targeted-toxins

Sorkin LS, Choi J-I, Koehrn FJ (2010) Carageenan evoked P-Akt in deep dorsal horn neurons is prevented by loss of neurokinin1 positive neurons in superficial dorsal horn. Neuroscience 2010 Abstracts 81.21/VV19. Society for Neuroscience, San Diego, CA.

Summary: P-Akt expression increases in dorsal horn after prolonged noxious stimulation and participates in synaptic strengthening. Recent work from our lab, showed separate p-Akt peaks in superficial (45 min) and deep dorsal horn (DH) neurons (2 hrs) after paw carrageenan. It has been suggested that NK1 receptor-expressing projection neurons (NK1+) in superficial DH are necessary for deep DH neuronal sensitization, possibly via a spino-bulbo-spinal loop. In this study, we examined whether the pattern of p-Akt expression was modulated by elimination of superficial DH NK1(+) neurons. Male Holtzman rats (250-275 g) were injected over the lumbar enlargement with substance P-saporin conjugate (SSP-SAP ([Sar9Met(O2)11] 100 ng/µl, n=8), SAP (n=8), or BSA vehicle (n=3). Catheters were removed 20 min post-injection. Two weeks later, carrageenan (2%, 100 µl) was injected into the hindpaw. Animals were perfused 45 min or 2 hrs after carrageenan (or sham) injection. Immunohistochemistry was performed on frozen sections (20mm). After correction for background density, NK-1 immunoreactivity was measured as number of bright pixels (intensity value >50 of 256)/total pixels within user-defined boxes in laminae I-III and IV-V. Neuronal p-Akt was measured using double labeling with rabbit anti-p-Akt ser 473 and mouse anti-NeuN; cells immunopositive for both were counted separately for lamina I-III and IV-V. For both measures, values from four random sections taken from segments L4 and 5 were averaged for each animal. The histologist was blinded as to spinal treatment. Density of NK-1 immunoreactivity was markedly reduced in laminae I-III in rats treated with SSP-SAP compared to SAP or BSA (24.7± 5.6% vs 78.3 ± 3.2%, 76.9 ± 2.0% respectively; p<0.01) with no differences in lamina IV-V (72.2 ± 3.4% vs 75.9 ± 1.7%, & 76.5 ± 3.4%. Counts of p-Akt neurons did not differ at any time point among animals with no pretreatment, BSA or SAP prior to carrageenan. However, the carrageenan-evoked increases in p-Akt neurons seen in laminae I-III at 45 min (9.3 ± 1.0 SAP and 2.4 ± 1.6 SSP-SAP at 45 min) and in laminae IV-V at 2 hour (11.3±1.0 SAP and 3.5± 0.9 SSP-SAP) were totally blocked by loss of NK1 neurons. Selective ablation of NK1+ neurons in superficial DH blocked peripheral inflammation-induced increase of p-Akt expression in both superficial and deep DH neurons. While some p-Akt reduction in laminae I-III was probably due to neuronal loss, i.e. NK1 receptor bearing neurons become p-AKT positive, we propose that reduction in deeper laminae was due to elimination of the first leg of a facilitatory spino-bulbo-spinal loop although loss of local NK+ interneurons could also have contributed.

Related Products: SSP-SAP (Cat. #IT-11)

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Zheng H, Rinaman LM (2010) Anxiety-like behavior in the elevated plus maze (EPMZ) depends on noradrenergic (NA) inputs to the anterolateral bed nucleus of the stria terminalis (alBST) in rats. Neuroscience 2010 Abstracts 90.7/FFF13. Society for Neuroscience, San Diego, CA.

Summary: The a2 adrenoceptor antagonist yohimbine (YO) increases transmitter release from NA neurons, activates the HPA stress axis, and increases anxiety-like behavior in rats. YO-induced HPA axis activation depends on collateralized projections from caudal brainstem NA neurons that innervate both the alBST and the medial parvocellular paraventricular nucleus of the hypothalamus (mpPVN) [Banihashemi & Rinaman, J. Neurosci., 2006]. The current study examined whether the same NA projections underlie the anxiogenic behavioral effects of YO. Adult male Sprague-Dawley rats were tested for baseline anxiety-like behavior in the EPMZ. Subsequently, anesthetized rats received bilateral microinjections of saporin toxin conjugated to an antibody against dopamine beta hydroxylase (DSAP) into the alBST to remove sources of NA input; sham control rats were similarly microinjected with vehicle. Two weeks after surgery, rats were re-tested in the EPMZ on three different days, with the first test conducted 30 min after i.p. YO (1.0 mg/kg BW), the second test conducted 30 min after i.p. saline, and the final test conducted without injection. As expected, the number of “anxiogenic” open arm entries was significantly reduced in sham control rats after YO compared to pre-surgery baseline behavior (P<0.01). Conversely, open arm entries were unaffected by YO in DSAP rats, and the YO-induced reduction of time spent in the open arms was significantly attenuated in DSAP rats vs. sham controls (P<0.01). Interestingly, in the final EPMZ test with no i.p. injections, sham control rats but not DSAP rats displayed increased anxiety-like behavior compared to their pre-surgery baseline (sham controls P<0.01; DSAP rats P=0.3). These findings support the view that caudal brainstem NA neurons projecting to the alBST are important for anxious behavior in the EPMZ, consistent with an earlier study utilizing pharmacological blockade of BST NA receptors [Cecchi et al., Neurosci., 2002]. Our ongoing studies are examining whether similar DSAP lesions attenuate conditioned and unconditioned fear and anxiety responses in rats under more ethologically relevant experimental conditions.

Related Products: Anti-DBH-SAP (Cat. #IT-03)

Weeden CS, Morris AM, Rossi CA, Roberts JM, Kesner RP (2010) The role of GABA-ergic interneurons in CA1 and dentate gyrus for sequence learning. Neuroscience 2010 Abstracts 99.26/KKK12. Society for Neuroscience, San Diego, CA.

Summary: The hippocampus (HPP) plays an important role in temporal and spatial memory. Lesion investigations of the CA1 region of HPP indicate the region’s importance in temporal processing and lesions of the dentate gyrus (DG) demonstrate an important role in spatial processing. It has been suggested that a subset of GABAergic interneurons that express Substance P mediate the inhibition of pyramidal and granule cells, which further affects the pattern of their output. This synchronizing action may directly affect information processing of CA1 pyramidal and DG granule cells. A form of temporal processing involves learning specific sequences of events for spatial locations, which incorporates both temporal and spatial qualities attributed to CA1 and DG, respectively. In order to investigate whether interneurons mediate CA1 and DG processing of newly learned locations of sequential patterns, Long-Evans male rats were randomly assigned to the following surgical groups: CA1 pyramidal cell (ibotenic acid), CA1 interneuron (peptidase-resistant substance P analog conjugated to the neurotoxin saporin) (SSP-Saporin), DG granule cell (colchicine), DG interneuron (SSP-Saporin) lesions and controls (PBS). Following recovery from surgery, rats were tested on a sequential learning task for spatial locations using an eight-arm radial maze. Six arm locations were pseudo-randomly assigned to a sequence; each of the arms was baited with a food reward. Doors remained closed until the rat oriented in front of the correct door in the sequence, at which time the door was opened and the rat was allowed access to the reward; the choice was scored as a correct response. However, if the rat oriented to an incorrect door in the sequence, the choice was scored as incorrect and the animal was allowed to reorient to the correct door. The same sequence was repeated ten times per day for a total of ten consecutive days. The percentage of correct choices per day was compared across all ten days. The results indicate that subjects with CA1 pyramidal cell, CA1 interneuron, and DG interneuron, but not DG granule cell lesions, had difficulty acquiring the sequential task when compared to controls. These results suggest an important role for CA1 pyramidal cells, and for interneurons in both CA1 and DG subregions of the HPP in temporal processing of spatial locations.

Related Products: SSP-SAP (Cat. #IT-11)

Easton A, Phil D, Fitchett A, Eacott MJ, Baxter MG (2010) Cholinergic innervation of the hippocampus is not neccesary for episodic memory, but is required for context-place learning in rats. Neuroscience 2010 Abstracts 99.27/KKK13. Society for Neuroscience, San Diego, CA.

Summary: Loss of cholinergic cortical input is associated with diseases in which episodic memory impairment is a prominent feature, but the degree to which this neurochemical lesion can account for memory impairment in humans with neurodegenerative diseases remains unclear. Removal of cholinergic input to hippocampus impairs some of its functions in memory, perhaps by reducing the plasticity of information representation within the hippocampus, but the role of cholinergic hippocampal input in episodic-like memories has not been investigated. To address this question we tested rats with selective lesions of basal forebrain neurons in the medial septum and vertical limb of the diagonal band (MS/VDB), which contains hippocampal-projecting cholinergic neurons, on a task of integrated memory for objects, places, and contexts (“what-where-which” memory). This task serves as a rodent model of human episodic memory (episodic-like memory) and is sensitive to damage to the hippocampal system. Rats with lesions of cholinergic MS/VDB neurons performed as well on the what-where-which task as controls, but were impaired in a task that simply required them to associate places with contexts (“where-which” memory). Thus, episodic-like memories that rely on the hippocampus do not require cholinergic neuromodulation to be formed. Nevertheless, some more specific aspects of where-which memory, which may be more dependent on the plasticity of hippocampal spatial representations, require acetylcholine. These results suggest that cholinergic projections to hippocampus are not necessary for episodic memory, and furthermore, that hippocampal spatial representations may be to some extent dissociable from episodic memory function.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Gompf HS, Mathai C, Fuller PM, Wood DA, Pedersen NP, Saper CB, Lu J (2010) Locus ceruleus and anterior cingulate cortex sustain wakefulness in a novel environment. J Neurosci 30(43):14543-14551. doi: 10.1523/JNEUROSCI.3037-10.2010

Objective: To examine arousal due to environmental stimuli after lesioning of the Locus ceruleus (LC).

Summary: Lesioned animals did not show sustained neurobehavioral and EEG arousal in response to a novel environment. The data suggest sustained attention requires an interaction between the LC and the anterior cingulate cortex.

Usage: Anti-DBH-SAP (0.25-1 µg) was injected into the lateral ventricle of rats.

Related Products: Anti-DBH-SAP (Cat. #IT-03)

Koenig J, Lecourtier L, Cosquer B, Pereira PM, Cassel J (2011) Spatial memory alterations by activation of septal 5HT(1A) receptors: no implication of cholinergic septohippocampal neurons. Psychopharmacology (Berl) 214(2):437-454. doi: 10.1007/s00213-010-2049-7

Summary: These experiments examined what effect damaged cholinergic neurons would have on memory deficits induced by the 5-HT1A/5-HT7 receptor agonist 8-OH-DPAT. Rats received 0.4 µg injections of 192-IgG-SAP (Cat. #IT-01) into the medial septum, delivered through an infusion device. Through use of a water maze test, the authors show that several neuronal populations are involved in processing hippocampal information, and non-cholinergic neurons in this region may be more important than the cholinergic ones for memory processing.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Q: In a previous question, you mentioned mixing anti-DBH-SAP (Cat. #IT-03) with a tracer to monitor drug delivery. Which tracer would you recommend? We were thinking of using FluoroGold. If we do not use a tracer, we were thinking of using a neutral red solution to dilute the stock of anti-DBH-SAP in order to be able to visibly see the toxin being injected into the spinal cord. Could there be an issue of pH if we used neutral red with anti-DBH-SAP? Our concern is that the toxin is not being ejected from the pipette tip or that it is not being taken up into the pipette tip as we can not see it (it’s the same color as the mineral oil). We are confident in the targeting of the spinal area for injection as we have previously used FluoroGold only and then were able to visualize it in the area of interest.

A: Our Scientific Advisor, Dr. Ronald G. Wiley, uses Fast Green dye (0.01-0.1% w/v) in the toxin injection solutions. He originally chose Fast Green because intracellular electrophysiologists had long used it while doing intracellular recordings and shown it was non-toxic. Fast Green has more contrast than Neutral Red (easier to see) and does not affect pH significantly. He has used it with many saporin-containing toxins with success.

Dr. Wiley says, “There are two issues when you talk about using “tracers” with targeted toxins: 1) tracing the acute injection volume to be sure it goes into the animal correctly, and 2) tracing the neurons that projected to the injection site and were therefore susceptible to being killed by the toxin.

Dr. Wiley does not use separate anatomic tracers for the immunotoxins, the only agents taken up and retrogradely transported efficiently. Since ATS immunotoxins are so efficient you have to use a high efficiency tracer such as cholera toxin B (but not WGA since it may not play well with saporin).

Dr. Wiley does not favor FluoroGold (a tin compound) because he has seen some local toxicity at FluoroG injection sites which might impair uptake and/or transport of a targeted toxin, and it is not clear if it is compatible with saporin-containing toxins.

Related: Anti-DBH-SAP (Cat. #IT-03), Anti-DBH-SAP Administration

Lujan HL, DiCarlo SE (2010) Featured Article: Targeted ablation of sympathetic neurons reduces ventricular arrhythmias and autonomic dysreflexia. Targeting Trends 11(4)

Related Products: CTB-SAP (Cat. #IT-14)

Read the featured article in Targeting Trends.

See Also:

• Lujan HL et al. Targeted Ablation of Cardiac Sympathetic Neurons Reduces the Susceptibility to Ischemia-Induced Sustained Ventricular Tachycardia in Conscious Rats. Am J Physiol Heart Circ Physiol 298:H1330-H1339, 2010.
• Lujan HL et al. Targeted Ablation of Mesenteric Projecting Sympathetic Neurons Reduces the Hemodynamic Response to Pain in Conscious Spinal Cord Transected Rats. Am J Physiol Regul Integr Comp Physiol 298(5):R1358-1365, 2010.

Do MTH, Yau K (2010) Intrinsically photosensitive retinal ganglion cells. Physiol Rev 90(4):1547-1581. doi: 10.1152/physrev.00013.2010

Summary: This review presents recent data that has established the importance of intrinsically photosensitive retinal ganglion cells (ipRPG) in nonimage visual functions. The use of melanopsin-SAP (Cat. #IT-44) in both mice and rats is discussed. It is of note that depletion of ipRPG’s using melanopsin-SAP resulted in deficits in communication to nonimage regions of the brain, but image vision appeared normal.

Related Products: Melanopsin-SAP (Cat. #IT-44)

Lyons AM, Thiele TE (2010) Neuropeptide Y conjugated to saporin alters anxiety-like behavior when injected into the central nucleus of the amygdala or basomedial hypothalamus in BALB/cJ mice. Peptides 31(12):2193-2199. doi: 10.1016/j.peptides.2010.09.009

Summary: Neuropeptide Y (NPY) in the hypothalamus is known to modulate feeding behavior. In this work the authors used bilateral 48 ng injections of NPY-SAP (Cat. #IT-28) into the central amygdala or basomedial hypothalamus (BMH) of rats to investigate the role of NPY in anxiety. Blank-SAP (Cat. #IT-21) was used as a control. Injections into the amygdala increased anxiety-like behavior, while injections into the BMH reduced anxiety-like behavior. BMH injections also initiated an increase of NPY-1 receptor expression in the basolateral nuclei of the amygdala.

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