targeted-toxins

Motooka Y, Kondoh T, Nomura T, Tamaki N, Nishizaki T (2001) Poster: Selective cholinergic denervation inhibits expression of long-term potentiation in the adult but not infant rat hippocampus. Neuroscience 2001 Abstracts 599.7. Society for Neuroscience, San Diego, CA.

Summary: The present study assessed the role of the cholinergic systems on the expression of perforant path long-term potentiation (LTP) in rat hippocampal slices from the infant and adult brain. To denervate the cholinergic systems, 192 1gG-saporin was injected into the lateral ventricle of the infant (2 weeks old) and adult (6 weeks old) rat brain. There, choline acetyltransferase immunoreactive fibers were little detectable two weeks and two months after injection for both the groups. For the infant rats, perforant path LTP was not affected by selective cholinergic denervation; the probability of LTP development was 0.83 (5/six slices) and 0.78 (7/nine slices) at 2 weeks and 2 months later in 192 IgG-saporin-treated slices, as compared with 0.83 at each period in control saline-treated slices. In contrast, the expression of the LTP was blocked by selective cholinergic denervation for the adult rats; the probability of LTP development was 0 (0/ten slices) and 0.38 (3/eight slices) at 2 weeks and 2 months later in 192 IgG-saporin-treated slices, as compared with 0.8 (8/ten slices) and 0.83 (5/six slices) at each period in control saline-treated slices. The results of the present study thus suggest that the cholinergic systems play a crucial role in the expression of LTP in the adult brain and that the denervated systems in the infant brain could be compensated by the sprouting of non-cholinergic fibers.

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

Kudoh M, Seki K, Watanabe S, Shibuki K (2001) Sound sequence discrimination requires cholinergic inputs and suppression of M-currents in the rat auditory cortex. Neuroscience 2001 Abstracts 621.2. Society for Neuroscience, San Diego, CA.

Summary: Synaptic potentiation after sequential heterosynaptic stimulation is dependent on the stimulus sequence in the auditory cortex. Atropine or antagonists of M1 receptors block this sequence dependence. However, it is rescued by linopirdine, an inhibitor of M-currents, even in the presence of atropine. In the present study, we investigated the role of cholinergic inputs and the resulting suppression of M-currents in sound sequence discrimination. Rats were trained to discriminate sequence of two sounds. Licking a spout during sound presentation of a particular sequence was rewarded with water. The sounds of the rewarded or unrewarded sequence were randomly presented in a trial, which was repeated every one minute for 12 hours in 4 days. The percentage of the trials with a licking response to sounds was calculated separately for the rewarded and unrewarded sequence. Test performance, estimated from the difference, was significantly increased in the 4 days. Atropine (10 mg/Kg, i.p.) suppressed the increase of test performance. A cholinergic immunotoxin, 192IgG-saporin, was injected into the auditory cortex (62-400 ng/1µl) 1 week before the test. Sound sequence discrimination, but not discrimination between the two sounds, was significantly suppressed in these rats. Linopirdine (5 mg/Kg, i.p.) rescued the test performance of sound sequence discrimination in the rats injected with 192IgG-saporin. These results suggest that sound sequence discrimination requires cholinergic inputs and the resulting suppression of M-currents in the auditory cortex.

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

Nattie EE, Li A (2001) Destruction by SP-SAP of rat retrotrapezoid nucleus (RTN) neurons expressing the neurokinin 1 receptor (NK1R) decreases breathing at rest and in response to hypercapnia. Neuroscience 2001 Abstracts 573.1. Society for Neuroscience, San Diego, CA.

Summary: Neurons in the RTN are hypothesized to provide both a tonic excitation for breathing and one location for central chemoreception (see Nattie, E., Prog. Neurobiol.1999). Lesions in anesthetized animals support the former while both lesions and focal acidification in unanesthetized animals support the latter. Application of substance P (SP) in the RTN increases respiratory output and immunohistochemistry for the SP (NK1) receptor shows extensive staining in the RTN. To destroy specifically these RTN neurons with NK1Rs we injected unilaterally in the RTN of the rat SP conjugated to the ribosomal toxin, saporin (SP-SAP; 100 nl; 1 uM; Advanced Targeting Systems). We measured ventilation by whole body plethysmography in the unanesthetized rat. At 6 to 15 days following SP-SAP injection, ventilation during air breathing was reduced by 19 to 24% and the response to 7% CO2 inhalation was reduced by 22 to 30%. Subsequent immunohistochemistry showed dramatically reduced NK1R staining in the area of the SP-SAP injection, which is difficult to quantify given the small number of RTN neurons and the extensive NK1R distribution along neuronal processes. RTN neurons with NK1Rs provide both a tonic excitation for breathing and a portion of the response to systemic hypercapnia.

Related Products: SP-SAP (Cat. #IT-07)

Sanders NM, Dinh TT, Pedrow C, Ritter S (2001) Selective immunotoxin lesions of hindbrain norepinephrine/epinephrine (NE/E) neurons impair feeding and corticosterone responses and Fos-immunoreactivity in hypothalamic sites during insulin-induced hypoglycemia (IIH). Neuroscience 2001 Abstracts 635.21. Society for Neuroscience, San Diego, CA.

Summary: Previously,we used the targeted immunotoxin saporin, conjugated to a monoclonal antibody against dopamine Beta-hydroxylase(DSAP),to destroy hypothalamic projecting NE/E neurons. Results showed that NE/E neurons are required for 2-deoxy-D-glucose induced feeding and Fos expression in the hypothalamus. In the present study,we used this same technique to determine if NE/E neurons play a similar role in mediating IIH responses.Rats were injected with DSAP or unconjugated saporin (SAP)into the hypothalamic paraventricular nucleus(PVH).Insulin reduced blood glucose to similar values in DSAP and SAP rats(15 and 17mg/dl, respectively). Glucagon responses to hypoglycemia were unaffected by DSAP,peaking at 597% and 504% of pre-drug levels after insulin in DSAP and SAP rats,respectively.In contrast,the corticosterone response was severely diminished in DSAP rats,peaking at only 123% of pre-insulin levels, compared to 353% in SAP rats.DSAP injections also abolished the feeding response to IIH.DSAP rats ate 0.9g of food during IIH while the SAP rats at 6.1g of food.DBH-ir was abolished in the A1/C1 overlap and reduced in A2,C2,C3 and A6 sites in DSAP rats. In the SAP rats,IIH induced Fos-ir in hindbrain NE/E neurons the PVH, LH and ARC.In DSAP rats, Fos-ir was reduced or abolished in these hypothalamic sites but was preserved in the adrenal medulla.These findings further support the role of hindbrain NE/E neurons in transmitting information from hindbrain glucoreceptive sites to hypothalamic circuits coordinating feeding and neuroendocrine responses to glucose deficit.

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

Palecek J, Paleckova V, Willis WD (2001) Dermorphin-saporin conjugate relieves inflammatory pain after peripheral application. Neuroscience 2001 Abstracts 508.10. Society for Neuroscience, San Diego, CA.

Summary: Opioid receptors have been shown to exist in specific population of DRG neurons signaling nociceptive information from peripheral tissues. In our study, we attempted to selectively destroy these neurons by using a peripheral application of the mu opioid agonist Dermorphin conjugated to ribosome inactivating toxin Saporin (DERM-SAP, Advanced Targeting Systems) in order to alleviate inflammatory pain. Intraarticular or intraplantar injection of carrageenan or CFA was used to induce inflammation in rats. The DERM-SAP conjugate was injected into the inflamed area 12-48h later. Responses of the animals to mechanical and thermal stimuli were tested before and after the inflammation and up to 21 days after the DERM-SAP application. The rats developed heat hyperalgesia in the affected paw 24 hours after the intraarticular CFA injection. In the saline injected group the hyperalgesia persisted for up to 19 days, but in the DERM-SAP injected group the signs of hyperalgesia were improving from day 7. Also mechanical allodynia tested with a VF filament (1.1g) was alleviated in the DERM-SAP group. In the carrageenan group, the DERM-SAP treatment decreased the heat hyperalgesia and prevented the development of hyperalgesia after repeated carrageenan application, 21days after the DERM-SAP treatment. Postmortem evaluation with a specific antibody showed presence of saporin in the DRG neurons. Our results show that peripheral application of DERM-SAP relieves inflammatory pain and suggest that peripheral application of neuropeptides conjugated to cell toxins or other substances such as antisense probes could be a useful tool for treating pain of peripheral origin. Supported by NIH grants NS09743 and NS11253.

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

Greco MA, Salin-Pascual R, Gerashchenko D, Blanco-Centurion C, Shiromani PJ (2001) Effects of 192-saporin lesion of the basal forebrain on sleep homeostasis and adenosine receptor (A1) mRNA levels. Neuroscience 2001 Abstracts 523.12. Society for Neuroscience, San Diego, CA.

Summary: Adenosine is hypothesized to be a mediator of sleep since adenosine levels in the basal forebrain increase after wakefulness and decrease following sleep. The inhibitory effect of adenosine on wake-active cholinergic neurons is thought to be mediated by the A-1 receptor subtype. We hypothesized that if adenosine inhibition of cholinergic neurons takes place via A-1A receptors on cholinergic neurons, the elimination of cholinergic cells should affect sleep homeostasis. To test this hypothesis, 192-saporin was used to selectively lesion basal forebrain cholinergic cells. 48h baseline sleep was recorded from male Sprague Dawley rats. Subsequently, the rats were kept awake for 12h and 24h recovery sleep was recorded. 192-saporin (4 ug) was then administered ICV. The rats were again continuously recorded for 3 weeks after the injection, a 12h prolonged waking period and during a 24h recovery sleep period. Brain sections processed for visualization of A1 mRNA and/or immunohistochemistry revealed that both ChAT- and parvalbumin-positive cells contained A-1A mRNA. 192-SAP eliminated ChAT immunoreactive cells in the basal forebrain. There were no differences in sleep-wakefulness up to 3 weeks after drug administration, a finding consistent with previous reports. In addition, there were no changes in recovery sleep following prolonged waking in lesioned rats. These results indicate that the cholinergic basal forebrain groups are not the primary mediators of wakefulness or of sleep homeostasis. We suggest that the effects of adenosine are mediated via binding to non-cholinergic neurons.

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

Shafi R, Berntson GG, Sarter M, Saurer T, Spino M (2001) Basal forebrain cholinergic system: Cortical activation, sleep/waking EEG and evoked potentials. Neuroscience 2001 Abstracts 533.16. Society for Neuroscience, San Diego, CA.

Summary: The role of the basal forebrain cholinergic system in cognitive functions such as arousal, attention and memory has been well documented. The purpose of the present study was to further elucidate the role of the basal forebrain in regulating cortical states and processes that may underlie these functions. Selective lesions of the cholinergic neurons of the basal forebrain were made using the immunotoxin 192 IgG-saporin, which selectively targets the p75 receptor on cholinergic neurons. The effects of these lesions on sleep structure and EEG activity and on afferent priming of cortical reactivity was evaluated. Specifically, we monitored behavioral activity and sleep states and examined the frequency distribution of power distribution in EEG frequency bands during these states. In separate sessions, we also recorded cerebral event-related potentials to auditory stimuli (100 ms, 1 K Hz at 60, 70 and 80 db) after intraperitoneal administration of saline or epinephrine (0.5 mg/kg, which we have previously found to result in priming or enhancement of the auditory evoked response). Compared to controls, lesioned animals showed a reduction in spontaneous activity, reduced power in higher frequency (primarily gamma) EEG bands during both sleep and waking, and altered sleep structure. In addition, lesioned animals displayed lower amplitude auditory evoked potentials and a loss of epinephrine-priming of the evoked response. Results support the view that the basal forebrain cholinergic system may play an important role in cortical activation and the regulation of sleep/waking states, as well as in cortical processing and its enhancement by visceral priming.

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

Zambon NJ, Nagle R, Pokala V, Gibbs RB, Johnson DA (2001) Low dose 192 IgG-saporin selectively destroys basal forebrain cholinergic neurons and impairs acquisition of a spatial memory task. Neuroscience 2001 Abstracts 534.13. Society for Neuroscience, San Diego, CA.

Summary: We previously showed that a high dose (1 μg) of the selective cholinergic immunotoxin 192 IgG-saporin (SAP), injected into the medial septum (MS) of Sprague-Dawley rats, impeded acquisition of a delayed matching-to-position (DMP) spatial memory task, whereas injections of ibotenate (5 μg in 1 μL) did not. The present study examined the effects of lower doses of SAP (0.22 and 0.45 μg in 1 μl) on DMP acquisition. Animals received either SAP or vehicle injected directly into the MS. Two weeks later, animals were food deprived and trained to the DMP task. Rats received 8 trial pairs/day until they reached a criterion of 15/16 correct choices. Seven days later, post-criteria testing for retention was performed. Brain tissues were analyzed for choline acetyltransferase (ChAT) activity, or were processed for immunohistochemical detection of ChAT and parvalbumin. Control rats required significantly fewer days (13.1) to reach criterion than rats that received 0.22 (22.0 days) or 0.45 (20.1 days) μg SAP. There was no effect of SAP treatment on post-criteria testing. Injections of SAP produced marked depletion of ChAT-positive cells and ChAT activity, but no apparent depletion of parvalbumin staining in the MS. In contrast, ibotenate injections used in the previous study were shown to produce marked depletion of parvalbumin staining in the MS, but no significant cognitive impairment. The data suggest that selective destruction of cholinergic neurons in the MS significantly impairs acquisition of the DMP task.

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

Chou TC, Gerashchenko D, Saper CB, Shiromani PJ (2001) Loss of histaminergic neurons does not produce hypersomnolence. Neuroscience 2001 Abstracts 522.21. Society for Neuroscience, San Diego, CA.

Summary: Electrolytic lesions of the posterior hypothalamus (PH) produce long-lasting hypersomnolence (1,2). The PH contains histaminergic neurons in the tuberomammillary nucleus (TMN) that project diffusely throughout the brain. Because histamine promotes wakefulness while antihistamines are sedating, the TMN is thought to be critically involved in maintaining wakefulness. To test this hypothesis, we placed cell-specific lesions in the PH and TMN of rats and measured sleep-wake behavior. Lesions were produced using either the conventional excitotoxin ibotenic acid, or the novel toxin orexin (hypocretin) conjugated to the ribosomal toxin saporin (ORX/HCRT-SAP). Ibotenic acid injections were ineffective at lesioning the TMN; most histaminergic neurons were selectively spared while neurons in surrounding regions such as the mammillary bodies and supramammillary area were completely lesioned. In contrast, ORX/HCRT-SAP injections into the TMN lesioned up to 95% of histaminergic neurons, as determined by adenosine-deaminase immunostaining, with a similar loss of neurons in adjacent areas. Surprisingly, neither group of rats showed changes in NREM or REM sleep time or circadian distribution of sleep relative to saline-injected controls for up to 2 weeks after surgery. Thus, the waking state may not be critically dependent on the PH or TMN in rats. Further research is needed to reconcile the sedating effects of antihistamines with the current findings. 1. Ranson 1939, Archiv Neurol and Psychiatry 41(1):1-23. 2. Swett and Hobson 1968, Arch Ital Biol 106(3):283-293.

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

Sauro KM, Sweeney M, Saari MJ (2001) Enriched housing, reticular thalamic nucleus and nucleus basalis: Mediators of attention and learning?. Neuroscience 2001 Abstracts 313.14. Society for Neuroscience, San Diego, CA.

Summary: Housing manipulations have been shown to alter behaviour, neural function and morphology. The Reticular Thalamic Nucleus (Rt) may be involved in thalamocortical relay loops possibly acting as a bi-directional filter for sensory information. Similarly, the Nucleus Basalis (NB) may play a role in attention via cholinergic projections to the cortex. In the current experiment 160 female Wistar rats received bilateral lesions of either the Rt (using ibotenic acid), the NB (using 192 IgG-saporin; Advanced Targeting Systems), or both. Following recovery, the rats were allocated to either an enriched or an isolated housing condition for two weeks. Following open field testing, a modification of Tolman’s latent learning paradigm was used to evaluate the effects of the treatments on attention. Thus, prior to behavioural testing half of the rats from each group were pre-exposed to the Spatial Orientation Task (SPOT) while the other half were not. Following sacrifice, brain slices were stained for metabolic activity using the cytochrome-oxidase method, for morphology using cresyl violet stain, and for a marker of cholinergic function, acetylcholinesterase. Data analysis revealed significant interactions among the lesions, housing and latent learning and provided some support for the suggestion that the Rt may filter incoming sensory information whereas the NB may mediate behaviourally relevant attention. Lesions and housing effects were confirmed by histology. (Approved by the Animal Care Committee; supported by Nipissing University).

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