targeted-toxins

Talk AC, Bernasconi D, Stevens Z, Grasby K, Edelstein L, Smythies J, Russell B (2013) The anterior claustrum and flexible behavior in the rat: A comparison of NMDA and dynorphin-saporin lesions. Neuroscience 2013 Abstracts 574.20. Society for Neuroscience, San Diego, CA.

Summary: The claustrum is a small structure of poorly understood function situated subcortically in the basal forebrain. The fact that it is extensively and reciprocally connected with the cerebral cortex has led to suggestions that it is involved in coordination of cortical activity. Here we created lesions to the anterior claustrum of rats and tested performance on tasks that involve neural processing in one or more frontal or limbic cortical structures. The excitotoxin NMDA was used to create partial lesions to the anterior claustrum. Lesions were constrained to the claustrum except in a minority of subjects. Taking into account the fact that some of the highest densities of kappa-opioid receptors in the mammalian brain are in the claustrum, in a separate study we used a custom dynorphin-saporin conjugate supplied by Advanced Targeting Systems to create lesions. We found that the claustral lesions using the targeted immunotoxin could be more complete than those provided by NMDA. In our first study, after excitotoxic lesions created by infusions of NMDA, we tested spatial reversal learning in a water maze. Lesioned rats were not impaired at acquiring the initial location of a goal platform in the maze, but were impaired at acquiring a switched location in the reversal phase. The lesioned rats also exhibited more perseverance errors compared to control rats during reversal. These same rats were not impaired at latent inhibition or working memory tasks, suggesting the effect of anterior claustral lesions may be related to behavioral flexibility. This finding is consistent with theories of claustral function that suggest it may help coordinate information necessary for cortical-dependent tasks. We are currently assessing the role of the anterior claustrum on other measures of behavioral flexibility using a cohort of rats that have been subjected to immunotoxic lesions.

Related Products: Dyno-SAP (Dynorphin-SAP) (Cat. #IT-68)

Nair DV, Al-Badri MM, Rogido M, Pacheco-Quinto J, Peng H, Iacono D, Eckman CB, Eckman EA (2013) Increased hippocampal neurogenesis and prolonged amelioration of memory deficits by chronic oxotremorine treatment in a rodent model of Alzheimer’s disease. Neuroscience 2013 Abstracts 599.01. Society for Neuroscience, San Diego, CA.

Summary: Cholinergic transmission plays a predominant role in memory processes, and loss of basal forebrain cholinergic innervation of the hippocampus has been correlated with memory impairment in Alzheimer’s disease (AD), as well as in decreased hippocampal neurogenesis in rats. Using a rat model of AD-like basal forebrain cholinergic cell loss, our lab has previously shown that central administration of a muscarinic receptor agonist, oxotremorine, for 4 weeks could reverse the spatial working memory deficit triggered by cholinergic denervation and induce hippocampal neurogenesis. The goal of the present study was to examine whether effects of chronic oxotremorine treatment persist beyond the treatment period, possibly indicating a disease-altering effect of the drug, particularly on memory function. Adult female Sprague Dawley rats were injected intracerebroventricularly (icv) with the immunotoxin 192-IgG-saporin (SAP), to induce AD-like basal forebrain cholinergic cell loss. After a 5 week recovery period, the rats then received 8 weeks of icv infusion of either oxotremorine or saline via osmotic minipump. Behavioral testing in a partially baited radial arm maze began 4 weeks after oxotremorine treatment was discontinued. To analyze cell proliferation, rats received intraperitoneal injections of BrdU either during the first 2 weeks of treatment, or at the end of behavioral testing. One month after oxotremorine treatment was discontinued, SAP-lesioned rats showed persistent improvements in radial arm maze acquisition, such that there was no difference in performance among sham/saline, sham/oxotremorine, and SAP/oxotremorine groups. SAP-lesioned rats treated with saline, however, still showed significant impairments compared to the other groups. Neuropathological and stereological analyses of these brains are ongoing, including analysis of hippocampal neurogenesis and neuronal cell counts in both basal forebrain and hippocampal regions. In a parallel cohort of rats analyzed at the end of the 8 week treatment period, initial results indicate no change in cholinergic cell density but a modest increase in the number of GABAergic cells in medial septum/diagonal band of lesioned rats treated with oxotremorine compared to saline. In the dentate gyrus (DG) of the hippocampus, increased numbers of cells labeled with BrdU during the first 2 weeks of treatment persisted to the end of the experiment, with an overall 1.5-fold increase in the number of BrdU labeled cells detected in the DG. These findings demonstrate that muscarinic stimulation is a promising target in the development of drugs to treat disorders involving cholinergic loss, such as AD.

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

Shin EJ, Rogers, J, Björklund A, Carta M (2013) The effect of noradrenaline depletion on motor impairment and dopamine cell loss in a rat model of Parkinson’s disease. Neuroscience 2013 Abstracts 623.12. Society for Neuroscience, San Diego, CA.

Summary: Objective: Parkinson’s disease (PD) has been mainly known as a neurodegenerative disease with loss of dopaminergic (DA) neurons in the substantia nigra. However, studies of post mortem PD brains have shown that not only DA neurons but also the noradrenergic (NA) neurons in the locus coeruleus degenerate, and that the NA neurodegeneration may be as profound, and also precedes degeneration of the midbrain DA neurons. The early involvement of the NA system is also in line with the caudal-to-rostral disease progression predicted by the model proposed by Braak et al. Hence, we have investigated the effect of NA depletion on motor deficits and DA cell loss in a rat PD model. Methods: To generate two lesion paradigms, rats were injected with a dopamine toxin, 6-OHDA in striatum and/or a NA toxin, DBH-saporin in lateral ventricles. Animals have been tested in a battery of behavioural tests to check the degree of motor impairment. Perfused tissues were then subjected to immunohistochemistry to assess the amount of degeneration in striatal DA fiber and nigral DA neurons. Results: In three motor tests (cylinder, amphetamine-induced rotation, and corridor tests) there was no significant difference in motor deficit between groups. However, the DA- and NA-lesioned animals showed more severe motor deficits than the DA-lesioned animals in stepping, staircase, and rotarod tests. Post mortem analysis revealed that NA depletion did not affect the degree of DA loss in striatum and substantia nigra determined by optical densitometry with tyrosine hydroxylase staining and stereological cell estimation with vesicular monoamine transporter staining, respectively. These results suggest that Parkinsonian-like motor symptoms could be worsened by NA degeneration but it is not due to more profound DA cell degeneration upon NA removal but maybe by dysregulated DA cell function.

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

Nguyen HN, Huppé-Gourgues F, Vaucher E (2013) Effect of medial prefrontal subregions electrical stimulation on the neuronal activity of the primary visual cortex and basal forebrain. Neuroscience 2013 Abstracts 639.04. Society for Neuroscience, San Diego, CA.

Summary: The cholinergic system and the medial prefrontal cortex (mPFC) play an important role in visual attention through the modulation of neuronal responses in the primary visual cortex (V1). The mPFC doesn’t project directly to V1, but does project to the horizontal limb of the diagonal band of Broca (HDB), which contains cholinergic neurons and projections to V1. Here, we investigated a possible involvement of the mPFC subregions in the activation of basal forebrain cholinergic neurons to enhance cholinergic transmission in V1. The different subregions of the mPFC, the anterior cingulate (Cg1), the prelimbic (PrL) and the infralimbic (IL) cortices, have anatomical and functional differences. Therefore the objective of this study was to determine if electrical stimulation of the different regions of the PFC activated V1 neurons in mice and if this activation was mediated through the HDB cholinergic pathway. The neuronal activity was evaluated by early gene c-Fos immunoreactivity in V1 and HDB after unilateral electrical stimulation of mPFC subregions (trains of 100 Hz for 0.3s every 2s, 50 μA, 30 mins) provided through a tungsten electrode in urethane anesthetized mice (n=4 mice per subregion). After the mPFC stimulation, mice were kept in darkness for 1h and brains were harvested after 4% paraformaldehyde intracardiac perfusion. The c-Fos expression was quantified on 35 µm coronal brain sections in V1 and HDB: an equivalent threshold was applied to all microphotographs and a c-Fos automated particle analysis tools was used (ImageJ). Moreover, the effect of the selective lesion of cholinergic fibers by the immunotoxin mu p75-saporin (1 μg/μl injections) was evaluated. The results show that electrical stimulation of PrL (Mann-Whitney U, p=0.021) and IL (p=0.021) cortices significantly induced a higher expression of c-Fos neurons in V1 of the stimulated hemisphere, but not in the HDB. Electrical stimulation of Cg1 did not elicit V1 nor HDB c-Fos immunoreactivity (p=0.248). Furthermore, selective lesions of basal forebrain cholinergic neurons did not eliminate the IL-induced c-Fos expression in the ipsilateral V1. Therefore, the mPFC stimulation seems to activate V1 neurons without the contribution of the HDB cholinergic neurons. This suggests there is a functional link between the mPFC and V1 independent from HDB cholinergic projections.

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

Sorge RE, Martin LJ, Alexander J, Beggs S, Rosen S, Zhang J, Salter MW, Mogil JS (2013) Male and female mice use distinct spinal immune cells to mediate chronic pain. Neuroscience 2013 Abstracts 642.11. Society for Neuroscience, San Diego, CA.

Summary: There are clear sex differences in the sensitivity to painful stimuli and analgesics in humans and animals. Some data suggest that pain processing is mediated by separable pathways in male and female mice, for example we recently demonstrated that spinal cord toll-like receptor 4 is used to mediate chronic pain in male, but not female mice. Here, we sought to investigate the sex-dependent pathways involved in spinal mediation of pain in male and female mice. First, we found that allodynia induced by complete Freund’s adjuvant (CFA) or spared nerve injury (SNI) was reversible via intrathecal glial inhibitors (minocycline, 0-300 μg; fluorocitrate, 0-1.5 nmol; propentofylline, 0-75 μg), or glial cell depletion using Mac1-saporin, only in male mice and never in intact female mice. This suggests that female mice utilize a microglia-independent spinal pathway to mediate chronic pain. To investigate whether T cells might mediate chronic pain in female mice, we used two strains of T cell-deficient animals; Rag1 (Rag1tm1Mom) and nude CD1 (Crl: CD1-Foxn1nu). In both strains, SNI- or CFA-induced allodynia was reversible in female mice by glial inhibitors, similar to male mice. This effect was prevented through adoptive transfer of wild type (C57BL/6) splenocytes to Rag1 female mice, suggesting T-cell involvement. T-cell infiltration into the CNS was reduced with an antibody to β1-integrin; this manipulation transiently reversed allodynia in female mice, but not male mice, further confirming that T cells mediate chronic pain in females. Finally, castration was found to reduce the anti-allodynic effect of glial inhibitors and enhanced the potential of anti-β1 integrin in male mice. In contrast, ovariectomy with testosterone replacement in female mice eliminated the effect of anti-β1 integrin and enhanced the effect of glial inhibitors. We have uncovered a robust, qualitative, and previously unknown sex difference in spinal mediation of chronic pain in mice. Attention to this critical sex difference in pain mediation may be vital to future pharmaceutical development and to interpretation of clinical pain treatments that focus on one system or the other in a mixed-sex population.

Related Products: Mac-1-SAP mouse/human (Cat. #IT-06)

Nodal FR, Leach ND, Keating P, Dahmen JC, King AJ, Bajo VM (2013) Spatial processing in the primary auditory cortex following cholinergic lesions of the basal forebrain in ferrets. Neuroscience 2013 Abstracts 353.09. Society for Neuroscience, San Diego, CA.

Summary: Cortical acetylcholine release has been implicated in different cognitive functions, including perceptual learning. We have recently shown that cortical cholinergic innervation is necessary for normal sound localization accuracy in ferrets, as well as for their ability to adapt with training to altered spatial cues (Leach et al., 2013, J Neurosci 33:6659-71). To explore whether these behavioral deficits are associated with changes in the spatial sensitivity of cortical neurons, we recorded neural activity in the primary auditory cortex (A1) from three animals in which cholinergic inputs had previously been reduced by making bilateral injections of the immunotoxin ME20.4-SAP in the nucleus basalis (NB). Neural activity was recorded from 146 penetrations in the left and right A1 under anesthesia (medetomidine/ketamine) using Neuronexus multi-site silicon probes. Histological analysis after the recording sessions revealed a mean loss of cholinergic neurons in the NB of 89.3±7.1% when compared to control animals, as well as a significant reduction in cholinergic fiber density across the auditory cortex, including the middle ectosylvian gyrus where A1 is located. On the basis of the location of the penetrations and electrophysiological characterization of the neural responses, which typically exhibited a mean latency of ≤20 ms, frequency tuning and onset responses with occasional weaker offset responses, we were able to assign the recordings to A1. The distribution of unit best frequencies was used to ensure that the tonotopic axis of A1 was evenly sampled. Spatial tuning was determined using virtual acoustic space stimuli comprising 200 ms broadband noise presented at three different levels (56, 70 and 84 dB SPL) from 12 locations separated by 30° in azimuth. Most of the units were broadly tuned, responding to all the virtual sound locations tested. Their spatial preferences were quantified by calculating the centroid direction vector from the variation in spike count with stimulus location within the onset response. This revealed a contralateral preference for most units, with the majority of the centroid azimuths located within the frontal hemifield. These data are consistent with the distribution of azimuth tuning previously described in the ferret, and initial comparisons with control animals have not shown any differences in spatial sensitivity in the animals with cholinergic lesions. Reduced cholinergic release therefore does not appear to influence the spatial response properties of A1 neurons in anesthetized animals, suggesting that any effects on sensory coding may only become apparent during behavior.

Related Products: ME20.4-SAP (Cat. #IT-15)

Parker RA, Wang F, Hayashida K, Martin TJ, Eisenach JC, Peters CM (2013) Descending noradrenergic control of conditioned pain modulation and postoperative pain trajectory in rats. Neuroscience 2013 Abstracts 461.10. Society for Neuroscience, San Diego, CA.

Summary: Aim of Investigation: Chronic pain after surgery (CPAS) is now recognized as a significant clinical problem that occurs in 10-50% of patients undergoing surgery. Recent clinical studies demonstrate the integrity of endogenous pain inhibitory controls are important for preventing CPAS, however this relationship and underlying mechanisms haven’t been examined in preclinical models. We hypothesized that a causal relationship exists between impaired endogenous analgesia and chronic pain after surgery that is in part dependent on descending noradrenergic pathways. Methods: We examined the integrity of endogenous analgesia in rats preoperatively used a previously described method for assessing conditioned pain modulation (CPM) in rats (Ferrari et al., 2010) involving injection of capsaicin (150μg/50μl) in the forepaw as a conditioning stimulus and detection of hindpaw mechanical thresholds (Randall-Selitto device) as the test stimulus. The partial L5 spinal nerve ligation (pSNL) model and mixed effects growth curve modeling of mechanical withdrawal thresholds assessed for 10 weeks following surgery were used to study resolution of postoperative mechanical hypersensitivity. The role of descending spinal noradrenergic pathways in CPM and postoperative trajectory was assessed by ablating spinal noradrenergic fibers with DβH-saporin or blocking their activity pharmacologically with adrenergic receptor antagonists. Results: Forepaw capsaicin resulted in release of NE in the lumbar spinal cord and this CPM paradigm was partially prevented by spinal idazoxan (30μg, i.t.). CPM assessed preoperatively resulted in pronounced but variable increases in hindpaw mechanical withdrawal thresholds at 30 minutes (range: 60-140g). Within individual rats, we observed a significant correlation between the degree of preoperative CPM and the slope of trajectory (P=0.006, r=0.610) as rats with lower endogenous analgesia had slower resolution of mechanical hypersensitivity. In support of a causal role between endogenous spinal noradrenergic activity and CPAS, depletion of spinal noradrenergic fibers prior to pSNL resulted in a significant reduction in the slope of trajectory within the ipsilateral hindpaw. Conclusions: Collectively, these studies suggest that the ability to engage descending endogenous noradrenergic pathways may be critical in determining whether CPAS develops. Furthermore, the use of growth curve modeling to study CPAS will allow us to examine the ability of socio-environmental conditions or pharmacological interventions to impair or improve aspects of postoperative pain trajectory as part of future studies.

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

Sengelaub DR, Cai Y, Chung M, Mnayarji (2013) Neuroprotection with gonadal steroids following partial motoneuron depletion: Dependence on steroid receptor activation and hormone action at the target musculature. Neuroscience 2013 Abstracts 467.12. Society for Neuroscience, San Diego, CA.

Summary: Aim of Investigation: Chronic pain after surgery (CPAS) is now recognized as a significant clinical problem that occurs in 10-50% of patients undergoing surgery. Recent clinical studies demonstrate the integrity of endogenous pain inhibitory controls are important for preventing CPAS, however this relationship and underlying mechanisms haven’t been examined in preclinical models. We hypothesized that a causal relationship exists between impaired endogenous analgesia and chronic pain after surgery that is in part dependent on descending noradrenergic pathways. Methods: We examined the integrity of endogenous analgesia in rats preoperatively used a previously described method for assessing conditioned pain modulation (CPM) in rats (Ferrari et al., 2010) involving injection of capsaicin (150μg/50μl) in the forepaw as a conditioning stimulus and detection of hindpaw mechanical thresholds (Randall-Selitto device) as the test stimulus. The partial L5 spinal nerve ligation (pSNL) model and mixed effects growth curve modeling of mechanical withdrawal thresholds assessed for 10 weeks following surgery were used to study resolution of postoperative mechanical hypersensitivity. The role of descending spinal noradrenergic pathways in CPM and postoperative trajectory was assessed by ablating spinal noradrenergic fibers with DβH-saporin or blocking their activity pharmacologically with adrenergic receptor antagonists. Results: Forepaw capsaicin resulted in release of NE in the lumbar spinal cord and this CPM paradigm was partially prevented by spinal idazoxan (30μg, i.t.). CPM assessed preoperatively resulted in pronounced but variable increases in hindpaw mechanical withdrawal thresholds at 30 minutes (range: 60-140g). Within individual rats, we observed a significant correlation between the degree of preoperative CPM and the slope of trajectory (P=0.006, r=0.610) as rats with lower endogenous analgesia had slower resolution of mechanical hypersensitivity. In support of a causal role between endogenous spinal noradrenergic activity and CPAS, depletion of spinal noradrenergic fibers prior to pSNL resulted in a significant reduction in the slope of trajectory within the ipsilateral hindpaw. Conclusions: Collectively, these studies suggest that the ability to engage descending endogenous noradrenergic pathways may be critical in determining whether CPAS develops. Furthermore, the use of growth curve modeling to study CPAS will allow us to examine the ability of socio-environmental conditions or pharmacological interventions to impair or improve aspects of postoperative pain trajectory as part of future studies.

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

Krajewski-Hall SJ, Mittelman-Smith, Mcmullen NT, Rance NE (2013) Ablation of arcuate KNDy neurons amplifies the LH surge in steroid-primed, ovariectomized rats. Neuroscience 2013 Abstracts 274.01. Society for Neuroscience, San Diego, CA.

Summary: KNDy (kisspeptin, neurokinin B and dynorphin) neurons in the arcuate nucleus play an important role in the reproductive axis. We have developed a method to selectively ablate KNDy neurons in the rat using NK3-SAP, a neurokinin 3 receptor agonist conjugated to saporin (Mittelman-Smith, Endocrinology 2012). Ablation of KNDy neurons results in cessation of estrous cycles, ovarian atrophy, a decrease in tonic LH secretion and loss of the rise of serum LH after ovariectomy. Given these profound effects, we tested if we could induce an LH surge in KNDy-ablated rats using a well-established steroid replacement regimen. Rats were maintained on a 14:10 light cycle (lights on at 0500). Using stereotactic surgery, NK3-SAP or Blank-SAP was injected in the arcuate nucleus and rats were allowed to recover for one month before ovariectomy. Seven days after ovariectomy they were implanted with silastic capsules containing 17β-estradiol. Two days later, they were implanted with progesterone capsules (~0830h). Rats were sacrificed in the afternoon at a time previously shown to exhibit peak LH surge levels (~1600h) and the brains were processed for immunohistochemistry. Ablation of KNDy neurons was verified by near complete loss of NKB-immunoreactive neurons in the arcuate nucleus in NK3-SAP rats. At 0830h, tonic levels of serum LH were significantly lower in KNDy ablated rats, consistent with our previous studies. Unexpectedly, the surge in serum LH at 1600h was more than 3-fold higher in NK3-SAP-treated rats compared to Blank-SAP controls (53.5 + 16.5 ng/ml vs 16.5 + 2.1 ng/ml, respectively). To determine if this change was associated with increased activation of GnRH neurons, dual-labeled GnRH-fos immunocytochemistry was performed in rostral hypothalamic sections. There was no significant difference in the total number of GnRH cells counted in 4 matched sections (NK3-SAP, 85.8 + 9.8 vs Blank-SAP, 84.9 + 4.6) or in the percentage of GnRH cells that were activated, as measured by GnRH-fos coexpression (NK3-SAP, 22.4 + 1.8% vs Blank-SAP, 16.6 + 4.9%). In addition, there was no difference between NK3-SAP and Blank-SAP controls in the number of fos-ir cells counted in the AVPV. These data indicate that arcuate KNDy neurons are not required for the induction of an LH surge. The marked increase in the LH surge in KNDy-ablated rats, however, suggests that KNDy neurons are important for regulating the magnitude of the surge.

Related Products: NKB-SAP (Cat. #IT-63), Blank-SAP (Cat. #IT-21)

Burk JA, Yonezaki K (2013) Loss of medial prefrontal cortical cholinergic projections increases preference for an immediately available reward in a delay discounting task. Neuroscience 2013 Abstracts 288.02. Society for Neuroscience, San Diego, CA.

Summary: Attentional control is thought to regulate numerous processes, including impulsive choices. The cholinergic projections to the medial prefrontal cortex are thought to be part of a distributed neural circuit that maintains attentional control. In the present experiment, male FBNF1 hybrid rats were trained in a delay discounting task that involved a choice between a small, immediately available reward and a larger reward. The larger reward was also immediately available at the beginning of the test session and then the delay to receive this reward was progressively increased within a session (0-40 s). After reaching stable performance levels, rats then either received infusions of the cholinergic immunotoxin, 192IgG-saporin, or vehicle into the medial prefrontal cortex. After recovering from surgery, rats were tested in the same delay discounting task that had been trained prior to surgery. Relative to sham-lesioned animals, rats with a loss of the cholinergic projections to the medial prefrontal cortex exhibited an increased preference for selecting the smaller, immediate reward. Subsequent administration of nicotine (0.0, 0.1, 0.2, 0.4 mg/kg, ip) did not substantially alter the effects of the lesion on delay discounting performance. The present results suggest that medial prefrontal cortical cholinergic projections contribute to choice behavior based upon delay to reward access and reward magnitude. Moreover, these results are consistent with the idea that disruption of attentional control can increase impulsive choices.

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