targeted-toxins

Chatterjee K, Lemons LL, Wiley RG (2010) Targeting inhibitory neurons in the superficial dorsal horn: Somatostatin-saporin. Neuroscience 2010 Abstracts 585.1/XX15. Society for Neuroscience, San Diego, CA.

Summary: Intrathecal injection of somatostatin (SST), or the long-acting congener, octreotide, have been reported analgesic in humans with intractable pain. The principal SST receptor, sst2a, is expressed by GABAergic neurons in the superficial dorsal horn of the spinal cord. In the present study, we sought to determine the nocifensive behavioral effects of selectively destroying sst2a-expressing dorsal horn neurons using intrathecal injection of the targeted toxin, SST-saporin. SST-sap (500-625 ng) was injected intrathecally into rats followed by thermal plate and thermal preference shuttle box testing for up to three weeks. One of three rats injected with 625 ng of SST-sap developed severe persistent scratching of its lower body. Compared to vehicle controls and rats injected with 500 ng of corticotrophin releasing factor (CRF)-saporin, the SST-sap rats showed initially prolonged latencies and decreased nocifensive reflex responses on the 44 C hotplate that persisted for up to 17 days before returning to control levels. SST-sap rats also showed decreased reflex responses on the 0.3 C cold plate. Lastly, SST-sap rats showed no change in thermal preference in a shuttle box with floor temperatures of 15 C vs 45 C. CRF-sap rats showed delayed onset (after 8 days) of decreased hotplate responding and increased hot side occupancy in the thermal preference shuttle box. These results suggest, at the doses used, that SST-sap produced incomplete depletion of target neurons followed by compensatory plasticity, whereas, CRF-sap produced no primary effect but induced secondary plasticity resulting in long term decrease in responses to aversive heat. Higher dose studies and anatomic analysis of lesions produced by these agents are planned.

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

Wiley RG, Lemons LL, Chatterjee K (2010) Targeting inhibitory neurons in the superficial dorsal horn: Neurotensin-saporin (NTS-sap) and neurotensin-cholera toxin A subunit (NTS-CTA). Neuroscience 2010 Abstracts 585.2/XX16. Society for Neuroscience, San Diego, CA.

Summary: Neurotensin (NTS) and high affinity neurotensin receptors (NTSR-1) are found in the superficial dorsal horn, primarily lamina II. Intrathecal NTS has been reported to be anti-nociceptive, naloxone does not block the anti-nociceptive effects of intrathecal NTS and NTS acting at the NTSR-1 is excitatory. Based on these facts, we hypothesized that intrathecal neurotensin produces anti-nociception by exciting inhibitory interneurons in the superficial dorsal horn. In the present study, we sought to determine the effects, on modified thermal plate responses, of lumbar intrathecal injections of NTS-saporin, that is expected to selectively kill NTSR-1-expressing dorsal horn neurons, and NTS-Cholera toxin A subunit (NTS-CTA), that is expected to excite the same neurons. NTS-sap (200-625 ng) produced sustained, remarkable, vigorous scratching of hindquarters, often to the exclusion of any other activity. 12-15 ng of NTS-sap produced no scratching and increased lick/guard responding on the 44 C hotplate. Lumbar intrathecal injections of NTS-CTA (500 ng) produced profound decrease in lick/guard responding on the 44.5 C hotplate that lasted for 100-150 hours. This unique pattern of effects is consistent with the hypothesis that NTSR-1-expressing lamina II dorsal horn neurons are both inhibitory and anti-nociceptive. These results also are consistent with the intrathecal injections of NTS-CTA producing sustained excitation of these inhibitory interneurons resulting in inhibition of nociceptive projection neurons. This strategy of exciting NTSR-1-expressing inhibitory interneurons of the superficial dorsal horn is a novel approach to achieve non-opioid-mediated analgesia which may prove valuable in treating refractory chronic pain.

Related Products: Neurotensin-SAP (Cat. #IT-56), Neurotensin-CTA (Cat. #IT-60)

Lemons LL, Chatterjee K, Wiley RG (2010) Neuropeptide receptor co-expression in superficial dorsal horn: Effects of galanin-saporin, neuropeptide y-saporin and dermorphin-saporin. Neuroscience 2010 Abstracts 585.5/XX19. Society for Neuroscience, San Diego, CA.

Summary: We have previously shown that the role of specific neurons in behavioral processes can be fruitfully studied using targeted toxins. Toxins composed of a targeting neuropeptide coupled to the ribosomal-inactivating toxin, saporin, are used to selectively destroy superficial dorsal horn neurons expressing the cognate peptide receptors followed by assessment of changes in pain behavior. In the present study, we sought to compare the anatomic effects of three closely related targeted toxins, each with different nocifensive behavioral effects. Rats were given single lumbar intrathecal injections of either galanin-saporin (Gal-SAP), neuropeptide Y-saporin (NPY-SAP), or dermorphin-saporin (Derm-SAP). Lumbar spinal cord sections from each rat were stained for each of the three receptors, GalR-1, Y1R and MOR (mu opiate) using standard immunoperoxidase technique. Each toxin produced a significant decrease in staining for its cognate receptor. Gal-SAP animals showed no change in either MOR or Y1R staining. NPY-SAP rats showed decreased staining for both GalR1 and MOR, and Derm-SAP rats were assessed for changes in expression of GalR1 and Y1R. These findings suggest overlaps between the populations of neurons that express the GalR1, Y1R, and MOR. Specifically, Y1R-expressing neurons also express GalR1 and MOR, probably by separate subpopulations of Y1R neurons. The results also suggest either that Gal-SAP only kills neurons that do not express either of the other two receptors, or some of the observed loss of receptors after NPY-SAP is due to secondary (transsynaptic) effects. Double- and triple-label fluorescent immunohistochemistry will be used to directly visualize receptor co-expression patterns and targeted toxin effects. These results will be valuable in interpreting the unique nocifensive behavioral effects of each of these targeted toxins.

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), Galanin-SAP (Cat. #IT-34), NPY-SAP (Cat. #IT-28)

Roland JJ, Janke KL, Savage LM, Servatius RJ, Pang KCH (2010) Damage of GABAergic neurons in the medial septum-diagonal band (MSDB) reduces behaviorally-activated hippocampal acetylcholine efflux and impairs spatial working memory. Neuroscience 2010 Abstracts 611.13/MMM64. Society for Neuroscience, San Diego, CA.

Summary: The septohippocampal pathway is mostly composed of cholinergic and GABAergic projections and has an established role in learning, memory and disorders of cognition. Most studies have focused on the role of the cholinergic system in learning, memory and disorders of cognition. Although MSDB cholinergic lesions do not result in learning impairments, changes in hippocampal acetylcholine (ACh) levels have been tied to memory functions where deficits or enhancements in memory were correlated with hippocampal ACh decreases or increases, respectively. The activity of MSDB cholinergic neurons is greatly influenced by GABAergic afferents, including those from GABAergic neurons within the MSDB. Recently, we’ve demonstrated that toxins that preferentially damage MSDB GABAergic neurons impair delayed match to position tasks, but not spatial reference memory. Interpretation of these results needs to take into account the fact that a MSDB GABAergic lesion would influence both septohippocampal cholinergic and GABAergic transmission. The current study examined the effect of MSDB GABAergic lesions on spontaneous alternation (Experiment 1) and a non-matching to position task (NMTP; Experiment 2) while concurrently using in vivo microdialysis to measure hippocampal ACh efflux. Adult male Sprague-Dawley rats received vehicle (PBS) or GABAergic (GAT-1 saporin) MSDB lesion and a hippocampal microdialysis cannula. In Experiment 1, treatment groups did not differ in terms of activity, alternation rates, or baseline and maze-activated ACh efflux. In Experiment 2, hippocampal ACh efflux was measured at two time points (early and late) across the acquisition of a delayed NMTP task. Overall, GAT1-saporin treated rats had lower accuracy scores across 10 days of maze training compared to the vehicle treated rats. Basal ACh release in the hippocampus was similar in vehicle and GAT1-saporin rats. During the two microdialysis sampling points, both groups of rats displayed significant increases in ACh efflux while performing the task. However, behaviorally activated ACh efflux was reduced in GABA-lesioned animals compared to vehicle treated rats. The results demonstrate that MSDB GABAergic lesions do not alter basal hippocampal ACh efflux, but can reduce ACh efflux when challenged cognitively. Future studies will attempt to determine whether reduced ACh efflux is due to damage of MSDB GABAergic neurons or a result of impaired working memory performance.

Related Products: GAT1-SAP (Cat. #IT-32)

Liu Y, Krencik R, Liu H, Ma L, Zhang X, Zhang S-C (2010) Functional cholinergic neurons from human embryonic stem cells. Neuroscience 2010 Abstracts 331.5/B19. Society for Neuroscience, San Diego, CA.

Summary: Basal forebrain cholinergic neurons play a critical role in regulating memory and cognition. Degeneration or dysfunction of these neurons is associated with neurological conditions including Alzheimer’s disease and dementia. In this study, we aimed at generating cholinergic neurons from human embryonic stem cells (hESCs) for therapeutic development. hESCs were first differentiated to primitive neuroepithelial cells in a chemically defined medium. In the presence of sonic hedgehog, over 97% of the differentiated cells became Nkx2.1-expressing ventral forebrain progenitors. These ventral progenitors further differentiated to cholinergic neurons with basal forebrain characteristics by expressing ChAT, VAChAT, FoxG1, Nkx2.1, Islet1, ßIII-tubulin, MAP2, P75, Synapsin but not GABA, Glutamate, or Mash2. The hESC-generated cholinergic neurons were electrophysiologically active in vitro. Following transplantation into the hippocampus of mice, in which cholinergic neurons in the medial septum were destroyed by IgG-P75-saporin, the grafted human cells produced large cholinergic neurons. The animals transplanted with cholinergic neurons demonstrated an improvement in learning and memory deficit. These results indicate that the human stem cell-generated cholinergic neurons are functional, thus providing a new source for drug discovery and cell therapy for neurological disorders that affect cholinergic neurons.

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

ATS Poster of the Year Winner

Rennie KE, Frechette M, Pappas BA (2010) The effects of neonatal cholinergic lesion on age-related changes in behaviour, neurogenesis and CA1 pyramidal cell morphology. Neuroscience 2010 Abstracts 349.8/J12. Society for Neuroscience, San Diego, CA.

Summary: Age-related cognitive decline is associated with dysfunction of the basal forebrain cholinergic (BFC) system, and cortico-hippocampal cholinergic denervation is a hallmark neurochemical feature of the Alzheimer’s-afflicted brain. It has been suggested that cognitive deficits that emerge with age may be rooted in early dysfunction of the BFC system and that impaired cholinergic transmission might interact with ageing-associated factors to produce cognitive decline. The purpose of this study was to examine the effects of neonatal cholinergic lesion on age-related changes in spatial working memory, neurogenesis and hippocampal CA1 pyramidal cell morphology. We have previously reported that neonatal cholinergic lesion results in only minor behavioural deficits, but impairs the birth and/or survival of new neurons and reduces CA1 dendritic complexity in the young adult rat. We hypothesized that memory impairments would become apparent in lesioned rats as they age, and that this impairment would be accompanied by more drastic reductions in neurogenesis and cytoarchitectural alterations than those that have been documented in the young adult animal after neonatal cholinergic lesion. Seven-day-old male Sprague-Dawley rats were subjected to basal forebrain cholinergic lesion by infusion of the cholinotoxin 192-IgG-Saporin into the lateral ventricles. At the age of 12 or 21 months, the rats were tested on a working memory version of the Morris water maze. While aging had only a slight effect on the memory performance of control rats, lesioned rats showed pronounced memory impairments with age. This occurred without CA1 cell loss or astrogliosis in 21-month-old lesioned rats when compared to age-matched controls. However, golgi analysis revealed that while cholinergic lesion did not alter the total dendritic length, branching, number of spines, or spine density of CA1 pyramidal cells in 21-month-old rats, the distribution of these parameters across branch orders was shifted. The lesion caused a slight reduction in apical branch length and spine density, and basal branch number, length and number of spines at low/middle branch orders, but increased these parameters at upper branch orders. Thus, perinatal cholinergic lesion precipitates spatial memory dysfunction during old age, and this seems to be associated with cytoarchitectural changes to neurons rather than neuronal loss.

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

Li A-J, Wang Q, Dinh TT, Ritter S (2010) Hindbrain catecholamine neurons are required for rapid switching of metabolic substrate utilization during glucoprivation. Neuroscience 2010 Abstracts 392.14/III1. Society for Neuroscience, San Diego, CA.

Summary: Glucoprivation is a metabolic emergency in which a rapid and effective system-wide switch to fat metabolism must occur to conserve any available glucose for use by the brain. Glucoprivation stimulates secretion of corticosterone, which is known to play an important role in promoting fat utilization. In previously published work, we showed that injections of the retrogradely transported catecholamine neuron immunotoxin, anti-dopamine beta-hydroxylase conjugated to saporin (DSAP) into the paraventricular nucleus of the hypothalamus (PVH) eliminate the corticosterone response to glucoprivation without impairing the response to a nonmetabolic stressor (swim stress), without altering the circadian rhythm of corticosterone secretion and without damaging the PVH CRF-secreting neurons. Here we microinjected DSAP into the PVH to selectively lesion hindbrain catecholamine neurons innervating this site, thus impairing the glucoprivation-induced corticosterone response. Using indirect calorimetry, we examined metabolic fuel utilization and other metabolic parameters in these lesioned rats under basal and glucoprivic conditions. Under basal conditions, energy expenditure and locomotor activity did not differ between DSAP rats and controls injected with unconjugated saporin (SAP). However, DSAP rats had a higher respiratory exchange ratio (RER) than SAPs, indicating their greater dependence on carbohydrate utilization. Glucoprivation induced by 2-deoxy-D-glucose (2DG, 250 mg/kg) reduced energy expenditure equally in SAP and DSAP rats. However, 2DG rapidly decreased RER to 0.8 (a value indicating ongoing fat metabolism) in the SAP group, but not in the DSAP group. Responses to 2DG persisted for about 6 hours. Adrenal dennervation, which eliminates the adrenal medullary response to glucoprivation, did not alter these responses to 2DG in either SAP or DSAP rats. Results indicate that in the absence of hypothalamically-projecting hindbrain catecholamine neurons, rats cannot efficiently switch their fuel utilization from carbohydrate to fat during glucoprivation, presumably due to a deficient corticosterone response. Results also suggest a previously unrecognized role for these catecholamine neurons in control of basal substrate utilization.

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

Li A-J, Dinh TT, Wang Q, Wiater MF, Ritter S (2010) Leptin-saporin lesion of hypothalamic arcuate neurons impairs circadian feeding rhythms. Neuroscience 2010 Abstracts 498.6/III29. Society for Neuroscience, San Diego, CA.

Summary: To examine the role of leptin receptor-expressing neurons in the arcuate nucleus (Arc) in circadian control of spontaneous feeding and energy expenditure, we injected a novel targeted toxin, leptin conjugated to saporin (Lep-SAP) into the Arc in rats. Lep-SAP effectively lesioned Arc neurons in a leptin-receptor dependent manner, indicated by an 80% reduction of agouti gene-related protein- or melanocyte-stimulating hormone-immunoreactive neurons in Sprague Dawley rats, but not in leptin receptor deficient Zucker fa/fa rats. Food intake and metabolism were monitored 3-5 weeks after Arc Lep-SAP and control blank-saporin (B-SAP) injections using an Oxymax system. Lep-SAP rats consumed 49% of their total daily intake during the day, compared to 34% in B-SAP rats. Eatograms (feeding actograms), cosinar analysis and Chi-square periodograms of continuous feeding records failed to detect a circadian oscillation in the feeding patterns of Lep-SAP rats, but did detect significant circadian rhythms in B-SAP controls. Unlike feeding, metabolic rate, respiratory exchange ratio and locomotor activity continued to exhibit significant circadian periodicity in both groups, though dampened in amplitude in Lep-SAPs, suggesting that rhythms of feeding and metabolism may be controlled by separate mechanisms. Expression of clock-related genes (Per1 and Bmal1) in hypothalamus, liver and white fat tissue was asynchronous in Lep-SAP rats. These results suggest that leptin-receptive neurons in the Arc exert a critical influence on the circadian patterning of food intake.

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

St Peters MM, Bruno JP, Sarter M (2010) Mesolimbic-basal forebrain circuitry mediating the motivational activation of attention. Neuroscience 2010 Abstracts 506.12/LLL52. Society for Neuroscience, San Diego, CA.

Summary: Prefrontal circuitry mediating cue detection is modulated by the tonic component of cholinergic activity. Performance-associated increases in tonic cholinergic activity are augmented by demands on the cognitive control of attention. Highest levels of tonic cholinergic activity are observed while animals perform below baseline as a result of, for example, a distractor, but while they remain motivated to stabilize and recover attentional performance. Cortico-mesolimbic-basal forebrain circuitry is thought to mediate such motivated activation of attentional performance. We previously observed that stimulation of ionotropic glutamate receptors in the shell of the nucleus accumbens (NAs) stimulates tonic cholinergic activity in the prefrontal cortex. Here we test the hypothesis that such stimulation benefits attentional performance while distractors evoke cognitive control. Rats were trained in an operant sustained attention task (SAT) before undergoing surgery for implantation of a bilateral guide cannula targeting the NAs or, in separate animals, the core of the NA (NAc). NMDA (0.01-0.15 µg/0.5 µL/hemisphere) or vehicle (0.9% saline) was infused bilaterally into task-performing animals during SAT and the more challenging distractor version (dSAT). For the dSAT, the operant chamber ceiling lights flashed on/off at 0.5 Hz during the middle block of three blocks of trials that constituted a session. NMDA infusions in the NAs, but not into the NAc, significantly improved the animals’ attentional performance in the presence of the distractor. These findings are consistent with the hypothesis that activation of the NAs mediates attentional performance under conditions that require top-down control. The next set of experiments determined whether the effects of NAs activation require the cortical cholinergic system. We infused the immunotoxin 192 IgG saporin into prefrontal or parietal regions, in addition to implantation of guide cannula targeting the NAs. Replicating the initial finding, NAs NMDA infusions enhanced dSAT performance. Both PFC and PPC cholinergic deafferentation prevented this effect of NMDA. These findings suggest that the motivated activation of the cholinergic attention system during demands on top-down control modulates fronto-parietal attention networks to optimize attentional performance.

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

Savage ST, Olson L, Mattsson A (2010) Novel object recognition and social interaction in rats lacking cortical cholinergic innervation; comparing manual and digital video tracking systems. Neuroscience 2010 Abstracts 506.9/LLL49. Society for Neuroscience, San Diego, CA.

Summary: Alterations in cholinergic signaling in the brain have been implicated as a contributing factor in the pathogenesis of schizophrenia. We have shown that cholinergic denervation of cortex cerebri by stereotaxic infusion of the immunotoxin 192 IgG-saporin into nucleus basalis magnocellularis in adult rats leads to an enhanced locomotor sensitivity to amphetamine, as well as, a potentiated dopamine release in nucleus accumbens. We have also shown that this cortical cholinergic denervation leads to an increased locomotor response to the NMDA receptor antagonist phencyclidine (PCP), suggesting that disruption of cortical cholinergic activity can lead to disturbances of glutamatergic transmission. We hypothesize that this loss of cortical cholinergic input alters the activity of cortical glutamatergic neurons and in turn, their regulation of subcortical dopamine neurons. In current studies we are investigating memory functions using the novel object recognition task (NOR) and social interaction in adult male Lister hooded rats with cholinergic denervation of neocortex. The behavioral tasks are being conducted under normal conditions and with a PCP-challenge. The data are analyzed both manually by a trained observer, and with a nose point digital video tracking system (Clever Sys Inc.). Manually scoring behavioral data requires extensive observer training, is subject to inter-observer variability, and is time consuming. An automated tracking system could potentially improve upon these issues, however is prone to other problems, including the difficulty of accurately tracking multiple body points. Furthermore, the Lister hooded fur has two different colors which proves difficult for computerized systems to accurately determine the body points. A comparison of the manual scoring and the computerized tracking system is being conducted to determine the most reliable method for each behavioral task. Preliminary results indicate that the cholinergically denervated rats performed the NOR task under normal conditions as well as the controls, however failed to show a preference for the novel object under PCP-challenge. These results were obtained through analysis with both the manual and automated system. Despite fur color difficulties, the video tracking system was able to analyze the NOR task and accurately calculate the distance traveled, which is not easily obtained through manual scoring. These initial results indicate that cortical cholinergic deficits, in addition to a potentiation of the locomotor response to PCP, can also lead to an enhanced sensitivity to PCP-induced cognitive impairments.

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