ELISA methods to measure cholinergic markers and nerve growth factor receptors in cortex, hippocampus, prefrontal cortex, and basal forebrain from rat brain

Gearhart, Debra A.; Middlemore, Mary Louise; Terry, Alvin V.

doi:10.1016/j.jneumeth.2005.06.009

Cited by 38 publications

(25 citation statements)

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“…Food (Standard Diet 4RF21; Charles River) and tap water were provided ad libitum. Animals were killed by decapitation between 9:00 and 11:00 A.M., trunk blood (5 ml) was collected in tubes containing EDTA (2 mg/ml blood), brains were removed from the skull, and the PFC was rapidly dissected using a standard procedure (Gearhart et al, 2006), frozen on dry ice, and kept at Ϫ80°C until processed. Similarly, the hippocampus and striatum were also dissected from the same rats as control regions.…”

Section: Methodsmentioning

confidence: 99%

Stress-Related Methylation of the Catechol-O-Methyltransferase Val¹⁵⁸Allele Predicts Human Prefrontal Cognition and Activity

Ursini¹,

Bollati²,

Fazio³

et al. 2011

J. Neurosci.

179

152

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DNA methylation at CpG dinucleotides is associated with gene silencing, stress, and memory. The catechol-O-methyltransferase (COMT) Val158 allele in rs4680 is associated with differential enzyme activity, stress responsivity, and prefrontal activity during working memory (WM), and it creates a CpG dinucleotide. We report that methylation of the Val 158 allele measured from peripheral blood mononuclear cells (PBMCs) of Val/Val humans is associated negatively with lifetime stress and positively with WM performance; it interacts with stress to modulate prefrontal activity during WM, such that greater stress and lower methylation are related to reduced cortical efficiency; and it is inversely related to mRNA expression and protein levels, potentially explaining the in vivo effects. Finally, methylation of COMT in prefrontal cortex and that in PBMCs of rats are correlated. The relationship of methylation of the COMT Val 158 allele with stress, gene expression, WM performance, and related brain activity suggests that stress-related methylation is associated with silencing of the gene, which partially compensates the physiological role of the high-activity Val allele in prefrontal cognition and activity. Moreover, these results demonstrate how stress-related DNA methylation of specific functional alleles impacts directly on human brain physiology beyond sequence variation.

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Section: Methodsmentioning

confidence: 99%

Stress-Related Methylation of the Catechol-O-Methyltransferase Val¹⁵⁸Allele Predicts Human Prefrontal Cognition and Activity

Ursini¹,

Bollati²,

Fazio³

et al. 2011

J. Neurosci.

179

152

View full text Add to dashboard Cite

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“…Tissues on the contralateral side of the brain were harvested as controls. The tissues were homogenized, and the soluble protein fraction was obtained by the slightly modified method of Gearhart et al (33). Tissues were homogenized in 10 vol (e.g., Ϸ10 ml of buffer per g of tissue) of supplemented modified radioimmunoprecipitation buffer (pH 8.0), which contained 150 mM NaCl, 1% (vol͞vol) Nonidet P-40, 0.5% (wt͞vol) sodium deoxycholate, 0.1% (wt͞vol) SDS, 50 mM Tris, and 10% (vol͞vol) glycerol (all from Sigma-Aldrich) and was supplemented with 200 l of PMSF, 100 l of protease inhibitor mixture, and 200 l each of phosphatase inhibitor mixtures 1 and 2 (all from Sigma-Aldrich) per 10 ml of ice-cold buffer.…”

Section: Mrimentioning

confidence: 99%

Noninvasive localized delivery of Herceptin to the mouse brain by MRI-guided focused ultrasound-induced blood–brain barrier disruption

Kinoshita

McDannold

Jólesz

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

601

451

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Antibody-based anticancer agents are promising chemotherapeutic agents. Among these agents, Herceptin (trastuzumab), a humanized anti-human epidermal growth factor receptor 2 (HER2͞ c-erbB2) monoclonal antibody, has been used successfully in patients with breast cancer. However, in patients with brain metastasis, the blood-brain barrier limits its use, and a different delivery method is needed to treat these patients. Here, we report that Herceptin can be delivered locally and noninvasively into the mouse central nervous system through the blood-brain barrier under image guidance by using an MRI-guided focused ultrasound blood-brain barrier disruption technique. The amount of Herceptin delivered to the target tissue was correlated with the extent of the MRI-monitored barrier opening, making it possible to estimate indirectly the amount of Herceptin delivered. Histological changes attributable to this procedure were minimal. This method may represent a powerful technique for the delivery of macromolecular agents such as antibodies to treat patients with diseases of the central nervous system. brain tumor ͉ microbubble A dvances in tumor cell biology have led to the availability of new types of anticancer chemotherapeutic agents that are superior to the conventional agents in that they can precisely target the signal-transduction system unique to malignant tumor cells, thereby lowering the toxic effects of anticancer agents on normal cells. Herceptin (trastuzumab; Genentech) is a humanized mAb that targets human epidermal growth factor receptor 2 (HER2͞c-erbB2) expressed in breast cancer cells. It has been used to treat breast cancer patients, and it has succeeded remarkably in controlling local and distal breast cancer lesions (1). Although breast cancer often metastasizes to the brain (2), Herceptin could only be used to treat extracranial lesions because there is currently no efficient method to deliver it to the CNS. The increased use of Herceptin to treat breast cancer patients has resulted in a higher incidence of brain metastasis from primary lesions (3, 4). When Herceptin was used as a first-line therapy in breast cancer patients, metastatic extracranial lesions responded to the agent in 71% of the patients who continued to develop metastatic lesions in the brain (3).The CNS is protected from the entry of foreign substances by the almost impenetrable blood-brain barrier (BBB) (5, 6), which hampers the delivery of potentially effective diagnostic or therapeutic agents and complicates the treatment of CNS diseases, including malignant brain diseases such as metastatic brain tumors. Because antibody-based agents with a molecular size of Ϸ150 kDa are easily blocked by the BBB, their delivery to the CNS requires the temporary suspension of the physiological role of the BBB to bar larger molecules from the CNS.Current advances in acoustic technology have made ultrasound a modality with therapeutic as well as diagnostic applicability. Focused ultrasound techniques facilitate the concentration of acoustic energy o...

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“…Brain lysates were evaluated using ELISA methods to measure the relative levels of ChAT, vesicular acetylcholine transporter (VAChT), p75 neurotrophin receptor (p75 NTR ), TrkA (nerve growth factor receptor), phospho-TrkA (P-TrkA), and NGF. The brain dissections, preparation of brain lysates, protein assay, and ELISA methods (except NGF) were performed according to Gearhart et al (2006), except that in the present study, different quantities of brain protein were analyzed by ELISA (see Table 2 for details). As an internal control for day-to-day variation in the ELISA methods, brain lysates (same amount of protein per well) from vehicle-, HAL-, and ZIP-treated rats were always assayed at the same time on the same ELISA plate.…”

Section: Enzyme-linked Immunosorbent Assay Experimentsmentioning

confidence: 99%

Time-Dependent Effects of Haloperidol and Ziprasidone on Nerve Growth Factor, Cholinergic Neurons, and Spatial Learning in Rats

Terry¹,

Parikh²,

Gearhart³

et al. 2006

J Pharmacol Exp Ther

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In this rodent study, we evaluated the effects of different time periods (7, 14, 45, and 90 days) of oral treatment with haloperidol (HAL; 2.0 mg/kg/day) or ziprasidone (ZIP; 12.0 mg/kg/day) on nerve growth factor (NGF) and choline acetyltransferase (ChAT) levels in the hippocampus, and we subsequently assessed water maze task performance, prepulse inhibition (PPI) of the auditory gating response, and several NGF-related proteins and cholinergic markers after 90 days of treatment. Seven and 14 days of treatment with either HAL or ZIP resulted in a notable increase in NGF and ChAT immunoreactivity in the dentate gyrus (DG), CA1, and CA3 areas of the hippocampus. After 45 days, NGF and ChAT immunoreactivity had abated to control levels in ZIP-treated animals, but it was markedly reduced in HAL-treated subjects. After 90 days of treatment, NGF and ChAT levels were substantially lower than controls in both antipsychotic groups. Furthermore, after 90 days of treatment and a drug-free washout period, water maze performance (but not PPI) was impaired in both antipsychotic groups, although the decrement was greater in the HAL group. Several NGFrelated and cholinergic proteins were diminished in the brains of subjects treated with either neuroleptic as well. These data support the premise that, although ZIP (given chronically) seems somewhat superior to HAL due to less pronounced behavioral effects and a more delayed appearance of neurochemical deficits, both antipsychotics produce time-dependent deleterious effects on NGF, cholinergic markers (i.e., important neurobiological substrates of memory), and cognitive function.The newer pharmacological treatments for schizophrenia, now commonly referred to as second generation antipsychotics (SGAs), offer several advantages over first generation antipsychotics (FGAs) such as greater improvements in negative symptoms, prevention of relapse, increased functional capacity and quality of life, and fewer movement-related side effects (for review, see Miyamoto et al., 2005). It is also generally believed that SGAs are superior to FGAs when their effects on cognition are considered (for reviews, see Keefe et al., 1999;Purdon, 1999), and some studies suggest that SGAs improve cognition in schizophrenia. This suggestion is of particular importance given that the degree of cognitive impairment in schizophrenia is recognized as an important predictor of social functioning, unemployment, and even relapse of psychiatric symptoms (for review, see Castner et al., 2004). It should be noted, however, that such conclusions regarding antipsychotics and cognitive function rely primarily on meta-analyses and short clinical trials (i.e., they rarely exceed a few months to 1 year in length). There-

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ELISA methods to measure cholinergic markers and nerve growth factor receptors in cortex, hippocampus, prefrontal cortex, and basal forebrain from rat brain

Cited by 38 publications

References 50 publications

Stress-Related Methylation of the Catechol-O-Methyltransferase Val¹⁵⁸Allele Predicts Human Prefrontal Cognition and Activity

Stress-Related Methylation of the Catechol-O-Methyltransferase Val¹⁵⁸Allele Predicts Human Prefrontal Cognition and Activity

Noninvasive localized delivery of Herceptin to the mouse brain by MRI-guided focused ultrasound-induced blood–brain barrier disruption

Time-Dependent Effects of Haloperidol and Ziprasidone on Nerve Growth Factor, Cholinergic Neurons, and Spatial Learning in Rats

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ELISA methods to measure cholinergic markers and nerve growth factor receptors in cortex, hippocampus, prefrontal cortex, and basal forebrain from rat brain

Cited by 38 publications

References 50 publications

Stress-Related Methylation of the Catechol-O-Methyltransferase Val158Allele Predicts Human Prefrontal Cognition and Activity

Stress-Related Methylation of the Catechol-O-Methyltransferase Val158Allele Predicts Human Prefrontal Cognition and Activity

Noninvasive localized delivery of Herceptin to the mouse brain by MRI-guided focused ultrasound-induced blood–brain barrier disruption

Time-Dependent Effects of Haloperidol and Ziprasidone on Nerve Growth Factor, Cholinergic Neurons, and Spatial Learning in Rats

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Product

Resources

About

Stress-Related Methylation of the Catechol-O-Methyltransferase Val¹⁵⁸Allele Predicts Human Prefrontal Cognition and Activity

Stress-Related Methylation of the Catechol-O-Methyltransferase Val¹⁵⁸Allele Predicts Human Prefrontal Cognition and Activity