targeted-toxins

Biggs LM, Simonton AR, Meredith M (2012) Intercalated nucleus modulates chemosensory processing in medial amygdala. Neuroscience 2012 Abstracts 781.07. Society for Neuroscience, New Orleans, LA.

Summary: The vomeronasal organ is necessary for interpretation by naive rodents (hamsters, mice) of many chemosensory signals. Information is relayed to medial amygdala (Me) via the accessory olfactory bulbs. FRA (Fos related antigen) responses in Me to chemosensory cues suggest this area is important for categorization of cues based on biological relevance to the animal. Anterior Me (MeA) is activated by all chemosensory cues (conspecific and heterospecific). Posterior Me (MeP) activates for conspecific and biologically relevant heterospecific stimuli only. Other heterospecific stimuli suppress MeP, apparently via GABA inhibition, while the adjacent medial-caudal intercalated nucleus (m-ICNc) is activated. Intercalated nuclei (ICNs) are groups of GABAergic cells between amygdala main-divisions. Those adjacent to basolateral and central amygdala (BLA, CeA) are known to mediate BLA, CeA responses via GABA inhibition, modulated by inhibitory DA-D1 receptors on ICN cells. We hypothesize that m-ICNc modulates MeP in a similar manner, as suggested by FRAs data, but this has not yet been tested directly. In hamsters, we show a hyperpolarization of MeP cells and suppression of ongoing spiking in whole-cell slice electrophysiology using in-slice stimulation of m-ICNc. The effect of dopamine and other modulators on this functional relationship is under study with agonists and antagonists. ICN, but not Me, cells carry mu-opioid receptors (MORs). In mice, we use Dermorphin-Saporin, a toxin that selectively destroys MOR+ neurons, to lesion m-ICNc to assess its role in chemosensory responses in MeP. Also in mice, a specific MUP protein in male urine (mMU) facilitates learning of a male’s chemosensory signature by females. We have quantified Me response to high and low molecular weight (HMW, LMW) fractions (LMW lacks protein) and whole mMU using FRAs immediate early gene expression. Preliminary results show no significant difference between HMW, LMW, or whole mMU in Me, however within the BLA (involved in volatile odor learning), there are significant differences in activity between stimuli in females without post-weaning exposure to male urine and no prior exposure to adult male urine.

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)

Carr F, Géranton SM, Hunt SP (2012) Descending facilitation contributes to changes in dorsal horn gene expression in a rat model of inflammatory joint pain. Neuroscience 2012 Abstracts 785.07. Society for Neuroscience, New Orleans, LA.

Summary: Chronic pain is associated with increased excitability and changes in gene expression within the dorsal horn. Descending facilitation from the rostral ventromedial medulla (RVM) is known to contribute to this excitability and behavioural hypersensitivity in a number of pain states. This would suggest that some of the gene changes associated with chronic pain could be driven by descending pathways terminating in the dorsal horn. We have previously demonstrated that ablation of a subset of RVM neurons expressing the mu opioid receptor (MOR) attenuates behavioural hypersensitivity following joint inflammation. The aim of the present study was to combine lesion of the RVM with microarray analysis of the dorsal horn to identify genes regulated by descending facilitation in this pain model. Selective lesion of MOR expressing cells of the RVM was carried out in rats by microinjection of the selective toxin dermorphin-saporin. Non-lesioned controls received vehicle microinjection. 4 weeks after the lesion procedure when depletion of the MOR+ cells was complete, both groups received an injection of 10μl Complete Freund’s Adjuvant to the left ankle joint. 7 days later the animals were sacrificed and the ipsilateral quadrant of the dorsal horn of the spinal cord lumbar region (L4-L6) removed. RNA was extracted and microarray analysis carried out using Affymetrix GeneChip Rat Gene 1.0 ST Arrays. Raw data was analysed in R using Bioconductor open source software. Limma testing was applied and a list of genes differentially regulated in animals with prior RVM lesion compared to non-lesioned controls was generated. The majority of differentially regulated genes (73%) were downregulated in the lesioned group. We used the DAVID bioinformatics resource to cluster the genes into groups with similar functional annotations (Huang et al., 2009). This analysis identified 16 gene clusters with significantly enriched functional annotations. Among these enriched functions were ribosomal function and biogenesis, inflammatory response (including the chemokine CXCL10), GPCR signalling (including the serotonin receptor 5HTR1D) and transcriptional regulation (including the transcriptional repressor RCOR2). Following on from identification of functional categories, validation of genes of interest was carried out using RT-qPCR. Our findings suggest that descending facilitation contributes to gene expression changes within the dorsal horn and that this may correlate with behavioural hypersensitivity observed in chronic pain.

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)

Wang F, Eisenach JC, Peters CM (2012) Depletion of spinal norepinephrine increases the duration of postoperative pain related behaviors following acute plantar incision and partial nerve injury in the rat. Neuroscience 2012 Abstracts 785.11. Society for Neuroscience, New Orleans, LA.

Summary: Background and Objective: The percentage of patients that develop chronic postsurgical pain can range from 10-50% depending on the type of surgery. The underlying mechanisms responsible for the transition from an acute to chronic postoperative pain state are unknown. Recent clinical studies suggest that the integrity of endogenous pain inhibitory circuits may be important for preventing this transition. The descending noradrenergic transmission has well-known inhibitory effects on spinal synaptic transmission and norepinephrine has anti-inflammatory effects on spinal glial activation. We hypothesized that disrupting spinal noradrenergic fibers in rats prior to peripheral tissue injury would enhance spinal glial activity and impair resolution of postoperative pain. Methods: To test this hypothesis, we used a model of acute pain (Brennan incision model) and a model of nerve injury involving partial L5 spinal nerve ligation. We intrathecally injected dopamine β hydroxylase conjugated to the ribosomal toxin saporin (DβH-sap, 5 μg) or control (IgG-sap) to Sprague-Dawley rats 14 days prior to surgery to deplete noradrenergic fibers. Sensitivity to mechanical stimuli (von Frey) and spontaneous guarding were assessed for several weeks. We used immunohistochemistry to assess microglial (IBA1) and astrocyte (GFAP) activation in spinal cord tissue. Results: Depletion of noradrenergic fibers resulted in a significant increase in the duration of mechanical hypersensitivity in the ipsilateral paw of rats with plantar incision (6 days in IgG-sap treated rats vs. at least 21 days in DβH-sap treated rats) and partial L5 spinal nerve ligation (42 days in IgG-sap treated rats vs. at least 70 days in DβH-sap treated rats). Depletion of noradrenergic fibers did not affect mechanical withdrawal thresholds in normal rats suggesting both tissue injury and spinal noradrenergic depletion were required for prolonged mechanical hypersensitivity. The duration of spontaneous guarding following plantar incision was not affected by DβH-sap treatment. Additionally, microglia and astrocyte activation was increased in the spinal cord 21 days following incision and 70 days after nerve injury in DβH-sap treated rats compared to IgG-sap treated rats. Conclusions: These findings highlight the crucial role of spinally projecting noradrenergic pathway in the resolution of incision and nerve injury induced hypersensitivity which may be due in part to inhibitory effect of norepinephrine on spinal glial activation. Future studies will focus on the adrenergic receptor subtypes and mechanisms responsible for the transition from acute to chronic postoperative pain in these models.

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

Schwartz MD, Dittrich L, Fisher SP, Lincoln W, Liu H, Miller MA, Warrier DR, Wilk AJ, Morairty SR, Kilduff TS (2012) Effects of a dual hypocretin receptor antagonist on sleep and wakefulness in rats. Neuroscience 2012 Abstracts 799.23. Society for Neuroscience, New Orleans, LA.

Summary: Benzodiazepine receptor agonists promote sleep by activating GABAA receptors, leading to generalized reduction in cortical activity. They are widely used as hypnotic medications, but have side effects including risk for tolerance and/or dependence, as well as cognitive impairment while under their influence. The excitatory hypocretin (HCRT) neuropeptides promote wakefulness by activating multiple subcortical wake-promoting neurotransmitter systems which, in turn, project to and regulate cortical activity. Blocking HCRT signaling should therefore promote sleep by acting specifically on subcortical brain areas regulating sleep and wake without adversely impacting cortical function. Here, we assessed the ability of the dual HCRT receptor antagonist almorexant (ALM) to promote sleep in rats following ablation of a major sleep-wake regulatory region, the cholinergic basal forebrain (BF). We predicted that ALM would be less effective at inducing sleep in BF-lesioned rats compared to neurologically-intact rats, whereas benzodiazepine-based compounds should be equally as effective in lesioned and intact rats. Male rats received bilateral stereotaxic injections of saline or the selective cholinergic neurotoxin192-IgG-saporin (SAP) directed at the BF and were implanted with telemetry for recording sleep EEG. Following recovery, animals were given increasing doses of ALM, the GABA-A receptor agonist zolpidem (ZOL), or vehicle. Spontaneous sleep/wake regulation and homeostatic recovery from sleep deprivation was also assessed. At baseline, NREM sleep in the dark (active) phase was reduced in SAP rats compared to intact rats; SAP rats also exhibited decreased NREM recovery sleep following 6 h sleep deprivation in the dark phase. Sleep in the light (rest) phase was unaffected by SAP. Analysis of ALM and ZOL administration in these animals is currently in progress.

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

Gulisano M, Parenti R, Gulino R (2012) Neural plasticity in injured spinal cord. Neuroscience 2012 Abstracts 846.09. Society for Neuroscience, New Orleans, LA.

Summary: Sonic hedgehog and Noggin are morphogenetic factors involved in neural induction and ventralization of the neural tube, but recent findings suggest that they could participate in regeneration and functional recovery after injury. Here, in order to verify if these mechanisms could occur in the spinal cord and involve synaptic plasticity, we measured the expression levels of Sonic hedgehog, Noggin, Choline acetyltransferase, Synapsin-I, and Glutamate receptor subunits (GluR1, GluR2, GluR4), in a motoneuron-depleted mouse spinal lesion model obtained by intramuscular injection of Cholera toxin-B saporin. The lesion caused differential expression changes of the analyzed proteins. Moreover, motor performance was found correlated with Sonic hedgehog and Noggin expression in lesioned animals. The results also suggest that Sonic hedgehog could collaborate in modulating synaptic plasticity. Together, these findings confirm that the injured mammalian spinal cord has intrinsic potential for repair and that some proteins classically involved in development, such as Sonic hedgehog and Noggin could have important roles in regeneration and functional restoration, by mechanisms including synaptic plasticity.

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

Jeong D, Lee J, Lee S, Kim S, Chang J (2012) Spatial memory facilitation by electrical stimulation of the medial septum in rats. Neuroscience 2012 Abstracts 851.01. Society for Neuroscience, New Orleans, LA.

Summary: Recently, deep brain stimulation has been used to treat various neurological disorders. Some studies support that DBS can be a strategy to treat Alzheimer’s disease. The aim of this study was to evaluate the effect of electrical stimulation in the medial septum using rat model mimicking basal forebrain cholinergic deficits of Alzheimer’s disease. Four experimental groups were composed of normal, lesion, lesion + implantation and lesion + stimulation. 192 IgG-saporin (Selective cholinergic toxin, 8ul of 0.63ug/ul) were bilaterally injected into the lateral ventricle. Electrode was stereotactically implanted into the left medial septum (AP +0.6, ML 0.16, DV -6). Stimulation parameters are 50Hz, 120us pulse width and 1 volt. One week after implantation, Stimulation started for 2 weeks. Two weeks after surgery, water maze was performed for 1 week and rats were sacrificed immediately after behavioral test. Features were verified by immunochemistry and AChE assay. During the training trials, latencies of lesion and implantation significantly increased in day3 and day4. In contrast, latency of stimulation group had no differences as compared to normal group but it decreased significantly when compared to lesion group in day4. In the probe test, lesion group had decreases in time in target quadrant, time in platform zone and the number of platform crossing. Although they did not perform as normal group, stimulation group showed tendency of recovery. IHC and AChE assay are ongoing. Spatial memory is associated with hippocampus. We had expected activation of hippocampus by stimulation of the medial septum. We confirmed that stimulation of the medial septum facilitates acquisition and recall of spatial memory. Currently we are studying the effects of medial septal stimulation on the hippocampus.

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

Rossi CA, Lehmkuhle MJ, Dudek FE (2012) Evidence that focal hippocampal interneuron loss disrupts theta- and gamma- band activity. Neuroscience 2012 Abstracts 918.06. Society for Neuroscience, New Orleans, LA.

Summary: Hippocampal theta (6-12 Hz) and gamma (40-100 Hz) activity are oscillatory local field potentials (LFPs) that are thought to play a critical role in the encoding and storage of new information. GABA-ergic interneurons are hypothetically involved in the generation and pacing of these oscillatory patterns of activity. The current study aimed to directly test the hypothesis that interneurons are responsible for local gamma and theta generation in the dorsal CA1 region of the hippocampus in mice. Selective focal interneuron lesions were made by intrahippocampal injection of the targeted neurotoxin SSP-Saporin into dorsal CA1 in the hippocampus of GAD67-GFP transgenic mice. Chronic recording electrodes were also implanted in the lesion area. LFPs were monitored continuously, along with video recordings of the subjects, for a period of several weeks. LFP recordings were analyzed over 24-hour periods for the occurrence of theta- and gamma-band activity. Analysis of LFP data revealed attenuation of both local theta and gamma activity in SSP-saporin-injected animals compared to controls. These results suggest a direct role of GABA-ergic interneurons in the generation of local rhythmic activity in these two frequency bands, and, by extension, an important role in learning and memory processes.

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

Ljubojevic V, Botly L, De Rosa E (2012) Cholinergic contributions to learned attentional suppression in the rat with touchscreens. Neuroscience 2012 Abstracts 729.13. Society for Neuroscience, New Orleans, LA.

Summary: One of the tasks of the attentional system is to filter environmental input according to its behavioral relevance. The neuromodulator acetylcholine (ACh) is thought to play a role in this process because of its ability to boost the signal-to-noise ratio of incoming sensory information. Cholinergic innervation of the attentional system has been shown to be necessary for successful selection of behaviorally-relevant stimuli (signal). However, it is not yet clear if ACh also plays a part in the attentional suppression of behaviorally-irrelevant information (noise). Thus, we examined the effect of cortical cholinergic deafferentation on attentional suppression in rats. To measure attentional suppression, we used a rat analog of the learning-to-ignore (LI) task originally designed for human participants (Dixon et al., 2009). The paradigm consisted of three stages of training (Prime1, Prime2, Probe; 10 sessions per stage), each of which involved stages of visual simultaneous discriminations between two stimuli. In both Prime conditions, individuals learned to respond to target stimuli (A+ and then C+ respectively), while ignoring the same distractor stimulus (B-). During Probe, the previously ignored stimulus became the target (B+) and a novel stimulus (D-) was introduced as a distractor. Eighteen male Long-Evans rats were trained to perform the touchscreen-based LI task. Like the human data, a behavioral decrement (lower accuracy) was observed during the Probe phase of the LI task when compared to Prime 1 and 2, which suggests that the ignored distractor stimulus was suppressed during Prime. We hypothesized that administration of the ACh-specific immunotoxin, 192 IgG-saporin, into the nucleus basalis magnocellularis (NBM) would lead to better performance during Probe condition relative to controls. Accordingly, the rats were subjected to either cholinergic immunotoxic (SAP, N=10) or sham lesion surgery (SHAM, N=8). After 2 weeks of post-surgical recovery, the rats were tested on the LI task with a new stimulus set. The two groups performed comparably during Prime1 and 2, with both SAP and SHAM rats successfully learning the discriminations. As predicted, during Probe SAP rats exhibited significantly less behavioral decrement than controls. Histological analysis revealed that the lesion was chemically and anatomically specific to cholinergic cells in the NBM. This counterintuitive finding suggests that the improved performance during Probe, due to reduced ACh input to the neocortex, was due to inefficient attentional suppression of the behaviorally-irrelevant stimulus.

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

Talman WT, Jones S, Nitschke Dragon D, Lin L-H (2012) Selective damage to glia in the nucleus tractus solitarii attenuates cardiovascular reflexes. Neuroscience 2012 Abstracts 524.05. Society for Neuroscience, New Orleans, LA.

Summary: Lesions of the nucleus tractus solitarii (NTS) are known to attenuate or abolish cardiovascular reflex responses. We have previously reported that lesions produced by saporin (SAP) conjugates and focused on neurons that express the neurokinin-1 (NK1) receptor or on other neurons that express both tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH), also attenuate baroreflex function in rats. We found that lesions of both types of neurons also led to loss of glia that stained with glial fibrillary acidic protein (GFAP). Further, we found that injection of SAP alone into the NTS led to loss of GFAP staining while leaving neurons in the region unaffected. Because both of the lesions directed at neurons were made by a toxic conjugate containing SAP, we sought to determine if SAP alone produced changes in cardiovascular reflex function. We found that injection of SAP (3 ng in 100 nl) into the NTS led to loss of the glial marker GFAP as well as connexin 43 (Cx43) immunofluorescent labeling in the NTS but did not affect the neuronal markers NMDAR1 (NMDA receptor subunit 1), GluR2 (AMPA receptor subunit 2), neuronal nitric oxide synthase (nNOS), TH, DBH, vesicular glutamate transporters (VGluTs), choline acetyl transferase (ChAT), NK1, and protein gene product 9.5 (PGP 9.5). In animals treated with bilateral injections of SAP into the NTS, reflex responses were decreased during testing of the baroreflex, the chemoreflex, or the von Bezold Jarisch reflex. Comparable decreases in baroreflex responses were seen in animals treated with SAP alone when compared with other animals treated with SAP conjugates that targeted and concentrated damage to TH/DBH neurons or NK1 neurons in NTS. In contrast, when TH/DBH neurons were targeted by the toxin 6-hydroxydopamine (6-OHDA) lability of arterial pressure did not occur as it did in the other SAP and SAP conjugate studies and reflex responses to the activation of the baroreflex, the chemoreflex, and the von Bezold Jarisch reflex did not differ from control. Furthermore, injections containing SAP or a SAP conjugate, but not those containing 6-OHDA, led to lability of arterial pressure as well as cardiac arrhythmias and cardiac myocytolysis. Our studies cannot exclude a physiological effect of SAP on neurons nor can it exclude an indirect effect of glial damage on NTS neurons. However, the similarity of responses when glia seem to have been targeted alone in contrast to those responses when select neuronal types seem to have been targeted suggests that each of the cardiovascular reflexes relies on intact glia in the NTS for full reflex expression.

Related Products: Anti-DBH-SAP (Cat. #IT-03), SSP-SAP (Cat. #IT-11)

Mallory CS, Paolone G, Koshy Cherian A, Blakely RD, Sarter M (2012) Paying attention with a compromised cholinergic system: Attenuated activation of cholinergic neurotransmission in attentional task-performing CHT+/- mice. Neuroscience 2012 Abstracts 536.08. Society for Neuroscience, New Orleans, LA.

Summary: Prefrontal cholinergic neurotransmission is necessary for sustained attentional performance. In rats, prefrontal acetylcholine (ACh) release reaches 140% over baseline during the performance of a sustained attention task (SAT; St. Peters et al., 2011a). SAT performance also increases the density of choline transporters (CHT) in synaptic plasma membranes (Apparsundaram et al., 2005), which we hypothesize is needed to sustain elevations of cholinergic activity and behavioral responses. Here we employed the SAT recently adapted for use in mice (St. Peters et al., 2011b) and developed new techniques that permit monitoring of ACh release via microdialysis of mice performing the SAT in order to determine the impact of genetically manipulated levels of choline transporter capacity. First, reverse dialysis of atropine (50 µM) increased ACh release levels in naive WT mice. In contrast, CHT+/- mice could not sustain these increases, consistent with changes observed in levels of muscarinic receptors in the CHT +/- mice (Bazalakova et al., 2007). However, SAT performance did not differ significantly between WT controls and CHT+/- mice. Furthermore, basal (absolute) levels of ACh release were comparable between strains. However, performance-associated increases in ACh release were strikingly attenuated in CHT+/- mice, reaching 40% over basal levels versus 130% in WT. Performance-associated increases in ACh release in CHT+/- mice were TTX-sensitive, similar to release monitored in WT mice (1 µM via reversed dialysis). To determine whether cholinergic activity was necessary for SAT performance in CHT+/- mice we then removed basal forebrain cholinergic neurons by infusing murine-p75NTR-saporin obtaining similar impairment on SAT performance in both strains. Finally, and because cholinergic activity modulates cortical circuitry primarily via nAChR, mecamylamine (MEC; 50 µM) was reverse dialyzed during SAT performance. WT mice were only moderately impaired in the SAT task, whereas the performance of CHT+/- mice rapidly declined and the performance-associated ACh levels rapidly returned to the pre-task levels. In summary CHT+/- mice are able to perform the basic SAT, despite attenuated levels of cholinergic neurotransmission, likely as a result of compensatory postsynaptic mechanisms. However, their attentional performance and underlying cholinergic signaling exhibit heightened sensitivity to behavioral and pharmacological challenges. Together, these findings suggest that CHT+/- mice are an important model for the impaired cognitive control of attentional performance that is a common symptom of ADHD, schizophrenia and other cognitive disorders.

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