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Amygdala intercalated neurons are required for expression of fear extinction.
Likhtik E, Popa D, Apergis-Schoute J, Fidacaro GA, Pare D (2008) Amygdala intercalated neurons are required for expression of fear extinction. Nature 454(7204):642-645. doi: 10.1038/nature07167
Summary: Scientists have been using fear learning in animals to study human anxiety disorders. In order to investigate the contribution of amygdala plasticity to fear learning, rats received 0.25-µl bilateral infusions of 3-µM dermorphin-SAP (Cat. #IT-12) into the amygdala. Blank-SAP (Cat. #IT-21) was used as a control. Lesioned rats displayed extinction expression deficits, indicating that the eliminated intercalated amygdala neurons play a large role in the extinction process.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), Blank-SAP (Cat. #IT-21)
Spinal mu-opioid receptor-expressing dorsal horn neurons: role in nociception and morphine antinociception.
Kline IV RH, Wiley RG (2008) Spinal mu-opioid receptor-expressing dorsal horn neurons: role in nociception and morphine antinociception. J Neurosci 28:904-913. doi: 10.1523/JNEUROSCI.4452-07.2008
Summary: The authors used Dermorphin-SAP (Cat. #IT-12) to investigate the function of spinal cord mu-opioid receptor (MOR)-expressing dorsal horn neurons in nociception and morphine analgesia. Rats were treated with 500 ng intrathecal injections of Dermorphin-SAP; 500 ng of Blank-SAP (Cat. #IT-21), and up to 1 µg of Saporin (Cat. #PR-01) were used as controls. The data indicate that MOR-expressing dorsal horn neurons are necessary for morphine action and play a role in nocifensive responses to persistent pain in the formalin test.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), Blank-SAP (Cat. #IT-21), Saporin (Cat. #PR-01)
Selective ablation of GABA neurons in the ventral tegmental area increases spontaneous locomotor activity.
Shank EJ, Seitz PK, Bubar MJ, Stutz SJ, Cunningham KA (2007) Selective ablation of GABA neurons in the ventral tegmental area increases spontaneous locomotor activity. Behav Neurosci 121:1224-1233. doi: 10.1037/0735-7044.121.6.1224
Summary: To further examine the importance of the ventral tegmental area (VTA) g-aminobutyric acid (GABA) neurons in behavioral function, the authors lesioned the VTA of rats with dermorphin-saporin (Cat. #IT-12). Lesioned animals received 1 or 2 pmol/200 nl bilateral injections of conjugate; blank-SAP (Cat. #IT-21) was used as a control. Rats treated with dermorphin-SAP displayed significantly elevated motility as compared to control animals. Rats receiving 1 pmol of dermorphin-SAP returned to normal motility 14 days after treatment, but rats receiving 2 pmol maintained the increased motility through day 14.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), Blank-SAP (Cat. #IT-21)
Lesioning mu opioid receptor-containing neurons in the ventrolateral periaqueductal gray attenuates morphine analgesia in male but not female rats
Loyd DR, Murphy AZ (2007) Lesioning mu opioid receptor-containing neurons in the ventrolateral periaqueductal gray attenuates morphine analgesia in male but not female rats. Neuroscience 2007 Abstracts 921.4/NN15. Society for Neuroscience, San Diego, CA.
Summary: Chronic pain will affect four out of five persons at some point across the lifespan. While the opioid-based narcotic morphine is the most prevalent treatment for chronic pain in clinical settings, it is becoming increasingly clear that morphine produces a significantly greater degree of analgesia in males compared to females. In both somatic and visceral pain models, the ED50 for morphine is generally two-fold higher for females than for males. The midbrain periaqueductal gray (PAG) and its descending projections to the rostral ventromedial medulla (RVM) is the primary circuit for opioid-based analgesia. We have recently shown that the PAG-RVM pathway is sexually dimorphic both in its anatomical organization and in its activation during persistent pain. Interestingly, while female rats have a greater number of PAG neurons that project to the RVM, inflammatory pain activates these cells to a greater degree in males. Additionally, systemic morphine inhibits the pain-induced activation of PAG neurons in males, but not females. Sex differences in neuronal activity during pain and morphine analgesia are prominent in the ventrolateral PAG, a region containing a large population of mu opioid receptor-containing neurons. We have recently shown that females have significantly lower levels of mu opioid receptors (MOR) in this region, however it is not known whether sex differences in MOR expression contribute to our observed sex differences in morphine analgesia. To test the role of ventrolateral PAG MOR in morphine analgesia, the cytotoxin saporin conjugated to the MOR agonist dermorphin (Der-Sap) was injected into the ventrolateral PAG to site-specifically lesion MOR-containing neurons. Twenty-eight days later, rats received an intraplantar injection of CFA to induce persistent pain and twenty-four hours later morphine was administered systemically using a cumulative dosing paradigm (1.8 -18mg/kg). Lesions of PAG MOR-containing neurons resulted in a two-fold rightward shift in morphine ED50 values in male rats compared to controls. Interestingly, in females no difference was noted in morphine ED50 for Der-Sap treated females versus controls suggesting that the PAG is not a critical site for morphine analgesia in females. Der-Sap treatment had no significant impact on baseline paw withdrawal latencies or CFA-induced hyperalgesia. These results indicate that the PAG is a primary locus for systemic morphine analgesia in males only and suggests the necessity for the development of sex-specific treatments for persistent pain in females.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Agonist Effects
Q: Do conjugated toxins (dermorphin-saporin in particular) exhibit agonist effects? I’ve generated behavioral and tissue time course effects but have not established agonist effects for this conjugated toxin.
A: The peptide ligand toxins should exhibit agonist effects. They are constructed purposely to retain complete agonist activity, including for us the most important: internalization. So, for instance, SP-SAP (Cat. #IT-07) causes receptor internalization similar to SP, as reported in Mantyh et al.
As to dermorphin-SAP (Cat. #IT-12) specifically, it has agonist activity very much like dermorphin. This is reported in Porreca et al. in which it’s stated:
The bilateral microinjection of 3 pmol of dermorphin or of dermorphin-saporin directly into the RVM produced a robust antinociceptive effect in the 52°C hot-water tail-flick test. The peak antinociceptive effect of dermorphin, 78 ± 13.2% MPE, was not significantly different from that of the dermorphin-saporin conjugate, which was 59 ± 4.7% MPE (p > 0.5, Student’s t test).
Usually the amount needed to give a response is lower than the amount needed to kill a cell. Depending on what your system is; it may be a peculiarity of that system, but I would be a little concerned about not seeing an agonist effect. On the other hand, if you have demonstration of specific toxicity, it may not be all that crucial.
See: Targeted Toxins
Role of spinal cord µ-opioid receptor expressing dorsal horn neurons in morphine analgesia
Kline IV RH, Wiley RG (2006) Role of spinal cord µ-opioid receptor expressing dorsal horn neurons in morphine analgesia. Neuroscience 2006 Abstracts 643.19. Society for Neuroscience, Atlanta, GA.
Summary: The role of spinal cord μ-opioid receptor expressing dorsal horn neurons in morphine analgesia is not clearly understood. Using lumbar intrathecal (i.t.) injections of the targeted toxin dermorphin-saporin to selectively destroy these cells, we sought to determine the effect of this lesion on the antinociceptive activity of systemic and i.t. morphine on the hotplate test. We examined the antinociceptive effects of morphine across a range of stimulus intensities (44, 47 & 52oC) in order to assess responses mediated by C or Aδ thermal nociceptors. Experiment 1 (systemic morphine): Sixteen Sprague Dawley male rats were injected with 500ng dermorphin-saporin i.t. or PBS and hotplate testing resumed one week after injections. Baseline hotplate responses were monitored for three weeks after which systemic morphine dose response curves (0, 2.5, 5, &10 mg/kg s.c.) were performed. Experiment 2 (spinal intrathecal morphine): Twelve Long Evans female rats were surgically implanted with indwelling lumbar i.t. catheters (8.5cm), underwent baseline hotplate testing for 7 days, had i.t. morphine dose response curves (0, 0.01, 0.1, & 1 μg) performed at 44 & 52oC seven days before and eight days after dermorphin-saporin injections. The dependent measures for the hotplate test were: 1) latencies to the first lick or guard response (all temperatures) and 2) the cumulative durations and amounts of licking and guarding events (44 and 47oC). Loss of lamina II MOR-expressing dorsal horn neurons after dermorphin-saporin was confirmed in spinal cord sections from each rat stained for MOR1 and MOR1C using standard immunoperoxidase techniques on adjacent 40 μm sections from the L4 spinal segment. Baseline responses to noxious heat did not decrease after i.t. dermorphin-saporin. The antinociceptive activity of systemic morphine was attenuated in dermorphin-saporin treated rats at 44 & 47oC; this effect was least striking on the 52oC hotplate and greatest on the 44oC hotplate. The dermorphin-saporin-induced lesion reduced the antinociceptive effects of intrathecal morphine more than systemic morphine. Based on the above findings are others not included here, we conclude that dorsal horn MOR expressing neurons are necessary for morphine to exert its maximum antinociceptive and analgesic effects.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
A selective lesioning method to probe the role of intercalated (ITC) amygdala neurons in the extinction of classically conditioned fear responses
Likhtik E, Apergis-Schoute J, Pare D (2006) A selective lesioning method to probe the role of intercalated (ITC) amygdala neurons in the extinction of classically conditioned fear responses. Neuroscience 2006 Abstracts 370.19. Society for Neuroscience, Atlanta, GA.
Summary: The acquisition of conditioned fear responses (CRs) is thought to involve the potentiation of synapses conveying information about the conditioned stimulus (CS) to the basolateral (BLA) amygdala. Expression of CRs would depend on transfer of potentiated CS inputs by the BLA to the central amygdala (CE). In contrast, the mechanisms of extinction remain controversial. It was proposed that ITC neurons, which receive BLA inputs and generate feedforward inhibition in CE, are in a key position to mediate extinction. In this view, potentiation of BLA inputs to ITC cells during extinction training, would dampen the impact of CS-related BLA activity on CE neurons, inhibiting CRs. However, this idea is difficult to test because ITC cells occur in small, lateromedially dispersed clusters, making conventional lesioning methods inadequate. The present study aimed to find an effective way of eliminating ITC cells, taking advantage of the fact that, compared to the rest of the amygdala, they exhibit strong immunoreactivity for mu opioid receptors (muORs). First, we performed electron microscopic observations to determine whether muORs are expressed by ITC cells vs. afferents to ITC cells. This test revealed that muORs are concentrated in the postsynaptic membrane density of asymmetric synapses found on ITC cells. Next, we tested whether it is possible to obtain selective ITC lesions by injecting the toxin saporin conjugated to the mu opioid agonist dermorphin (DER-SAP) in the proximity of ITC cells. Thus, rats received intra-amygdaloid pressure injections of DER-SAP in one hemisphere and of vehicle on the contralateral side. Seven days later, the animals were perfused and the tissue processed to reveal muOR. DER-SAP injections produced a marked reduction in muOR immunoreactivity at the BLA-CE border, where ITC cells are usually located. Thus, selective lesioning of ITC cells can be achieved using this method. We are currently testing the impact of such ITC lesions on extinction learning.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Descending facilitation from the rostral ventromedial medulla maintains nerve injury-induced central sensitization.
Vera-Portocarrero LP, Zhang ET, Ossipov MH, Xie JY, King T, Lai J, Porreca F (2006) Descending facilitation from the rostral ventromedial medulla maintains nerve injury-induced central sensitization. Neuroscience 140(4):1311-1320. doi: 10.1016/j.neuroscience.2006.03.016
Summary: Rats were treated with 1.5 pmol of dermorphin-SAP (Cat. #IT-12) or saporin (Cat. #PR-01) into each side of the rostral ventromedila medulla, followed by spinal nerve ligation. The data indicate that mu opioid-expresing neurons are necessary to maintain nerve injury-induced central sensitization.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), Saporin (Cat. #PR-01)
Descending facilitation from the rostral ventromedial medulla maintains visceral pain in rats with experimental pancreatitis.
Vera-Portocarrero LP, Yie JX, Kowal J, Ossipov MH, King T, Porreca F (2006) Descending facilitation from the rostral ventromedial medulla maintains visceral pain in rats with experimental pancreatitis. Gastroenterology 130(7):2155-2164. doi: 10.1053/j.gastro.2006.03.025
Summary: Here the authors investigated the role of ascending or descending pathways in the mediation of pain caused by pancreatitis. Rats received 1.5 pmol injections of dermorphin-SAP (Cat. #IT-12) into each side of the rostral ventromedial medulla. Abdominal hypersensitivity was tested using von Frey filaments. Although the ablation of mu-opioid receptor-expressing neurons by dermorphin-SAP did not prevent the initial expression of pancreatitis pain, maintenance of this pain was absent. The data link maintenance of pancreatitis pain to descending pathways.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Retrograde Transport
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
- 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.
- 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.
- 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.
- 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.
- Llewellyn-Smith IJ et al. Retrogradely transported CTB-saporin kills sympathetic preganglionic neurons. Neuroreport 10(2):307-312, 1999.
- Vulchanova L et al. Cytotoxic targeting of isolectin IB4-binding sensory neurons. Neuroscience 108(1):143-155, 2001.
- Wiley RG et al. Molecular neurosurgery with targeted toxins. , 2005. Humana Press, Totowa, New Jersey