control-conjugates

Oyamaguchi A, Abe T, Sugiyo S, Niwa H, Takemura M (2016) Selective elimination of isolectin B4-binding trigeminal neurons enhanced formalin-induced nocifensive behavior in the upper lip of rats and c-Fos expression in the trigeminal subnucleus caudalis. Neurosci Res 103:40-47. doi: 10.1016/j.neures.2015.07.007

Summary: In adult rats non-peptidergic neurons and peptidergic neurons innervate different areas and layers of the lamina. It is thought that these two neuronal populations play different roles in nociceptive processing, but the specific function of each group is not well understood. In order to investigate peptidergic and non-peptidergic neurons in orofacial pain processing the authors injected the cisterna magna of rats with 2.9 μg of rIB4-SAP (Cat. #IT-10). Blank-SAP (Cat. #IT-21) was used as a control. The lesioned animals displayed more frequent face-rubbing responses on the administration of formalin, indicating that IB4-binding neurons in the trigeminal nerve play an anti-nociceptive role in response to this type of pain.

Related Products: IB4-SAP (Cat. #IT-10), Blank-SAP (Cat. #IT-21)

King T, Ruyle B, Kline D, Heesch C, Hasser E (2015) Catecholaminergic neurons projecting to the paraventricular nucleus of the hypothalamus are essential for cardiorespiratory adjustments to hypoxia. Am J Physiol Regul Integr Comp Physiol 309:R721-731. doi: 10.1152/ajpregu.00540.2014

Summary: Catecholaminergic neurons in the brainstem are known to be involved in cardiorespiratory control and to modulate sensory function. Some of the projections from these neurons are to the paraventricular nucleus (PVN), and are involved in cardiorespiratory and neuroendocrine responses to hypoxia. While data have shown the PVN-projecting neurons are activated by hypoxia, their function in this context is not known. In this work the authors bilaterally injected 42 ng of Anti-DBH-SAP (Cat. #IT-03) into the PVN of rats. Mouse IgG-SAP (Cat. #IT-18) was used as control. Respiratory measurements of the lesioned animals indicates that PVN-projecting catecholaminergic neurons are involved in peripheral and central chemoreflex and arterial oxygen levels during exposure to hypoxic stimuli.

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

Akiyama T, Nguyen T, Curtis E, Nishida K, Devireddy J, Delahanty J, Carstens M, Carstens E (2015) A central role for spinal dorsal horn neurons that express neurokinin-1 receptors in chronic itch. Pain 156:1240-1246. doi: 10.1097/j.pain.0000000000000172

Summary: Chronic itch is caused by increased sensitivity of itch-signaling pathways. It can be generated by normally itchy stimuli (hyperknesis) and by normally non-itchy light touch (alloknesis). The authors used an ovalbumin-induced atopic dermatitis model to study chronic itch in mice. The mice received 400-ng intrathecal injections of Bombesin-SAP (Cat. #IT-40), SSP-SAP (Cat. #IT-11), or the control Blank-SAP (Cat. #IT-21). While Bombesin-SAP significantly attenuated hyperknesis, it had no effect on spontaneous scratching or alloknesis. SSP-SAP reduced all behavioral signs of chronic itch.

Related Products: Bombesin-SAP (Cat. #IT-40), SSP-SAP (Cat. #IT-11), Blank-SAP (Cat. #IT-21)

Bourassa E, Stedenfeld K, Sved A, Speth R (2015) Selective C1 lesioning slightly decreases angiotensin II type I receptor expression in the rat rostral ventrolateral medulla (RVLM). Neurochem Res 40:2113-2120. doi: 10.1007/s11064-015-1649-3

Summary: Exogenous angiotensin II administered to the RVLM produces a significant pressor response that can be countered by angiotensin II type I receptor antagonists. In this work the authors examined the relative contribution of C1 and non-C1 neurons in the RVLM to this angiotensin II response. Rats received 10 or 15 ng of Anti-DBH-SAP (Cat. #IT-03) as unilateral injections into the RVLM. Mouse IgG-SAP (Cat. #IT-18) was used as control. The data indicate that the majority of angiotensin II type 1 receptors are expressed on non-C1 neurons or glia.

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

Peters C, Hayashida K, Suto T, Houle T, Aschenbrenner C, Martin T, Eisenach J (2015) Individual differences in acute pain-induced endogenous analgesia predict time to resolution of postoperative pain in the rat. Anesthesiology 122:895-907. doi: 10.1097/ALN.0000000000000593

Summary: The authors investigated the relationship between preoperative Conditioned Pain Modulation (CPM) and the time course of recovery from surgery. CPM was evaluated using forepaw capsaicin injections into rats. During the study, lesioned rats received 5-μg intrathecal injections of anti-DBH-SAP (Cat. #IT-03), followed 14 days later by a partial L5 spinal nerve ligation surgery. Mouse-IgG-SAP (Cat. #IT-18) was used as a control. CPM was partially blocked in the lesioned animals, suggesting descending noradrenergic signaling is important in the time course of recovery from surgery.

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

Mittelman-Smith M, Krajewski-Hall S, McMullen N, Rance N (2015) Neurokinin 3 receptor-expressing neurons in the median preoptic nucleus modulate heat-dissipation effectors in the female rat. Endocrinology 156:2552-2562. doi: 10.1210/en.2014-1974

Summary: Kisspeptin and Neurokinin B (NKB) expression in the infundibular, or arcuate, nucleus is increased after menopause. Here the authors investigate whether KNDy (kisspeptin, NKB, and dynorphin expressing) neurons are able to influence cutaneous vasodilation through Neurokinin 3 (NK3)-expressing projections from the median preoptic nucleus (MnPO). Rats received two 10-ng injections of NK3-SAP (Cat. #IT-63) into the MnPO. Blank-SAP (Cat. #IT-21) was used as a control. The data indicate that NK3-expressing neurons in the MnPO facilitate vasodilation.

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

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.

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)

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.