targeted-toxins

Xu, WZhu JPQ, Angulo JA (2005) Ablation of NK-1 receptor-expressing interneurons prevents methamphetamine-induced apoptosis but not dopamine terminal toxicity in the striatum of mice. Neuroscience 2005 Abstracts 337.9. Society for Neuroscience, Washington, DC.

Summary: Pharmacological evidence from our laboratory demonstrates that the neurokinin-1 (NK-1) receptor mediates methamphetamine (METH)-induced toxicity of the dopamine terminals and the apoptosis of some striatal neurons. We have shown that systemic administration of the NK-1 receptor antagonist, WIN 51,708, prior to METH exposure, can protect the striatum from METH-induced damage at pre- and post-synaptic sites. To further assess the role of the NK-1 receptor on METH-induced striatal neural damage, NK-1 receptor-expressing interneurons were selectively ablated by means of intrastriatal injections of [Sar9,Met(O2)11]substance P conjugated to the ribosomal-inactivating cytotoxin saporin (SSP-SAP). TUNEL-labeling showed that ablation of striatal neurons that express NK-1 receptors provided protection against METH-induced apoptosis of some striatal neurons. However, ablation of NK-1 receptor-expressing interneurons did not provide protection against METH-induced depletion of tyrosine hydroxylase, a reliable marker of the dopamine terminals of the strtiatum. These results suggest that METH-induced apoptosis and dopamine terminal toxicity occur via distinct mechanisms in the mouse striatum.

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

Gerashchenko D, Murillo-Rodriguez E, Blanco-Centurion C, Lin L, Nishino S, Mignot E, Shiromani PJ (2005) Adenosine levels do not increase with 6 h waking in rats with lesions of the lateral hypothalamus. Neuroscience 2005 Abstracts 63.9. Society for Neuroscience, Washington, DC.

Summary: The hypocretin neurons in the lateral hypothalamus (LH) have been implicated in wakefulness, but it is not clear which projection is responsible for the arousal. One possibility is that the LH neurons induce wakefulness by driving the basal forebrain (BF) wake-active neurons (Gerashchenko and Shiromani, Cellular & Molec Neurosci, 29: 41, 2004). Here we measure adenosine (AD) levels in the BF as a marker of arousal and test the LH-BF circuit in Sprague-Dawley rats with lesions of the LH induced by hypocretin-2-saporin. 64 days after lesions the rats were kept awake (gentle handling) for six hours (ZT 3-9) and microdialysis samples (5ul) were collected hourly for 9 hours (24h after probe stabilization). AD levels were assessed using HPLC. Hypocretin-saporin ablated 95% of the hypocretin neurons and reduced CSF hypocretin levels (-75% versus control). AD levels increased with 6h waking in saline control rats (n=9), consistent with previous studies in cats (Strecker et al., Behav Brain Res 115: 183, 2000) and rats (Murillo-Rodriguez et al., Neuroscience 123: 361, 2004). However, in rats with LH lesions (n=5) such an increase with waking did not occur. Sleep drive was measured by conducting a rodent version of a multiple sleep latency test (MSLT). In this test, conducted over 10h (from ZT2-ZT12) the rats were kept awake for 20min and then allowed 20min to sleep. The lesioned rats had more sleep during the 20min sleep periods indicating a higher sleep drive. These results suggest that in narcolepsy when the HCRT LH neurons die, there is a loss of stimulation of the wake-active BF neurons and the decline in this pathway may be the cause of the increased sleep attacks. Supported by VA Medical Research and NIH

Related Products: Orexin-B-SAP (Cat. #IT-20)

Gerashchenko D, Blanco-Centurion CA, Miller JD, Shiromani PJ (2006) Insomnia following hypocretin2-saporin lesions of the substantia nigra. Neuroscience 137(1):29-36. doi: 10.1016/j.neuroscience.2005.08.088

Objective: To investigate which regions of major arousal areas might be responsible for the changes in sleep-wake architecture

Summary: It is known that orexin (also known as hypocretin) is involved in waking. The results suggest that motor activity is under inhibitory control of the substantia nigra.

Usage: Bilateral injection of Orexin-SAP (92 and 184 ng/ml, 0.25 ml in the ventral tegmental area and 0.5 ml in the substantia nigra) of rats induced insomnia, as well as hyperactivity and stereotypic movements.

Related Products: Orexin-B-SAP (Cat. #IT-20)

Mattsson A, Lindqvist E, Ogren SO, Olson L (2005) Increased phencyclidine-induced hyperactivity following cortical cholinergic denervation. Neuroreport 16(16):1815-1819. doi: 10.1097/01.wnr.0000185018.29316.87

Summary: A potential contribution to schizophrenia is altered cholinergic function. The authors investigated how lesioning cholinergic corticopetal projections might affect glutaminergic activity. Rats were injected with 0.134 µg of 192-IgG-SAP (Cat. #IT-01) into the nucleus basalis magnocellularis. The authors found that cholinergic lesioning of the neocortex led to enhanced sensitivity to phencyclidine, which has been shown to induce clinical symptoms similar to those of schizophrenia. These data suggest that glutaminergic dysfunction may be relevant to schizophrenia pathophysiology.

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

Q: We just completed surgeries where we implanted third ventricular cannulas and temporary bilatera cannulas directed into the nucleus tractus solitarius in the brainstem of animals. We injected either the Blank-SAP control toxin (Cat. #IT-21) or the experimental material Oxytocin-SAP into the bilateral NTS cannulae over a 30-second period. However, within the next week — two weeks post-surgery, we lost 13 of the 19 animals treated; they appeared not to be able to groom properly and lost over 20% of their body weight. This was apparent in both the Blank-SAP and the Oxytocin-SAP groups. We gave a dose of 40 ng/300 nl for each of the reagents. This dose was determined based on a published article using another of ATS’s targeted toxins. I’m very surprised by my results. Can you offer any explanation/advice?

A: This is a particularly disturbing result; it appears that a dose was chosen by comparison to one used with another targeted toxin. Although this can be a good approximating tool to begin a dose-ranging study, it usually doesn’t take into account the tissue, system, target molecule — so many parameters that are important to determining the proper dosage. The literature is quite extensive on targeted toxins, and so there may be a comparable starting dose that has been published. Let’s use, for example, 4 mg. Reduce that amount by 20% quantities (4, 3.2, 2.4) and test in a small number of animals to determine a value that is safe and effective. If no trouble is seen at the highest dose, and the effect is minimal, that would indicate a higher dose may be acceptable. You can then test doses in 20% increased increments (4.8, 5.6, 6.4). The effects you see in your animals should only be reflective of the particular cell type you are eliminating. In the case of control reagents, such as Blank-SAP, no cell type is being targeted, so if you are seeing any kind of result, then you are certainly over-dosing.

Q: Is there some kind of formula that one can use that will help determine a starting point for establishing a range of doses to test in animals prior to initiating a study? For example, if the targeted toxin is administered intravenously, does it take more or less material than when administered directly into tissue?

A: Start with a few animals and do dose-ranging as discussed in the previous question. The various modes of application are really too wide to discuss in any detail here, but I, a biochemist by training, always like the approach of thinking about what sort of concentration will be needed to have a cytotoxic effect. Generally, these molecules have an ED50 in the nanomolar to picomolar range. Obviously if you inject systemically, the material from the first becomes greatly diluted, relative to an injection directly into tissue, and so you’ll need a lot more. If you inject directly into tissue the local concentration can be quite high.

See: Targeted Toxins, Control Conjugates

Thinschmidt JS, Frazier CJ, King MA, Meyer EM, Papke RL (2005) Septal innervation regulates the function of alpha7 nicotinic receptors in CA1 hippocampal interneurons. Exp Neurol 195(2):342-352. doi: 10.1016/j.expneurol.2005.05.006

Summary: The authors examined whether hippocampal innervation by medial septum/diagonal band of Broca projections is necessary for normal a7 receptor function. 1 µg of 192-Saporin (Cat. #IT-01) was injected into the medial septum of rats. Various methods, including whole-cell patch clamping and immunohistochemistry, were used to evaluate the effects of these lesions. Lesioning with 192-Saporin did not affect a7 receptor currents, indicating that cholinergic neurons are not linked to a7 function.

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

Pang K, Smith H (2005) Featured Article: Effects of intraseptal orexin-saporin on spatial memory. Targeting Trends 6(4)

Related Products: Orexin-B-SAP (Cat. #IT-20)

Read the featured article in Targeting Trends.

See Also:

• Smith HR et al. Orexin-saporin lesions of the medial septum impair spatial memory. Neuroscience 132(2):261-271, 2005.

Suzuki R, Rahman W, Rygh LJ, Webber M, Hunt SP, Dickenson AH (2005) Spinal-supraspinal serotonergic circuits regulating neuropathic pain and its treatment with gabapentin. Pain 117(3):292-303. doi: 10.1016/j.pain.2005.06.015

Summary: The anticonvulsant, gabapentin, is thought to modulate calcium channel function. In animals, it also affects abnormal pain function. 10 µl of 1 µM SP-SAP (Cat. #IT-07) was injected into the subarachnoid space of rats. It was found that the effects of gabapentin were blocked when NK-1r expressing neurons in the dorsal horn were eliminated. The results suggest that not only is the NK-1r pathway a determinant of neuronal and behavioral manifestations of neuropathy, it is also involved in the action of gabapentin.

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

Sherren N, Pappas BA (2005) Selective acetylcholine and dopamine lesions in neonatal rats produce distinct patterns of cortical dendritic atrophy in adulthood. Neuroscience 136(2):445-456. doi: 10.1016/j.neuroscience.2005.08.053

Summary: In this work the authors examined lesions of acetylcholine afferents in 7-day-old rat pups, and the effect on dendritic development. 600 ng of 192-IgG-SAP (Cat. #IT-01) were administered to the ventricles of test animals. Various morphological changes in the retrosplenial cortex were observed, including smaller apical tufts and fewer basilar dendritic branches in layer V medial prefrontal cells. The data demonstrate that ascending acetylcholine afferents are very important in the development of cortical cytoarchitecture.

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

Schechter LE, Smith DL, Rosenzweig-Lipson S, Sukoff SJ, Dawson LA, Marquis K, Jones D, Piesla M, Andree T, Nawoschik S, Harder JA, Womack MD, Buccafusco J, Terry AV, Hoebel B, Rada P, Kelly M, Abou-Gharbia M, Barrett JE, Childers W (2005) Lecozotan (SRA-333): a selective serotonin 1A receptor antagonist that enhances the stimulated release of glutamate and acetylcholine in the hippocampus and possesses cognitive-enhancing properties. J Pharmacol Exp Ther 314(3):1274-1289. doi: 10.1124/jpet.105.086363

Related Products: ME20.4-SAP (Cat. #IT-15)