targeted-toxins

Grupe M, Paolone G, Jensen AA, Nielsen KS, Christensen JK, Grunnet M, Sarter M (2012) Positive allosteric modulation of 4 2* nicotinic acetylcholine receptors augments the amplitudes of prefrontal nicotine-evoked glutamatergic transients. Neuroscience 2012 Abstracts 696.15. Society for Neuroscience, New Orleans, LA.

Summary: α4β2* nicotinic acetylcholine receptors (nAChR) are a promising target for cognition enhancement. These receptors have been demonstrated to mediate the modulatory effects of the tonic component of cholinergic neurotransmission on fast prefrontal glutamatergic-cholinergic interactions. Specifically, α4β2* nAChR are expressed by thalamic glutamatergic afferents and amplify cue-evoked glutamatergic release events, thereby initiating a chain of neuronal events required for the detection of cues in attention tasks (Hasselmo & Sarter, 2011). In this study we investigated the effect of NS9283, a potent and selective positive allosteric modulator of low-sensitivity α4β2 nAChR (Timmermann et al., 2012), on nicotine-evoked glutamatergic release events in the mPFC of anaesthetized rats. Glutamatergic transients were recorded using amperometric measures of currents generated by the oxidation of glutamate and, subsequently, peroxide, on Platinum electrodes equipped with immobilized glutamate oxidase (see Parikh et al., 2010). Nicotine was pressure-ejected (0.040-2 nmol in 40-100 nL, respectively) into the vicinity of the recording electrode situated in the thalamic input layer of the prelimbic cortex. Systemic (i.p.) administration of NS9283 (3.0 mg/kg; administered 30 min prior to nicotine) enhanced the amplitude of glutamatergic transients evoked by the lowest dose of nicotine (40 pmol) by 72%. The modulator did not increase the efficacy of nicotine. Local pressure-ejections of NS9283 (400 pmol in 40 nL) per se were capable of evoking glutamatergic release events, presumably reflecting modulation of the effects of endogenous acetylcholine at these nAChRs. Accordingly, 192 IgG saporin-induced removal of cholinergic projections to the recording region abolished NS9283-evoked glutamatergic transients. Collectively, this evidence substantiates the identification of NS9283 as a positive modulator of nAChRs and its potency in vivo to modulate evoked glutamatergic release events. These results are consistent with the hypothesis that such compounds facilitate cue detection processes and thereby enhance attentional performance. Supported by NIH grant MH080332 and The Ministry of Science, Innovation and Higher Education, Denmark, PhD grant 10-084289.

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

Lee J, Jeong D, Chang J (2012) The effects of basal forebrain cholinergic neuron of recognition tests. Neuroscience 2012 Abstracts 345.10. Society for Neuroscience, New Orleans, LA.

Summary: The cholinergic neurons of the Medial septum and the basal nucleus areas of the basal forebrain project to the frontal cortex and the Hippocampus, and degeneration of the cholinergic basal forebrain neuron is a common feature of Alzheimer’s disease(AD) and vascular dementia and it has been correlated with cognitive decline. This research studied to verify the effects of cholinergic neuron in basal forebrain and the role of hippocampus and frontal cortex on recognition through recognition test and immunohistochemistry after damaging cholinergic neuron of the basal forebrain by intraventricular injection of 192 IgG-saporin. 192 IgG-saporin of 8ul (0.63ug/ul) was injected to the bilateral lateral ventricle of rats. After 2 weeks, Novel object recognition (NOR) test and Object in place (OIP) test was conducted to elucidate damage of cholinergic neuron. After completing the behavioral test, the ChAT cholinergic neuron in the brain was ascertained to confirm with immunohistochemistry if cholinergic neuron was damaged. In NOR test, the lesion group with 192 IgG-saporin showed 10% lower novel object preference than normal group. In OIP test, the normal group showed 50% novel object preference and the lesion group with 192 IgG-saporin showed 30% novel object preference in an hour delay test. On the other hand, the normal group and the lesion group with 192 IgG-saporin shoed 33% and 35% novel object preference respectively in a day delay test. However, this rate is not that significant value enough to elucidate behavioral difference between normal group and lesion group. In immunohistochemistry, the number of cholinergic neuron was remarkably decreased in basal forebrain. According to both of the behavioral tests, lesion group seem to less remember novel object than normal group. Also, they searched less the novel object that changed its location than normal group in the short term condition. However, there was no significant difference in the long term condition. These results suggest that the lesion with 192 IgG-saporin can damage spatial working memory.In the Immunohistochemistry result of the lesion condition, cholinergic input to hippocampus in basal forebrain affects recognition. However, the effect is not so essential.

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

Lee S-Y, Ma J, Chung C, Han J-S (2012) Effects of chronic stress on alterations of GR-PKA-NF-kappa B signaling and spatial learning in rats with cholinergic deafferentation. Neuroscience 2012 Abstracts 345.20. Society for Neuroscience, New Orleans, LA.

Summary: Aging and Alzheimer’s disease (AD) is associated with diminished integrity of the cholinergic innervations of the hippocampus and cortex. Previously, we demonstrated that removal of the cholinergic innervations impaired regulation of the HPA axis with response to acute stress and induced changes in the interaction among glucocorticoid receptor (GR), nuclear factor-κB (NF- κB) p65, and the cytoplasmic catalytic subunit of protein kinase A (PKAc) in the hippocampus. The current research examined effects of chronic stress on the altered signaling induced by cholinergic deafferentation. Young adult rats received immunotoxic lesions of basal forebrain cholinergic neurons by intracranial injections of 192 IgG-saporin into the medial septum/vertical limb of the diagonal band and substantia innominata/nucleus basalis. After 2 weeks recovery from surgery, rats with cholinergic lesions and vehicle-injected control rats were subjected to 1 hr restraint stress per day for 2 weeks. Rats with only cholinergic deafferentation or sham-operated rats with chronic stress showed intact spatial learning. Rats with cholinergic deafferentation that received chronic stress showed impairments of spatial learning. And we examined that cholinergic deafferentation induced alterations in GR and NF- κB p65 expression in hippocampus and prefrontal cortex. Thus the loss of cholinergic integrity during aging and in AD may increase proneness to chronic stress.

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

Fricks-Gleason AN, Keefe KA (2012) Poster: Evaluating the role of neuronal nitric oxide synthase-containing striatal interneurons in methamphetamine-induced dopamine neurotoxicity. Neuroscience 2012 Abstracts 360.06. Society for Neuroscience, New Orleans, LA.

Summary: It is well established that exposure to multiple high doses of methamphetamine (METH) produces damage to central monoamine systems. A number of factors, including the production of nitric oxide (NO), have been implicated in this neurotoxicity. While it is relatively clear that NO contributes to METH-induced neurotoxicity to the dopamine (DA) nerve terminal, the source of this NO has not been clearly delineated. There is considerable evidence suggesting that the generation of NO arises a consequence of the activation of neuronal nitric oxide synthase (nNOS). In striatum, nNOS is located post-synaptic to the DA nerve terminal in a subpopulation of striatal interneurons. Thus, we have hypothesized that DA-mediated activation of the nNOS-containing striatal interneurons is necessary for METH-induced neurotoxicity. These interneurons, along with the cholinergic neurons of striatum, selectively express the neurokinin-1 (NK-1) receptor, which is activated by the neuropeptide Substance P. Consequently, toxins targeted to NK-1 receptor-containing neurons can be used to lesion this population of striatal interneurons. One such toxin, a conjugate of Substance P to the ribosome inactivating protein saporin (SSP-SAP), has been shown to be effective in selectively destroying neurons expressing the NK-1 receptor in striatum. Therefore, using targeted deletion of the nNOS-containing interneurons via SSP-SAP, we examined the extent to which impairing post-synaptic production of NO attenuates METH-induced neurotoxicity. The SSP-SAP lesions resulted in a significant and selective loss of nNOS-containing interneurons throughout the striatum, although it was not possible to completely eliminate all of the neurons. Surprisingly, however, this marked deletion of nNOS-containing interneurons did not confer resistance to METH-induced DA neurotoxicity, even in areas completely devoid of nNOS-positive cell bodies and histochemical detection of NOS activity with NADPH diaphorase histochemical staining. Furthermore, these lesions did not attenuate NO production, as assessed via nitrotyrosine immunohistochemistry, even in areas devoid of nNOS. Taken together, these data suggest that nNOS-containing interneurons either are not necessary for METH-induced DA neurotoxicity, leaving open the potential contribution of other sources of NO, such as endothelial NOS (eNOS), or produce NO/RNS that can diffuse extensively through striatal tissue and thereby still mediate the neurotoxicity.

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

Koppen JR, Stuebing SS, Winter SS, Cheatwood JL, Wallace DG (2012) Infusion of GAT1-Saporin into the medial septum spares mnemonic function and impairs self-movement cue processing. Neuroscience 2012 Abstracts 394.25. Society for Neuroscience, New Orleans, LA.

Summary: Spatial orientation depends on multiple neural systems processing environmental and self-movement cues. Previous research has demonstrated a relationship between cholinergic function in the medial septum and processing of self-movement cues. The medial septum also sends GABAergic projections to the hippocampus that synapse on interneurons, thereby producing a strong dis-inhibitory effect on hippocampal pyramidal neurons. Initial studies using non-selective lesion techniques have supported the involvement of this system in spatial orientation; however, the development of an immunotoxin (i.e., GAT1-Saporin) that selectively targets GABAergic neurons, allows for a novel technique in which to study this relationship. The current study examined the effect of infusing GAT1-Saporin or saline into the medial septum on performance during multiple spatial tasks. Environmental and self-movement cue processing was evaluated using the food hoarding paradigm, whereas mnemonic function was evaluated using several water maze tasks. Although GAT1-Saporin spared performance on water maze tasks, impaired performance was observed when rats were restricted to using self-movement cues (i.e., testing under dark conditions) during food hoarding. The current study adds to the growing literature that supports a role for components of the septohippocampal system in self-movement cue processing during spatial navigation.

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

Nichols TW (2012) Emerging roles of pathogens in alzheimer’s and moderate magnetic field therapy: dc emf 0.5 tesla. Neuroscience 2012 Abstracts 438.10. Society for Neuroscience, New Orleans, LA.

Summary: Chronic spirochetal infection can cause slowly progressive dementia, cortical atrophy and amyloid deposition in the atrophic form of general paresis. There is a significant association between Alzheimer disease (AD) and various types of spirochete (including the periodontal pathogen Treponemas and Borrelia burgdorferi), and other pathogens such as Chlamydophyla pneumoniae and herpes simplex virus type-1 (HSV-1). (Miklossy 2011 Exp Rev Mol Med) Miklossy’s lab exposed mammalian glia & neuronal cells in vitro to Borrelia burgdorferi spirochetes and bacterial lipopolysaccharides (LPS). Morphological changes analogous to amyloid deposits were observed at 2-8 wks exposure. Increased levels of ß-amyloid precursor protein and hyperphosphorylated tau were detected by WB.The frequency of spirochetes is significantly higher in the brains of Alzheimer patients compared to controls.The statistical analysis is based on the cumulative data of the literature. (P=1.5×10-17,OR=20, 95%CI=8-60! Seven out of ten brains from the Harvard McLean Brain bank were positive for Borrelia DNA. Alan Mac Donald MD. “Borrelia Infection is the root cause of at least 70% of Alzheimer’s disease, based on the detection of positive In situ DNA hybridization results in the cytoplasic GVB sites of hippocampal neurons ( with no positive signals detected in the nucleus) for flagellin B DNA sequences of Borrelia burgdorferi.” Antibiotics in Alzheimer’s disease: A randomized controlled trial of doxycycline and rifampin for patients with Alzheimer’s disease 2004. Cognitive decline was statically improved in treatment over placebo. Minocycline protects basal forebrain cholinergic neurons from mu p 75-saporin immunotoxic lesioning 2004 in animal model. Minocycline attenuates neuronal cell death and improves cognitive impairment in Alzheimer’s disease models 2007. Minocycline does not affect amyloid ß phagocytosis by human microglia cells. (Minocycline attenuates the release of TNF-α by human microglia upon exposure to Abeta, SAP and C1q) 2007. Moderate Magnetic Field Therapy (0.5 Tesla) in 15 Alzheimer’s patients. Results; Cognition Improved: group average hours = 184.Mechanism hypothesis: Overview of crosstalk between SMF & IL-6.Wang, Z, Sarje A, Che PL, Yarema K. Moderate strength (0.23-0.28T) static magnetic fields (SMF) modulate signaling and differentiation in human embryonic cells. BMC Genomic 2009;10:356

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

Kalinchuk AV, Kim S, Mccarley RW, Basheer R (2012) Cholinergic basal forebrain neurons contribute to the biochemical and electrophysiological changes in the cortex during sleep deprivation. Neuroscience 2012 Abstracts 486.24. Society for Neuroscience, New Orleans, LA.

Summary: Short term sleep deprivation (SD) (2-3h) increases the levels of inducible nitric oxide (NO) synthase (iNOS)-mediated NO and adenosine (AD) in the basal forebrain (BF) (Basheer et al., 1999; Kalinchuk et al., 2006). We showed recently that the prolongation of SD for 5h triggers similar biochemical changes in the prefrontal/frontal cortex (PFC/FC), the area which receives projections from the BF (Kalinchuk et al., 2010). Lesion of the BF cholinergic cells using immunotoxin 192-IgG saporin attenuates SD-induced AD increase in the BF and recovery non-rapid eye movement (NREM) sleep response (Kalinchuk et al., 2008). However, it is not known whether the cholinergic cells play a role in SD-induced biochemical changes in the cortex. In the current study we lesioned BF cholinergic cells, and compared SD-induced biochemical changes simultaneously in the PFC and BF in the same animals before and after the lesion. We correlated the changes in the biochemical markers, NO and adenosine, with the changes in electrophysiological markers of homeostatic sleep pressure, encephalogram (EEG) theta power during SD and delta power during recovery NREM sleep after SD. Male rats were implanted with electrodes for EEG/electromyogram (EMG) recording and 2 guide cannulae for microdialysis probes targeting BF and PFC. Microdialysis samples were collected simultaneously from both areas every 30 min during 8h SD. Dialysates were analyzed for AD using high performance liquid chromatography (HPLC)/fluorescent detection and for NO metabolites nitrate and nitrite (NOx) using Fluorimetric Assay Kit (Cayman). The lesion of the BF cholinergic cells was performed using the local injections of 192-IgG saporin into the BF, and similar experiment was repeated 2 weeks after the injection. Histochemical analysis confirmed the localization of the probes in the BF and PFC and the quality of the lesion procedure. Before saporin injection, SD induced increases in the levels of NOx and AD, which became significant after 1h (NOx) and 2h (AD) of SD in the BF and after 4h (NOx) and 5h (AD) of SD in the FC. EEG recording detected increases in the intensity of theta power during SD and delta power during following recovery NREM sleep. 2 weeks after saporin injection, SD-induced changes in NOx and AD were significantly attenuated both in the BF and the PFC. Also the increases in theta and delta power were significantly attenuated. We conclude that cholinergic neurons of the BF, which provide strong activating input to the PFC, contribute to the generation of homeostatic sleep pressure during SD, including its biochemical and electrophysiological correlates.

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

Zushida K, Light K, Uchida S, Hevi C, Shumyatsky GP (2012) The GRP peptide and the GRPR-positive interneurons control fear acquisition and extinction. Neuroscience 2012 Abstracts 496.03. Society for Neuroscience, New Orleans, LA.

Summary: The gastrin releasing peptide (GRP) is the marker of the neural circuits relaying fear-related conditioned stimulus (CS) information to the amygdala. The GRP is expressed by principal cells and the GRP-receptor (GRPR) is expressed by interneurons. The GRPR is expressed in the amygdala and hippocampus. To examine the role of the GRPR-positive interneurons in these two brain areas, we performed local injections of the bombesin-saporin (SAP)-toxin, which selectively eliminates the GRPR-expressing cells. The intra-BLA [lateral (LA) and basal nuclei (BA) of amygdala] injection of bombesin-SAP before fear conditioning significantly enhanced cued, but not contextual fear memory. We did not observe any significant effect of post-training intra-BLA injections of bombesin-SAP on fear memory recall. Also, there were no significant effects of bombesin-SAP on acquisition of contextual and cued fear memory in mice injected bombesin-SAP into LA, BA and central amygdala (CeA), respectively. Also, we examined cued fear memory in the GRP knockout mice and found significant enhancement in their cued fear memory. These results support the idea that GRPR-expressing interneurons play an inhibitory role in acquisition of fear memory and suggested inhibitory effect by the GRPR-expressing GABA interneurons on fear memory requires both LA and BA but not CeA.

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ATS Poster of the Year Winner

Mathis C, Moreau P-H, Zerbinatti C, Goutagny R, Cosquer B, Geiger K, Kelche C, Cassel J-C (2012) Combined loss of entorhinal and basal forebrain cholinergic hippocampal inputs deeply impairs spatial navigation memory in C57BL/6J and hAPPxapoE mice. Neuroscience 2012 Abstracts 203.28. Society for Neuroscience, New Orleans, LA.

Summary: The hippocampus plays a key role in spatial learning and memory. Major inputs provided by the cholinergic basal forebrain (CBF) and the entorhinal cortex (EC) neurons are expected to modulate hippocampal functions. Surprisingly, the selective lesion of one or the other produces only moderate performance degradation in spatial navigation tasks, suggesting possible compensation provided by other hippocampal inputs. We therefore assessed the effects of single versus combined lesions of the EC (NMDA excitotoxin) and the CBF (mu-p75 saporin immunotoxin) on several forms of memory in C57BL/6 mice. Single lesions had moderate or no effects, while the combined lesions completely abolished long-term spatial memory retention in the water-maze and the Barnes-maze navigation tasks. Object recognition memory was selectively and profoundly affected by the loss of cholinergic neurons, whereas object location memory was only marginally affected by the lesions. These results suggest that the integrity of both the CBF and the EC is critical to establish an enduring spatial navigation memory. The synergistic interaction between the two lesions is particularly relevant to Alzheimer’s disease (AD) since both structures undergo severe degeneration in parallel to dramatic impairments in spatial navigation tasks. The apolipoprotein E4 (apoE4) allele, a major genetic risk factor for AD, has been proposed as a cholinergic deficit predictor and has been associated with larger EC atrophy in AD patients. Thus, the effects of single and combined EC and CBF lesions were evaluated on Barnes maze navigation performance in hAPPxapoE mice knocked-in for the human apoE3 or apoE4 gene allele on a (normal) human APP YAC transgenic background. Long-term spatial memory performances of hAPPxapoE3 and hAPPxapoE4 mice were dramatically affected by the CBF lesion and the combined lesions, but not by the EC lesion. A similar pattern of deficit was observed on learning performances in apoE4 not in apoE3 mice; the latter were only affected by the combined lesions. In conclusion, the apoE4 genotype had no effect on the consequences of EC and combined lesions, but it worsened the outcome of CBF lesions compared to the apoE3 genotype. Since the mice of the two genotypes showed similar loss of cholinergic neurons, our data may reflect a deleterious impact of apoE4 on the activity of the few surviving neurons (about 20%). Alternatively, our findings would also be consistent with impaired compensatory mechanisms following cholinergic loss which could depend on other hippocampal inputs such as the entorhinal cortex. Further analyses are underway to clarify this issue.

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

Hayward LF (2012) Neurotoxic lesion of CRF-R1 neurons in the amygdala selectively attenuates the heart rate response to acute stress in the spontaneously hypertensive rat. Neuroscience 2012 Abstracts 281.28. Society for Neuroscience, New Orleans, LA.

Summary: The magnitude of a person’s autonomic response to mental stress is predictive of one’s risk for the development of cardiovascular disease and has been linked to indicators of exaggerated neuronal activity in the amygdala. Recent evidence from our lab identified a link between changes in the expression of the neuropeptide corticotrophin-releasing factor (CRF) within the central nucleus of the amygdala (CEA) to exaggerated cardiovascular responses to acute stress in the spontaneously hypertensive rat (SHR). The present study was undertaken to evaluate the impact of selective lesion of CRF-R1 neurons in the amygdala on the cardiovascular response to acute air jet stress (AJS) in the SHR. Male SHR rats underwent local bilateral microinjections of 10 nanograms/200 nl per side of blank-saporin (n=4) or CRF-receptor (R1) targeting saporin (n=4) into the region of the CEA. Following 7-10 days of recovery and two days following arterial catheter instrumentation, animals underwent AJS testing. CRH-R1 lesion in the amygdala produced a small reduction in resting systolic blood pressure (160±6 vs 173±4 mmHg, p<0.1) but not change in heart rate (354±16 vs 352±+4 bpm). CRH-R1 lesion also significantly attenuated the mean rise heart rate in response to AJS (72±21 vs 130±13 bpm) and facilitated a more rapid heart rate recovery independent of any effect on the blood pressure response to AJS. The findings demonstrate for the first time that CRF-R1 activation in the amygdala selectively contributes to the elevated heart rate response to stress in individuals with hypertension, thus providing a link between the exaggerated activity in the amygdala and a specific cardiovascular response to stress.

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