control-conjugates

Kozicz T, Xu L, Geenen B, Gaszner B, Kovacs K, Roubos E (2011) Leptin-receptor-expressing neurons in the non-preganglionic Edinger-Westphal nucleus regulate white and brown adipose tissue. Neuroscience 2011 Abstracts 822.19. Society for Neuroscience, Washington, DC.

Summary: Leptin, produced by white adipose tissue (WAT), is a key factor that regulates food intake and energy expenditure in vertebrates. It conveys information about fat storage in the periphery to the brain. The leptin receptor long form (LepRb) can be found in the non-preganglionic Edinger-Westphal nucleus (npEW) in the midbrain, which is the main site of urocortin 1 (Ucn1) production in the brain. In both mice and rats, intraperitoneal administration of leptin induces an increase in Ucn1 expression in the npEW whereas in mice that lack LepRb (db/db mice), the npEW contains considerably reduced amount of Ucn1. The npEW also responds to acute thermal exposure, indicating a role of this nucleus in thermoregulation. Brown adipose tissue (BAT) is critical to maintain homoeothermia and is centrally controlled via sympathetic outputs. A recent study demonstrates a projection from EW to BAT by using retrograde tracer pseudorabies virus (PRV). In our study, using PRV injection into the WAT of rats, we identified PRV-labeled Ucn1 neurons in the npEW, indicating a connection from npEW to WAT. In order to analyze the involvement of the npEW in the regulation of sympathetic WAT and BAT outputs, we performed the experiment using the neurotoxin saporin. When conjugated to leptin (Lep-SAP), Lep-SAP can selectively kill LepRb-expressing neurons. Wister rats were given injection of either Lep-SAP or a control blank saporin (B-SAP) into the left npEW Results showed that injection of Lep-SAP significantly blunted Ucn1 expression in the npEW. The weights of WAT and BAT were analyzed on both sides. The WAT and BAT weights were increased significantly on the contralateral side in Lep-SAP compared with B-SAP injected rats, however not different on the ipsilateral side. Interestingly, we observed that both WAT and BAT weighed more on the ipsilateral than the contralateral side only in the B-SAP animals. We will further test the effect of lesioning npEW neurons on the function of WAT and BAT by assessing specific WAT and BAT markers by RT-PCR and histology. Taken these data together, we provide evidence that LepRb-expressing neurons in the npEW regulate BAT and WAT most probably via sympathetic circuits.

Related Products: Leptin-SAP (Cat. #IT-47), Blank-SAP (Cat. #IT-21)

Talman WT, Nitschke Dragon D, Jones S, Moore SA, Lin L-H (2011) Cardiovascular dysfunction and cardiac injury result from selective glial damage in the nucleus tractus solitarii. Neuroscience 2011 Abstracts 664.14. Society for Neuroscience, Washington, DC.

Summary: In man, extensive CNS dysfunction as may occur after subarachnoid hemorrhage may lead to cardiac damage and cardiac arrhythmias. We have shown that highly selective and restricted lesions of the nucleus tractus solitarii (NTS) may lead to similar cardiac and cardiovascular compromise. For example, using conjugates including the cytotoxin saporin (SAP) to selectively damage NTS neurons that express NK1 receptors or those that express tyrosine hydroxylase (TH) leads to cardiac dysfunction and associated lability of arterial pressure. In continuing efforts to better characterize cellular changes produced by introducing into the NTS conjugates containing SAP, we have studied the effect of anti-dopamine-beta-hydroxylase (anti-DBH)-SAP, stabilized substance P (SSP)-SAP, SAP (unconjugated), blank-SAP (non-targeted peptide conjugate), IgG-SAP (non-targeted immunoglobulin conjugate), and 6-hydoxydopamine (6-OHDA) as a control without SAP injected into NTS. We assessed effects of the injected agents both on cellular markers [NMDAR1 (NMDA receptor subunit 1), GluR2 (AMPA receptor subunit 2), gamma-aminobutyric acid (GABA) receptor type a and b, neuronal nitirc oxide synthase (nNOS), TH, vesicular glutamate transporters (VGluTs), choline acetyl transferase (ChAT), glial fibrillary acidic protein (GFAP), connexin 43 (Cx43), DBH and protein gene product 9.5 (PGP 9.5)] and on cardiovascular and cardiac function. We have found that each compound containing SAP (including blank-SAP, IgG-SAP, unconjugated SAP) led to loss of GFAP and Cx43 immunofluorescent labeling in the NTS as well as lability of arterial pressure, cardiac arrhythmias, and cardiac myocytolysis. Those outcomes occurred despite neuronal specificity for each of the SAP conjugates. For example, anti-DBH-SAP led to a decrease in TH and DBH staining as well as a profound loss in GFAP and Cx43. In contrast, SSP-SAP led to loss of NK1 as well as GFAP, Cx43, and glutamate receptor markers but did not lead to loss of DBH or GABA. SSP-SAP also caused a loss in PGP9.5 which was not observed in all other agents. SAP and blank-SAP, on the other hand, led to loss of GFAP and Cx43 while 6-OHDA led to loss of TH and DBH, increased GFAP and decreased Cx-43. We are still investigating the effects of 6-OHDA on lability of arterial pressure and cardiac events but preliminary data suggest that, in doses used, it led to loss of TH and DBH but did not lead to either lability or cardiac events that were seen with each of the conjugates containing an SAP moiety. This study suggests that glial dysfunction may alone interefere with cardiovascular control through the NTS and may lead to cardiac damage and cardiovascular dysfunction.

Related Products: Anti-DBH-SAP (Cat. #IT-03), SSP-SAP (Cat. #IT-11), Mouse IgG-SAP (Cat. #IT-18), Blank-SAP (Cat. #IT-21), Saporin (Cat. #PR-01)

Smith MA, Williams H, Krajewski SJ, Mcmullen NT, Rance NE (2011) Arcuate NK3 receptor-expressing KNDy neurons are essential for estrogen modulation of LH secretion and body weight in the female rat. Neuroscience 2011 Abstracts 712.07. Society for Neuroscience, Washington, DC.

Summary: Arcuate kisspeptin, neurokinin B, and dynorphin (KNDy) neurons have been proposed to mediate estrogen negative feedback in multiple species. To determine if these neurons are essential for this feedback, we ablated KNDy neurons in the arcuate nucleus of female rats using [MePhe7]Neurokinin B, a selective NK3 receptor (NK3R) agonist, conjugated to Saporin ([MePhe7]NKB-SAP, Advanced Targeting Systems, San Diego, CA). The specificity of this conjugate for NK3R-expressing KNDy neurons is described in a separate abstract (see Krajewski et al., Soc. Neurosci. Abstr. 2011). Twenty-four female rats were ovariectomized (OVX) and received bilateral arcuate microinjections of either [MePhe7]NKB-SAP or a scrambled peptide conjugated to Saporin (Blank-SAP controls). 20-23 days later, animals were implanted with s.c. silastic capsules containing 17β-estradiol (E2), and animals were sacrificed 11 days later. Blood samples for RIA of serum LH were taken at time of OVX and injections (baseline), 20-23 days post-OVX, and 11 days after E2-treatment. Because OVX and E2-treatment have well-described effects on body weight, animals were weighed at the same three time points. In control animals, OVX induced a 13-fold rise in serum LH, which returned to baseline 11 days after E2 replacement. In contrast, OVX had no effect on serum LH in [MePhe7]NKB-SAP animals. There was a small decrease in serum LH 11 days after E2 replacement in [MePhe7]NKB-SAP animals, but the magnitude of this change was much less than seen in control animals. Control animals also exhibited a 20% increase in body weight 20-23 days after OVX, followed by a significant reduction after E2 replacement. Surprisingly, neither OVX nor E2 replacement affected body weight in [MePhe7]NKB-SAP-treated animals. Rather, these animals showed a steady increase in body weight throughout the experiment, at rates comparable to intact female rats or OVX rats treated with E2 (Williams et al., Endocrinology, 2010). Immunohistochemical studies showed near-complete destruction of KNDy neurons in the arcuate nucleus of [MePhe7]NKB-SAP animals. There was preservation of proopiomelanocortin and neuropeptide Y immunoreactivity in the arcuate nucleus and GnRH-immunoreactive fibers in the median eminence. These data provide compelling evidence that arcuate KNDy neurons play an essential role in estrogen negative feedback on LH secretion as well as the estrogen modulation of body weight.

Related Products: Blank-SAP (Cat. #IT-21), NKB-SAP (Cat. #IT-63)

ATS Poster of the Year Winner

Krajewski SJ, Smith MA, Williams H, Ciofi P, Lai JY, Mcmullen NT, Rance NE (2011) Ablation of NK3 receptor-expressing KNDy neurons in the rat arcuate nucleus using [MePhe7]Neurokinin B-Saporin. Neuroscience 2011 Abstracts 712.09. Society for Neuroscience, Washington, DC.

Summary: A subpopulation of neurons expressing kisspeptin, neurokinin B and dynorphin (KNDy neurons) has been shown to reside within the arcuate nucleus of many mammalian species. Although these peptides are critical for reproductive function, the precise role of the arcuate KNDy neurons is not fully understood. Here we describe a method to ablate KNDy neurons based on their co-expression of the Neurokinin 3 receptor (NK3R, Burke et al., J. Comp. Neurol, 2006). Saporin, a molecular neurotoxin, was conjugated to [MePhe7]Neurokinin B, a selective NK3R agonist ([MePhe7]NKB-SAP, Advanced Targeting Systems, San Diego, CA). Binding studies revealed that the conjugation of saporin did not alter the affinity of [MePhe7]NKB to NK3R in rat cerebral cortex membranes. To investigate the specificity of this conjugate for ablation of NK3R neurons, stereotaxic surgery was used to bilaterally inject [MePhe7]NKB-SAP into the arcuate nucleus of female rats. Control rats were injected with saporin conjugated to a scrambled peptide (Blank-SAP, Advanced Targeting Systems). Rats were sacrificed 31-34 days later and the brains were processed for immunohistochemical studies. Nissl stained sections from [MePhe7]NKB-SAP-treated rats showed no signs of inflammation at the injection sites and no qualitative changes in cell density compared to Blank-SAP control rats. Immunohistochemistry revealed near-complete loss of NK3R-immunoreactive (ir) neurons throughout the arcuate nucleus of [MePhe7]NKB-SAP rats. When the injection site was dorsal to the arcuate nucleus, there was also variable loss of NK3R-ir cells in the lateral hypothalamus and zona incerta. In the arcuate nucleus, [MePhe7]NKB-SAP injections resulted in a 98% and 94% reduction in the number of kisspeptin and neurokinin B-ir neurons, respectively, compared to Blank-SAP controls. The number of dynorphin-ir neurons in the arcuate nucleus of [MePhe7]NKB-SAP-treated rats was reduced by 67%, a value consistent with the co-expression of NK3R on dynorphin neurons in our previous study (Burke et al., J. Comp. Neurol, 2006). In contrast, arcuate proopiomelanocortin and neuropeptide Y immunoreactivity were preserved in [MePhe7]NKB-SAP rats. Moreover, there was no difference in GnRH-ir fiber density in the median eminence between the two groups. These results document the utility of [MePhe7]NKB-SAP for selective ablation of NK3R-expressing KNDy neurons in rat hypothalamus. These rats were used to examine the role of KNDy neurons in the estrogen regulation of LH secretion and body weight in the female rat (see Smith et al., Soc. Neurosci. Abstr. 2011).

Related Products: Blank-SAP (Cat. #IT-21), NKB-SAP (Cat. #IT-63)

ATS Poster of the Year Winner

Wiater MF, Jansen H, Oostrom M, Li A-J, Dinh T, Ritter S (2011) Lesions targeting leptin-sensitive neurons in the mediobasal hypothalamus dissociate activity and temperature circadian rhythms. Neuroscience 2011 Abstracts 396.11. Society for Neuroscience, Washington, DC.

Summary: Previously we investigated the role of NPY and leptin sensitive networks in the mediobasal hypothalamus in sleep and feeding and found profound regulatory and circadian deficits. We propose that the MBH, particularly the arcuate nuclei (Arc), is required for the integration of homeostatic circadian systems including temperature and activity. We tested this hypothesis with the use of the saporin toxin conjugated to leptin (Lep-SAP) or a blank molecule with no known biological function or receptor (B-SAP) directed to the Arc. Lep-SAP binds to, is internalized by and destroys leptin receptor expressing neurons at the injection site. Lep-SAP rats became obese and hyperphagic and progressed through a dynamic phase to a static phase of growth similar to a ventromedial lesioned rat. Activity and temperature data were collected using intraperitoneal PDT-4000 Emitters with Vital View Data Acquisition Software (Mini Mitter, Philips Respironics, Bend, OR). Circadian rhythms were examined over 49 days during the static phase of obesity in B-SAP (n=10) and Lep-SAP (n=12) rats. Rats were maintained on a 12:12 light:dark (LD) schedule for 13 days and thereafter maintained in continuous dark (DD). After the first thirteen days of DD, food was restricted to four hours per day from 9AM until 1PM for ten days. Immediately thereafter, rats were fasted for three days to evaluate persistence of food-entrained rhythms. Using ClockLab software (Natick, MA) actograms and tempograms were generated as double raster plots. Lomb-Scargle periodograms were used to assess rhythms and their robustness. We found that Lep-SAP rats were arrhythmic for activity in DD, but that food anticipatory activity was nevertheless entrainable to the restricted feeding schedule and the entrained rhythm persisted during the subsequent 3-day fast. Thus, for activity, the light-entrainable oscillator, but not the food entrainable oscillator, was disabled by the MBH lesion. In contrast, temperature remained rhythmic in DD in the Lep-SAP rats, but did not entrain to restricted feeding. We conclude that the leptin-sensitive network of the Arc and MBH is required for entrainment of activity by photic cues and for entrainment of temperature by food and for the integration of these rhythms.

Related Products: Leptin-SAP (Cat. #IT-47), Blank-SAP (Cat. #IT-21)

Liu XY, Liu ZC, Sun YG, Ross M, Kim S, Tsai FF, Li QF, Jeffry J, Kim JY, Loh HH, Chen ZF (2011) Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids. Cell 147(2):447-458. doi: 10.1016/j.cell.2011.08.043

Summary: Recent work has begun to define the different pathways used by itch and pain. This study was designed to investigate whether opioids cause the itch sensation by gastrin releasing peptide receptor activation. Mice received intrathecal injections of bombesin-SAP (Cat. #IT-40) in order to investigate the coexpression of various signaling molecules in the spinal cord. Blank-SAP (Cat. #IT-21) was used as a control. The data suggest that opioid-induced itch is independent of opioid analgesia, and is controlled through a mu-opioid receptor isoform.

Related Products: Bombesin-SAP (Cat. #IT-40), Blank-SAP (Cat. #IT-21)

King T, Porreca F (2011) Featured Article: Contribution of afferent pathways to nerve injury-induced spontaneous pain and evoked hypersensitivity. Targeting Trends 12(4)

Related Products: SSP-SAP (Cat. #IT-11), Blank-SAP (Cat. #IT-21)

Read the featured article in Targeting Trends.

See Also:

• King T et al. Contribution of afferent pathways to nerve injury-induced spontaneous pain and evoked hypersensitivity. Pain 152(9):1997-2005, 2011.

Wiater MF, Mukherjee S, Li AJ, Dinh TT, Rooney EM, Simasko SM, Ritter S (2011) Circadian integration of sleep-wake and feeding requires NPY receptor-expressing neurons in the mediobasal hypothalamus. Am J Physiol Regul Integr Comp Physiol 301(5):R1569-R1583. doi: 10.1152/ajpregu.00168.2011 PMID: 21880863

Summary: Feeding and sleep/wake states interact rhythmically across the circadian cycle. It is suspected that the mediobasal hypothalamic area (MBH) is the site where these rhythms are integrated. The authors administered bilateral 24-ng injections of NPY-SAP (Cat. #IT-28) into the arcuate nucleus in order to eliminate NPY receptor-expressing neurons in the MBH of rats. Blank-SAP (Cat. #IT-21) was used as a control. The results indicate that these neurons are required for the interaction of feeding and sleep/wake timing.

Related Products: NPY-SAP (Cat. #IT-28), Blank-SAP (Cat. #IT-21)

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Lemons LL, Wiley RG (2011) Galanin receptor-expressing dorsal horn neurons: role in nociception. Neuropeptides 45(6):377-383. doi: 10.1016/j.npep.2011.08.002

Summary: This work examines the mociceptive role of galanin receptor-1-expressing neurons found in the superficial dorsal horn. 500 ng of galanin-SAP (Cat. #IT-34) was injected into the lumbar intrathecal space of rats; blank-SAP (Cat. #IT-21) was used as a control. The rats were then tested in a series of thermal nociception models. Lesioned animals were less sensitive to heat, suggesting that loss of the gal1r-expressing excitatory interneurons disrupted the pain transmission pathway.

Related Products: Galanin-SAP (Cat. #IT-34), Blank-SAP (Cat. #IT-21)

Lin Y, Sarfraz Y, Jensen A, Dunn AJ, Stone EA (2011) Participation of brainstem monoaminergic nuclei in behavioral depression. Pharmacol Biochem Behav 100(2):330-9. doi: 10.1016/j.pbb.2011.08.021

Summary: While the classical model states reductions in central noradrenergic activity produces depression, more recent work has indicated that higher activity in this brain region directly correlates with depression. Using a dopamine-ß-hydroxlase targeted toxin to lesion the locus coeruleus of mice, along with goat-IgG-SAP (Cat. #IT-19) as a control, the authors found that treated animals showed increased resistance to depressive behavior in several tests. The results suggest that monoaminergic lesions are greatly affected by mouse strain, lesion size, and involvement of other neuronal systems.

Related Products: Goat IgG-SAP (Cat. #IT-19)