Datta S, Chatterjee K, Wiley R (2010) Decreasing abnormal nocifensive responses in the bilateral chronic constriction injury (bCCI) model of neuropathic pain: Effects of lumbar intrathecal CCK-saporin. Neuroscience 2010 Abstracts 175.22/MM12. Society for Neuroscience, San Diego, CA.

Summary: The bCCI model produces long lasting -cold hyperalgesia (at least 100 days) along with decreases in staining for cholecystokinin (CCK) in the dorsal horn (DH). Spinal cholecystokinin (CCK) has anti-opiate activity, and selective destruction of DH neurons expressing CCK receptors by injection of intrathecal CCK-saporin, in naïve rats decreases thermal nocifensive reflex responses and is additive with morphine in decreasing nocifensive responses to heat. In the present study, we sought to determine the effects of intrathecal CCK-sap in the bCCI model of neuropathic pain in Long Evans female rats. bCCI rats underwent bilateral ligation of the sciatic nerves with chromic gut sutures. Controls underwent sham surgery with no ligation. Rats were tested on 0.3 C cold plate, thermal preference task (TPT) (shuttle box with floor temperatures of 15 C vs 45 C) and mechanical stimulation (von Frey). bCCI produced increased responses on the cold plate. 21 days after the bCCI surgery, the rats were injected with 1500 ng CCK-sap into the lumber CSF. Then, thermal and mechanical testing was repeated at intervals. Intrathecal CCK-sap injections decreased abnormal nocifensive responding of bCCI rats on the cold plate. CCK-sap reduced withdrawal responses to mechanical stimulation in bCCI rats. In TPT testing, the bCCI animals were hyperalgesic to cold (reduced cold side occupancy). After intrathecal CCK-sap injections, thermal preference was reversed (increased cold side occupancy). We interpret these results as showing that CCK-sap reverses abnormal nocifensive responses of bCCI in rats to aversive cold and mechanical stimuli. These results suggest that silencing CCK receptor-expressing superficial DH neurons is a potential strategy for development of new treatments for chronic neuropathic pain.

Related Products: CCK-SAP (Cat. #IT-31)

Datta S, Chatterjee K, Kline IV RH, Wiley RG (2009) CCK receptor- expressing dorsal horn neurons: Role in pain and morphine analgesia. Neuroscience 2009 Abstracts 265.13/Z37. Society for Neuroscience, Chicago, IL.

Summary: Spinal intrathecal cholecystokinin (CCK) has anti-opiate activity, and the CCK antagonist, proglumide potentiates opiate analgesia. In the present study, we sought to determine the effects of selectively destroying CCK receptor-expressing lumbar dorsal horn neurons using the targeted cytotoxin, CCK-saporin on reflex and operant nocifensive responses to heat, and on the actions of systemic morphine and naloxone. Exp. 1: Adult, female rats were injected into the lumbar CSF with either 1500 ng of CCK-sap (n=7) or blank (control nonsense peptide)-saporin (n=6). Exp. 2: rats were pre-injected intrathecally with 1 ug of proglumide (CCK antagonist) followed by 1500 ng CCK-sap (n=4) or only CCK-sap (1500 ng; n=4). Rats were then tested on the hotplate at 44°C and 47°C and on an operant thermal preference task (TPT) using a shuttle box where the floor on one side was 15°C and the other 45°C. Morphine was tested in the TPT using 0, 0.5, 1.5 and 2.5 mg/kg s.c. 4-8 weeks post-toxin. Naloxone (0 vs 0.8 mg/kg s.c) was also tested in the TPT. In Exp. 1, the CCK- sap group showed decreased hotplate reflex responses, but decreased time on the 45°C side in the TPT. In Exp. 2, CCK-sap only rats also showed greater heat aversion in the TPT. In both Exps, CCK-sap groups demonstrated greater heat aversion (less analgesia) than either control group after morphine in the TPT. After naloxone, both control groups, but not the CCK-sap rats, showed increased heat aversion (hyperalgesia). We interpret these results as showing that selective destruction of CCK receptor- expressing superficial dorsal horn neurons increases nocifensive reflex responses to aversive heat and produces thermal hyperalgesia while decreasing the effects of both morphine and naloxone suggesting a complex role for CCK receptor-expressing dorsal horn neurons in modulation of nociception and opiate drug action.

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Zhang W, Gardell S, Zhang D, Xie JY, Agnes RS, Badghisi H, Hruby VJ, Rance N, Ossipov MH, Vanderah TW, Porreca F, Lai J (2009) Neuropathic pain is maintained by brainstem neurons co-expressing opioid and cholecystokinin receptors. Brain 132:778-787. doi: 10.1093/brain/awn330

Summary: It has been hypothesized that a subset of rostral ventromedial medulla (RVM) neurons co-expressing the cholecystokinin type 2 receptor and the mu-opioid receptor are responsible for the maintenance of neuropathic pain. Rats were treated with 50-ng bilateral RVM injections of Dermorphin-SAP (Cat. #IT-12), CCK-SAP (Cat. #IT-31), or saporin (Cat. #PR-01) as a control. Lesion of the RVM neurons prevented hyperalgesia in response to CCK treatment, and shortened abnormal pain states caused by sciatic nerve injury.

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), CCK-SAP (Cat. #IT-31), Saporin (Cat. #PR-01)

Datta S, Chatterjee K, Kline IV RH, Wiley RG (2008) Lumbar intrathecal CCK-saporin: anatomic and nociceptive effects. Neuroscience 2008 Abstracts 773.4/MM32. Society for Neuroscience, Washington, DC.

Summary: Lumbar intrathecal CCK (cholecystokinin) appears anti-opiate in nocifensive reflex testing and may be important in opiate-resistant neuropathic pain states suggesting a role for CCK receptor-expressing dorsal horn neurons in nociception. In the present study, we sought to determine if selective destruction of CCK receptor-expressing superficial dorsal horn neurons alters pain sensitivity or the analgesic potency of morphine using the targeted cytotoxic conjugate (CCK-sap) of CCK to saporin, a ribosome inactivating protein. 28 adult Sprague Dawley rats were injected via lumbar intrathecal catheter with CCK-sap in doses of 500 ng (n=2), 350 ng (n=3), 700 ng (n=3), 1000 ng (n=4), 1500 ng (n=4), or 3000 ng (n=4). Controls included PBS (n=4) or 1500 ng of plain, unconjugated saporin (n=4). 2 weeks later rats were sacrificed. Lumbar spinal cords were frozen sectioned at 40 µm. One-in-six series of transverse sections at L4-6 were immunostained for CCK. Two rats were injected with 1500 ng of CCK-sap followed by transcardiac aldehyde perfusion in 72 hours. L5 Dorsal root ganglia (DRG) sections were stained with cresyl violet and examined for signs of acute cytotoxicity (chromatolysis and karyohexis). 350 to 1500 ng of intrathecal CCK-sap were well tolerated with no obvious signs of any toxicity. 3000 ng of intrathecal saporin led to motor signs within 72 hours including increased muscle tone, leading to tonic hind limbs extension. Subsequently, twelve Long Evans female rats were tested before and after intrathecal injection of either PBS (n=8) or CCK-sap, 1500 ng (n=4) on: 1 – cold plate (15 °C); 2 – thermal preference shuttle box testing (15/45°C); 3 – hotplate at 44°C, 47°C and 52°C and 4 – thermal preference after morphine (0.5, 1 and 2.5 mg/kg s.c). Anatomical analysis revealed that 1500 ng of CCK-sap decreased CCK immunostaining in the L4-6 Dorsal horn. No acute cytotoxicity was seen in the DRG with1500 ng CCK-sap. Intrathecal CCK-sap was well tolerated at doses ≤1500 ng. CCK-sap produced increased hot side time and decreased crossovers in the thermal preference test. In contrast, CCK-sap decreased latency to first hindpaw lift and increased total responding on the 44 °C hotplate. CCK-sap rats also showed increased hot side time at 45° C at all morphine doses (0, 1 and 2.5 mg/kg s.c.) also with decreased crossovers. We interpret these observations to indicate that CCK-sap produced increased nocifensive reflex responding on the 44° C hotplate consistent with positive modulation of motor responsiveness, and CCK-sap reduced aversion to 45° C heat consistent with an analgesic effect that was additive with morphine.

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Edelmayer RM, Vanderah TW, Majuta L, Fioravanti B, De Felice M, Chichorro JG, Ossipov MH, King T,Lai J, Kori SH, Nelsen AC, Cannon KE, Heinricher MM, Porreca F (2008) A brainstem generator for cutaneous allodynia associated with migraine headache. Neuroscience 2008 Abstracts 171.15/LL16. Society for Neuroscience, Washington, DC.

Summary: Migraine patients often demonstrate cutaneous allodynia that begins unilaterally and intracranially and spreads, via unknown mechanisms, to contralateral and extracranial body regions. As cutaneous allodynia likely reflects the development of central sensitization, we hypothesized that descending facilitatory influences from the rostral ventromedial medulla (RVM) might underlie the generalized expression of this phenomenon. We employed a modified model of application of inflammatory mediators (IM) to the dura of unanesthetized animals and explored the possible requirement of a brainstem site for expression of generalized cutaneous allodynia. Rats were surgically implanted with two cannulas, one of which permitted the application of IM to the surface of the dura and the other for administration of compounds to the RVM, 7 days after surgery. Tactile withdrawal thresholds of the peri-ocular region of the face as well as the hindpaws were tested pre-surgery, post-surgery, and up to 6 hr after application of IM. Bupivacaine or YM022 (CCK2 receptor antagonist) were administered to the RVM at various times after IM. In some studies dermorphin-saporin was administered as a single microinjection to elicit a cytotoxic effect on presumed pain facilitation cells in the RVM; these rats were tested with IM after a further 28 days. Recordings of RVM “ON” and “OFF” cell activity were also performed in separate groups of naïve animals prior to, and after, IM application to the dura. Dural IM produced robust facial and hindpaw allodynia which peaked after approximately 3 hr and recovered to baseline thresholds by approximately 6 hr. RVM bupivacaine, YMO22, or cytotoxic destruction of pain facilitation cells had no effects on sensory thresholds alone, but prevented or significantly attenuated the expression of IM-induced cutaneous allodynia. In addition, IM applied to the dura produced a sustained increase in the discharge of RVM ON cells while transiently inhibiting OFF cells. Facial and hindpaw allodynia associated with dural stimulation may be a useful surrogate of migraine-associated pain which may be exploited mechanistically for the development of novel therapeutic strategies. The data demonstrate the requirement of descending facilitation from the RVM for the expression of cranial and extracranial cutaneous hypersensitivity and offer direct evidence of brainstem involvement in cutaneous allodynia associated with headache pain.

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Lai J, Zhang W, Badghisi H, Hruby VJ, Porreca F (2006) Featured Article: The biologically active cholecystokinin (26-33) peptide, [Tyr2-SO3]CCK-8, retains high affinity for CCK2 receptors after covalent conjugation to saporin. Targeting Trends 7(1)

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Read the featured article in Targeting Trends.

See Also:

• Lai J et al. Featured Article: The biologically active cholecystokinin (26-33) peptide, [Tyr2-SO3]CCK-8, retains high affinity for CCK2 receptors after covalent conjugation to saporin. Targeting Trends 7(1), 2006.

Zhang W, Gardell SE, Xie Y, Luo M, Rance NE, Vanderah TW, Porreca F, Lai J (2005) Pain facilitatory cells in the rostral ventromedial medulla coexpress opioid-μ receptors and cholecystokinin type 2 receptors. Neuroscience 2005 Abstracts 394.17. Society for Neuroscience, Washington, DC.

Summary: Pain transmission can be modulated by descending input to the spinal dorsal horn from the rostral ventromedial medulla (RVM). RVM neurons that facilitate nociception are termed “ON-cells”, which are inhibited by mu-opioids, suggesting that they express opioid mu receptors (MOR). Focal application of cholecystokinin (CCK8(s)) into the RVM elicits acute thermal and tactile hypersensitivity and induces ON-cell activity. In situ hybridization using riboprobes for either rat MOR or rat cholecystokinin type-2 receptor (CCK-2) confirms the expression of these receptors in the RVM. Pretreatment with a toxin conjugate, CCK8(s)-saporin results in a significant loss of CCK-2 positive cells in the RVM, concomitant with a blockade of CCK8(s) induced hyperalgesia. The pretreatment also significantly reduces the number of neurons labeled for MOR in the RVM, suggesting that MOR and CCK-2 may be co-localized in some RVM cells. Consistent with these data, similar pretreatment with the toxin conjugate, dermorphin-saporin, which selectively targets MOR expressing neurons, significantly reduces the number of MOR labeled cells in the RVM, blocks RVM CCK8(s) induced hyperalgesia and reduces the number of CCK-2 positive cells in the RVM. In situ hybridization using 35S-labeled CCK-2 riboprobes and Digoxigenin-labeled MOR riboprobes shows that over 80% of labeled RVM neurons co-express both MOR and CCK-2, ~15% express only CCK-2, and very few cells express only MOR. These findings represent the first direct demonstration of the phenotype of pain facilitatory neurons in the RVM. Together with previous studies showing that RVM CCK-2 antagonists reverse nerve injury-induced pain, this phenotype provides strong support for the view that endogenous CCK is a critical mediator of the descending pain facilitation, particularly in the maintenance of experimental neuropathic pain. Support Contributed By: NIDCR R01 DE016458

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), CCK-SAP (Cat. #IT-31)

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Zhang J, Speth RC, Simasko S, Ritter RC (2003) Hypothalamic injection of targeted toxin for cholecystokinin receptive neurons leads to increased 24 hour food intake and weight gain. Neuroscience 2003 Abstracts 830.3. Society for Neuroscience, New Orleans, LA.

Summary: Peptides conjugated to the ribosomal toxin, saporin, bind to their specific G-protein coupled receptors, and are internalized. Once internalized, saporin inactivates ribosomes, selectively killing the receptive cells. We are using cholecystokinin (CCK)-saporin to selectively destroy CCK receptive neurons that may participate in the control of food intake and body weight. We have demonstrated that CCK-saporin binds to CCK-A receptors (Approximate IC50, 3nM), and evokes an increase in cytosolic calcium, which is blocked by the CCK-A receptor antagonist, lorglumide. Thus CCK-saporin has properties that recommend it as a targeted toxin of CCK-receptive neurons. We injected CCK-saporin (138 nM in 500 nl) bilaterally into the medial hypothalamus, an area where CCK-A receptors are expressed. CCK-saporin did not change 24h chow intake or weight gain. However, when rats were fed high fat diet, CCK-saporin treated rats increased their 24h food intake and gained nearly twice the weight as control rats during 14 days on this diet. Following an overnight fast CCK-saporin injected rats ate significantly more high fat diet than controls during the first 30 min after return of food. Nonetheless, both the CCK-saporin injected rats and controls reduced their food intake in response to intraperitoneal CCK-8. Our results suggest that ventromedial hypothalamic CCK receptors participate in control of 24h food intake and body weight gain. Our results also suggest that CCK-saporin may be a valuable tool for investigating the participation of discrete populations of CCK-sensitive neurons in various physiological responses.

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Treece BR, Speth RC, Ritter RC, Burns GA (2003) Altered CCK binding in the dorsal vagal complex following cytotoxic lesion of the nodose ganglion. Neuroscience 2003 Abstracts 830.5. Society for Neuroscience, New Orleans, LA.

Summary: CCK reduces meal size by activating a specific subpopulation (30%-38%) of vagal afferent neurons. MK-801, attenuates reduction of food intake by CCK and increases meal size. We hypothesized that vagal afferents that are sensitive to excitatory amino acids might express CCK receptors and thereby mediate effects of both CCK and MK-801. Therefore we examined I125CCK-8 binding in the dorsal vagal complex, following unilateral nodosectomy or cytotoxic lesion of nodose cell bodies. To destroy vagal afferent cell bodies and their central projections we made unilateral intranodose injections of NMDA/kainic acid, which has been shown to destroy about 40% of vagal afferents. We also made intranodose injections of CCK-saporin (CCK-SAP), a novel cytotoxin, which appears to destroy vagal afferents when the ribosomal toxin, saporin, is selectively internalized along with the CCK/receptor complex. In some rats we completely eliminated vagal afferents on one side via nodosectomy. We found that this produced significant ipsilateral reduction in CCK binding in the nucleus of the solitary tract (NTS). Intranodose injection of NMDA/kainate or CCK-SAP also resulted in significantly reduced CCK binding in the NTS, ipsilateral to the injected nodose. The reduction of NTS CCK binding following nodosectomy, NMDA/kainate or CCK-SAP injection did not differ significantly. Interestingly, CCK binding appeared to be reduced in the vagal dorsal motor nucleus, ipsilateral to nodose removal, but was increased ipsilateral to CCK-SAP injection. We conclude that CCK-SAP destroys a subpopulation of vagal afferents that express CCK receptors and that intranodose NMDA/kainate destroys a partially overlapping afferent subpopulation that also expresses CCK receptors. Finally, our data suggest that expression of CCK binding in the dorsal motor nucleus may be controlled by feedback from CCK sensitive vagal afferents.

Related Products: CCK-SAP (Cat. #IT-31)

Xie Y, Vanderah TW, Ossipov MH, Lai J, Porreca F (2003) A single rostral ventromedial medulla (RVM) treatment with cholecystokinin-saporin (CCK-sap) prevents the development of opioid-induced paradoxical pain and spinal morphine antinociceptive tolerance. Neuroscience 2003 Abstracts 177.4. Society for Neuroscience, New Orleans, LA.

Summary: Sustained morphine elicits tactile and thermal hypersensitivity (opioid-induced paradoxical pain) and antinociceptive tolerance which are mediated through the time-dependent activation of descending facilitation from the RVM. With morphine exposure, CCK expression and/or release may be altered to activate pain facilitatory neurons of the RVM, manifesting as diminished spinal morphine antinociception (antinociceptive tolerance). To explore a possible role of RVM CCK in morphine-induced paradoxical pain and tolerance, CCK-SAP conjugate was used to selectively lesioned RVM neurons expressing CCK receptors. Male S-D rats received a single RVM injection of CCK, SAP or CCK-SAP. Behavioral responses to tactile (von Frey) and thermal (radiant heat) stimuli were normal 3,7,14 and 28 days after injection. RVM CCK microinjection produced tactile and thermal hypersensitivity in uninjured rats 28 days after receiving RVM CCK or SAP, but not in those receiving CCK-SAP, suggesting the probable loss of RVM CCK receptor-expressing cells. 28 days after RVM CCK, SAP or CCK-SAP injections, rats were implanted with placebo or morphine pellets. Morphine pelleted rats pretreated with RVM CCK or SAP developed tactile and thermal hypersensitivity and spinal antinociceptive tolerance. In contrast, animals pretreated with RVM CCK-SAP did not show morphine induced tactile or thermal hypersensitivity and antinociceptive tolerance was not present. Moreover, CCK-SAP, but not CCK or SAP, pretreatment significantly attenuated the antinociceptive effect of RVM morphine. This suggests that RVM CCK activates tonic descending facilitation driving morphine-induced abnormal pain and spinal antinociceptive tolerance. Moreover, these results suggest the possibility that CCK and opioid receptors may colocalize on some RVM neurons which may act to facilitate pain transmission.

Related Products: CCK-SAP (Cat. #IT-31)