targeted-toxins

Hayashida K, Clayton B, Ma W, Eisenach J (2006) Brain-derived neurotrophic growth factor from p75-expressing sensory afferents drives spinal noradrenergic fiber sprouting following nerve injury in rats. Neuroscience 2006 Abstracts 248.19. Society for Neuroscience, Atlanta, GA.

Summary: We previously showed that peripheral nerve injury in mice results in sprouting of noradrenergic (NA) fibers in the spinal cord, possibly reflecting a substrate for increased efficacy of α2-adrenoceptor agonists such as clonidine. Here we tested whether spinal NA fiber sprouting also occurs in rats after peripheral nerve injury and examined the role of brain derived neurotrophic factor (BDNF) for such sprouting. Ligation of L5 and L6 spinal nerves unilaterally in rats resulted in mechanical hypersensitivity of the paw ipsilateral to injury and sprouting of NA fibers in the dorsal horn of the lumbar spinal cord. BDNF content increased in L4-L6 dorsal root ganglia (DRG) ipsilateral to injury and in lumbar spinal cord following nerve injury and intrathecal infusion of BDNF antiserum prevented spinal NA sprouting. Pro-BDNF immunoreactivity increased in L4-L6 DRG neurons ipsilateral to injury, especially in large-size neurons, and was highly co-localized with the low affinity neurotrophin receptor, p75NTR. Intrathecal injection of anti-p75NTR linked to saporin destroyed p75NTR expressing afferents and reversed NA sprouting after nerve injury. Manipulations which blocked NA sprouting (BDNF antiserum, anti-p-75NTR saporin) also prevented the increased analgesic efficacy of intrathecal clonidine observed after nerve injury. These results suggest that increased BDNF synthesis and release from p75NTR expressing injured and uninjured sensory afferents drives spinal NA sprouting following nerve injury and this sprouting increase the capacity for analgesia from drugs which utilize the NA pathway.

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

Tait DS, Brown VJ, Farovik A, Theobald DE, Dalley JW, Robbins TW (2006) Lesions of the dorsal noradrenergic bundle impair attentional set-shifting in the rat. Neuroscience 2006 Abstracts 264.4. Society for Neuroscience, Atlanta, GA.

Summary: Rats with medial prefrontal cortex (mPFC) lesions are impaired in attentional set shifting (Birrell and Brown, 2000, J Nsci, 20:4320-4324). The mPFC receives multiple projections, but norepinephrine (NE) has previously been reported to modulate attention by its action in the mPFC (for review see Dalley et al., 2004, Nsci Biobeh Rev, 28:771-784), including shifting attentional set. Indeed, there is recent evidence that increasing NE in the mPFC by autoreceptor antagonism improves set-shifting performance in rats (Lapiz and Morilak, 2006, Nsci, 137:1039-1049). Furthermore, reduction of prefrontal NE by infusion of anti-DBH-saporin into PFC has been shown to impair attentional set-shifting in rats (Eichenbaum et al., 2003, SfN Abstract 940.7). The main source of noradrenergic input to the mPFC is from locus coerulus via the dorsal noradrenergic bundle (DNAB). This study examined the effect of lesions of the DNAB on the acquisition, maintenance and shifting of attentional set. Eleven male Lister-hooded rats received bilateral DNAB lesions by infusion of 6-hydroxydopamine (4μg in 2μl each side) at (nosebar -2.4mm) AP -6.0mm, ML ±1.0mm, DV -5.0mm (from dura). Twelve control rats received injections of vehicle. Rats learned to dig for bait in bowls then learned two simple discriminations – based on the bowls odor or the digging substrate – to a criterion of six consecutive correct trials. The next day, a series of discriminations tested acquisition of novel discriminations (both intra (ID) and extradimensional (ED)) and reversal learning. Trials to criterion, incorrect trials and dig-latencies were recorded and analysed. At conclusion of testing, brain tissue samples were analysed for NE content by HPLC-ECD. All rats required more trials to reverse previously learned associations, and to learn new discriminations when attentional refocusing was required (ED shift). Rats with DNAB lesions were unimpaired at reversal stages, but were impaired at the ED acquisition stage. Lesioned rats showed reductions of NE levels in mPFC (up to 95% in the infralimbic region, 89% in the prelimbic region and 93% in cingulate area Cg1). These data provide further evidence for the role of NE in attentional set-shifting, and combine with previous data to elucidate the mechanisms by which mPFC mediates attentional set-shifting in the rat.

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

Chauhan NB (2006) Cholinergic immunolesioning produced tangle-like inclusions in TgCRND8 brain. Neuroscience 2006 Abstracts 271.8. Society for Neuroscience, Atlanta, GA.

Summary: Today’s Alzheimer’s disease (AD) research lacks a “complete” model that would represent both plaque and tangle pathology together with correlative memory deficits. Although currently developed transgenic model including APP/PS1/tau mutations do not “truly” represent AD because tangles observed in AD brain are independent of tau mutations. Subtly increased β-amyloid (Aβ) levels either due to familial mutations or sporadic causes, primarily targets pre-tangle cytopathology and degeneration of basal forebrain cholinergic neurons (BFCN) via deranged signaling of glygogen synthase kinase 3-beta (GSK3β)-, protein kinase A (PKA)-, and extracellular signal-regulated kinase (ERK2) of ERK-mitogen-activated protein kinase (MAPK) cascade, leading to reduced phosphorylation of cAMP responsive element binding protein (CREB) that results in synaptic and memory deficits much earlier than the emergence of classic AD-pathology. Thus, subtly elevated Aβ, together with BFCN deficits resulting from Aβ-induced deranged signaling, set up a vicious feedback loop to produce characteristic plaque- and tangle-pathology observed in AD. Based on these facts, we wished to test if selective lesioning of basal fore brain cholinergic neurons during the early stages of amyloid build-up will exacerbate tau phosphorylation and produce tangle-like inclusions in transgenic mice with APP mutations. We produced selective immunotoxic lesions of BFCN by injecting the BFCN-specific cholinergic immunotoxin, which is known to specifically target p75-expressing BFCN and spare p75-expressing cerebellar neurons (Mu-p75-Saporin, Advanced Targeting Systems, #IT-16), intracerebroventricularly (ICV) in TgCRND8 mice harboring Swedish (KM670/671NL) and Indiana (V717F) mutations. This model exhibited tangle-like inclusions, provoked already existing plaque pathology, and worsened already impaired behavioral deficits.

Related Products: mu p75-SAP (Cat. #IT-16)

ATS Poster of the Year Winner. Read the featured article in Targeting Trends.

Cortez AM, Amodeo D, Chavez C, Flesher M, Balbous M, Butt AE (2006) Selective 192 IgG-saporin lesions of the cholinergic basal forebrain impair negative patterning discrimination learning in rats. Neuroscience 2006 Abstracts 162.7. Society for Neuroscience, Atlanta, GA.

Summary: We have previously argued that the cholinergic nucleus basalis magnocellularis (NBM) is necessary for complex or “configural” association learning, but is not necessary for simple association learning. The current experiment further tests the hypothesis that the cholinergic basal forebrain is involved in configural association learning by examining the respective contributions of the NBM projections to neocortex and the medial septal (MS) projections to hippocampus in separate groups of rats. Rats with bilateral 192 IgG-saporin lesions of either the NBM or MS were tested in a negative patterning operant discrimination task. Rats were food-reinforced (+) for responding in the presence of a light (L+) or a tone (T+), but were not reinforced (-) for responding in the presence of the configural stimulus comprised of the light and tone presented simultaneously (LT-). We have previously shown that NBM lesions cause a transient but significant impairment in negative patterning discrimination learning. Consequently, we hypothesized a similar NBM lesion-induced impairment in the current experiment. Because hippocampus lesions cause dramatic disruptions in the acquisition of the negative patterning task, it was hypothesized that lesions of the cholinergic neurons of the MS would cause a greater degree of impairment than NBM lesions. Consistent with our hypotheses, NBM lesions retarded but did not prevent acquisition. MS lesions, in contrast, caused significantly greater impairments than NBM lesions. Rats in both lesion groups responded normally to L+ and T+ but responded more often to LT-. These findings demonstrate intact simple association learning but disrupted configural association following damage to the cholinergic neurons of the NBM or MS. Results suggest that cholinergic basal forebrain modulation of neocortex and hippocampus contributes to configural association learning.

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

Martin MM, Schultz MD, Winter SS, Wallace DG (2006) Selective cholinergic lesions of the medial septum disrupt dead reckoning-based navigation. Neuroscience 2006 Abstracts 66.10. Society for Neuroscience, Atlanta, GA.

Summary: Recent investigations using selective lesion techniques have suggested that the septohippocampal cholinergic system may not be critical for spatial orientation. These studies employ spatial tasks that provide the animal with access to both allothetic and idiothetic cues; therefore, the spared performance may reflect intact spatial orientation or compensatory mechanisms associated with one class of spatial cues. The present study examined the contribution of the septohippocampal cholinergic system to spatial behavior by examining performance in foraging tasks in which cue availability was manipulated. Female Long-Evans rats were either given a sham surgery or a selective medial septum/ vertical limb of the diagonal band cholinergic lesion using the neurotoxin 192 IgG-saporin. Rats were then trained to find food pellets randomly located on an open field which they then carried back to a visible home base (“cued”) to eat. Once they became proficient at returning to their home base location, cued training was alternated with probes. The three probes included 1) replacing the visible home base with a hidden home base to measure ability to use cues not associated with the home base (“uncued”); 2) moving the hidden home base to a new location to pit use of allothetic cues against idiothetic cues (“reversal”); and 3) testing under completely dark conditions thereby limiting access only to idiothetic cues (“dark”). Although both groups could use allothetic cues as evidenced by intact performance on cued and uncued probes, rats with compromised septohippocampal cholinergic systems were impaired during the reversal and dark probes. These observations are consistent with a selective role for the septohippocampal cholinergic system in idiothetic cue processing necessary for dead reckoning based navigation.

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

Masawaki A, Sugiyo S, Shimoda T, Sakai Y, Watanabe M, Moritani M, Yoshida A, Niwa H, Takemura M (2006) Formalin-induced pain-related responses in rat lacking neurokinin-1 receptor neurons in the trigeminal nucleus caudalis. Neuroscience 2006 Abstracts 50.8. Society for Neuroscience, Atlanta, GA.

Summary: This study examines the effect of intra cisterna magna injection of substance P (SP) conjugated to saporin (SP-Sap; 5µM, 5µl) on formalin-induced pain-related behavior (PRB; face scrubbing behavior ) and c-Fos expression in the trigeminal nucleus caudalis (SpVc). In SP-Sap-treated rats, the numbers of NK-1-immunoreactive neurons in lamina I of the SpVc decreased compared with those in saline- or blank Sap-treated rats. The mean numbers ±SEM of PRB /5 min at the first phase (0-5 min after For injection) were 58.2±19.2 in the SP-Sap-treated rats, 115.6±14.0 in the saline treated rats and 86.9±45.7 in the blank-Sap-treated rats. The numbers at the quiescent period (5-10 min) were 45.2±26.3 in the SP-Sap- treated rats, 93.6±26.5 in the saline treated rats and 69.4±16.3 in the blank-Sap-treated rats. These at the former second phase (10-50 min) were 58.1±22.3 in the SP-Sap-treated rats, 133.6±26.1 in the saline treated rats and 95.8±29.6in the blank-Sap-treated rats. These at the latter second phase (55-90 min) were 7.0±5.6 in the SP-Sap-treated rats,13.7±12.4 in the saline treated rats and 10.4±22.5 in the blank-Sap-treated rats. These results indicate that formalin-induced nociceptive responses in the SP-Sap-treated rats are reduced.

Related Products: SP-SAP (Cat. #IT-07), Blank-SAP (Cat. #IT-21)

Q: We injected anti-DBH-SAP (Cat. #IT-03) into the hypothalamus of Sprague-Dawley rats and sacrificed them 2 weeks later. We did not see any reduction in the DBH fiber staining.

When the drug arrived, we aliquoted it in 1-µl snap-cap tubes on ice, and stored them at -80°C. For injections, a 1-µl aliquot was diluted to a little over 10 µl so that we had a final concentration of 1 µg/10 µl. We administered two injections of 100 nl on each side (with 10 ng of anti-DBH-SAP) using a 0.5-µl Hamilton syringe attached to a stereotax. The needle was a 33-gauge with a blunt tip. I tried previously to use glass micropipette tips attached to a Hamilton syringe with the line filled with mineral oil, but found that the actual volume displacement was too unreliable.

A: We have not had any problems related to the stability of anti-DBH-SAP. In our work, failure to lesion is nearly always associated with a misplaced injection. From the information conveyed, I would suggest the following:

(1) It is possible that no drug was actually delivered to the brain. Two things could be done to ensure drug delivery. The first would be to add a tracer to the saporin solution that could be identified histologically. The second would be to visually monitor drug delivery using a calibrated tip. Air bubbles, pressure leaks and compression of the liquid can interfere with accurate delivery.

(2) It is possible that the anti-DBH-SAP was not delivered to the correct site, so that the expected uptake into the targeted terminals did not occur. Again, marking the site so it is clear where the injection was would help evaluate your accuracy. Establishing a reliable set of stereotaxic coordinates that work in your lab, in your rats and with your equipment and then using a dye to estimate the diffusion radius of your selected injection volume are always good ways to start. However, that being said, it should not be difficult to locate the injection site with such a large injector (33 g) – so #1 seems more likely to be the problem in the case you describe. Also, I would add that the larger the injector, the more nonspecific damage there will be. Glass capillary micropipettes are by far preferable to stainless steel cannulas in providing more reliable delivery of small volumes and in producing less nonspecific damage. Chronically implanted cannulas should be avoided, in my opinion, because gliosis at the cannula tip is apt to occur and this may alter the diffusion pattern of the injected substance, as well as interfering with lesion analysis.

(3) Try a different anesthetic. We have not tested a lot of anesthetics, but we have had problems getting a good lesion that we think are attributable to use of a ketamine/xylazine/acepromazine anesthetic cocktail. So we routinely avoid that one.

(4) I assume you are looking at fibers in the area of the injection. If not, it would be important to make sure the fibers being evaluated are associated with the same neurons innervating the terminal field at the injection site. Secondly, the 2-week wait mentioned between toxin injection and histology is critical for evaluating the lesion to assure that immunoreactive products are no longer present. Making sure that tissue processing controls are stringently adhered to so that controls and lesioned animals are run together in the same batch is also important.

(5) You might try injecting only one side and comparing terminal staining with the non-injected side in the same animal. This would not be a good idea, however, if the injection site is too close to the midline, so that both sides might be damaged from a unilateral injection.

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

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)

Related Products: Anti-DBH-SAP (Cat. #IT-03), NPY-SAP (Cat. #IT-28), Saporin (Cat. #PR-01), Blank-SAP (Cat. #IT-21)

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.

Turner LH, Lim CE, Heinrichs SC (2007) Antisocial and seizure susceptibility phenotypes in an animal model of epilepsy are normalized by impairment of brain corticotropin-releasing factor. Epilepsy Behav 10(1):8-15. doi: 10.1016/j.yebeh.2006.08.013

Summary: There appears to be an inverse relationship between seizure susceptibility and social interaction. This work examines the role that CRF may play in this system. 2.5 µg of CRF-SAP (Cat. #IT-13) was administered to the lateral ventricle of rats, and the lesioned animals were assessed in terms of social investigation times as well as handling-induced seizures. The results support the involvement of CRF systems in facilitating evoked seizures and suppression of social activity.

Related Products: CRF-SAP (Cat. #IT-13)

van der Staay FJ, Bouger P, Lehmann O, Lazarus C, Cosquer B, Koenig J, Stump V, Cassel JC (2006) Long-term effects of immunotoxic cholinergic lesions in the septum on acquisition of the cone-field task and noncognitive measures in rats. Hippocampus 16(12):1061-1079. doi: 10.1002/hipo.20229

Summary: 192-Saporin (Cat. #IT-01) has been used to make extremely specific lesions in the septohippocampal cholinergic system of the brain. The specificity of these lesions is allowing researchers to more accurately map the involvement of the septohippocampal cholinergic system in spatial learning and memory. Here, rats received 0.8 µg of 192-Saporin in the medial septum and the vertical limb of diagonal band of Broca. Lesioned animals only exhibited deficits in attentional learning.

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