control-conjugates

Fu C, Xue J, Wang R, Chen J, Ma L, Liu Y, Wang X, Guo F, Zhang Y, Zhang X, Wang S (2017) Chemosensitive Phox2b-expressing neurons are crucial for hypercapnic ventilatory response in the nucleus tractus solitarius. J Physiol 595:4973-4989.. doi: 10.1113/JP274437

Objective: To investigate whether paired-like homeobox 2b  (Phox2b)-expressing NTS neurons are recruited in hypercapnic ventilatory response (HCVR)  and whether these neurons exhibit intrinsic chemosensitivity.

Summary: Respiratory deficits caused by injection of SSP-SAP into the NTS are attributable to proportional lesions of CO2/H+-sensitive Phox2b-expressing neurons.

Usage: Two protocols were applied for SSP-SAP injections. In immunostaining experiments, to determine how many Phox2b-containing cells were destroyed, a total volume of 100 nl of PBS containing 6 ng of SSP-SAP (3 ng in 50 nl; 2 injections) was injected into one side of the NTS and the contralateral NTS was used as a control (no injection).  For in vivo experiments, to determine whether loss of Phox2b cells led to impaired HCVR, bilateral injections with a total volume of 200 nl of PBS containing 6 ng (1.5 ng in 50 nl per injection; 2 injections per side) or 12 ng (3 ng in 50 nl per injection; two injections per side) of toxin into NTS.  Breathing was studied in conscious, freely moving mice treated with SSP-SAP and Blank-SAP.

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

Q: Your targeted toxin kits come with different controls. I’m not sure of the best way to use them; there is included unconjugated antibody, unconjugated saporin, and a control conjugate, mouse IgG-SAP. Should I use them all in the same experiment or for different purposes?

A: For mouse IgG-containing conjugates, the ideal control is Mouse IgG-SAP (Cat. # IT-18). Mouse IgG-SAP — that is, saporin conjugated to mouse IgG — that has no specific antigen for targeting is the best control. Unconjugated saporin is still considered a second good control, useful in cases where down-regulation by the antibody is a concern.

Q: What about for the peptide toxins?

A: We have produced Blank-SAP as a control for the peptide ligand toxins. Blank-SAP (Cat. #IT-21) is a peptide that has the usual common amino acids that are found in peptide neurotransmitters, but arranged in a sequence that is random and not detected in homology searches. So, it should never find an amenable receptor.  This is quite an important control; the peptide ligand toxins are often delivered directly to tissue, and there are cases in which there will be no toxicity or non-specific toxicity.

One of the best uses we have seen for Blank-SAP has been in: Bugarith K et al. Basomedial hypothalamic injections of neuropeptide Y conjugated to saporin selectively disrupt hypothalamic controls of food intake. Endocrinology 146(3):1179-1191, 2005.

As any journal reviewer will tell you, it’s very important to document the specificity, and with Blank-SAP as a control, you can definitively show that toxicity is due to proper targeting, rather than non-specific cytotoxicity. This should provide the information needed so the reviewer doesn’t have to make you go back and document specificity with further experimental work!

See a complete list of control conjugates here.

Havelin J, Imbert I, Sukhtankar D, Remeniuk B, Pelletier I, Gentry J, Okun A, Tiutan T, Porreca F, King T (2017) Mediation of movement-induced breakthrough cancer pain by IB4-binding nociceptors in rats. J Neurosci 37:5111-5122.. doi: 10.1523/JNEUROSCI.1212-16.2017

Objective: To define a novel preclinical measure of movement-induced breakthrough pain (BTP) that is observed in the presence of morphine controlling ongoing pain.

Summary: Novel compounds targeting IB4-binding nociceptors may improve pain management for cancer pain patients and other patient populations suffering from BTP that is inadequately treated by currently available medications.

Usage: To determine the effect of eliminating input from IB4-binding fibers, separate groups of rats received spinal administration of IB4-SAP or the control, Blank-SAP (3.2 mcg/20 mcl saline) followed by a 10 mcl flush of saline. Movement of an air bubble placed between drug solution and saline was used to monitor progress of the injection.

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

Ye J-H, Fu R, He W (2017) The rostromedial tegmental nucleus and alcohol addiction. Oncotarget 8:18624-18625.. doi: 10.18632/oncotarget.15822

Summary: The authors discuss their work with Dermorphin-SAP (Cat. #IT-12) and their demonstration that damage of RMTg MOR-expressing GABAergic neurons by Dermorphin-SAP increased the intake and preference for alcohol, boosted the expression and slowed down the extinction of alcohol conditioned place preference, and increased locomotion. Microinjection of DS into the RMTg substantially reduced the number of RMTg cells. Importantly, the rats that received DS injection elevated their alcohol intake and preference compared to those that received an injection of Blank-SAP (Cat. #IT-21), which did not cause neuronal damage.

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

Kelly SC, Nelson PT, Counts SE (2017) Featured Article: The locus coeruleus: a potential link between cerebrovascular and neuronal pathology in Alzheimer’s disease. Targeting Trends 18

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

Read the featured article in Targeting Trends.

See Also:

• Kelly SC et al. The locus coeruleus: a potential link between cerebrovascular and neuronal pathology in Alzheimer’s disease. Neuroscience 2016 Abstracts 786.11 / H7, 2016. Society for Neuroscience, San Diego, CA

Turnbull M, Coulson E (2017) Cholinergic basal forebrain lesion decreases neurotrophin signaling without affecting tau hyperphosphorylation in genetically susceptible mice. J Alzheimers Dis 55:1141-1154.. doi: 10.3233/JAD-160805

Summary: Alzheimer’s disease(AD) is a progressive, irreversible neurodegenerative disease that destroys memory and cognitive function. Aggregates of hyperphosphorylated tau protein are a prominent feature in the brain of patients with AD, and area major contributor to neuronal toxicity and disease progression. However, the factors that initiate the toxic cascade that results in tau hyperphosphorylation in AD are unknown. The authors investigated whether degeneration of basal forebrain cholinergic neurons (BFCNs) and/or resultant decrease in neurotrophin signaling cause aberrant tau hyperphosphorylation. Two-month-old male and female pR5 mice were infused with murine p75-SAP (Cat. #IT-16) at a concentration of 0.4 mg/ml or 0.4 mg/ml of control Rabbit IgG-SAP (Cat. #IT-35) using a 30G needle attached to a 5 ml Hamilton syringe and pump. The needle was lowered into the medial septum according to coordinates in a mouse brain atlas, and the toxin was infused at a rate of 0.4 ul/min (1.5 u total volume). The results reveal that the loss of BFCNs in pre-symptomatic pR5 tau transgenic mice results in a decrease in hippocampal brain-derived neurotrophic factor levels and reduced TrkB receptor activation. However, there was no exacerbation of the levels of phosphorylated tau or its aggregation in the hippocampus of susceptible mice. Furthermore the animals’ performance in a hippocampal-dependent learning and memory task was unaltered, and no changes in hippocampal synaptic markers were observed. This suggests that tau pathology is likely to be regulated independently of BFCN degeneration and the corresponding decrease in hippocampal neurotrophin levels, although these features may still contribute to disease etiology.

Related Products: mu p75-SAP (Cat. #IT-16), Rabbit IgG-SAP (Cat. #IT-35)

Kaushal R, Taylor B, Jamal A, Zhang L, Ma F, Donahue R, Westlund K (2016) GABA-A receptor activity in the noradrenergic locus coeruleus drives trigeminal neuropathic pain in the rat; contribution of NAα1 receptors in the medial prefrontal cortex. Neuroscience 334:148-159. doi: 10.1016/j.neuroscience.2016.08.005

Summary: The goal of this study was to investigate the role of the locus coeruleus (LC) in a rat orofacial pain model of trigeminal neuropathy induced by chronic constrictive injury of the infraorbital nerve (CCI-ION). Mechanical thresholds to von Frey filaments were tested on whisker pads to evaluate neuropathic pain behavior; pain was indicated by development of mechanical hypersensitivity. Noradrenergic (NA) neurons were lesioned with 5-mcg injections of Anti-DBH-SAP (Cat. #IT-03) into the left lateral ventricle. Mouse-IgG-SAP (Cat. #IT-18) was used as a control. After ablation of NA neurons there was a notable increase in the mechanical threshold compared to control animals. They also targeted coeruleotrigeminal NA neurons by injecting Anti-DBH-SAP into the trigeminal brainstem nuclei bilaterally in one animal and saw similar results. Injecting a GABAA receptor antagonist into the LC after injury had an inhibitory effect on nerve injury induced hypersensitivity. Injection of a NAα1 receptor antagonist, but not a NAα2 receptor antagonist, into the medial prefrontal cortex (mPFC) alleviates mechanical hypersensitivity. They conclude that GABAA-mediated activation of NA neurons during CCI-ION can facilitate hypersensitivity through NAα1 receptors in the mPFC, and that the LC is a chronic pain generator.

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

Young J (2016) Hunger, thirst, sex, and sleep: How the brain controls our passions. Rowman & Littlefield Publishers.

Summary: The author reviews what has been learned about feeding, “Other clever techniques have confirmed that leptin specifically acts upon NPY-containing neurons to depress feeding. One technique is to infuse the hypothalamus with a form of NPY that is linked to a poisonous molecule called saporin. Neurons that normally contain NPY usually release it into their surroundings and then specifically take it back up into the cell so that it is not wasted. When NPY-containing neurons are tricked into taking up NPY linked to saporin, they die but leave other adjacent neurons completely unaffected. Rats treated in this way become completely insensitive to the feeding-restraining effects of leptin because they lack NPY-containing neurons in the arcuate nucleus.”

Related Products: Blank-SAP (Cat. #IT-21), NPY-SAP (Cat. #IT-28)

See Also:

• Bugarith K et al. Basomedial hypothalamic injections of neuropeptide Y conjugated to saporin selectively disrupt hypothalamic controls of food intake. Endocrinology 146(3):1179-1191, 2005.

Kumar J, Rajkumar R, Lee L, Dawe G (2016) Nucleus incertus contributes to an anxiogenic effect of buspirone in rats: Involvement of 5-HT1A receptors. Neuropharmacology 110:1-14. doi: 10.1016/j.neuropharm.2016.07.019

Summary: The nucleus incertus (NI) is involved in stress and anxiety responses. The NI is a cluster of GABAergic neurons in the brainstem, and coexpresses CRF, 5-HT1A and D2 receptors. Buspirone is a partial agonist of 5-HT1A receptors, an antagonist of D2 receptors, and activates the NI. Buspirone is an anti-anxiety drug, but preclinical studies showed that it induces anxiety at high doses. To see if the NI is necessary for the anxiogenic effects of high doses of buspirone, rats were bilaterally injected with 86 ng of CRF-SAP (Cat. #IT-13) into the NI. Blank-SAP (Cat. #IT-21) was used as a control. NI lesioning alone had an anxiogenic effect in several anxiety screening tests compared to sham-lesioned rats, which suggests that the NI reduces anxiety physiologically. Lesioning with CRF-SAP reduced the anxiogenic effects of intra-NI injections of buspirone. No significant difference in the anxiety screening tests resulted from injecting quinpiole, a D2 agonist, which suggests that the 5HT1A receptors in the NI are involved in the anxiogenic effects of buspirone.

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

Read the featured article in Targeting Trends.

Matsuura T, Kawasaki M, Hashimoto H, Yoshimura M, Motojima Y, Saito R, Ueno H, Maruyama T, Ishikura T, Sabanai K, Mori T, Ohnishi H, Onaka T, Sakai A, Ueta Y (2016) Possible involvement of the rat hypothalamo-neurohypophysial/-spinal oxytocinergic pathways in acute nociceptive responses. J Neuroendocrinol 28(6) doi: 10.1111/jne.12396

Summary: It has been suggested that the amplification of GABAergic neurons in the inhibitory system induces the selective inhibition by Oxytocin (OXT) of excitability in the spinal cord, and the pain transmitted from the periphery to the dorsal horn of the spinal cord by this action may be attenuated at the spinal cord level. Rats were injected IT with Oxytocin-SAP (Cat. #IT-46) dissolved in saline (0.06 μg/μl), Blank-SAP (Cat. #IT-21) dissolved in saline (0.06 μg/μl), or saline. Formalin-induced acute nociception activated OXT-containing cells in both the magnocellular and parvocellular divisions of hypothalamus, and that the parvocellular division remains activated longer than the magnocellular division. Acute nociception-induced activation of the hypothalamo-neurohypophysial system caused elevation of plasma OXT levels. In addition, the OXTergic spinal pathway may be involved in pain modulation via OXTRs in the spinal cord.

Related Products: Oxytocin-SAP (Cat. #IT-46), Blank-SAP (Cat. #IT-21)