targeted-toxins

Bhide NS, Dickinson S, Feinberg E, Mohamed M, Dupre K, Eskow-Jaunarajs K, Lindenbach D, Ostock C, Bishop C (2011) Norepinephrine denervation by dopamine beta-hydroxylase saporin impacts L-DOPA efficacy and side effects in a hemi-parkinsonian rat model. Neuroscience 2011 Abstracts 883.20. Society for Neuroscience, Washington, DC.

Summary: Dopaminergic neurodegeneration in Parkinson’s disease (PD) is accompanied by concomitant loss in the norepinephrine (NE) system. The exact contribution of NE denervation in the development of PD remains elusive. Recently, we demonstrated that NE neurons may contribute to the efficacy and side effects of L-DOPA, however, to better mimic NE loss observed in PD we employed the selective NE neurotoxin dopamine beta hydroxylase saporin (DHB saporin) and evaluated its effects on the anti-parkinsonian efficacy of L-DOPA and the development & expression of L-DOPA induced dyskinesia (LID). To do so, hemiparkinsonian adult Sprague-Dawley rats were exposed to intraventricular injections of either vehicle or DHB saporin. Three weeks later, animals were primed with L-DOPA (4mg/kg) for days 1-7 and L-DOPA (12 mg/kg) for days 9-15. During this period animals were monitored for motor-performance, a marker for L-DOPA’s anti-parkinsonian efficacy, and dyskinesia measured using Abnormal Involuntary Movements (AIMs) scale. Further, sensitivity of primed animals to different doses of L-DOPA (ranging from 2 to 12 mg/kg) was assessed. Results indicate that NE denervation resulted in reduced anti-parkinsonian efficacy of L-DOPA, but not the development of dyskinesia. In fully primed rats, NE denervation attenuated dyskinetic responses to L-DOPA when compared to animals with an intact NE innervation. These findings suggest that the NE system significantly modulates the anti-parkinsonian effects of L-DOPA and the expression of LID and indicate the importance of understanding the mechanisms by which NE modifies basal ganglia function in PD.

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

Lee C, Rajkumar R, Suri S, Chin WM, Dawe GS (2011) The nucleus incertus contributes to the anxiety-like behaviour in rats. Neuroscience 2011 Abstracts 901.09. Society for Neuroscience, Washington, DC.

Summary: The nucleus incertus (NI), the principal source of relaxin-3 (Rln3) in the brain, is found in the periventricular gray, ventral and medial to the posterodorsal tegmental nucleus (PDTg). Several neuroanatomical studies have indicated that the NI projects to putative correlates of anxiety, especially the amygdala. Relaxin family peptide receptor type-3 (Rxfp3), the native receptor for Rln3, is expressed in the amygdala. These studies have hence predicted that the NI is strategically located to control neural circuits that underlie anxiety-like behaviour in rodents. Presence of Rln3-immunoreactive nerve fibres in the amygdala suggested the involvement of the Rln3/Rxfp3 system. Corticotrophin-releasing factor receptors type-1 (Crfr-1), one of the important anxiolytic drug targets, are prominently expressed in the NI neurons. Based on the aforesaid anatomical and receptor distribution reports, the present investigation was designed to clarify the function of the NI in anxiety-like behaviour of rats. We hypothesized that lesioning of the NI and the resulting decrease in Rln3 would affect the regulation of stress and anxiety response in rats. Firstly, the effect of NI neuron ablation, by CRF-Saporin toxin, on fear conditioning and elevated plus maze (EPM) exploration paradigms was evaluated. Secondly, the firing rates of NI neurons as the rat explored the EPM were assessed. Lastly, the effects of high frequency simulation of the NI on the expression of immediate early genes (IEG) in the amygdala were studied. The results revealed that, in a cued fear conditioning paradigm, NI-lesioned rats exhibited greater fear, indicated by longer freezing periods in the test phase, than sham-lesioned rats. Likewise, in the EPM, NI-lesioned rats made fewer entries into and spent less time in the open arms demonstrating an anxious phenotype. In addition, the NI also showed distinct firing patterns in the open and closed arms of the EPM. Stimulation of the NI activated the medial amygdaloid (MeA) nucleus as indicated by the increased expression of markers of neuronal activation. To sum up, the present study shows a significant contribution of the NI and NI-MeA pathway in the anxiety-like behaviour of rats. It also suggests that the NI and/or Rln3 have a role in the regulation of anxiety-like behaviour, implicating them as targets for anxiety-related disorders.

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

Li AJ, Wang Q, Ritter S (2011) Fourth ventricular glucosamine-induced feeding is catecholamine-dependent. Neuroscience 2011 Abstracts 600.17. Society for Neuroscience, Washington, DC.

Summary: Glucokinase has been identified as a glucose-sensor for detecting glucose changes both in the brain and periphery. Previous reports have shown that lateral ventricular injection of a glucokinase inhibitor, glucosamine, stimulates glucoprivic feeding in rats. Other work has demonstrated involvement of hindbrain glucokinase in glucoregulation. Here we compared the effects of lateral (LV) and fourth ventricular (4V) injections of glucosamine on food intake in rats. We found that glucosamine injected into 4V (0, 0.2, 0.6, and 1.0 mg/rat) enhanced food intake in a dose-dependent manner and that LV and 4V injections were of similar potency. Glucosamine did not elevate blood glucose under the conditions of our test. We also found that enhancement of feeding by 4V glucosamine was abolished by medial hypothalamic injections of anti-dopamine beta hydroxylase saporin, a retrogradely transported catecholamine immunotoxin that selectively lesions norepinephrine and epinephrine neurons that innervate the injection site. Furthermore, 4V injection of glucosamine increased Fos expression in catecholamine populations responsible for key glucoregulatory responses. These results demonstrate that glucokinase in hindbrain catecholamine neurons is a mediator of food intake and possibly a transduction mechanism for stimulation of glucoregulatory feeding by these neurons.

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

Roach EB, Hussain Shuler MG (2011) Cholinergic reinforcement and temporal learning in rodent visual cortex. Neuroscience 2011 Abstracts 608.16. Society for Neuroscience, Washington, DC.

Summary: The idea that neuromodulators act as reinforcement signals has an intricate scientific history, including the well-characterized analogue of prediction error relayed by midbrain dopamine neurons. Neuromodulators released from discrete nuclei are poised to broadcast to many brain regions at once, and so it is an appealing concept to investigate other neuromodulatory systems within a reinforcement learning framework. Reward timing activity, a neural reflection of operantly learned stimulus-reward intervals in the primary visual cortex (V1), offers a tractable in vivo model to examine the role of candidate neuromodulators in temporal reward learning. Reward timing was first characterized in rats trained to lick a delivery tube to receive water rewards, where stimulation to one eye indicated reward availability after x licks, while stimulation to the other eye required y licks. Simultaneously recorded activity in V1 indicated that single unit responses evolve from reporting only visual characteristics to showing persistent increased/decreased firing or peak activity corresponding to the time of anticipated reward. Individual neurons report one interval or the other, even those with binocular peri-stimulus responses, arguing that reward timing is learned locally within V1. Theoretical work suggests that the local expression of reward associated intervals requires an interaction between the visually-evoked network response and a reinforcement signal conveying the time of reward. Based on anatomical and neurophysiological evidence, we hypothesized that cholinergic input from the basal forebrain (BF) could provide such a reward signal to V1. To test its necessity, BF cholinergic innervation in V1 was lesioned — using the selective neurotoxin 192 IgG-saporin — prior to changing the experimental policy between cues and associated reward delays. This allowed an examination of two potential roles for BF cholinergic input: in expressing previously learned intervals and in acquiring information about new intervals. We found that neurons from saline-infused controls, but not lesioned animals, shifted as a population to report the new, behaviorally relevant intervals (Kolmogorov-Smirnov, p < 0.05). Importantly, neurons from lesioned animals continued to report the previously learned intervals, suggesting that BF cholinergic input is required to learn, but not express, reward timing. These results support the notion that acetylcholine released from BF afferents acts as a reinforcement signal that guides cortical network plasticity.

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

Paolone G, Lamy D, Sarter M, Lee T (2011) Cognitive performance-associated increases in cholinergic neurotransmission also serve as a circadian signal to sustain performance-induced diurnal activity patterns. Neuroscience 2011 Abstracts 610.12. Society for Neuroscience, Washington, DC.

Summary: Daily practice of a sustained attention task (SAT) during the light phase of the light/dark cycle causes a stable, entrained, diurnal behavioral activity pattern (Gritton et al. 2009). As SAT performance is mediated by increases in cortical cholinergic neurotransmission, this experiment assessed levels of acetylcholine (ACh) release across the light and dark cycle of animals that previously performed the SAT at a fixed time. Circadian behavioral activity was recorded, and prefrontal ACh release was measured, using microdialysis, beginning on the third day following the last SAT session. SAT practice took place in either the light phase [ZT4], the dark phase [ZT16], or in a constant light condition [LL]. A control group practiced a daily fixed interval [FI-9] schedule of reinforcement at ZT4. A second control group was handled at randomly selected times but was neither water-deprived nor performed a task [NP]. Dialysates were collected, in a new environment, for 180 min total, beginning 90 min before the onset of prior task practice and again during the equivalent time period twelve hours later. For all animals, ACh release levels were higher during the dark phase. In SAT-performing animals, ACh levels increased for 45 min at ZT4 and ZT16. In addition, the ZT4 animals’ behavioral activity was robustly increased during this interval. Animals trained at ZT 4 reversed back to a nocturnal activity pattern 8-10 days after cessation of SAT practice, coinciding with the loss of the task time-synchronized cholinergic activity. In order to determine the necessity of these prior task period-synchronized release events for maintaining diurnal activity patterns, basal forerbain cholinergic neruons were lesioned by intra-basalis infusion of 192 IgG-saporin. As was expected, this lesion impaired SAT performance. Furthermore, following cessation of daily SAT practice, prior performance-period synchronized cholinergic release events were abolished in lesioned animals. Moreover, the lesion triggered a rapid post-performance return to a nocturnal acitvity pattern. Collectively, these results indicate that SAT performance-associated increases in prefrontal cholinergic activity not only support SAT performance but also contribute to cognition-induced diurnality. Furthermore, circadian control of cholinergic activation optimizes task performance as well as the generation of a cholinergic zeitgeber signal. In conclusions, the brain’s clocks and increases in cortical cholinergic neurotransmission interact bidirectionally to sustain cognitive performance and performance-evoked diurnal activity patterns.

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

Talman WT, Nitschke Dragon D, Jones S, Moore SA, Lin L-H (2011) Cardiovascular dysfunction and cardiac injury result from selective glial damage in the nucleus tractus solitarii. Neuroscience 2011 Abstracts 664.14. Society for Neuroscience, Washington, DC.

Summary: In man, extensive CNS dysfunction as may occur after subarachnoid hemorrhage may lead to cardiac damage and cardiac arrhythmias. We have shown that highly selective and restricted lesions of the nucleus tractus solitarii (NTS) may lead to similar cardiac and cardiovascular compromise. For example, using conjugates including the cytotoxin saporin (SAP) to selectively damage NTS neurons that express NK1 receptors or those that express tyrosine hydroxylase (TH) leads to cardiac dysfunction and associated lability of arterial pressure. In continuing efforts to better characterize cellular changes produced by introducing into the NTS conjugates containing SAP, we have studied the effect of anti-dopamine-beta-hydroxylase (anti-DBH)-SAP, stabilized substance P (SSP)-SAP, SAP (unconjugated), blank-SAP (non-targeted peptide conjugate), IgG-SAP (non-targeted immunoglobulin conjugate), and 6-hydoxydopamine (6-OHDA) as a control without SAP injected into NTS. We assessed effects of the injected agents both on cellular markers [NMDAR1 (NMDA receptor subunit 1), GluR2 (AMPA receptor subunit 2), gamma-aminobutyric acid (GABA) receptor type a and b, neuronal nitirc oxide synthase (nNOS), TH, vesicular glutamate transporters (VGluTs), choline acetyl transferase (ChAT), glial fibrillary acidic protein (GFAP), connexin 43 (Cx43), DBH and protein gene product 9.5 (PGP 9.5)] and on cardiovascular and cardiac function. We have found that each compound containing SAP (including blank-SAP, IgG-SAP, unconjugated SAP) led to loss of GFAP and Cx43 immunofluorescent labeling in the NTS as well as lability of arterial pressure, cardiac arrhythmias, and cardiac myocytolysis. Those outcomes occurred despite neuronal specificity for each of the SAP conjugates. For example, anti-DBH-SAP led to a decrease in TH and DBH staining as well as a profound loss in GFAP and Cx43. In contrast, SSP-SAP led to loss of NK1 as well as GFAP, Cx43, and glutamate receptor markers but did not lead to loss of DBH or GABA. SSP-SAP also caused a loss in PGP9.5 which was not observed in all other agents. SAP and blank-SAP, on the other hand, led to loss of GFAP and Cx43 while 6-OHDA led to loss of TH and DBH, increased GFAP and decreased Cx-43. We are still investigating the effects of 6-OHDA on lability of arterial pressure and cardiac events but preliminary data suggest that, in doses used, it led to loss of TH and DBH but did not lead to either lability or cardiac events that were seen with each of the conjugates containing an SAP moiety. This study suggests that glial dysfunction may alone interefere with cardiovascular control through the NTS and may lead to cardiac damage and cardiovascular dysfunction.

Related Products: Anti-DBH-SAP (Cat. #IT-03), SSP-SAP (Cat. #IT-11), Mouse IgG-SAP (Cat. #IT-18), Blank-SAP (Cat. #IT-21), Saporin (Cat. #PR-01)

Carr F, Géranton SM, Hunt SP (2011) The role of descending facilitation in the initiation and maintenance of mechanical hypersensitivity following inflammation. Neuroscience 2011 Abstracts 702.10. Society for Neuroscience, Washington, DC.

Summary: Central sensitisation is the key mechanism involved in the generation of mechanical hypersensitivity associated with tissue injury. Dorsal horn excitability is subject to regulation by descending modulation via the rostral ventromedial medulla (RVM) and enhanced descending facilitation under conditions of persistent nociceptive input contributes to the maintenance of mechanical hypersensitivity in chronic pain states. Depletion of mu-opioid receptor expressing (MOR+) cells of the RVM and depletion of spinal serotonin have been used previously to demonstrate the contribution of descending facilitation to the maintenance of neuropathic pain. Here we have used the same ablation techniques to investigate the contribution of descending pathways to the initiation and maintenance of mechanical hypersensitivity associated with ankle joint inflammation. Male Sprague-Dawley rats (215-220g at the time of injection) received bilateral microinjections of the selective cytotoxin dermorphin-saporin (1.5pM each side). 28 days later the animals received either an injection of 10μl Complete Freund’s Adjuvant (CFA) to the left ankle joint or underwent a sham procedure. Mechanical hypersensitivity of the hindpaw plantar surface was assessed using von Frey hairs from 2 hours up to 8 days post CFA injection. In a separate group of rats (160-180g at the time of injection) depletion of spinal serotonin was out carried out by intrathecal administration of 5,7-dihydroxytrptamine (5,7-DHT). Animals received either 10 μl of 5,7-DHT in saline (6μg/μl) or vehicle control. 6 days later animals received either CFA injection or underwent a sham procedure and mechanical hypersensitivity was assessed as in the dermorphin-saporin experiment. Depletion of the MOR+ cells of the RVM and of spinal serotonin was confirmed using immunohistochemistry. Dermoprhin-saporin pre-treatment resulted in significantly increased paw withdrawal thresholds from 6 hours up to 8 days following CFA injection (p < 0.01, ANOVA with repeated measures). In contrast depletion of spinal serotonin by 5,7-DHT led to a smaller attenuation of mechanical hypersensitivity at 24 hours and 48 hours following inflammation (LSD post hoc test, p < 0.01) but did not result in significantly increased paw withdrawal thresholds at the earlier time points.

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

Clausen B, Klipec W, Bastlund J, Collins M (2011) P300-like event related potentials in IgG192-saporin induced rat model of Alzheimer´s disease. Neuroscience 2011 Abstracts 550.01. Society for Neuroscience, Washington, DC.

Summary: The P300 event-related potential (ERP) is a time-locked response to rare, response-relevant stimuli. Decreased ERP amplitude is correlated with decreased memory function. Not surprisingly, alterations in P300 ERP amplitude are commonly associated with the progressive disruption of cognitive function in human Alzheimer’s disease. Here, a rat model of Alzheimer’s disease was created by injecting the antibody-linked toxin, IgG192-saporin, into the basal forebrain, producing a progressive degeneration of cholinergic cells to mimic the cholinergic degeneration that is part of Alzheimer’s disease. The goal of this experiment was to investigate the rat model by examining the relationship between the expected degenerative deficits and possible changes in the EEG patterns. Following preliminary training that has produced reliable P300-like ERPs in prior experiments in our lab, half of the rats were injected with IgG192-saporin (lesioned), while the other half were injected with saline (controls). Recording electrodes were surgically implanted on the surface of the brain and in the prefrontal cortex (PFC) and ventral hippocampus (vHipp). Following recovery, P300-like ERP data was recorded for three weeks, after which a ChAT analysis of choline acetyltransferase activity confirmed the extent of cholinergic damage in PFC and Hipp. While no systematic increases in latency were found, surprisingly, significant increases in P300-like ERP amplitude occurred in PFC and vHipp in the lesioned compared to the control rats. The implication of these findings for a rat model of Alzheimer’s disease will be discussed.

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

Dinh TT, Smith BR, Wiater MF, Jansen H, Li A-J, Ritter S (2011) Lesions targeting leptin-sensitive neurons in the ventromedial and suprachiasmatic nuclei differentiate sites for circadian control of feeding. Neuroscience 2011 Abstracts 600.13. Society for Neuroscience, Washington, DC.

Summary: We have previously investigated the role of leptin sensitive networks in the mediobasal hypothalamus (MBH) for feeding using the saporin toxin conjugated to leptin (Lep-SAP) and a control conjugate, blank-saporin (B-SAP). Lep-SAP binds to, is internalized by and destroys leptin receptor expressing neurons at the injection site. We found that injections of Lep-SAP directed at the arcuate nuclei (Arc) caused profound regulatory and circadian deficits including arrhythmia for feeding. We have proposed that leptin-sensitive circuitry within the MBH, particularly the arcuate nuclei (Arc), is required for the maintenance of feeding rhythms. Here we tested this hypothesis further by examining effects of Lep-SAP injections into two additional hypothalamic nuclei in the vicinity of the Arc, the ventromedial nucleus (VMN) and the suprachiasmatic nucleus (SCN). In an additional group, the SCN was lesioned electrolytically. Feeding data were collected using BioDAQ computerized meal monitors (Research Diets, Inc) and analyzed using ClockLab software to generate double raster eatogram plots. Lomb-Scargle periodograms were used to assess rhythms and their robustness. Feeding was monitored under light:dark (LD) and dark:dark (DD) conditions in all groups except SCN Lep-SAPs. Unlike Arc-directed injections of Lep-SAP, VMN injections did not alter the diurnal distribution of feeding in either LD or DD and lesioned rats did not become obese or hyperphagic. Lomb-Scargle analysis and eatograms indicated that VMN rats have intact circadian rhythms for feeding. Both Lep-SAP and electrolytic lesions of the SCN caused a slight reduction body weight, compared to controls. Total 24h food intake was unchanged, but light-period food intake was increased. Rats with electrolytic lesions of the SCN were arrhythmic for feeding under both LD and DD conditions. Together with results from Arc Lep-SAP injections, these findings strongly implicate leptin-sensitive circuitry in the MBH in control of circadian feeding rhythms. In addition, they point to the particular significance of the Arc and its connections with the SCN in this circuitry. Leptin-sensitive neurons in the VMN appear to be of less importance in this role.

Related Products: Leptin-SAP (Cat. #IT-47)

Winter SS, Köppen JR, Stout JM, Cameron HA, Wallace DG, Cheatwood JL (2011) Growth factor infusion increases BrdU-positive cells in the denervated medial septum following 192-IgG-saporin lesion. Neuroscience 2011 Abstracts 331.04. Society for Neuroscience, Washington, DC.

Summary: During the progression of Alzheimer’s Disease, degeneration of basal forebrain structures is associated with a decline in mnemonic function and frequently results in episodes of wandering behavior. Previous work has demonstrated that the septohippocampal cholinergic system uniquely contributes to rat spatial orientation. Enhancement of endogenous adult neurogenesis represents one potential method to restore function to the septohippocampal system. Therefore, we tested the hypothesis that co-infusion of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) would increase the number of new cells in the medial septum following a lesion of the cholinergic system produced by focal injection of the ribosome-inactivating selective immunotoxin 192-IgG-saporin in rats. For this, rats received injections of 192-IgG-saporin into the medial septum. At the same time, a cannula was placed in the lateral ventricle and attached to a subcutaneously-placed osmotic minipump containing either 1) EGF, bFGF, and bromodeoxyuridine (BrdU), or 2) BrdU alone. Infusion of growth factors and BrdU continued for a period of two weeks, at which point the pumps were removed. At 21 days following 192-IgG-saporin injury, rats were perfused following standard protocols. Cryostat sections were collected at 40 microns and were processed via double-fluorescent immunochemistry (IHC) using antibodies against BrdU and doublecortin (DCX). Photomicrographs of BrdU and DCX immunofluorescence were captured under epifluorescence and the number of BrdU-positive and DCX-positive cells was quantified. We detected significantly higher numbers of BrdU-positive cells in the medial septum of rats that received growth factors compared to rats that received BrdU-only (p<0.05). These results indicate that infusion of growth factors following 192-IgG-saporin lesion of the medial septum resulted in an increase in the number of new immature neurons in the medial septum. Studies aimed at determining the fate of these young neurons and their influences on spatial orientation are ongoing.

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