Toward discovering a novel family of peptides targeting neuroinflammatory states of brain microglia and astrocytes.

Microglia are immune-derived cells critical to the development and healthy function of the brain and spinal cord, yet are implicated in the active pathology of many neuropsychiatric disorders. A range of functional phenotypes associated with the healthy brain or disease states has been suggested from in vivo work and were modeled in vitro as surveying, reactive, and primed sub-types of primary rat microglia and mixed microglia/astrocytes. It was hypothesized that the biomolecular profile of these cells undergoes a phenotypical change as well, and these functional phenotypes were explored for potential novel peptide binders using a custom 7 amino acid-presenting M13 phage library (SX7) to identify unique peptides that bind differentially to these respective cell types. Surveying glia were untreated, reactive were induced with a lipopolysaccharide treatment, recovery was modeled with a potent anti-inflammatory treatment dexamethasone, and priming was determined by subsequently challenging the cells with interferon gamma. Microglial function was profiled by determining the secretion of cytokines and nitric oxide, and expression of inducible nitric oxide synthase. After incubation with the SX7 phage library, populations of SX7-positive microglia and/or astrocytes were collected using fluorescence-activated cell sorting, SX7 phage was amplified in Escherichia coli culture, and phage DNA was sequenced via next-generation sequencing. Binding validation was done with synthesized peptides via in-cell westerns. Fifty-eight unique peptides were discovered, and their potential functions were assessed using a basic local alignment search tool. Peptides potentially originated from proteins ranging in function from a variety of supportive glial roles, including synapse support and pruning, to inflammatory incitement including cytokine and interleukin activation, and potential regulation in neurodegenerative and neuropsychiatric disorders.

Fluoxetine and citalopram decrease microglial release of glutamate and D-serine to promote cortical neuronal viability following ischemic insult.

Depression is one of the most common disorders appearing following a stroke, and is also a major factor limiting recovery and rehabilitation in stroke patients. Antidepressants are the most common prescribed treatment for depression and have shown to have anti-inflammatory properties within the central nervous system (CNS). The major source of pro-inflammatory factors within the CNS is from activated microglia, the innate immune cells of the CNS. Antidepressants have been shown to promote midbrain and hippocampal neuronal survival following an ischemic insult and this survival is mediated through the anti-inflammatory effects on microglia, but the effects on cortical neuronal survival after this insult have yet to be investigated. The present study aimed to test and compare antidepressants from three distinct classes (tricyclics, monoamine oxidase inhibitors, and selective serotonin-reuptake inhibitors [SSRIs]) on the release of inflammatory factors and amino acids from activated microglia and whether altering this release could affect cortical neuronal viability after an ischemic insult. Primary microglia were treated with 1 µg/ml LPS and/or 10 µM antidepressants, and the various factors released into medium were assayed. Co-cultures consisting of microglia and primary cortical neurons were used to assess the effects of antidepressant-treated activated microglia on the viability of ischemic injured neurons. Of the antidepressants tested, most decreased the release of the proinflammatory factors nitric oxide, tumor necrosis factor-alpha, and interleukin 1-beta from activated microglia. Fluoxetine and citalopram, the SSRIs, decreased the release of the amino acids glutamate and d-serine from LPS-activated microglia. oxygen-glucose deprived (OGD) cortical neurons cocultured with LPS-activated microglia pre-treated with fluoxetine and citalopram showed greater survival compared to injured neurons co-cultured with untreated activated microglia. Microglial release of glutamate and d-serine was shown to be the most important factor mediating neuronal survival following antagonism studies. To our knowledge, our results demonstrate for the first time that fluoxetine and citalopram decrease the release of glutamate and d-serine from LPS-activated microglia and this causes an increase in the survival of OGD-injured cortical neurons after co-culture.

Biomaterials and glia: Progress on designs to modulate neuroinflammation.

Microglia are multi-functional cells that play a vital role in establishing and maintaining the function of the nervous system and determining the fate of neurons following injury or neuropathology. The roles of microglia are diverse and essential to the capacity of the nervous system to recover from injury, however sustained inflammation can limit recovery and drive chronic disease processes such as neurodegenerative disorders. When assessing implantable therapeutic devices in the central nervous system, an improved lifetime of the implant is considered achievable through the attenuation of microglial inflammation. Consequently, there is a tremendous underexplored potential in biomaterial and engineered design to modulate neuroinflammation for therapeutic benefit. Several strategies for improving device compatibility reviewed here include: biocompatible coatings, improved designs in finer and flexible shapes to reduce tissue shear-related scarring, and loading of anti-inflammatory drugs. Studies about microglial cell cultures in 3D hydrogels and nanoscaffolds to assess various injuries and disorders are also discussed. A variety of other microglia-targeting treatments are also reviewed, including nanoparticulate systems, cellular backpacks, and gold plinths, with the intention of delivering anti-inflammatory drugs by targeting the phagocytic nature of microglia. Overall, this review highlights recent advances in biomaterials targeting microglia and inflammatory function with the potential for improving implant rejection and biocompatibility studies. STATEMENT OF SIGNIFICANCE: Microglia are the resident immune cells of the central nervous system, and thus play a central role in the neuroinflammatory response against conditions than span acute injuries, neuropsychiatric disorders, and neurodegenerative disorders. This review article presents a summary of biomaterials research that target microglia and other glial cells in order to attenuate neuroinflammation, including but not limited to: design of mechanically compliant and biocompatible stimulation electrodes, hydrogels for high-throughput 3D modelling of nervous tissue, and uptake of nanoparticle drug delivery systems. The goal of this paper is to identify strengths and gaps in the relevant literature, and to promote further consideration of microglia behaviour and neuroinflammation in biomaterial design.

Supporting microglial niches for therapeutic benefit in psychiatric disorders.

Inflammation is an essential tissue response to injury, stress, or infection resulting in debris and/or pathogen clearance intended to promote healing and recovery. Due to the status as an immune 'privileged' tissue, microglia serve as endogenous regulators of inflammation in the central nervous system, but maintain communication with peripheral immune system to enable recruitment of peripheral immune cells in case of injury or infection. While microglia retain the functional capacity for a full range of inflammatory functions - microglia express a range of pattern-recognition receptors and function as innate immune cells, carry out phagocytosis of pathogens, and act as antigen presenting cells - in the healthy central nervous system (CNS) these functions are rarely engaged. Subsequently microglia are being recognized to occupy an increasing number of homeostatic niches, and in many cases have adopted immune or inflammatory mechanisms to carry out these niche functions absent immune activation. These sterile inflammatory functions are challenging long-held views of the role of inflammation in the central nervous system while simultaneously expanding the potential for the development of truly novel therapeutic interventions for a range of neuroinflammatory, neurodegenerative, and neuropsychiatric disorders. In the present review we discuss recent preclinical evidence for conserved niche functions for microglia whose disruption may causally contribute to various psychiatric disorders, and prospective targets for restoring disrupted niches.

A comparison of ventral tegmental neurons projecting to optic flow regions of the inferior olive vs. the hippocampal formation.

The ventral tegmental area (catecholaminergic group A10) is a midbrain region characterized by concentrated dopaminergic immunoreactivity. Previous studies in pigeons show that the ventral tegmental area provides a robust projection to the hippocampal formation and to the medial column of the inferior olive. However, the distribution, morphology, and neurochemical content of the neurons that constitute these projections have not been resolved. In this study, we used a combination of retrograde tracing techniques and immunofluorohistochemistry to address these issues. Retrograde tracers were used to demonstrate that the distribution of ventral tegmental area neurons projecting to the hippocampus and the inferior olive overlap in the caudo-ventral ventral tegmental area. The hippocampus- and inferior olive-projecting ventral tegmental area neurons could not be distinguished based on morphology: most neurons had small- to medium-sized multipolar or fusiform soma. Double-labeling with fluorescent retrograde tracers revealed that the hippocampus- and medial column of the inferior olive-projecting neurons were found intermingled in the ventral tegmental area, but no cells were double labeled; i.e. individual ventral tegmental area neurons do not project to both the hippocampal formation and medial column of the inferior olive. Finally, we found that a minority (8.2%) of ventral tegmental area neurons providing input to the hippocampus were tyrosine hydroxylase-immunoreactive, whereas none of the inferior olive-projecting neurons were tyrosine hydroxylase positive. Combined, our findings show that the projections to the hippocampus and olivocerebellar pathway arise from intermixed subpopulations of ventral tegmental area neurons with indistinguishable morphology but only the hippocampal projection involves dopaminergic neurons. We suggest that equivalent projections from the ventral tegmental area to the hippocampal formation and inferior olive exist in mammals and discuss their potential role in the processing of optic flow and the analysis of self-motion.

Brain biocompatibility and microglia response towards engineered self-assembling (RADA)4 nanoscaffolds.

(RADA)4-based nanoscaffolds have many inherent properties making them amenable to tissue engineering applications: ease of synthesis, ease of customization with bioactive moieties, and amenable for in situ nanoscaffold formation. There is a dearth in the literature on their biocompatibility in brain tissues; where the glia response is key to regulating the local host response. Herein, nanoscaffolds composed of (RADA)4 and (RADA)4-IKVAV mixtures were evaluated in terms of their effect on primary microglia in culture and general tissue (in vivo) biocompatibility (astrocyte and migroglia). Laminin-derived IKVAV peptide was chosen to promote beneficial cell interaction and attenuate deleterious glial responses. Microglia remained ramified when cultured with these nanoscaffolds, as observed using TNF-α and IL-1ß, NO, and proliferation assays. Evidence suggests that cultured microglia phagocytise the matrix whilst remaining ramified and viable, as shown visually and metabolically (MTT). Nanoscaffold intracerebral injection did not lead to microglia migration or proliferation, nor were glial scarring and axonal injury observed over the course of this study. IKVAV had no affect on microglia activation and astrogliosis. (RADA)4 should be advantageous for localized injection as a tuneable-platform device, which may be readily cleared without deleterious effects on tissue-resident microglia. STATEMENT OF SIGNIFICANCE: Self-assembling nanoscaffolds have many inherent properties making them amenable to tissue engineering applications: ease of synthesis, ease of customization with bioactive moieties, and amenable for in situ nanoscaffold formation. A dearth of literature exists on their biocompatibility in brain tissues; where the glia response is key to regulating the local host response. Herein, nanoscaffolds composed of the peptides (RADA)4 and (RADA)4-IKVAV mixtures were evaluated in terms of their effect on microglia cells in culture and general tissue (in vivo) biocompatibility (astrocyte and migroglia). Laminin-derived IKVAV peptide was chosen to promote beneficial cell interaction and attenuate deleterious glial responses. (RADA)4 nanoscaffolds showed no adverse effect from these cell types and should be advantageous for localized injection as a tuneable-platform device.

Antisense knockdown of the glial glutamate transporter GLT-1, but not the neuronal glutamate transporter EAAC1, exacerbates transient focal cerebral ischemia-induced neuronal damage in rat brain.

Transient focal cerebral ischemia leads to extensive neuronal damage in cerebral cortex and striatum. Normal functioning of glutamate transporters clears the synaptically released glutamate to prevent excitotoxic neuronal death. This study evaluated the functional role of the glial (GLT-1) and neuronal (EAAC1) glutamate transporters in mediating ischemic neuronal damage after transient middle cerebral artery occlusion (MCAO). Transient MCAO in rats infused with GLT-1 antisense oligodeoxynucleotides (ODNs) led to increased infarct volume (45 +/- 8%; p < 0.05), worsened neurological status, and increased mortality rate, compared with GLT-1 sense/random ODN-infused controls. Transient MCAO in rats infused with EAAC1 antisense ODNs had no significant effect on any of these parameters. This study suggests that GLT-1, but not EAAC1, knockdown exacerbates the neuronal death and thus neurological deficit after stroke.

Patency of cerebral microvessels after focal embolic stroke in the rat.

In patients with thrombotic stroke, the occluded artery often reopens over time. This results through a natural dissolution of the occluding material, and fragments of the material may move downstream to obstruct distal arteries. The current study was undertaken to investigate the patency of brain microvessels at varying time intervals after injection of a preformed clot into the right internal carotid artery of rats. Cerebral microvessels in brain sections were visualized using immunohistochemistry for fibronectin (detecting existing microvessels) and Evans blue (visualizing perfused microvessels). The percentage of patent microvessels was calculated as the number of Evans blue-positive microvessels divided by the number of fibronectin-positive microvessels. In normal control animals, results showed that 98% +/- 3% (mean +/- SD) of microvessels in the cortex and 94% +/- 14% in the striatum were patent. In the ischemic animals, immediately after clot injection, microvessels in the cortex and striatum were occluded, mainly in the territory irrigated by the middle cerebral artery. One hour after clot injection, microvessels had reopened in most of the cortex but remained occluded in some portions of the striatum, possibly as a result of downstream movement of fragments formed from the original clot. By 3 hours after clot injection, microvessels in the cortex were patent in all animals, whereas in the striatum microvessels were patent in 50% of the animals. In the other 50%, small striatal perfusion deficits persisted. At 24 hours after clot injection, microvessels were patent in both the cortex and striatum of all animals except one. These findings suggest that intracerebral clots dissolve spontaneously in a relatively short period of time, but that fragments formed from the clot may obstruct more distal blood vessels. It is likely that clot fragments lodge in arteries with lower blood flow and poor collateral perfusion, where they continue to cause ischemia for a longer duration. These results may in part explain the resistance of the striatum to neuroprotective strategies used for the treatment of focal cerebral ischemia.

In vivo biodistribution, pharmacokinetic parameters, and brain uptake of 5-halo-y-methoxy(or ethoxy)-5,6-dihydro-3'-azido-3'-deoxythymidine diastereomers as potential prodrugs of 3'-azido-3'-deoxythymidine.

A new class of 5-halo-6-alkoxy-5,6-dihydro-3'-azido-3'-deoxythymidine diastereomers (5-x-6-OR -5,6-dihydro-AZTs; X = I, Br, Cl; R = Me, Et) were evaluated as potential anti-AIDS prodrugs of 3'-azido-3'-deoxythimidine (AZT). In vivo regeneration of AZT from these 5-X-6-OR-5,6-dihydro-AZTs was examined in Balb/c mice after intravenous tail vein injection. The (5R,6R)- and (5S,6S)-5-bromo(or iodo)-6-methoxy-5,6-dihydro derivatives of AZT (BMAZT, IMAZT) were rapidly converted to AZT, resulting in AZT plasma concentrations after a 144 micromol/kg dose similar to those after an equivalent dose (144 microg/kg, 38.5 mg/kg) of AZT, whereas AZT was not detectable by HPLC after the same dose of the chloro diastereomer (5R,6R)-CMAZT. The interaction of AZT and the 5-X-6-methoxy-5,6dihydro-AZT diastereomers with the 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine equilibrative-sensitive nucleoside transporter in murine erythrocytes was also studied. The (5R,6R)- and (5S,6S)-5-X-6-OMe-5,6-dihydro-AZT diastereomers demonstrated a high affinity (K(i) = 0.2-0.5 mM) for the transporter relative to AZT (K(i) = 1.3 mM), with the exception of (5S,6R)-5-chloro-6-methoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (CMAZT) which has a K(i) value larger than 1.5 mM. [2-(14)C]-Labeled (5R,6R)- and (5S,6S)-5-bromo-6-methoxy(or ethoxy)- 5,6-dihydro-3'-azido-3'-deoxythymidines were synthesized by the regiospecific addition of methyl hypobromite or ethyl hypobromite to the 5,6-olefinic bond of [2-(14)C]-AZT in high radiochemical yield [(5R,6R)-BMAZT, 48%, and (5S,6S)-BMAZT, 33%; (5R,6R)-BEAZT, 61%, and (5S,6S)-BEAZT, 15%), high radiochemical purity (>98%), and high specific activity (56mCi/mmol)]. The amounts of radioactivity in mouse brain after iv injection of [2-(14))C]-labeled (5R,6R)-BMAZT, (5S,6S)-BMAZT, or (5R,6R)-BEAZT were 2-4 fold higher that that for [2-(14)C]-AZT (P < 0.05). The radioactivity remaining in blood after dosing with these 5-bromo-6-alkoxy-5,6-dihydro-AZTs was up to 20-fold higher than after injection of [2-(14)C]-AZT at longer time intervals after injection. The amounts of radioactivity present in femoral bone following injection of [2(-14)C]-AZT, or these 5-bromo-6-alkoxy-5,6-dihydro-AZTs, were similar. Subcellular and regional distributions of [2-(14)C]-labeled AZT, (5R,6R)-BMAZT, or (5R,6R)-BEAZT in mouse brain after jugular vein injection did not show preferential concentration in any particular subcellular fraction nor a marked preferential regional localization for either AZT or these 5,6-dihydro prodrugs of AZT.

The additive effects of quinine on antidepressant drugs in the forced swimming test in mice.

The aim of this study was to investigate if quinine plus antidepressant drugs (ADS) leads to an additive effect in the forced swimming test. Quinine (0.125, 0.5 mg/kg) and ADS (subactive doses) were given IP 45 and 30 min, respectively, before the test. When combined with QUIN, all drugs that act via inhibition of 5-HT uptake (imipramine, amitriptyline, citalopram, paroxetine, fluoxetine and fluvoxamine) significantly increased the swimming time of mice. Among trazodone, mianserin and iprindole (atypical ADS), only iprindole combined with quinine decreased the immobility (increased swimming) of the animals. The specific noradrenaline (NA) uptake inhibitors, desipramine and viloxazine, but not maprotiline, were also found to reduce the immobility time when pretreated with quinine. The mixed monoamine oxidase (MAO) inhibitor (pargyline) and MAO-A inhibitor (moclobemide) also shortened the period of immobility whereas the MAO-B inhibitor (nialamide) and the dopamine (DA) uptake inhibitor (bupropion) did not. Quinine's additive effects on several types of ADS is likely a result of blockade of potassium channels.

Effects of AMPA/kainate receptor blockade on responses to dopamine receptor agonists in the core and shell of the rat nucleus accumbens.

The present experiments were conducted to investigate effects of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleprionic acid (AMPA)/kainate receptor blockade (CNQX, NBQX) on locomotor responses to D2/3 (7-OH-DPAT) and D1 [(+)-SKF 38393] dopamine receptor agonists in the nucleus accumbens (NAS) core and shell. CNQX (0.25-0.5 microgram) microinjected into the NAS core or shell did not affect baseline locomotor activity. 7-OH-DPAT (2.5-5 micrograms) decreased locomotor activity. Co-administration of CNQX (0.5 microgram) increased the effects of 7-OH-DPAT (5 micrograms) in the NAS core and shell. A similar increase was observed with NBQX (0.5 microgram) in the NAS shell. (+)-SKF 38393 (5 micrograms) into the NAS core and shell increased locomotor activity after 30 min; this effect was not altered by CNQX (0.5 microgram). As the D2/3 dopamine agonist (-)-quinpirole (2 micrograms) increased effects of (+)-SKF 38393 (5 micrograms) in NAS shell but not core, lack of site-selective effects of (+)-SKF-38393 and of 7-OH-DPAT within NAS is not attributable to drug diffusion. The previous observation that glutamate effects on locomotor activity depend on the relative involvement of D1 or D2/3 dopamine receptors in the NAS was based on the dopamine-depletion model. The present results demonstrate differential interactions of AMPA receptor blockade with dopamine agonists in "dopamine-intact" animals.

Increased "peripheral-type" benzodiazepine receptor sites and mRNA in thalamus of thiamine-deficient rats.

"Peripheral-type" benzodiazepine receptors (PTBRs) are highly expressed on the outer mitochondrial membrane of several types of glial cells. In order to further elucidate the nature of the early glial cell changes in thiamine deficiency, PTBR sites and PTBR mRNA were measured in thalamus, a brain structure which is particularly vulnerable to thiamine deficiency, of thiamine-deficient rats at presymptomatic and symptomatic stages of deficiency. PTBR sites were measured using an in vitro binding technique and the selective radio ligand [3H]-PK11195. PTBR gene expression was measured by RT-PCR using oligonucleotide primers based upon the published sequence of the cloned rat PTBR. Microglial and astrocytic changes in thalamus due to thiamine deficiency were assessed using immunohistochemistry and antibodies to specific microglial (ED-1) and astrocytic (GFAP) proteins respectively. Significant increases of [3H]-PK11195 binding sites and concomitantly increased PTBR mRNA were observed in thalamus at the symptomatic stage of thiamine deficiency, coincident with severe neuronal cell loss and increased GFAP-immunolabelling (indicative of reactive gliosis). Positron Emission Tomography using 11C-PK11195 could provide a novel approach to the diagnosis and assessment of the extent of thalamic damage due to thiamine deficiency in humans with Wernicke's Encephalopathy.

Neuroprotection by memantine, a non-competitive NMDA receptor antagonist after traumatic brain injury in rats.

This study investigated whether memantine, a non-competitive NMDA receptor antagonist is neuroprotective after traumatic brain injury (TBI) induced in adult rats with a controlled cortical impact device. TBI led to significant neuronal death in the hippocampal CA2 and CA3 regions (by 50 and 59%, respectively), by 7 days after the injury. Treatment of rats with memantine (10 and 20 mg/Kg, i.p.) immediately after the injury significantly prevented the neuronal loss in both CA2 and CA3 regions. This is the first study showing the neuroprotective potential of memantine to prevent the TBI-induced neuronal damage.

GABA-elevating effects of the antidepressant/antipanic drug phenelzine in brain: effects of pretreatment with tranylcypromine, (-)-deprenyl and clorgyline.

The antidepressant/antipanic drug phenelzine (PLZ) is both an inhibitor of, and a substrate for, monoamine oxidase (MAO). PLZ also causes an elevation of brain levels of the amino acid neurotransmitter gamma-aminobutyric acid (GABA); this action can be reversed by pretreatment with the MAO inhibitor tranylcypromine (TCP), suggesting that the GABA-elevating effect is largely the result of a metabolite of PLZ formed by MAO. In the present report, rats were pretreated with the nonselective MAO inhibitor TCP, the MAO-A inhibitor clorgyline and the MAO-B inhibitor (-)-deprenyl: at the doses used, clorgyline and (-)-deprenyl caused selective inhibition of MAO-A and MAO-B, respectively. Both TCP and (-)-deprenyl caused a greater reduction in the GABA-elevating action of PLZ than did clorgyline, suggesting that MAO-B is more important than MAO-A in the formation of the active metabolite of PLZ. The results also suggest that an effect other than, or in addition to, inhibition of GABA transaminase by the metabolite may be important in the GABA-elevating action.

Anti-conflict effect of 5-HT1A agonists in rats: a new model for evaluating anxiolytic-like activity.

A new conflict procedure was developed to study the potential anti-punishment effects of 5-HT( 1A) agonists as compared to diazepam. In this paradigm, the opportunity existed for rats to choose during punished periods between immediate, punished reinforcement and delayed, non-punished reinforcement. The results confirm that, for non-sedative doses (1 mg/kg), diazepam increases the number of punished responses. Furthermore, the present paradigm seems sensitive for the detection of 5-HT(1A) activity. Buspirone, gepirone, ipsapirone, zalospirone and 8-OH-DPAT increased responding for immediate but punished reinforcement. 1-(2-pyrimidinyl)piperazine, the common metabolite of the azapirones, does not participate in their anti-conflict effect. NAN 190, a 5-HT(1A) antagonist, was shown to block the 5-HT(1A) agonists. The findings of the present study suggest that benzodiazepines and 5-HT( 1A) agonists reduce the capacity to tolerate delays in reward. Abnormality in serotonin systems may be associated with poor impulse control.

Effects of two substituted hydrazine monoamine oxidase (MAO) inhibitors on neurotransmitter amines, gamma-aminobutyric acid, and alanine in rat brain.

Time- and dose-response analyses were undertaken to investigate the effects of the substituted hydrazine monoamine oxidase (MAO) inhibitors iproniazid and nialamide on the following: MAO-A and -B activity; levels of gamma-aminobutyric acid (GABA), alanine (ALA), and the neurotransmitter amines dopamine, noradrenaline, and 5-hydroxytryptamine (serotonin) and their acid metabolites; and the activity of GABA-transaminase and ALA-transaminase. The results showed that these drugs are relatively potent MAO inhibitors but, unlike the unsubstituted hydrazine MAO inhibitor phenelzine, they do not produce increased GABA and ALA levels in brain. These experiments suggest that a free hydrazine group is necessary for MAO inhibitors to also have marked effects on GABA and ALA.

Effects of D1 and D2 dopamine antagonists on behavior of polydipsic rats.

The behavioral and neurochemical effects of SCH3390 (SCH), a dopamine (DA) D1 antagonist, and haloperidol (HAL), a DA D2 receptor antagonist, on schedule-induced polydipsia (SIP) were examined. Once animals were made polydipsic, a vehicle or one of three doses of SCH or HAL were administered to seven groups of rats in a series of three five-session blocks in a drug condition, no-drug condition, drug condition design. Detailed behavioral measures and brain regional levels of monoamine neurotransmitters and their major acidic metabolites were analyzed. The volume of water consumed and the percent of time spent drinking was reduced dose dependently by both SCH and HAL. As drinking decreased, the time spent chewing increased for both drugs. The total amount of time animals engaged in all oral behaviors was not changed, suggesting that chewing was substituted for drinking. Neurochemical analysis revealed that HAL increased striatal DA significantly. The polydipsic paradigm may be an advantageous model for examining neuroleptics due to SIP's sensitivity to extrapyramidal side effects.

Distinct activation profiles in microglia of different ages: a systematic study in isolated embryonic to aged microglial cultures.

Microglia have been implicated in disease progression for several age-related brain disorders. However, while microglia's contribution to the progression of these disorders is accepted, the effect of aging on their endogenous cellular characteristics has received limited attention. In fact, a comprehensive study of how the structure and function of microglia changes as a function of developmental age has yet to be performed. Here, we describe the functional response characteristics of primary microglial cultures prepared from embryonic, neonatal (Neo), 2-3month-old, 6-8month-old, 9-11month-old, and 13-15month-old rats. Microglial morphology, glutamate (GLU) uptake, and release of trophic and inflammatory factors were assessed under basal conditions and in microglia activated with adenosine 5'-triphosphate (ATP) or lipopolysaccharide. We found that microglia from different age groups were both morphologically and functionally distinct. Upon activation by ATP, Neo microglia were the most reactive, upregulating nitric oxide, tumor necrosis factor-α, and brain-derived neurotrophic factor release as well as GLU uptake. This upregulation translated into neurotoxicity in microglia-neuron co-cultures that were not observed with microglia of different developmental ages. Interestingly, 13-15month-old microglia exhibited similar activation profiles to Neo microglia, whereas microglia from younger adults and embryos were activated less by ATP. Our data also identify age-dependent differences in purinergic receptor subtype expression that contribute to the regulation of neuronal survival. Combined, our data demonstrate that microglial activation and purinergic receptor profiles vary non-linearly with developmental age, a potentially important finding for studies examining the role of microglia in neurodegenerative disorders.

Inhibition of ß-amyloid1-42 internalization attenuates neuronal death by stabilizing the endosomal-lysosomal system in rat cortical cultured neurons.

A number of recent studies have indicated that accumulation of ß amyloid (Aß) peptides within neurons is an early event which may trigger degeneration of neurons and subsequent development of Alzheimer's disease (AD) pathology. However, very little is known about the internalization and/or subcellular sites involved in trafficking of Aß peptides into the neurons that are vulnerable in AD pathology. To address this issue we evaluated internalization of fluoroscein conjugated Aß1-42 (FAß1-42) and subsequent alteration of endosomal-lysosomal (EL) markers such as cathepsin D, Rab5 and Rab7 in rat cortical cultured neurons. It is evident from our results that internalization of FAß1-42, which occurred in a dose- and time-dependent manner, triggered degeneration of neurons along with increased levels and/or altered distribution of cathepsin D, Rab5 and Rab7. Our results further revealed that FAß1-42 internalization was attenuated by phenylarsine oxide (a general inhibitor of endocytosis) and sucrose (an inhibitor of clathrin-mediated endocytosis) but not by antagonists of N-methyl-d-aspartate (NMDA) glutamate receptors. Additionally, inhibition of FAß1-42 endocytosis not only protected neurons against toxicity but also reversed the altered levels/distributions of EL markers. These results, taken together, suggest that internalization of exogenous Aß1-42, which is partly mediated via a clathrin-dependent process, can lead to degeneration of neurons, possibly by activating the EL system. Inhibition of FAß endocytosis attenuated toxicity, thus suggesting a potential strategy for preventing loss of neurons in AD pathology.