Wiley RG, Lemons LL, Kline IV RH (2009) Neuropeptide Y receptor-expressing dorsal horn neurons: role in nocifensive reflex responses to heat and formalin. Neuroscience 161:139-147. doi: 10.1016/j.neuroscience.2008.12.017

Summary: This work examines the effect of lumbar intrathecal administration of NPY-SAP (Cat. #IT-28), and the role of Y1 NPY receptor-expressing neurons (Y1R) in response to thermal and chemical stimulation. Rats received 500 ng or 750 ng intrathecal injections of NPY-SAP. Blank-SAP (Cat. #IT-21) was used as a control. Lesioned animals displayed a specific loss of Y1R in the dorsal horn, as well as reduced nocifensive reflex responses.

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

Wiley RG, Moore SA, Kline IV RH (2008) Analysis of inhibitory phase of formalin test: Effects of specific neural lesions. Neuroscience 2008 Abstracts 772.4/MM19. Society for Neuroscience, Washington, DC.

Summary: The formalin test has been widely used as a model of persistent pain. The 90 mins of formalin-induced nocifensive responding can be divided into two phases (phase 1, first ~10 mins; phase 2, last ~60 mins) separated by a period of reduced responding (interphase, IP), that has received relatively little attention. Behavioral inhibition during the IP of the formalin test has been associated with electrophysiological evidence of inhibition of dorsal horn nociceptive neurons (Henry et al, Pain, 82:57, 1999), probably due, at least in part, to local spinal mechanisms. Behavioral inhibition during IP has been shown to be enhanced by morphine and suppressed by naloxone. In the present study, we sought to determine the effect of selective depletion of specific dorsal horn interneurons known to be involved in nociception, i.e. neurons expressing NPY1R, GalR1 or MOR, or selective destruction of cerebral noradrenergic neurons or spinal cord projecting 5-HT neurons on formalin-induced nociceptive behavior, with particular attention to IP. Type-selective lesions were produced by lumbar intrathecal injection of NPY-saporin, galanin-saporin or dermorphin-saporin, respectively. Cerebral noradrenergic neurons and spinally projecting 5-HT neurons were destroyed using the immunotoxins, anti-DBH-saporin (intracerebroventricular) or anti-SERT-saporin (lumbar intrathecal), respectively. Partial loss of dorsal horn interneurons expressing NPY1R or GalR1 decreased nocifensive responding during IP and phase 2 of the formalin test, while partial loss of MOR-expressing dorsal horn interneurons increased nocifensive responding during IP and during phase 2. Both antiDBH-sap and antiSERT-sap decreased responding during IP, without effects on either phase 1 or 2. These results suggest that the apparent anti-nociception during IP and phase 2 produced by loss of NPY1R- and GalR1-expressing dorsal horn neurons is due to increased inhibition over excitation/facilitation of nociceptive projection neurons, whereas depletion of MOR-expressing interneurons produces the opposite effect. The apparent enhanced nociception during IP, but not phase I and II, produced by anti-DBH-sap and anti-SERT-sap suggests that these neural systems serve to enhance the excitability of nociceptive projection neurons during the formalin IP. Electrophysiologic and pharmacologic studies of formalin IP in selectively lesioned animals combined with the above behavioral findings may reveal new insights into endogenous modulation of nocifensive motor responses and/or nociception.

Related Products: NPY-SAP (Cat. #IT-28), Anti-SERT-SAP (Cat. #IT-23), Galanin-SAP (Cat. #IT-34), Anti-DBH-SAP (Cat. #IT-03), Dermorphin-SAP / MOR-SAP (Cat. #IT-12)

Mack SO, Wu M, Xu G (2008) Chemoresponsiveness of the hypothalamic paraventricular nucleus (PVN) is influenced by neuropeptide Y. Neuroscience 2008 Abstracts 383.7/RR74. Society for Neuroscience, Washington, DC.

Summary: The hypothalamic paraventricular nucleus (PVN) initiates autonomic responses to stress and behavioral changes. Neuropeptide Y-containing neurons primarily from the arcuate nucleus and, to a lesser extent, from the brainstem innervate preautonomic oxytocin-containing neurons in the PVN. The role of the PVN in mediating the effects of NPY on energy balance has been studied extensively; however, whether NPY influences respiratory drive via the PVN is not known. Previously, we demonstrated that stimulation of the PVN modulates cardiorespiratory responses via oxytocinergic innervation of neurons in the rostral ventrolateral region of the medulla oblongata where rhythm generating neurons are located. In this study, we selectively lesioned neurons in the PVN bilaterally with neurotoxin neuropeptide Y-saporin (NPY-SAP; 50 ng/100 μl per side) or blank-saporin (control vehicle). Both groups of rats showed similar (P>0.05) increases in body weight gain and intake of food and water over an 8 week period after lesioning. Core body temperature, measured at the same time every day, was also similar for both groups (P>0.05). At 4 weeks post lesioning, NPY-SAP treatment had no effect (P>0.05) on respiratory frequency (fR), tidal volume (VT) and minute ventilation (VE ) in awake, unrestrained animals breathing room air. During exposure to a hypercapnic challenge (5% CO2) for 10 minutes, fR (135 ± 7 vs 114 ±5 breaths min-1) and VE (154 ±13 vs 114± 5 ml min-1 100g-1 ) for the treated animals were significantly elevated (P<0.05) above responses for the control rats. Tidal volume for the treated (1.1 ±0.06) and the control (1.0 ± 0.03) groups was not different (P>0.05). Sensitivity to CO2 with respect to fR in the treated animals reached a peak at 4 weeks and declined thereafter over the next 4 weeks. While there were no apparent changes in morphology or number of parvocellular oxytocin-containing neurons 4 weeks after lesioning, abnormal morphology and a significant (P<0.05) reduction in oxytocin immunoreactive cells were prominent by 8 weeks post treatment. These findings indicate that NPY plays a role in modulating the respiratory response to hypercapnic stress through oxytocin neurons in the PVN. Further studies are needed to determine whether alterations in this pathway may be involved in the onset of hypoventilation associated with obesity.

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

Li AJ, Dinh TT, Ritter S (2008) Hyperphagia and obesity produced by arcuate injection of NPY-saporin do not require upregulation of lateral hypothalamic orexigenic peptide genes. Peptides 29(10):1732-1739. doi: 10.1016/j.peptides.2008.05.026

Summary: It has already been shown that lesioning NPY receptor-expressing cells in the arcuate nucleus (Arc) and basomedial hypothalamus produces obesity in rats. The authors examined the contribution of orexigenic peptides, orexins, and melanocortin-concentrating hormone to the lesion effects. Rats received bilateral 24 ng injections of NPY-SAP (Cat. #IT-28) into the dorsal border of the Arc. Blank-SAP (Cat. #IT-21) was used as a control. The data suggest that obesity produced by NPY-SAP lesion is different than dietary obesity or obesity associated with leptin or leptin receptor deficiency.

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

Kline IV RH, Lemons LL, Wiley RG (2007) Antinociceptive effects of lumbar intrathecal neuropeptide y-saporin. Neuroscience 2007 Abstracts 821.5/FF20. Society for Neuroscience, San Diego, CA.

Summary: Spinal intrathecal (i.t.) neuropeptide Y (NPY) has been shown to be antinociceptive in the rat. Using lumbar i.t. NPY, coupled to the ribosomal inactivating protein, saporin, to selectively destroy spinal dorsal horn cells that express NPY receptors, we sought to determine the effect of this lesion on nocifensive behaviors in the hotplate and formalin tests and on NPY1R staining in the lumbar dorsal horn. Twenty Sprague Dawley male rats were injected i.t. with either saline or 500ng NPY-sap and then were tested on the hotplate for 30 days. Fifteen Long Evans female rats were injected i.t. with either saline or NPY-sap (500ng or 750ng) and then tested on the hotplate for two weeks followed by hindpaw formalin injection. In order to assess responses mediated by C or A-delta thermal nociceptors, hotplate testing used three temperatures: 44C (600 sec trial duration), 47C (200 sec trials), and 52C (first response or 30 sec). In male rats, lumbar i.t. NPY-sap increased hindpaw withdrawal latencies to 44, 47 and 52C, with the greatest effect on 44C. NPY-sap also reduced the total amount of hindpaw lick/guard responding (duration and number of responses) on the 44 and 47C hotplates. Female rats injected with 750ng of NPY-sap showed a decrease in the number of hindpaw lick/guard events on the 44C hotplate. Female rats also showed a decrease in the total number of hindpaw lick/guard events during the interphase (7-21min) and phase II (22-90min) of the formalin test. Additionally operant thermal place preference testing (45C vs 12C) was compared to hotplate reflex testing. Selectivity of NPY-sap was assessed by immunocytochemistry for cells expressing NPY1R and non-selectivity was assessed by staining for NK-1R. Based on the above findings we conclude that selective destruction of dorsal horn NPY1R-expressing neurons produces decreased thermal nociception to a range of noxious heat and also decreases responses to persistent noxious chemical stimulation during the formalin test. In summary, reduced nocifensive behaviors after NPY-sap were more prominent when assessing responses elicited by input from predominately C fiber activation (44C and formalin). This study was supported by the Department of Veterans Affairs.

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

Li A-J, Dinh TT, Ritter S (2007) Destruction of NPY receptor expressing neurons in the arcuate nucleus causes obesity and hyperphagia without increasing lateral hypothalamic orexigenic peptide gene expression. Neuroscience 2007 Abstracts 524.20/BBB20. Society for Neuroscience, San Diego, CA.

Summary: NPY-SAP, a conjugate of neuropeptide Y (NPY) and the ribosomal inactivating toxin, saporin (SAP), is a compound that selectively lesions NPY receptor-expressing neurons. Previously we showed that injection of NPY-SAP into the hypothalamic arcuate nucleus (ARC) induces hyperphagia and obesity in rats. To further investigate the mechanisms responsible for NPY-SAP-induced obesity, we injected NPY-SAP or blank-saporin (B-SAP) control into the ARC and subsequently examined the expression of two orexigenic neuropeptide genes in the lateral hypothalamic area (LHA), which is densely innervated by ARC neurons. Our hypothesis was that loss of leptin-sensitive neurons in the ARC in the NPY-SAP injected rats would lead to increased expression of orexigenic neurons elsewhere in the hypothalamic feeding circuitry. Body weight gain and food intake were dramatically increased in the NPY-SAP group. In addition, expression of NPY and cocaine- and amphetamine-regulated transcript (CART) mRNA was significantly reduced in the ARC of obese rats, indicating a loss of NPY receptor-expressing NPY and CART neurons in this region. In contrast, NPY and CART gene expression in the dorsomedial hypothalamic nucleus was unchanged in NPY-SAP rats, indicating that the NPY-SAP-induced lesion was limited to the ARC. However, contrary to our hypothesis, expression of the orexigenic neuronpeptides, melanin-concentrating hormone (MCH) or prepro-orexin mRNA in LHA was not enhanced, but was slightly reduced in the NPY-SAP rats. These results indicate that an enhancement of MCH or orexin expression in the LHA is not necessary for the hyperphagia and obesity observed after NPY-SAP lesions in the ARC. Supported by PHS grant #DK 40498.

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

Sparrow AW, Lowery EG, Thiele TE (2007) Amygdalar neuropeptide Y (NPY) signaling modulates stress-induced reductions of food intake in Balb/cJ mice. Neuroscience 2007 Abstracts 270.10/X24. Society for Neuroscience, San Diego, CA.

Summary: The existing literature suggests that NPY signaling in the amygdala modulates anxiety-like behaviors and ethanol consumption in rodents, but does not modulate food intake. On the other hand, NPY signaling within the hypothalamus controls food intake but does not influence anxiety-like behavior. Based on these observations, the current study tested the hypothesis that attenuation of NPY signaling within the amygdala would increase anxiety-like behavior and augment stress-induced increases of ethanol consumption while at the same time have no effect of feeding behavior. To address this hypothesis, male Balb/cJ were given bilateral injection (48 ng/5-min/side) into the central nucleus of the amygdala (CeA) of NPY conjugated to the neurotoxin saporin (NPY-SAP) or saporin alone (Blank-SAP). NPY-SAP is a ribosome inactivating neurotoxin that targets and kills cells expressing NPY receptors. After recovery, mice were first tested for anxiety-like behavior using the zero maze test. They were then given access to 8% (v/v) ethanol versus water in a two-bottle test. After ethanol intake stabilized, half the NPY-SAP and Blank-SAP mice were subjected to a 5-min forced swim stress sessions, once a day over 5-days. Ethanol, water and food consumption were measured for 4-weeks following the forced swim procedures. At the end of the experiment, ethanol was removed for two-weeks and all mice were given a 24-hour open-field locomotor activity test. The results showed that mice treated with NPY-SAP in the CeA spent significantly less time in the open portion of the zero maze reflecting elevated anxiety-like behavior. Contrary to predictions, neither neurotoxin treatment nor stress condition altered ethanol intake. Interestingly, NPY-SAP treated mice that experienced forced swim stress consumed significantly less food than non-stressed NPY-SAP treated mice and stress and non-stressed mice treated with the Blank-SAP. Reduced feeding by NPY-SAP stressed mice was not associated with reduced body weight, suggesting possible alterations of energy metabolism. Further, reduced feeding was not attributable to reductions of activity. This study provides novel evidence that amygdalar NPY signaling modulates feeding/energy balance in mice with a history of stress exposure.

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

Bugarith K, Dinh TT, Li AJ, Speth RC, Ritter S (2006) Featured Article: Basomedial hypothalamic injections of neuropeptide Y conjugated to saporin selectively disrupt hypothalamic controls of food intake. Targeting Trends 7(4)

Related Products: Anti-DBH-SAP (Cat. #IT-03), NPY-SAP (Cat. #IT-28), Saporin (Cat. #PR-01), Blank-SAP (Cat. #IT-21)

Read the featured article in Targeting Trends.

See Also:

• Bugarith K et al. Basomedial hypothalamic injections of neuropeptide Y conjugated to saporin selectively disrupt hypothalamic controls of food intake. Endocrinology 146(3):1179-1191, 2005.

Q: I spoke with someone from your technical service over the phone and got the impression that your product dermorphin-SAP (Cat. #IT-12) is not a retrograde and will only affect the terminals or the cells that express mu opioid receptors in the injection site in the brain. I have three questions:

1) Do you have any written document on this issue?

A: That the peptide-toxins don’t undergo retrograde transport is an example of negative data, so people haven’t really been publishing too much on that. But two articles deal specifically with it: Lappi and Wiley[1] and Bugarith et al.[2] The latter, in particular, presents solid data on the inability of the peptide ligand toxin NPY-SAP (Cat. #IT-28) to undergo retrograde transport. I don’t think we have a single example of a peptide-ligand toxin that undergoes retrograde transport. In order for a peptide-toxin to kill cells, the cell body must have the receptor and the toxin must be injected within reach of the cell body. We’ve made a mistake in not putting that in the data sheets, and will begin to change that.

2) Will dermorphin-SAP also kill terminals in the injection site or just cell bodies?

A: Let me cite for you: Tokuno et al., Efferent projections from the striatal patch compartment: anterograde degeneration after selective ablation of neurons expressing mu-opioid receptor in rats.[3] As the title implies, they address the issue of elimination of processes following cell body destruction.

3) If it also kills terminals, will it affect their remote cell bodies?

A: I’m not sure I understand this question, but that won’t stop me from trying to answer it: The situation is the contrary, because the destruction of processes comes from the action taking place in the cell body. Our experience is that once the cell body is gone, it’s just a matter of time for the process to go away. This makes these toxins a little different than others. In fact, we recommend that you wait two weeks at least to see immunohistological evidence of a toxic effect after injection of a saporin toxin in vivo. That’s how long it takes the removal process to get rid of all the antigens that you might want to use for evidence of cell loss.

Q: Can I inject NPY-SAP to destroy projections through retrograde transport?

A: Regarding NPY-SAP, a peptide-toxin, see previous response. The antibody-toxins such as 192-IgG-SAP (Cat. #IT-01) or anti-DBH-SAP (Cat. #IT-03) will undergo retrograde transport from terminals to cell bodies. Thus, you can put 192-IgG-SAP into the cortex and it will destroy neurons in the basal forebrain, because the saporin (probably the whole conjugate) is transported from the projection to the cell body. Likewise, anti-DBH-SAP in the spinal cord destroyed hindbrain catecholaminergic neurons by retrograde transport.[4] All the antibody-toxins appear to undergo retrograde transport. 

Finally, the lectin-toxins, CTB-SAP (Cat. #IT-14) and IB4-SAP (Cat. #IT-10) undergo retrograde transport, just like the native lectins do. CTB-SAP is well-described in Llewellyn-Smith et al.[5] and several others. For IB4-SAP, Vulchanova et al.[6] describe use, along with several other articles on our reference page. In addition, detailed discussions are available in the book Molecular Neurosurgery with Targeted Toxins,[7] available from Humana Press.

See: Targeted Toxins

References

1. Lappi DA et al. Entering through the doors of perception: characterization of a highly selective Substance P receptor-targeted toxin. Neuropeptides 34(5):323-328, 2000.
2. Bugarith K et al. Basomedial hypothalamic injections of neuropeptide Y conjugated to saporin selectively disrupt hypothalamic controls of food intake. Endocrinology 146(3):1179-1191, 2005.
3. Tokuno H et al. Efferent projections from the striatal patch compartment: anterograde degeneration after selective ablation of neurons expressing mu-opioid receptor in rats. Neurosci Lett 332(1):5-8, 2002.
4. Ritter S et al. Immunotoxic destruction of distinct catecholamine subgroups produces selective impairment of glucoregulatory responses and neuronal activation. J Comp Neurol 432(2):197-216, 2001.
5. Llewellyn-Smith IJ et al. Retrogradely transported CTB-saporin kills sympathetic preganglionic neurons. Neuroreport 10(2):307-312, 1999.
6. Vulchanova L et al. Cytotoxic targeting of isolectin IB4-binding sensory neurons. Neuroscience 108(1):143-155, 2001.
7. Wiley RG et al. Molecular neurosurgery with targeted toxins. , 2005. Humana Press, Totowa, New Jersey

Bugarith K, Dinh TT, Li AJ, Speth RC, Ritter S (2005) Basomedial hypothalamic injections of neuropeptide Y conjugated to saporin selectively disrupt hypothalamic controls of food intake. Endocrinology 146(3):1179-1191. doi: 10.1210/en.2004-1166

Summary: The authors examined the effect of 48 ng injections of NPY-SAP (Cat. #IT-28) into the basomedial hypothalamus (BMH) on glucoprivic feeding in rats. While there was no evidence of retrograde transport, the lesions inhibited responses to intracerebroventricular leptin and ghrelin. Neither the feeding nor the hyperglycemic response to 2-deoxy-D-glucose was affected by the lesion, indicating that these hindbrain processes do not utilize neurons in the BMH. This work also describes dosing and injection parameter studies for the use of NPY-SAP.

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

Read the featured article in Targeting Trends.