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A high efficacy selection method for transfected cells utilizing recombinant isolectin B4-saporin
Galvan MA, Shramm PA, Bouajram R, Lappi DA, Ancheta LR (2019) A high efficacy selection method for transfected cells utilizing recombinant isolectin B4-saporin. Neuroscience 2019 Abstracts 794.10. Society for Neuroscience, Chicago, IL.
Summary: Transfection protocols often rely on the use of antibiotics for the selection of transfected cells and has become the accepted approach for in vitro research and therapeutic applications. Antibiotics have several shortcomings such as cost, continuous use, and harmful effects — even on the transfected cell population. In addition, selection pressures are often inefficient and fail to provide a population of cells that express the gene of interest (GOI) at high levels. We have used three separate GOI’s to select for solely high-expressing transfectants using targeted toxin selection pressure. Normal Rat Kidney Cells (KNRK) were individually transfected to express green fluorescent protein (GFP), melanopsin or the low-affinity nerve growth factor receptor (p75) using an innovative new transfection delivery vector called pGEI. The results from various assays were utilized to visually determine the expression rate and pattern of the targeted toxin selection method. Melanopsin and p75 — a photopigment and nerve growth factor, respectively — were of great interest to express in our transfected cells as a means to study their role in the development and function of neurons. The delivery vector, pGEI, removes resident Galalpha(1-3)Gal epitopes from non- human mammalian cell surfaces. This residue is the target of recombinant Isolectin B4 – Saporin (IB4-SAP), a selective targeted toxin. IB4-SAP is extremely potent, with an EC50 in the low picomolar range for alpha-D-galactopyranoside expressing cells in vitro. The cells with the highest expression of the inserted vector, and therefore the GOI, will have these residues removed. Those that fail to express the vector or do not express the vector in high enough amounts, will not have all the residues removed, and will be targeted and eliminated via IB4-SAP. This method of selection provides a means of purifying the highest- expressing transfected populations using a more cost-effective and time-saving approach.
Related Products: IB4-SAP (Cat. #IT-10)
An acetylcholine-dopamine interaction in the rat nucleus accumbens and its tentative involvement in ethanol’s dopamine-liberating effect
Andrén A, Adermark L, Söderpalm B, Ericson M (2019) An acetylcholine-dopamine interaction in the rat nucleus accumbens and its tentative involvement in ethanol’s dopamine-liberating effect. Neuroscience 2019 Abstracts 079.08. Society for Neuroscience, Chicago, IL.
Summary: Alcohol use disorder is a chronic, relapsing brain disorder associated with serious medical consequences leading to preterm death. Although few in number, cholinergic interneurons (CIN) have arisen as an important cell population within the nucleus accumbens (nAc) that may exert a regulatory impact on dopamine (DA) neurotransmission locally. A defect in CIN have been suggested to be involved in psychiatric diseases such as alcohol addiction. The mechanisms through which endogenous cholinergic activity modulates DA release in response to ethanol administration and its role in development of addiction is not known. In this project, the aim was to study if acetylcholine (ACh) can influence DA release locally in the nAc and if so, through which receptor population(s) this effect is mediated. Further, we wanted to determine the role of ACh in ethanol-induced DA elevation.Using reversed in vivo microdialysis, the acetylcholinesterase inhibitor physostigmine was administered locally in the nAc of male Wistar rats followed by addition of either the muscarinic ACh receptor inhibitor scopolamine or the nicotinergic ACh receptor inhibitor mecamylamine. Subsequently, ethanol was perfused following local pretreatment with scopolamine or mecamylamine, using the same methodology. An immunotoxin, anti-ChAT-saporine, was infused locally into the nAc of a subset of male Wistar rats to selectively lesion CIN, followed by local ethanol administration via reversed in vivo microdialysis. Local administration of physostigmine induced a DA elevation within the nAc, an effect blocked by scopolamine but not by mecamylamine. Local administration of ethanol increased DA levels. Scopolamine pretreatment non-significantly attenuated the ethanol-induced DA elevation, whereas pretreatment with mecamylamine had no effect. Preliminary results indicate a minor attenuation of the DA elevation observed after local administration of ethanol in toxin-treated animals, as compared to sham-treated controls. Taken together, these results suggest that ACh increases extracellular DA levels in nAc in vivo, an effect mediated by muscarinic ACh-receptors and not by nicotinic ACh-receptors. Considering that scopolamine moderately attenuated ethanol-induced DA output and that lesioning of CIN appeared to hamper DA release in response to ethanol, ACh release from CIN within the nAc may be partially involved in ethanol-induced DA release in nAc.
Related Products: Anti-ChAT-SAP (Cat. #IT-42)
Exercise is neuroprotective following partial motoneuron depletion via androgen action at the target muscle
Chew C, Sengelaub DR (2019) Exercise is neuroprotective following partial motoneuron depletion via androgen action at the target muscle. Neuroscience 2019 Abstracts 134.13. Society for Neuroscience, Chicago, IL.
Summary: We have previously demonstrated that partial depletion of motoneurons innervating the quadriceps muscles induces dendritic atrophy in remaining motoneurons. Furthermore, systemic treatment with supplemental androgens is neuroprotective, and dendritic atrophy following partial motoneuron depletion is attenuated. Blockade of the androgen receptor at the target muscle prevents the neuroprotective effects on motoneuron dendrites in rats treated with supplemental androgens. We have recently shown that exercise is also neuroprotective on motoneuron dendrites following partial motoneuron depletion, and circulating levels of androgens have previously been shown to increase following exercise. Together, these results suggest that exercise may be neuroprotective via androgen action at the muscle. In the present study, we examine whether blockade of androgen receptors at the target musculature would prevent the neuroprotective effects of exercise on dendrites following partial motoneuron depletion. Motoneurons innervating the vastus medialis muscle in adult male rats were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Simultaneously, some saporin-injected rats were given implants of the androgen receptor antagonist hydroxyflutamide, either directly at the quadriceps musculature or interscapularly as a systemic control. Following saporin injections, some animals were allowed free access to running wheels attached to their home cages. Four weeks later, motoneurons innervating the ipsilateral vastus lateralis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Compared with untreated males, partial motoneuron depletion resulted in decreased dendritic length in remaining quadriceps motoneurons. Early data suggests that following partial motoneuron depletion, exercised males with androgen receptor blockade at the quadriceps show dendritic lengths that are significantly shorter than those of exercised males with no treatment, while dendritic lengths in exercised males with interscapular implants do not differ from those of exercised animals without implants. These findings suggest that exercise may be protective against dendritic atrophy via androgens binding at the target musculature.
Related Products: CTB-SAP (Cat. #IT-14)
The retrotrapezoid nucleus: Central chemoreceptor and regulator of breathing automaticity.
Guyenet PG, Stornetta RL, Souza GMPR, Abbott SBG, Shi Y, Bayliss DA. (2019) The retrotrapezoid nucleus: Central chemoreceptor and regulator of breathing automaticity. Trends Neurosci 42(11):807-824. doi: 10.1016/j.tins.2019.09.002
Summary: This review describes the neurons of the retrotrapezoid nucleus (RTN), their transcriptome, developmental lineage, and anatomical projections. The authors also review their contribution to CO2 homeostasis and to the regulation of breathing automaticity during sleep and wake.
Usage: Local injection of SSP-SAP to kill RTN neurons.
Related Products: SSP-SAP (Cat. #IT-11)
RGS4 maintains chronic pain symptoms in rodent models.
Avrampou K, Pryce KD, Ramakrishnan A, Sakloth F, Gaspari S, Serafini RA, Mitsi V, Polizu C, Swartz C, Ligas B, Richards A, Shen L, Carr FB, Zachariou V (2019) RGS4 maintains chronic pain symptoms in rodent models. J Neurosci 39(42):8291-8304. doi: 10.1523/JNEUROSCI.3154-18.2019 PMID: 31308097
Usage: western
Related Products: Metabotropic Glutamate Receptor 2 (mGluR2) Mouse Monoclonal (Cat. #AB-N32)
Lesions of the patch compartment of dorsolateral striatum disrupt stimulus-response learning.
Jenrette TA, Logue JB, Horner KA (2019) Lesions of the patch compartment of dorsolateral striatum disrupt stimulus-response learning. Neuroscience 415:161-172. doi: 10.1016/j.neuroscience.2019.07.033
Objective: To investigate whether enhanced activation of the patch compartment contributes to habitual behavior.
Summary: The dorsolateral patch compartment may mediate habit formation by altering information flow through basal ganglia circuits.
Usage: A volume of 2 ul of Dermorphin-SAP (17 ng/ul or an equivalent amount of unconjugated SAP (as a control) was infused bilaterally, at a rate of 0.5 ul/min.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), Saporin (Cat. #PR-01)
Role of nociceptor toll-like receptor 4 (TLR4) in opioid-induced hyperalgesia and hyperalgesic priming.
Araldi D, Bogen O, Green PG, Levine JD (2019) Role of nociceptor toll-like receptor 4 (TLR4) in opioid-induced hyperalgesia and hyperalgesic priming. J Neurosci 39(33):6414-6424. doi: 10.1523/JNEUROSCI.0966-19.2019
Objective: To evaluate the hypothesis that hyperalgesia and priming induced by opioids are mediated by similar nociceptor mechanisms.
Summary: Treatment with isolectin B4 (IB4)-saporin or SSP-saporin (which deplete IB4! and peptidergic nociceptors, respectively), or their combination, prevented systemic LDM-induced hyperalgesia, but not priming. HDM-induced priming, but not analgesia, was markedly attenuated in both saporin-treated groups.
Usage: IB4-SAP was diluted in saline and a dose of 3.2 μg in a volume of 20 μl administered intrathecally. SSP-SAP was diluted in saline and a dose of 100 ng in a volume of 20 μl was administered intrathecally.
Related Products: IB4-SAP (Cat. #IT-10), SSP-SAP (Cat. #IT-11)
3D reconstruction of the neurovascular unit reveals differential loss of cholinergic innervation in the cortex and hippocampus of the adult mouse brain.
Nizari S, Carare RO, Romero IA, Hawkes CA (2019) 3D reconstruction of the neurovascular unit reveals differential loss of cholinergic innervation in the cortex and hippocampus of the adult mouse brain. Front Aging Neurosci 11:172. doi: 10.3389/fnagi.2019.00172
Objective: To further characterize the effect of the loss of cholinergic innervation on the NVU (neurovascular unit) in Alzheimer’s Disease.
Summary: Significantly less ChAT staining was detected in the medial septum of saporin-treated mice at 45 days post-surgery. This was accompanied by a significant decrease in cholinergic nerve fiber density in the hippocampus and the cortex. As expected, p75 NTR-negative neurons in the striatum were not affected by mu p75-SAP treatment.
Usage: In this study, the mu-p75-SAP was used to induce death of basal forebrain cholinergic neurons and their fiber projections. mu p75-SAP 0.5 µL (0.596 µg/µL) or 0.9% saline (n = 19) was injected into each ventricle.
Related Products: mu p75-SAP (Cat. #IT-16)
Conditioned aversion and neuroplasticity induced by a superagonist of extrasynaptic gabaa receptors: Correlation with activation of the oval BNST neurons and CRF mechanisms.
de Miguel E, Vekovischeva O, Elsilä LV, Panhelainen A, Kankuri E, Aitta-aho T, Korpi ER (2019) Conditioned aversion and neuroplasticity induced by a superagonist of extrasynaptic gabaa receptors: Correlation with activation of the oval BNST neurons and CRF mechanisms. Front Mol Neurosci 12:130. doi: 10.3389/fnmol.2019.00130 PMID: 31178693
Usage: immunohistochemistry (1:250)
Related Products: Corticotropin Releasing Hormone Rabbit Polyclonal (Cat. #AB-02)
Featured Article: Impaired reach-to-grasp responses in mice depleted of striatal cholinergic interneurons
Abudukeyoumu N, Garcia-Munoz M, Nakano Y, Arbuthnott GW (2018) Featured Article: Impaired reach-to-grasp responses in mice depleted of striatal cholinergic interneurons. Targeting Trends 19
Related Products: Anti-ChAT-SAP (Cat. #IT-42)
Read the featured article in Targeting Trends.
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