Jenny L. Fiedler

Improving the brain delivery of gold nanoparticles by conjugation with an amphipathic peptide

BACKGROUND & AIMS Gold nanoparticles (GNPs) have promising applications for drug delivery as well as for the diagnosis and treatment of several pathologies, such as those related to the CNS. However, GNPs are retained in a number of organs, such as the liver and spleen. Owing to their negative charge and/or processes of opsonization, GNPs are retained by the reticuloendothelial system, thereby decreasing their delivery to the brain. It is therefore crucial to modify the nanoparticle surface in order to increase its lipophilicity and reduce its negative charge, thus achieving enhanced delivery to the brain. RESULTS In this article, we have shown that conjugation of 12 nm GNPs with the amphipathic peptide CLPFFD increases the in vivo penetration of these particles to the rat brain. The C(GNP)-LPFFD conjugates showed a smaller negative charge and a greater hydrophobic character than citrate-capped GNPs of the same size. We administered intraperitoneal injections of citrate GNPs and C(GNP)-LPFFD in rats, and determined the gold content in the tissues by neutron activation. Compared with citrate GNPs, the C(GNP)-LPFFD conjugate improved the delivery to the brain, increasing the concentration of gold by fourfold, while simultaneously reducing its retention by the spleen 1 and 2 h after injection. At 24 h, the conjugate was partially cleared from the brain, and mainly accumulated in the liver. The C(GNP)-LPFFD did not alter the integrity of the blood-brain barrier, and had no effect on cell viability.

Glutamate in rat brain cortex synaptic vesicles: influence of the vesicle isolation procedure.

Rat brain cortex synaptic vesicles have been isolated by 3 different procedures. The one of Hata et al. (J. Neurochem., 27 (1976) 139) gave synaptic vesicles with a high glutamate content, but also, as judged by [3H]ouabain binding and electron microscopy, with considerable contamination by plasma membrane vesicles. This did not allow a precise estimation of the glutamate content of each synaptic vesicle. The second procedure used (Life Sci., 21 (1977) 1075), in which the tissue is homogenized with an all glass homogenizer, yielded vesicles of higher purity, but with no glutamate. A slightly modified Kadota and Kadota procedure (J. Cell Biol., 58 (1973) 135) gave synaptic vesicles of a very high purity that were filtered on a Sepharose 4B column, and there, the synaptic vesicle fraction of highest purity was estimated to contain 3640 glutamate molecules in each glutamatergic vesicle. This is equivalent to an intravesicular concentration of 0.21 M, that is, at least 10 times higher than the glutamate concentration in the rat brain cortex.

Desipramine prevents stress-induced changes in depressive-like behavior and hippocampal markers of neuroprotection.

Extracellular signal-regulated kinases (ERKs) are widely implicated in multiple physiological processes. Although ERK1/2 has been proposed as a common mediator of antidepressant action in naive rodents, it remains to be determined whether the ERK1/2 pathway plays a role in depressive disorder. Here, we investigated whether chronic restraint stress (14 days) and antidepressant treatment [desipramine (DMI), 10 mg/kg intraperitoneally] induce changes in animal behavior and hippocampal levels of phospho-ERK1/2 and its substrate phospho-cAMP response element-binding protein (CREB). The results indicated that stress-induced depressive-like behaviors were correlated with an increase in P-ERK1/2 and P-CREB in the hippocampus evaluated by immunoblot analysis. As an indication of CREB activity, we evaluated changes in mRNA levels of its target genes. Brain-derived neurotrophic factor (BDNF) mRNA was reduced by stress, an effect prevented by DMI only in the CA3 area of hippocampus. Bcl-2 mRNA was reduced in all hippocampal regions by stress, an effect independent of DMI treatment. However, immunoblot from hippocampal extracts revealed that stress increased BCL-2 levels, an effect prevented by chronic DMI. These results suggest that ERKs and BDNF may be altered in depressive disorder, modifications that are sensitive to DMI action. In contrast, the stress-induced increase in BCL-2 may correspond to a neuroprotective response.

Novel aspects of glucocorticoid actions.

Normal hypothalamic‐pituitary‐adrenal (HPA) axis activity leading to the rhythmic and episodic release of adrenal glucocorticoids (GCs) is essential for body homeostasis and survival during stress. Acting through specific intracellular receptors in the brain and periphery, GCs regulate behaviour, as well as metabolic, cardiovascular, immune and neuroendocrine activities. By contrast to chronic elevated levels, circadian and acute stress‐induced increases in GCs are necessary for hippocampal neuronal survival and memory acquisition and consolidation, as a result of the inhibition of apoptosis, the facilitation of glutamatergic neurotransmission and the formation of excitatory synapses, and the induction of immediate early genes and dendritic spine formation. In addition to metabolic actions leading to increased energy availability, GCs have profound effects on feeding behaviour, mainly via the modulation of orexigenic and anorixegenic neuropeptides. Evidence is also emerging that, in addition to the recognised immune suppressive actions of GCs by counteracting adrenergic pro‐inflammatory actions, circadian elevations have priming effects in the immune system, potentiating acute defensive responses. In addition, negative‐feedback by GCs involves multiple mechanisms leading to limited HPA axis activation and prevention of the deleterious effects of excessive GC production. Adequate GC secretion to meet body demands is tightly regulated by a complex neural circuitry controlling hypothalamic corticotrophin‐releasing hormone (CRH) and vasopressin secretion, which are the main regulators of pituitary adrenocorticotrophic hormone (ACTH). Rapid feedback mechanisms, likely involving nongenomic actions of GCs, mediate the immediate inhibition of hypothalamic CRH and ACTH secretion, whereas intermediate and delayed mechanisms mediated by genomic actions involve the modulation of limbic circuitry and peripheral metabolic messengers. Consistent with their key adaptive roles, HPA axis components are evolutionarily conserved, being present in the earliest vertebrates. An understanding of these basic mechanisms may lead to novel approaches for the development of diagnostic and therapeutic tools for disorders related to stress and alterations of GC secretion.

Chronic Intermittent Cold Stress Activates Ovarian Sympathetic Nerves and Modifies Ovarian Follicular Development in the Rat

Abstract We studied the effects of a chronic intermittent cold stress regime on sympathetic nerve activation and ovarian physiology. This paradigm (4°C for 3 h/day, Monday–Friday, for 3 or 4 wk) does not affect basal plasma levels of corticosterone. After 3 wk of stress, we detected a decrease in noradrenaline (NA) in the ovary, but after 4 wk, this ovarian neurotransmitter concentration increased over that of unstressed control rats. To analyze whether this effect on NA is preceded by an activation of the neurotrophic factor system responsible for growth and survival of sympathetic neurons, we measured both nerve growth factor (NGF) (by enzyme immunoassay) and the intraovarian levels of its low affinity receptor mRNA (by reverse transcription-polymerase chain reaction). The activation of sympathetic nerves was followed by an increase in NGF concentration without affecting the ovarian levels of either NGF or the mRNA of its receptor. Interestingly, follicular development changed during the stress procedure; after 3 or 4 wk of stress, we found a decrease in preantral healthy follicles without a compensatory increase in atresia. Concomitantly with the increase in NA and NGF in the ovary, we observed that a new population of follicles with hypertrophied thecal cell layers appeared after 4 wk of stress. These results suggest that chronic stress, through an intraovarian neurotrophin-mediated sympathetic activation, produces changes in follicular development that could lead to an impairment of reproductive function.

Stress promotes development of ovarian cysts in rats : The possible role of sympathetic nerve activation ()

AbstractActivation of the sympathetic innervation precedes the induction of polycystic ovaries in rats given estradiol valerate (EV). The mechanism of induction by EV may thus involve both direct and neurogenic components. We tested this hypothesis using a combined cold and restraint stress to induce an increase in sympathetic tone, including that of the ovarian sympathetic nerves. Three weeks after the start of stress we found:1.An increase in the content of norepinephrine (NE) in the celiac ganglion.2.An increase in the release of NE from the ovary.3.An unchanged NE uptake by the ovary.4.An unchanged content of NE in the ovary. The ovarian content of neuropeptide Y (NPY) (colocalized with NE) was significantly decreased. These results suggest that NE synthesis and its secretion are increased during this period and correlate with the increase in secretion of androgens and estradiol, the development of precystic follicles, and a decrease in the ovulatory rate. After 11 wk, NE release had returned to control values, whereas the ovarian NE content had risen significantly, suggesting a maintained high rate of NE synthesis. In the ovary, NPY contents, steroid secretion, morphology, and ovulation had returned to the control state. These results suggest the participation of an extraovarian factor that might act locally to control the release of NE from the ovary, and further support the hypothesis that increased sympathetic activity plays a role in the development and maintenance of ovarian cysts.

Plasticity of hippocampus following perinatal asphyxia: Effects on postnatal apoptosis and neurogenesis

Asphyxia during delivery produces long‐term deficits in brain development, including hippocampus. We investigated hippocampal plasticity after perinatal asphyxia, measuring postnatal apoptosis and neurogenesis. Asphyxia was performed by immersing rat fetuses with uterine horns removed from ready‐to‐deliver rats into a water bath for 20 min. Caesarean‐delivered pups were used as controls. The animals were euthanized 1 week or 1 month after birth. Apoptotic nuclear morphology and DNA breaks were assessed by Hoechst and TUNEL assays. Neurogenesis was estimated by bromodeoxyuridine/MAP‐2 immunocytochemitry, and the levels and expression of proteins related to apoptosis and cell proliferation were measured by Western blots and in situ hybridization, respectively. There was an increase of apoptosis in CA1, CA3, and dentate gyrus (DG) and cell proliferation and neurogenesis in CA1, DG, and hilus regions of hippocampus 1 week after asphyxia. The increase of apoptosis in CA3 and cell proliferation in the suprapyramidal band of DG was still observed 1 month following asphyxia. There was an increase of BAD, BCL‐2, ERK2, and bFGF levels in whole hippocampus and bFGF expression in CA1 and CA2 and hilus at P7 and P30. There was a concomitant decrease of phosphorylated‐BAD (Ser112) levels. The increase of BAD levels supports the idea of delayed cell death after perinatal asphyxia, whereas the increases of BCL‐2, ERK2, and bFGF levels suggest the activation of neuroprotective and repair pathways. In conclusion, perinatal asphyxia induces short‐ and long‐term regionally specific plastic changes, including delayed cell death and neurogenesis, involving pro‐ and antiapoptotic as well as mitogenic proteins, favoring hippocampal functional recovery.

Comparison of the antidepressant sertraline on differential depression-like behaviors elicited by restraint stress and repeated corticosterone administration

Depressive disorder involves emotional, cognitive, autonomic and endocrine alterations and also evidences support the role of stress in the development of this disorder. Because the hypothalamic-pituitary-adrenal axis is involved in the stress response with a concomitant rise in plasma corticoids, the present study compares the antidepressant effects of sertraline (10mg/kg, i.p.) on behavioral changes elicited by (i) restraint stress (2.5h/day for 13days) and (ii) corticosterone injections (30mg/kg, s.c., for 13days). Stressed animals, but not corticosterone-treated animals displayed anxiety behavior and a reduction in the acquisition of a conditioned avoidance response to 25% of control levels (8.0±2.2 vs. 31.7±3.2), being this effect partly sensitive to sertraline. Stressed, but not corticosterone-treated, animals displayed an increased escape failure compared with the control group (24.6%±3.5 vs. 1.6±0.7), an effect partly prevented by sertraline treatment (7.3%±2.0). Both stressed rats and corticosterone-treated rats showed an increase in immobility in the forced swim test, an effect prevented by sertraline. These results suggest that the altered behaviors elicited by stress and corticosterone can be explained by neural modifications that are sensitive to the sertraline antidepressant.

Regional alteration of cholinergic function in central neurons of trisomy 16 mouse fetuses, an animal model of human trisomy 21 (Down syndrome)

The trisomy-16 (TS16) mouse is considered to be a model of human trisomy 21 (Down syndrome) because of genetic homology between mouse chromosome 16 and human chromosome 21. We examined cholinergic function of brain and spinal cord tissue and in cultured neurons from TS16 mouse compared with that of age matched controls. Mean acetylcholinesterase activity in both tissue types did not differ between trisomic and control conditions. Acetylcholine (ACh) synthesis, measured as choline O-acetyltratransferase (acetyl-CoA) activity, was reduced to 67% of control in TS16 brain but not in TS16 spinal cord. Steady-state accumulation of ACh precursor, [3H]choline, was measured in primary cell cultures. Steady-state choline uptake was reduced to 35% and to 61% in neurons of TS16 brain and spinal cord, respectively, when compared with controls. Kinetics experiments in TS16 brain cells showed a 50% reduction of the maximal velocity of choline uptake when compared to controls. Further, the ACh release induced by KCl depolarization in TS16 spinal cord neurons did not differ from control neurons but was reduced in TS16 brain neurons. This effect cannot be explained solely by a reduction in ACh synthesis. The results indicate that the TS16 condition in mice significantly modified the cholinergic function in brain, and to a lesser degree in spinal cord, suggesting that the higher gene dosage inherent to the trisomic condition affects cholinergic neurons in different regions of the central nervous system in a differential fashion.

Serum brain-derived neurotrophic factor and glucocorticoid receptor levels in lymphocytes as markers of antidepressant response in major depressive patients: a pilot study.

Depressive patients often have altered cortisol secretion, an effect that likely derives from impaired activity of the glucocorticoid receptor (GR), the main regulator of the hypothalamus-pituitary-adrenal (HPA) axis. Glucocorticoids reduce the levels of brain-derived neurotrophic factor (BDNF), a downstream target of antidepressants. Antidepressants promote the transcriptional activity of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), a regulator of BDNF expression. To identify potential biomarkers for the onset of antidepressant action in depressive patients, GR and phospho-CREB (pCREB) levels in lymphocytes and serum BDNF levels were repeatedly measured during the course of antidepressant treatment. Thirty-four depressed outpatients (10 male and 24 female) were treated with venlafaxine (75mg/day), and individuals exhibiting a 50% reduction in their baseline 17-Item Hamilton Depression Rating Scale score by the 6th week of treatment were considered responders. Responders showed an early improvement in parallel with a rise in BDNF levels during the first two weeks of treatment. Non-responders showed increased GR levels by the third week and reduced serum BDNF by the sixth week of treatment. In contrast, venlafaxine did not affect levels of pCREB. We conclude that levels of BDNF in serum and GR levels in lymphocytes may represent biomarkers that could be used to predict responses to venlafaxine treatment.