Targeting Trends Newsletter 06q4

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Newsletter Highlights

  • ATS Hits the Field (page 2)
  • Teaser Winners (page 2)
  • Anti-DBH-SAP (page 5)
  • Anti-6-His and Anti-SAP-HRP (page 7)

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Basomedial Hypothalamic Injections of Neuropeptide Y Conjugated to Saporin Selectively Disrupt Hypothalamic Controls of Food Intake – read article
(continued on page 6)

This article is a summary of data presented in the reference below. Figures 1-4 are taken from that article. This work was funded by NS045520 and DK40498 to S. Ritter.
Bugarith K, Dinh TT, Li AJ, Speth RC, Ritter S. (2005) Endocrinology 146:1179-1191.

Product information related to cover article: NPY-SAP (Cat. #IT-28), Anti-DBH-SAP (Cat. #IT-03), Saporin (Cat. #PR-01), Blank-SAP (Cat. #IT-21)

Targeting Teaser Winners

Upcoming Events

  • Society for Neuroscience, Atlanta GA — October 10-14, Booth #1240
  • American Society for Cell Biology, San Diego CA — December 10-13, Booth #734

ATS Hits the Field

Recent Scientific References – read online

Targeting Talk: Anti-DBH-SAP Administration

  • We injected anti-DBH-SAP 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 alittle 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.

NPY- SAP Selectively Disrupts Hypothalamic Controls of Food Intake – read article
(continued from page 1)

Targeting Tools

Targeting Technology Tutorial

Targeting Teaser (Jumble)

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Targeting Tools: Anti-6-His

Anti-6-His Mouse Monoclonal, Cat. #AB-20 [see also Cat. #AB-213]

The use of polyhistidine tags has become a popular method for protein purification, commonly used in the screening process as a tag for your protein or peptide of interest. Whether the material you are screening for is affinity purified or in crude bacterial extract, you will find our antibody suitable to your needs.

This antibody was created as a mouse monoclonal generated to recognize a 6 Histidine (6-His) amino acid sequence, independent of its location. It will recognize C-terminal, N-terminal, or internal 6-His epitopes, with very high sensitivity and low background (Fig. 1).

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Lane 1: Molecular weight standards (See-Blue)
Lane 2: Crude bacterial extract containing a 6- His-tag expressing protein
probed with Anti-6-His antibody at a 1:5,000 dilution.
Band of interest is visualized at 25-28 kDa.

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Targeting Tools: HRP-labeled Saporin

HRP-labeled Saporin Goat Polyclonal, Cat. #AB-15HRP

HRP-labeled Anti-SAP can be used to verify binding specificity of a targeted toxin to a cell line expressing the target molecule. By first binding the targeted toxin to protein extract or plate-bound antigen, then binding HRP-labeled Anti-SAP to the targeted toxin, specificity can be confirmed through the use of competing molecules or a control cell line.

This antibody recognizes saporin. Saporin was used as the immunogen. The antibody was coupled to Horseradish Peroxidase (HRP) and dialyzed against PBS.

Saporin (200, 100, 50, and 25 ng) was run on a 10% SDS- PAGE gel and transferred to PVDF membrane. The blot was blocked with 4% NFM/TBS, then incubated overnight with 0.5 μg/ml (Lanes 11-15), 1 μg/ml (Lanes 6-10), or 5 μg/ml (Lanes 1-5) of antibody. The blot was washed and developed with 4- chloro-1-naphthol and hydrogen peroxide.
Lane 1, 6, 11: Molecular weight standards (Invitrogen See-Blue)
Lane 2, 7, 12: 200 ng Saporin
Lane 3, 8, 13: 100 ng Saporin
Lane 4, 9, 14: 50 ng Saporin
Lane 5, 10, 15: 25 ng Saporin

Targeting Talk: Anti-DBH-SAP Administration

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.

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.

Targeting Topics 06q4

CD70 (TNFSF7) is expressed at high prevalence in renal cell carcinomas and is rapidly internalised on antibody binding.

Adam PJ, Terrett JA, Steers G, Stockwin L, Loader JA, Fletcher GC, Lu LS, Leach BI, Mason S, Stamps AC, Boyd RS, Pezzella F, Gatter KC, Harris AL.

Br J Cancer 95(3):298-306, 2006.

Renal cell carcinoma (RCC) is usually resistant to chemotherapy. The authors found a potential target for immunotherapy. An antibody against CD70 was combined with Hum-ZAP (Cat. #IT-22). The complex was then added to an RCC-derived cell-line in vitro, demonstrating significant killing at several different concentrations.

Adenosine and sleep homeostasis in the Basal forebrain.

Blanco-Centurion C, Xu M, Murillo-Rodriguez E, Gerashchenko D, Shiromani AM, Salin-Pascual RJ, Hof PR, Shiromani PJ.

J Neurosci 26(31):8092-8100, 2006.

The authors investigated whether basal forebrain cholinergic neurons are involved in adenosine regulation of sleep. 6 µg of 192-IgG-SAP (Cat. #IT-01) was administered to the lateral ventricle of rats. In treated animals, adenosine levels did not increase with prolonged waking.

Secondary hyperalgesia in the monoarthritic rat is mediated by GABAB and NK1 receptors of spinal dorsal horn neurons: a behavior and c-fos study.

Castro AR, Pinto M, Lima D, Tavares I.

Neuroscience 141(4):2087-2095, 2006.

Hallmarks of secondary hyperalgesia in a rat model of monoarthritic pain are: decreased activation of GABA(B) neurons, and increased activation of NK-1r neurons. Using 10-µl injections of 1-µM SP-SAP (Cat. #IT-07) into T(13)-L(1) the authors looked at the role of each receptor. Results indicate that both GABA(B) and NK-1r are involved in secondary hyperalgesia.

High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells.

Collins BE, Blixt O, Han S, Duong B, Li H, Nathan JK, Bovin N, Paulson JC.

J Immunol 177(5):2994-3003, 2006.

CD22, a member of the siglec subgroup of the Ig superfamily, is a potential target for immunotherapy of B cell lymphomas. The authors demonstrate that a biotinylated probe specific for CD22 combined with streptavidin-ZAP (Cat. #IT-27), can eliminate several different lymphoma cell lines.

Immunolesions of glucoresponsive projections to the arcuate nucleus alter glucoprivic-induced alterations in food intake, luteinizing hormone secretion, and GALP mRNA, but not sex behavior in adult male rats.

Fraley GS.

Neuroendocrinology 83(2):97-105, 2006.

In this work the author looked at the role hypothalamic glucose may play in reproductive function. 42 ng of anti-DBH-SAP (Cat. #IT-03) was injected dorsal of the arcuate nucleus of rats, which were then given glucoprivic challenges. The data demonstrate the involvement of A1/C1 efferents to the ventromedial hypothalamus in glucostatic regulation of various processes.

The nuclear DNA repair protein Ku70/80 is a tumor-associated antigen displaying rapid receptor mediated endocytosis.

Fransson J, Borrebaeck CA.

Int J Cancer 119(10):2492-2496, 2006.

In this study, the authors show that Ku70/80 is internalized into pancreatic carcinoma cells upon binding of the antibody INCA-X. INCA-X was combined with Mab-ZAP (Cat. #IT-04) and applied to several pancreatic carcinoma cell lines in vitro. Cell death in some of the treated lines demonstrates the potential of Ku70/80 as a therapeutic target.

Lack of neurogenesis in the adult rat cerebellum after Purkinje cell degeneration and growth factor infusion.

Grimaldi P, Rossi F.

Eur J Neurosci 23(10):2657-2668, 2006.

Although neurogenesis occurs in very specific areas of the mammalian brain, neural progenitors can be found in many central nervous system sites. Here the authors examined neurogenesis in the rat cerebellum. 2.2 µg of 192-IgG-SAP (Cat. #IT-01) was injected into each lateral ventricle, and some animals were given exogenous EGF, bFGF, or FGF8. In this model, the local environment was not sufficient to direct neuronal differentiation, even with the addition of growth factors.

Ameliorating effect of saporin-conjugated anti-CD11b monoclonal antibody in a murine T-cell-mediated chronic colitis.

Kanai T, Uraushihara K, Totsuka T, Nemoto Y, Fujii R, Kawamura T, Makita S, Sawada D, Yagita H, Okumura K, Watanabe M.

J Gastroenterol Hepatol 21(7):1136-1142, 2006.

Using SCID mice, the authors evaluated the effects of Mac-1-SAP (Cat. #IT-06) on the development of chronic colitis. After transfer of T cells to the mice, 12.5 µg of Mac-1-SAP was injected into the intraperitoneal space. The reduction in CD4(+) T-cell infiltration of the colon, and suppression of IFNγ and TNFα production indicate that macrophages play a significant role in the pathogenesis of Crohn’s disease.

Neurokinin-1 receptor-expressing neurons in the ventral medulla are essential for normal central and peripheral chemoreception in the conscious rat.

Nattie E, Li A.

J Appl Physiol 101(6):1596-1606, 2006.

The authors ask if neurokinin-1 receptor (NK-1r)-positive cells scattered throughout the ventral medulla are involved in central and peripheral chemoreception. Rats received 250-280 ng of SSP-SAP (Cat. #IT-11) into the cisterna magna; mouse IgG-SAP (Cat. #IT-18) was used as a control. The results indicate that NK-1r neurons in the ventral medulla are involved in both central and peripheral chemoreception, during both waking and sleep states.

Purkinje cell loss by OX7-saporin impairs acquisition and extinction of eyeblink conditioning.

Nolan BC, Freeman JH.

Learn Mem 13(3):359-365, 2006.

This work examines the effect of a global depletion of Purkinje cells in the cerebellar cortex on delay eyeblink conditioning in rats. 15 µg of OX7-SAP (Cat. #IT-02) was infused into the left lateral ventricle 2 weeks prior to training. Purkinje cell loss in the anterior lobe and lobule HVI correlated with impaired acquisition and extinction of delay eyeblink conditioning.

Cortical choline transporter function measured in vivo using choline-sensitive microelectrodes: clearance of endogenous and exogenous choline and effects of removal of cholinergic terminals.

Parikh V, Sarter M.

J Neurochem 97(2):488-503, 2006.

The authors investigated the role of high-affinity choline transporters (CHT) in the clearance of exogenous choline, as well as choline from newly released acetylcholine. 0.085 µg of 192-IgG-SAP (Cat. #IT-01) was injected into each hemisphere of the basal forebrain of rats (mouse IgG-SAP, Cat. #IT-18, was used as a control). The results demonstrate that no matter the source, increases in choline concentrations are cleared by CHT’s.

Hindbrain catecholamine neurons control multiple glucoregulatory responses.

Ritter S, Dinh TT, Li AJ.

Physiol Behav 89(4):490-500, 2006.

The authors focus on mechanisms eliciting glucoregulatory responses; in particular the catecholaminergic neurons in the hindbrain. Rats received injections of anti-DBH-SAP (Cat. #IT-03) into epinephrine (E) and norepinephrine (NE) terminal areas of hypothalamus and spinal cord. The data suggest that E/NE neurons coordinate various components of the behavioral response to glucoprivation.

Local and descending circuits regulate long-term potentiation and zif268 expression in spinal neurons.

Rygh LJ, Suzuki R, Rahman W, Wong Y, Vonsy JL, Sandhu H, Webber M, Hunt S, Dickenson AH.

Eur J Neurosci 24(3):761-772, 2006.

Long-term potentiation (LTP) has been shown to occur in sensory areas of the spinal cord and may be one of the mechanisms by which acute pain is transformed into chronic pain. 10 µl of 1µM SP-SAP (Cat. #IT-07) or saporin (Cat. #PR-01) were injected into the subarachnoid space (L4-L5) of rats. The authors demonstrate that dorsal horn neuron generation of LTP may transform acute pain into chronic pain.

Descending facilitation from the rostral ventromedial medulla maintains nerve injury-induced central sensitization.

Vera-Portocarrero LP, Zhang ET, Ossipov MH, Xie JY, King T, Lai J, Porreca F.

Neuroscience 140(4):1311-1320, 2006.

Rats were treated with 1.5 pmol of dermorphin-SAP (Cat. #IT-12) or saporin (Cat. #PR-01) into each side of the rostral ventromedial medulla, followed by spinal nerve ligation. The data indicate that mu opioid-expressing neurons are necessary to maintain nerve injury-induced central sensitization.

Cover Article: Basomedial Hypothalamic Injections of Neuropeptide Y Conjugated to Saporin Selectively Disrupt Hypothalamic Controls of Food Intake

This article is a summary of data presented in reference #1. Figures 1-4 are taken from that article. This work was funded by NS045520 and DK40498 to S. Ritter.

Neuropeptide Y (NPY) conjugated to saporin (SAP), a ribosomal toxin, is a compound designed to selectively target and lesion NPY receptor-expressing cells. We conducted competitive binding studies using I 125 -NPY to evaluate the binding of NPY-SAP (Cat. #IT-28) to rat forebrain homogenates (1). Results indicate that NPY-SAP binds to and has a higher binding affinity than NPY for the NPY receptor (Fig. 1). The binding results, in combination with previous studies demonstrating agonist-driven NPY receptor internalization (2, 3), indicated that this peptide-saporin conjugate would produce effective lesions of NPY receptor-expressing neurons. Accordingly, when we injected NPY-SAP (48 ng in 100 nl) bilaterally into the arcuate nucleus (ARC) of the hypothalamus, we found a profound reduction of NPY Y1 receptor-immunoreactivity (-ir) in the ARC (Fig. 2). We also found a nearly complete loss of NPY, AGRP and CART mRNA expression and α-MSH-ir in the ARC and mediobasal hypothalamus, showing that these NPY receptor-expressing neurons were lesioned by NPY-SAP (Fig. 3).

To date, there is no evidence that any of the available peptide-saporin conjugates are retrogradely transported. To determine whether NPY-SAP is retrogradely transported, we injected the conjugate into the ARC and examined catecholamine cell bodies in the A1/C1 region of the ventrolateral medulla. Nearly all of the catecholamine neurons in this area co-express NPY and project to the medial hypothalamus. A1/C1 neurons are almost completely destroyed by medial hypothalamic injections of the retrogradely transported immunotoxin, anti-dopamine-beta-hydroxylase-saporin (anti-DBH-SAP, Cat. #IT-03) (4-6). However, there was no loss of cells in this area in the NPY-SAP injected rats (Fig. 4), indicating that NPY-SAP is not internalized by NPY terminals or that, once internalized, there is no mechanism for retrograde transport of the conjugate. Supporting these findings, we also showed that NPY terminals in the area of cell body loss, though initially reduced, were not obliterated, as they would be if all NPY neurons innervating that area had been retrogradely destroyed.

In previous work, we used anti-DBH-SAP to examine the importance of hindbrain catecholamine neurons that innervated the hypothalamus for glucoregulation (7-10). We found that these neurons (many of which co-express NPY) are required for a number of glucoregulatory responses, including feeding, corticosterone secretion and suppression of estrous cycles in response to glucose deficit. In addition, we found that catecholamine neurons with projections to the spinal cord, which are distinct from those that project to the hypothalamus, are required for the adrenal medullary hyperglycemic response to glucoprivation. The goal of our work with NPY-SAP (1) was to determine whether the ARC NPY neurons, which co-express agouti-related protein (AGRP), are required for systemic glucoregulation. Gene knockout studies indicate that the NPY gene is required for glucoprivic feeding (11). However, there are multiple, presumably functionally heterogeneous, NPY populations in the brain. Furthermore, in the medial hypothalamus, the terminals of ARC NPY neurons are co-extensive with those of the hindbrain catecholamine neurons, making it difficult to distinguish the separate functions of these two NPY cell populations. NPY-SAP was useful in addressing this question. To date, we have examined feeding (Fig. 5), hyperglycemic and corticosterone responses to glucoprivation. None of these responses were impaired by ARC NPY-SAP injections that destroyed NPY receptor-expressing neurons, including the NPY/AGRP neurons, in the ARC and basomedial hypothalamus. However, these same lesions severely reduced feeding and body weight responses to leptin and feeding responses to ghrelin, which are known to depend upon ARC NPY receptor-expressing neurons. Thus, using anti-DBH-SAP and NPY-SAP we have been able to functionally differentiate the hindbrain NPY/catecholamine and the ARC NPY/AGRP co-expressing neuronal populations and to establish the primacy of the hindbrain NPY/catecholamine neurons for elicitation of systemic glucoregulatory responses.

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Figure 1.
Top: Competitive binding of NPY and NPY-SAP with I125-NPY in rat forebrain tissue homogenates. Duplicate determinations were made for each concentration.
Bottom: Bars show IC50 for NPY and NPY reduced NPY-SAP binding. Data show that NPY-SAP has a binding affinity for NPY receptors that is equal to or greater than NPY at the concentrations examined.

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Figure 2. Coronal sections through the arcuate nucleus of the hypothalamus showing effects of Blank-SAP (B-SAP) control (A) and NPY-SAP (B) injections into the arcuate nucleus on NPY-Y1 receptor immunoreactivity.

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Figure 3. Pseudocolor representation of OD of NPY, AGRP and CART mRNA expression in the arcuate nucleus after bilateral injection of Blank-SAP (B-SAP, left) or NPY-SAP (right) into the arcuate nucleus. NPY-SAP significantly reduced NPY, AGRP and CART hybridization signal at the injection site.

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[twocol_one][/twocol_one] [twocol_one_last]Figure 4. Coronal sections showing tyrosine hydroxylase-ir in the ventrolateral medullary catecholamine cell column ventral to the area postrema in rats injected into the arcuate nucleus with Blank-SAP (B-SAP, control) or NPY-SAP. Nearly all cells in this area co-express NPY and project to the hypothalamus. Arcuate NPY-SAP injections did not cause retrograde destruction of the hindbrain catecholamine/NPY neurons.[/twocol_one_last]

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Figure 5. Food intake in response to glucoprivation induced by the antiglycolytic agent, 2-deoxy-D-glucose (2DG, 200 mg/kg, s.c.) in rats injected into the medial hypothalamus with anti-DBH-SAP (top) or NPY-SAP (bottom) or their respective control solutions (unconjugated saporin and Blank-SAP, respectively). Anti-DBH-SAP, but not NPY-SAP, caused impairment of the glucoprivic feeding response.
*2DG vs saline for the same group, P<.001

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References:     (back to top)

  1. Bugarith K, Dinh TT, Li AJ, Speth RC, Ritter S. (2005) Endocrinology 146:1179-1191.
  2. Parker MS, Parker SL, Kane JK. (2004) Regul Pept 118:67-74.
  3. Parker SL, Parker MS, Lundell I, Balasubramaniam A, Buschauer A, Kane JK, Yalcin A, Berglund MM. (2002) Regul Pept 107:49-62.
  4. Li AJ, Ritter S. (2004) Eur J Neurosci 19(8):2147-2154.
  5. Li AJ, Wang Q, Ritter S. (2006) Endocrinology 147(7):3428-3434.
  6. Ritter S, Bugarith K, Dinh TT. (2001) J Comp Neurol 432(2):197-216.
  7. Ritter S, Watts AG, Dinh TT, Sanchez-Watts G, Pedrow C. (2003) Endocrinology 144(4):1357-1367.
  8. Ritter S, Dinh TT, Sanders NM, Pedrow C. (2001) Soc Neuroscience Abstracts 27:947.3.
  9. I’Anson H, Sundling LA, Roland SM, Ritter S. (2003) Endocrinology 144(10):4325-4331.
  10. Hudson B, Ritter S. (2004) Physiol Behav 82(2-3):241-250.
  11. Sindelar DK, Ste Marie L, Miura GI, Palmiter RD, McMinn JE, Morton GJ, Schwartz MW. (2004) Endocrinology 145(7):3363-3368.