significant reductions in current amplitudes. These findings are the first to show functional changes in alpha7 receptors following hippocampal denervation and suggest that MS/DB hippocampal innervation regulates functional aspects of hippocampal alpha7 receptors. The results confirm hippocampal alpha7 nicotinic receptors as viable therapeutic targets in diseases that involve degradation of the septohippocampal pathway and may indicate that GABAergic MS/DB hippocampal input plays a more substantial role in the regulation of alpha7 nicotinic receptor function than MS/DB hippocampal cholinergic input.

The therapeutic targeting of nicotinic receptors in the brain will benefit from the identification of drugs that may be selective for their ability to activate or inhibit a limited range of nicotine acetylcholine receptor subtypes. In the present study, we describe the effects of 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), a novel compound that is a potent inhibitor of neuronal nicotinic receptors. Evaluation of nicotinic acetylcholine receptor (nAChR) subunits expressed in Xenopus laevis oocytes indicated that TMPH can produce a potent and long-lasting inhibition of neuronal nAChR formed by the pairwise combination of the most abundant neuronal alpha (i.e., alpha3 and alpha4) and beta subunits (beta2 and beta4), with relatively little effect, because of rapid reversibility of inhibition, on muscle-type (alpha1beta1gammadelta) or alpha7 receptors. However, the inhibition of neuronal beta subunit-containing receptors was also decreased if any of the nonessential subunits alpha5, alpha6, or beta3 were coexpressed. This decrease in inhibition is shown to be associated with a single amino acid present in the second transmembrane domain of these subunits. Our data indicate great potential utility for TMPH to help relate the diverse central nervous system effects to specific nAChR subtypes.

Human alcoholics and animals that have received chronic ethanol treatment (CET) display memory deficits. Previous work in our laboratory has shown that CET produces damage to the hippocampus and a reduction in the magnitude of hippocampal long-term synaptic potentiation. In the present report we examined the effects of CET on hippocampal long-term depression (LTD). We used in vitro hippocampal slices to examine LTD after rats received 38–41 weeks of paired feeding on liquid diets containing ethanol or isocaloric sucrose. Stimulation delivered through electrodes in the CA3-CA1 Schaffer collateral pathway activated synaptic population responses in CA1. LTD of CA1 stratum radiatum evoked field potential slope was not induced by 900 single pulses at 1 Hz, but was elicited by 900 pulse pairs separated by 50 or 200 msec delivered at 1 Hz (pLFS50, pLFS200). LTD evoked by pLFS200, but not by pLFS50, was significantly reduced in slices from ethanol-treated rats. The N-methyl D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5) (50 μM) blocked LTD induced by pLFS50 and pLFS200 equally, but the L-type calcium channel blocker nimodipine (10 μM) had no effect on either type of LTD. Thus, direct effects on these channels cannot explain how CET selectively reduces the magnitude of pLFS200 LTD. Finally, we describe a novel and robust LTD of the presynaptic afferent volley that is resistant to CET, NMDAR antagonists, GABA-A receptor blockade, and blockade of L-type calcium channels. Synapse 48:189–197, 2003. © 2003 Wiley-Liss, Inc.

Age-related macular degeneration (AMD) is a leading cause of visual impairment worldwide. Genetics and diet contribute to the relative risk for developing AMD, but their interactions are poorly understood. Genetic variations in Complement Factor H (CFH), and dietary glycemic index (GI) are major risk factors for AMD. We explored the effects of GI on development of early AMD-like features and changes to central nervous system (CNS) inflammation in Cfh-null mice.Aged 11-week-old wild type (WT) C57Bl/6J or Cfh-null mice were group pair-fed high or low GI diets for 33 weeks. At 10 months of age, mice were evaluated for early AMD-like features in the neural retina and RPE by light and electron microscopy. Brains were analyzed for Iba1 macrophage/microglia immunostaining, an indicator of inflammation.The 10-month-old WT mice showed no retinal abnormalities on either diet. The Cfh-null mice, however, showed distinct early AMD-like features in the RPE when fed a low GI diet, including vacuolation, disruption of basal infoldings, and increased basal laminar deposits. The Cfh-null mice also showed thinning of the RPE, hypopigmentation, and increased numbers of Iba1-expressing macrophages in the brain, irrespective of diet.The presence of early AMD-like features by 10 months of age in Cfh-null mice fed a low GI diet is surprising, given the apparent protection from the development of such features in aged WT mice or humans consuming lower GI diets. Our findings highlight the need to consider gene-diet interactions when developing animal models and therapeutic approaches to treat AMD.

GHB produced a concentration-dependent depression of evoked synaptic field potentials (EFPs) recorded extracellularly in the CA1 region of the in vitro rat hippocampal slice. The concentration/response function revealed a threshold near 1 mM, with IC50 of 10.85 mM and a Hill coefficient of 1.29. The gamma-aminobutyric acid B-receptor (GABA-B) agonist baclofen also depressed the EFP, but even maximally effective concentrations of the GABA-B antagonist 2-hydroxy-saclofen (800 microM) could not completely block the GHB-induced EFP depression. Nor was GHB-induced EFP depression blocked by the GHB receptor "antagonist" NCS-382, which does not displace GABA-B receptor ligands. However, NCS-382 produced a concentration-dependent increase in EFP slope. The threshold concentration was about 100 microM but the maximally effective concentration, and thus the IC50, could not be determined in the perfusion slice system. NCS-382 may be an inverse agonist at hippocampal GHB receptors, or else endogenous hippocampal GHB receptor ligands medicate a tonic inhibition in CA1. At concentrations sufficient to induce EFP depression GHB did not alter pH. Although isosmotic sucrose did depress CA1 EFPs it was essentially ineffective at the IC50 for GHB. Gamma-butyrolactone, a prodrug of GHB, was only 1/20th as effective as GHB. This is consistent with previous data suggesting that GBL is freely permeable (does not substantially disturb tonicity) and that brain has very little capacity to either enzymatically convert the lactone to GHB or respond to the lactone itself.

In order to separate the effect of different theta generators in the hippocampus and to characterize the pattern of relationships between them, in this study, we calculated the coherence that remains between EEG signals, recorded (1) in the stratum oriens of the CA1 region and (2) close to the hippocampal fissure in the dentate gyrus of the right or left hippocampus, after the variations, common also for a third recording site is eliminated (partialization). We found that in both anesthetized and freely moving rats, there is a selective high correlation (coherence) between theta rhythmic activities of contralateral homonymous sites of the hippocampus. The coherence between field potentials recorded in ipsilateral superficial and deep layers was eliminated when allowance was made for any of the contralateral hippocampal recordings. On the other hand, coherence between contralateral homonymous theta dipoles did not decrease when partialized by a heteronymous hippocampal EEG signal. The present results support earlier findings on multiple hippocampal theta dipoles and indicate that they can be separated using partial coherence analysis. The left and right superficial and deep dipoles oscillate as if they formed two separate systems one extending over the superficial CA1 layers on both sides and the other consisting of the left and right deep hippocampal theta dipoles. The results also suggest an important role of the commissural projections in interhemispheric theta synchronization.

Background To date, recombinant tissue plasminogen activator (rtPA) is the only approved drug for ischemic stroke. It is intravenously administered functioning as a thrombolytic agent and is used to obtain reperfusion of the affected area of the brain. Excitotoxicity, inflammation and apoptosis are all involved in delayed neuronal death following stroke and offer multiple opportunities to intervene with neuroprotective agents. Gelsolin (GSN) is an actin- and calcium-binding protein mediating the disassembly of actin filaments and activity of calcium channels. It also functions as a regulator of apoptosis and inflammatory responses. This study tests the hypothesis that increasing the concentration of the form of GSN known as plasma GSN (pGSN) near an infarct will provide neuroprotection following ischemic stroke. Methods We induced middle cerebral artery occlusion (MCAO) in male rats via intracranial injection of endothelin-1 (ET-1), a potent vasoconstrictor, and then treated with local delivery of pGSN. Whole brain laser Doppler perfusion imaging was performed through the skull to assess MCAO effectiveness. Cylinder and vibrissae tests evaluated sensorimotor function before and 72 h after MCAO. Infarct volumes were examined 72 h after MCAO via 2, 3, 5-triphenyltetrazolium chloride (TTC) assay. Results Estimates of relative cerebral perfusion were significantly decreased in all groups receiving MCAO with no differences detected between treatments. Despite equivalent initial strokes, the infarct volume of the pGSN treatment group was significantly reduced compared with the untreated MCAO rats at 72 h. ET-1 induced significant deficits in both cylinder and vibrissae tests while pGSN significantly limited these deficits. Conclusion Gelsolin could be a promising drug for protection against neurodegeneration following ischemic stroke.

One approach for the identification of therapeutic agents for Alzheimer's disease has focused on the research of alpha7 nAChR-selective agonists such as the partial agonists 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) and, more recently, 2-[2-(4-bromophenyl)-2-oxoethyl]-1-methyl pyridinium (S 24795). An alternative approach for targeting alpha7 nAChR has been the development of positive modulators for this receptor. In this study we examined the interactions between full or partial agonists and positive modulators of alpha7 nAChRs in situ in brain tissue. Three positive modulators were used, 5-hydroxyindole (5-HI), 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596), and genistein. Whole-cell recordings were performed in stratum radiatum interneurons from rat brain slices. Hippocampal interneurons were stimulated by ACh, choline, S 24795, or 4OH-GTS-21, before and after bath perfusion with the positive modulators. 5-HI was not effective at potentiating 200 microM 4OH-GTS-21-evoked responses, however 5-HI induced a sustained potentiation of responses evoked by 30 microM 4OH-GTS-21. When 1 mM ACh and 200 microM 4OH-GTS-21 were applied alternately alpha7-mediated responses to both agonists were reduced, suggesting that high concentration of 4OH-GTS-21 produces residual inhibition or desensitization and that 5-HI is not effective at overcoming receptor desensitization. Similar results were obtained with alpha7 receptors expressed in Xenopus oocytes. Interestingly, responses evoked by S 24795 were potentiated by 5-HI but not by genistein. Additionally, PNU-120596 was able to potentiate alpha7-mediated responses, regardless of the nature of the agonist. We demonstrated that the potentiation of alpha7 nAChR response would depend on the nature and the effective concentration of the agonist involved and its particular interaction with the positive modulator.

Altered hippocampal synaptic plasticity may underlie age-related memory impairment. In acute hippocampal slices from aged (22–24 mo) and young adult (1–12 mo) male Brown Norway rats, extracellular excitatory postsynaptic field potentials were recorded in CA1 stratum radiatum evoked by Schaffer collateral stimulation. We used enhanced Ca2+to Mg2+ratio and paired-pulse stimulation protocol to induce maximum changes in the synaptic plasticity. Six episodes of theta-burst stimulation (TBS) or nine episodes of paired low-frequency stimulation (pLFS) were used to generate asymptotic long-term potentiation (LTP) and long-term depression (LTD), respectively. In addition, long-term depotentiation (LTdeP) or de-depression (LTdeD) from maximal LTP and LTD were examined using two episodes of pLFS or TBS. Multiple episodes of TBS or pLFS produced significant LTP or LTD in aged and young adult rats; this was not different between age groups. Moreover, there was no significant difference in the amount of LTdeP or LTdeD between aged and young adult rats. Our results show no age differences in the asymptotic magnitude of LTP or LTD, rate of synaptic modifications, development rates, reversal, or decay after postconditioning. Thus impairment of the basic synaptic mechanisms responsible for expression of these forms of plasticity is not likely to account for decline in memory function within this age range.

S 24795 evoked methyllycaconitine-sensitive inward currents in voltage-clamped hippocampal interneurons with maximum amplitude about 14% that of ACh-evoked responses. Experiments with rat alpha7 receptors expressed in Xenopus oocytes confirmed that S 24795 is a partial agonist of alpha7 nAChR with an EC(50) of 34+/-11 microM and I(max) of approximately 10% relative to ACh. When 60 microM ACh was co-applied to alpha7-expressing oocytes along with increasing concentrations of S 24795, there was a progressive decrease in response compared to the responses to 60 microM ACh alone (IC(50) 45+/-9 microM). The positive allosteric modulator 5-hydroxyindole potentiated ACh- and S 24795-evoked responses of alpha7 receptors in both oocytes and hippocampal interneurons. In hippocampal slice experiments, depending on the ACh concentrations in the application pipette and the ratio of ACh to S 24795, co-application of S 24795 with ACh variously increased, decreased, or had no effect on responses, compared to ACh alone. In order to estimate the effective dilution factor for the pressure application experiments, we tested alpha7 receptors in oocytes with ACh alone and in co-application with S 24795 at the same ratios as in the slice experiments, but at varying dilution factors. The pattern of interaction seen in the slice experiments was most closely matched under the conditions of a 3:100 dilution, suggesting that the pipette solution was diluted approximately 30-fold at the site of action. This dilution factor was consistent with the potency of ACh and S 24795 in the oocyte expression system (EC(50)s approximately 30 microM).

The medial septum-diagonal band (MS/DB) contains primarily cholinergic and GABAergic neurons that project to the hippocampus, and are important for learning and memory. Whole-cell patch clamp methods with brain slices from p11–p20 rats were used to measure MS/DB cell responses to focal somatic application of 1 mM acetylcholine (ACh) and a series of current pulses was applied in order to assess firing frequencies and the presence of hyperpolarization-activated currents (Ih). We identified three types of cells: (1) cells with fast inward currents blocked by methyllycaconitine (MLA) with slow firing rates (3–12 Hz), accommodating action potentials, and no Ih ( n = 20); (2) cells with currents that had both fast (MLA-sensitive) and slow components that were blocked with mecamylamine (MEC) that showed fast firing (up to 60 Hz) and slow firing (up to 3 Hz), with accommodating and non-accommodating action potentials ( n = 46), 33% of which had Ih; and (3) cells not responsive to ACh with moderate firing rates (10–42 Hz), some with accommodating action potentials and some without ( n = 19), of which 92% had Ih. These results are among the first to demonstrate functional nicotinic receptors in the MS/DB. The data suggest that these receptors include 7 and non-7 subtypes and that the expression of each is correlated with firing frequency and the presence of Ih. Responses to ACh were not affected by tetrodotoxin (TTX) and CdCl2 but were blocked by MLA or MLA and MEC, suggesting that these currents involve direct activation of nicotinic receptors. © 2005 Elsevier Ireland Ltd. All rights reserved.

Perfusion with high millimolar levels of taurine evoked a long-lasting potentiation (LLP-TAU) of synaptic transmission in the Schaffer-collateral CA1 region of the rat hippocampus. Although LLP-TAU showed some correlations to increases in the total taurine content of hippocampal slices, it could not be blocked by the taurine transport inhibitor guanidinoethanesulfonic acid (GES), which was able to significantly reduce total slice taurine uptake. Inhibition of GABA transport by either nipecotic acid or beta-guanidinopropionate failed to abolish LLP-TAU and had no significant effect on taurine uptake. The combination of GES and nipecotic acid also had no significant effect on LLP-TAU. Experiments with transportable structural analogs of taurine (beta-aminoisobutyric acid, homotaurine, and isethionic acid) suggest that activation of classical taurine transport pathways does not always yield a robust LLP-TAU. Hippocampal LLP-TAU could be significantly attenuated, however, by pre-incubation with submillimolar levels of taurine. In summary, the development of LLP-TAU in the rat hippocampus appears to be associated with the intracellular accumulation rather than the activation of known transporters of taurine, but the precise means of its accumulation remains to be identified.

We characterized the development and pharmacology of Ca(2+) channel currents in NGF-treated embryonic day 21 cultured rat septal cells. Using standard whole-cell voltage clamp techniques, cells were held at -80 mV and depolarized to construct current-voltage relations in conditions that eliminated Na(+) or K(+) currents. Barium (10 mM) was used as the charge carrier. Maximum current was produced when cells were depolarized to 0 or +10 mV. Recordings from 77 cells revealed that Ca(2+) channel current density increases over time in culture from nearly 0 pA/pF on day 2 in vitro (0.65+/-0.65 pA/pF) to (6.95+/-1.59 pA/pF) on days 6-8. This was followed by a period where currents became nearly 3 times more dense (21.05+/-7.16 pA/pF) at days 9-17. There was little or no evidence for low voltage activated currents. Bath application of 50-100 microM CdCl(2) abolished approximately 95% of the current. Application of 10 microM nimodipine produced a 50.5+/-3.22% reduction in current, 2 microM omega-CTx-GVIA produced a 32.4+/-7.3% reduction, and application of 4 microM omega-Aga-IVA produced a 29.5+/-5.73% reduction in current. When all three inhibitors (10 microM nimodipine, 2 microM omega-CTx-GVIA, and 4 microM omega-Aga-IVA) were applied simultaneously, a residual current remained that was 18.0+/-4.9% of the total current and was completely abolished by application of CdCl(2). This is the first report to characterize Ca(2+) channel currents in cultured embryonic septal cells. These data indicate that there is a steady increase in Ca(2+) channel expression over time in vitro, and show that like other cultured neuronal cells, septal cells express multiple Ca(2+) channel types including L, N, P/Q and R-type channels.

Multipotential progenitor cells grown from central nervous system (CNS) tissues in defined media supplemented with epidermal growth factor (EGF), when attached to a suitable substratum, differentiate to express neural and glial histochemical markers and morphologies. To assess the functional characteristics of such cells, expression of voltage-gated Na+ and K+ currents (INa, IK) was studied by whole-cell patch clamp methods in progenitors raised from postnatal rat forebrain. Undifferentiated cells were acutely dissociated from proliferative "spheres," and differentiated cells were studied 1-25 days after plating spheres onto polylysine/laminin-treated coverslips. INa and IK were detected together in 58%, INa alone in 11%, and IK alone in 19% of differentiated cells recorded with K(+)-containing pipettes. With internal Cs+ (to isolate INa), INa up to 45 pA/pF was observed in some cells within 1 day after plating. I Na ranged up to 150 pA/pF subsequently. Overall, 84% of cells expressed I Na, with an average of 38 pA/pF. INa had fast kinetics, as in neurons, but steadystate inactivation curves were strongly negative, resembling those of glial INa. Inward tail currents sensitive to [K+]out were observed upon repolarization after the 10-ms test pulse with internal Cs+, indicating the expression of K+ channels in 82% of cells. In contrast to the substantial currents observed in differentiating cells, little or no INa or Ik-tail currents were detected in recordings from cells acutely dissociated from spheres. Thus, in the presence of EGF, ionic currents develop early during differentiation induced by attachment to an appropriate substratum. Cells switched from EGF to basic fibroblast growth factor (bFGF) when plated onto coverslips showed greatly reduced proliferation and developed less neuron-like morphologies than cells plated in the presence of EGF. INa was observed in only 53% of bFGF-treated cells, with an average of 9 pA/pF. Thus, in contrast to reports that bFGF promotes neuronal differentiation in some CNS progenitor populations, our EGF-generated postnatal rat CNS progenitors do not develop neuronal characteristics when switched to medium containing bFGF. Thus, differentiated CNS progenitors can express a mix of neuronal and glial molecular, morphological, and electrophysiological properties that can be modified by culture conditions.