targeted-toxins

Tai S, Leung L (2009) Facilitation of dentate gyrus population spike may involve septohippocampal GABAergic input. Neuroscience 2009 Abstracts 566.18/EE33. Society for Neuroscience, Chicago, IL.

Summary: The medial septum (MS) is known to modulate the neural circuitry in the hippocampus. It have been demonstrated that, in anaesthetized rats, MS stimulation prior to perforant path stimulation facilitated the population spike in the dentate gyrus and such facilitation was unaffected by muscarinic and nicotinic cholinergic antagonists, suggesting that this facilitation may be mediated by septohippocampal GABAergic neurons. To study the effects of septohippocampal neurons in modulating neural circuitry in the dentate gyrus, selective lesion of septohippocampal GABAergic cells was made by infusion of orexin-saporin in the MS. Evoked field potentials were recorded in the dentate gyrus following stimulation of the medial perforant path in urethane-anesthetized rats using multichannel silicon probes and analyzed as current source density. High-frequency stimulation of the pontis oralis (PnO) activated a hippocampal theta rhythm. Theta peak power was attenuated in lesion rats as compared to control (sham lesion) rats. PnO stimulation resulted in a larger average increase in dentate population spike (pSpike) in control rats compared to lesion rats (26.4 ± 8.8 % vs 5.1 ± 8.7 %) but the difference was not significant (P = 0.1082; control n = 9, lesion n = 8). The number of choline acetyltransferase-immunopositive (cholinergic) cells in the MS was not significantly different (P = 0.88, unpaired t-test) between lesion and control rats, while a significant decrease (about 50%) of the number of parvalbumin-immunopositive GABAergic cells in the MS was observed in lesion as compared to control rats (P<0.05, unpaired t-test). The effect of PnO stimulation on the paired-pulse pSpike response versus interpulse interval (30 - 400 ms) was not different between control and lesion rats. No difference between control and lesion rats was detected in the pSpike enhancement after PnO stimulation following scopolamine (5 mg/kg i.p.). Additional studies were needed to demonstrate the facilitation of dentate gyrus population spike by septohippocampal GABAergic neurons.

Related Products: Orexin-B-SAP (Cat. #IT-20)

Gaykema RP, Thacker GC, Shapiro NJ, Goehler LE (2009) Immunotoxic lesion of hypothalamic noradrenergic/adrenergic input ameliorates the effects of peripheral LPS challenge on sickness behavior and associated brain c-Fos expression. Neuroscience 2009 Abstracts 570.11/EE120. Society for Neuroscience, Chicago, IL.

Summary: Caudal medullary catecholamine neurons that innervate the hypothalamus play a major role in the activation of paraventricular neurons that drive pituitary adrenocorticotropin and adrenal corticosteroid release in response to peripheral pro-inflammatory challenges with interleukin-1 or lipopolysaccharide (LPS). Pro-inflammatory challenges also lead to marked behavioral changes, including fatigue, loss of social interest, anorexia, somnolence, but the precise neuronal mechanisms that underlie sickness behavior remain elusive. We reasoned that the medulla-hypothalamic catecholaminergic pathway may also contribute to the behavioral manifestations in illness. To investigate such possible role, we applied a targeted lesion approach in rats to determine whether or not caudal brainstem catecholaminergic neurons that innervate the hypothalamus are also necessary for the expression of sickness behavior. Anti-dopamine beta hydroxylase antibodies conjugated to saporin (DSAP), when injected into a target region, selectively poisons and destroy noradrenergic/adrenergic neurons that innervate the target. DSAP was micro-injected bilaterally into the hypothalamic paraventricular nucleus (PVN), whereas control rats received unconjugated saporin (SAP controls). Fourteen days later the animals were injected intraperitoneally with either LPS or saline, and 2h later were submitted to the open field to record their exploratory behavior, 1h after which the rats were sacrificed for brain immunohistochemical analyses. LPS-treated SAP control rats showed drastic reduction in exploratory behavior (reduced locomotion distance and velocity). Prior DSAP microinjections largely reversed the LPS-induced reduction in locomotor behavior. The brains of these DSAP rats showed a dramatic loss of noradrenergic innervation of the PVN but also in other parts of the medial, tuberal and tuberomammilary regions of the hypothalamus. The behavioral resilience to LPS coincided with diminished LPS-related c-Fos staining in the PVN, and increased c-Fos staining in the lateral and tuberomammillary regions related to behavior and/or arousal. In summary, our findings support the hypothesis that hypothalamic catecholaminergic projections originating in the lower brainstem play a critical role in the expression of sickness behavior in the context of novelty-induced exploratory activity, but we cannot determine with precision in which part of the hypothalamus the noradrenergic/adrenergic input contributes to the expression of sickness behavior due to extensive collateralization of the ascending projections throughout the hypothalamus.

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

Huang T-Y, Lin L-S, Chen H-I, Jen C (2009) Chronic treadmill exercise improves cerebellar functions: Alterations in mitochondrial protein expression, rotarod performance, and toxin resistance. Neuroscience 2009 Abstracts 660.18/CC34. Society for Neuroscience, Chicago, IL.

Summary: The effects of exercise on cerebellar functions were studied. Five-week-old male Wistar rats were divided into exercise and sedentary groups. For exercise groups, rats were subjected to 8 weeks of treadmill exercise at moderate intensity. In some groups, rats were administered with OX7-saporin, a cerebellar Purkinje cell toxin, into the lateral ventricle during the 5th week of training. At the end of training period, they were tested for rotarod performance. Brain tissues were obtained for measurement of mitochondria-related protein, including Mfn2, OPA1, Drp1 and CcOx-IV. The morphology of Purkinje cells was also examined by two photon microscopy. Our results showed that exercise training improve rotarod performance, and increased cerebellar protein levels of Mfn2 and OPA1 (mitochondrial fusion proteins) but not Drp1 (mitochondrial fission protein) or CcOx-IV (a mitochondrial complex IV marker). The dendritic field of Purkinje cells was significant modified in exercise groups. OX7-saporin application impaired the rotarod performance and decreased cerebellar Purkinje cell number only in sedentary rats. In summary, chronic exercise enlarged dendritic field of Purkinje cells and improved cerebellar function, including the rotarod performance, the mitochondrial fusion protein expression, and the resistance to toxin insult.

Related Products: OX7-SAP (Cat. #IT-02)

Freiria-Oliveira AH, Blanch GT, De Paula PM, Colombari E, Menani JV, Colombari DS (2009) Role of A2 noradrenergic neurons and angiotensinergic mechanisms on hypotension induced by hemorrhage. Neuroscience 2009 Abstracts 467.18/DD70. Society for Neuroscience, Chicago, IL.

Summary: The A2 catecholaminergic neurons in the commissural subdivision of the nucleus tractus solitarii (cNTS) are activated by hemorrhage. However, the role of these neurons on the cardiovascular adjustments to hemorrhage is not fully understood. In the present study we investigated the effects of A2 noradrenergic neuron lesion alone or combined with the blockade of angiotensinergic mechanisms on the recovery of blood pressure after hemorrhage. Male Holtzman rats (280-320 g) anesthetized with ketamine combined with xylazine were submitted to lesions of dopamine-beta-hydroxilase (DβH)-containing neurons in the cNTS achieved with injections of anti-DβH-saporin (12.6 ng/60 nl, n=6-8) or sham lesions (injection of immunoglobulin-G-saporin, 12.6 ng/60 nl, n=6). Changes in blood pressure to hemorrhage were tested 30 days after lesions. Immunohistochemistry for tyrosine-hydroxilase was performed to confirm the efficacy of DβH neuron lesion in the cNTS. Two days before tests, femoral artery and vein were cannulated under ketamine and xylazine anesthesia. Hemorrhage consisted in four blood withdrawals (2 ml/300 g body weight, every 10 min) in conscious rats. Immediately after the 4th blood withdrawal, the hypotension was similar in A2-lesioned and sham-lesioned rats (-62 ± 7 mmHg and -73±7 mmHg, respectively). However, A2-lesioned rats rapidly (20 min) recovered from hypotension (-7±2 mmHg), while sham rats did not completely recover from hypotension until the end of experiment (60 min after the 4th blood withdrawn, -20±3 mmHg). The pre-treatment with losartan (angiotensin type 1 receptor antagonist, 10 mg/kg of body weight, iv) impaired the recovery of blood pressure by A2-lesioned rats (-29 ± 4 mmHg and -28 ± 3 mmHg, 20 and 60 min after the 4th blood withdrawal). In sham rats, the treatment with losartan also reduced the partial recovery of blood pressure at the end of the test (-39±6 mmHg, vs. sham control: -20±3mmHg), however, losartan did not affect the hypotension 20 min after the 4th blood withdrawal (-30± 6 mmHg vs. sham control: -35 ± 9 mmHg). The results suggest that A2 noradrenergic neuron lesion in the cNTS facilitates the recovery of hypotension after hemorrhage, probably increasing the action of angiotensinergic mechanisms.

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

Macleod JE, Ackerman CM, Bucci DJ (2009) Contributions of the medial prefrontal cortex to negative occasion setting. Neuroscience 2009 Abstracts 477.2/FF61. Society for Neuroscience, Chicago, IL.

Summary: The medial prefrontal cortex of rats has a role in many aspects of cognitive function, and especially forms of inhibitory learning. Recent research has revealed heterogeneous functions of the prelimbic (PL) and infralimbic (IL) regions of the medial prefrontal cortex in modulating response inhibition. In a recent study, we tested the effects of separate neurotoxic lesions of the PL or IL in a serial feature negative discrimination paradigm (negative occasion setting). Rats received daily training sessions consisting of 16 trials: on 4 trials in each session, a tone was presented and followed by food reward; on the remaining trials, the tone was preceded by a visual stimulus and not reinforced. Our results indicate that PL but not IL is necessary for learning the discrimination. A second study was conducted to investigate the effects of these lesions on rats that were first extensively trained in this task. We found that rats that had been trained for 30 days prior to receiving PL or IL lesions were still able to perform the task as well as controls. Therefore, PL lesions disrupt acquisition but not performance of a serial feature negative discrimination. This same task has been used in our laboratory to investigate the effects of nicotine on learning. We have shown that nicotine-treated rats exhibit greater discrimination between the two trial types as evidenced by less frequent responding during non-reinforced trials, and learn the discrimination in fewer sessions than control rats. In addition, rats receiving nicotine showed an increase in rearing behavior during the presentation of the light, suggesting nicotine enhanced attention to the visual stimulus. One possible critical site of action for nicotine’s effects is the medial prefrontal cortex. Research in other laboratories utilizing other training procedures suggest that cholinergic activity in the medial prefrontal cortex is critical for attending to behaviorally relevant stimuli, and have implicated the rat PL in visual attention as well as inhibiting prepotent, goal oriented responses. We investigated the contribution of the cholinergic PL to learning the serial feature negative discrimination task by training rats that had received infusions of 192-IgG-saporin into PL to remove cholinergic input from the basal forebrain. No differences between control and lesion rats were observed. Taken together, the results suggest that PL is necessary for acquisition of a serial feature negative discrimination, although the basal forebrain cholinergic input into this region is not required to sufficiently learn the task.

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

Laplante FP, Dufresne M, Lappi DA, Sullivan RM (2009) Depletion of cholinergic neurons in the nucleus accumbens impairs dopamine function in the prefrontal cortex in the rat. Neuroscience 2009 Abstracts 341.7/O16. Society for Neuroscience, Chicago, IL.

Summary: Studies of post mortem schizophrenic brains have revealed a selective loss of cholinergic interneurons, most pronounced in the ventral striatal region. We have previously shown in the rat, that a novel saporin immunotoxin coupled with an antibody targeting choline acetyltransferase (ChAT) and microinjected (0.5 _g/_l; 0.5 _l) into the nucleus accumbens (N. Acc) of adult rats, reduces the number of cholinergic neurons in N. Acc. by 40-50 %. Such lesions result in a markedly heightened response to the locomotor activating effects of amphetamine and impair prepulse inhibition of the acoustic startle response. We proposed that this local cholinergic deficit leads to a hyperresponsiveness in subcortical dopamine (DA) systems of relevance to schizophrenic symptomatology. Presently, we hypothesize that the same local cholinergic defect may trigger broader changes in cortical/subcortical networks, specifically prefrontal cortex (PFC) deficits in DA-mediated functions, also proposed in schizophrenia. Young adult male Srpague-Dawley rats were injected bilaterally in the N. Acc. as described above with either the cholinergic immunotoxin or vehicle. Two weeks later, they were trained in a working memory task dependent on PFC function, using the delayed alternation paradigm in the T-maze. Lesioned rats took significantly longer to reach criterion performance during training than controls. During testing, lesioned rats were significantly impaired in the percentage of correct arm choices across delay intervals, but especially with longer (40 sec) delays. The same animals were then implanted with voltammetric recording electrodes in the ventromedial PFC to examine the increases in in vivo extracellular DA release in response to a brief tail pinch stress. Lesioned rats showed a significantly reduced activation of the mesocortical DA system compared to controls. Taken together, the data suggest that reduction in the density of cholinergic neurons in the N. Acc also triggers deficits in prefontally-mediated function known to be under mesocortical DAergic regulation. This raises the possibility that ventral striatal cholinergic deficits may be causally linked to cortical/subcortical functional imbalances proposed to exist in schizophrenia.

Related Products: Anti-ChAT-SAP (Cat. #IT-42)

Brink TS, Khasabov SG, Fliss PM, Simone DA (2009) Ablation of NK-1 expressing neurons in the rostral ventromedial medulla attenuates inflammatory hyperalgesia. Neuroscience 2009 Abstracts 361.5/BB31. Society for Neuroscience, Chicago, IL.

Summary: Substance P (SP) is a neuropeptide synthesized by many nociceptive primary sensory neurons and is released into the spinal cord following noxious stimulation where it binds to neurokinin-1 (NK-1) receptors, mostly located on ascending spinal neurons. Spinal NK-1 receptors are involved in the development of hyperalgesia and central sensitization. NK-1 expressing neurons are also present in the rostral ventromedial medulla (RVM), a brainstem area involved in descending modulation of nociceptive transmission in the spinal cord. ON cells in the RVM are involved in facilitation of nociceptive transmission and their activity may be modulated by SP. SP injected into the RVM excites ON cells through NK-1 receptors, and NK-1 receptor antagonists into the RVM attenuate hyperalgesia produced by capsaicin. Here, we studied the role of RVM NK-1 positive neurons in modulating hyperalgesia following acute (intraplantar capsaicin injection) or sustained (complete Freund’s adjuvant (CFA) in the hindpaw) inflammation. We used the ribosomal toxin saporin (SAP) conjugated to a stable agonist of SP (SSP) to selectively ablate RVM cells that possess NK-1 receptors. In male Sprague-Dawley rats, withdrawal responses to noxious heat and mechanical stimuli were obtained using the Hargreaves method and a 15 g von Frey monofilament applied to the plantar hindpaw, respectively. Rats were treated with either the SSP-SAP toxin (0.5 µg/0.5 µl) or blank-SAP, and were tested 10-24 days after injection, when NK-1 expressing RVM neurons are ablated. In control rats, injection of capsaicin (10 µl of 0.1%) produced a 63% decrease in withdrawal latency to heat and an increase in withdrawal response frequency evoked by the monofilament from 16% up to 87%. However, SSP-SAP attenuated capsaicin-evoked hyperalgesia to heat (15% decrease in withdrawal latency) and mechanical (increase to 44% withdrawal frequency) stimuli. Elimination of NK-1 positive neurons in the RVM also attenuated the development of hyperalgesia following CFA. Whereas control rats exhibited a 60% decrease in withdrawal latency to heat and an increase in withdrawal frequency the monofilament from 10% up to 78%, withdrawal latency decreased 27% and withdrawal frequency increased to only 46% in rats treated with SSP-SAP. We conclude that neurons in the RVM that contain NK-1 receptors are pronociceptive and contribute to the hyperalgesia produced by capsaicin or CFA.

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

Li A-J, Wang Q, Dinh TT, Ritter S (2009) Leptin-saporin injection into the arcuate nucleus lesions NPY/AGRP and POMC neurons and produces hyperphagia, obesity and changes in diurnal feeding patterns in rats. Neuroscience 2009 Abstracts 374.5/EE116. Society for Neuroscience, Chicago, IL.

Summary: Leptin-saporin (Lep-SAP), a conjugate of leptin with a ribosomal inactivating toxin, saporin (Advanced Targeting Systems), is a novel toxin designed to destroy leptin receptor-expressing cells selectively in vitro. However, its lesioning properties in vivo are currently unknown. Here, we injected Lep-SAP into the arcuate nucleus (Arc), to examine its effects on feeding behavior and on leptin receptor-expressing NPY/AGRP and POMC neurons in this area. Immunohistochemical studies showed unilateral injection of Lep-SAP into the Arc dramatically reduced numbers of NPY-Y1- and α-MSH- positive neurons compared to the contralateral side injected with SAP control. Real-time PCR revealed only 11-21% of Agrp and Pomc expression remaining in the Arc after Lep-SAP injection into this region. Rats injected bilaterally with Lep-SAP were unresponsive to central leptin administration and showed dramatic increases in feeding, body weight and light-phase feeding, compared pre-injection baseline. Two weeks after injection, total daily feeding was increased by 75%, light phase feeding by 359% and dark phase feeding by 33%. Control SAP injections did not produce these changes. Clock gene expression in homogenates of whole hypothalamus and liver were quantified at ZT 5-7. Bmal1 expression in hypothalamus and liver of Lep-SAP rats was decreased, while hepatic Per1 expression was increased compared to control. Results demonstrate that Lep-SAP effectively lesions Arc leptin receptor-expressing NPY/AGRP and POMC neurons in vivo, and that rats with this lesion are hyperphagic and obese, possibly due to enhanced hunger drive, lack of responsiveness to leptin and/or changes in circadian control of feeding behavior.

Related Products: NPY-SAP (Cat. #IT-28), Leptin-SAP (Cat. #IT-47)

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

Carvalho AF, Reyes AS, Van Bockstaele EJ (2009) The role of limbic norepinephrine in cannabinoid-induced aversion. Neuroscience 2009 Abstracts 449.3/V29. Society for Neuroscience, Chicago, IL.

Summary: The endocannabinoid system has been implicated in diverse physiological mechanisms including modulation of pain and analgesia, learning and memory and feeding, among others. Thus, targeting the cannabinoid system has risen to the forefront in the development of novel treatments for a number of pathophysiological processes. Consistent with this, agonists of the cannabinoid receptor type 1 (CB1R) have been successfully used in the treatment of severe anorexia in patients with AIDS and in alleviating nausea and vomiting in patients undergoing chemotherapy. However, significant side effects have been observed in clinical trials raising concerns regarding the potential clinical utility of cannabinoid-based agents. Disturbances in mood and affect, including paranoia, anxiety and nervousness, have been reported in patients. Understanding the neural circuits and neurochemical substrates impacted by cannabinoids will provide a better means of gauging their actions within the central nervous system that contribute to the expression of unwanted side effects. We have previously shown an increase in anxiety-like behaviors in rats receiving repeated administration of cannabinoid agonists. This increase in anxiety was accompanied by increases in indices of noradrenergic activity. In the present study, we investigated whether norepinephrine in the limbic forebrain of rats is required for cannabinoid-induced aversion using an immunotoxin lesion approach combined with behavioral analysis using a place conditioning paradigm. Male Sprague Dawley rats received bilateral injections of a ribosomal toxin, saporin (SAP) conjugated to an antibody that specifically recognizes the enzyme dopamine-beta-hydroxylase (DSAP), into the limbic forebrain. Control rats received saporin alone. As previously reported, administration of the synthetic cannabinoid receptor agonist, WIN 55,212-2 (3.0mg/kg), induced aversion in a place conditioning paradigm in SAP-only treated rats. The rats’ spatial memory was also evaluated using the Morris Water Maze. Depletion of norepinephrine using DSAP in specific limbic regions impaired cannabinoid-induced aversion to WIN 55,212-2 without affecting learning and memory processes. Taken together, noradrenergic projections to the limbic forebrain may be critical in the manifestation of aversive behaviors associated with cannabinoid agonist exposure.

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

Lima T-Z, Blanco MM, Bueno MA, Dos Santos Junior JG, Bargieri DY, Mello LE (2009) The influence of cholinergic degeneration on the progression of Alzheimer’s disease and its action in determining the outcome of lithium treatment. Neuroscience 2009 Abstracts 139.26/D36. Society for Neuroscience, Chicago, IL.

Summary: A substantial loss of cholinergic innervation in the hippocampus and cerebral cortex is universally accepted as a typical feature of Alzheimer’s disease (AD). Cholinergic deafferentation is an often, but not a constant phenomenon in AD and its contribution to the progression of disease is not completely understood. The present work was aimed to evaluate the effect of cholinergic deafferentation on cognitive decline and on Amyloid-b (A_) metabolism and how this outcome is modulated by lithium. To this end rats were subjected to neonatal intracerebroventricular injection of 192 IgG-saporin (an immunotoxin selective to cholinergic neurons). Three months after surgery animals were evaluated in Morris Water Maze (MWM) and then entered a three months long lithium (LiCl) or control treatment. At the end of treatment, animals were once again tested in MWM and their cerebral cortical A_ levels were measured. We found that working memory impairment arises earlier and it is also more severe than reference memory related deficits. The cognitive performance was only slightly influenced by LiCl treatment. Furthermore we found that the cholinergic denervation produced by neonatal IgG-sap infusion did not modify A_ levels or its aggregation state. Moreover lithium increased the levels of A_1-42 despite decreasing the amount of A_1-40, an effect that is dependent of cholinergic integrity. These data suggest that the contribution of cholinergic deafferentation, which occurs over the progression of disease, to the amyloigenesis is likely to be modest in AD brain. In addition the effects of lithium treatment presented here imply in avoiding its use as prophylactic propose for AD and in AD cases without marked cholinergic degeneration.

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