Pramlintide Antagonizes Beta Amyloid (Aß)- and Human Amylin-Induced Depression of Hippocampal Long-Term Potentiation.

Accumulation of amyloid-ß peptide (Aß) is a pathological hallmark of Alzheimer's disease (AD). We have previously demonstrated that electrophysiological and neurotoxic effects of Aß and human amylin are expressed via the amylin receptor. Recently, pramlintide, a synthetic analog of amylin, has been reported to improve cognitive function in transgenic AD mouse models. In this study, we examined the effects of pramlintide on Aß1-42 and human amylin-evoked depression of long-term potentiation (LTP) at Schaeffer collateral-CA1 hippocampal synapses. In mouse hippocampal brain slices, field excitatory postsynaptic potentials (fEPSPs) were recorded from the stratum radiatum layer of the CA1 area in response to electrical stimulation of Schaeffer collateral afferents and LTP induced by 3-theta-burst stimulation (TBS) protocol. Aß1-42 (50 nM) and human amylin (50 nM), but not Aß42-1 (50 nM), depressed LTP. Pre-application of pramlintide (250 nM) blocked Aß- and human amylin-induced reduction of LTP without affecting baseline transmission or LTP. We also examined the effects of pramlintide on LTP in transgenic mice (TgCRND8) that over-express amyloid precursor protein. In contrast to wild-type controls, where robust LTP was observed, 10- to 12-month-old TgCRND8 mice show blunted LTP. In TgCRND8 mice, basal LTP is enhanced by application of pramlintide. Our data indicate that pramlintide acts as a functional amylin receptor antagonist to reverse the effects of Aß1-42 and human amylin on LTP and also increases LTP in transgenic mice that demonstrate increased ambient brain amyloid levels. Amylin receptor antagonists may thus serve as potentially useful therapeutic agents in treatment of AD.

Activity and metabolism-related Ca2+ and mitochondrial dynamics in co-cultured human fetal cortical neurons and astrocytes.

Neurons and neighboring astrocytic glia are mostly studied in nervous tissues from rodents whereas less is known on their properties and interactions in the human brain. Here, confocal/multiphoton fluorescence imaging for several hours revealed that co-cultured fetal human cortical neurons and astrocytes show pronounced spontaneous rises of cytosolic Ca(2+) which last for up to several minutes without concomitant changes in either movements or membrane potential of mitochondria. Similar Ca(2+) rises were evoked mainly in neurons by bath-applied glutamate or γ-aminobutyric acid (GABA) acting via N-methyl-d-aspartate (NMDA)+AMPA/Kainate and GABAA receptors, respectively. Predominantly in astrocytes, Ca(2+) baseline was elevated by adenosine diphosphate (ADP) and adenosine triphosphate (ATP) acting via P2Y1 and P2X7 receptors, likely causing the release of glutamate and glutamine. Mainly astrocytes responded to histamine, whereas the activation of muscarinic acetylcholine (ACh) receptors raised Ca(2+) in both cell types. Evoked neuronal and astrocytic Ca(2+) rises could last for several minutes without affecting mitochondrial movements or membrane potential. In contrast, reversible depolarization of mitochondrial membrane potential accompanied neuronal Ca(2+) rises induced by cyanide-evoked chemical anoxia or the uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP). During such metabolic perturbation, mitochondrial depolarization also occurred in astrocytes, whereas Ca(2+) was largely unaffected. In summary, fetal human cortical neurons and astrocytes show distinct patterns of neuro/glio-transmitter- and metabolically-evoked Ca(2+) rises and possess active mitochondria. One aspect of our discussion deals with the question of whether the functional mitochondria contribute to cellular Ca(2+) homeostasis that seems to be already well-developed in fetal human cortical brain cells.

Role of calpain and caspase in beta-amyloid-induced cell death in rat primary septal cultured neurons.

The invariant characteristic features associated with Alzheimer's disease (AD) brain include the presence of extracellular neuritic plaques composed of amyloid beta (Abeta) peptide, intracellular neurofibrillary tangles containing hyper-phosphorylated tau protein and the loss of basal forebrain cholinergic neurons. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that in vivo accumulation of Abeta(1-42) may initiate the process of neurodegeneration observed in AD brains. However, the cause of degeneration of the basal forebrain cholinergic neurons and their association to Abeta peptides or phosphorylated tau protein have not been clearly established. In the present study, using rat primary septal cultures, we have shown that Abeta(1-42), in a time (1-48 h) and concentration (0.01-20 microM)-dependent manner, induce toxicity in cultured neurons. Subsequently, we have demonstrated that Abeta toxicity is mediated via activation of cysteine proteases, i.e., calpain and caspase, and proteolytic breakdown of their downstream substrates tau, microtubule-associated protein-2 and alpha II-spectrin. Additionally, Abeta-treatment was found to induce phosphorylation of tau protein along with decreased levels of phospho-Akt and phospho-Ser(9)glycogen synthase kinase-3beta. Exposure to specific inhibitors of caspase or calpain can partially protect cultured neurons against Abeta-induced toxicity but their effects are not found to be additive. These results, taken together, suggest that Abeta peptide can induce toxicity in rat septal cultured neurons by activating multiple intracellular signaling molecules. Additionally, evidence that inhibitors of caspase and calpains can partially protect the cultured basal forebrain neurons raised the possibility that their inhibitors could be of therapeutic relevance in the treatment of AD pathology.

Central administration of neuropeptide FF causes activation of oxytocin paraventricular hypothalamic neurones that project to the brainstem.

Neuropeptide FF (NPFF), a morphine modulatory peptide, is emerging as an important neuromodulator in the context of central autonomic and neuroendocrine regulation. NPFF immunoreactivity and receptors have been identified in discrete autonomic regions within the brain and spinal cord, including the hypothalamic paraventricular nucleus (PVN). In this study, we examined the effects of intracerebroventricular (i.c.v.) administration of NPFF on activation of chemically identified PVN neurones that project to the brainstem nucleus of the solitary tract (NTS). In conscious rats, i.c.v. NPFF at a dose of 10 micro g, but not 8 micro g, caused an increase in arterial blood pressure. Immunohistochemical analysis revealed a dose-dependent increase in activated (Fos positive) PVN neurones following i.c.v. NPFF administration compared to controls receiving i.c.v. saline. Activated PVN neurones were located predominantly in the parvocellular compartment of the nucleus with relatively few Fos positive cells in the magnocellular subdivision. Chemical identification of activated neurones revealed significant number of activated cells to be oxytocin positive, whereas only few vasopressin, tyrosine hydroxylase (TH) or corticotrophin-releasing factor (CRF) neurones were double-labelled. Injection of the retrograde tracer fluorogold into the NTS resulted in labelling of significant numbers of parvocellular oxytocin, but not vasopressin, TH or CRF, PVN neurones. We conclude that centrally administered NPFF stimulates brainstem-projecting oxytocin PVN neurones. Oxytocin released from terminals within the NTS oxytocin thus modulate the activity of ascending visceral autonomic pathways that synapse initially within the NTS.

Nociceptin/orphanin FQ modulation of ionic conductances in rat basal forebrain neurons.

Nociceptin/orphanin FQ (N/OFQ) is an endogenous opioid-like heptadecapeptide that plays an important role in a variety of physiological functions. N/OFQ and its receptor opioid receptor-like orphan receptor-1 are abundant in the diagonal band of Broca (DBB), a basal forebrain nucleus where the loss of cholinergic neurons is linked to memory and spatial learning deficits. In the whole animal, central injections of N/OFQ have been shown to disrupt spatial learning. In this study, we investigated the basis for these behavioral observations by examining the cellular effects of N/OFQ on chemically identified DBB neurons. Whole cell patch-clamp recordings were performed on enzymatically dissociated DBB neurons. Under voltage-clamp conditions, bath application of N/OFQ (10 pM-1 microM) resulted in a dose-dependent depression of whole cell currents. Single cell reverse transcription-polymerase chain reaction analysis identified cholinergic and fewer GABAergic cells to be N/OFQ-responsive. [Nphe(1)]nociceptin-(1-13)-NH(2) and CompB (J-113397) antagonized the N/OFQ response, but both compounds also displayed partial agonist activity. Using a combination of channel blockers we determined that the effects of N/OFQ were mediated via a suite of Ca(2+) (N- and L-type) and Ca(2+)-dependent K(+) (iberiotoxin-sensitive) conductances. In addition, biophysical analysis of voltage subtraction protocols revealed that N/OFQ reduces transient outward and the delayed rectifier K(+) currents. Because N-type and L-type Ca(2+) channels are important in the context of neurotransmitter release, our observations indicate that N/OFQ inhibition of Ca(2+)-dependent conductances in cholinergic neurons would be expected to result in depression of acetylcholine release, which may explain the behavioral actions of N/OFQ in the brain.

Cellular mechanisms for amyloid beta-protein activation of rat cholinergic basal forebrain neurons.

The deposition of amyloid beta-protein (Abeta) in the brain and the loss of cholinergic neurons in the basal forebrain are two pathological hallmarks of Alzheimer's disease (AD). Although the mechanism of Abeta neurotoxicity is unknown, these cholinergic neurons display a selective vulnerability when exposed to this peptide. In this study, application of Abeta(25-35) or Abeta(1-40) to acutely dissociated rat neurons from the basal forebrain nucleus diagonal band of Broca (DBB), caused a decrease in whole cell voltage-activated currents in a majority of cells. This reduction in whole cell currents occurs through a modulation of a suite of potassium conductances including calcium-activated potassium (I(C)), the delayed rectifier (I(K)), and transient outward potassium (I(A)) conductances, but not calcium or sodium currents. Under current-clamp conditions, Abeta evoked an increase in excitability and a loss of accommodation in cholinergic DBB neurons. Using single-cell RT-PCR technique, we determined that Abeta actions were specific to cholinergic, but not GABAergic DBB neurons. Abeta effects on whole cell currents were occluded in the presence of membrane-permeable protein tyrosine kinase inhibitors, genistein and tyrphostin B-44. Our data indicate that the Abeta actions on specific potassium conductances are modulated through a protein tyrosine kinase pathway and that these effects are selective to cholinergic but not GABAergic cells. These observations provide a cellular basis for the selectivity of Abeta neurotoxicity toward cholinergic basal forebrain neurons that are at the epicenter of AD pathology.

Effects of restraint stress and spontaneous hypertension on neuropeptide Y neurones in the brainstem and arcuate nucleus.

Neuropeptide Y (NPY) is found in autonomic neurones and participates in regulation of autonomic functions. To investigate the role of NPY in the stress response in normo- and hypertensive rats, activation of brainstem and arcuate nucleus (ARC) NPY neurones and levels of NPY mRNA in the ARC were measured in response to restraint stress in adult spontaneously hypertensive rats (SHRs) and two strains of normotensive rats. Controls from each strain were not restrained. Sections of the brain were prepared for Fos immunohistochemistry and NPY in-situ hybridization to identify activated NPY neurones in the nucleus of the tractus solitarii (NTS), ventrolateral medulla (VLM), and ARC. NPY mRNA levels were quantified in the ARC. In the NTS and VLM of restrained rats, approximately 33% and 75%, respectively, of NPY neurones were activated. No differences among strains were found. In the ARC, about 36% of neurones activated by restraint contained NPY mRNA with no differences found among strains. In unrestrained rats, NPY mRNA levels were significantly elevated in SHRs compared to the normotensive rats. Restraint led to significant decreases in mRNA levels in all strains and mRNA levels among strains were no longer different from one another. These data show that NPY likely participates as a neurotransmitter in the autonomic pathways utilized during stress and originating in the NTS, VLM, and ARC. On the other hand, the decreased gene expression of NPY in the ARC in response to restraint stress argues against a role for activation of autonomic pathways or the hypothalamo-pituitary-adrenal (HPA) axis by NPY from the ARC of stressed rats. The elevated NPY gene expression in resting SHRs compared to normotensive rats is abrogated after restraint, suggesting that this gene is differentially regulated in SHRs compared to normotensive rats.

Hypertensive rats exhibit heightened expression of corticotropin-releasing factor in activated central neurons in response to restraint stress.

To test the hypothesis that chronically elevated sympathetic drive is associated with hyperreactiveness of autonomic centers in the brain to stress, adult spontaneously hypertensive rats (SHRs) and two strains of normotensive rats (Wistar Kyoto [WKY] and Sprague Dawley [SD] rats) were acutely exposed to restraint stress; controls from each strain were not stressed. Brain sections were prepared for Fos immunohistochemistry to identify activated neurons in the paraventricular nucleus of the hypothalamus, Barrington's nucleus of the pons, nucleus of the tractus solitarius, and ventrolateral medulla, or for combined Fos immunohistochemistry and corticotropin-releasing factor (CRF) in situ hybridization in the paraventricular nucleus and Barrington's nucleus. Restraint led to increased activation of neurons in all nuclei. Strain differences were found only in the caudal and rostral paraventricular nucleus where restraint resulted in greater numbers of activated neurons in SHRs compared to either normotensive strain. Levels of CRF mRNA in Barrington's nucleus of unrestrained rats were similar among strains. After restraint, mRNA levels and double labeled neurons were increased in Barrington's nucleus of SHRs. In unstressed rats, CRF mRNA levels were elevated in some regions of the paraventricular nucleus in SHRs. After restraint, mRNA levels increased throughout the paraventricular nucleus of SHRs. Significantly greater numbers of double labeled neurons were found in the dorsolateral medial and ventral medial parvocellular paraventricular nucleus of stressed SHRs compared to WKY or SD rats. These data show that chronic elevation in sympathetic activity, present in SHRs, is associated with hyperreactiveness of the paraventricular and Barrington's nucleus including recruitment of neurons to express CRF, and may have important implications for the response of the hypothalamo-pituitary-adrenal axis during stress.

Atrazine and metolachlor residues in Brookston CL following conventional and conservation tillage culture.

Atrazine and metolachlor are extensively used in Ontario, Canada for control of broadleaf weeds and annual grasses in corn. Conservation tillage may alter the physical and biological environment of soil affecting herbicide dissipation. The rate of dissipation of these two herbicides in soil from conventional, ridge and no-tillage culture was followed. Herbicide dissipation was best described by first order reaction kinetics. Half life, the time for herbicide residues to dissipate to half their initial concentration, was unaffected by tillage. Half life for atrazine and metolachlor was similar and ranged from 31 to 66 d. The rate of dissipation decreased in dry years when soil moisture content was low. In a dry year, herbicide residues during the growing season were significantly greater on ridge tops than in the other tillage treatments. However, after harvest no differences in herbicide residues were detected among tillage treatments. Residues of atrazine (6 to 9% of applied) and metolachlor (4 to 6%) were detected in soil before planting a year after application. De-ethyl atrazine, the primary degradation product of atrazine, increased in concentration during the growing season with the greatest concentrations measured at harvest and in years when atrazine dissipated fastest. De-ethyl atrazine one year after application accounted for about 12% of the remaining triazine residue. These herbicide residues would not be phytotoxic to subsequent crops but are a potential source for leaching to ground and surface waters.

Activation by hypotension of neurons in the hypothalamic paraventricular nucleus that project to the brainstem.

To investigate the involvement of neuronal nitric oxide (NO) in the response of the brain to changes in blood pressure, we studied the activation of putative NO-producing neurons in the paraventricular nucleus of the hypothalamus (PVN) in rats whose mean arterial pressures (MAPs) were decreased by 40-50% with hemorrhage (HEM) or infusion of sodium nitroprusside (NP). Activation was assessed on the basis of expression of the immediate early gene, c-fos; putative NO-producing neurons were identified with the histochemical stain for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d); and the proportions of neurons projecting to the nucleus of the tractus solitarius (NTS) and/or caudal ventrolateral medulla (CVLM) were determined with retrograde tracing techniques. No differences were found for results obtained from HEM and NP animals. Three to four percent of activated PVN neurons projected to the NTS or CVLM. Conversely, approximately 33% and 16% of neurons projecting to the NTS and CVLM, respectively, were activated. About 43% of NADPH-d neurons in the PVN were activated. Of PVN neurons projecting to the NTS or CVLM, 38% and 32%, respectively, were NADPH-d positive. About 11% of NADPH-d PVN neurons projected to the NTS or CVLM. An average of 3 NADPH-d neurons per section were activated and projected to either target. Finally, 7 PVN cells per section sent collateral branches to the NTS and CVLM; 2 or 3 of these cells per section were also activated by decreases in arterial pressure. No NADPH-d cells were found that sent collateral branches to the NTS and CVLM. This study shows that decreases in MAP activate PVN neurons that project, singly and through collaterals, to the NTS and CVLM. A relatively high proportion of the singly projecting neurons is NADPH-d positive. These results support the contention that descending projections from the PVN to the brainstem play an important role in the physiological response to decreases in arterial pressure and suggest that NO may participate in this response.

Changes in blood volume and pressure induce c-fos expression in brainstem neurons that project to the paraventricular nucleus of the hypothalamus.

Immunohistochemistry for c-fos was combined with retrograde tracing techniques to study the effects of acute reductions in arterial blood pressure due to hemorrhage (HEM) in conscious rats on activated neurons in the brainstem nucleus of the tractus solitarius (NTS) or ventrolateral medulla (VLM) which project to the paraventricular nucleus (PVN) of the hypothalamus. In an attempt to separate blood pressure effects from those associated with changes in blood volume, a similar approach was used to study the effects of drug-evoked hypotension using peripheral infusions of sodium nitroprusside (NP). Few differences were found in patterns or numbers of activated neurons (Fos-immunoreactive) in the NTS or VLM after HEM or NP treatment; only in the NTS at the level of the area postrema were significantly higher numbers of neurons that expressed Fos found in NP rats. In addition, a large proportion of PVN-projecting neurons in the NTS and VLM was activated whereas many activated neurons in the NTS and VLM did not project to the PVN. These results show that a decrease in blood pressure leads to the activation of NTS and VLM neurons but that a change in blood volume does not activate significantly greater numbers of neurons in these areas that project to the PVN or to other targets. Whereas substantial numbers of neurons in the NTS and VLM appear to transmit cardiovascular information to the PVN, many others likely transmit this information to other central targets.

Hypotension induces Fos immunoreactivity in NADPH-diaphorase positive neurons in the paraventricular and supraoptic hypothalamic nuclei of the rat.

Double staining for Fos and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-D) was used to study the distribution of activated neurons that synthesize nitric oxide in the paraventricular (PVN) and supraoptic nuclei (SON) following hypotensive stimulation in conscious rats. Fos was detected in many magno- and parvocellular NADPH-D positive neurons in response to haemorrhage or drug-evoked hypotension using i.v. infusions of sodium nitroprusside. However, quantitative analysis did not reveal any differences in the number of Fos positive PVN neurons following either mode of stimulation. These results suggest that a subpopulation of hypothalamic NADPH-D positive neurons is activated following hypotensive challenge. This activation of NADPH-D neurons may occur indirectly through other CNS structures that influence the excitability of hypothalamic SON and PVN. Furthermore, the lack of a difference in activated neurons within the PVN following either haemorrhage or nitroprusside infusion suggests that while a drop in blood pressure causes activation of neurons that produce nitric oxide, a decrease in blood volume, which accompanies haemorrhage, does not.

Residues of nonylphenol in spruce foliage, forest soil, stream water and sediment after its aerial application.

Nonylphenol, a major component of the Matacil (aminocarb) formulation, was mixed with insecticide diluent 585 and applied at 0.47 l/ha by a fixed-wing aircraft to a mixed coniferous forest in Algoma District, Ontario. Residues in white spruce foliage were the highest 1 h after spraying (18.90 ppm) and declined to 60% in the next two hours. After 30 days only 3% of the highest concentration recorded was observed. No detectable levels (less than 0.20 ppm) of nonylphenol were found after 62 days. No residues were detected in any forest soil sample collected after spraying. Nonylphenol residues were about 9 ppb in the stream water 1 h after its application and declined to 50% in the next two hours. Only trace amounts (less than 2.0 ppb) were detected after 5 days and no residues were detected in any water sample taken beyond this time. Residues at trace level (less than 0.10 ppm) were detected in only one sediment sample taken 4 hours after spraying.