targeted-toxins

Masawaki A, Sugiyo S, Shimoda T, Sakai Y, Watanabe M, Moritani M, Yoshida A, Niwa H, Takemura M (2006) Formalin-induced pain-related responses in rat lacking neurokinin-1 receptor neurons in the trigeminal nucleus caudalis. Neuroscience 2006 Abstracts 50.8. Society for Neuroscience, Atlanta, GA.

Summary: This study examines the effect of intra cisterna magna injection of substance P (SP) conjugated to saporin (SP-Sap; 5µM, 5µl) on formalin-induced pain-related behavior (PRB; face scrubbing behavior ) and c-Fos expression in the trigeminal nucleus caudalis (SpVc). In SP-Sap-treated rats, the numbers of NK-1-immunoreactive neurons in lamina I of the SpVc decreased compared with those in saline- or blank Sap-treated rats. The mean numbers ±SEM of PRB /5 min at the first phase (0-5 min after For injection) were 58.2±19.2 in the SP-Sap-treated rats, 115.6±14.0 in the saline treated rats and 86.9±45.7 in the blank-Sap-treated rats. The numbers at the quiescent period (5-10 min) were 45.2±26.3 in the SP-Sap- treated rats, 93.6±26.5 in the saline treated rats and 69.4±16.3 in the blank-Sap-treated rats. These at the former second phase (10-50 min) were 58.1±22.3 in the SP-Sap-treated rats, 133.6±26.1 in the saline treated rats and 95.8±29.6in the blank-Sap-treated rats. These at the latter second phase (55-90 min) were 7.0±5.6 in the SP-Sap-treated rats,13.7±12.4 in the saline treated rats and 10.4±22.5 in the blank-Sap-treated rats. These results indicate that formalin-induced nociceptive responses in the SP-Sap-treated rats are reduced.

Related Products: SP-SAP (Cat. #IT-07), Blank-SAP (Cat. #IT-21)

Q: We injected anti-DBH-SAP (Cat. #IT-03) into the hypothalamus of Sprague-Dawley rats and sacrificed them 2 weeks later. We did not see any reduction in the DBH fiber staining.

When the drug arrived, we aliquoted it in 1-µl snap-cap tubes on ice, and stored them at -80°C. For injections, a 1-µl aliquot was diluted to a little over 10 µl so that we had a final concentration of 1 µg/10 µl. We administered two injections of 100 nl on each side (with 10 ng of anti-DBH-SAP) using a 0.5-µl Hamilton syringe attached to a stereotax. The needle was a 33-gauge with a blunt tip. I tried previously to use glass micropipette tips attached to a Hamilton syringe with the line filled with mineral oil, but found that the actual volume displacement was too unreliable.

A: We have not had any problems related to the stability of anti-DBH-SAP. In our work, failure to lesion is nearly always associated with a misplaced injection. From the information conveyed, I would suggest the following:

(1) It is possible that no drug was actually delivered to the brain. Two things could be done to ensure drug delivery. The first would be to add a tracer to the saporin solution that could be identified histologically. The second would be to visually monitor drug delivery using a calibrated tip. Air bubbles, pressure leaks and compression of the liquid can interfere with accurate delivery.

(2) It is possible that the anti-DBH-SAP was not delivered to the correct site, so that the expected uptake into the targeted terminals did not occur. Again, marking the site so it is clear where the injection was would help evaluate your accuracy. Establishing a reliable set of stereotaxic coordinates that work in your lab, in your rats and with your equipment and then using a dye to estimate the diffusion radius of your selected injection volume are always good ways to start. However, that being said, it should not be difficult to locate the injection site with such a large injector (33 g) – so #1 seems more likely to be the problem in the case you describe. Also, I would add that the larger the injector, the more nonspecific damage there will be. Glass capillary micropipettes are by far preferable to stainless steel cannulas in providing more reliable delivery of small volumes and in producing less nonspecific damage. Chronically implanted cannulas should be avoided, in my opinion, because gliosis at the cannula tip is apt to occur and this may alter the diffusion pattern of the injected substance, as well as interfering with lesion analysis.

(3) Try a different anesthetic. We have not tested a lot of anesthetics, but we have had problems getting a good lesion that we think are attributable to use of a ketamine/xylazine/acepromazine anesthetic cocktail. So we routinely avoid that one.

(4) I assume you are looking at fibers in the area of the injection. If not, it would be important to make sure the fibers being evaluated are associated with the same neurons innervating the terminal field at the injection site. Secondly, the 2-week wait mentioned between toxin injection and histology is critical for evaluating the lesion to assure that immunoreactive products are no longer present. Making sure that tissue processing controls are stringently adhered to so that controls and lesioned animals are run together in the same batch is also important.

(5) You might try injecting only one side and comparing terminal staining with the non-injected side in the same animal. This would not be a good idea, however, if the injection site is too close to the midline, so that both sides might be damaged from a unilateral injection.

Related: Anti-DBH-SAP (Cat. #IT-03)

Bugarith K, Dinh TT, Li AJ, Speth RC, Ritter S (2006) Featured Article: Basomedial hypothalamic injections of neuropeptide Y conjugated to saporin selectively disrupt hypothalamic controls of food intake. Targeting Trends 7(4)

Related Products: Anti-DBH-SAP (Cat. #IT-03), NPY-SAP (Cat. #IT-28), Saporin (Cat. #PR-01), Blank-SAP (Cat. #IT-21)

Read the featured article in Targeting Trends.

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.

Turner LH, Lim CE, Heinrichs SC (2007) Antisocial and seizure susceptibility phenotypes in an animal model of epilepsy are normalized by impairment of brain corticotropin-releasing factor. Epilepsy Behav 10(1):8-15. doi: 10.1016/j.yebeh.2006.08.013

Summary: There appears to be an inverse relationship between seizure susceptibility and social interaction. This work examines the role that CRF may play in this system. 2.5 µg of CRF-SAP (Cat. #IT-13) was administered to the lateral ventricle of rats, and the lesioned animals were assessed in terms of social investigation times as well as handling-induced seizures. The results support the involvement of CRF systems in facilitating evoked seizures and suppression of social activity.

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

van der Staay FJ, Bouger P, Lehmann O, Lazarus C, Cosquer B, Koenig J, Stump V, Cassel JC (2006) Long-term effects of immunotoxic cholinergic lesions in the septum on acquisition of the cone-field task and noncognitive measures in rats. Hippocampus 16(12):1061-1079. doi: 10.1002/hipo.20229

Summary: 192-Saporin (Cat. #IT-01) has been used to make extremely specific lesions in the septohippocampal cholinergic system of the brain. The specificity of these lesions is allowing researchers to more accurately map the involvement of the septohippocampal cholinergic system in spatial learning and memory. Here, rats received 0.8 µg of 192-Saporin in the medial septum and the vertical limb of diagonal band of Broca. Lesioned animals only exhibited deficits in attentional learning.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Hawkes C, Kabogo D, Amritraj A, Kar S (2006) Up-regulation of cation-independent mannose 6-phosphate receptor and endosomal-lysosomal markers in surviving neurons after 192-IgG-saporin administrations into the adult rat brain. Am J Pathol 169(4):1140-1154. doi: 10.2353/ajpath.2006.051208

Summary: The cation-independent mannose 6-phosphate receptor (CI-MPR) plays a major role in the endosomal-lysosomal (EL) system. One of the tasks carried out by the EL system is clearance of abnormal proteins after injury. By administering 2.0 µg bilateral injections of 192-Saporin (Cat. #IT-01) to rats, the researchers were able to increase CI-MPR expression levels, as well as other EL markers in response to the lesion. The upregulation of EL components suggests that the EL system may be able to repair neuronal abnormalities induced by injury.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Marcos B, Gil-Bea FJ, Hirst WD, Garcia-Alloza M, Ramirez MJ (2006) Lack of localization of 5-HT6 receptors on cholinergic neurons: implication of multiple neurotransmitter systems in 5-HT6 receptor-mediated acetylcholine release. Eur J Neurosci 24(5):1299-1306. doi: 10.1111/j.1460-9568.2006.05003.x

Summary: The authors investigated a potential link between 5-HT6 receptors, cholinergic activity, and learning. After 0.067 µg of 192-Saporin (Cat. #IT-01) was injected into each hemisphere of the nucleus basalis magnocellularis in the basal forebrain of rats, 5-HT6 receptor mRNA and protein expression were measured. Results demonstrate the involvement of multiple neurotransmitter systems in neurochemical actions following 5-HT6 receptor blockade.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Castro AR, Pinto M, Lima D, Tavares I (2006) Secondary hyperalgesia in the monoarthritic rat is mediated by GABA(B) and NK1 receptors of spinal dorsal horn neurons: A behavior and c-fos study. Neuroscience 141(4):2087-2095. doi: 10.1016/j.neuroscience.2006.05.048

Summary: Hallmarks of secondary hyperalgesia in a rat model of monoarthritic pain are: decreased activation of GABA(B) neurons, and increased activation of NK1r neurons. Using 10-µl injections of 1-µM SP-SAP (Cat. #IT-07) into T(13)-L(1) the workers looked at the role of each receptor. Pain thresholds increased after treatment with SP-SAP or baclofen, a selective GABA(B) receptor agonist. Fos immunoreactivity was also decreased in treated animals, indicating that both GABA(B) and NK1r are involved in secondary hyperalgesia.

Related Products: SP-SAP (Cat. #IT-07)

Scattoni ML, Adriani W, Calamandrei G, Laviola G, Ricceri L (2006) Long-term effects of neonatal basal forebrain cholinergic lesions on radial maze learning and impulsivity in rats. Behav Pharmacol 17(5-6):517-524. doi: 10.1097/00008877-200609000-00018

Summary: Work in the last decade has focused on clarifying the role of cholinergic dysfunction in Alzheimer’s disease. 7 day-old rats received 0.21 µg of 192-Saporin (Cat. #IT-01) administered to the third ventricle, and were tested at 5 months of age in delay tolerance and a radial maze. Test results suggest that prolonged basal forebrain cholinergic hypofunction is detectable only when using highly complex tasks.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Rygh LJ, Suzuki R, Rahman W, Wong Y, Vonsy JL, Sandhu H, Webber M, Hunt S, Dickenson AH (2006) Local and descending circuits regulate long-term potentiation and zif268 expression in spinal neurons. Eur J Neurosci 24(3):761-772. doi: 10.1111/j.1460-9568.2006.04968.x

Summary: Long-term potentiation (LTP) has been shown to occur in sensory areas of the spinal cord. This modification of synaptic strength may be one of the mechanisms by which acute pain is transformed into chronic pain. 10 µl of 1-µM SP-SAP (Cat. #IT-07) or control saporin (Cat. #PR-01) was injected into the subarachnoid space (L4-L5) of rats. Using electrophysiological recording, immunohistochemistry, behavioral assessment, and antisense experiments, the authors demonstrate that dorsal horn neuron generation of LTP may transform acute pain into chronic pain.

Related Products: SP-SAP (Cat. #IT-07), Saporin (Cat. #PR-01)