Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjac... more Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models ...
Disruption of the precise balance of positive and negative molecular regulators of blood and lymp... more Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessel growth can lead to myriad diseases. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessel growth has not to our knowledge been previously described. We report the existence of a splice variant of the gene encoding vascular endothelial growth factor receptor-2 (Vegfr-2) that encodes a secreted form of the protein, designated soluble Vegfr-2 (sVegfr-2), that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c function. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting blood vasculature. Administration of sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 thus acts as a molecular uncoupler of blood and lymphatic vessels; modulation of sVegfr-2 might have therapeutic effects in treating lymphatic vascular malformations, transplantation rejection and, potentially, tumor lymphangiogenesis and lymphedema (pages 993-994).
Age-related macular degeneration (AMD) is the leading cause of blindness in the United States. Al... more Age-related macular degeneration (AMD) is the leading cause of blindness in the United States. Although AMD shares multiple risk factors with coronary artery disease (CAD), the association between AMD and CAD has not been established. The objective of our study was to demonstrate an association between the diagnosis of AMD and CAD and/or major cardiovascular risk factors. We performed a retrospective chart review of >13,000 patients at the Lexington Veterans Affairs Medical Center. Patients diagnosed as having AMD served as cases, and patients diagnosed with cataract and no AMD served as controls. We examined the prevalence of CAD and associated risk factors in both groups using univariate analysis followed by multivariate analyses to examine the association between AMD and CAD after adjusting for known common risk factors. We identified 3950 patients with AMD and 9166 controls. Patients with AMD were on average 6 years older than controls (P < 0.001) and had a significantly h...
INTRODUCTION. Corneal avascularity, which is required for optical clarity and optimal vision, is ... more INTRODUCTION. Corneal avascularity, which is required for optical clarity and optimal vision, is still an active subject of mechanistic inquiry (1). We hypothesized that the cornea remains avascular despite the presence of vascular endothelial growth factor (VEGF)-A due to expression of soluble VEGFR-1 (sVEGFR-1), an endogenous VEGF-A trap. METHOD. VEGF-A and sVEGFR-1 expression were assayed by ELISA, western blotting, immunostaining, and in situ hybridization in corneas of normal humans and mice, in corn1 mice and manatees (which have spontaneously vascularized corneas), and in patients with neovascular keratopathies. Corneal vascularization was assessed by CD31-stained flat mounts. sVEGFR-1 was suppressed in vivo in mice by subconjunctival injection of neutralizing anti-VEGFR-1 antibodies, by intracorneal injection of plasmids (2) encoding short hairpin RNA (shRNA) against sVEGFR-1, or by Ad-Cre injection into the corneas of Vegfr1 loxP/loxP mice. Various controls were employed in...
To examine the expression and regulation of an injury-related protein, heat shock protein (Hsp) 2... more To examine the expression and regulation of an injury-related protein, heat shock protein (Hsp) 27, in retinal pigment epithelium (RPE), since RPE injury may be an important feature of age-related macular degeneration (ARMD). Retinal cross sections from eyes of Lewis rats were examined for Hsp27 in vivo by immunohistochemistry, and in vitro expression of Hsp27 in human ARPE-19 cells was determined by Northern and Western blot analysis. Oxidant-mediated injury was performed by exposing ARPE-19 cells to myeloperoxidase and hydrogen peroxide. Cell lines stably expressing green fluorescent protein (GFP) targeted to the cell membrane were used to study injury-induced membrane blebbing, and XTT conversion was used to detect cell viability. High level of Hsp27 expression was detected in vivo in ganglion cells, RPE, and photoreceptor outer segments of rat retina. ARPE-19 cells also expressed high levels of Hsp27 in vitro. Oxidative injury in ARPE-19 cells resulted in transcriptional and tra...
Advances in Experimental Medicine and Biology, 2008
Toward a Higher Fidelity Model of AMD Brian J. Raisler, Miho Nozaki, Judit Baffi, William W. Haus... more Toward a Higher Fidelity Model of AMD Brian J. Raisler, Miho Nozaki, Judit Baffi, William W. Hauswirth, and Jayakrishna Ambati ... Sci. 39, 24742478. Rasmussen H., Chu KW, Campochiaro P., Gehlbach PL, Haller JA, Handa JT, Nguyen QD, & Sung JU (2001) Clinical protocol. ...
ABSTRACT In the study discussed in this chapter, immunohistochemical, in situ hybridization histo... more ABSTRACT In the study discussed in this chapter, immunohistochemical, in situ hybridization histochemistry, tract-tracing, and in vivo microdialysis techniques have been used to investigate the participation of the central catecholaminergic (CA) systems in the organization of responses to an acute neurogenic (pain) stress: subcutaneous injection of 0.5 ml of 4% formalin into the hind paw of rats. This single injection produces a local pain that develops immediately after injection and results in four- to fivefold increase in corticosterone and adrenocorticotropic hormone (ACTH) levels in the plasma and a similar increase in norepinephrine levels in the paraventricular nucleus (PVN). Formalin also induced a rapid expression of c-fos and corticotropin-releasing hormones (CRHs) mRNA in the PVN. The possible crossover of the nociceptive spinal afferents to the medullary CA neurons and the crossover of the ascending CA neurons to the PVN were investigated after spinal and medullary surgical hemisections. Hemisection between the spinal cord and the medulla oblongata did not influence formalin-induced elevation of plasma corticosterone and ACTH levels, and strong c-fos immunoreactivity was observed in the medullary CA cell groups and in the PVN bilaterally, as in the sham-operated animals. Hemisection between the medullary CA cell groups and the PVN (unilateral knife cut at the pons-medulla oblongata border) decreased CRH immunoreactivity and CRH gene expression in the PVN ipsilateral to the transection but remained unchanged contralateral to the knife cut. Although, unilateral pontomedullary hemisection reduce TH and CRH immunoreactivity and CRH gene expression in the PVN, it does not influence the high plasma corticosterone and ACTH levels in response to formalin stress. However, acute immobilization stress in hemisected animals is able to induce CRH expression. These findings suggest that in brain stem-hemisected animals, the remaining intact ascending pathways are sufficient to allow normal, or almost normal, activation of the hypothalamopituitary-adrenal axis during stress.
Lactoperoxidase (LPO) is an enzyme with antimicrobial properties present in saliva, milk, tears, ... more Lactoperoxidase (LPO) is an enzyme with antimicrobial properties present in saliva, milk, tears, and airway secretions. Although the formation of microbicidal oxidants by LPO has been recognized for some time, the source of hydrogen peroxide (H2O2) for LPO-catalyzed reactions remains unknown. Reactive oxygen species produced by the phagocyte NADPH oxidase (phox) play a critical role in host defense against pathogens; however, analogous oxidant-generating systems in other tissues have not been associated with antimicrobial activity. Several homologues of gp91phox, the catalytic core of this enzyme, were described recently; dual oxidase (Duox)1/thyroid oxidase 1 and Duox2/thyroid oxidase 2 were identified in the thyroid gland and characterized as H2O2 donors for thyroxin biosynthesis. We examined Duox1 and Duox2 expression in secretory glands and on mucosal surfaces and give evidence for their presence and activity in salivary glands, rectum, trachea, and bronchium. Epithelial cells in salivary excretory ducts and rectal glands express Duox2, whereas tracheal and bronchial epithelial cells express Duox1. Furthermore, we detected Duox1-dependent H2O2 release by cultured human bronchial epithelial cells. Our observations suggest that Duox1 and Duox2 are novel H2O2 sources that can support LPO-mediated antimicrobial defense mechanisms on mucosal surfaces.
Castro et al. (1) reported that bone marrow cells (BMCs) fail to generate neural cells in vivo. T... more Castro et al. (1) reported that bone marrow cells (BMCs) fail to generate neural cells in vivo. The bone marrow they injected came from mice in which LacZ expression was driven by a widely expressed trapped promoter. In principle, such cells and their progeny should readily be ...
Retinal vascular disease is the most common cause of macular edema (ME). While there are several ... more Retinal vascular disease is the most common cause of macular edema (ME). While there are several etiologies of vascular compromise and subsequent macular leakage, diabetic retinopathy is the most prevalent and continues to challenge ophthalmologists and frustrate patients due to its refractory nature. In response to this epidemic, diabetic ME (DME) along with cystoid ME (CME) have been areas of active investigation both in the clinic and the laboratory. Several decades of basic science research have revealed a growing and complex array of cytokine growth factors and proinflammatory mediators which are capable of inciting the cellular changes that result in accumulation of fluid within the retina. Much of this new molecular foundation provides the current and fundamental scaffold for understanding the pathologic process of ME while simultaneously identifying potential therapeutic targets. Whereas CME has classically been treated with corticosteroids and nonsteroidal antiinflammatory drugs, recent clinical studies have demonstrated improved visual outcomes for DME treatment with light focal/grid laser, corticosteroids and anti-vascular endothelial growth factor antibodies. Yet, each of these treatments has differential effects on the multifactorial mechanisms of ME. This article reviews the anatomical, cellular and molecular derangements associated with ME and highlights specific pathways targeted by current treatments.
The orphan nuclear receptor Nurr1 is a transcription factor that belongs to the steroid/thyroid h... more The orphan nuclear receptor Nurr1 is a transcription factor that belongs to the steroid/thyroid hormone receptor superfamily and is expressed in many regions of the brain. To determine the physiological role of Nurr1, we previously generated mice with a null mutation in the Nurr1 gene. Nurr1-null mice appear to develop normally but die within 12 h after birth. Subsequent analysis revealed the absence of neurotransmitter dopamine and tyrosine hydroxylase immunoreactivity in the central dopaminergic area of newborn pups. Herein, using in situ hybridization histochemistry, we show that Nurr1 is expressed only in subset of catecholamine producing neurons (A2 partly, A8-A10 and A11 catecholaminergic cell groups), and is excluded from the norepinephrine producing neurons (A1, A2, A5-A6 catecholaminergic cell groups). Nurr1 was not expressed in the dopamine synthesizing cell groups (A12-A16 catecholaminergic cell groups) of the diencephalon and the olfactory bulb. As previously shown and confirmed in this study, tyrosine hydroxylase immunoreactivity was absent in the substantia nigra and ventral tegmental area of Nurr1-deficient mice. However, the loss of Nurr1 expression in A2 and A11 dopaminergic neurons did not affect their tyrosine hydroxylase immunoreactivity. This study begins to dissect cues necessary for understanding the complex regulation of the catecholaminergic biosynthetic pathway with regard to local, chemical and developmental changes in the brain.
The postsynaptic AMPA/kainate and N-methyl-D-aspartate-selective glutamate receptors are formed b... more The postsynaptic AMPA/kainate and N-methyl-D-aspartate-selective glutamate receptors are formed by several different subunits and the overall subunit composition of the receptor appears to determine its physiological and pharmacological properties. Although glutamatergic mechanisms have been implicated in various forms of hippocampal stress responses, the impact of stress on glutamate receptor subunit composition has not yet been elucidated. We have used cell-by-cell quantitative in situ hybridization to assess stress-induced changes in transcript levels of N-methyl-D-aspartate and AMPA receptor subunit genes in subdivisions of the rat hippocampus and hypothalamus that are implicated in the stress response. We found that 24 h after a single immobilization stress there was a significant increase in the cellular level of NR1 subunit messenger RNA (about 35-45% above control values) in hippocampal CA3 and CA1 pyramidal cells as well as in neurons of the hypothalamic supraoptic and paraventricular nuclei. Moreover, in the CA3 area we have detected a concomitant increase (50% above controls) in the level of NR2B subunit messenger RNA, while the expression of NR2A subunit gene did not change after stress. Stress induced a selective decrease in the level of AMPA receptor subunit glutamate receptor A messenger RNA in neurons of both the CA3 and CA1 areas (18 and 24%, respectively, below control values). These results suggest that the regulation of specific subunit messenger RNAs of the N-methyl-D-aspartate and AMPA receptors may be involved in altered hippocampal and hypothalamic responsiveness to glutamate and thus could play a critical role in stress-induced changes in their function.
Cellular levels of vasopressin (VP) and corticotropin-releasing factor (CRF) mRNAs were determine... more Cellular levels of vasopressin (VP) and corticotropin-releasing factor (CRF) mRNAs were determined in the rat hypothalamic paraventricular nucleus after acute immobilization stress. Messenger RNA levels were measured by in situ hybridization histochemistry using 35S-labeled synthetic DNA oligonucleotide probes and quantitative autoradiography. Analysis of histograms related to density values revealed that two hours after immobilization the average cellular VP and CRF mRNA levels were significantly increased (145 +/- 30.3 and 68 +/- 21.3%, respectively, above control values). Moreover, the number of VP-expressing parvicellular neurons was doubled in stressed rats relative to controls. These results indicate that single acute immobilization stress is sufficient to up-regulate VP as well as CRF mRNA production in the hypothalamic CRF-neurosecretory system.
Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjac... more Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models ...
Disruption of the precise balance of positive and negative molecular regulators of blood and lymp... more Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessel growth can lead to myriad diseases. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessel growth has not to our knowledge been previously described. We report the existence of a splice variant of the gene encoding vascular endothelial growth factor receptor-2 (Vegfr-2) that encodes a secreted form of the protein, designated soluble Vegfr-2 (sVegfr-2), that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c function. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting blood vasculature. Administration of sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 thus acts as a molecular uncoupler of blood and lymphatic vessels; modulation of sVegfr-2 might have therapeutic effects in treating lymphatic vascular malformations, transplantation rejection and, potentially, tumor lymphangiogenesis and lymphedema (pages 993-994).
Age-related macular degeneration (AMD) is the leading cause of blindness in the United States. Al... more Age-related macular degeneration (AMD) is the leading cause of blindness in the United States. Although AMD shares multiple risk factors with coronary artery disease (CAD), the association between AMD and CAD has not been established. The objective of our study was to demonstrate an association between the diagnosis of AMD and CAD and/or major cardiovascular risk factors. We performed a retrospective chart review of >13,000 patients at the Lexington Veterans Affairs Medical Center. Patients diagnosed as having AMD served as cases, and patients diagnosed with cataract and no AMD served as controls. We examined the prevalence of CAD and associated risk factors in both groups using univariate analysis followed by multivariate analyses to examine the association between AMD and CAD after adjusting for known common risk factors. We identified 3950 patients with AMD and 9166 controls. Patients with AMD were on average 6 years older than controls (P < 0.001) and had a significantly h...
INTRODUCTION. Corneal avascularity, which is required for optical clarity and optimal vision, is ... more INTRODUCTION. Corneal avascularity, which is required for optical clarity and optimal vision, is still an active subject of mechanistic inquiry (1). We hypothesized that the cornea remains avascular despite the presence of vascular endothelial growth factor (VEGF)-A due to expression of soluble VEGFR-1 (sVEGFR-1), an endogenous VEGF-A trap. METHOD. VEGF-A and sVEGFR-1 expression were assayed by ELISA, western blotting, immunostaining, and in situ hybridization in corneas of normal humans and mice, in corn1 mice and manatees (which have spontaneously vascularized corneas), and in patients with neovascular keratopathies. Corneal vascularization was assessed by CD31-stained flat mounts. sVEGFR-1 was suppressed in vivo in mice by subconjunctival injection of neutralizing anti-VEGFR-1 antibodies, by intracorneal injection of plasmids (2) encoding short hairpin RNA (shRNA) against sVEGFR-1, or by Ad-Cre injection into the corneas of Vegfr1 loxP/loxP mice. Various controls were employed in...
To examine the expression and regulation of an injury-related protein, heat shock protein (Hsp) 2... more To examine the expression and regulation of an injury-related protein, heat shock protein (Hsp) 27, in retinal pigment epithelium (RPE), since RPE injury may be an important feature of age-related macular degeneration (ARMD). Retinal cross sections from eyes of Lewis rats were examined for Hsp27 in vivo by immunohistochemistry, and in vitro expression of Hsp27 in human ARPE-19 cells was determined by Northern and Western blot analysis. Oxidant-mediated injury was performed by exposing ARPE-19 cells to myeloperoxidase and hydrogen peroxide. Cell lines stably expressing green fluorescent protein (GFP) targeted to the cell membrane were used to study injury-induced membrane blebbing, and XTT conversion was used to detect cell viability. High level of Hsp27 expression was detected in vivo in ganglion cells, RPE, and photoreceptor outer segments of rat retina. ARPE-19 cells also expressed high levels of Hsp27 in vitro. Oxidative injury in ARPE-19 cells resulted in transcriptional and tra...
Advances in Experimental Medicine and Biology, 2008
Toward a Higher Fidelity Model of AMD Brian J. Raisler, Miho Nozaki, Judit Baffi, William W. Haus... more Toward a Higher Fidelity Model of AMD Brian J. Raisler, Miho Nozaki, Judit Baffi, William W. Hauswirth, and Jayakrishna Ambati ... Sci. 39, 24742478. Rasmussen H., Chu KW, Campochiaro P., Gehlbach PL, Haller JA, Handa JT, Nguyen QD, & Sung JU (2001) Clinical protocol. ...
ABSTRACT In the study discussed in this chapter, immunohistochemical, in situ hybridization histo... more ABSTRACT In the study discussed in this chapter, immunohistochemical, in situ hybridization histochemistry, tract-tracing, and in vivo microdialysis techniques have been used to investigate the participation of the central catecholaminergic (CA) systems in the organization of responses to an acute neurogenic (pain) stress: subcutaneous injection of 0.5 ml of 4% formalin into the hind paw of rats. This single injection produces a local pain that develops immediately after injection and results in four- to fivefold increase in corticosterone and adrenocorticotropic hormone (ACTH) levels in the plasma and a similar increase in norepinephrine levels in the paraventricular nucleus (PVN). Formalin also induced a rapid expression of c-fos and corticotropin-releasing hormones (CRHs) mRNA in the PVN. The possible crossover of the nociceptive spinal afferents to the medullary CA neurons and the crossover of the ascending CA neurons to the PVN were investigated after spinal and medullary surgical hemisections. Hemisection between the spinal cord and the medulla oblongata did not influence formalin-induced elevation of plasma corticosterone and ACTH levels, and strong c-fos immunoreactivity was observed in the medullary CA cell groups and in the PVN bilaterally, as in the sham-operated animals. Hemisection between the medullary CA cell groups and the PVN (unilateral knife cut at the pons-medulla oblongata border) decreased CRH immunoreactivity and CRH gene expression in the PVN ipsilateral to the transection but remained unchanged contralateral to the knife cut. Although, unilateral pontomedullary hemisection reduce TH and CRH immunoreactivity and CRH gene expression in the PVN, it does not influence the high plasma corticosterone and ACTH levels in response to formalin stress. However, acute immobilization stress in hemisected animals is able to induce CRH expression. These findings suggest that in brain stem-hemisected animals, the remaining intact ascending pathways are sufficient to allow normal, or almost normal, activation of the hypothalamopituitary-adrenal axis during stress.
Lactoperoxidase (LPO) is an enzyme with antimicrobial properties present in saliva, milk, tears, ... more Lactoperoxidase (LPO) is an enzyme with antimicrobial properties present in saliva, milk, tears, and airway secretions. Although the formation of microbicidal oxidants by LPO has been recognized for some time, the source of hydrogen peroxide (H2O2) for LPO-catalyzed reactions remains unknown. Reactive oxygen species produced by the phagocyte NADPH oxidase (phox) play a critical role in host defense against pathogens; however, analogous oxidant-generating systems in other tissues have not been associated with antimicrobial activity. Several homologues of gp91phox, the catalytic core of this enzyme, were described recently; dual oxidase (Duox)1/thyroid oxidase 1 and Duox2/thyroid oxidase 2 were identified in the thyroid gland and characterized as H2O2 donors for thyroxin biosynthesis. We examined Duox1 and Duox2 expression in secretory glands and on mucosal surfaces and give evidence for their presence and activity in salivary glands, rectum, trachea, and bronchium. Epithelial cells in salivary excretory ducts and rectal glands express Duox2, whereas tracheal and bronchial epithelial cells express Duox1. Furthermore, we detected Duox1-dependent H2O2 release by cultured human bronchial epithelial cells. Our observations suggest that Duox1 and Duox2 are novel H2O2 sources that can support LPO-mediated antimicrobial defense mechanisms on mucosal surfaces.
Castro et al. (1) reported that bone marrow cells (BMCs) fail to generate neural cells in vivo. T... more Castro et al. (1) reported that bone marrow cells (BMCs) fail to generate neural cells in vivo. The bone marrow they injected came from mice in which LacZ expression was driven by a widely expressed trapped promoter. In principle, such cells and their progeny should readily be ...
Retinal vascular disease is the most common cause of macular edema (ME). While there are several ... more Retinal vascular disease is the most common cause of macular edema (ME). While there are several etiologies of vascular compromise and subsequent macular leakage, diabetic retinopathy is the most prevalent and continues to challenge ophthalmologists and frustrate patients due to its refractory nature. In response to this epidemic, diabetic ME (DME) along with cystoid ME (CME) have been areas of active investigation both in the clinic and the laboratory. Several decades of basic science research have revealed a growing and complex array of cytokine growth factors and proinflammatory mediators which are capable of inciting the cellular changes that result in accumulation of fluid within the retina. Much of this new molecular foundation provides the current and fundamental scaffold for understanding the pathologic process of ME while simultaneously identifying potential therapeutic targets. Whereas CME has classically been treated with corticosteroids and nonsteroidal antiinflammatory drugs, recent clinical studies have demonstrated improved visual outcomes for DME treatment with light focal/grid laser, corticosteroids and anti-vascular endothelial growth factor antibodies. Yet, each of these treatments has differential effects on the multifactorial mechanisms of ME. This article reviews the anatomical, cellular and molecular derangements associated with ME and highlights specific pathways targeted by current treatments.
The orphan nuclear receptor Nurr1 is a transcription factor that belongs to the steroid/thyroid h... more The orphan nuclear receptor Nurr1 is a transcription factor that belongs to the steroid/thyroid hormone receptor superfamily and is expressed in many regions of the brain. To determine the physiological role of Nurr1, we previously generated mice with a null mutation in the Nurr1 gene. Nurr1-null mice appear to develop normally but die within 12 h after birth. Subsequent analysis revealed the absence of neurotransmitter dopamine and tyrosine hydroxylase immunoreactivity in the central dopaminergic area of newborn pups. Herein, using in situ hybridization histochemistry, we show that Nurr1 is expressed only in subset of catecholamine producing neurons (A2 partly, A8-A10 and A11 catecholaminergic cell groups), and is excluded from the norepinephrine producing neurons (A1, A2, A5-A6 catecholaminergic cell groups). Nurr1 was not expressed in the dopamine synthesizing cell groups (A12-A16 catecholaminergic cell groups) of the diencephalon and the olfactory bulb. As previously shown and confirmed in this study, tyrosine hydroxylase immunoreactivity was absent in the substantia nigra and ventral tegmental area of Nurr1-deficient mice. However, the loss of Nurr1 expression in A2 and A11 dopaminergic neurons did not affect their tyrosine hydroxylase immunoreactivity. This study begins to dissect cues necessary for understanding the complex regulation of the catecholaminergic biosynthetic pathway with regard to local, chemical and developmental changes in the brain.
The postsynaptic AMPA/kainate and N-methyl-D-aspartate-selective glutamate receptors are formed b... more The postsynaptic AMPA/kainate and N-methyl-D-aspartate-selective glutamate receptors are formed by several different subunits and the overall subunit composition of the receptor appears to determine its physiological and pharmacological properties. Although glutamatergic mechanisms have been implicated in various forms of hippocampal stress responses, the impact of stress on glutamate receptor subunit composition has not yet been elucidated. We have used cell-by-cell quantitative in situ hybridization to assess stress-induced changes in transcript levels of N-methyl-D-aspartate and AMPA receptor subunit genes in subdivisions of the rat hippocampus and hypothalamus that are implicated in the stress response. We found that 24 h after a single immobilization stress there was a significant increase in the cellular level of NR1 subunit messenger RNA (about 35-45% above control values) in hippocampal CA3 and CA1 pyramidal cells as well as in neurons of the hypothalamic supraoptic and paraventricular nuclei. Moreover, in the CA3 area we have detected a concomitant increase (50% above controls) in the level of NR2B subunit messenger RNA, while the expression of NR2A subunit gene did not change after stress. Stress induced a selective decrease in the level of AMPA receptor subunit glutamate receptor A messenger RNA in neurons of both the CA3 and CA1 areas (18 and 24%, respectively, below control values). These results suggest that the regulation of specific subunit messenger RNAs of the N-methyl-D-aspartate and AMPA receptors may be involved in altered hippocampal and hypothalamic responsiveness to glutamate and thus could play a critical role in stress-induced changes in their function.
Cellular levels of vasopressin (VP) and corticotropin-releasing factor (CRF) mRNAs were determine... more Cellular levels of vasopressin (VP) and corticotropin-releasing factor (CRF) mRNAs were determined in the rat hypothalamic paraventricular nucleus after acute immobilization stress. Messenger RNA levels were measured by in situ hybridization histochemistry using 35S-labeled synthetic DNA oligonucleotide probes and quantitative autoradiography. Analysis of histograms related to density values revealed that two hours after immobilization the average cellular VP and CRF mRNA levels were significantly increased (145 +/- 30.3 and 68 +/- 21.3%, respectively, above control values). Moreover, the number of VP-expressing parvicellular neurons was doubled in stressed rats relative to controls. These results indicate that single acute immobilization stress is sufficient to up-regulate VP as well as CRF mRNA production in the hypothalamic CRF-neurosecretory system.
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