targeted-toxins

Furlong TM, Carrive P (2005) Hypocretin/orexin neurons and the perifornical hypothalamus play a more important role in contextual fear than in restraint stress. Neuroscience 2005 Abstracts 304.11. Society for Neuroscience, Washington, DC.

Summary: We investigated the role of the neuropeptide hypocretin (Hcrt; also known as orexin) in two different types of stress: conditioned fear to context and restraint stress. For contextual fear, male Wistar rats were tested by re-exposure to a chamber where electric footshocks had previously been administered. For restraint stress, the rats were restrained in tight Plexiglas tubes. In the first study, lesions of the perifornical region of the hypothalamus (PeF; where Hcrt neurons are located) were made with a Hcrt-saporin toxin prior to testing. The cardiovascular response was measured using radio-telemetry. The pressor and tachycardic responses to the context were reduced by 77% and 74% respectively, compared to an intact group (p<0.001, for both comparisons). The lesioned group also displayed significant reductions in freezing (by 67%) and ultrasonic vocalisations (by 74%). In contrast, the cardiovascular response to restraint stress did not differ between the two groups (p>0.5). In the second study, two hours after the tests, the rats were euthanased (200 mg/kg sodium pentobarbitone, i.p) and their brains removed and processed for double immunohistochemical detection of Hcrt and Fos. There was a higher percentage of Hcrt neurons double labeled with Fos after contextual fear (17%) than after restraint stress (6%), which indicates that more Hcrt neurons were active during contextual fear (p=0.024). These studies suggest that the PeF region and Hcrt neurons play a more important role in contextual fear than in restraint stress.

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

Kalinchuk AV, Stenberg D, Rosenberg PA, Porkka-Heiskanen T (2005) On the role of the basal forebrain cholinergic neurons in regulation of recovery sleep. Neuroscience 2005 Abstracts 308.8. Society for Neuroscience, Washington, DC.

Summary: Basal forebrain (BF) is an critical site in regulation of propensity for sleep (Porkka-Heiskanen et al., 2000; Kalinchuk et al., 2003).We have recently shown that development of recovery sleep after sleep deprivation (SD) might be mediated by release of nitric oxide (NO) in the BF during SD (Kalinchuk et al., 2003; 2004). To further elucidate the role of BF neuronal mechanisms in regulation of NO-mediated recovery sleep we selectively destroyed BF cholinergic neurons and compared effects of SD and pharmacologically increased NO level (induced by NO donor infusion) to the effects observed in intact animals. Male rats were implanted with electrodes for EEG/EMG recording and guide cannulae for microdialysis probes targeting the BF. The experimental schedule for each rat included: recording of natural sleep-waking cycle; SD for 3h; infusion of NO donor (DETA NONOate) for 3h. In separate group of rats immunotoxin 192 IgG-saporin was injected into the BF and the same experimental schedule was performed. After the end of experiments brains were taken for validation of the quality of cholinergic cells lesion and/or probes locations. In all intact rats SD induced significant increase in subsequent NREM sleep by 30.2±3%. Infusion of DETA NONOate into the BF increased sleep by 35.2±4%. Relative delta power was increased by 44.4±8% and 44.1±19%, respectively. After lesion of the BF cholinergic cells recovery NREM sleep after SD was significantly attenuated (9.5±3% increase as compared with baseline). Effect of DETA NONOate infusion was also inhibited (3.1±4% decrease as compared with baseline). Increases in relative delta power were totally abolished. Our data allow to conclude that cholinergic neurons in the BF play an important role in regulation of SD-induced recovery sleep which is mediated by release of NO.

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

Coolen LM, Amstalden KAZ, Allard J (2005) Involvement of lumbar spinothalamic cells in relay of sensory cues related to vaginocervical stimulation in female rats. Neuroscience 2005 Abstracts 321.13. Society for Neuroscience, Washington, DC.

Summary: The rat lumbar spinal cord contains a population of galanin containing spinothalamic (LSt) cells, which in male rats play a pivotal role in the control of ejaculation. However, the function of LSt cells in female rats is unknown. LSt cells project to the parvocellular subparafascicular thalamic nucleus (SPFp), where Fos is expressed following vaginocervical stimulation (VCS). Hence, we hypothesize that LSt cells are involved in relay of sensory cues related to VCS to the brain. To test this hypothesis, the effects of LSt lesions were investigated on two parameters that are dependent on relay of VCS-related cues: pseudopregancy and Fos expression in the SPFp. Adult female Sprague Dawley rats received infusions of substance P-conjugated (SSP-SAP) or unconjugated saporin (control) in lumbar levels 3-4. Females were investigated for: estrous cyclicity, expression of sexual behavior, and induction of pseudopregnancy by mating with vasectomized male partners including 5, 10, or 15 intromissions. For the final test, females received 10 or 15 intromissions from male partners, were perfused one hour later, and brains and spinal cords were examined for Fos expression and LSt lesions. SSP-SAP treatment resulted in severe reduction of LSt cells, but did not affect cyclicity or expression of sexual behavior, suggesting that LSt cells are not involved in regulation of these functions. In contrast, LSt lesions significantly reduced mating-induced Fos expression in the SPFp, supporting the involvement of LSt cells in relay of VCS-related sensory information to the SPFp. However, LSt lesions did not prevent mating-induced pseudopregancy and only partly blocked mating-induced neural activation in SPFp, indicating the possible involvement of alternate pathways. Alternatively, the few remaining LSt cells in lesioned females are sufficient for induction of Fos in SPFp and pseudopregnancy.

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

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.