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.

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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.

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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.

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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.

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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.

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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)

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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.

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.

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Read the featured article in Targeting Trends.

Bugarith KH, Li A, Dinh TT, Ritter S (2004) Injection of the targeted-toxin, neuropeptide Y-saporin (NPY-SAP), into the basomedial hypothalamus (BMH) disrupts leptin and ghrelin signaling. Neuroscience 2004 Abstracts 893.17. Society for Neuroscience, San Diego, CA.

Summary: NPY-SAP, a conjugate of the peptide NPY and saporin, a ribosomal inactivating toxin, specifically lesions NPY receptor-expressing cells. We injected NPY-SAP into the BMH and examined the effects of various inhibitory (leptin, 5ug/5ul/day, icv; GLP-1, 5ug/5ul, icv, CCK, 4ug/kg, ip;) and stimulatory (ghrelin, 2ug/5ul, icv; NPY, 500ng/100nl, icv; 2-DG, 100, 200 and 400 mg/kg; MA, 68mg/kg, ip) peptide and metabolic signals that influence food intake. We also examined the effect of NPY-SAP on NPY, CART and AGRP mRNA expression in NPY/AGRP and POMC/CART neurons known to express the NPY receptor, and the effect of NPY and NPY Y1 receptor immunoreactivity in the arcuate (Arc) nucleus. We found that the anorectic effects of leptin and the orexigenic effects of ghrelin were abolished by NPY-SAP. The stimulation of feeding induced by NPY, 2-DG and MA, and the suppression of deprivation-induced feeding by GLP-1 and CCK were not attenuated by NPY-SAP injection. There was a profound but localized reduction of NPY Y1 receptor-, and NPY fiber and terminal immunoreactivity, and NPY, AGRP and CART mRNA expression in the Arc. NPY-SAP did not appear to be retrogradely transported in hindbrain NPY neurons with hypothalamic terminals. Leptin and ghrelin are thought to act primarily on Arc NPY/AGRP and POMC/CART neurons to mediate their ingestive effects, whereas the effects of 2-DG, MA, CCK and GLP-1 are thought to be mediated in part by mechanisms outside the Arc. Present results show that BMH injections of NPY-SAP selectively impair controls mediated by Arc neural circuitry without causing widespread disruption of other ingestive behaviors. Results also reveal important ingestive controls that do not require Arc NPY/AGRP and POMC/CART neurons.

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Wiley RG, Kline IV, RHLappi DA (2004) Intrathecal galanin-saporin and NPY-saporin reduce nocifensive responses to noxious heat and formalin. Neuroscience 2004 Abstracts 292.15. Society for Neuroscience, San Diego, CA.

Summary: Although the precise circuitry of the dorsal horn underlying nociception is not fully understood, there is evidence that regulation of the excitability of nociceptive projection neurons is influenced/modulated by excitatory interneurons. The present study sought to determine if selectively destroying presumed excitatory interneurons in the superficial dorsal horn would alter nocifensive responses to noxious thermal or chemical stimuli. The strategy chosen was to inject saporin (SAP) conjugates of either galanin (GAL) or neuropeptide Y (NPY) into the lumbar subarachnoid space and then test rats on the hotplate and observe the nocifensive responses to hindpaw formalin injection. After hotplate testing for 2 weeks, staining for c-fos expression in the dorsal horn was performed 2 hrs after hindpaw formalin injection. Lumbar intrathecal injection of 500 ng of either GAL-SAP or NPY-SAP produced no obvious change in appearance, body weight or spontaneous activity of adult male Sprague-Dawley rats. Both toxins reduced responses on the 44 C hotplate but not at 52 C. Nocifensive responses to the 47 C hotplate also were reduced but not as strikingly as at 44 C. Responses to hindpaw formalin were remarkably different. Toxin-injected rats held the injected foot close to the body, off the floor, throughout the 90 minute observation period but otherwise ignored the injected paw. Unlike controls, toxin-injected rats did not shake, lick or bite the injected hindpaw and showed normal exploratory behavior. These results are interpreted as showing that these two toxins likely destroy excitatory interneurons in the superficial dorsal horn resulting in decreased excitability of nociceptive projection neurons, and therefor reduced sensitivity to noxious thermal and chemical stimuli.

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Bugarith K, Ritter S, Dinh T (2001) Hyperphagia and obesity results from the injection of the immunotoxin neuropeptide Y (NPY)-saporin (NPY-sap) into the paraventricular hypothalamus (PVH) of rats. Neuroscience 2001 Abstracts 948.2. Society for Neuroscience, San Diego, CA.

Summary: NPY is a peptide implicated in the control of numerous physiological processes. A variety of G-protein coupled receptors, Y1, Y2, Y4, and Y5, mediate the actions of NPY. Recently a new immunotoxin, NPY-SAP, has been developed that is potentially of great importance for the study of NPY function. NPY-SAP is a conjugate of the peptide NPY and saporin, a plant toxin that inactivates ribosomes. The proposed mechanism of toxicity involves immunotoxin binding NPY receptors and destruction of neurons containing these receptors. In this study we injected different doses of NPY-sap or unconjugated saporin (SAP) into the PVH to test the effects of this new toxin. We found that at low doses, there was no effect of NPY-SAP on cytoarchitecture or immunoreactivity of select peptides in the PVH. There was also no difference in body weight between the groups. At high doses, there was histologically-detectable damage in the hypothalamus of NPY-SAP animals. We also found a major difference in body weight between the NPY-SAP rats and SAP controls. Ten weeks after the injections, the NPY-SAP rats (654.3g ± 39.04g) were much heavier than the SAP rats (410.6g ± 15.29g). Further, daytime (0800 – 1700H) food intake was about twice as much in NPY-SAP (9.53g ± 0.996g) as in SAP (5.74g ± 0.476g) rats, with no difference in overnight (1700 – 0800H) feeding. Work is ongoing to determine the specificity of the lesion and the mechanism of action of NPY-SAP.

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