targeted-toxins

Savage ST, Lundströmer K, Olson L, Mattsson A (2009) Alterations in dopaminergic and glutamatergic systems following cortical cholinergic denervation. Neuroscience 2009 Abstracts 839.14/M21. Society for Neuroscience, Chicago, IL.

Summary: Alterations in cholinergic signaling in the brain have been implicated as a contributing factor in the pathogenesis of schizophrenia. We have previously shown that cholinergic denervation of cortex cerebri by stereotaxic infusion of the immunotoxin 192 IgG-saporin in the nucleus basalis magnocellularis (nbm) in adult rats leads to an enhanced sensitivity to both amphetamine and the NMDA receptor antagonist phencyclidine (PCP). The enhanced sensitivity to amphetamine shown as a potentiated dopamine release in nucleus accumbens, along with a marked increase in locomotor activity in response to both amphetamine and PCP, suggested that the disruption of cortical cholinergic activity can lead to disturbances of glutamatergic and dopaminergic transmission. To further evaluate the consequences of cortical cholinergic denervation on the dopamine and glutamate systems, we are conducting an in depth in situ hybridization and immunohistochemistry analysis of nbm 192 IgG-saporin lesioned rats. Preliminary data from these investigations show an enhancement of expression levels of TH and DAT mRNA in the VTA and substantia nigra of the cholinergically denervated rats. The data suggests that cortical levels of NMDAR1 mRNA are not altered in the lesioned animals. However, preliminary data indicate that the induction of c-fos mRNA expression in cortex following PCP administration is reduced in denervated animals as compared to sham lesioned controls. These data may suggest hypofunction of NMDA receptors as a consequence of loss of cholinergic innervations. To evaluate the behavioral consequences of cortical cholinergic denervation, we are employing three behavioral paradigms (Locomotor and Rearing behavior, Social Interaction, and the Novel Object Recognition (NOR) task) under normal and drug challenged conditions. Preliminary social interaction studies have found that the saporin lesioned rats spend a significantly less amount of time interacting with each other as compared to control sham operated rats. We are currently investigating how this impairment is effected under drug challenge. Furthermore, we have found that the degree of lesion affects the performance to the novel object recognition task under saline and drug challenged conditions. Our results from the in situ hybridization and behavioral studies indicate that the loss of cortical acetylcholine can lead to alterations in glutamatergic and dopaminergic signaling. These observations are compatible with a possible role of cholinergic deficits in schizophrenia, and provide a possible link between different hypotheses of the disorder.

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

Chatterjee K, Kline IV RH, Wiley RG (2009) Role of brainstem noradrenergic neurons in modulation of operant nocifensive responses to heat: Pharmacology and hyperalgesia. Neuroscience 2009 Abstracts 855.10/X15. Society for Neuroscience, Chicago, IL.

Summary: Many spinal dorsal horn neurons are under direct modulation from various brainstem nuclei which act to modulate nociceptive activity. Nocifensive reflex response modulation by spinally projecting noradrenergic brainstem nuclei has been extensively categorized. Strong evidence supports a role for these neurons in the modulation of reflex nocifensive responses but the role of noradrenergic neurons in the cerebral component of nociception remains to be defined in rats. In the present study, we sought to determine the effects of selectively destroying noradrenergic rostral brainstem neurons (A5,A6,A7) on operant escape from 44°C floor heat under several conditions: 1-baseline (after i.c.v. toxin/vehicle injection), 2- after s.c. injection of morphine, clonidine or yohimbine, 3- three hours after bilateral dorsal hindpaw application of mustard oil (secondary hyperalgesia), and 3- three hours after bilateral plantar application of 0.9% capsaicin cream (primary hyperalgesia). Rats were tested daily until steady operant escape responding (~1 month), then injected i.c.v. with 10µl of PBS (vehicle control, n=8) or antiDBH-saporin (10µg, n=8). After recovery from toxin injection, escape responses decreased in the antiDBH-sap rats. Morphine (0, 0.5, 1.0, 2.0 mg/kg s.c.) 20 min prior to testing, dose dependently attenuated escape from the noxious thermal plate at 44oC for all treatment groups. antiDBH-sap treated rats, however, showed an enhanced morphine effect (more prolonged occupancy of the noxious thermal plate). Three hours after plantar capsaicin, or mustard oil to the dorsal surface of both hindpaws, PBS but not antiDBH-sap rats showed enhanced escape. Systemic clonidine (0.125mg/kg) decreased escape for both PBS and antiDBH-sap treated rats, but the anti-nociceptive effect was greater in antiDBH-sap rats. Systemic yohimbine (1.0, 2.5, 5.0mg/kg) had no effect on escape in antiDBH-sap rats but enhanced escape in PBS rats. In direct contrast to effects on escape responding, antiDBH-sap did not affect hotplate lick/guard initial latencies to nociceptive heat at 44° or 47oC. Escape responses to aversively bright light were also decreased in antiDBH-sap rats suggesting generally decreased responsiveness to aversive stimuli. These results support a significant role for rostral brainstem noradrenergic neurons in modulation of pain and highlight important differences between reflex nocifensive responses (hotplate) and operant (escape) responses.

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

Bukowski RK, Duffy TE, Ryu V, Covasa M, Czaja K, Ritter RC (2009) Attenuated CCK-induced satiation and increased weight gain following destruction of abdominal vagal afferents by intravagal OX7-saporin conjugate. Neuroscience 2009 Abstracts 870.5/DD2. Society for Neuroscience, Chicago, IL.

Summary: Bilateral subdiaphragmatic vagotomy attenuates reduction of food intake by cholecystokinin (CCK) and other GI satiation signals. However, abdominal vagotomy also is associated with mild to moderate reductions of food intake and body weight gain. These sequels of vagotomy may be due to surgical trauma, gastroesphageal dysmotility or, perhaps, hypersensitivity of residual or regenerating afferent vagal fibers and terminals. In an attempt to selectively destroy the abdominal vagal afferents and their cell bodies, we injected the abdominal vagal trunks with OX7-saporin (OX7), a conjugate of the ribosomal toxin, saporin, and a monoclonal antibody against Thy1. This conjugate has been shown to destroy vagal afferent cell bodies in the ipsilateral nodose ganglion following unilateral injection into a cervical vagal trunk. In our study rats received an IP injection of fast blue (FB) which retrogradely labeled cell bodies of abdominal vagal afferents, enabling us to verify their destruction. OX7 was injected into both dorsal and ventral abdominal vagal trunks using a picospritzer and capillary pipettes. Beginning two weeks after OX7, the rats were tested for reduction of food intake by IP injection of CCK-8 (4ug/kg). Subsequently, nodose ganglia from the treated rats and their controls were examined to determine the number of FB-labeled nodose neurons remaining in the ganglia. Successful destruction of nodose neurons varied between animals. However, in OX7-treated rats the number of FB-labeled nodose neurons was reduced by approximately 60%, compared to vehicle injected controls. While CCK injection significantly reduced food intake in control rats, CCK-induced reduction of intake by the OX7 treated group was significantly attenuated. Interestingly, the OX7-treated rats did not exhibit the chronically reduced body weight that is typical of surgically vagotomized rats. In fact OX7 rats actually gained more weight than control rats over the 30 period following vagal injections. Our data indicate that immunotoxic destruction of the abdominal vagal innervation mimics surgical vagotomy in its attenuation of CCK-induced satiation, but does not cause sustained reduction of body weight.

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

Botly LC, De Rosa E (2009) Using visual search to examine cholinergic contributions to feature binding in the rat. Neuroscience 2009 Abstracts 873.26/EE13. Society for Neuroscience, Chicago, IL.

Summary: According to the feature integration theory of attention, feature binding is an attention-dependent process whereby the different features of an object are simultaneously integrated to form a unified whole. Using a rat digging paradigm that was faithful to this theory of attention, we have previously demonstrated that acetylcholine is critical to the attention-dependent processes required for both crossmodal and intramodal feature binding. Moreover, we demonstrated that cholinergic cells in brain regions that have been implicated in human feature binding, specifically frontal and parietal cortices, supported feature binding in rats. We have now translated the gold-standard test of human feature binding, visual search (VS), for rats. In the present study, sixteen male Long-Evans rats were trained to perform VS using touchscreen-equipped operant chambers and black-and-white shapes. Testing sessions comprised Feature-Search (no feature binding required) and Conjunctive-Search (feature binding required) trials using set sizes of four, six, and eight stimuli. Following acquisition of the VS task, eight rats received bilateral 192 IgG-Saporin immunotoxic lesions of the nucleus basalis magnocellularis (NBM) of the basal forebrain to reduce cholinergic afferentation of the neocortex. Importantly, there was no significant effect of lesion on accuracy for selecting the target stimulus. As expected, relative to sham-lesioned rats, NBM-lesioned rats took significantly longer to locate the target stimulus on Conjunctive-Search but not Feature-Search trials; thus reflecting a less efficient VS. These data confirm that cholinergic contributions from the NBM support feature binding using a rat analog of the VS paradigm.

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

Gulino R, Gulisano M (2009) Poster: Expression of cell fate determinants and plastic changes after neurotoxic lesion of adult mice spinal cord by cholera toxin-B saporin. Neuroscience 2009 Abstracts 563.15/DD51. Society for Neuroscience, Chicago, IL.

Summary: Recent studies have attempted to achieve recovery after spinal cord (SC) injury or disease by either increase neurogenesis or stimulate neuroplasticity. Sonic hedgehog (Shh) Notch-1 and Numb are involved in the regulation of stem cell function. Additionally, Notch-1 has a role as modulator of synaptic plasticity. Little is known about the role of these proteins in the adult SC after selective removal of motoneurons. We injected Cholera toxin-B saporin into the gastrocnemius muscle to induce a selective depletion of motoneurons within lumbar mice SC and analysed the expression levels of Shh, Notch-1, Numb, Choline acetyltransferase (ChAT) and Synapsin-I proteins. The functional outcome of the lesion was monitored by grid walk test and rotarod. The neurotoxin lesion determined a motoneuron depletion and a decrease of ChAT and Synapsin-I protein levels in the lumbar SC. ChAT and Synapsin-I appeared correlated each other and with the motor performance, suggesting that the recovery of locomotion could depend from synaptic plasticity. Moreover, we observed a number of proliferating cells within the depleted SC, which were identified as active astrocytes. Shh and Notch-1 appeared reduced in the lesioned tissue and correlated with ChAT and Synapsin-I levels, suggesting a role in modulating synaptic plasticity. Numb expression was also reduced after lesion and appeared correlated with motor performance Therefore, given the role of these proteins in adult neurogenesis, we presume their involvement also in the observed glial reaction. The in vivo manipulation of Shh, Notch-1 and Numb signalling after lesion could be a way to reduce glial reaction and improve functional recovery.

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

Jeong D, Hwang Y, Lee D, Chang J (2009) Behavioral and histological characteristics of 192 IgG-saporin injected rats depending on injection site and dose. Neuroscience 2009 Abstracts 526.23/H8. Society for Neuroscience, Chicago, IL.

Summary: Cholinergic neuronal deficits are evident in both Alzheimer’s disease dementia (AD) and vascular dementia (VaD). Forebrain Cholinergic neurons in the nucleus basalis magnocellularis (NBM) project primarily to the neocortex, and those in the medial septum project to the hippocampus and they make an important role in memory function. We used 192 IgG-saporin to mimic deficits of cholinergic neurons at AD and VaD. 192 IgG-saporin is composed with monoclonal antibody had a low affinity to the rat nerve growth factor receptor p75 and ribosomal inactivating protein, called saporin. When injected intracerebroventricularly or directly into the basal forebrain cholinergic complex, 192 IgG-saporin selectively destroys cholinergic neurons. Many experimenters had used 192 IgG-saporin to investigate cholinergic function but it had been used in different doses and sites of lesion. This makes it difficult to compare the degrees of impairment produced by different lesions. Consequently, our aim is observation of behavioral and histological changes depending on injection site and dose of 192 IgG-saporin. We injected 192 IgG-saporin (0.63ug/ul) in medial septum (dose: 0.05ul, 0.1ul, 0.2ul) or lateral ventricle (dose: 6ul, 8ul, 10ul). 192 IgG-saporin injected rats were compared with Dulbecco’s phosphate buffered saline injected rats. Neurological deficit and functional outcome were determined by immuohistochemistry using anti-cholineacetyltransferase antibody and behavioral test, called water maze. In immunohistological study, the extent of the cholinergic lesion was showed in the basal forebrain complex region of 8ul and 10ul of 192 IgG-saporin injected rats. In behavioral study, sham and lesion groups were able to learn the reference aspect of the water maze within 5day of training. In probe test, we observed significant decrease in time in target quadrant, platform and platform crossings, and increase in latency to first crossing at 8ul and 10ul of 192 IgG-sapoin injected rats (p<0.05). Therefore, our study evaluated that 8ul 192 IgG-saporin injections were sufficient to make an AD mimic dementia model.

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

Matchynski JJ, Lowrance S, Rossignol J, Puckett N, Derkorver N, Radwan J, Trainor K, Sandstrom M, Dunbar G (2009) Intracerebroventricular injections of mu-P-75 saporin can produce memory deficits without impairing motor deficits in a mouse model of Alzheimer’s disease. Neuroscience 2009 Abstracts 528.1/H34. Society for Neuroscience, Chicago, IL.

Summary: Intracerebroventricular injections of mu-P-75 saporin (Advanced Targeting Systems, San Diego, CA) effectively and efficiently destroys cholinergic neurons and creates memory deficits in mice, mimicking some of the key symptoms of Alzheimer’s disease. Early attempts to use mu-P-75 saporin in mice required a relatively high mean effective dose (ED50) of 3.6 µg in order to create behavioral deficits (Berger-Sweeney et al., 2001, The Journal of Neuroscience, 21: 8164-8173; Hunter et al, 2004, European Journal of Neuroscience, 19: 3305-3316). Recent advances in producing the saporin have lowered the ED50 to doses to 0.4 µg, although the resulting memory deficits are transient, and doses above 0.8 µg can cause motor deficits (Moreau et al., 2008, Hippocampus, 18: 610-622). In an effort to elucidate the behavioral effects of a higher (0.8 µg) dose, we gave bilateral intracerbroventricular injections of mu-P-75 saporin (n=6) or sterile phosphate buffered saline (n=3) into C57/BL6 mice and assessed their cognitive abilities on both a Morris water maze (MWM) and an object-recognition task, while monitoring their motor abilities using a rotarod task. Mice receiving the mu-P-75 saporin performed significantly worse than sham animals on an object recognition task and tended to have longer latencies and swim paths during the seven days of MWM testing. Importantly, no between-group differences were observed for latency to fall on the rotarod task. Collectively, these results suggest that the 0.8 µg dose of saporin is both safe and effective for mimicking AD-like memory deficits, without causing significant motor deficits.

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

Hernandez MA, Pineda JB, Del Valle-Mondragón L, Alcaraz-Zubeldia M, Ríos C, Pérez-Severiano F (2009) Evaluation of the effect of molsidomine on nitregic system in an experimental model of cognitive impairment. Neuroscience 2009 Abstracts 529.24/J10. Society for Neuroscience, Chicago, IL.

Summary: The relationship between nitric oxide (NO) and cholinergic system in brain has been evidenced by using inhibitors of the nitric oxide synthase (NOS) that blocked cognition, while NO donors can facilitate it. Nevertheless, the participation of NO in the recovery of cholinergic deficit due to the administration of a selective cholinergic immunotoxin, 192 IgG saporin (SAP) has not been studied. The aim in this work was to evaluate the modulation of the nitrergic system after the damage induced by SAP and to measure the response to the administration of a NO donor, molsidomine (MOL). We used adult male Wistar rats allocated into either one of 4 groups: 1) vehicle PBS, 0.1M pH 7.4, 2) intraseptal administration of SAP (0.22 µg), 3) MOL ip 4 mg/kg, 4) SAP+MOL. Striatum, prefrontal cortex and hippocampus were dissected out at different times after treatment and quantification of nitrites, NOS activity and expression were performed. Our results show that SAP induces a reduction on the constitutive NOS activity in prefrontal cortex and striatum (54%, 64% respectively compared with control p<0.05); while hippocampal cNOS tended to decrease. MOL alone improved NOS activity in those regions. Neuronal and endothelial NOS expression (nNOS, eNOS) in the same regions did not change significantly. When the nitrites levels were analyzed, changes were region-specific. We conclude that administration of the NO donor promotes the recovery of cNOS activity in the model of cholinergic denervation associated to 192 IgG SAP. Further cognitive studies are being carried out in order to demonstrate the cholinergic recovery by MOL.

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

Thomsen MS, Hay-Schmidt A, Hansen HH, Mikkelsen JD (2009) Poster: Distinct neural pathways mediate alpha7 nicotinic acetylcholine receptor-dependent activation of the forebrain. Neuroscience 2009 Abstracts 646.2/V14. Society for Neuroscience, Chicago, IL.

Summary: α7 nicotinic acetylcholine receptor (nAChR) agonists are novel drugs candidates for the treatment of cognitive deficits in schizophrenia, which have shown pro-cognitive effects in clinical trials. Selective α7 nAChR agonists, such as SSR180711, activate neurons in the medial prefrontal cortex (mPFC) and shell of the nucleus accumbens (ACCshell) in rats, regions which are important for cognitive function. However, the neural substrates involved in these effects remain elusive. Using retrograde tracing from the mPFC with Cholera Toxin B and immunoreactivity of the immediate-early gene c-Fos, a marker of neuronal activation, we identify the horizontal limb of the diagonal band of broca (HDB) in the basal forebrain as an important site of α7 nAChR activation. Approximately 26% of the cortically projecting neurons in the HDB are activated by acute administration of SSR180711 (10 mg/kg), and the neurons activated by SSR180711 in the HDB are cholinergic. Selective depletion of these cholinergic neurons with 192 IgG-Saporin abolishes the SSR180711-induced activation of the mPFC, but not the ACCshell, demonstrating their critical importance for α7 nAChR-dependent activation of the mPFC. Contrarily, selective depletion of dopaminergic neurons in the ventral tegmental area (VTA) with 6-OHDA abolishes the SSR180711-induced activation of the ACCshell, but not the mPFC or HDB. These results indicate that two distinct neural pathways are activated by SSR180711, involving HDB-to-mPFC and VTA-to-ACCshell projections, respectively. The basal forebrain and mPFC are important for attentional function, and may subserve the pro-cognitive effects of α7 nAChR agonists, whereas activation of the ACCshell is implicated in beneficial effects on the positive symptoms of schizophrenia.

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

Kline IV RH, Wiley RG (2009) Spinal µ-opiate receptor (MOR)-expressing dorsal horn neurons: Role in modulating pain and opiate analgesia. Neuroscience 2009 Abstracts 560.13/CC72. Society for Neuroscience, Chicago, IL.

Summary: Selective destruction of MOR-expressing interneurons in lamina II of the dorsal horn of the spinal cord increases reflex nocifensive responses to formalin and decreases the anti-nociceptive effects of morphine on the hotplate and in the formalin test. The interpretation of these studies is limited because reflex-based assays may not accurately reflect the cerebral component of nociception. Therefore, we sought to determine the effects of selectively destroying MOR-expressing dorsal horn neurons on baseline operant responses to aversive thermal and mechanical stimuli in a shuttle box task and effects of systemic morphine and naloxone in the same task. The preference apparatus consisted of a 15 X 15 X 30 cm smoked Plexiglas vented chamber placed upon two adjoining temperature-controlled smooth aluminum floor plates (thermal preference task) or one smooth temperature-controlled floor plate adjoined to a room temperature surface covered with 40 grit sandpaper (mechanical preference task). For both preference tasks, response functions were obtained by pairing a 44°C plate or the sandpaper surface with either 11°, 16°, 25°, 38° or 46°C. Rats were intrathecally injected over the lumbar cord with either 625ng of derm-sap (n=7) or blank-sap (n=6) followed by daily thermal or mechanical preference testing on a randomized schedule. Derm-sap treated rats showed enhanced avoidance of aversive thermal stimuli and the aversive mechanical stimulus. Morphine and naloxone significantly altered responses of control rats (blank-sap), but not derm-sap rats, in both thermal and mechanical preference tasks. We interpret these results as showing that the derm-sap lesion produces hyperalgesia/allodynia, impairs the anti-nociceptive and analgesic effects of morphine and therefore indicating that postsynaptic dorsal horn MOR-expressing neurons play a key role in modulating nociception, pain and opiate analgesia. Dysfunction of these neurons may also play a role in pathological pain states.

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