McGaughy J, Ross RS, Eichenbaum H (2008) Noradrenergic, but not cholinergic, deafferentation of prefrontal cortex impairs attentional set-shifting. Neuroscience 153:63-71. doi: 10.1016/j.neuroscience.2008.01.064
Summary: Norepinephrine and acetylcholine are involved in the mediation of attention, however, it is not yet clear whether the roles of these molecules are unique. This work utilizes a specific task shown to dissociate the roles played by the dorsolateral prefrontal cortex and the orbitofrontal cortex in primates. Rats received 5-ng infusions of anti-DBH-SAP (Cat. #IT-03) or 192-IgG-SAP (Cat. #IT-01) into each hemisphere. The type of lesion had an effect on attentional shifts and reaction to irrelevant stimuli.
Rahman W, Suzuki R, Hunt SP, Dickenson AH (2008) Selective ablation of dorsal horn NK(1) expressing cells reveals a modulation of spinal alpha2-adrenergic inhibition of dorsal horn neurones. Neuropharmacology 54:1208-1214. doi: 10.1016/j.neuropharm.2008.03.014
Summary: In this work the spinal origin of the major descending noradrenergic inhibitory pathway is examined with the help of SP-SAP (Cat. #IT-07). Rats received a 10-µl infusion of 1-mM SP-SAP (saporin, Cat. #PR-01, was used as a control) into the sub-arachnoid space terminating in the L4-5 region. Results from examining neuronal responses under the influence of the alpha2-adrenoceptor antagonist atipamezole suggest that NK1 expressing cells are involved with activity in noradrenergic pathways and descending facilitation.
One of the tests you can use to test your targeting agent for internalization is the in vitro Cytotoxicity Assay. Protocols to assist in preparing for, executing and interpreting results are now posted on our website.
There are several protocols available.
Preparing for a Cytotoxicity Assay using Secondary Conjugates. This protocol will be helpful when using our secondary antibody-saporin conjugates with your primary antibody. These include Anti-M-ZAP (Cat. #IT-30), Goat-ZAP (Cat. #IT-36), Hum-ZAP (Cat. #IT-22), Mab-ZAP (Cat. #IT-04), Rab-ZAP (Cat. #IT-05), and Rat-ZAP (Cat. #IT-26).
Preparing for a Cytotoxicity Assay using Streptavidin-ZAP. This protocol will be helpful when using our streptavidin-saporin conjugate (Streptavidin-ZAP, Cat. #IT-27) with your biotinylated targeting agent (peptide, ligand, cytokine, growth factor, antibody, etc.).
Concentration Calculation: Convert molarity to mg/ml and mg/ml to molarity. This protocol will help in determining the correct amount of material to use in your assay. There is also a link to an Online Calculator.
Cytotoxicity Assay for Targeted Toxins in vitro. This protocol includes photos of what your plates should look like during the assay process. It takes five days to complete this assay. Start on a Monday and develop on Friday. There are many factors that go into a successful cytotoxicity assay. This protocol should help you design and execute appropriately.
Preparing Cytotoxicity Data. This protocol will give an example of how to process the data from a Cytotoxicity Assay. ATS uses SOFTMax Pro software connected to a plate reader to determine the A490 value. Then we import this data into Prism software (GraphPad) to conduct further data analysis. Here is a figure generated with Prism.
We hope these protocols will be helpful to you in your research. If there are additional protocols or tutorials we can provide, please do not hesitate to ask.
This graph gives important information about how the potency of your targeted toxin. The ED50 is the Median Effective Dose (produces desired effect in 50% percent of population). The lower this number is, the more potent the targeted toxin.
Moreau PH, Cosquer B, Jeltsch H, Cassel JC, Mathis C (2008) Featured Article: Selective lesion of basal forebrain cholinergic neurons in mice with the mu p75-saporin immunotoxin: Neuroanatomy and behavior. Targeting Trends 9(2)
Wiley RG (2008) Substance P receptor-expressing dorsal horn neurons: Lessons from the targeted cytotoxin, substance P-saporin. Pain 136:7-10. doi: 10.1016/j.pain.2008.03.010
Summary: This review covers some of the more recent work utilizing SP-SAP (Cat. #IT-07) and SSP-SAP (Cat. #IT-11) in the dorsal horn. Specific answers to experimental questions are discussed, as well as some of the questions generated by the research. The potential of SP-SAP and SSP-SAP as pain therapeutics is also explored, along with potential clinical applications of other targeted toxins in pain therapy.
Joseph EK, Chen X, Bogen O, Levine JD (2008) Oxaliplatin acts on IB4-positive nociceptors to induce an oxidative stress-dependent acute painful peripheral neuropathy. J Pain 9:463-472. doi: 10.1016/j.jpain.2008.01.335
Summary: Oxaliplatin is a platinum-based chemotherapy agent. Use of this reagent produces various pathological pain states, depending on the dosage site. The authors administered 3.2-µg intrathecal injections of IB4-SAP (Cat. #IT-10), using saporin (Cat. #PR-01) as a control. Lesioning IB4-binding neurons in the dorsal horn completely prevented oxaliplatin-induced hyperalgesia, indicating that the IB4-positive nociceptor neuronal subset is crucial to this type of neuropathy.
Newman LA, McGaughy J (2008) Cholinergic deafferentation of prefrontal cortex increases sensitivity to cross-modal distractors during a sustained attention task. J Neurosci 28:2642-2650. doi: 10.1523/JNEUROSCI.5112-07.2008
Summary: The authors injected 5 ng of 192-IgG-SAP (Cat. #IT-01) into the prefrontal cortex of rats to investigate the effect of cholinergic loss on distractors to attentional demand. Where all animals experienced impaired performance in the presence of visual distractions, lesioned animals were more sensitive to auditory distractions. While these results indicate compromised top-down processing, lesioned animals showed improved performance in bottom-up processing, possibly caused by a shift in circuit dynamics after the lesion.
Moreau PH, Cosquer B, Jeltsch H, Cassel JC, Mathis C (2008) Neuroanatomical and behavioral effects of a novel version of the cholinergic immunotoxin mu p75-saporin in mice. Hippocampus 18(6):610-622. doi: 10.1002/hipo.20422
Summary: 192-IgG-SAP (Cat. #IT-01) has been used for over a decade to examine the cholinergic system in the basal forebrain of rats. Establishing the same reagent for mice has been problematic. Here the authors describe the use of a mouse-specific lesioning agent, mu p75-SAP (Cat. #IT-16). After deciding on a dosage of 0.4 µg administered in the form of bilateral intracerebroventricular injections, mice were lesioned and tested. Lesioned animals displayed increased locomotor activity, and spatial learning and memory deficits, with minimal side effects.
Pijpers A, Winkelman BH, Bronsing R, Ruigrok TJ (2008) Selective impairment of the cerebellar C1 module involved in rat hind limb control reduces step-dependent modulation of cutaneous reflexes. J Neurosci 28:2179-2189. doi: 10.1523/JNEUROSCI.4668-07.2008
Summary: The cerebellar cortex is arranged in a series of modules. Elucidation of module-specific function has been difficult because of the closely arranged structure of these modules. Here the authors lesioned the C1/C3 hindlimb module of the rat with CTB-SAP (Cat. #IT-14). Rats received 75-125 ng injections of CTB-SAP into the C1 zone of the copula pyramidis or the paramedian lobule of the right cerebellar hemisphere. C1-injected animals displayed marked diminishment of cutaneously induced reflexes with no significant impact on walking or stepping pattern.
Tait DS, Brown VJ (2008) Lesions of the basal forebrain impair reversal learning but not shifting of attentional set in rats. Behav Brain Res 187:100-108. doi: 10.1016/j.bbr.2007.08.035
Summary: The authors compared specific lesions of the basal forebrain using 192-IgG-SAP (Cat. #IT-01) with non-specific lesions generated by ibotenic acid. Rats were given 0.12 µg per 0.5 µl bilateral injections of 192-IgG-SAP. The treated animals were then tested in food reward tasks involving two-choice discriminations and reversal of stimulus-reward. Animals with specific lesions did not show impairment with any of the tasks suggesting that non-cholinergic neurons are involved in reversal learning. This work also demonstrates the similarities between monkey and rodent basal forebrain function.
