Clyde F. Phelix

Orexin-A (Hypocretin-1) and leptin enhance LTP in the dentate gyrus of rats in vivo

Orexin-A (Hypocretin-1) has been localized in the posterior and lateral hypothalamic perifornical region. Orexin containing axon terminals have been found in hypothalamic nuclei and many other parts of the brain; for example, the hippocampus. Two types of orexin receptors have been discovered. Orexin 1 type of receptors have been described and been shown to be widely distributed in the rat brain including the hippocampus. Subsequently Orexin-A was found to impair both water maze performance and hippocampal long term potentiation (LTP). Leptin is expressed in adipose tissue and released into the blood where it affects food intake and can also produce widespread physiological changes mediated via autonomic preganglionic neurons, pituitary gland, and cerebral cortex. Immunoreactivity for leptin receptors has been found in various hypothalamic nuclei including the lateral hypothalamic area as well as the hippocampus especially in the dentate gyrus and CA1. Leptin receptor deficient rats and mice also show impaired LTP in CA1 and poor performance in the water maze. The present study was conducted to determine the effects of 0.0, 30, 60, 90, and 100 nM, orexin-A, and leptin, 0.0, 1.0, 100 nM, 1, and 10 microM, in 1.0 microl of ACSF, applied directly into the dentate gyrus, on LTP in medial perforant path dentate granule cell synapses in urethane anesthetized rats. Orexin-A specifically enhanced LTP at the 90 nM dose; and it was possible to block the enhancement by pretreating the animals with SB-334867, a specific orexin 1 receptor antagonist. Leptin enhanced normal LTP at 1.0 microM but inhibited LTP at lower and higher doses. These results and previous data indicate that the same peptide could possibly have different modulatory post synaptic effects in different hippocampal synapses dependent upon different types of post synaptic receptors.

Monoamine innervation of bed nucleus of stria terminalis: An electron microscopic investigation

Immunocytochemical studies showed distinctive monoamine input to the bed nucleus of the stria terminalis (BST). A comparison of axons immunoreactive (IR) for a catecholamine synthetic enzyme [tyrosine hydroxylase (TH) or dopamine beta-hydroxylase (DBH) or phenylethanolamine-N-methyl transferase (PNMT)] or serotonin (5-HT) was performed. TH-IR axons had a greater density in the lateral BST, but DBH-IR and 5-HT-IR axons had a greater density in the medial BST. PNMT-IR axons were dense in the intermediate BST. TH-IR axons had a greater density than DBH- and PNMT-IR axons in the dorsolateral BST, but DBH-IR axons had the greatest density in the ventrolateral BST. Ultrastructural studies revealed that TH-IR terminals formed synapses with soma, dendrites, spines, and axons in the dorsolateral BST. DBH-IR terminals formed synapses with dendritic shafts and spines, and 5-HT-IR terminals formed synapses with dendrites in the ventrolateral BST. Only some 5-HT-IR axons were myelinated. The medial vs. lateral organization of the noradrenergic and dopaminergic afferents in the BST of the rat brain is now evident and is similar to the human brain. The medial-lateral functional subdivision of the BST is supported by the pattern of dopaminergic, noradrenergic, and serotonergic afferents. This demonstration of epinephrine-producing afferents in the BST is the first detailed description of adrenergic input to the BST and aided the determination that catecholaminergic innervation of the ventrolateral BST is predominantly noradrenergic as has been proposed for many years. However, the additional demonstration of rich dopaminergic innervation of the dorsolateral subnucleus suggests further division of the BST into dorsal and ventral functional subgroups.

Angiotensin IV enhances LTP in rat dentate gyrus in vivo

Angiotensins have been shown to play a significant role in a variety of physiological functions including learning and memory processes. Relatively recent evidence supports the increasing importance of angiotensin IV (Ang IV), in many of these functions previously associated only with Ang II, including learning and memory. An interesting hypothesis generated by these results has been that Ang II is a precursor for the production of a more active peptide fragment, Ang IV. Since Ang II impairs learning and memory, when administered directly or released into the hippocampal dentate gyrus, and inhibits long term potentiation (LTP) in medial perforant path-dentate granule cell synapses, as well; it remained to be seen what effects Ang IV had on LTP in these same synapses. Results of this study show clearly that Ang IV significantly enhances LTP, and the enhancement is both dose and time dependent. The following solutions of Ang IV were administered over a five min period, at the end of baseline and before the first tetanus was applied: 2.39, 4.78, and 9.56 nM. An inverted U-type dose related effect was observed. A complex time related effect was observed with a maximum at 5 min, a return to normal LTP at 30 min and a minimum below normal at 90 min, and a return to normal LTP at 120 min. The effects of the 4.78 nM solution were determined at the following intervals between administration and the first tetanus: 5, 15, 30, 60, 90, and 120 min. The enhancement of LTP can be prevented by pretreatment with Divalinal, an Ang IV antagonist, without any effect on normal LTP. Two solutions of Divalinal were used; 5 nM and 5 microM, and the 5 microM was more effective and completely blocked the enhancement of normal LTP. Results were also obtained with 4.78 nM Nle1-Ang IV (Norleucine), an Ang IV agonist. Norleucine was less effective than Ang IV in the enhancement of normal LTP and displayed a similar time course of activity. Both Ang IV and Norleucine produced a significant suppression of normal LTP at 90 min; that remains to be explained. However, the inhibition by Ang IV was dose dependent and was blocked by Divalinal. The fact that the Ang IV enhancement of normal LTP was blocked by losartan, an Ang II AT1 receptor antagonist, is puzzling since Divalinal had no effect on the inhibition of LTP by Ang II.

Catecholamine-CRF synaptic interaction in a septal bed nucleus: Afferents of neurons in the bed nucleus of the stria terminalis

Projections of catecholamine neurons to the bed nucleus of the stria terminalis (BST), especially its corticotropin releasing factor (CRF)-producing neurons, are implicated as being major contributors to the neurochemically mediated central regulation of the stress response. The purpose of the present study was to examine in the BST of the rat brain the morphological characteristics of interactions between two neuron populations of the brain, catecholaminergic and CRF neurons. A double-label immunocytochemical, light and electron microscopic technique allowed the demonstration of the synaptic interaction between dopamine (DA, i.e., tyrosine hydroxylase-containing) and norepinephrine (NE, i.e., dopamine-beta-hydroxylase-containing) axons and CRF neurons in the BST. DA terminals formed synapses with dendrites and soma of CRF neurons in the dorsolateral BST. NE terminals formed synapses with dendrites of CRF neurons in the ventrolateral BST. In conclusion, catecholamine afferents can directly affect the contribution of CRF neurons of the BST to an animals response to stress.

Serotonin-CRF interaction in the bed nucleus of the stria terminalis: A light microscopic double-label immunocytochemical analysis

The purpose of the present study was to examine in the bed nucleus of the stria terminalis (BST) of the rat brain the morphological characteristics of interactions between corticotropin-releasing factor (CRF)-producing neuron populations and serotonin (5-HT) axons. A double-label immunocytochemical, light microscopic technique was used to demonstrate axosomatic and axodendritic interactions between 5-HT axons and CRF neurons in the BST. Both the dorsolateral and ventrolateral subpopulations of CRF neurons were targets for the 5-HT afferents. Projections of monoamine neurons to the BST and the CRF neurons that are in the BST are implicated as being major contributors to the neurochemically mediated central regulation of the stress response.

Light microscopic immunocytochemical evidence of converging serotonin and dopamine terminals in ventrolateral nucleus accumbens

The mesencephalic tegmentum contains monoaminergic neurons that project to the nucleus accumbens (NAcc). These monoaminergic neurons consist of the serotonergic (5-HT) neurons of the dorsal and median raphe and the dopaminergic (DA) neurons of the ventral tegmental area (VTA). Recent neurochemical reports describe cocaine-induced alterations in dopamine and serotonin release in NAcc that has coincidental occurrence both spatially and temporally, as shown by in vivo voltammetry. There is a functional role for 5-HT-DA interactions within the NAcc in the underlying mechanism of action of cocaine as well as for 5-HT in A10 DA neurons in the basal or endogenous state whether or not cocaine-relevant reward circuits are involved. Our objective was to study the neuroanatomic localization of tyrosine hydroxylase-containing (TH) and 5-HT-containing axons in the ventrolateral region of the rat NAcc, where codetection of monoamines had been assessed. The significance of this vINAcc is its reciprocal connectivity with VTA, which contains the somatodendritic portions of the mesoacumbens DA neurons. The results showed that, in the vINAcc, the core contained a dense terminal field of TH axons that had an extensive overlap with 5-HT axons in the periphery within the core. Because the in vivo electrochemical codetection of DA and 5-HT assessed in the ventral-most aspect of this overlap zone can be correlated with terminal release, a functional interaction of 5-HT and DA at postsynaptic sites in vINAcc is possible.

Thiol Oxidative Stress Induced by Metabolic Disorders Amplifies Macrophage Chemotactic Responses and Accelerates Atherogenesis and Kidney Injury in LDL Receptor–Deficient Mice

Background—Strengthening the macrophage glutathione redox buffer reduces macrophage content and decreases the severity of atherosclerotic lesions in LDL receptor–deficient (LDLR−/−) mice, but the underlying mechanisms were not clear. This study examined the effect of metabolic stress on the thiol redox state, chemotactic activity in vivo, and the recruitment of macrophages into atherosclerotic lesions and kidneys of LDL-R−/− mice in response to mild, moderate, and severe metabolic stress. Methods and Results—Reduced glutathione (GSH) and glutathione disulfide (GSSG) levels in peritoneal macrophages isolated from mildly, moderately, and severe metabolically-stressed LDL-R−/− mice were measured by HPLC, and the glutathione reduction potential (Eh) was calculated. Macrophage Eh correlated with the macrophage content in both atherosclerotic (r2=0.346, P=0.004) and renal lesions (r2=0.480, P=0.001) in these mice as well as the extent of both atherosclerosis (r2=0.414, P=0.001) and kidney injury (r2=0.480, P=0.001). Compared to LDL-R−/− mice exposed to mild metabolic stress, macrophage recruitment into MCP-1–loaded Matrigel plugs injected into LDL-R−/− mice increased 2.6-fold in moderately metabolically-stressed mice and 9.8-fold in severely metabolically-stressed mice. The macrophage Eh was a strong predictor of macrophage chemotaxis (r2=0.554, P<0.001). Conclusion—Thiol oxidative stress enhances macrophage recruitment into vascular and renal lesions by increasing the responsiveness of macrophages to chemoattractants. This novel mechanism contributes at least in part to accelerated atherosclerosis and kidney injury associated with dyslipidemia and diabetes in mice.

Cholesterol homeostasis markers are localized to mouse hippocampal pyramidal and granule layers

Changes in brain cholesterol homeostasis are associated with multiple diseases, such as Alzheimers and Huntingtons; however, controversy persists as to whether adult neurons produce their own cholesterol, or if it is outsourced to astrocytes. To address this issue, we analyzed 25 genes most immediately involved in cholesterol homeostasis from in situ data provided by the Allen Brain Mouse Atlas. We compared the relative mRNA expression in the pyramidal and granule layers, populated with neurons, with the rest of the hippocampus which is populated with neuronal processes and glia. Comparing the expression of the individual genes to markers for neurons and astrocytes, we found that cholesterol homeostasis genes are preferentially targeted to neuronal layers. Therefore, changes in gene expression levels might affect neuronal populations directly.

Ethanol affects hypothalamic neurons projecting to the hippocampus and inhibits dentate granule cell LTP

In previous studies we demonstrated that ethanol inhibition of hippocampal granule cell long-term potentiation (LTP) is mediated by angiotensin II (AII), and the inhibition can be blocked by losartan, a specific AII receptor antagonist. The purpose of the present study was to demonstrate that this low-dose ethanol inhibition of dentate granule cell LTP induction is mediated by lateral hypothalamic (LH) afferents that project to the granule cells. In urethane anesthetized rats, we compared the effects of ethanol infusion, 6.0 microliter/30 min, by means of an open-ended push-pull type cannula, in both the LH and the dentate gyrus, on dentate granule cell LTP. Results demonstrate a dose-dependent inhibition of LTP induction when the LH is perfused that can be blocked by losartan, 10 mg/kg i.p.. Four doses of ethanol were used: 5, 10, 20, and 30 mM. There was no effect when the dentate gyrus was infused with 30 mM ethanol and normal granule cell LTP was observed. Also, these results demonstrate for the first time a low-dose ethanol effect on a physiological function, LTP in a specific neural pathway, directly related to the anterograde amnesia produced by ethanol on short-term memory. Therefore, these data support our hypothesis that ethanol inhibition of LTP induction at the medial perforant path-granule cell synapse can be attributed to a presynaptic release of AII and cannot be explained in terms of a direct postsynaptic effect on the granule cells.

C-terminal fragment of transforming growth factor beta-induced protein (TGFBIp) is required for apoptosis in human osteosarcoma cells

Transforming growth factor beta-induced protein (TGFBIp), is secreted into the extracellular space. When fragmentation of C-terminal portions is blocked, apoptosis is low, even when the protein is overexpressed. If fragmentation occurs, apoptosis is observed. Whether full-length TGFBIp or integrin-binding fragments released from its C-terminus is necessary for apoptosis remains equivocal. More importantly, the exact portion of the C-terminus that conveys the pro-apoptotic property of TGFBIp is uncertain. It is reportedly within the final 166 amino acids. We sought to determine if this property is dependent upon the final 69 amino acids containing the integrin-binding, EPDIM and RGD, sequences. With MG-63 osteosarcoma cells, transforming growth factor (TGF)-beta1 treatment increased expression of TGFBIp over 72 h (p<0.001). At this time point, apoptosis was significantly increased (p<0.001) and was prevented by an anti-TGFBIp, polyclonal antibody (p<0.05). Overexpression of TGFBIp by transient transfection produced a 2-fold increase in apoptosis (p<0.01). Exogenous purified TGFBIp at concentrations of 37-150 nM produced a dose dependent increase in apoptosis (p<0.001). Mass spectrometry analysis of TGFBIp isolated from conditioned medium of cells treated with TGF-beta1 revealed truncated forms of TGFBIp that lacked integrin-binding sequences in the C-terminus. Recombinant TGFBIp truncated, similarly, at amino acid 614 failed to induce apoptosis. A recombinant fragment encoding the final 69 amino acids of the TGFBIp C-terminus produced significant apoptosis. This apoptosis level was comparable to that induced by TGF-beta1 upregulation of endogenous TGFBIp. Mutation of the integrin-binding sequence EPDIM, but not RGD, blocked apoptosis (p<0.001). These pro-apoptotic actions are dependent on the C-terminus most likely to interact with integrins.