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Targeting Trends Newsletter 01q4

Below are links to view the quarterly newsletter Targeting Trends. If you would like to be added to the mailing list, please complete the information on our contact page.

Newsletter Highlights

  • Surfers with a Cause (page 2)
  • Time Course of Action (page 5)
  • Orexin-SAP (page 7)

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Immunolesioning Hippocampal Inhibitory Interneurons

Dr. Robert Sloviter, University of Arizona, contributes this issue’s article from the laboratories of ATS customers. Dr. Sloviter summarizes his research with SSP- saporin, which he and his graduate student Jennifer Martin used to examine the role of inhibitory neurons in maintaining normal network excitability.

Product information related to cover article: SSP-SAP (Cat. #IT-11)

ATS Receives $900,000 in NIH Funding

Recent Scientific References

Targeting Talk: Time Course of Targeted Toxins

  • How long does it take to see the cell death occurring from the use of targeted toxins using saporin? Is there a time course of hours or days?
  • Will this time course be the same regardless of the targeted toxin used or the method of administration?
  • What are some assays/methods to use to be able to graphically demonstrate cell death?

Targeting Ticklers (Jokes)

Targeting Teaser Winners from last issue

Targeting Tools: Featured Products

Targeting Technology Tutorial

Targeting Teaser (Jumble)

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Targeting Tools: Orexin-SAP

Work on hypocretin, or orexin, as it is also known, is some of the most interesting in all of biology these days. When first characterized a mere three years ago by two groups, it was thought to be involved in feeding (hence the name orexin from the Greek orexis meaning appetite). But two articles exploded onto the scene in 1999 and indicated a fundamental role in sleep for the hypocretin/orexin receptor axes. Chemelli et al. reported that knocking out the orexin gene produced mice that suffered from narcolepsy.1 In a prominent model of narcolepsy, some dobermans are found to lapse into a cataplectic state while doing things like running after a ball thrown by their master. Lin et al. used positional cloning to determine that narcolepsy in this astounding model is due to a mutation in the hypocretin/orexin receptor gene.2 These reports clearly put orexin/hypocretin into the narcolepsy mix. Thannickal et al. then reported that missing neurons were the problem in the human disease; that hypocretin/orexin neurons were depleted.3 These new discoveries make the sleep field into something to watch for incredible new results.

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Orexin-SAP (50 ng/0.5 μl) delivered to the lateral hypothalamus kills the orexin/hypocretin receptor-positive neurons. The asterisk marks the site of injection. VMH=ventromedial hypothalamus; F=fornix; 3v=third ventricle

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Loss of histaminergic neurons in the tuberomammillary nucleus (TMN) after unilateral injection of orexin-SAP (50 ng/0.5 μl). The TMN neurons contain the orexin/hypocretin receptor and are heavily innervated by hypocretin fibers.3 HCRT2-SAP (hypocretin 2-Saporin) = orexin-SAP

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Because of the new, exciting work on this system, Peter Shiromani approached Advanced Targeting Systems to request that we construct a molecule that would remove orexin/hypocretin receptor-expressing neurons. He reports on the properties of this targeted cytotoxin in recent issues of Brain Research and the Journal of Neuroscience.4,5 It turns out that hypocretin-2/orexin B-SAP (Orexin- SAP, Cat. #IT-20) is able to eliminate specifically orexin receptor-expressing neurons. Interestingly, these neurons also contain orexin, probably for some sort of feedback loop mechanism, so that, like in the human disease, orexin neurons are lost also. The rats treated with this material injected in the lateral hypothalamus have narcoleptic symptoms and will fall directly into REM sleep while happily munching on rat chow (videos available at www.jneurosci.org).5

This new tool binds best to the hypocretin-2/orexin 2 receptor, but still has affinity for hypocretin-1/orexin 1 receptor. These are G protein-coupled receptors, and, as seen with the other peptide toxins from ATS that target these receptors—SP- SAP (Cat. #IT-07), SSP-SAP (Cat. #IT-11), dermorphin-SAP (Cat. #IT-12) and corticotropin releasing factor- SAP (Cat. #IT-13)—upon ligand binding, the complex is rapidly internalized. The internalized saporin then inhibits protein synthesis by ribosomal inactivation and the target cell dies. The figures provided here by Dr. Shiromani show the remarkable specificity and potency of orexin-SAP. It provides a simple model for narcolepsy and is destined to become an invaluable tool for the study of the role of hypocretin/orexin receptor- expressing neurons wherever they may occur.

References

  1. Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C, Richardson JA, Williams SC, Xiong Y, Kisanuki Y, Fitch TE, Nakazato M, Hammer RE, Saper CB, Yanagisawa M (1999) Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98:437-451.
  2. Lin L, Faraco J, Li R, Kadotani H, Rogers W, Lin X, Qiu X, de Jong PJ, Nishino S, Mignot E (1999) The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 98:365-376.
  3. Thannickal TC, Moore RY, Nienhuis R, Ramanathan L, Gulyani S, Aldrich M, Cornford M, Siegel JM (2000) Reduced number of hypocretin neurons in human narcolepsy. Neuron 27(3):469-474.
  4. Gerashchenko D, Salin-Pascual R, Shiromani PJ (2001) Effects of hypocretin-saporin injections into the medial septum on sleep and hippocampal theta. Brain Res 913:106-115.
  5. Gerashchenko D, Kohls MD, Greco M, Waleh NS, Salin-Pascual R, Kilduff TS, Lappi DA, Shiromani PJ (2001) Hypocretin-2-saporin lesions of the lateral hypothalamus produce narcoleptic-like sleep behavior in the rat. J Neurosci 21(18):7273-7283.
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Targeting Talk: Time Course of Targeted Toxins

How long does it take to see the cell death occurring from the use of targeted toxins using saporin? Is there a time course of hours or days?

Details of the time course of early events have not been extensively studied. After ricin injections into the cervical vagus nerve, the proximal nerve becomes unresponsive to electrical stimulation between 36 and 48 hours. After septal injection of 192-Saporin (192-IgG-SAP, Cat. #IT-01), hippocampal theta rhythm begins to diminish on the third postoperative day and reaches a minimum by 7 days which is maintained indefinitely. Anatomical disintegration is complete within 10-14 days after injection of most toxins.

Will this time course be the same regardless of the targeted toxin used or the method of administration?

Presumably, injection of toxin into the vicinity of target cell bodies and dendrites should produce effects somewhat sooner than toxin injections into axonal terminal fields where retrograde axonal transport must first deliver toxin to the perikarya. In the cervical vagus, based on transport times for ricin and inhibition of toxin transport by vincristine, we concluded that fast axonal transport is involved. Colchicine coinjected intraventricularly with 192-Saporin (192-IgG-SAP, Cat. #IT-01) prevents destruction of cholinergic basal forebrain neurons suggesting that fast axonal transport also is involved with i.c.v. toxin injections. Consequently, the delay introduced by injecting toxin into axon terminal fields is usually a few hours at most.

What are some assays/methods to use to be able to graphically demonstrate cell death?

Toxin-induced cell death can be observed and documented with a variety of techniques. Often the easiest is simple Nissl staining because all of the RIP toxins (ricin, volkensin, saporin) produce profound chromatolysis that is readily apparent in Nissl stains (i.e. cresyl violet).

Electron microscopy can demonstrate details of neuron degeneration including loss of axon terminals at a distance from the cell body which can be useful in anatomic tracing studies.

Typically, target neurons express proteins that can be visualized with immunocytochemical techniques. Thus, immunofluorescence or peroxidase immunohistochemistry can be useful in detecting loss of staining for target molecules and co-expressed molecules in the neurons being targeted. The use of multiple markers is recommended to insure that cell loss occurred rather than down regulation of marker expression.

See also: Targeted Toxins Catalog

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Targeting Topics 01q4

Focal inhibitory interneuron loss and principal cell hyperexcitability in the rat hippocampus after microinjection of a neurotoxic conjugate of saporin and a peptidase-resistant analog of Substance P.

Martin JL, Sloviter RS.

J Comp Neurol 436(2):127-152, 2001. PMID: 11438920

The authors used SSP-SAP (0.4 ng/10 nl; Cat. #IT-11). See Cover Story.

Selective cholinergic denervation inhibits expression of long-term potentiation in the adult but not infant rat hippocampus.

Motooka Y, Kondoh T, Nomura T, Tamaki N, Tozaki H, Kanno T, Nishizaki T.

Brain Res Dev Brain Res 129(1):119-123, 2001. PMID: 11454420

The authors studied the possible role of cholinergic systems in long-term potentiation (LTP), which is one of the most intensively studied models of learning and memory. 192-Saporin (4.2 μg/5 μl, Cat. #IT-01) injections were made in both infant and adult rats and the probability of LTP development was studied in hippocampal slices from animals treated 2 weeks or 2 months before. Cholinergic denervation by 192- Saporin did not affect LTP expression in the infant brain, however, the results strongly suggest that cholinergic systems in the adult brain participate in an LTP pathway.

Effects of hypocretin-saporin injections into the medial septum on sleep and hippocampal theta.

Gerashchenko D, Salin-Pascual R, Shiromani PJ.

Brain Res 913(1):106-115, 2001. PMID: 11532254

Hypocretin, also known as orexin, neurons are located only in the lateral hypothalamus. Recently, the loss of these neurons was shown to be associated with narcolepsy. The authors used orexin- SAP (100 ng/0.5 μl; Cat. #IT-20) to eliminate parvalbumin and cholinergic neurons (orexin B receptor-expressing) in the rat medial septum. They used 192- Saporin (1 μg/ 1 μl; Cat. #IT-01) to contrast the effect and eliminate only cholinergic neurons (NGF/p75 receptor- expressing). Hippocampal theta activity was completely eliminated in orexin- SAP treated rats by day 12, suggesting that orexin neurons influence cognitive processes critical for survival.

Transneuronal tracing from sympathectomized lumbar epaxial muscle in female rats.

Daniels D, Miselis RR, Flanagan-Cato LM.

J Neurobiol 48(4):278-290, 2001. PMID: 11500841

The authors use pseudorabies virus (PRV) to study central neural networks such as the one controlling the lordosis reflex (increased curvature of the spine). To aid in the separation of the sympathetic nervous system and higher order systems, rats were treated with lumbar injections of anti-DBH-SAP (156 ng to 5 μg; Cat. #IT-03), then labeled with PRV. PRV labeling in the brain was absent in areas associated with vasomotor tone, but persisted in areas implicated in control of the lordosis response.

Hippocampal sympathetic ingrowth occurs following 192-IgG-Saporin administration.

Harrell LE, Parsons D, Kolasa K.

Brain Res 911(2):158-162, 2001. PMID: 11511384

Electrolytic lesions of the medial septal region in rats cause peripheral sympathetic fibers from the superior cervical ganglia to grow into the cholinergically-denervated areas of the hippocampus. This lesioning method is non-specific and disrupts several other cell types in the area of the lesion. The authors infused 192-Saporin (1 μg/10 μl saline into medial septum; Cat. #IT-01) to eliminate only the cholinergic neurons, leaving other cell types intact. Hippocampal sympathetic ingrowth still occurs when only the cholinergic neurons are eliminated, indicating that this occurrence is in response to the loss of cholinergic projections from the medial septum.

Selective antibody-induced cholinergic cell and synapse loss produce sustained hippocampal and cortical hypometabolism with correlated cognitive deficits.

Browne SE, Lin L, Mattsson A, Georgievska B, Isacson O.

Exp Neurol 170(1):36-47, 2001. PMID: 11421582

The authors used 192-Saporin (two 2.5- μg bilateral injections of 1 μg/μl; Cat. #IT-01) to eliminate cholinergic neurons in the rat, then measured cerebral rates of glucose utilization. The findings show sustained reduction in glucose utilization in the brain regions showing loss of cholinergic neurons, specifically the frontal cortical and hippocampal regions. These same animals demonstrated impaired performance in a Morris water maze. The results reinforce the theory that cholinergic systems influence metabolism and cognition in the cortex and hippocampus.

Selective loss of cholinergic neurons projecting to the olfactory system increases perceptual generalization between similar, but not dissimilar, odorants.

Linster C, Garcia PA, Hasselmo ME, Baxter MG.

Behav Neurosci 115(4):826-833, 2001. PMID: 11508721

Selective cholinergic lesioning of the basal forebrain has been linked to attentional and cognitive deficits. 192-Saporin (Cat. #IT-01) was administered to the horizontal limb of the diagonal band of Broca (0.3 μl at 0.175 μg/μl in each hemisphere) destroying projections to the olfactory bulb and cortex. The results demonstrate cholinergic lesions affect the perceptual qualities of odors, and may possibly represent a general mechanism for cholinergic effects on information processing.

Contribution of the cholinergic basal forebrain to proactive interference from stored odor memories during associative learning in rats.

De Rosa E, Hasselmo ME, Baxter MG.

Behav Neurosci 115(2):314-327, 2001. PMID: 11345957

Proactive interference (PI) is the damaging effect of previously learned information on the acquisition of new, related information. Human patients with basal forebrain (BF) damage due to aneurysms are sensitive to PI. The authors administered 192-Saporin (Cat. #IT-01) to the horizontal limb of the diagonal band of Broca (two 0.2-μl injections of 0.175 μg/μl in each hemisphere) in rats and evaluated performance in an olfactory discrimination task. The treated rats had more difficulty acquiring an overlapping odor pair when muscarinic receptors were blocked by scopalomine. These results indicate that cholinergic neurons have a role in the modulation of PI in associative learning.

It’s enough to raise your blood pressure!

Deuchars J, Deuchars S.

Trends Neurosci 24(4):200, 2001. PMID: 11249993

The authors review studies completed by Schreihofer and Guyenet using anti- DBH-SAP (Cat. #IT-03) to eliminate C1 adrenergic neurons. The results show that, although C1 neurons play a role in some sympathoexcitatory responses, they are probably not responsible for maintaining sympathetic tone.

Effects of selective immunotoxic lesions on learning and memory.

Baxter MG.

Methods Mol Biol 166:249-265, 2001. PMID: 11217371

Dr. Baxter presents a brief review of studies using immunotoxins to study learning and memory. In particular, this chapter (from the book entitled “Immunotoxin Methods and Protocols”) focuses on the use of 192-Saporin (Cat. #IT-01) for elimination of basal forebrain cholinergic neurons and cerebellar Purkinje cells.

Distribution and co-localization of choline acetyltransferase and p75 neurotrophin receptors in the sheep basal forebrain: implications for the use of a specific cholinergic immunotoxin.

Ferreira G, Meurisse M, Tillet Y, Levy F.

Neuroscience 104(2):419-439, 2001. PMID: 11377845

ME20.4 is a monoclonal antibody (Cat. #AB-N07) that has been shown to bind the p75 receptor in rabbit, sheep, dog, cat, raccoon, pig, and several primate species. Ferreira et al. investigate ME20.4-SAP (bilateral, 150 μl per ventricle, 50-150 μg total; Cat. #IT-15) use in sheep to assess distribution and localization of p75. The authors demonstrate 80-95% loss of basal forebrain cholinergic neurons and acetylcholinesterase-positive fibers in the hippocampus, olfactory bulb, and entorhinal cortex.

Hypocretin-2-saporin lesions of the lateral hypothalamus produce narcoleptic-like sleep behavior in the rat.

Gerashchenko D, Kohls MD, Greco M, Waleh NS, Salin-Pascual R, Kilduff TS, Lappi DA, Shiromani PJ.

J Neurosci 21(18):7273-7283, 2001. PMID: 11549737

Orexin (also knows as hypocretin) peptides are produced exclusively by neurons in the lateral hypothalamus, however non-specific lesioning in this region has not produced narcoleptic-like sleep. Gerashchenko et al. use orexin- SAP (490 ng/0.5 μl; Cat. # IT-20) to specifically eliminate orexin neurons in rats. The treated rats displayed several sleep disturbances found in narcolepsy, including increased slow-wave sleep, and sleep-onset REM sleep periods. The data suggest that orexin-SAP can be used to create a model for narcolepsy in rats (see page 7, Featured Products).

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Targeting Article: Surfing to Help Spinal Cord Injuries

On Saturday, September 22, I attended the Tony Mezzadri Surf Contest in Ocean Beach, California. A number of years ago Tony, surfing at the Ocean Beach pier, was paralyzed with no movement in his legs and limited movement in his hands and arms. The Ocean Beach community rose to help Tony and this contest has been held yearly since 1994. The proceeds from entry fees, T-shirt sales, raffles, a spaghetti dinner, and donations from sponsor companies were originally to help Tony with this catastrophic event, but now enough money is collected that funds go to support spinal cord research. Dr. Mark Tuszynski’s laboratory at UC San Diego has benefited from thousands of dollars of contributions. Advanced Targeting Systems has been a donor to this event for the past four years. The surf contest is a wonderful grass roots effort in which we are proud and happy to participate. Unlike last year’s monstrous ten-foot surf that was striking the bottom of the pier, this year there was three- to four-foot surf that allowed the contestants to have fun and show their stuff. At the end, money goes to a great cause in supporting work by an excellent scientist.

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Targeting Article: ATS Receives $900,000 in NIH Funding

In September, Advanced Targeting Systems received two Small Business Innovation Research (SBIR) awards from the National Institutes of Health. The first is a Phase II grant from the National Institute of Neurological Disorders and Stroke. This project continues a collaboration with Drs. Joanne Berger-Sweeney (Wellesley College) and Mark Baxter (Harvard University) to further develop the mouse p75 immunotoxin. More than three-quarters of a million dollars will be invested in characterizing this lesioning agent for use in modeling and studying neurodegenerative diseases such as Alzheimer’s disease (AD). Part of the project will include use of the immunotoxin in a transgenic mouse model of AD.

The second award issued to ATS is a Phase I grant from the National Institute of Dental & Craniofacial Research. This $134,000 award will support research to develop an expression system using Substance P as the targeting agent. The purpose of this six-month study is to demonstrate that an expression plasmid can be introduced into Substance P receptor-bearing neurons and that the protein can be observed. If this is successful, then other expression systems will be tested for delivery of bioactive molecules that could diminish the transmission of the chronic pain signal.

Dr. Patrick Mantyh (University of Minnesota) is collaborating with ATS on this project. His laboratory will be testing the system on spinal cord neurons. Dr. Mantyh has been an important collaborator in the development of Substance P-Saporin (SP- SAP), a targeted toxin currently being tested in toxicology/safety studies as a possible therapeutic for chronic pain.

Since it’s first SBIR grant was funded in 1994, ATS has received nearly three million dollars in support for research to develop innovative new products. The SBIR program is a valuable resource for small companies to be able to expand and enhance their in-house R&D efforts. ATS appreciates the ability to collaborate with some of the finest academic institutions and their scientists to meet the goals of these SBIR projects and meet the needs of research scientists throughout the world.

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Cover Article: Immunolesioning Hippocampal Inhibitory Interneurons

Dr. Robert Sloviter, University of Arizona, contributes this issue’s article from the laboratories of ATS customers. Dr. Sloviter summarizes his research with SSP-saporin, which he and his graduate student Jennifer Martin used to examine the role of inhibitory neurons in maintaining normal network excitability.

The mammalian hippocampus is perhaps the most intensely studied brain region for a variety of reasons. Hippocampal structure and function are highly conserved among mammalian species, and its highly laminar organization greatly facilitates experimental design and interpretation. However, its greatest attractions are its involvement in the normal functions of learning and memory, and in a variety of neurological disorders including stroke, Alzheimer’s Disease, and epilepsy. One of the major issues of hippocampal research involves the structure and function of hippocampal inhibitory interneurons, and how they determine the behavior of excitatory hippocampal principal cells. We and others have sought to determine whether certain network behaviors might be the result of inhibitory neuron dysfunction or loss, but it has always been difficult to remove or disable inhibitory neurons selectively, without producing significant collateral damage.

Selective loss of Substance P receptor (SPR)-immunoreactive cells after intrahippocampal injection of SSP-SAP.(A) All SPR-positive cells and dendrites have been ablated on the left side of the photograph. (B) Calretinin (CR)-immunoreactive cells and fibers in an adjacent section survive in the SPR depletion zone. (C) In another adjacent section, zinc transporter-3 (ZnT3)-positive terminals are similarly unaffected in the SPR depletion zone.

After some failed attempts to lesion specific neuronal populations, we were excited to read the paper by Pat Mantyh and his colleagues,1 in which they reported the efficacy of Substance P-saporin (SP-SAP) for removing SP receptor- positive cells in the spinal cord. Because some hippocampal inhibitory interneurons had been reported to express SP receptors (SPRs),2 we purchased SP-SAP, hoping to use this approach in the hippocampus. However, we discovered in pilot experiments that SP-SAP, when injected directly into the hippocampal parenchyma, did not diffuse sufficiently far from the injection site to destroy interneurons in an area large enough for our purposes. Fortunately, ATS had just developed a conjugate using a peptidase- resistant SP analog (SSP-SAP), which we obtained and tested while we conducted an anatomical study designed to determine exactly which hippocampal interneurons constitutively express SPRs, and should therefore be vulnerable to SSP-SAP. That study demonstrated that most inhibitory neurons of all hippocampal subregions expressed SPRs, and that no excitatory principal cells or glia were SPR- positive.3

We found that 10 nl of a solution containing less than 1 ng of SSP-SAP was capable of selectively eliminating all SPR-positive neurons within a 2-mm diameter sphere of tissue. The survival of SPR-negative elements within the SPR depletion zone was remarkable and included excitatory neurons, glia, myelinated fibers, and a number of afferent fiber systems originating outside the hippocampus. Selective loss of SPR- positive inhibitory interneurons was associated with a highly focal disinhibition and hyperexcitability4 that was clearly not caused by a global neurological insult that invariably causes a myriad of non-specific pathologies. Our results indicate that epileptiform behavior is intrinsic to the hippocampal network and does not require the principal cell loss or synaptic reorganization that other models of network hyperexcitability exhibit as a result of less specific neurological injuries. At the least, our results clearly indicate that SSP-SAP will be an extremely useful tool for a wide variety of studies in the hippocampus and other SPR-positive brain regions.

References

1. Mantyh PW, Rogers SD, Honore P, Allen BJ, Ghilardi JR, Li J, Daughters RS, Lappi DA, Wiley RG, Simone DA (1997) Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor. Science 278:275-279.

2. Acsády L, Katona I, Gulyas AI, Shigemoto R, Freund TF (1997) Immunostaining for substance P receptor labels GABAergic cells with distinct termination patterns in the hippocampus. J Comp Neurol 378:320-336.

3. Sloviter RS, Ali-Akbarian L, Horvath KD, Menkens KA (2001) Substance P receptor expression by inhibitory interneurons of the rat hippocampus: enhanced detection using improved immunocytochemical methods for the preservation and colocalization of GABA and other neuronal markers. J Comp Neurol 430:283-305, 2001.

4. Martin JL, Sloviter RS (2001) Focal inhibitory interneuron loss and principal cell hyperexcitability in the rat hippocampus after microinjection of a neurotoxic conjugate of saporin and a peptidase-resistant analog of Substance P. J Comp Neurol 436:127-152.