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Distinct roles of P2X receptors in modulating glutamate release at different primary sensory synapses in rat spinal cord.
Nakatsuka T, Tsuzuki K, Ling JX, Sonobe H, Gu JG (2003) Distinct roles of P2X receptors in modulating glutamate release at different primary sensory synapses in rat spinal cord. J Neurophysiol 89(6):3243-3252. doi: 10.1152/jn.01172.2002
Summary: P2X receptors are important modulating neurons in the spinal cord. These authors used IB4-SAP (Cat. #IT-10) to target a neuronal subset, those neurons expressing P2X3 receptors. 2 µg of IB4-SAP were injected directly into the sciatic nerve on one side. Histological examination showed efficient removal of IB4 and P2X3-staining ipsilaterally in the dorsal horn outer laminae. Behavioral experiments showed intact modulation of glutamate release in the absence of P2X3-positive neurons, indicating involvement by other P2X neurons.
Related Products: IB4-SAP (Cat. #IT-10)
Retrograde Transport
Q: I’m interested in using SAP to eliminate cells through retrograde transport, like OX7-SAP (Cat. #IT-02) and IB4-SAP (Cat. #IT-10) have been used. Can you explain how retrograde transport works and if it is possible for this to work with dermorphin-SAP (Cat. #IT-12)? What determines whether a targeted toxin will be able to be used in retrograde transport?
A: Current evidence indicates that effective suicide transport agents undergo endocytosis at nerve terminals followed by retrograde axonal transport of the endocytic vesicles containing the toxin. Experiments using vincristine have shown that the retrograde axonal transport of suicide transport toxins utilizes the fast transport system (microtubules). However, it is not known what determines whether or not a specific toxin-ligand undergoes axonal transport after internalization.
Empirically, it has been observed that immunotoxins (OX7-SAP, 192-Saporin or 192-IgG-SAP [Cat. #IT-01], anti-DBH-SAP [Cat. #IT-03]) and lectin-toxins (ricin, volkensin, IB4-SAP) all undergo retrograde axonal transport and are therefore effective suicide transport agents. This is not true, however, for neuropeptide-toxin conjugates, such as dermorphin-SAP. For example, in an unpublished study, we injected large doses (1-2 µg) of dermorphin-SAP into the lumbar intrathecal space of rats. After 2-3 days, rats were sacrificed and lumbar dorsal root ganglia examined for evidence of toxin effect (striking chromatolysis). None was found after examining numerous ganglia and >15,000 primary afferent neurons. Apparently, dermorphin-SAP is not retrogradely transported even if it is taken into the primary afferent terminals that express the mu opioid receptor (MOR).
Q: If a targeted toxin cannot be used in retrograde transport, will it only kill cell bodies in the injection site or will it also kill terminals?
A: Current evidence suggests that applying dermorphin-SAP (Cat. #IT-12) to the population of MOR-expressing neurons in the dorsal horn of the spinal cord results in destruction only of the neurons in lamina II and not the primary afferent terminals that also express MOR. This may be a general principle but it has not been tested in any other situation for dermorphin-SAP, nor have SP-SAP (Cat. #IT-07) and SSP-SAP (Cat. #IT-11) been evaluated for terminal uptake and suicide transport. Any saporin taken into a nerve terminal should not be toxic unless retrogradely transported to the cell body since there are no ribosomes (site of saporin action) or protein synthesis in the nerve terminal.
See: Targeted Toxins
Suggested Reading:
- Oeltmann TN, Wiley RG (1986) Wheat germ agglutinin-ricin A-chain conjugate is neuronotoxic after vagal injection. Brain Res 377:221-228.
- Wiley RG, Stirpe F, Thorpe P, Oeltmann TN (1989) Neuronotoxic effects of monoclonal anti-Thy 1 antibody (OX7) coupled to the ribosome inactivating protein, saporin, as studied by suicide transport experiments in the rat. Brain Res 505:44-54.
- Contestabile A, Fasolo A, Virgili M, Migani P, Villani L, Stirpe F (1990) Anatomical and neurochemical evidence for suicide transport of a toxic lectin, volkensin, injected in the rat dorsal hippocampus. Brain Res 537(1-2):279-286.
- Pangalos MN, Francis PT, Pearson RC, Middlemiss DN, Bowen DM (1991) Destruction of a sub-population of cortical neurones by suicide transport of volkensin, a lectin from Adenia volkensii. J Neurosci Methods 40(1):17-29.
- Wiley RG (1992) Neural lesioning with ribosome-inactivating proteins: suicide transport and immunolesioning. Trends in Neurosci 15:285-290.
- Roberts RC, Harrison MB, Francis SMN, Wiley RG (1993) Differential effects of suicide transport lesions of the striatonigral or striatopallidal pathways on subsets of striatal neurons. Exp Neurol 124:242-252.
- Contestabile A, Stirpe F (1993) Ribosome-inactivating proteins from plants as agents for suicide transport and immunolesioning in the nervous system. Eur J Neurosci 5:1292-1301.
- Wiley RG, Lappi DA(1994) Suicide Transport and Immunolesioning. R.G. Landes, Houston.
- Roberts RC, Strain-Saloum C, Wiley RG (1995) Effects of suicide transport lesions of the striatopallidal or striatonigral pathways on striatal ultrastructure.Brain Res 710:227-237.
- Wiley RG, Kline IVRH (2000) Neuronal lesioning with axonally transported toxins. J Neurosci Methods 103:73-82.
Featured Article: Biotinylated targeting: A viable option?
Lappi DA (2003) Featured Article: Biotinylated targeting: A viable option?. Targeting Trends 4(2)
Related Products: IB4-SAP (Cat. #IT-10)
Loss of IB4 staining in dorsal root ganglion neurons after spinal nerve ligation is not the result of cell death.
Arunkumar R, Ackerman LL, Jones III R, Holdsworth R, Proudfit HK, Hammond DL (2002) Loss of IB4 staining in dorsal root ganglion neurons after spinal nerve ligation is not the result of cell death. IASP 2002 Abstracts International Association for the Study of Pain, San Diego, CA.
Related Products: IB4-SAP (Cat. #IT-10)
Loss of IB4-positive sensory neurons mitigates the consequences of nerve injury in the rat.
Tarpley JW, MacIntyre E, Martin WJ (2002) Loss of IB4-positive sensory neurons mitigates the consequences of nerve injury in the rat. IASP 2002 Abstracts International Association for the Study of Pain, San Diego, CA.
Related Products: IB4-SAP (Cat. #IT-10)
Toxin Safety
Safety Instructions
Good laboratory technique must be employed for the safe handling of this product. This requires observation of the following practices:
1. Wear appropriate laboratory attire, including lab coat, gloves and safety glasses.
2. Do not pipet by mouth, inhale, ingest or allow product to come into contact with open wounds. Wash thoroughly any part of the body which comes into contact with the product.
3. Avoid accidental autoinjection by exercising extreme care when handling in conjunction with any injection device.
4. This product is intended for research use by qualified personnel only. It is not intended for use in humans or as a diagnostic agent. Advanced Targeting Systems is not liable for any damages resulting from the misuse or handling of this product.
For disposal: autoclave, or expose to 0.2 M NaOH, materials that come into contact with the toxin.
Q: We’re submitting a protocol to our IACUC to use IB4-SAP (Cat. #IT-10). We plan to inject the targeted toxin and then sacrifice the animal ten days later. What, if any, are the safety issues here?
A: The only danger to lab personnel from IB4-SAP would be accidental self-injection, and even then, at the doses typically used in rats, it would only produce very localized effects at the injection site.
Once injected into animals, the agent is rapidly rendered inaccessible to anyone else by binding, internalization and eventual catabolism. It is extremely unlikely that intact toxin would ever be excreted or recoverable from the rats. The components of the toxin, IB4 and saporin, by themselves are no toxic threat. We use no special precautions with such rats except appropriate care for whatever neurologic deficits they develop, i.e. foot drop, autotomy, etc.
One caveat: To the best of my knowledge the above statements are accurate, but I do not know of any experimental data that directly addresses the issues. I base my comments on our long experience with similar agents including ricin and volkensin which are much more toxic and unstable.
See: Targeted Toxins
Cytotoxic targeting of isolectin IB4-binding sensory neurons.
Vulchanova L, Olson TH, Stone LS, Riedl MS, Elde R, Honda CN (2001) Cytotoxic targeting of isolectin IB4-binding sensory neurons. Neuroscience 108(1):143-155. doi: 10.1016/s0306-4522(01)00377-3 PMID: 11738138
Summary: Vulchanova et al. examine the role of IB4-binding neurons in nociception. IB4-SAP (Cat. #IT-10) was injected into rats (2 µg in left sciatic nerve). The resulting ablation of IB4-binding neurons provides evidence for their role in nociceptive processing and demonstrates a rapid compensatory response to signalling of acute pain.
Related Products: IB4-SAP (Cat. #IT-10), Saporin Goat Polyclonal (Cat. #AB-15), Saporin Goat Polyclonal, HRP-labeled (Cat. #AB-15HRP)
Capsaicin-sensitive inhibitory pathway in rat spinal cord dorsal horn.
Gu JG, Nakatsuka T, Tanaka E, Takeda D, Jennifer LX (2001) Capsaicin-sensitive inhibitory pathway in rat spinal cord dorsal horn. Neuroscience 2001 Abstracts 158.13. Society for Neuroscience, San Diego, CA.
Summary: The inhibitory system in the spinal cord plays an important role in regulating nociceptive sensory inputs. Here we examined inhibitory synaptic activity in lamina V neurons of the spinal dorsal horn following the activation of capsaicin VR1 receptors. Experiments were performed with spinal cord slice preparations and inhibitory postsynaptic currents (IPSCs) were recorded using patch-clamp technique. Bath application of capsaicin (2 μM) increased the amplitude and frequency of GABAergic and glycinergic spontaneous IPSCs in the majority of lamina V neurons tested. The effects of capsaicin were completely antagonized by capsazepine (10 μM), and were also blocked in the presence of tetrodotoxin (0.5 μM). However, when CNQX (20 μM) and APV (100 μM) were used to block glutamatergic synaptic transmission, the effects of capsaicin were not abolished. Furthermore, after the injection of IB4-saporin into sciatic nerve to remove IB4-positive C-primary afferent terminals, capsaicin still increased sIPSC frequency in the presence of CNQX and APV. These results suggest that inhibitory pathway could be recruited in the absence of glutamatergic inputs from primary afferents. The release of neuropeptides from capsaicin-sensitive C-primary afferents may activate GABAergic and glycinergic interneurons in superficial laminae, and the inhibitory activity may be further forwarded to lamina V neurons. The capsaicin-sensitive inhibitory pathway may play an important role in the control of nociceptive transmission in the spinal cord.
Related Products: IB4-SAP (Cat. #IT-10)
α,β-methylene ATP sensitive P2X receptor mediated enhancement of glutamate release from the central terminals of Aδ primary afferents onto lamina V neurons in rat spinal cord.
Nakatsuka T, Takeda D, Gu JG (2001) α,β-methylene ATP sensitive P2X receptor mediated enhancement of glutamate release from the central terminals of Aδ primary afferents onto lamina V neurons in rat spinal cord. Neuroscience 2001 Abstracts 158.16. Society for Neuroscience, San Diego, CA.
Summary: We examined the role of αβmATP-sensitive P2X receptors in modulating glutamate release from sensory synapses of the spinal cord by using whole-cell patch-clamp recordings from dorsal horn neurons in lamina V region. The majority of lamina V neurons synapsed with terminals expressing αβmATP-sensitive P2X receptors. Application of P2X receptor agonist 100 μM αβmATP resulted in a large increase in mEPSC frequency. The increases in mEPSC frequency by αβmATP were completely abolished by the P2X receptor antagonist 10 μM PPADS, but were not blocked by Ca2+ channel blocker 30 μM La3+. αβmATP remained to be effective in increasing mEPSC frequency after the removal of superficial dorsal horn (lamina I-III) or after the injection of IB4-saporin into sciatic nerve to remove P2X3 expressing afferent terminals. Furthermore, we found that αβmATP-sensitive synapses of lamina V neurons were associated with central terminals derived from Aδ primary afferents. The EPSCs evoked by dorsal root stimulation at Aδ-fiber intensity were potentiated by 1 μM αβmATP as well as by the ecto-ATPase inhibitor 10 μM ARL67156, and depressed in the presence of 10 μM PPADS and 5 μM suramin. These results suggest that αβmATP-sensitive P2X receptors play a significant role in modulating excitatory synaptic transmission in the spinal cord.
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Contribution of IB4-positive sensory neurons to NGF-induced hyperalgesia in the rat.
Tarpley JW, Martin WJ, Baldwin BS, Forrest MJ, MacIntyre DE (2000) Contribution of IB4-positive sensory neurons to NGF-induced hyperalgesia in the rat. Neuroscience 2000 Abstracts 633.18. Society for Neuroscience, New Orleans, LA.
Summary: Sensory information is transmitted from the periphery to the spinal cord by distinct subsets of primary afferent neurons, including two major classes of C-fibers that are distinguished by their ability to bind the lectin IB4. IB4-positive neurons are primarily non-peptidergic, express the receptor tyrosine kinase, Ret, and are preferentially sensitive to the neurotrophic factor, GDNF. By contrast, the nerve growth factor (NGF) receptor tyrosine kinase, trkA, is predominantly expressed in IB4-negative neurons that contain substance P. Previous work suggested that depletion of IB4-positive neurons increases acute nociceptive thresholds to noxious thermal stimuli. The extent to which these non-peptidergic neurons mediate changes in nociception after injury is unknown. Here, we examined the contribution of IB4-positive, non-peptidergic, neurons to thermal and mechanical sensitivity after acute tissue injury. Baseline thermal (radiant heat) and mechanical (von Frey) thresholds were measured in rats prior to injection of IB4-saporin (5 ug/5 ul) into the left sciatic nerve. By two weeks, IB4-saporin-treated animals exhibited pronounced increases in their nociceptive thresholds to thermal and mechanical stimuli. At this time, treatment with NGF (5 ug/50 ul, i.pl.) increased paw thickness in both control and IB4-saporin-treated rats. However, nociceptive thresholds were significantly lowered in control rats, but not in those treated with IB4-saporin. This suggests that IB4-positive neurons contribute to injury-induced changes in thermal and mechanical sensitivity and provide insight into the function of this unique set of primary afferent neurons.
Related Products: IB4-SAP (Cat. #IT-10)