control-conjugates

Xu M, Kobets A, Du J, Lennington J, Li L, Banasr M, Duman R, Vaccarino F, DiLeone R, Pittenger C (2015) Targeted ablation of cholinergic interneurons in the dorsolateral striatum produces behavioral manifestations of Tourette syndrome. Proc Natl Acad Sci U S A 112:893-898. doi: 10.1073/pnas.1419533112

Summary: Postmortem studies of Tourette syndrome patients has revealed a reduction in the number of specific striatal interneurons. The authors explored the hypothesis that this neuronal deficit is enough to produce the symptoms of Tourette syndrome in mice. Animals received 90-ng injections of Anti-ChAT-SAP (Cat. #IT-42) into the striatum. Rabbit IgG-SAP (Cat. #IT-35) was used as a control. The data suggest that loss of the striatal interneurons is enough to produce some, but not all, of the symptoms caused by Tourette syndrome.

Related Products: Anti-ChAT-SAP (Cat. #IT-42), Rabbit IgG-SAP (Cat. #IT-35)

Q: Our lab is getting ready to begin a project using one of your targeted toxins. We already did a preliminary experiment to try out the material, but we have a couple of questions before we start the larger project. First, do you have any protocols or references for injecting intrathecally?

A: Thank you for your inquiry. We appreciate the opportunity to get involved in projects before they begin. At Advanced Targeting Systems, we do not do any in vivo work, just in vitro, however we have collaborated with many fine laboratories that have good experience with intrathecal injections. If you search PubMed with the keywords ‘saporin’ and ‘intrathecal’ you will be able to view references that will give you good information on techniques and protocols. Prior to beginning your project you will want to submit your animal care guidelines to your IACUC committee. Turner et al. published an article that will be helpful regarding intrathecal injections. [1]

Q: The second question is in two parts: 1) how do we determine the appropriate dose, and 2) how do we know saporin is not killing indiscriminately at that dose?

A: You should always use a control when determining the appropriate dose. A basic premise of the ATS targeting technology is that if a control (saporin alone or a control conjugate) evokes a response, then the dose is too high. Whenever a new shipment of targeted toxin is received, the proper working dilution should be ascertained before beginning a project. The targeted toxin data sheet states:

“There may be lot-to-lot variation in material; working dilutions must be determined by end user. If this is a new lot, assess the proper working dilution before beginning a full experimental protocol.”

If you search on the ATS website for the species and route of administration you plan to use, you can look through the publication summaries and see the dose that was used for that particular study. That will give you a ballpark range in which to start your dose titration. Just keep in mind: if the control kills cells, the dose is too high.

References

1. Turner et al. Administration of Substances to Laboratory Animals: Routes of Administration and Factors to Consider. J Am Assoc Lab Anim Sci 50(5): 600–613, 2011.

Cetas J, McFarlane R, Kronfeld K, Smitasin P, Liu J, Raskin J (2015) Brainstem opioidergic system is involved in early response to experimental SAH. Transl Stroke Res 6:140-147. doi: 10.1007/s12975-014-0378-2

Objective: To determine the cause of poor long-term outcomes after Subarachnoid hemorrhage (SAH).

Summary: Failure of the RVM μ-opioid receptor cells to initiate the compensatory CBF response sets the stage for acute and delayed ischemic injury following SAH.

Usage: To lesion medullary neurons expressing the μ-opioid receptor, Dermorphin–SAP was microinjected as a bilateral dose of 0.5 pmol in 500 nL per side (1 pmol in 1 μL total dose and injection volume). Blank–SAP or vehicle was injected in equal volumes and dose as controls.

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), Blank-SAP (Cat. #IT-21)

Q: I ordered a control conjugate to use alongside my targeted conjugate, but the two products are at different concentrations. How much control conjugate should I use?

A: Conjugate products are often of differing protein concentrations, meaning dilution of one is usually necessary to ensure comparable amounts of control conjugate and targeted conjugate are used. This adjustment can be done on a molar basis or a protein concentration basis. The data sheet shipped with each Advanced Targeting System conjugate specifies the molecular weight of the product. There are various calculators available on the ATS website.

By using these tools, calculations can be done that will ensure the same number of molecules of both control and targeted conjugate are used in your experiment. Alternatively, if the molecular weights of the two products are similar, calculations can be done to use the same amount of control protein as targeted conjugate protein in your experiment.

Zhao Z, Wan L, Liu X, Huo F, Li H, Barry D, Krieger S, Kim S, Liu Z, Xu J, Rogers B, Li Y, Chen Z (2014) Cross-inhibition of NMBR and GRPR signaling maintains normal histaminergic itch transmission. J Neurosci 34:12402-12414. doi: 10.1523/JNEUROSCI.1709-14.2014

Summary: After itch detection, the itch pathway moves through an array of G-protein coupled receptors and transient receptor potential channels in dorsal root ganglion neurons into dorsal horn neurons which integrate and transduce these signals, sending them to the somatosensory cortex. The purpose of this work is to clarify whether gastrin-releasing peptide (GRP) or B-type natriuretic peptide regulates histaminergic itch. Several strains of knockout mice received 200, 300, or 400 ng intrathecal injections of bombesin-SAP (Cat. #IT-40). Blank-SAP (Cat. #IT-21) was used as a control. The data further define the respective functions of the neuromedin B receptor and GRP receptor in itch, and reveals a working relationship between the different interneuron populations.

Related Products: Bombesin-SAP (Cat. #IT-40), Blank-SAP (Cat. #IT-21)

Lee CL, Rajkumar R, Dawe GS (2014) Featured Article: Corticotropin releasing factor-saporin conjugate selectively lesions nucleus incertus. Targeting Trends 15(2)

Related Products: CRF-SAP (Cat. #IT-13), Blank-SAP (Cat. #IT-21)

Read the featured article in Targeting Trends.

See Also:

• Lee LC et al. Selective lesioning of nucleus incertus with corticotropin releasing factor-saporin conjugate. Brain Res 1543:179-190, 2014.

Jokiaho A, Donovan C, Watts A (2014) The rate of fall of blood glucose determines the necessity of forebrain-projecting catecholaminergic neurons for male rat sympathoadrenal responses. Diabetes 63:2854-2865. doi: 10.2337/db13-1753

Summary: Different sets of glucosensors detect insulin-induced hypoglycemia depending on the onset rate. This detection controls the activation of sympathoadrenal counterregulatory responses (CRRs). Slow onset hypoglycemia, common with insulin therapy, is detected by glucosensors in the portal-mesenteric veins. Fast onset is detected by brain elements. The authors lesioned hindbrain catecholaminergic neurons to determine which set of responses-they interact with. Rats received 42 ng bilateral injections of Anti-DBH-SAP (Cat. #IT-03) into the paraventricular nucleus of the hypothalamus. Mouse IgG-SAP (Cat. #IT-18) was used as a control. The data indicate that these neurons are critical for detection of slow-onset insulin-induced hypoglycemia.

Related Products: Anti-DBH-SAP (Cat. #IT-03), Mouse IgG-SAP (Cat. #IT-18)

Ogawa S, Nathan FM, Parhar IS (2014) Habenular kisspeptin modulates fear in the zebrafish. Proc Natl Acad Sci U S A 111(10):3841-3846. doi: 10.1073/pnas.1314184111

Summary: The peptide kisspeptin can be found in several areas of the brain, but its role in regions other than the hypothalamus has not been studied. Zebrafish express kiss1 mRNA which is a conserved ortholog of the mammalian KISSI/KissI making zebrafish a viable model for investigating the role of kisspeptin in various brain systems. Animals received 1 μg of the custom conjugate kiss-SAP (see NK3-SAP, Cat. #IT-63) via an intracranial injection. Blank-SAP (Cat. #IT-21) was used as a control. Reducing Kiss1 immunoreactivity in the habenula and the raphe reduced an invoked fear response, indicating a role for kisspeptin in fear inhibition.

Related Products: Blank-SAP (Cat. #IT-21), NKB-SAP (Cat. #IT-63), Custom Conjugates

Lee SC, Seo KW, Kim HJ, Kang SW, Choi HJ, Kim A, Kwon BS, Cho HR, Kwon B (2015) Depletion of alloreactive T cells by anti-CD137-saporin immunotoxin. Cell Transplant 24:1167-1181. doi: 10.3727/096368914X679327

Summary: The ability to selectively remove T cells that mediate graft-versus-host disease (GVHD) would prevent graft rejection as well as preserve the T cells that mediate graft-versus-leukemia (GVL) effect – especially in cases of hematopoietic stem cell transplantation. In this work the authors used a custom conjugate of anti-mouse CD137 and saporin to eliminate alloreactive T cells during a T cell donor transfer in mice. Rat IgG-SAP (Cat. #IT-17) was used as a control. Transfer of T cells after the immunotoxin treatment did not cause GVHD, but the GVL effect was left intact, indicating the potential of selective T cell depletion for transplantation.

Related Products: Rat IgG-SAP (Cat. #IT-17)

Suto T, Severino AL, Eisenach JC, Hayashida KI (2014) Gabapentin increases extracellular glutamatergic level in the locus coeruleus via astroglial glutamate transporter-dependent mechanisms. Neuropharmacology 81C:95-100. doi: 10.1016/j.neuropharm.2014.01.040

Summary: Gabapentin is effective in reducing acute and chronic pain, but the mechanisms by which it works are not well understood. The authors assessed extracellular glutamate levels and glutamate interaction with several different cellular membrane proteins. Rats received a 0.25 μg injection of anti-DBH-SAP (Cat. #IT-03) into the locus coeruleus (LC) in order to deplete noradreline levels. Mouse IgG-SAP (Cat. #IT-18) was used as a control. The gabapentin-induced glutamate increase in the LC was not affected by the lesion, supporting data indicating that gabapentin induces glutamate release from astrocytes to stimulate descending inhibition.

Related Products: Anti-DBH-SAP (Cat. #IT-03), Mouse IgG-SAP (Cat. #IT-18)